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Overweight (bmi 85th percentile for age and sex) but otherwise healthy youth, 71 african american and 80 caucasian (aged 817 years), underwent assessment of glucose tolerance at the pediatric clinical and translational research center at children's hospital of pittsburgh . Body composition was measured by dual - energy x - ray absorptiometry (dexa; lunar, madison, wi). Plasma glucose, insulin, lipid profile, adiponectin, leptin, and the nontraditional cvd risk markers intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and e - selectin were measured as described previously (911). Proinsulin, fibrinogen, and high - sensitivity c - reactive protein (hs - crp) were measured at the esoterix endocrinology laboratory (calabasas hills, ca). For hs - crp, values> 10 mg / l were excluded because they may be a sign of occult infection or other systemic inflammatory process (10 of 151 participants) (12). Data are presented as means sd for normally distributed continuous variables, median (interquartile range) for non - normal continuous variables, and n (%) for categorical variables . Independent student t tests were used to compare normally distributed continuous subject characteristics, and pearson test was used to compare proportions . The study population was categorized according to quartiles of fasting and 2-h glucose and insulin levels to assess differences between the quartiles in cvd risk factors . Ancova, adjusted for age, sex, race, tanner stage (prepubertal and pubertal), and percent body fat (measured by dexa), was used to compare metabolic variables between the quartiles of glucose and insulin groups . All statistical tests were two - tailed, and p values of 0.05 were considered to be statistically significant . Plasma glucose, insulin, lipid profile, adiponectin, leptin, and the nontraditional cvd risk markers intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and e - selectin were measured as described previously (911). Proinsulin, fibrinogen, and high - sensitivity c - reactive protein (hs - crp) were measured at the esoterix endocrinology laboratory (calabasas hills, ca). For hs - crp, values> 10 mg / l were excluded because they may be a sign of occult infection or other systemic inflammatory process (10 of 151 participants) (12). Data are presented as means sd for normally distributed continuous variables, median (interquartile range) for non - normal continuous variables, and n (%) for categorical variables . Independent student t tests were used to compare normally distributed continuous subject characteristics, and pearson test was used to compare proportions . The study population was categorized according to quartiles of fasting and 2-h glucose and insulin levels to assess differences between the quartiles in cvd risk factors . Ancova, adjusted for age, sex, race, tanner stage (prepubertal and pubertal), and percent body fat (measured by dexa), was used to compare metabolic variables between the quartiles of glucose and insulin groups . All statistical tests were two - tailed, and p values of 0.05 were considered to be statistically significant . Of the 151 subjects, 71 were african american (59% female, 41% male; mean age 12.3 2 years; tanner stage 22.9% prepubertal, 77.1% pubertal; mean bmi 32.1 5.8 kg / m; bmi percentile 98.2 2.0; percent body fat 43.2 7.5%) and 80 were caucasian (54% female, 46% male; mean age 13.1 2.6 years; tanner stage 21.9% prepubertal, 78.8% pubertal; mean bmi 33.0 6.8 kg / m; bmi percentile 98.4.2 1.6; percent body fat 43.8 6.1%). There were no significant associations between quartiles of glucose levels, either fasting (mean glucose 77 4, 85 1, 90 1, and 94 2 mg / dl, respectively, in the lowest, 2nd, 3rd, and highest quartiles) or at 2 h (mean glucose 94 7, 108 3, 118 3, and 129 4 mg / dl, respectively, in the lowest, 2nd, 3rd, and highest quartiles), with any of the cvd risk factors (p for trend> 0.05) after adjusting for age, sex, race, tanner stage, and percent body fat . The median (interquartile range) values for fasting insulin levels was 13.7 (12.015.3), 21.0 (18.422.0), 28.7 (26.230.7), and 43.8 (37.853.7) iu / ml for quartiles 1, 2, 3, and 4, respectively . For 2-h insulin levels the values were 48.1 (30.752.9), 76.4 (69.184.2), 125.4 (107.0155.4), and 222.8 (202.0300.0) iu / ml for quartiles 1, 2, 3, and 4, respectively . Quartiles of insulin, fasting and at 2 h, were associated with heightened cvd risk factors . Systolic and diastolic blood pressure, triglycerides, vldl cholesterol, intracellular adhesion molecule-1, and leptin increased significantly with increasing fasting insulin; triglycerides, vldl - cholesterol, and leptin increased with increasing 2-h insulin . Hdl cholesterol decreased with increasing quartiles of fasting insulin (44 12, 42 9, 40 7, 39 9 mg / dl for quartiles <25, 25<50, 50<75 and 75, respectively; p for trend = 0.04) cvd risk markers according to fasting and 2-h insulin quartiles . Dbp, diastolic blood pressure; icam, intracellular adhesion molecule-1; sbp, systolic blood pressure . Our findings in overweight nondiabetic children demonstrate that 1) an increase in insulin quartiles, fasting and at 2 h, was associated with higher levels of a number of cvd risk factors; and 2) glucose quartiles were not associated with increased risk factors for cvd . With childhood obesity reaching epidemic proportions, there is a need to determine the best marker for early identification of cvd morbidity in this population . In our study, quartiles of glucose, both fasting and at 2 h, were not associated with heightened risk factors for cvd . It is arguable that, in children, glucose levels may need to be in the impaired fasting glucose or impaired glucose tolerance range in order to begin to observe a relationship with cvd risk factors . Insulin levels have also been described to play a significant role with regard to risk for cvd . In our study, increases in insulin quartiles, fasting and at 2 h, it has been postulated that diabetes and cvd may share an underlying cause, a theory known as the common soil hypothesis (1314). Insulin resistance has been proposed as central to both progression to type 2 diabetes and cvd . Our data, although cross - sectional, suggest that hyperinsulinemia may well be the first and earlier abnormality that presents in children at risk . We could speculate that this may lead to an increase in cvd risk factors and, with time, to glucose dysregulation . Prospective studies are needed, and interventions should be initiated early in life in overweight at - risk youths.
We herein report the exceptional case of a patient who died because of very early, disseminated kaposi sarcoma (ks) without skin lesions after allogeneic kidney transplantation . The unusual course as well as the absence of cutaneous metastases led to a challenging diagnostic workup of the patient . Moreover, ks developed under an immunosuppressive regimen using mechanistic target of rapamycin (m - tor) inhibition which is considered to be an effective treatment for ks . Ultimately, [f]2-fluoro-2-deoxy - d - glucose (fdg) positron emission tomography / computed tomography (pet / ct) allowed diagnosis of disseminated malignancy and might therefore be considered early in the management of patient at risk . A 52-year - old white with end - stage renal failure secondary to rapid progressive glomerulonephritis presented 4 months after first renal transplantation with undulating fever, acute gastroenteritis, axillary abscesses, and a strong reduction of his general state . Laboratory results revealed thrombocytopenia (108 10/l, reference range 166308 10/l), anemia (hemoglobin 87 g / l), acidosis, urinary tract infection with enterococcus faecalis, as well as acute kidney injury . Immunosuppressive induction therapy included tacrolimus, everolimus, steroids, and basiliximab whereas maintenance treatment (athena study protocol) consisted of tacrolimus (35 ng / ml), everolimus (38 ng / ml), and prednisolone 5 mg / day . Barr virus (ebv), hepatitis c virus, parvo b19, and human immunodeficiency virus was negative . The patient s general state improved, he was without fever, and thrombocytes and hemoglobin increased while creatinine decreased . Two months after discharge, the patient was hospitalized because of acute gastroenteritis, sepsis, and acute kidney injury again . On physical examination, no suspicious skin lesions or lymph nodes were noted . Laboratory analyses showed increased procalcitonin (2.9 ng / ml, reference range <0.5 ng / ml), c - reactive protein (10.5 mg / dl, reference range <0.5 mg / dl), creatinine (2.2 mg / dl, baseline creatinine 1.5 mg / dl), and lactate dehydrogenase (328 u / l, reference range 135255 u / l). G / l), thrombocytopenia (60 10/l), mild lymphocytopenia (0.95 10/l, reference range 1.263.35 10/l), and monocytosis (1.84 10/l, reference range 0.290.95 10/l). Staphylococcus epidermidis was isolated from multiple blood cultures and from the relapsed axillary abscess (shown by asterisk in figure 1b). Moreover, ebv polymerase chain reaction (pcr) testing was slightly positive (354 iu / ml) and clostridium difficile was identified in the stool . Sonography revealed splenomegaly (18 7.6 cm, also seen in ct, shown by asterisk in figure 1a) and cervical as well as reactive inguinal lymph nodes (2.3 cm). As the patient complained of progressive intolerance of everolimus, immunosuppressive therapy was modified (everolimus was stopped and tacrolimus was reduced). Persisting fever and coughing led to the performance of ct of the thorax excluding everolimus - induced pneumonitis and showing pulmonary emphysema and multiple enlarged but calcified mediastinal and hilar lymph nodes . Interestingly, the patient s condition improved, but persisting thrombocytopenia and anemia led us to puncture and biopsy the bone marrow (iliac crest biopsy). Toxic or infectious bone marrow suppression as well as folic acid deficiency was suspected . Two weeks later, the patient developed fever, massive thrombocytopenia (12 10/l), and acute kidney failure . Further diagnostics included combined pet / ct with fdg . Besides of the very intensive uptake measured in nearly all lymph node stations, in particular cervical, axillary, mediastinal, paraaortic, and inguinal, an pathological uptake was documented in the tongue, thyroid, and lung (figure 1b and c); the uptake pattern was indicative for malignancy (coronal slice of ct, maximum intensity projection (mip)pet, and fused coronal slice of fdg pet / ct). Extirpation of an inguinal lymph node (shown by asterisk in figure 1b) revealed fast proliferating ks . Slices of fluorodeoxyglucose pet combined with computed tomography (a: ct; b: pet; c: fusion of pet / ct). Besides a splenomegaly (yellow asterisk) a very intensive uptake was measured in nearly all lymph node stations . In particular, pathologic [f]2-fluoro-2-deoxy - d - glucose accumulation was detected in cervical, axillary, mediastinal, paraaortic, and inguinal lymph nodes (red asterisks) as well as in the tongue, thyroid, and lung (mip pet). Ct = computed tomography, mip = maximum intensity projection, pet = positron emission tomography . Although extremely rare (incidence below 1% within 15 years after renal transplantation), ks has been described to occur early (mean time to diagnosis: 426 days after renal transplantation). Ks is a vascular low - grade malignant tumor that is associated with human herpesvirus-8 (hhv-8) infection . Interestingly, in our patient, hhv-8 staining of the lymph node was positive, whereas serum pcr was negative (figure 2b). It typically manifests in mucocutaneous sites such as the skin or the oropharyngeal mucosa, in lymph nodes, and in visceral organs, most frequently in the respiratory and gastrointestinal tract . In our patient, typical, for example, lymph nodes, and atypical manifestations, for example, thyroid, were seen (only 5 cases worldwide). In the absence of skin lesions (only 5% of cases and exceptional in metastatic disease), ks often proves to be a challenging diagnosis because of missed recognition on routine imaging studies, unspecific systemic manifestations, for example, anemia, thrombocytopenia, and fever, or infectious complications, for example, abscesses, diarrhea, ebv reactivation, urinary tract infection, or sepsis, especially in the setting of our immunocompromised host . Awareness of ks may avoid delayed or potential misdiagnosis with deleterious consequences . In our patient, the combination of hhv-8 and immunosuppression promoted the development of ks . Notably, ks occurred under maintenance therapy with an m - tor inhibitor (everolimus). M - tor inhibition is considered to be effective for the treatment of ks because it is antiangiogenetic by reduction of vascular endothelial growth factor secretion and inhibition of formation of tumor vasculature . Histological evaluation of an inguinal lymph node (gross specimen, a) revealed only residual lymphatic tissue (low power 10, b) and showed a diffuse endothelial neoplasm with scattered lymphocytes and plasma cells . Immunohistochemistry for hhv-8 showed intense hhv-8 nuclear positivity and the final diagnosis of ks was established (40, d). Ks can be clearly visualized as areas of intense fdg uptake on a pet scan (figure 1b). As bone marrow biopsy specimens were unspecific in ks, unexplained cytopenias should lead to further work - up . Thus, fdg pet imaging can be advantageous in the management of patients with suspicious malignancy or unclear inflammatory / infectious diseases . Pet provides noninvasive whole - body diagnostics considerably assessing the extent of the disease (useful for both staging and restaging), that is, for the detection of further sites involved yet uncovered by conventional diagnostic methods . It might, therefore, be considered early in the management of patient at risk to reveal underlying disease (for timeline of the diagnostic workup and for key findings, see table 1). Herein, combined fdg pet / ct ultimately allowed diagnosis of disseminated malignancy . However, because of the late stage of ks on the time of diagnosis, it was too late for initiation of chemotherapy.
Intra - radicular disc herniations are rare disorders with only few cases reported in literature . In most of these cases there we present a case of completely intra - radicular disc which was misdiagnosed as nerve root tumor as there was no evidence of disc prolapse at the time of diagnosis . 51 year old male presented with history of severe back pain radiating to right lower limb since 11/2 month . Mri showed hypointense lesion completely inside the s1 root and a provisional diagnosis of nerve root tumor was done . At surgery, post operatively all symptoms of patient was relieved except dysesthesia in sole which lasted for a year post surgery . At 5 year conclusion: a diagnosis of intra - radicular disc should be considered in differential of nerve root tumor . Surgical excision of intra - radicular disc gives good clinical and functional results . Intervertebral disc herniations are very common although intra dural and intra radicular disc herniation are rare . More than 100 intra dural disc herniations are reported till date but only 17 cases of intra radicular disc herniations are reported [1 - 15] (table 1). All the reported cases show some evidence of disc prolapse in the adjacent areas indicating sequestered disc that is then engulfed into the dural sac . The mechanism of dural penetration in these cases is unclear, however some indication of a disc disease is always visible on radiological investigations (table 1). Many cases had history of prior spinal surgery, which may generate free disc material that was missed during surgery and cause intra - radicular herniation (table1). This is the first case of intra radicular disc herniation mimicking a nerve root tumor without any evidence of disc prolapsed or any surgery in recent past . This case was suspected as a case of nerve root tumor and intraoperatively the lesion was completely intraradicular with a clear space between the dura and the nerve root . We report details of this unique case review of literature showing details of cases of intra - radicular disc herniation reported till 2011 . 51 year old male presented with low back ache with radiation of pain to the right lower limb for 1 months duration . Patient was given epidural injection 3 weeks before he presented to us at another center . Currently he did not have relief with analgesics and physiotherapy and had no relief from epidural injection . There was no history of any previous interventions to the spine or bowel or bladder symptoms . Plantar reflex was equivocal on the right side and down going on the left side . Mri done showed hypointense lesion that appeared intraradicular in s1 root (arrow mark) with a clear layer of csf all around the lesion (fig . There was a clear gap between the dura and the fusiform swelling in the nerve root with no evidence of disc prolapse . The upper edge of the s1 lamina was nibbled and the swelling exposed . Due to the long intra spinal course of s1 root, the swelling could be very well exposed . The swelling was seen at a distance from the dura with a definite segment of normal looking nerve root in between (fig . Histopathology report was consistent with degenerated disc material with collagenous tissue showing characteristic cell clusters (fig . Bhistopathology of the specimen confirms it to be degenerated intervertebral disc with characteristic cell clusters post operatively the patient was relieved of his symptoms except for dysesthesia at the sole of foot which remained for one year post surgery . At 5 years follow up the patient has no complains and is doing all his functional activities normally . We could locate 17 cases of intra radicular disc prolapse that are reported in the literature (table 1). These were seen in relatively younger population with only three cases in age more than 60 years . Another important point was the level of such lesions which most commonly was seen at l5-s1 level . A probable reason may be that inracanicular length of s1 is much longer and thus more prone to disc herniation . Mut et al divided these lesions into two types; a- herniationin dural sac, b- herniation in preganglionic nerve root . In our case the lesion was of type b. in type b lesions the disc material may either lie between the two leaflets od dura (inter - dural or pseudointradural) or pierce both dural leaflets to be true intra - radicular . In pseudo - intradural cases there is no leakage of csf when the dura is excised to remove the disc material . In our case there was no csf leak indicating it to be pseudo - intradural variety of intra - radicualr disc herniation . Most of the cases there were evidences of previous interventions or evidences of disc prolapse in the nearby area (table1). Bilkra suggested that there are localized adhesions which may limit mobility of the nerve roots and thus allow for intra - radicular penetration by disc material . These adhesions probably occur as a result of several mechanisms, including traumatic irritation from a herniated disc, previous surgery and chronic local in - ammation . However many reported cases did not have previous surgery and in our case we could not find any adhesions and nerve root was really free from all sides . Dandy suggested that acute prolapse may cause acute pressure on the anterior wall of dura and may eventually erode it to reach intra - radicular . A combined hypothesis was suggested by mut et al where adhesions between anterior nerve root dura and the posterior longitudinal ligament lead to ischemia and thinning of dura which is then easily torn by extruding disc . Preoperative diagnosis of intra - radicular disc herniation is difficult, however some suggest to suspect it if mri shows rounded or fusiform shape of extruded fragment . Mainly the diagnosis in intraoperative and in cases where no extruded disc material is found, an intra - radicular herniation should be suspected . In present case there was no previous interventions and no evidence of disc prolapse in the nearby area . The swelling in the nerve root was at a distance from the dura, leaving a normal segment of root free . The disc material was found in between the nerve fibers rather than to one side . Again in our case mri was suggestive of an intraneural tumor and hence only a histopathological examination could provide a definitive final answer . As seen from table 1, all lesions were treated surgically and had uniformly good result except in two cases (one with infection and other continued to have neurodeficit although pain relief was complete). In our case too there was immediate relief of pain, however sensory dysesthesia of sole was seen postoperative till one year . At final this case represents the first histopathologically confirmed herniated disc sequestration in the nerve root in between the nerve fibers, at a distance from the dura mimicking a nerve root tumor . The surgeons must be aware of this condition, when they suspect nerve root tumor intra - radicular disc herniations can present without other signs of disc herniations and can mimic nerve root tumor.
Press - through packages (ptps) are commonly used to enclose drugs and are also increasingly seen in cases of foreign body in the digestive tract . Although commonly considered too small and soft to cause bowel damage, ptps tend to become caught and to pose a potential risk for perforation of the bowel because of their sharp edges . Removal should be attempted for a ptp in the esophagus or stomach, while computed tomography (ct) and early laparotomy should be considered if it passes through the pyloric ring and the patient develops symptoms . Efforts to prevent the swallowing of ptps should be an essential part of our everyday practice, particularly for elderly patients . Intestinal perforation resulting from accidental ingestion of a ptp has occasionally been reported . However, a search of the literature revealed no other cases of combined abdominal incisional hernia and perforative peritonitis due to accidental ptp ingestion . A 90-year - old woman, who had been aware of repeated abdominal herniation over several months, was admitted to the emergency department of national hakodate hospital with severe abdominal pain . She had a history of hysterectomy about 30 years previously and had developed swelling and redness in the resultant lower median abdominal scar . Serum biochemistry revealed the following: white cell count 3,700/mm (normal <9,500) (segmented: 90%), c - reactive protein 14.79 mg / dl (normal <0.5 mg / dl), and creatine phosphokinase 230 iu / l (normal <119 iu / l). Abdominal ct showed an edematous intestinal tract image over the median abdominal wall (fig . 1). Incarceration of abdominal incisional hernia was suspected and emergency surgery was performed . The small intestine was herniated and incarcerated about 100 cm orally from the terminal ileum, and a 2-mm perforation was present in the incarcerated small intestine . In addition, some white areas measuring 1 mm, which were considered to be the stimulation to intestinal wall by sharp corners of a ptp, were found in the intestinal wall . A ptp (fig . After dilation of the perforation to pass through, a ptp was removed through the perforation . After surgery, when we retrospectively reviewed the preoperative abdominal ct, we detected a high - density shadow that appeared to represent the ptp (fig . After physical rehabilitation she was discharged in stable condition on the 40th day of hospitalization . They consist of a lid coated with heat - sealed material on an aluminum leaf and a dome of vinylchloride . The dome is pushed externally to expel a tablet or capsule; therefore, it is called a press - through package . The mean size of a square ptp for a tablet is about 18 16 mm, and the mean size of a rectangular ptp for a capsule is about 30 16 mm . Since small square pieces of ptps with sharp edges may break off, they tend to be caught and to pose a potential risk of bowel perforation when inadvertently swallowed . In addition to lodging in the esophagus, mistakenly swallowed ptps have caused distal intestinal and colonic perforation or obstruction [2, 3]. Although previous reports have noted that 80 - 90% of swallowed foreign bodies are spontaneously passed and that less than 1% cause perforation, ptps might be associated with a greater risk of perforation . As in the present case, some patients may initially ignore accidental swallowing of a ptp and be subsequently admitted to hospital after deteriorating symptoms and signs [1, 5]. Radiological tests play a very important role in revealing the presence, location and nature of an ingested foreign body, thus enabling the best therapeutic approach . Hence, in most of the reported cases, the correct diagnosis was not made until operation or endoscopic examination [1, 3, 6, 7, 8]. However, hou et al . Emphasized that air trapped in the ptp can make it visible on x - ray . Their cases were diagnosed after early correct interpretation of these specific radiographic findings . In a study by traub et al . Fulford and tooley reported that a previously unnoticed ptp was visible when the preoperative abdominal radiograph was reviewed . In the present case, however, the ptp was not visible on the preoperative abdominal radiograph, even when it was reviewed postoperatively . Early ct examination should therefore be considered in symptomatic patients to evaluate possible complications, as ct is more sensitive than plain radiography in detecting foreign body ingestion [5, 10]. It has been reported that patients with a history of abdominal surgery are at risk of obstruction from a foreign body that may lodge at a pre - existing stenosis and perforate the intestine [1, 3]. However, no other cases of perforative peritonitis from an incarcerated incisional hernia associated with an inadvertently swallowed ptp have been reported . The ptp reached the incarcerated intestine while it was prolapsed, and then irritated and perforated the wall . Physical pressure of the incisional hernia by the clothes may also have contributed to bowel perforation by the ptp . Accidental ingestion of foreign bodies is not uncommon, particularly in older patients and among those who are blind or mentally impaired . Having seniors cut up sheets of ptp by themselves when preparing their medications is a real hazard because of the possibility that they will not take the medication out of the package, and this practice should be avoided . We therefore suggest that elderly people should be given their tablets after they have been unwrapped by a nurse or family member . Further, changes in packaging design might help prevent accidental ingestion or minimize adverse effects if ingestion does occur . Therefore abdominal ct should be performed routinely when a patient with acute abdominal pain is admitted . In addition, it is essential to adequately explain to patients how to take their medications . Improved design to avoid accidental ingestion and better training of patients in the use of ptps should decrease the incidence of this problem . In conclusion, although relatively rare, ptps are being seen increasingly in cases of foreign body in the digestive tract . Improved design to avoid accidental ingestion and better training of patients in the use of ptps could avoid complications of this condition, which is most likely to occur in elderly patients.
Generally, the common method for straightening the patient's convex profile in cases with bimaxillary dentoalveolar protrusion is orthodontic treatment with an extraction of the upper and lower first premolars and a retraction of the anterior segments under maximum anchorage [13]. Maximum anchorage means less than 25 per cent of space closure in the extraction space via posterior anchorage loss . Therefore, an accurate prediction of the amount of anchorage loss during retraction of the anterior teeth is critical for treatment planning and selection of the appropriate mechanics . Traditionally, conventional anchorage reinforcements (cars), such as cervical or high pull headgear (hg) and/or transpalatal arch (tpa), have been used for this purpose . Recently, there has been a dramatic increase in the use of orthodontic mini - implants (omis), (also known as temporary anchorage devices (tads)) to allow maximum anchorage, decrease the need for patient compliance, and simplify the treatment procedure [512]. Although there are several reports on the comparative evaluation between skeletal anchorage and conventional anchorage during an en masse retraction of the anterior teeth [1316], the subjects in these studies were heterogeneous due to use of patients with class ii malocclusion and class i malocclusion / bimaxillary dentoalveolar protrusion . In cases with class ii malocclusion, the correction of class ii molar relationship to class i key requires distalization of the upper molars or mesialization of the lower molars hence, the effect of anchorage preservation of the posterior teeth cannot be compared in combined samples with class ii malocclusion and class i bimaxillary dentoalveolar protrusion . Therefore, this study compared the anchorage loss of the upper posterior teeth and the amount of retraction of the upper anterior teeth in class i malocclusion patients with bimaxillary dentoalveolar protrusion and minimal crowding between car and omi . The null hypothesis was that there would be no significant differences in the anchorage loss of the posterior teeth and the amount of retraction of the anterior teeth using either omi or car . The initial sample in this retrospective study consisted of korean female young adult patients (n = 125; mean age = 23.32 years; range = 18 ~ 35 years) who had class i malocclusion with dentoalveolar protrusion and minimal crowding that required maximum posterior anchorage . The final samples (n = 40, table 1) were selected from the initial ones according to the following criteria: cases with craniofacial deformities including a cleft lip and palate and other syndromes, facial asymmetry (chin point deviation> 4 mm), and supernumerary or more than two missing teeth were excluded; women older than 17 years to eliminate the gender- and growth - related bias; class i molar relationship, normal overbite (> 0 mm, <4 mm), labioversed upper incisor (u1 to fh plane> 115), minimal crowding in each arch (<4 mm), and a skeletal class i malocclusion (0 <anb <4); lip protrusion (lower lip to ricketts' esthetic line> 2 mm); fixed appliance and archwire: mbt brackets (.022 slot, 3m - unitek, monrovia, ca, usa) with .019 .025 inch stainless steel wire for en masse retraction of the upper anterior teeth.in group 1 (n = 20), the upper and lower first premolars were extracted, car, such as hg (1214 hours / day, 350 gram / side) and tpa were applied, and the sliding mechanics described in the mbt technique [1719] were performed . All the patients with hg showed good compliance by the doctor's instruction.in group 2 (n = 20), the upper and lower first premolars were extracted, an omi (dual - top anchor system, jeil medical co, seoul, korea; 1.6 mm diameter, 8 mm length, self - drilling type) was applied and the sliding mechanics were performed . The omi was placed in the buccal attached gingiva areas between the upper second premolar and first molar adjacent to the mucogingival junction of the upper arch after leveling and alignment . Ni - ti closed coil springs (medium, 9 mm, jinsung, seoul, korea) stretched from the omi head to the hook on the archwire between the upper lateral incisor and canine were used from two weeks after omi installation . If the omi was unstable or failed, a new one was installed at the buccal attached gingiva between the upper first and second molars . Fixed appliance and archwire: mbt brackets (.022 slot, 3m - unitek, monrovia, ca, usa) with .019 .025 inch stainless steel wire for en masse retraction of the upper anterior teeth . In group 1 (n = 20), the upper and lower first premolars were extracted, car, such as hg (1214 hours / day, 350 gram / side) and tpa were applied, and the sliding mechanics described in the mbt technique [1719] were performed . All the patients with hg showed good compliance by the doctor's instruction . In group 2 (n = 20), the upper and lower first premolars were extracted, an omi (dual - top anchor system, jeil medical co, seoul, korea; 1.6 mm diameter, 8 mm length, self - drilling type) was applied and the sliding mechanics were performed . The omi was placed in the buccal attached gingiva areas between the upper second premolar and first molar adjacent to the mucogingival junction of the upper arch after leveling and alignment . Ni - ti closed coil springs (medium, 9 mm, jinsung, seoul, korea) stretched from the omi head to the hook on the archwire between the upper lateral incisor and canine were used from two weeks after omi installation . If the omi was unstable or failed, a new one was installed at the buccal attached gingiva between the upper first and second molars . Finished with a class i canine and molar relationship.normal overbite and overjet (> 2 mm and <4 mm, resp .) Normal overbite and overjet (> 2 mm and <4 mm, resp . ). Lateral cephalometric radiograms taken before (t0) and after treatment (t1) were traced and digitized using a graphic tablet (wacom co ltd, vancouver, bc, canada) and v - ceph program (cybermed, seoul, korea) by one operator (yh kim). Seven skeletal and dental (figure 2) and ten anchorage variables (figure 3) were measured to the nearest 0.01 mm and 0.01. six randomly selected sets of cephalograms from groups 1 and 2 were retraced and redigitized after two weeks to determine the error of measurement . There was no significant difference between the two measurements (p>.05; error of the linear measurement, <0.98 mm; error of the angular measurement, <1.01). Mann - whitney test was performed to compare the differences at t0 and t1 stages between two groups and to assess the amount of changes in the anchorage variables during the t0 and t1 stages between the two groups . The subjects' age, amount of crowding of the upper arch, inclination of the upper incisor to fh plane at t0 stage, and treatment duration were not different between the two groups (table 1). The anteroposterior skeletal variables, such as sna, snb, anb, apdi, and downs' facial plane angle at the t0 stage, did not show significant difference between two groups (table 1). However, group 2 had a more hyperdivergent pattern than group 1 (fma, p <.05 and bjrk sum, p <.01, table 1). At the t0 stage, the anchorage variables, which describe the perpendicular distance from the upper central incisor or upper first molar to the vertical reference line (u1e - sag, u1a - sag, u6m - sag, and u6a - sag), and the angulation of the upper first molar to the horizontal reference line (u6 to pp) were not different between two groups (table 2). The amounts of retraction of the upper incisor edge was significantly larger in group 2 than group 1 (9.5 mm: 7.1 mm, p <.05, table 3). However, the upper incisor moved in a relatively controlled tipping manner in both two groups because the sagittal changes in the root apex of the upper incisor revealed 1.1 to 1.6 mm of lingual movement (table 3). In terms of the posterior anchorage loss, group 2 showed significantly less posterior anchorage loss than group 1 (u6m - sag, 0.2 mm: 2.2 mm, p <.05; u6a - sag, 0.3 mm: 2.4 mm, p <.01; table 3). In the vertical aspect, there was opposite vertical movement in u1 and u6 between groups 1 and 2: intrusion of the upper central incisor and first molar in group 2 and extrusion of these teeth in group 1 (u1e - ver, 0.9 mm intrusion: 0.7 mm extrusion, p <.05; u6f - ver, 1.0 mm intrusion: 0.9 mm extrusion, p <.05; table 3). This study examined how much omi could provide better posterior anchorage preservation than car and compared the amount of retraction of the upper anterior teeth during en masse retraction of the upper anterior teeth in cases with class i malocclusion which needed maximum anchorage . In contrast to the subjects of the other comparative studies about the effect of omi and car during en masse retraction of the anterior teeth [1316], the subjects of this study were homogenous (samples with class i malocclusion with bimaxillary dentoalveolar protrusion and minimal crowding only). Owing to the different treatment strategies and biomechanics for distalization of the upper molars in class ii malocclusion, the effect of anchorage preservation of the posterior teeth cannot be evaluated precisely in the combined samples with class ii and class i malocclusion . Lack of a significant difference in the treatment duration between groups 1 and 2 (28.0 months versus 25.0 months, table 1) suggests that omi might not reduce the treatment duration significantly in patients who require the maximum posterior anchorage, which is in accordance with ma et al . . The more hyperdivergent pattern in group 2 than group 1 (table 2) means that the omi produced more stable posterior anchorage in this type . Patients with a hyperdivergent facial type generally have weaker natural posterior anchorage than those with a hypodivergent one [20, 21]. However, in the present study, there was significantly less posterior anchorage loss in group 2 than group 1 (u6m - sag, 0.2 mm versus 2.2 mm, p <.05; u6a - sag, 0.3 mm versus 2.4 mm, p <.01; table 3). This suggests that omi can produce superior anchorage preservation in spite of the hyperdivergent pattern . When considering the upper premolar extraction space as 8.3 to 8.4 mm, the levels of posterior anchorage loss in group 2 (0.2 mm, table 3) were extremely low, whereas the levels were more than 25% of the extraction space in group 1 (2.2 mm, table 3). Also demonstrated the effect of omi for maximum posterior anchorage loss in the treatment of maxillary dentoalveolar protrusion, and upadhyay et al . Reported no anchorage loss in either the horizontal or vertical direction in mini - implants compared to conventional methods . In the present study, after treatment, the upper incisors were slightly more upright in the omi group than the car group (u1-fh, p <.01, table 2 and u1-pp, p <.01, table 3), which is in accordance with ma et al . . The findings that the upper incisor showed significantly larger amounts of retraction in group 2 than group 1 (9.5 mm: 7.1 mm, p <.05, table 3) and that the upper incisor was intruded in group 2 and extruded in group 1 (0.9 mm intrusion: 0.7 mm extrusion, u1e - ver, p <.05; table 3) suggest that the omi can demonstrate its ability to intrude the upper anterior teeth during retraction of the upper anterior teeth due to distal and intrusive force vector, which is in accordance with previous reports [15, 16]. Group 2 showed simultaneous intrusion of the upper first molar and upper central incisor (u6f - ver, 1.0 mm intrusion, p <.05; u1e - ver, 0.9 mm intrusion, p <.05, table 3). Omi might apply a distal and intrusive vector to the entire upper arch during en masse retraction of the upper anterior teeth . This appears to be due to the direction of pull by the ni - ti closed coil spring from the omi head to the hooks on the upper archwire and eventually can help correct the gummy smile and counterclockwise rotation of the mandible, particularly in patients with a hyperdivergent face . Further studies will be needed to determine the effect of omi on the anchorage preservation in various clinical situations, such as deep overbite, open bite, and severe overjet, negative overjet . Although the omi could not reduce the treatment duration, it provided better maximum posterior anchorage and greater retraction of the upper anterior teeth than car in spite of hyperdivergent pattern . Therefore, the null hypothesis was rejected . In addition, omi led to an intrusion of the upper central incisor and first molar, whereas car resulted in extrusion of these teeth.
Hemodialysis (hd) sustains life for more than 1 million patients with end - stage renal disease (esrd) worldwide and has been doing so since 1980 . Therapeutic hd removes undesired solutes (potassium, urea, and phosphorus) primarily via diffusion and partially via convection through a semipermeable membrane . Dry weight is defined as the lowest body weight that a patient can tolerate without the symptoms of hypotension until the next dialysis . Dry weight is achieved by removing interdialytic weight gain via ultrafiltration during each hd session . The ultrafiltration rate and the degree of blood volume refill from the interstitial compartment during hd might cause intradialytic hypotension (idh), which is defined as a relative or absolute decline in blood pressure (bp), as well as the presence of specific symptoms . Patients undergoing hd cannot tolerate as great a decrease in blood volume as healthy people can . The presence of cardiac disease and autonomic neuropathy can exacerbate idh and can compromise peripheral blood circulation . It has been reported that the patients undergoing hd showed choroidal perfusion defect on fundus angiography (ffa) and indocyanine green angiography (icga) [46]. Traditional imaging modalities, such as ultrasonography (usg), ffa, and icga, are insufficient for quantitative assessment of the choroid . More recent studies have focused on changes in choroidal thickness (ct) and retinal thickness following hd using different types of commercially available spectral domain optical coherence tomography (sd - oct) in patients with esrd . Although jung et al . Reported a significant increase in ct after hd, two other studies reported a significant decrease in ct after hd [9, 10]. These inconsistent findings indicate that the effect of hd on ct is not clear . The present study, therefore, aimed at measuring changes in ct in esrd patients after a hd session . This prospective, cross - sectional study included 41 patients with stage 5 esrd who were undergoing hd . Because of the high correlation between all parameters in the right and left eyes, only the patients' right eyes were analyzed . Pathologic conditions that could alter choroidal structure and ct, such as macular degeneration, smoking, ocular trauma, ocular surgery, ocular inflammation, and refractive errors outside 5 to + 3 d, were excluded . The bicarbonate used was diazole with a dialysate flow rate of 500 ml min and blood flow rate of 250300 ml min . Systolic and diastolic blood pressure (sbp and dbp, resp .) Were measured every hour during hd . Induced hypoglycemia, all patients received 200 cc of 5% dextrose solution at the end of hd . All patients underwent detailed ophthalmologic examination, including best - corrected visual acuity, intraocular pressure (iop) before and after hd (tonopachy nt-530p, nidek co., ltd ., tokyo, japan), and lens status . Ct scanning was performed using rtvue sd - oct system (rtvue - xr 100 avanti software v.6.1, optovue, inc ., the repeated oct measurements were obtained by an operator (a) at approximately 0700 and 1100, respectively, to avoid diurnal variations . The system utilized a wavelength of 840 10 nm and was capable of 26,000 a - scans s. tissue depth resolution was 5.0 m with transverse resolution of 15 microns . The patients were informed to fixate the internal fixation light of the rtvue sd - oct during image acquisitions . After the patients' heads were correctly positioned, a fovea - centered, 6 mm, horizontal high definition line scan (4096 a - scan / frame line) was obtained with the chorioretinal line mode . Postdialytic scans were obtained using the follow - up mode to obtain the images from the previous location . All images underwent manual segmentation by two researchers (sinan alkan and tolga bier) who were blinded to the patients . Ct was measured perpendicularly from the outer edge of the retina pigment epithelium to the inner sclera, as previously reported . Ct was measured at the fovea and at 1.5 mm nasal, 3.0 mm nasal, 1.5 mm temporal, and 3.0 mm temporal to the fovea . The study was performed in accordance with the tenets of the declaration of helsinki; the study protocol was approved by the dkapi yildirim beyazit training and research hospital ethics committee, ankara, turkey . Written informed consent the normality of the distribution of data was determined using the kolmogorov - smirnov test . There was a high correlation between the patients' right and left eyes; therefore, only right - eye data were used for further analysis . Descriptive statistics are presented as mean sd . For continuous data, student's paired t - test and pearson's coefficient test were utilized to compare pre - hd and post - hd data . The level of statistical significance was set at p <0.05 . The right eyes of 41 patients (61% female and 39% male) with esrd were included . Mean age of the patients was 53.2 14.6 years and mean duration of hd treatment was 42.81 30.38 months . Eight of the 41 patients had diabetes mellitus (dm) (table 1). Mean body weight decreased significantly from 65.77 10.50 kg to 63.65 10.41 kg after hd (p <0.001). Sbp and dbp decreased significantly following hd; the differences were 21.63 23.06 mmhg and 14.00 21.87 mmhg, respectively . Mean subfoveal ct (sfct) decreased significantly from 254.59 84.66 m to 229.34 77.79 m (p <0.001) after hd . There was also a significant decrease in all measurement points from the fovea (table 3). There was no difference in ct between the patients with and without dm before and after hd . The mean sfct was 241.88 99.57 m and 257.67 82.09 m, respectively, in the patients with dm and those without dm (p = 0.285). After hd, the mean sfct was 203.0 84.96 m and 235.73 75.96 m, respectively, in patients with dm and those without dm (p = 0.707). Associations between the difference in sfct and the other parameters, including age, gender, dm, duration of hd, dry body weight, serum osmolarity, plasma osmotic colloid pressure, ultrafiltration volume, and the percentage of the change in iop, sbp, dbp, and body weight, were evaluated (table 4). There was a moderate positive correlation between changes in sfct and the percentage of the changes in dbp (r = 0.311, r = 0.097, and p = 0.047). Stepwise multivariate linear regression analysis showed that only change in dbp could explain 14% of the changes in sfct (r = 0.137; p = 0.022). The primary goal of hd is to maintain the kidneys' excretory functions in esrd patients . During a hd session, ultrafiltration removes excess fluid from plasma, which leads to blood volume depletion, an increase in the plasma protein concentration (increase in plasma colloid osmotic pressure), and a decrease in serum osmolarity . Blood volume depletion is equilibrated by vascular refilling from the interstitial and intracellular space [1, 12]. The choroid has a rich vascular network and the highest blood supply per organ area . In the present study, ct decreased significantly in the subfoveal, nasal, and temporal choroidal regions after hd . Studied 28 eyes in 19 patients with esrd and reported that sfct increased following a hd session and was correlated with a decrease in sbp . They proposed that the increase in ct may be associated with the choroidal autoregulatory control of ocular hemodynamics, shifting of fluid, and molecules between the plasma and choroidal interstitium . The difference between two studies may be related to the methodology used to measure ct and the demographic variation of the studies' population . Chen and coauthors reported that image j software showed better repeatability and agreement than the heidelberg eye explore software . To the best of our knowledge, there was not any study which was comparing the intrinsic softwares of two oct devices and image j software . Secondly, coexisting systemic diseases such as dm may have the influence on the changes in ct after hd . It may be related to the difference in the incidence of dm in two studies . While 46.4% of patients had dm in the first study, only 19.5% of the patients had dm in our study . On the other hand, yang et al . Observed a decrease in sfct and iop following hd . They suggested that choroidal blood flow is poorly autoregulated; a decrease in volume in the choroidal vascular bed may lead to a decrease in ct . Likewise, ula et al . Reported a significant decrease in sfct and nasal and temporal ct after hd but not in iop levels . The researchers posited that the observed choroidal thinning may have been related to ultrafiltration - induced hypovolemia and increased plasma colloid osmotic pressure . The thinning of ct after hd may be caused by the changes in autoregulation of the choroidal blood flow and increased plasma colloid osmotic pressure . Additionally, in the present study, the ct thinning was correlated with a decrease in dbp . Furthermore, stepwise multivariate analysis showed that dbp is associated with 14% of the changes in sfct . Based on the association between the changes in ct and dbp, we hypothesized that the ct thinning may be a result of the increase in choroidal vascular and nonvascular smooth muscle contraction due to activation of the sympathetic autonomic nervous system . It is well known that retinal vascular circulation is autoregulatory and that choroidal circulation is controlled primarily by the extrinsic autonomic system . The parasympathetic efferent nerves acting on vascular and nonvascular smooth muscle of the choroid tissue may lead to the increase in the choroidal blood flow . Sympathetic activation of choroidal smooth muscles leads to vasoconstriction and can cause ct to decrease . It is well known that under physiological circumstances loss of blood volume activates the sympathetic system, initially leading to an increase in peripheral vascular resistance (due to constriction of resistance vessels), an increase in heart rate and myocardial contractility, and constriction of capacitance vessels . During hd sympathetic activation it is necessary to initiate compensatory mechanisms and to maintain blood pressure, especially via an increase in the heart rate and peripheral vasoconstriction . Although healthy individuals can tolerate a loss of 20% of blood volume before hypotension occurs, patients undergoing hd have a greater risk of hypotension and exhibit high interindividual variability and intraindividual variability in response to blood volume depletion . The main causes of hypotension as a complication of hd are thought to be acute hypovolemia during ultrafiltration and inadequate compensatory mechanisms, including autonomic dysfunction, diastolic and systolic dysfunction, decreased plasma osmolality, a decrease in extracellular fluid volume with inadequate plasma, impaired venous compliance, and decreased cardiac reserve [3, 16, 17]. Consequently, the choroidal vascular tonus changes lead to a decrease in ct . In the present study, it is known that blood volume depletion causes diastolic dysfunction, whereas contractility dysfunction of the heart leads to systolic dysfunction . Reported that the mean dbp at the start of hd treatment and mean ultrafiltration volume were associated with intradialytic morbid events (mostly in hypotension - prone patients). We suggested that the decrease in dbp may be related to the idh which activates sympathetic nervous system to avoid hypotension . Recently, farias et al . Reported significantly thinner ct in dm patients with microalbuminuria and suggested that decreased ct may be a sign of microvascular choroidal damage to the eye even before clinical signs become apparent . Yang et al . Observed that although ct was thinner in dm patients, the difference was insignificant in the patients with dm and without dm . We agree with earlier reports that suggest that a decrease in ct following hd may be associated with vascular damage and autonomic nervous system disruption in dm patients . The effect of hd on iop remains unclear [2325]. In the present study, plasma colloid osmotic pressure and serum osmolarity may have an effect on iop changes, but additional research is needed to determine the precise effect of hd on iop . The present study has several limitations . Only predialysis serum osmolarity and plasma colloid serum osmotic pressure were measured, which limited our ability to analyze the correlation between changes in those parameters and sfct . In a previous study, ct was shown to have a negative correlation with axial length . In addition, axial length and anterior segment parameters such as anterior chamber depth that may have an influence on ct were not measured . In conclusion we suggest that sympathetic activation triggered by blood volume depletion to prevent hypotension might cause choroidal vascular and nonvascular smooth muscle constriction, which leads to a decrease in ct . The decrease in dbp and sfct following hd may be a consequence of systemic compensatory mechanisms . Additional large - scale randomized studies are needed to more fully determine the effect of hd on ct and the relationship between ct and changes in systemic parameters.
The online version of this article (doi:10.1007/s40261 - 014 - 0260 - 8) contains supplementary material, which is available to authorized users . Administration of miglitol to four patients with type 2 diabetes receiving ongoing insulin treatment showed beneficial effects on postprandial hyperglycemia.based on the continuous glucose monitoring results, the coadministration of anagliptin with miglitol resulted in additional improvements in glycemic control in three of the patients.c - peptide, glucagon, and total and active glucagon - like peptide-1 and glucose - dependent insulinotropic peptide responded differently to the study medications for each patient . Dipeptidyl peptidase-4 (dpp-4) inhibitors, by inhibiting dpp-4 enzymatic activity, increase active glucagon - like peptide-1 (glp-1) and glucose - dependent insulinotropic polypeptide (gip) levels and improve hyperglycemia in a glucose - dependent manner by increasing serum insulin and decreasing serum glucagon levels in diabetic patients [16]. Alpha - glucosidase inhibitors (a - gis), another type of oral antidiabetic drug, also attenuate postprandial blood glucose fluctuations by delaying the absorption of digested carbohydrates from the small intestine [79]. Considering the different mechanisms of dpp-4 inhibitors and -gis, their use in combination therapy is promising for improving glycemic control [10, 11]. The present case study aimed at examining the efficacy, through the use of a continuous glucose monitoring system (cgms) [1214] and hormone measurements, of a dpp-4 inhibitor (anagliptin), and an -gi (miglitol) when added to ongoing insulin treatment in patients with type 2 diabetes mellitus . The baseline characteristics of the four japanese inpatients with type 2 diabetes in this study are summarized in table 1 . The study protocol was approved by the ethics committee of the national center for global health and medicine (ncgm), and written informed consent was obtained from each subject . The study was conducted in accordance with the ethical principles stated in the declaration of helsinki and guidelines for good pharmacoepidemiology practice . All patients were admitted to the diabetes ward at the ncgm and equipped with a cgms device (medtronic minimed, cgms - gold, minneapolis, mn, usa). Prior to study initiation, oral medications that might affect the results of the study, such as -gis or glinides, were discontinued for at least 5 days . A once - daily injection of insulin glargine was continued for all patients throughout the study without changing the dosage and timing of injections (table 1). The first blood samples, as for baseline data, were collected on day 1 . Subsequently, miglitol (50 mg three times a day, just before each meal) was administered on days 2 and 3 . On days 4 and 5, anagliptin (100 mg / day) was given before breakfast, in addition to miglitol . Blood samples were collected on the first, third, and fifth days of the study . Measurements of plasma glucose, serum c - peptide, plasma glucagon, total and active glp-1, and total and active gip levels were conducted using blood samples that were drawn at five time points (0, 15, 30, 60, and 120 min) after breakfast following a 14-h overnight fast . Active glp-1 concentrations were measured using an enzyme - linked immunosorbent assay (elisa) kit (millipore corp . 2) assay kit with an electrochemiluminescence (ecl) method (meso scale discovery, rockville, md, usa). Active gip142 and inactive gip342 were simultaneously measured using nanoflow liquid chromatography tandem mass spectrometry (lc ms / ms). All sample measurements, except those for total and active gip, were performed by mitsubishi chemical medience corporation (tokyo, japan). Total and active gip were measured by sanwa kagaku kenkyusyo co., ltd (aichi, japan). Serum c - peptide levels were measured using a chemiluminescent immunoassay (clia), and plasma glucagon was measured using a double - antibody radioimmunoassay (ria). The area under the curve (auc) values for these hormones after meal ingestion were calculated using the trapezoidal rule.table 1patient characteristicscharacteristiccase 1case 2case 3case 4age (years)63807783sexmalemalemalefemalebmi (kg / m)26.226.522.124.8diabetes duration (years)815228hba1c (%) 7.18.07.78.724-h urinary c - peptide (g / day)45.236.49.759.8oad use before the studymetformin-gi + glinide-ginoneinsulin use before the studyins l + ins gins gins l + ins gins goad use during the studymetforminnonenonenonedosage and timing of insulin injection during the studyins g (10 units) before bedtimeins g (5 units) before breakfastins g (5 units) before lunchins g (5 units) before bedtime bmi body mass index, hba1c glycosylated hemoglobin, oad oral antidiabetic drug, -gi -glucosidase inhibitor, ins l insulin lispro, ins g insulin glargine patient characteristics bmi body mass index, hba1c glycosylated hemoglobin, oad oral antidiabetic drug, -gi -glucosidase inhibitor, ins l insulin lispro, ins g insulin glargine figure 1 shows the representative cgm results of each patient . In case 1, the glucose levels and fluctuations after miglitol administration were not remarkably attenuated but were attenuated when miglitol and anagliptin were coadministered . In case 2, the glucose levels and fluctuations were not remarkably attenuated even after the administration of miglitol alone or coadministration of miglitol and anagliptin . In cases 3 and 4, the glucose levels and fluctuations after miglitol administration were attenuated, and this attenuation was even more pronounced with the combination of miglitol and anagliptin . The accuracy of these measurements was reflected in the mean and standard deviation (sd) values of the cgm measurements (table 2).fig . 1continuous glucose monitoring (cgm) results for cases 14 under the conditions of insulin administration, coadministration of insulin and miglitol, and coadministration of insulin, miglitol, and anagliptintable 224-h glucose levels measured by continuous glucose monitoring (cgm)treatmentglucose level (mg / dl)case 1case 2case 3case 4insulin alone176 36189 42234 91261 37insulin + miglitol184 30216 52165 36220 26insulin + miglitol + anagliptin137 20208 37162 27198 16values are expressed as mean sd continuous glucose monitoring (cgm) results for cases 14 under the conditions of insulin administration, coadministration of insulin and miglitol, and coadministration of insulin, miglitol, and anagliptin 24-h glucose levels measured by continuous glucose monitoring (cgm) values are expressed as mean sd the time - course levels of plasma glucose, serum c - peptide, plasma glucagon, total and active glp-1, and total and active gip and the auc values for these hormones for up to 120 min after meal ingestion are summarized in supplementary figures 25 and table 3 . Under the fixed dosage and timing of the insulin injection, miglitol administration attenuated the postprandial increments in plasma glucose levels in all of the patients . When anagliptin was coadministered with miglitol, further attenuation of postprandial increments in glucose was observed in case 1, but no remarkable changes in postprandial glucose levels were observed in the other patients . Miglitol administration attenuated the postprandial increments in c - peptide levels in all of the patients . When anagliptin was coadministered with miglitol, compared with the sole administration of miglitol, no remarkable changes in postprandial c - peptide levels were observed in cases 1 and 2, but slight increases were observed in cases 3 and 4 although the c - peptide levels in cases 3 and 4 were much lower than in the other patients . Miglitol administration had no observable effect on blood glucagon levels after meal ingestion in all patients except case 1 . When anagliptin was administered in addition to miglitol, compared to miglitol alone, no remarkable changes in glucagon levels were observed in cases 2 and 3, but a reduction in glucagon levels was observed in cases 1 and 4.table 3area under the curve (auc) values during the 120-min meal testparametercase 1case 2case 3case 4glucose auc (10 mg / dl120 min) insulin22.231.623.833.9 insulin + miglitol18.924.414.722.2 insulin + miglitol + anagliptin15.324.014.625.8c - peptide auc (nmol / l120 min) insulin132.4109.535.567.3 insulin + miglitol93.686.116.942.5 insulin + miglitol + anagliptin92.188.624.661.3glucagon auc (10 ng / l120 min) insulin17.517.312.411.9 insulin + miglitol15.416.813.012.3 insulin + miglitol + anagliptin13.718.411.810.8total glp-1 (10 pmol / l120 min) insulin26.829.97.314.4 insulin + miglitol21.419.811.616.9 insulin + miglitol + anagliptin22.129.39.917.0active glp-1 (10 pmol / l120 min) insulin5.13.51.43.2 insulin + miglitol3.11.91.43.6 insulin + miglitol + anagliptin8.710.72.56.8total gip (10 pmol / l120 min) insulin278.6318.2131.773.0 insulin + miglitol92.0120.558.661.6 insulin + miglitol + anagliptin150.773.628.637.7active gip (10 pmol / l120 min) insulin109.660.031.931.0 insulin + miglitol19.224.28.48.1 insulin + miglitol + anagliptin97.544.09.818.1 glp-1 glucagon - like peptide-1, gip glucose - dependent insulinotropic polypeptide area under the curve (auc) values during the 120-min meal test glp-1 glucagon - like peptide-1, gip glucose - dependent insulinotropic polypeptide miglitol administration decreased active glp-1 levels in cases 1 and 2, but not in cases 3 and 4 . However, when anagliptin was administered in addition to miglitol, all of the patients showed increased active glp-1 levels compared to when miglitol alone was added . The auc values for active glp-1 with the coadministration of miglitol and anagliptin tended to be larger than the auc values for active glp-1 when miglitol alone was added (p = 0.07; wilcoxon signed - rank test). Compared to when miglitol alone was added, coadministration of miglitol and anagliptin showed higher active gip levels in all patients, with varying patterns . The active: total glp-1 ratio and active: total gip ratio in each patient are shown in supplementary figures 6 and 7, respectively . Miglitol administration showed lower or at least similar active: total glp-1 ratios and active: total gip ratios in all patients, whereas coadministration of miglitol and anagliptin showed remarkably higher active: total glp-1 ratios and active: total gip ratios in all patients . In the present study, administration of miglitol to four patients with type 2 diabetes receiving ongoing insulin treatment showed beneficial effects on postprandial hyperglycemia . Based on the cgm results, the coadministration of anagliptin with miglitol resulted in additional improvements in glycemic control over the entire day in three of the patients, including lower glucose levels and attenuated glucose fluctuations . In addition, administration of only miglitol or in combination with anagliptin reduced postprandial c - peptide levels in all patients we studied, which might be explained by the associated decrease in blood glucose levels . The reason underlying the lack of a further effect on glycemic control with anagliptin co - administration in case 2 is not clear . A previous study conducted in japanese patients with type 2 diabetes indicated that treatment with a different dpp-4 inhibitor, sitagliptin, lowered glycosylated hemoglobin (hba1c) levels, especially in those with higher baseline hba1c levels, lower body mass indices (bmis), and shorter durations of diabetes . Therefore, the combination of a high hba1c level, relatively high bmi, and long history of diabetes in case 2 may have affected the efficacy of anagliptin in this patient to some extent . Regarding glucagon levels, the effect of miglitol - only administration was varied; however, when anagliptin was coadministered with miglitol, all of the patients, except case 2, showed a tendency for suppressed glucagon secretion . Theoretically, dpp-4 inhibitors, including anagliptin, increase glp-1 levels, and glp-1 lowers glucose levels not only by its potent insulinotropic action but also by its ability to suppress glucagon secretion [1921]. It remains uncertain and controversial whether glp-1 directly suppresses glucagon release by binding to glp-1 receptors expressed on the alpha cell [19, 22]. Indirectly, other intraislet paracrine factors, including insulin [23, 24], somatostatin [19, 25, 26], -aminobutyric acid (gaba), and zinc ions (zn), are considered to modulate glucagon secretion . In addition, a recent report suggested that increased portal glp-1 levels activate brain - derived neurotrophic factor (bdnf) and bdnf modulates glucagon secretion from pancreatic cells via the central and peripheral nervous systems . In patients with diabetes, miglitol is also reported to induce enhanced and prolonged glp-1 release after meal ingestion [3037]. A possible mechanism may be increased exposure of nutrients to the distal small intestine, where l - cells are located [32, 3436]. To confirm these results, we previously studied postprandial active glp-1 levels in patients with type 2 diabetes treated with or without insulin and found that miglitol administration increased active glp-1 levels in some, but not all, patients [10, 11]. The effect of miglitol administration in the present study resulted in varying total or active glp-1 levels in each patient, but no typical pattern was observed . As we expected, coadministration of miglitol and anagliptin showed increased postprandial active glp-1 levels in all patients of various magnitudes, reflecting the pharmacologic effect of anagliptin as a dpp-4 inhibitor . Despite increased active glp-1 levels, miglitol administration is reported to decrease gip levels, possibly via inhibited glucose absorption in the proximal small intestine ., the coadministration of miglitol and anagliptin showed increased active gip levels in all of our patients, reflecting the pharmacologic effect of anagliptin as a dpp-4 inhibitor; however, there was a decrease in total gip levels in all patients, except case 1 . As previously suggested, this phenomenon may be explained by a feedback inhibitory mechanism; the elevated concentrations of endogenous biologic active (intact) forms of the incretins may themselves restrict further secretion from the k- and l - cells [5, 37]. In cases 1 and 2, both total and active gip levels were higher than in the other patients, even before anagliptin administration . It is well known that gip promotes the efficient storage of ingested fat and directly links overnutrition to obesity [3840]. Furthermore, recent research suggests that gip receptor antagonists may offer a new therapeutic option for obesity in diabetes . As we only examined four patients, our results do not necessarily generalize to the majority of the patients with type 2 diabetes . Through the use of cgms, we determined that a combination of -gi, miglitol, and a dpp-4 inhibitor, anagliptin, was effective in reducing glucose fluctuations and stabilizing postprandial blood glucose levels in three of the four patients . Furthermore, c - peptide, glucagon, and total and active glp-1 and gip responded differently to the study medications for each patient, suggesting that hormonal responses to these drugs differ among individuals and may be complicated by multifactorial effects . Supplementary material 1 (docx 14 kb) supplementary material 2 (pdf 263 kb) supplementary material 3 (pdf 277 kb) supplementary material 4 (pdf 250 kb) supplementary material 5 (pdf 259 kb) supplementary material 6 (pdf 190 kb) supplementary material 7 (pdf 194 kb)
We evaluated a total of 129 cases that had the histologic diagnosis of primary pulmonary high - grade ne carcinoma . The tumors were originally diagnosed as sclc (n=35) or lcnec (n=94). Immunohistochemical staining for such general ne markers as chromogranin, synaptophysin, and cd56 was performed if necessary at the time when the original diagnosis was made at each institute . The tissues were obtained from patients who underwent surgery between 1999 and 2008 at two university hospitals, including seoul samsung hospital and dong - a university medical center . To ensure that there would be enough specimens for pathologic examination, only surgical cases were considered . Since we used existing data that did not identify individual subjects, informed consent by the study participants was not necessary and waived for this study . A representative h&e - stained slide from each case was circulated among four pathologists and independently reviewed based on the 2004 who criteria.1 of the four pathologists that participated in this study, one pathologist had 22 years of experience in pathology, one had 20 years of experience, and the remaining two pathologists had 17 years of experience each . They were all experienced pulmonary pathologists . Agreement was regarded as " unanimous " if all four pathologists agreed on a particular diagnosis, as a " majority " if three or more of four pathologists agreed, and as a " lack of consensus " if two pathologists had opposite diagnoses . Since only four observers participated in this study and they were all experienced pulmonary pathologists, we considered cases with no unanimous agreement to be " debated cases . " To measure the interobserver agreement between four pathologists, the generalized kappa value was calculated using spss ver we adopted the generally accepted convention for interpreting kappa values; values from 0.0 - 0.20 corresponded to slight agreement, 0.21 - 0.40 fair agreement, 0.41 - 0.60 moderate agreement, 0.61 - 0.80 substantial agreement, and 0.81 - 1.00 almost perfect agreement . We carried out morphometric analysis to measure the maximal diameter of cells that discriminate between sclc and lcnec using a previously described method.5 among 129 studied cases, we further evaluated 5 m h&e - stained sections from 66 high - grade pulmonary ne carcinomas, including 24 unanimous lcnec, 10 unanimous sclc, and 32 debated cases (tumors with lack of unanimous agreement; 16 majority lcnec, 6 majority sclc and 10 tumors with lack of consensus) after pathology review by four observers for morphometric analysis . Images were captured using a dp70 digital camera (olympus, tokyo, japan) attached to a bx51 microscope with a 40 objective . The final image captured on the monitor had a magnification of 400 . The cell diameters of 200 randomly selected tumor cells and the cell diameters of 20 mature lymphocytes were measured for each case . The actual measurements of the morphometric parameters were done using an i - solution image analysis system ver . 8.4 (imt i - solution inc ., coquitlam, bc, canada). The system segmented the nuclei automatically from the background, and the investigator needed only to manually edit adjacent nuclei that overlapped with each other . Receiver operating characteristic (roc) curve analysis was performed to identify the most useful cut - off value of cell diameter that provided the greatest sum of sensitivity and specificity in predicting a unanimous diagnosis of high - grade ne carcinomas based on cell size . The area under the roc curve (auc) and 95% confidence interval that did not include the 0.5 value was considered to be the cell diameter that had some ability to distinguish between the groups . We evaluated a total of 129 cases that had the histologic diagnosis of primary pulmonary high - grade ne carcinoma . The tumors were originally diagnosed as sclc (n=35) or lcnec (n=94). Immunohistochemical staining for such general ne markers as chromogranin, synaptophysin, and cd56 was performed if necessary at the time when the original diagnosis was made at each institute . The tissues were obtained from patients who underwent surgery between 1999 and 2008 at two university hospitals, including seoul samsung hospital and dong - a university medical center . To ensure that there would be enough specimens for pathologic examination, only surgical cases were considered . Since we used existing data that did not identify individual subjects, informed consent by the study participants was not necessary and waived for this study . A representative h&e - stained slide from each case was circulated among four pathologists and independently reviewed based on the 2004 who criteria.1 of the four pathologists that participated in this study, one pathologist had 22 years of experience in pathology, one had 20 years of experience, and the remaining two pathologists had 17 years of experience each . They were all experienced pulmonary pathologists . Agreement was regarded as " unanimous " if all four pathologists agreed on a particular diagnosis, as a " majority " if three or more of four pathologists agreed, and as a " lack of consensus " if two pathologists had opposite diagnoses . Since only four observers participated in this study and they were all experienced pulmonary pathologists, we considered cases with no unanimous agreement to be " debated cases . " To measure the interobserver agreement between four pathologists, the generalized kappa value was calculated using spss ver . 18.0 (spss inc ., we adopted the generally accepted convention for interpreting kappa values; values from 0.0 - 0.20 corresponded to slight agreement, 0.21 - 0.40 fair agreement, 0.41 - 0.60 moderate agreement, 0.61 - 0.80 substantial agreement, and 0.81 - 1.00 almost perfect agreement . We carried out morphometric analysis to measure the maximal diameter of cells that discriminate between sclc and lcnec using a previously described method.5 among 129 studied cases, we further evaluated 5 m h&e - stained sections from 66 high - grade pulmonary ne carcinomas, including 24 unanimous lcnec, 10 unanimous sclc, and 32 debated cases (tumors with lack of unanimous agreement; 16 majority lcnec, 6 majority sclc and 10 tumors with lack of consensus) after pathology review by four observers for morphometric analysis . Images were captured using a dp70 digital camera (olympus, tokyo, japan) attached to a bx51 microscope with a 40 objective . The final image captured on the monitor had a magnification of 400 . The cell diameters of 200 randomly selected tumor cells and the cell diameters of 20 mature lymphocytes were measured for each case . The actual measurements of the morphometric parameters were done using an i - solution image analysis system ver . 8.4 (imt i - solution inc ., coquitlam, bc, canada). The system segmented the nuclei automatically from the background, and the investigator needed only to manually edit adjacent nuclei that overlapped with each other . Receiver operating characteristic (roc) curve analysis was performed to identify the most useful cut - off value of cell diameter that provided the greatest sum of sensitivity and specificity in predicting a unanimous diagnosis of high - grade ne carcinomas based on cell size . The area under the roc curve (auc) and 95% confidence interval that did not include the 0.5 value was considered to be the cell diameter that had some ability to distinguish between the groups . After the four pathologists independently performed a pathology review, unanimous agreement was achieved in 71 of 129 cases (55.0%), and a majority agreement was achieved in 102 of 129 cases (79.1%). Based on the original diagnosis, unanimous agreement occurred for 52 (55.3%) of 94 lcnecs and for 19 (54.3%) of 35 sclcs . A majority agreement occurred for 73 (77.7%) of 94 lcnecs and for 29 (82.9%) of 35 sclcs . Of the originally diagnosed cases of lcnec, 21 (22.3%) lacked a consensus (two observers had opposite diagnoses), and 6 (17.1%) of 36 sclcs lacked a consensus . The kappa statistics for comparing the diagnosis of the four observers for the overall group of evaluated tumors are summarized in table 2 . The kappa values ranged from 0.35 to 0.81; one of the values fell into the almost perfect agreement category, one of the values fell into the substantial agreement category, three of the values fell into the moderate agreement category, and one fell into the fair agreement category . Roc analysis indicated that cell diameter for unanimous lcnec yielded an auc of 0.773 (95% confidence interval, 0.654 to 0.891). At the cut - off value of 33.8 m, cell diameter had 70.8% sensitivity and 73.8% specificity in discriminating unanimous lcnec from other high - grade ne carcinomas (unanimous sclc and debated cases). On the contrary, cell diameter for unanimous sclc yielded an auc of 0.905 (95% confidence interval, 0.826 to 0.985). At the cut - off value of 28.6 m, cell diameter had 80.0% sensitivity and 92.9% specificity in discriminating unanimous sclc from other high - grade ne carcinomas (unanimous lcnec and debated cases) (fig . 2). The kappa value for comparing the diagnosis based on the cut - off value of morphometric analysis and unanimous diagnosis evaluated by four observers was 0.44 . Of the 32 debated cases that lacked unanimous agreement, 18 (56.2%) cases fell into cell diameter between 28.6 m and 33.8 m (table 3). Moreover, seven of 10 tumors with lack of consensus (70.0%) fell into cell diameter between 28.6 m and 33.8 m (table 4). After the four pathologists independently performed a pathology review, unanimous agreement was achieved in 71 of 129 cases (55.0%), and a majority agreement was achieved in 102 of 129 cases (79.1%). Based on the original diagnosis, unanimous agreement occurred for 52 (55.3%) of 94 lcnecs and for 19 (54.3%) of 35 sclcs . A majority agreement occurred for 73 (77.7%) of 94 lcnecs and for 29 (82.9%) of 35 sclcs . Of the originally diagnosed cases of lcnec, 21 (22.3%) lacked a consensus (two observers had opposite diagnoses), and 6 (17.1%) of 36 sclcs lacked a consensus . The kappa statistics for comparing the diagnosis of the four observers for the overall group of evaluated tumors are summarized in table 2 . The kappa values ranged from 0.35 to 0.81; one of the values fell into the almost perfect agreement category, one of the values fell into the substantial agreement category, three of the values fell into the moderate agreement category, and one fell into the fair agreement category . Roc analysis indicated that cell diameter for unanimous lcnec yielded an auc of 0.773 (95% confidence interval, 0.654 to 0.891). At the cut - off value of 33.8 m, cell diameter had 70.8% sensitivity and 73.8% specificity in discriminating unanimous lcnec from other high - grade ne carcinomas (unanimous sclc and debated cases). On the contrary, cell diameter for unanimous sclc yielded an auc of 0.905 (95% confidence interval, 0.826 to 0.985). At the cut - off value of 28.6 m, cell diameter had 80.0% sensitivity and 92.9% specificity in discriminating unanimous sclc from other high - grade ne carcinomas (unanimous lcnec and debated cases) (fig . The kappa value for comparing the diagnosis based on the cut - off value of morphometric analysis and unanimous diagnosis evaluated by four observers was 0.44 . Of the 32 debated cases that lacked unanimous agreement, 18 (56.2%) cases fell into cell diameter between 28.6 m and 33.8 m (table 3). Moreover, seven of 10 tumors with lack of consensus (70.0%) fell into cell diameter between 28.6 m and 33.8 m (table 4). Our results provided an objective explanation for the considerable levels of interobserver variability in the diagnosis of high - grade pulmonary ne carcinomas, with kappa values that ranged from 0.35 (fair agreement) to 0.81 (almost perfect agreement). Den bakker et al.4 reported that there was striking variability amongst observers in diagnosing sclc and lcnec with variable agreement, from weak agreement (kappa value=0.19) to good agreement (kappa value=0.54), and the overall level of agreement for all cases was fair (kappa value=0.40). In their study, unanimity of diagnosis was achieved in 11.8% of 170 studied cases, and no consensus diagnosis was reached in 20.6% . A study by travis et al.6 revealed that unanimous diagnostic agreement occurred in 70% of sclc and 40% of lcnecs . Similarly, our study also demonstrated that it was difficult to reach a unanimous agreement, which was achieved in only 50.0% of the 129 studied cases . These studies of interobserver variability thus raised questions about whether the distinction between sclc and lcnec in the lung based who categories is truly reproducible . Difficulty of diagnosis in high - grade ne carcinoma is thought to occur for a variety of reasons, including the continuum of cell size and morphology from sclc to lcnec . Moreover, it may be true that the criteria for differentiating sclc and lcnec are subjectively interpreted by pathologists . In this study, the ratio of lcnec to sclc diagnoses varied from 0.8 to 4.8 according to observers . As shown in fig . 1c and d, some pathologists may place emphasis on the cell size in diagnosing pulmonary ne carcinomas, whereas other pathologists may diagnose a tumor focusing on the shape of the tumor cell, organoid pattern, nuclear / cytoplasmic ratio, or nucleoli . Practically, all the features except for nuclear size are qualitative rather than quantitative . For this reason, our attention shifted to morphometric analysis of cell size that is objective and a graphical method to compare discriminating power . In this study, the kappa value for comparing the diagnosis according to the cut - off point of morphometric analysis and unanimous diagnosis by the four observers was 0.44 . Of the 32 debated tumors that lacked unanimous agreement, 18 (56.2%) fell into nuclear diameter between 28.6 m and 33.8 m, which corresponds to three to four times the size of a lymphocyte . Moreover, seven of 10 tumors with lack of consensus (70.0%) fell into having a cell diameter between 28.6 m and 33.8 m . The morphological who criteria for diagnosing sclc and lcnec have proposed the use of an arbitrary cut - off that is three times the size of a lymphocyte to distinguish' small' from' large' cells.1 however, marchevsky et al.7 reported that 5 of 16 sclcs exhibited a predominant number of neoplastic cells that were larger than three normal lymphocytes, while 4 of 12 lcnecs had a predominant number of small cells classified by cell size alone, and these results were similar to those of our study . Therefore, we demonstrated that there is a continuum of cell sizes from sclc to lcnec with substantial cell size overlap and indicated that cell size alone is insufficient for reliable distinction between sclc and lcnec, and those cases falling in the middle cell size are difficult to categorize . Meanwhile, to determine the biological identity, similarity, and difference among high - grade pulmonary ne carcinomas, there is an obvious need for well - defined criteria . It is well - known that misclassification of a tumor could potentially mask differences in biological characteristics and clinical outcome . Due to the lack of a clear understanding of these high - grade ne carcinomas, studies that have reported inconsistency in clinical management of high - grade ne carcinomas suggest that lcnec should be treated as a non - small cell lung cancer or sclc.8,9 this is a critical point, especially in clarifying the histology - specific sensitivity to treatment . Although a constellation of morphological features distinguishing sclc and lcnec clearly exists, the dilemma of how to classify tumors that have features intermediate between sclc and lcnec must be resolved . It has been suggested that an alternative approach to classifying sclc and lcnec may be adopted by combining them into a single group of high - grade ne carcinoma.7 however, there is insufficient data to justify combining all the variants as a single entity . Therefore, further studies of high - grade pulmonary ne carcinomas would need to define more stringent and objective definitions of cytologic and architectural characteristics that would enable a pathologist to reliably distinguish between sclc and lcnec.
The foxp protein family (foxp1 - 4) is a group of transcription factors that play an important role in embryological, immunological, hematological, and speech and language development 1 . 2 reviewed a total of 10 patients with de novo mutations of foxp1 and showed that haploinsufficiencies of foxp1 are associated with global developmental delay / mental retardation with moderate / severe speech delay . Here, we report the case of a japanese female patient with severe speech delay and the identification of a de novo foxp1 missense mutation by exome analysis . The patient is a 22-year - old female and is the second - born child of nonconsanguineous japanese parents . She was born by normal vaginal delivery at 34 weeks gestation, and her birthweight was 2100 g (+ 0.1 sd). Her developmental delay was noted by a pediatrician when the patient was 1 year old . The patient's hearing and vision were normal, and no autistic features, developmental regression, or history of seizure were present . She began to menstruate at the age of 13 . At the time of examination (22 years old), she displayed a short stature (141 cm, 3.2 sd, body weight 44.3 kg, 1.1 sd) and delayed speech (she was unable to speak), but her receptive language abilities were relatively developed as indicated by her understanding of relational concepts . She required assistance with routine daily activities, and hyperextension of her joints was observed . Chromosome analysis with g - banding showed a 46, xx karyotype . Her father's height is 171 cm and mother's height is 154 cm . Table1 shows the neurodevelopmental features of the patient in comparison with data presented by le fevre et al . Comparison of the neurodevelopmental features reported by le fevre et al . 2 and the patient in this study this study was approved by the ethical committee at the university of tsukuba and was conducted according to the principles of the declaration of helsinki . Exome sequencing was performed, following the protocol described in the sureselect library prep kit (post - pool version 4; agilent technologies, inc ., santa clara, ca). The dna library was subjected to emulsion pcr (solid ez bead emulsifier kit; life technologies, carlsbad, ca) to generate clonal dna fragments on beads, followed by bead enrichment (solid ez bead enrichment kit; life technologies). Enriched template beads were sequenced on a solid 5500xl sequencer as single - end, 60-bp reads (life technologies). The solid 5500xl output reads were aligned against the human genome reference sequence (hg19) using lifescope version 2.5.1 (life technologies) to generate bam files . Variant calling was performed following the best practices specified in the genome analysis toolkit 3 (gatk, version 2.7.4), picard (http://picard.sourceforge.net) and samtools 4, and only reads that mapped to a unique position in the reference genome were used . We first filtered out the variants with low - quality values generated by gatk output, resulting in a new total of 62,200 variants . To distinguish potentially pathogenic variants from other variants, we filtered out variants in our in - house references (57 exome samples), public data from dbsnp (http://www.ncbi.nlm.nih.gov/snp/, version 137), and a 1000-genome database 5 . We then used annovar software to filter out synonymous variants and intronic variants because they are less likely to be pathogenic 6, which resulted in 84 remaining variants . We then used sift 7, polyphen2 8, lrt 9, or mutationtaster 10 software to predict the potential impact of an amino acid substitution on the function of human proteins, and we filtered out benign missense mutations as defined by the above - mentioned software . A total of 13 single - nucleotide variants and one frame - shift variant remained after this step (table2). Results of the exome sequence and confirmation by sanger sequencing not detected; mutations detected in the exome data analysis but not confirmed by sanger sequencing, de novo; mutations detected in the patient but not present in either parent . We performed direct sequencing to evaluate these 14 candidate mutations using dna obtained from the patient and her parents . Among these, two mutations (spert and grp52) were not confirmed by direct sequencing . Ten of the mutations existed in at least one of the healthy parents, suggesting that they are unlikely to be pathogenic . The patient is a 22-year - old female and is the second - born child of nonconsanguineous japanese parents . She was born by normal vaginal delivery at 34 weeks gestation, and her birthweight was 2100 g (+ 0.1 sd). Her developmental delay was noted by a pediatrician when the patient was 1 year old . The patient's hearing and vision were normal, and no autistic features, developmental regression, or history of seizure were present . She began to menstruate at the age of 13 . At the time of examination (22 years old), she displayed a short stature (141 cm, 3.2 sd, body weight 44.3 kg, 1.1 sd) and delayed speech (she was unable to speak), but her receptive language abilities were relatively developed as indicated by her understanding of relational concepts . She required assistance with routine daily activities, and hyperextension of her joints was observed . Chromosome analysis with g - banding showed a 46, xx karyotype . Her father's height is 171 cm and mother's height is 154 cm . Table1 shows the neurodevelopmental features of the patient in comparison with data presented by le fevre et al . Comparison of the neurodevelopmental features reported by le fevre et al . 2 and the patient in this study this study was approved by the ethical committee at the university of tsukuba and was conducted according to the principles of the declaration of helsinki . Exome sequencing was performed, following the protocol described in the sureselect library prep kit (post - pool version 4; agilent technologies, inc ., the dna library was subjected to emulsion pcr (solid ez bead emulsifier kit; life technologies, carlsbad, ca) to generate clonal dna fragments on beads, followed by bead enrichment (solid ez bead enrichment kit; life technologies). Enriched template beads were sequenced on a solid 5500xl sequencer as single - end, 60-bp reads (life technologies). The solid 5500xl output reads were aligned against the human genome reference sequence (hg19) using lifescope version 2.5.1 (life technologies) to generate bam files . Variant calling was performed following the best practices specified in the genome analysis toolkit 3 (gatk, version 2.7.4), picard (http://picard.sourceforge.net) and samtools 4, and only reads that mapped to a unique position in the reference genome were used . We first filtered out the variants with low - quality values generated by gatk output, resulting in a new total of 62,200 variants . To distinguish potentially pathogenic variants from other variants, we filtered out variants in our in - house references (57 exome samples), public data from dbsnp (http://www.ncbi.nlm.nih.gov/snp/, version 137), and a 1000-genome database 5 . We then used annovar software to filter out synonymous variants and intronic variants because they are less likely to be pathogenic 6, which resulted in 84 remaining variants . We then used sift 7, polyphen2 8, lrt 9, or mutationtaster 10 software to predict the potential impact of an amino acid substitution on the function of human proteins, and we filtered out benign missense mutations as defined by the above - mentioned software . A total of 13 single - nucleotide variants and one frame - shift variant remained after this step (table2). Results of the exome sequence and confirmation by sanger sequencing not detected; mutations detected in the exome data analysis but not confirmed by sanger sequencing, de novo; mutations detected in the patient but not present in either parent . We performed direct sequencing to evaluate these 14 candidate mutations using dna obtained from the patient and her parents . Among these, two mutations (spert and grp52) were not confirmed by direct sequencing . Ten of the mutations existed in at least one of the healthy parents, suggesting that they are unlikely to be pathogenic . It has been demonstrated that the use of next - generation sequencing techniques provides a high success rate in the diagnosis of unidentified genetic conditions . Performed exome sequencing on dna from 12 patients with unexplained and apparent genetic conditions, which resulted in a diagnosis of a likely genetic origin of the condition in six of the 12 patients 11 . Exome sequencing has also been applied in autopsies of patients with sudden unexplained death and has been used to successfully identify mutations related to cardiac arrhythmia and cardiomyopathy 12 . Therefore, exome is becoming a powerful tool for the diagnosis of patients with unexplained conditions . The foxp protein family (foxp1 - 4) is a group of transcription factors that play an important role in embryological, immunological, hematological, and speech and language development 1 . It was identified using a three - generation pedigree in which a severe speech and language disorder was transmitted as an autosomal - dominant monogenic trait 13 . Subsequently, many de novo and familial cases of severe speech disorders associated with foxp2 mutations have been reported, and mutations in foxp2 are well known to cause developmental speech and language disorders 14 . Because foxp1 and foxp2 form heterodimers for transcriptional regulation, it has been suggested that they cooperate in common neurodevelopmental pathways through the coregulation of common targets 15 . Vernes et al . Screened for mutations in foxp1 genes with a denaturing high - performance liquid chromatography method using dna from 49 patients with developmental verbal dyspraxia . They found one missense mutation (p215a) in one patient, but p215a was also identified in an unaffected sibling of the patient 16, which suggests that p215a is unlikely to be pathogenic . Identified two patients with de novo foxp1 mutations that caused haploinsufficiency and suggested that decreased expression of foxp1 has a more global impact on brain development than does decreased expression of foxp2 17 . Subsequently, mutations in foxp1 have been reported to be associated with global developmental delay, intellectual disability, and speech defects 2,1721 . As noted by le fevre et al ., the most consistent feature of a foxp1 mutation is global developmental delay with prominent speech delay, which was also observed in the present case study . To the best of our knowledge, this is the first report of a foxp1 de novo mutation in an individual with severe speech delay who belongs to a non - caucasian population . All of the previously reported foxp1 mutations as well as the one in this study occurred de novo, suggesting that haploinsufficiency of foxp1 reduces fitness . In addition, some characteristic features of foxp1 mutations, such as speech delay, a prominent forehead, a short nose with a broad tip, and frontal hair upsweep, were concordant with the phenotype of the patient in this study . Prkaa1 is a catalytic subunit of the 5-prime - amp - activated protein kinase (ampk). Ampk is a cellular energy sensor conserved in all eukaryotic cells, and ampk regulates the activities of a number of key metabolic enzymes through phosphorylation . Variations in prkaa1 have been reported to be associated with diabetes 22, cancer 2325, coronary artery disease in type 2 diabetes 26, and open - angle glaucoma 27 . Although mutations in prkaa1 or other genes may contribute to the patient's symptoms, it appears likely that the patient's severe speech delay may have been caused by a de novo missense mutation of the foxp1 gene based on results of previous studies . Next - generation sequencing techniques can provide information that is essential for the molecular diagnosis of patients with unexplained conditions.
It is known that children with cerebral palsy (cp) show neurodevelopmental disorders including spasticity, contracture, reduced coordination, selective voluntary motor control, and muscle weakness1 . Even when spasticity and contractures are more apparent impairments, deficits in selective motor control can produce an increased negative effect on function2, 3 . Selective voluntary motor control (svmc) is the ability to isolate the activation of muscles in a selected pattern by voluntary movement or posture4 . Evaluative tools widely used for the effects of physical therapy intervention for children with cp in clinics to date are the gross motor function measurement (gmfm), timed up and go test, pediatric balance scale (pbs), functional independence measure (weefim), etc . Abnormal movement patterns occur in children with cp due to the absence or insufficiency of the ability to control coordination . However, the assessment of motor impairment is poorly considered, even though it is the most important factor for determining motor function . The selective control assessment of the lower extremity (scale) was designed for clinical assessment of selective motor control . In the scale, the scale score for each limb is the sum of all the points given to each joint, up to a maximum of 10 points per limb . Significant inverse correlation (spearman s r = 0.83, p<0.001) between scale scores and gross motor function classification system (gmfcs) levels indicates construct validity for the scale . The scale also shows high interrater reliability, with intraclass correlation coefficients ranging from 0.88 to 0.915 . Therefore, it is applicable to participants with the ability to follow simple motor commands . It is not suitable for participants under 4 years of age or those with severe motor and intellectual impairments (e.g., gmfcs level v). It does not require any equipment5 . Svmc at the ankle strongly predicts functional movement ability in children with cp and is used for determining candidates for selective posterior rhizotomy6, 7 . Reduced selective motor control caused by flexor or extensor synergies interfering with isolated joint movement impairs functional movements such as gait and reaching8 . Impaired selective motor control usually occurs with muscle weakness, spasticity, and short muscle - tendon length . These motor deficits are associated with injury to the corticospinal tract (cst) and other descending tracts and the subsequent loss of descending signals . The pbs was developed by modifying the berg balance scale (bbs) to apply to children with cp who have impaired balance ability to respond to postural stability and external changes . The pbs has been further developed as a balance measure to be used for children with mild or severe motor deficits . It has been reported that the modified pbs shows both high intrarater and interrater reliabilities (intraclass correlation coefficient = 0.998 and interrater correlation coefficient = 0.997)9 . It is necessary to develop a method with high reliability and validity for proper evaluation . Therefore, the purpose of this study was to investigate the correlation between the scale (especially measuring the degree of motor impairment in the lower extremity) and pbs (measuring functional balance) and thus further examine whether the scale is a valid tool to predict the pbs score . The subjects of this study were 23 children with cp treated in local clinics in south korea . The inclusion criteria were diagnosis of spastic cp, the ability to independently maintain a standing position for at least four seconds, level i to iii according to the gmfcs, and the ability to understand and follow verbal commands . Before conducting this study, consent forms that described the purpose and detailed procedures of the study, were provided to the participants and their legal guardians . All participants signed the informed consent forms, which were approved by the institutional review board of dankook university, prior to participation in this study . Following evaluation, scores were given by one pediatric physical therapist with 12 years of experience . The scale and pbs were performed in triplicate, and the highest scores were chosen to be analyzed further . The scale tool was used to bilaterally assess the movements of hip, knee, ankle, subtalar, and toe joints within 15 minutes without specialized equipment . Evaluations were performed in the sitting position, except for hip flexion, which was tested in the side - lying position for proper joint excursion . For each joint, the participants were asked to perform the task by passively moving their limbs through the desired movement sequence using a three - second verbal count . The scale was graded at each joint as normal (2 points), impaired (1 points), or unable (0 points). A grade of normal was given when the participant completed the desired movement sequence within the verbal count without movement of untested ipsilateral or contralateral lower extremity joints . A grade of impaired was given when the participant isolated the motion during the task but showed any of the following errors: only one directional movement, less than 50% of movement achieved, movement of a non - tested joint (including mirror movements), or the time exceeding a three - second verbal count . A grade of unable was given when the participant showed no initiation of the requested movement or when the participant performed a synergistic mass flexor or extensor pattern . The pbs is a standard tool to measure standing balance and is used to evaluate functional balance for children with cp having mild to severe motor impairment . It is composed of a total of 14 items in 3 dimensions, including sitting, standing, and postural change . A score from 0 to 4 points was given to each item, with a higher score representing a better balance ability . Means and standard deviations of age, gender, and scale and pbs scores were obtained . An independent t - test was used to determine the differences between pbs and scale total scores in the subjects with spastic hemiplegia and spastic diplegia . Spearman s rank correlation coefficients were calculated to estimate the correlation between each scale item and the pbs total score, and that between each scale item and pbs dimension . Twenty - three children (6 to 15 years old) diagnosed with spastic cp were selected for this study . This included 5 children with spastic hemiplegia (2 males and 3 females) and 18 children with spastic diplegia (12 males and 6 females). The general characteristics of the participants are shown in table 1table 1.general characteristics of the participantsspastic typenumber (gender)age (meansd)gmfcs levelhemiplegia5 (2 males, 3 females)8.001.58i = 5 (100%)diplegia18 (12 males, 6 females)9.672.32i = 8 (44.4%)ii = 7 (38.9%)iii = 3 (16.7%)total239.302.26gmfcs: gross motor function classification system . Gmfcs: gross motor function classification system the average total scores of the scale and pbs in the children with spastic hemiplegia type were 15.001.58 and 53.801.10, respectively . In the children with spastic diplegia, the average scale and pbs total scores were 9.946.13 and 45.678.90, respectively . The scores obtained from the children with spastic hemiplegia were higher than those from the children with spastic diplegia, and the differences were statistically significant (p<0.05) (table 2table 2.scale and pbs total scoresassessmentsspastic typehemiplegia (n=5)diplegia (n=18)scale15.01.589.96.13pbs53.81.1045.78.90p<0.01.scale: selective control assessment of the lower extremity; pbs: pediatric balance scale). Scale: selective control assessment of the lower extremity; pbs: pediatric balance scale the numbers of points obtained from each scale item were highest for the knee and lowest for the subtalar joint . The numbers of points obtained from each pbs item were lowest for pbs item 8 (table 3table 3.average scores of the scale and pbs itemsscale items (score)hipkneeanklesubtalartoe(2.6)(3.1)(2.0)(1.6)(1.7)pbs items (score)12345(3.8)(3.8)(3.9)(4.0)(4.0)678910(3.9)(3.8)(2.0)(2.2)(3.0)11121314(3.2)(3.8)(3.0)(3.0)). Low numbers of points for pbs items (pbs 8, 9, 10, and 14) indicated that these items were difficult for participants to perform . The correlation coefficient between the scale and pbs scores was 0.797 and found to be statistically significant (p<0.05). When examining the correlation between each scale item and pbs scores, all scale items were significantly correlated with the pbs (most items, p<0.01; hip joint item, p<0.05). The correlation coefficients between each scale item and the pbs were 0.672, 0.795, 0.790, 0.747, and 0.626, respectively (table 4table 4.correlation between scale and pbs assessmentsscale itemtotalhipkneeanklesubtalartoepbs0.797 0.672 * 0.7950.7900.7470.626p<0.05; * p<0.01). When comparing scale items with pbs dimensions, all scale items were significantly correlated with two pbs dimensions (standing and postural change) (p<0.01) (table 5table 5.comparison of scale items and pbs dimensionsscale itempbs dimensionstandingpostural changehip0.6760.570knee0.7880.733ankle0.7780.707subtalar0.7540.629toe0.6190.563**p<0.01). Children with cp show various muscle recruitment patterns and magnitudes when compared with normal children . These differences can affect voluntary muscle recruitment, leading to impairments in motor ability . Tedroff et al . Reported that during maximal voluntary contractions, children with spastic cp showed activation of muscles other than the intended muscles, particularly when the intended muscles were located at a more distal part10 . In addition, children with spastic cp show 52% or less maximum contractile force in the main lower extremity muscles in ankle dorsiflexion and plantar flexion compared with the same - aged normally developing children11 . It has also been reported that svmc impairment of distal joints of the lower limb was increased12 . These results indicate decreased ability to perform joint movements isolated from the distal part of the limb . As individuals with impaired svmc lack normal interjoint coordination13, children with good svmc are more capable of moving out of synergy during the swing phase of gait14 . Therefore, this study was conducted to examine the correlation between the newly developed scale and the well - established pbs methods and to test whether the scale is useful as a tool to predict pbs outcome . In the present study, the scale and pbs scores obtained from the spastic hemiplegia group were 5.06 (15.001.58 versus 9.946.13) and 8.13 (53.801.10 versus 45.678.90) points higher than those from the spastic diplegia group, respectively . These results agree with a report indicating that the pbs can be used to predict the gmfcs level, due to the presence of differences between the pbs total score and gmfcs level . The main reason for the difference in total pbs between gmfcs levels i and ii is that individuals assigned to level i are capable of performing more complex motor tasks such as running and jumping, maintaining balance when standing without support, and walking long distances15 . The children in the spastic hemiplegia group were assigned to gmfcs level i, indicating that they were more capable of following verbal commands and performing tasks . Therefore, it is assumed that the scale and pbs scores obtained from the spastic hemiplegia group were higher than those from the spastic diplegia group in this study . When ranked by sacle items scores in order from highest to lowest, the knee joint was the highest, followed by the hip, ankle, toe, and subtalar joint . It was difficult for participants to perform pbs dimension tasks, with the highest difficulty experienced for item 8, followed by items 9, 10, and 14 . It has been reported that svmc impairments are more severe in the distal part of the limb than in the proximal part12 . Similar results were shown in this study except with the knee and subtalar joints . Fowler et al.12 reported that one participant (15 years old) with spastic diplegia (gmfcs level i) had a subtalar score of 2 and an ankle joint score of 1, owing to a restricted range of motion . Therefore, it is likely that because the average age of the participants in this study was 9.3 years, contracture developed . The most frequent exceptions for proximal to distal concordance occurred when the toes were graded as 1 and the subtalar joint was graded as 0, indicating absence of subtalar svmc with sparing at the toes16 . Similar exceptions were observed in the present study . These exceptions could be explained by the fact that the origin of the muscles controlling the ankle, subtalar joint, and toes are similar, although the insertions of the toe musculature are more distal . In addition, eversion was described as a challenging movement in adults after stroke, and it is an indicator of the highest level of recovery for the lower extremity16 . Furthermore, isolated movement of the subtalar joint was very difficult for children with cp to understand and perform12 . In a previous study, the scale hip test was performed for participants with spastic cp showing crouch gait in an antigravity side - lying position17 . In this position, it was difficult for the participants to raise their hip joints due to muscle weakness, and thus they had limitations of hip extension because of hamstring shortness . Therefore, this is likely why higher scores were observed for the knee joint than the hip joint in the scale test in this study . It has been reported that performance of pbs tasks involving items 7, 8, 9, 10, 11, and 13 reveals significant differences between individuals assigned to gmfcs levels i and ii15 . Similar results were observed in this study; that is, the standing test in pbs items 8, 9, 10, and 14 was the hardest task for the participants to perform . It is assumed that the lowest score, which was found for item 8 (standing with one foot in front), was the result of absence of postural stability for maintaining balance due to a narrow base of support . All scale items were significantly correlated with the pbs (most items, p<0.01; hip joint item, p<0.05). Total scale scores were very useful for explaining the overall functional capacity of the participants . There are many previous reports showing that pbs assessment for children with cp was capable of predicting the gmfcs level, and the correlations between the total pbs score and gmfm and those between muscle strength and gmfm were significantly higher15, 18,19,20,21,22 . Most of the participants in this study were assigned to gmfcs levels i and ii (~83.0%) indicating high correlation between the scale and pbs tests . All scale items were significantly correlated with two pbs dimensions (standing and postural change). The ability to perform svmc is dependent on the capability to perform the movements of hip and knee joints uncoupled during the swing period . Therefore, it is assumed that improvement of the scale score increases gross motor function but decreases the synergistic pattern and thus improves pbs . To examine the correlation between scale and pbs dimensions, the present study included only subjects who had the ability to stand for at least 4 seconds and a gmfcs level of i to iii . Therefore, there were limitations regarding the number of subjects and exclusion of severely disabled children . The present study was conducted to investigate the correlation between the scale (especially measuring the degree of motor impairment in the lower extremity) and pbs (measuring functional balance), and to examine if the scale is a valid tool for predicting the pbs . In scale assessment, based on these results, it is likely that the scale is a useful tool to predict pbs outcome.
Large - scale clinical trials have created a robust evidence base to inform much of what is now standard acute - stroke practice.13 the classical clinical trial is designed to test efficacy of a particular intervention over a comparator, for example, placebo or usual care . To facilitate comparison between the groups requires a standard measure of outcome that is relevant and suited to the clinical question, valid for the population studied, and meaningful to the research team . In those trials that describe interventions designed to impact on quantifiable physiological variables, such as glycemia or blood pressure, choice of end - point assessment choice of assessment strategy is more challenging for a chronic, nonprogressive, or variably progressive disorder with potential multisystem effects such as cerebrovascular disease . Hard clinical end points such as stroke mortality or stroke recurrence are useful, but do not fully capture the potential devastating effect of a disabling but survivable stroke . As stroke represents the leading global cause of adult disability,4 an important consideration for any study of stroke interventions this is recognized by regulatory authorities, who now recommend a measure of functional recovery / disability as primary or coprimary end point for stroke intervention trials . Although the focus of this review will be functional assessment tools for stroke trials, these instruments also have utility in clinical practice . As functional assessment scales give a numerical value to abstract concepts such as disability, they can be used to objectively quantify deficits and track change over time . In clinical practice, an appreciation of how to describe stroke recovery in terms of common stroke scales allows for development of a common language between professionals caring for stroke survivors that facilitates comparisons of patients and services . Within a single review, it would be impossible to review all stroke - specific and generic scales that may be needed in a stroke survivor s journey (figure 1). For the interested reader, we recommend a number of reference works.57 we recognize there is also extensive literature on assessment strategies for cognitive function poststroke . We will not review cognitive testing, suffice to say that there are a multitude of tools available with little consistency in choice of assessment.8 a large number of stroke - assessment scales are described, with novel scales frequently appearing (and often subsequently disappearing) in the literature . For those who are new to functional assessment, the large and varied nature of available scales and tools may seem daunting . The world health organization s international classification of functioning, disability and health (who - icf)9 gives a conceptual framework that can aid classification of the scales and help decide on the appropriate measure for a particular purpose . Who - icf describes levels of pathology (in this case, the stroke lesion), impairments (the direct loss of function), activity limitation (formerly called disability), and societal participation (formerly called handicap). The who - icf grades do not exist in isolation; they interact and often create feedback loops . For example, an ischemic stroke (pathology) may cause a hemianopia (impairment); this may lead to poor mobility (activity limitation) and may restrict the stroke survivor from driving (societal participation limitation). These problems may result in a fall with soft - tissue injury (impairment), and fear of falling may cause the stroke survivor to forgo usual hobbies and activities (societal participation limitation) (figure 2). Measures of pathology (for example, size of infarct on imaging) or impairment (for example the medical research council motor assessment scale) are straightforward to perform and interpret, but give little useful information on how stroke affects the individual . For this reason, impairment scales are often used in early phase trials . For phase iii studies, activity measures or measures of participation although not part of the who - icf, a further concept of quality of life (qol) is also described, and tools exist for its measurement . Measures of qol give a far more detailed assessment, but as a result can be more burdensome to the patient and are often more difficult to interpret (figure 3). Clinimetrics is the study of properties of clinical assessment tools;10 the term is derived from the theory of psychometrics.11 classical test theory describes important properties such as validity and reliability.12 other important factors for clinical scales are acceptability, both to patient and to assessor, and responsiveness to change . Although in psychometrics, classical test measures are increasingly being superseded by contemporary theories of item response, the measures of validity, reliability, and responsiveness remain important for understanding clinical scales, and we will discuss them in turn . The clinimetric property of validity seeks to assess whether a scale measures the concept it purports to measure . Adequate validity is essential for a stroke scale to have clinical utility, as a functional assessment tool that does not measure function is meaningless . Validity can be assessed in various complementary ways.57 there is no gold standard for poststroke function, so assessment of criterion validity, where a scale is compared to a reference standard, is not possible . However, concurrent validity can be applied to a stroke scale by comparing it with another measure that purports to measure a similar construct; for example, comparing a novel impairment scale with an established scale . Face validity is an assessment of whether a priori the scale should measure the concept of interest, usually assessed by experts in the field . Content validity asks whether the various items of a scale can adequately describe the concept of interest . Prognostic or predictive validity for a stroke scale may be examined by, for example, studying if an impairment scale is associated with longer - term stroke outcomes . Important reliability measures include the reproducibility of repeat scoring by the same observer (intraobserver reliability or test retest reliability) and between scorers (interobserver variability). Whether all items within a scale measure the same construct is a further measure of reliability, usually termed internal consistency . In contemporary stroke trials, where many thousands of stroke survivors may be assessed by hundreds of international research teams, reliability of assessment is clearly paramount . Whereas validity of a scale is inherent, reliability of assessment may be modified . Various methods to improve consistency of assessment are employed in large - scale trials, including training in use of scales, certification exams, and use of standardized protocols . While validity is relative, reliability can be objectively described . There is no consensus on the optimal method to measure reliability, although kappa statistics are frequently used in the biomedical literature to assess agreement . A kappa of 0 would imply no agreement other than that expected by chance, and perfect agreement is scored as 1.0 . Traditionally, a kappa greater than 0.6 is taken as sufficient agreement to justify use of a scale . Various forms of statistical weighting of kappa values can be used to give a measure of the degree of difference between raters.13,14 increasingly, more sophisticated analyses, such as that of bland altman, are being used to assess reliability.15 responsiveness can be thought of as the ability to detect meaningful change over time . Meaningful change is clearly a subjective term, and will vary with the context in which the scale is used . The issue of responsiveness and the ability to detect small but meaningful change is especially important for a condition with high incidence and prevalence, such as stroke . If a scale does not pick up change in function, treatment effects that are modest for the individual but potentially important at a population level could be missed . The ideal scale would be easy and quick to administer, acceptable to patients and researchers, valid for its chosen purpose, reliable, and responsive to meaningful clinical change . There is no ideal stroke measure that fulfills all these criteria (nor is there ever likely to be). Although some guidance on stroke assessment for trials is emerging, debate continues as to the relative strengths and limitations of differing assessment strategies, and there is no consensus as to the optimal outcome measure(s) for use . The stroke literature describes a variety of instruments, generic and specific to stroke, for functional assessment of recovery . A recent analysis of tools used in stroke trials suggests substantial heterogeneity in choice of assessment measure and in method of application.16 use of bespoke, nonvalidated assessments is still seen, although less commonly than previously . Certain assessments are used more frequently than others and are increasingly recommended by specialist societies.17 for the non - stroke specialist, a basic knowledge of the more prevalent stroke scales will allow for improved understanding and critical analysis of stroke studies . We will describe three common stroke assessments: the national institutes of health stroke scale (nihss), the modified rankin scale (mrs) and the barthel index (bi). For each scale, we will discuss history, development, and application, and use the scales to further discuss the importance of clinimetric properties . The scientific study of assessment scales, particularly stroke assessment, is rapidly expanding and it would be impossible to comprehensively cover all areas . In this review, we will not describe the optimal analysis of functional scales for stroke trials . Debate continues as to the relative merits of various statistical techniques, including dichotomization and use of the complete range of a scale, with the differing approaches having vocal proponents.18,19 equally, we will not consider the literature on outcome assessment in animal models of stroke.20,21 the nihss is a 15-item scale that standardizes and quantifies the basic neurological examination, paying particular attention to those aspects most pertinent to stroke . The nihss provides an ordinal, nonlinear measure of acute stroke - related impairments by assigning numerical values to various aspects of neurological function.22 the scale incorporates assessment of language, motor function, sensory loss, consciousness, visual fields, extraocular movements, coordination, neglect, and speech.22 it is scored from 0 (no impairment) to a maximum of 42 . A standardized approach to assessment, starting with fundamental assessments such as level of consciousness, is recommended, and guidance is given on how to score where the stroke survivor is not able to respond to commands . The nihss was developed in the early 1980s as a research tool to allow consistent reporting of neurological deficits in acute - stroke studies, particularly the early trials of thrombolysis and putative neuroprotectants.22 the nihss was developed through a robust consensus approach, taking the most informative measures from existent stroke - examination scales (toronto stroke scale, oxbury initial severity scale, and cincinnati stroke scale) and creating a composite scale that was further reviewed by a panel of stroke researchers and amended (further items were added to ensure the assessment was as comprehensive as possible). The resulting scale was piloted and refined in a controlled trial of naloxone in acute stroke . It has since been used as primary or coprimary end point in landmark trials of thrombolytic agents and is commonly used in clinical acute - stroke practice . Using a factor - analysis process, utility of individual components of the nihss has been assessed.23 this work has formed the basis for development of the modified nihss or mnihss, which removed components deemed unreliable.24 the resulting 11-point mnihss has been prospectively assessed, and improved reliability is described.25 as the standard nihss is already fairly quick to perform and reliable, it is debatable whether a shorter scale is needed, and at present the mnihss is not frequently used in trials or practice . Further amendments to the nihss have been described to facilitate use of the scale in prehospital settings.26 a pediatric nihss (pednihss) is also described.27 the nihss has many advantages as a stroke outcome - assessment tool . It is relatively straightforward and takes around 6 minutes to perform, with no need for additional equipment . In the acute - stroke environment, it has been suggested that a change in the nihss of more than 2 points represents clinically relevant early improvement or deterioration.28 nihss scores are reliable across observers, and this has been demonstrated both in cohorts of neurology - trained and non - neurologist raters . The availability of a reliable method for neurological exam that is suitable for nonspecialists is a particular strength of the nihss . Reliability and validity has also been demonstrated for remote nihss assessment via telemedicine.29 the interobserver reliability of the nihss is further improved by the various training materials available . Training resources now exist, such as dvds and online educational aids, as well as pocket - sized nihss summary scales . Practitioners can undergo certification to demonstrate their proficiency in assessment and interpretation of the nihss.30 content validity of the nihss has been demonstrated, although high internal consistency suggests that certain items of the nihss may be redundant . The nihss has predictive validity, as initial score is a robust predictor of in - hospital complication and outcome at 3 months.31,32 correlations with objective measures of stroke severity, such as size of infarct on imaging, provide further evidence of nihss validity.23,33,34 compared with bi and mrs, the nihss is the more sensitive outcome score, requiring potentially smaller sample sizes to detect relevant therapeutic effects.23,35 the nihss is responsive to change and can measure impairment throughout the expected range of stroke severity.36 a criticism of the nihss relates to its validity in certain nondominant - hemisphere stroke syndromes . It is well recognized that an individual can score 0 on the nihss, despite having evidence of ischemic stroke, particularly in the posterior circulation territory.37 examination of the component subscales of the nihss reveals a focus on limb and speech impairments and relatively little attention to, for example, cranial nerve lesions . Similarly, when the nihss is used to predict dependent living, lower scores are seen in posterior circulation events compared to anterior circulation.38 there are radiological correlates, when quantifying extent of cerebral damage for a specified nihss score, the median volume of right - hemisphere strokes is larger than the volume of left - hemisphere strokes, suggesting nondominant strokes are required to be more severe to reach the same grading on the nihss.39 as an impairment scale, the nihss can give only limited information on how stroke has affected the individual stroke survivor . Excellent outcome from stroke; a hemianopia that precludes driving and may necessitate loss of employment would score nihss 1, but for the individual this may not seem an excellent result . Adapted from the maryland disability index, the bi authors florence i mahoney and dorothea w barthel intended their scale for use as a simple index of independence, useful in scoring improvement in rehabilitation.40 first described in the 1950s and published in 1965, the bi was developed to assist in discharge planning from long - term care wards . With time, the bi has been adopted by other disciplines and is a recommended assessment in older adult care.41 the bi is the most commonly used functional measure in stroke - rehabilitation settings and the second most commonly used functional outcome measure across stroke trials.16,42 many scales have been described that take the name some authors have sought to modify or adapt the bi from the original; these include reducing the number of items,43 extending it with the addition of cognitive and social domains,44 and attempts to further subdivide the outcomes to include different degrees of assistance.45 however, each of these requires independent validation, as it is known that even comparatively minor adaptations alter the validity of a tool and accuracy of responses.46 for consistency, it is recommended that a single bi measure is used; the scale as described by wade and collin47 has been used in many trials . Scores range from 0 and 100, with a higher score indicating greater independence (table 1). While this can be useful for statistical analysis, it is more informative in practice to present the scores for the individual domains . An unresolved issue for trials is how to define a good bi outcome, with significant heterogeneity within the published literature48 and attempts to subcategorize, based on total score.49,50 a popular interpretation of bi scores is that subjects with bi> 80 are generally independent and should be able to return home; while subjects with bi <40 are very dependent.51 other interpretations of favorable and unfavorable bi outcomes have been described: statistical modeling looking at differing bi scores as a trial end point suggested a score of 95/100 was the optimal descriptor of an excellent outcome and that 75/100 was the best cut point for defining a poor outcome.52 validity of bi is well described . The scale is recognized as a valid prognostic tool following stroke, in particular as predictor of recovery, level of care required,53 and duration of rehabilitation required following stroke.54 bi scores correlate with other stroke - assessment scales,55 including other more detailed adl scales.56 interobserver reliability is usually quoted as a strength of the bi, and reliability has been demonstrated in nonstroke populations.57 systematic review of reliability of bi in stroke also suggests reasonable reliability, although few multicenter reliability studies are available.58 the original bi is not without its limitations . As a scale of primarily physical function, it does not reflect the burden on the individual of communication and cognitive deficits that can result from a stroke event.59 for clinical trials, the bi lacks a result to represent stroke mortality, and this can complicate analysis of results . However, the major limitation of the bi for clinical trial use is its responsiveness to change . Although in certain stroke - care settings, the bi is as sensitive to change as other scales,60 a score must be able to represent changes throughout the entire spectrum of potential functional outcomes . It is in this regard that the foor and ceiling effects of the bi become apparent.50 floor and ceiling describes the phenomenon by which the score does not change from minimum or maximum despite clinical change.61 for example, a stroke patient in a neurointensive care setting can make significant gains but still score a total 0 on the bi; conversely, a patient who is discharged from hospital and independent may still have substantial functional problems but will score 100 on the bi . Given this limitation, the bi may be best suited to stroke survivors requiring inpatient rehabilitation, while other scales may be needed to assess functional change in those with more major or minor stroke symptoms.62 the bi can be considered as a measure of basic adl (self - care and mobility). Extended activities of daily living (e - adl) measures.63 the term instrumental adl was first used in lawton and brody s work, and a lawton i - adl scale is described.64 a validated measure that has been used with stroke survivors is the nottingham extended adl scale, which asks participants to reflect their actual activities over the preceding weeks, rather than simply what they have the capability to do.65,66 the nottingham extended adl scale compares favorably to the bi, and is less susceptible to the ceiling effects described.67 the mrs is a 6-point, ordinal hierarchical scale that describes global disability with a focus on mobility (table 2). The original rankin scale was developed by the scottish physician john rankin to describe the positive outcomes he was achieving in his prototypic stroke unit.68 although not originally intended as an assessment for clinical trials, a slightly modified version of rankin s eponymous scale was used as end point in the first multicenter stroke trial (the uk tia study).69 since this time, the mrs has grown in popularity and is now the most commonly used functional measure in stroke trials, and has been the primary or coprimary outcome in most recent large - scale stroke trials.16 a further variation of the mrs, the oxford handicap scale, has been described but is not commonly used by trialists . In contemporary stroke studies, the mrs is often used both as a measure of premorbid ability to assist in selection of patients and as final outcome measure . The mrs has many potential strengths, and it is acceptable to patient and assessor, with nonstandardized interviews taking around 5 minutes to complete.70 concurrent validity is demonstrated by strong correlation with measures of stroke pathology (for example, infarct volumes) and agreement with other stroke scales.71,72 the six potential scores on the mrs (05) describe a full range of stroke outcomes, with a score of 6 usually added to denote death . With a limited number of scores, the mrs may be less responsive to change than some other scales; however, a single - point change on the mrs will always be clinically relevant . The principle limitation of the mrs is its reliability, with the potential for substantial interobserver variability . A study describing the interobserver variability of the mrs is available; indeed, in the first clinical studies that used the mrs as end point, the trialists described interobserver variability for a third of subjects interviewed by paired assessors.73 a systematic review and meta - analysis of studies describing interobserver variability of the mrs reports pooled reliability across ten published studies (n = 587 patients) of kappa = 0.46.74 those studies that assessed mrs reliability with multiple raters and centers (ie, similar to a contemporary clinical trial) revealed a worryingly low agreement of kappa = 0.21.75 this level of inconsistency will impact on the validity of the trial results and conclusions . The statistical noise created by the interobserver variability will increase the possibility of a type ii error, ie, a beneficial treatment effect is missed . It has been postulated that problems with mrs reliability may have partly explained a series of unexpected neutral results in large - scale neuroprotectant studies . There are published examples of nonstroke studies whose results were fundamentally altered when statistical analysis accounted for observer variation.76 recognizing the problems of reliability in standard mrs assessments, trialists have explored various interventions to improve consistency in scoring . Usual mrs interviews are unstructured, and researchers vary considerably in their length of interview and number of questions asked . More structured approaches to assessment have been described, from a comprehensive scripted interview75 to use of anchoring questions that require a yes / no answer.77 the groups that developed these assessments describe substantial improvements in reliability . However, improvements have not been seen when the structured interviews have been tested by independent centers.78 training in use of the mrs can also offer potential to improve consistency . As with the nihss and bi, an online training resource is available with an accompanying certification exam.79 a further trial modification that may improve reliability is to record mrs interviews and have a remote consensus grading by experienced stroke trialists . Two other modifications to mrs assessment are commonly used and deserve some discussion: using proxies to substitute for stroke survivors in the mrs interview and calculating a prestroke mrs . Stroke survivors often have physical, language, or cognitive impairments that may complicate a standard face - to - face interview . In this situation, an informant who knows the patient often supplements the interview or substitutes it completely . While this approach makes intuitive sense, we should not assume validity, and clinimetric analysis is still required . A recent systematic review of proxy stroke scales (the mrs was not included) suggested that the properties of certain proxy - based assessments may differ from equivalent standard assessments.80 a study of proxy mrs described suboptimal reliability and validity, and recommended that direct mrs interview with the patient should be the preferred assessment if possible.81 in stroke trials, traditional statistical analyses assess numbers achieving a good functional outcome . To improve trial power, prestroke mrs has been used in many landmark stroke trials, where prestroke mrs> 2 is used as the exclusion criterion during participant selection . The wording of the mrs grades is not suited to such prestroke assessment, and it is perhaps unsurprising that when formally assessed, prestroke mrs had only moderate reliability and validity.82 in view of improving longer - term survival and functional outcomes following stroke,83 it could be argued that assessments against participation or qol will become increasingly important.84 certainly evaluations of health - related qol in stroke survivors can provide a rich description of the multifaceted effects of a stroke, providing insights above those recorded with traditional impairment and activity measures.85 measuring health - related qol in stroke presents particular challenges . Important predictors and components of qol following stroke will vary at different periods following the event.86 thus, we must balance having a suitably comprehensive assessment that is sensitive to the nuances of qol against the time and burden required for this assessment . Carer / family - based assessments of the patient s qol are often biased, with the proxies reporting poorer outcomes than the subject.87 various qol scales have been proposed, some generic and some specific to stroke / brain injury . Qol scales should be subject to the same rigor of clinimetric assessment as any other scale . It is evident from the published literature that for qol there is a propensity to generate new scales rather than validating existing ones.88,89 it has been argued that qol can be assessed by asking just two questions assessing dependency and problems.90 an alternative approach is to apply existing health - related qol scales or to use disease - specific scores . The short form 36 (sf-36) is a generic scale intended for patient completion that assesses eight domains of health - related qol derived from the medical outcomes study (table 3).91 although the sf-36 is validated for stroke patients,92 noncompletion bias and marked floor and ceiling effects may limit its utility.93,94 the generic qol scale, euro - qol, was developed based on the findings of an international postal survey.95 the self - completion questionnaire requires assessment across five domains complemented by a visual analog scale (table 3).96 euroqol has been validated in stroke populations.97 however, noncompletion bias is recognized: in one study, only 61% of stroke survivors could complete the scale without external assistance.97 the stroke - specific qol scale was developed based on interviews with stroke survivors.98 it is based on twelve domains (table 3).93 the scale is validated in stroke populations, and values for minimal detectable change and clinically important difference99 are established . Many assessment tools, spanning various functional domains, are available to clinicians and researchers working with stroke survivors . We have given a favor of the marked heterogeneity in use of assessment scales . Lack of consistency in outcome assessment has hindered comparative research and meta - analysis, and so we would recommend that future researchers use a common set of outcome assessments . No perfect stroke - assessment scale exists, and in this review we have deliberately avoided suggestions that one scale is better than an other . We have focused on the three most commonly used stroke scales (mrs, bi, nihss) as exemplars . These scales have been validated, are familiar to many, and have proven utility, with each suited to differing assessment scenarios . Assessment, we would recommend continuing use of the three core assessment scales: the mrs as an outcome if the study is describing global disability, the nihss for studies looking at neurological impairment, and the bi for studies looking at basic adl . Trialists and clinicians can supplement these core assessments with specific tools suited to the clinical scenario / research question . Increasing awareness of the importance of clinimetric properties has highlighted deficiencies and potential limitations with stroke functional assessment . Clinicians and researchers should always select their assessment tool(s) based on the question of interest and the evidence base around clinimetric properties . Where, as is often the case, the research around clinimetric properties of a scale is sparse, we would encourage researchers to design and conduct their own clinimetric studies.
Most fish bones stick in the oropharynx and are within the province of the ear nose throat surgeon . Thoracic complications due to esophageal penetration by an ingested foreign body are rare, with the reported incidence being 1% to 4% . Incidence of complications by ingested fish bones can be of a more significant rarity because only approximately 9% to 45% ingested foreign bodies are fish bones . Fatal hemorrhage followed by aortoesophageal fistula, aortic pseudoaneurysm, and severe mediastinitis is the most common cause of death . Lung abscess caused by ingested fish bones is very rare . To date, less than ten similar cases have been reported in the literature . To our knowledge, there have been no cases reported in which this condition was treated using video - assist thoracic surgery (vats). In november 2014, a 47-year - old male public official was admitted to the department of thoracic surgery with the complaint of continuous cough and fever . The patient recalled that he had swallowed a bowl of fish soup in a drunken state twenty days before . He felt that a fish bone was impacted in his throat, and he tried several ways to pick it out . He made no gain even after having suffered great pains of trying inducing vomiting with his fingers, drinking a cup of vinegar, chewing gum and swallowing repeatedly . Before deciding to seek medical intervention his sensation of foreign body impaction was relieved at that time by this self - service approach, but several days later, he began to suffer from the throat itching, dry cough, and a feeling of being very ill . He went to see doctor a week later but no abnormalities could be found by endoscopic and barium meal x - ray examinations . The dry cough continued and so did the fever that began fourteen days after the accident . A computed tomography (ct) scan at a local hospital revealed that a lung abscess and a foreign body embedded in his right upper lobe . After double - lumen intubation and in the lateral position, exploration by vats was carried out and a dissection of the lung abscess located in the right upper lobe tip section was performed . The 4-cm - long, sharp - blade shaped fish bone was successfully removed . We spent a bit more time treating hemorrhage of adhesion resulting from the stimulation of inflammation on the posterior chest wall . The surgical exploration revealed that there was no observable mediastinitis and the enlargement of the mediastinum seen in the ct film was proved to be the peripheral lung abscess . The surgical time was 50 minutes, and there was about 50 ml blood loss . After antibiotic therapy followed by observation, the patient was discharged with an uneventful recovery on the seventh day after surgery . There were no pulmonary or esophageal abnormalities at a follow - up appointment 3 months later . The case study was approved by the ethics committee of the second xiangya hospital, central south university, changsha, hunan, china . Complications such as aortoesophageal fistula, aortic pseudoaneurysm and mediastinitis are extremely difficult to treat surgically . Strategies using a thoracotomy with or without cardiopulmonary bypass or intravascular stent have been reported in the literature but often the erosion of the walls of the great vessels is too extensive to be repaired . Potential life - threatening sequela such as severe thorax / mediastinal infection, massive hemoptysis or latent hemorrhage caused by erosion can occur, although these complications are not as urgent as aortic perforation . Fish bones are the common kind of foreign bodies, which are easy to ingest because of their shape and size . When a fish bone stuck in the throat, many people adopt a strategy of swallowing a compact mass of food to press the fish bone downward . However, this approach can backfire if the fish bone has already penetrated the esophageal mucosa . In this patient, we suspect that it was the swallowed rice that forced the sharp blade - shaped fish bone completely into the right upper lobe parenchyma . The process was so quick as the patient complained of no initial esophageal symptoms and it could not be found by the endoscopic or barium x - ray examinations . Fortunately for the patient there was no great vessel involvement during injury . Although the diagnosis was supported by routine ct scanning, ct angiography before surgery is recommended to clarify the situation of vessel injury so as to reduce the probability to take adventure . There was no evidence of a visible injury to the esophagus based on ct scanning, barium x - ray and endoscopy, although there was a long period of time between the injury and treatment . These factors enable us to choose vats, which has not been reported before, to treat this patient . The good clinical outcome showed it to be safe, minimally invasive, and feasible (figure 1). (a and b) a 4-cm - long fish bone is shown embedded in the abscess in right upper lobe . In conclusion, vats procedure can be useful to obtain a good clinical outcome in a case of lung abscess from an ingested fish bone . However, it is also important to educate the public of the risks of trying to force an ingested object down into the stomach.
As a representative musculoskeletal disease in the orofacial region, temporomandibular disorder (tmd) is one of the most frequent causes of non - odontogenic pain in the orofacial region . Nowadays, the number of tmd patients is on the rise, visiting dental clinics complaining of diverse symptoms . Yang and kim1 examined the incidence of korean tmd patients and treatment modes . According to them, from 2003 to 2005, there was an increase in patients every year, with teenagers showing a high incidence average with 22.5% . In korea, numerous epidemiological studies on tmd have been reported after the 1970s1 - 3 . In particular, the ratio of teenage patients who are under high pressure due to their studies tends to rise; hence the need to evaluate not only the symptoms of teenage tmd patients but also their behavior and psychological factors . The research diagnostic criteria for tmd (rdc / tmd) used in our study was prepared to examine patients with tmd by objective and standardized methods . Since it was introduced by dworkin and leresche4 in 1992, it has been translated into numerous languages worldwide and used widely to investigate tmd and maxillofacial pain . In clinical studies in particular, it contributes to systemic investigation and standardization, defines the investigation of an area that is difficult to measure, and proves the reliability of clinically standardized research methods . Rdc / tmd consists of a dual - axis system; axis i is mostly composed of the physical diagnosis of tmd, and axis ii, the evaluation of the behavioral, psychological and psychosocial factors associated with tmd . Specifically, rdc / tmd axis ii consists of a scale that evaluates depression; thus, it is also referred to as the rdc / tmd axis ii depression scale . This study sought to evaluate the severity and pattern of tmd particularly the behavioral, psychological, and psychosocial factors present among korean teenage tmd patients by analyzing their responses in the initial rdc / tmd axis ii questionnaire . The subjects were teenagers (between 11 and 19 years of age) who first visited seoul national university bundang hospital from january 2006 to december 2010 with chief complaint of tmd symptoms . Patients were asked to fill out the rdc / tmd questionnaire at the first visit . Only patients who completed the rdc / tmd questionnaire were included in this study; others who did not answer some of the questions were excluded . This study was conducted after obtaining approval from the institutional review board of seoul national university bundang hospital (b-1110 - 138 - 115). By analyzing the medical records of patients, radiological examinations, and rdc / tmd axis ii questionnaires prepared by patients at the initial examination, the symptoms and patterns exhibited by patients were evaluated retrospectively. (table 1) of the entire group of patients, the frequency of incidence of each diagnosis group, chief complaint of patients, level of impairment experienced by patients during daily activities, level of psychological pain (depression and vegetative symptoms), pain due to somatization, and graded chronic pain score (0-iv) were examined . The diagnosis groups were classified according to the classification of tmd suggested by the japanese society for temporomandibular joint (tmj)5 . They were classified as types 1 - 5 depending on the area of the major lesion, pathology, symptoms, and radiological results . Type 1: the major lesion area is the masticatory muscles . It is manifested as muscle tone, myositis, and muscle spasm . Type 2: the major lesion areas are the articular capsule, articular ligaments, and articular disc . It is manifested as the extension and contusion of the articular capsule, ligaments, and articular disc . Type 3: the major lesion area is the synovial membrane . It is manifested as the displacement of articular disc, degeneration of articular disc, perforation, and fibrosis . Patients experience tmj pain as well as a clicking sound . Magnetic resonance imaging (mri) type 4: the major lesion areas are articular cartilages, articular disc, synovial membrane, mandibular condyle, and mandibular fossa . It is manifested as cartilage destruction, bone resorption, and osteolysis . Type 5: patients who are not included in any of the above groups . Patients were asked what their chief complaint was for visiting, and their responses were classified into pain, trismus, clicking sound, tmj discomfort, and bruxism . The questionnaire asked whether discomfort was experienced during chewing, drinking, brushing teeth or face washing, exercise, yawning, eating of hard food, swallowing of food, eating of soft food, speaking, smiling or laughing, and routine facial expression, answerable by either " yes " or " no . " In the questionnaire, a question that evaluates depression (0 - 4) was added, and the average was obtained . According to severity, in addition, the level of severity of the presented symptoms according to the questionnaire was also evaluated . Pain due to somatization was examined; the average value of the responses (0 - 4) was obtained, and they were classified into 3 levels: normal, moderate, and severe . Graded chronic pain score: 0 (no pain during the past 6 months), i, ii, iii, and iv . Characteristic pain intensity was calculated as the average of the value of visual analogue scale (vas) described in the 7th, 8th, and 9th questions of rdc10 . Disability points were calculated as disability days (0 - 3)+disability score (0 - 3, the average of the value of vas described in the 11th, 12th, and 13th questions of rdc10). The diagnosis groups were classified according to the classification of tmd suggested by the japanese society for temporomandibular joint (tmj)5 . They were classified as types 1 - 5 depending on the area of the major lesion, pathology, symptoms, and radiological results . Type 1: the major lesion area is the masticatory muscles . It is manifested as muscle tone, myositis, and muscle spasm . Type 2: the major lesion areas are the articular capsule, articular ligaments, and articular disc . It is manifested as the extension and contusion of the articular capsule, ligaments, and articular disc . Type 3: the major lesion area is the synovial membrane . It is manifested as the displacement of articular disc, degeneration of articular disc, perforation, and fibrosis . Patients experience tmj pain as well as a clicking sound . Magnetic resonance imaging (mri) type 4: the major lesion areas are articular cartilages, articular disc, synovial membrane, mandibular condyle, and mandibular fossa . It is manifested as cartilage destruction, bone resorption, and osteolysis . Patients were asked what their chief complaint was for visiting, and their responses were classified into pain, trismus, clicking sound, tmj discomfort, and bruxism . The questionnaire asked whether discomfort was experienced during chewing, drinking, brushing teeth or face washing, exercise, yawning, eating of hard food, swallowing of food, eating of soft food, speaking, smiling or laughing, and routine facial expression, answerable by either " yes " or " no . " In the questionnaire, a question that evaluates depression (0 - 4) was added, and the average was obtained . According to severity, in addition, the level of severity of the presented symptoms according to the questionnaire was also evaluated . Pain due to somatization was examined; the average value of the responses (0 - 4) was obtained, and they were classified into 3 levels: normal, moderate, and severe . Graded chronic pain score: 0 (no pain during the past 6 months), i, ii, iii, and iv . Characteristic pain intensity was calculated as the average of the value of visual analogue scale (vas) described in the 7th, 8th, and 9th questions of rdc10 . Disability points were calculated as disability days (0 - 3)+disability score (0 - 3, the average of the value of vas described in the 11th, 12th, and 13th questions of rdc10). Of a total of 194 subjects who completed the rdc / tmd questionnaire, 77 were male (39.6%) and 117 were female (60.4%); their mean age was 16.22.05 years . Among the first - visit patients from 2006 to 2010, approximately 18 - 22% of tmd patients were teenagers. (table 2) for the evaluation of teenage patients' distribution according to age, the ratio of patient from 17 years to 19 years was high (53%) in the male group . In the case of the female group, however, patient distribution was relatively constant from 13 years to 19 years. (fig . 1) at the first visit, the number of patients who visited with chief complaint of clicking sound (34.5%) or tmj pain (36.6%) was the highest. (table 3) as a result of the analysis of chief complaint according to sex, the ratio of clicking sound was highest in the male group (34 patients, 44.1%). In the female group, however, the percent ratio of tmj pain appeared highest among others (49 patients, 41.8%). At the time of classification into the diagnosis groups, the incidence of temporomandibular internal derangement (53%) was the highest. (table 4, fig . 2) when the level of impairment during daily life was assessed, patients were found to experience discomfort while chewing, yawning, and eating hard food. (table 5) in evaluating the depression index, 75.8% of the total subjects were normal, and 12.9% were moderate; 11.3% belonged to the severe group. (table 6) additionally, the subjects noted a decrease in energy . Regarding the non - specific physical symptoms (including pain), 66.5% of the subjects were normal, 17.0% were moderate, and 16.5% were severe. (table 6) when it was examined in more detail, symptoms of " headache, " " vertigo or dizziness, " and " back pain " were prominent . Regarding non - specific physical symptoms (excluding pain), 70.6% of the subjects were normal, 14.4% were moderate, and 15.0% were severe. (table 6) among them, the level of discomfort was high due to " weakness in a part of the body " and " heaviness in the arm or leg . " With regard to the graded chronic pain score, high disability (grade iii or iv) was shown to be 9.3%. (table 7) for the comparison of the average of depression index and non - specific physical symptoms including pain and those excluding pain between male and female, there was no statistical significant difference (independent t test, p>0.05). The chief complaints of tmd are chronic pain, muscular pain during palpation, trismus, and clicking sound . Among them, chronic pain is most common . Chronic pain appears to be associated with psychological, behavioral, and social factors as well as physical lesions . For treatment, behavioral therapy there has been a trend showing that pressure, sorrow, weakness, and abnormal function manifest abundantly in tmd patients6,7 . In our study, chief complaints of pain, clicking sound, and trismus were most frequent among all teenage tmd patients . In other studies, similar results have been reported8 . As a result of classifying the diagnosis groups at first visit, type 3 or cases accompanied with type 3 recorded the highest ratio (76%), similar to the results of previous studies2 . The incidence of tmd has been reported to be high among persons in their 30s-40s9 . According to recent studies, the ratio of patients in their teens and 20s makes up 50% of tmd patients, and the onset age of tmd becomes lower7,10 . In our study, among the first - diagnosed tmd patients, the ratio of teens was found to be 18 - 22% annually . Regarding the ratio of male and female tmd patients, females have been reported to be more prevalent than males by 2 - 5 times7 . According to magnusson et al.11, the manifestation of temporomandibular symptoms--such as clicking sound, fatigue in the jaw, trismus, and pain during mastication--was higher among females than males . In a study that examined clicking sounds in children and teenagers, torii12 reported that the rate of manifestation of clicking sound was significantly higher in girls than boys . In our study, 39.6% of the subjects were males and 60.4% were females . As a result of chief complaint analysis according to sex, our study found that the ratio of clicking at first visit appeared highest in the male group . On the other hand, females have been reported to visit hospitals more frequently than males because the former have higher sensitivity to pain . Likewise, due to cultural background, females complaining of pain were deemed more acceptable than males13 . As to the reason for the higher incidence of tmd in females than males, hatch et al.14 suggested the role of the sex hormone . The hypothesis stated that the value of the sex hormone is associated with the menstrual cycle; thus possibly wielding influences on pain perception . Another study that examined the association of temporomandibular arthritis with females reported that the polymorphism of the estrogen receptor is associated with pain sensitivity15 . In addition, certain symptoms of tmd--for example, trismus--show a tendency to have high prevalence of a specific estrogen receptor . On the other hand, the association with tmd symptoms has been reported to be not statistically significant; thus, the association of estrogen with the etiology of tmd is deemed to require more investigation17 . Non - specific physical symptoms (somatization) are assessed to evaluate pain, fatigue, and level of non - specific symptoms pertinent to the cardiopulmonary circulation system . The chronic pain index is based on the description of the level of pain experienced by patients and impairment of function during daily life due to pain described as vas and is classified into 5 grades (0 - 4)18 . Kim et al.19 have reported that, in tmd patients, somatization and tmd may be manifested in close association with the strength of pain and level of impairment . Therefore, comprehensive understanding of emotional pressure experienced by patients and appropriate management of dysfunction are very important for the successful treatment of tmd patients . In our study, 33.5% of patients exhibited moderate to severe non - specific physical symptoms . Chewing was found to require strong force of the masticatory muscles; yawning or eating hard food caused patients great discomfort . In addition, the results of the depression scale show that 24.2% of patients were in the moderate or severe group . At the time of clinical examination, patients who ranked high on the depression scale were deemed to require collaborative treatments with psychologists . The sensitivity of the rdc / tmd axis ii depression scale in the korean version has been reported to be high; thus, it could be applied to examine the presence or absence of depression symptoms in korea20 . Lee and kim8 mentioned that bruxism, clenching, chewy food, history of trauma, and pressure were major causes . In particular, when the difference in bruxism and clenching between genders was examined, males showed relatively higher ratio in bruxism, and females, in clenching . Nevertheless, bruxism has also been reported not to be associated with age and gender13 . In another study, the incidence of unilateral mastication, sleeping on one side, or tongue or lip biting was found to be higher than bruxism or clenching21 . Yet another study reported that the incidence of tmd caused by traffic accidents was lower than yawning and masticatory function, and that chronic pain in the temporomandibular area after traffic accidents was due to psychological factors rather than physical impacts in many cases22 . According to yun and kim23, major trauma in the facial area should be considered an important causative factor of tmd . Kamisaka et al.24 have also emphasized the association of history of trauma with the manifestation of tmd symptoms . Teenage koreans, in addition to the task of ego - identity development, are considered to experience high pressure due to the task of overloaded study . According to a 2009 report of the statistics bureau of the department of social welfare, the ratio of children / teenagers who experienced pressure routinely was " very high " or " high " with 46.5% . It was higher in female students than male students; as they became older, they experienced more pressure25 . Similarly, our study showed that the late teens ratio was relatively high in male patients, and that ratio over 15 years was high in female patients . Therefore, pressure may be a closely associated factor that contributes to the development of tmd among teenage patients . The results of our study show that patients experienced great discomfort in the following categories of tmd: chewing, yawning, and eating hard food . The results of chronic pain and depression scale show that 24.2% of the patients were classified under the moderate or severe group . For the graded chronic pain score, high disability (grade iii or iv) was seen in 9.3% of the subjects . Among teenage tmd patients, clinical symptoms and psychological pressure were very severe in some patients; thus, they are deemed to require aggressive attention and treatment . Understanding of psychological pressure and appropriate treatments for dysfunction are important and should be considered.
Extraskeletal osteosarcoma (esos) is a rare aggressive tumor, which commonly presents with disseminated metastases . Lungs and bones are common sites of metastatic involvement in osteosarcoma at initial presentation as well as after completion of treatment . Computed tomography (ct) scan of the chest and bone scan are routinely used to detect lung and bone metastases in osteogenic sarcomas . With the advent of f - fluoro - deoxyglucose (f - fdg) positron emission tomography / computed tomography (pet / ct) imaging, unusual and often rare sites of metastatic spread have been uncovered in several malignancies . In this report, we describe an extremely rare combination of unusual metastatic sites from esos on fdg pet / ct . A 32-year - old female patient presented to our institution with a bulky left supraclavicular mass . The swelling was noticed 3 months back and had rapidly grown in size over the past month, with no accompanying pain . On examination, a hard, non - tender, mobile mass was felt in the left supraclavicular fossa, separate from the adjacent bones, in a relaxed posture, which became taut on shrugging or moving left shoulder . A biopsy specimen from the mass, on histopathological examination revealed osteoid deposition by tumor cells, with osteoclastic giant cells and pleomorphic malignant cells within the osteoid, suggestive of osteosarcoma [figure 1]. Thus, correlation of clinical, imaging and histopathological findings confirmed the diagnosis of esos . Patient was then referred for a whole body f - fdg pet / ct study for pre - treatment staging . Maximum intensity projection (mip) images [figure 2a] revealed a large focus of tracer concentration in left supraclavicular region [figure 2a arrow], with multiple foci of uptake in the right hemithorax, region of the right kidney, left pelvis, mid thorax and abdomen [figure 2a all arrowheads]. Fused axial pet / ct images showed tracer uptake in a bulky soft - tissue mass arising from the left trapezius muscle, [figure 2b arrow], which was the site of primary malignancy . There was no involvement of left scapula noted on the ct component of the study, confirmed on coronal oblique ct [figure 2c arrow] and pet / ct [figure 2d arrow] images . Furthermore seen was intense fdg uptake in the right lung mass, left iliac bone marrow [figure 3b arrow], d12 vertebra [figure 3c arrow], within a soft - tissue deposit in the left internal oblique muscle [figure 3d arrow], in right renal cortex [figure 3e arrow] and in medial end of left clavicle . Also noted was tracer uptake in the brain, which correlated to a soft - tissue lesion in right paraventricular region, with specks of calcification within, seen on axial fused pet / ct [figure 3a arrow]. Thus, in addition to the common sites of metastases in the lungs and bones, fdg pet / ct detected metastatic deposits at unusual sites such as brain, renal parenchyma, marrow and skeletal muscle . Photomicrograph of pathology section showing osteoclastic giant cells and pleomorphic malignant cells amidst osteoid (h and e, 200) (a) maximum intensity projection image showing tracer uptake in left supraclavicular region (2a - arrow), in the region of mid thorax, mid abdomen, right lung, right kidney, left pelvis (all arrow heads) (b) axial fused pet / ct images show fdg avid mass arising from left trapezius muscle (arrow) (c) oblique coronal ct and fused pet / ct (d) images show origin of the mass from left trapezius, with no involvement of left scapula axial fused pet / ct images show fdg avid metastatic lesions in brain (a arrow), left iliac marrow (b arrow), d12 vertebral body (c arrow), left internal oblique muscle (d arrow) and right renal cortex (e arrow) esos is a rare tumor, accounting for 1% of all soft - tissue sarcomas and 4% of osteogenic osteosarcomas . Though it is histologically similar to osteogenic osteosarcoma, it is demographically, clinically and therapeutically different from it . It most commonly presents as a rapidly growing soft - tissue mass, either with or without pain; with lower extremity being the most common site and thigh accounting for 48% of the lesions, followed by upper extremity (8 - 23%), retroperitoneum (8 - 17%) and trunk (10 - 11%). Lung is the most common site of metastases (80 - 88%), similar to osteogenic osteosarcoma; other metastatic sites being soft - tissue, bone (8 - 19%), liver (8 - 17%), peritoneum and adrenal glands (<5%). Since esos is generally a high grade spindle cell malignancy, the primary tumor as well as metastatic sites demonstrate fdg avidity . Fusion with ct helps to characterize the lesions, which show the presence of mineralized material within the soft - tissue and also provides an anatomical dimension to the fdg uptake . The existing literature on fdg pet / ct in esos confirms the fact that these tumors are fdg avid, however all these reports demonstrate a single metastatic site, unlike multiple and rare sites demonstrated in our report . Metastases to the brain are extremely rare, with only a single case detected on histopathology, after surgical resection was performed in 21 patients with intraparenchymal brain metastases from sarcoma . Though bone involvement is commonly seen, discrete marrow metastases have not been reported yet . Thus, a whole body metabolic imaging modality like fdg pet / ct is the investigation of choice in such high grade rare malignancies, which are aggressive at presentation . It is very well - known that the management of metastatic disease is not altered based on the number of sites of distant metastases . Fdg pet / ct detects rare metastatic sites, which are not only an interesting finding, but also a valuable addition to the existing literature on metastatic sites of esos . Such documentation is of value, for a rare subgroup of patients who can present with metastases, from an unknown primary site, wherein, esos can emerge as one of the likely primary.
It has correctly been called as the great masquerader due to its protean manifestations and its many faces . The 81 manifestations of this tumor may challenge a physician's acumen because it can present in so many disguises, suggesting a large variety of diagnostic possibilities . Cerebral (pseudo) vasculitis, renal artery stenosis, coronary vasospasm and intestinal ischemia due to mesenteric vasospasm are the frequently described vascular and/or vasculitic manifestations of pheochromocytoma . Although medium - sized vessels are the most commonly involved, small vessel vasculitis (cutaneous leucocytoclastic vasculitis, which was glucocorticoid resistant but reversed promptly with excision of pheochromocytoma) is also described . However, aortoarteritis (large vessel vasculitis) as a manifestation of catecholamine - induced vasculitis is not described in the literature . We report two cases of pheochromocytoma with aortoarteritis, where we propose a role for catecholamine excess status in the etiopathogenesis of aortoarteritis . A 21-year - old lady, mother of two children, presented with recurrent episodes of palpitations, excessive sweating and dyspnea for 3 months . She had a family history of pheochromocytoma [figure 1a]. On examination, she had hypertension in the right upper limb (160/100 mmhg) but pulses were feeble in the left upper limb and both lower limbs . Further evaluation revealed elevated plasma free normetanephrine (1405 pg / ml) with normal plasma free metanephrine (23 pg / ml). Magnetic resonance angiogram [figure 2] showed dilatation of aortic root and the proximal ascending aorta (largest diameter = 41 mm). Brachiocephalic trunk showed dilatation just before its bifurcation into the right common carotid artery and right subclavian artery, which were normal . The left subclavian artery was occluded from its origin . A short segment, high - grade stenosis of the celiac artery was seen at its origin . Her erythrocyte sedimentation rate (56 mm/1 hour) was elevated while antinuclear antibody and rheumatoid factor were negative . After appropriate preoperative management (blood pressure was 130/86 mmhg on prazosin 20 mg / day and atenolol 50 mg / day), she underwent laparoscopic bilateral adrenalectomy . Postoperatively, she had hypocortisolism and was started on prednisolone (1 mg / kg / day) and fludrocortisone (50 g / day). Currently, she is under regular follow - up by a cardiovascular surgeon and is normotensive on 10 mg / day of amlodipine . (a) family pedigree of case 1, (b) family pedigree of case 2 bilateral pheochromocytoma, left sided pheochromocytoma with pancreatic neuroendocrine carcinoma the numbers in squares and circles represent the age of diagnosis of pheochromocytoma in each affected member magnetic resonance angiogram of case 1 (a) showing dilatation of brachiocephalic trunk (b), dilatation of aortic root (c), stenosis of the celiac artery at its origin (d) and stenosis of right renal artery (e) a 17-year - old girl presented with recurrent episodes of palpitation, headache and excessive sweating for 6 months, which were associated with abdominal pain, and dyspnea for the last 1 month . At the time of presentation to hospital, she complained of severe dyspnea, generalized body swelling and right - sided weakness for the last 3 days . Magnetic resonance imaging revealed hypertensive encephalopathy with infarct in the left middle cerebral artery territory . Ct from head to pelvis showed bilateral adrenal pheochromocytomas and a paraganglioma at left renal hilum with calcification of abdominal aorta and coronary artery . Ct angiogram showed thickening of the aortic wall with characteristic beading of aorta suggestive of aortoarteritis [figure 3]. Her plasma free normetanephrine (1070 pg / ml) was elevated . After appropriate preoperative preparation (blood pressure was 128/84 mmhg on prazosin 20 mg / day and atenolol 25 mg / day), she underwent right adrenalectomy, left cortical sparing adrenalectomy and excision of paraganglioma at left renal hilum . She had transient postoperative hypertension, which was managed on 5 mg of amlodipoine until 3 months after surgery . Postoperatively, she had normal serum cortisol (16 g / dl) levels and did not require glucocorticoid or mineralocorticoid replacement . Family screening revealed bilateral pheochromocytomas in one sibling and her father [figure 1b]. Computerized tomography of case 2 showing calcification of aorta (a), calcification of coronary artery (b), beaded appearance of aorta on ct angiogram with calcification of both common iliac arteries (c) and right adrenal pheochromocytoma and paraganglioma at left renal hilum (d) a 21-year - old lady, mother of two children, presented with recurrent episodes of palpitations, excessive sweating and dyspnea for 3 months . She had a family history of pheochromocytoma [figure 1a]. On examination, she had hypertension in the right upper limb (160/100 mmhg) but pulses were feeble in the left upper limb and both lower limbs . Further evaluation revealed elevated plasma free normetanephrine (1405 pg / ml) with normal plasma free metanephrine (23 pg / ml). Magnetic resonance angiogram [figure 2] showed dilatation of aortic root and the proximal ascending aorta (largest diameter = 41 mm). Brachiocephalic trunk showed dilatation just before its bifurcation into the right common carotid artery and right subclavian artery, which were normal . The left subclavian artery was occluded from its origin . A short segment, high - grade stenosis of the celiac artery was seen at its origin . Her erythrocyte sedimentation rate (56 mm/1 hour) was elevated while antinuclear antibody and rheumatoid factor were negative . After appropriate preoperative management (blood pressure was 130/86 mmhg on prazosin 20 mg / day and atenolol 50 mg / day), she underwent laparoscopic bilateral adrenalectomy . Postoperatively, she had hypocortisolism and was started on prednisolone (1 mg / kg / day) and fludrocortisone (50 g / day). Currently, she is under regular follow - up by a cardiovascular surgeon and is normotensive on 10 mg / day of amlodipine . (a) family pedigree of case 1, (b) family pedigree of case 2 bilateral pheochromocytoma, left sided pheochromocytoma with pancreatic neuroendocrine carcinoma the numbers in squares and circles represent the age of diagnosis of pheochromocytoma in each affected member magnetic resonance angiogram of case 1 (a) showing dilatation of brachiocephalic trunk (b), dilatation of aortic root (c), stenosis of the celiac artery at its origin (d) and stenosis of right renal artery (e) a 17-year - old girl presented with recurrent episodes of palpitation, headache and excessive sweating for 6 months, which were associated with abdominal pain, and dyspnea for the last 1 month . At the time of presentation to hospital, she complained of severe dyspnea, generalized body swelling and right - sided weakness for the last 3 days . Magnetic resonance imaging revealed hypertensive encephalopathy with infarct in the left middle cerebral artery territory . Ct from head to pelvis showed bilateral adrenal pheochromocytomas and a paraganglioma at left renal hilum with calcification of abdominal aorta and coronary artery . Ct angiogram showed thickening of the aortic wall with characteristic beading of aorta suggestive of aortoarteritis [figure 3]. After appropriate preoperative preparation (blood pressure was 128/84 mmhg on prazosin 20 mg / day and atenolol 25 mg / day), she underwent right adrenalectomy, left cortical sparing adrenalectomy and excision of paraganglioma at left renal hilum . She had transient postoperative hypertension, which was managed on 5 mg of amlodipoine until 3 months after surgery . Postoperatively, she had normal serum cortisol (16 g / dl) levels and did not require glucocorticoid or mineralocorticoid replacement . Family screening revealed bilateral pheochromocytomas in one sibling and her father [figure 1b]. Computerized tomography of case 2 showing calcification of aorta (a), calcification of coronary artery (b), beaded appearance of aorta on ct angiogram with calcification of both common iliac arteries (c) and right adrenal pheochromocytoma and paraganglioma at left renal hilum (d) aortoarteritis in pheochromocytoma patients may be due to triggering of an autoimmune process by excess catecholamines . The first report of association between a catecholamine excess status and a vasculitis was published in 1989 . However, the coexistence of bechet's disease and pheochromocytoma in this report was thought to be a chance phenomenon . This report was closely followed by another similar case where behcet's disease symptoms were persistent even after high - dose steroids but rapidly resolved after excision of pheochromocytoma . This observation suggested a definitive role for catecholamine excess in the pathogenesis of bechet's disease . In addition, a few other reports have suggested a definitive role for catecholamine excess status in other autoimmune disorders such as systemic lupus erythematosus and rheumatoid arthritis. [57] hence, we hypothesize that a similar mechanism might have operated in our patients, leading to aortoarteritis . Pheochromocytoma has been described to be associated with cerebral vasculitis and renal artery stenosis (either permanent or transient) where catecholamine excess status has been implicated in their etiology . In animal studies, adrenaline injections caused medial necrosis, destruction of the elastic lamellae and atrophic scarring of the walls of large vessels . These changes may be associated with aneurismal dilatation and involvement of the medium - sized vessels . Catecholamine - induced vascular damage may be due to constriction of the vasa vasorum with resultant medial ischemia or the mechanical trauma to the vessel wall by the induced rise in blood pressure . It appears that catecholamine excess status leads to direct damage to the vessel wall as well as triggering of autoimmune mechanism, both of which act in conjunction to produce aortoarteritis . Both patients had elevated normetanephrine (suggesting excess production of norepinephrine) with normal metanephrine . Norepinephrine being a more powerful vasoconstrictor than epinephrine, it may also lead to more vascular damage . Hence, norepinephrine - secreting pheochromocytoma / paraganglioma patients may be at higher risk for all vessel - related complications including aortoarteritis . In conclusion, we report two cases with coexisting pheochro - mocytoma and aortoarteritis where we have suggested a role for excess catecholamines in the etiopathogenesis of aortoarteritis.
A review on attrition from behavioral medicine treatments showed that about one - third drops out from weight loss programs . Others, however, suggest that drop - out rates in weight loss studies might be as high as 80% [2, 3]. Attrition research has tried to understand the reasons for dropping out from weight loss studies . Various reasons for drop - out have been examined in the past . For many years, drop - out has been tried to link to demographic variables, such as age, social class, occupational factors, life stress, and financial factors . The differences between study completers and drop - outs with regard to social class, occupational factors, life stress, and financial factors might represent a more general socio - economic difference between obese patients who finish treatment and those who drop out of treatment . Although these studies show that demographic variables have successfully been related to drop - out, davis and addis argued that in order to improve attrition research, more attention should be paid to theoretically grounded psychological and behavioral predictors of drop - out . Their review on predictors of attrition in weight loss treatment showed that psychological and behavioral variables are the most predictive variables of drop - out from weight loss programs . Psychological variables that were found to relate to treatment drop - out were having too high treatment expectancies, low self - efficacy expectancies, low perceived success of weight loss, and low expectations of stress . Behavioral variables that were associated with study drop - out were a low number of previous diets, a high frequency of weight loss attempts, a low amount of weight loss, little exercise, and high cigarette consumption . The value of assessing psychological predictors of drop - out was confirmed by more recent research by [6, 7]. Grossi and colleagues studied complexity of attrition in the treatment of obesity . By means of structured telephone interviews, 940 obese patients were interviewed about their reasons for dropping out from weight loss programs . Except for having a university degree, practical problems, such as organizational or physical barriers, were the most mentioned reasons for drop - out . Furthermore, lack of motivation, dissatisfaction with the study (weight loss) results, lack of self - confidence, and sense of abandonment were the most important psychological reasons for drop - out as mentioned by patients . More recently, mental disorders and personality characteristics have been examined as possible psychological predictors of drop - out . Psychiatric comorbidity, such as depression or anxiety, has been shown common in treatment seeking obese subjects [8, 9] and was found to relate to both attrition and weight loss success [10, 11]. De panfilis and colleagues examined personality features as possible predictors of obesity treatment drop - out, controlling for comorbid psychopathology . Results showed that study completers presented with higher scores on harm avoidance. Non - completers were more likely than study completers to show psychiatric comorbidity, more specifically anxiety disorders . Although all of the abovementioned demographic, practical, psychological, and behavioral factors have added to existing knowledge on weight loss drop - outs, the concepts are quite diverse and difficult to compare . It seems as if the call for theoretically grounded psychological predictors of weight loss drop - out has remained unanswered, so far . Therefore, the aim of this study is to add a grounded psychological theory to research on attrition in weight loss studies . The self - regulatory predictors of drop - out will be examined next to demographic and behavioral predictors of attrition . Self - regulation (s - r) or goal theory provides a framework for differentiation between relevant motivational cognitions . S - r theory states that human actions are goal - oriented, and that goal pursuit and attainment are more likely if goals are personally relevant (autonomous or own goals), if individuals feel competent to attain them (goal efficacy), receive the necessary social support (goal support), and have an adequate plan for goal attainment (goal planning). There is evidence that autonomous regulation (goal ownership) is associated with better diabetes regulation . Goal support has been associated with better diabetes regulation and goal planning proved to be related to diabetes self - care and weight - related behaviors . These s - r variables have thus been proven to be predictors of treatment success, but they have not been used as potential predictors of attrition . The aim of this study is therefore to examine whether s - r variables predict attrition from a weight reduction intervention in diabetes type 2 patients, next to socio - demographic (age, gender, educational level, having a partner, and hours of employment), somatic (bmi, waist, and hba1c), distress and lifestyle (eating habits and physical activity) variables . A dataset of 1,316 diabetes type 2 patients from a general dutch hospital was screened for study inclusion . Based on inclusion criteria, 304 patients were invited for study participation by doctors, nurses, and dieticians during hospital visits . Inclusion criteria were: type 2 diabetes, body mass index (bmi) between 27 and 45, age between 21 and 70, caucasian, and proficient in the dutch language . Patients with co - morbidity (except for cardiovascular diseases) or under psychological or psychiatric treatment were excluded from the study . At baseline (t1), a total of 101 adult overweight (bmi 27 - 45) diabetes type 2 patients were included in the study . Of these 101 patients, 39 patients (39%) dropped out from t1 to t2 . Study non - completers were defined as patients who either actively withdrew from study participation or stopped attending study meetings . These study meetings could be intervention meetings (for the patients in the intervention group) or meetings for measuring weight, waist circumference, and blood pressure control (for those in the control groups). Patients who would miss out on one or several study meetings, but then started attending again, were not defined as drop - outs . Of course, several subgroups of drop - outs could be identified, e.g., early versus late drop - outs, passive versus active drop - outs . However, the only subgroup of drop - outs that was taken into account in the analyses of this study was membership of the intervention or control group. All patients were randomly assigned to (a) a self - regulatory weight reduction intervention in addition to standard care, or (c) an active control condition consisting of a self - help diabetes lifestyle manual in addition to standard care, and (c) a passive control condition consisting of standard care for diabetes type 2, including weight management . Psychosocial measures were s - r cognitions (goal ownership, goal efficacy, goal support, and goal planning), diabetes distress, and diabetes self - efficacy, all with good reliability and validity estimates . Bio - medical measures included weight, bmi, waist circumference, and glycemic control (hba1c). Healthy eating and exercise behavior assessed with eight items regarding the frequency of various nutrition and exercise behaviors within the past week . The variable salt, red meat, and sweets and snacks. Exercise behavior was assessed with the item on how many days in the past week did you have at least 30 minutes of moderate physical activity? Demographic variables that were assessed were age, gender, educational level, hours of employment, and having a partner . All statistical analyses were conducted with spss 16.0 . For power reasons, the active and passive condition formed one control group in the analyses . Analysis of covariance (ancovas) were conducted on the variables hba1c, demographic variables, diabetes self - efficacy, and diabetes distress to detect possible differences between study drop - outs and completers . Multivariate analysis of covariance (mancovas) was used to analyze differences between drop - outs and completers on the variables bmi and waist, the various s - r cognitions, and all of the lifestyle variables . The significant variables that were found in the (m)ancova analyses were entered, together with the dichotomous variable allocated to intervention or control group in a multiple logistic regression analysis to predict drop - out at t2 . Socio - demographic, somatic, psychological, and lifestyle baseline characteristics of the 101 patients are described in table 1 . Furthermore, baseline characteristics are described for patients from the intervention and the control group, separately . From the 101 patients who participated in the study at t1, 39 patients (39%) dropped - out at t2, more specifically this concerned ten patients from the intervention and 29 patients from the control group . Table 1baseline characteristics (means and standard deviations) of study completers and noncompletersoverallintervention groupcontrol groupcompletersnoncompleterscompletersnoncompleterscompletersnoncompleterssomatic variablesbmi (kg / m)34.62 (5.27)36.24 (5.51)35.01 (6.17)37.09 (5.46)34.29 (4.47)36.00 (5.62)n = 57n = 31n = 26n = 7n = 31n = 24waist (cm)117.42 (11.52)118.98 (12.15)120.17 (13.63)116.43 (10.86)115.03 (8.88)119.70 (12.61)n = 56n = 32n = 26n = 7n = 30n = 25hba1c (%) 7.26 (1.07)7.57 (0.86)7.39 (1.25)7.07 (0.77)7.15 (0.89)7.70 (0.85)n = 56n = 30n = 26n = 6n = 30n = 24socio - demographic variablesage (y)59.21 (7.40)56.67 (10.23)60.71 (6.55)57.67 (8.78)57.67 (7.99)56.33 (10.80)n = 61n = 36n = 31n = 9n = 30n = 27gender (m / f)28/3320/1916/156/412/1814/15having a partner (yes / no)52/931/826/57/326/424/5educational level (low / medium / high)32/14/1425/6/813/8/96/2/219/6/519/4/6hours of employment8.07 (15.56)14.87 (18.46)6.52 (14.04)12.90 (17.39)9.67 (17.08)15.55 (19.07)n = 61n = 39n = 31n = 10n = 30n = 29psychological variablesgoal ownership (1 = low, 5 = high)4.06 (0.64)2.18 (0.95)4.09 (0.59)2.61 (1.11)4.02 (0.70)2.04 (0.87)n = 59n = 37n = 30n = 9n = 29n = 28goal planning (1 = low, 5 = high)3.20 (0.68)3.70 (0.90)3.25 (0.60)3.68 (1.10)3.16 (0.76)3.71 (0.83)n = 57n = 34n = 31n = 9n = 29n = 25goal efficacy (1 = low, 5 = high)3.41 (0.56)3.50 (0.66)3.44 (0.58)3.50 (0.70)3.39 (0.55)3.50 (0.66)n = 60n = 37n = 31n = 9n = 29n = 28goal support (1 = low, 5 = high)3.17 (0,40)2.24 (1.03)3.12 (0.42)2.41 (0.85)3.22 (0.39)**2.18 (1.09)n = 54n = 36n = 27n = 9n = 27n = 27diabetes self - efficacy (1 = low, 10 = high)7.56 (1.08)6.65 (2.32)7.44 (1.09)7.56 (2.21)7.66 (1.06)6.42 (2.33)n = 57n = 35n = 28n = 7n = 29n = 28diabetes distress (paid; 1 = low, 100 = high)38.07 (13.14)37.00 (12.54)36.79 (13.11)40.00 (13.31)39.36 (13.28)35.88 (12.34)n = 56n = 33n = 28n = 9n = 28n = 24lifestyle variableshealthy eating (1 = 1 day a week, 7 = 7 days a week)5.21 (1.23)4.87 (1.26)5.15 (1.21)5.06 (1.28)5.27 (1.26)4.81 (1.27)n = 57n = 34n = 28n = 8n = 29n = 26unhealthy eating (1 = 1 day a week 7 = 7 days a week)2.93 (1.24)3.32 (1.35)3.17 (1.24)3.00 (1.36)2.69 (1.21)3.43 (1.37)n = 58n = 30n = 29n = 8n = 29n = 221 = 1 day a week of> 30 min . Physical activity, 7 = 7 days a week)4.69 (2.39)3.65 (2.63)4.71 (2.38)3.67 (2.18)4.67 (2.44)3.64 (2.79)n = 61n = 37n = 31n = 9n = 30n = 28p <.05, * p <.01, * * * p <.001 baseline characteristics (means and standard deviations) of study completers and noncompleters * p <.05, * * p <.01, * * * p <.001 ancovas (hba1c, demographic variables, diabetes self - efficacy, and diabetes distress) and mancovas (bmi and waist, s - r cognitions, and lifestyle variables; table 1) indicated that study non - completers were employed for more hours [t (98) = 1.98, p = .050] and scored lower on goal ownership [t (94) = 11.53, p <.000], goal support [t (88) = 5.99, p = .000] and diabetes self - efficacy [t(90) = 2.55, p = .013]. Interestingly, study non - completers scored higher on goal planning [t (89) = 2.99, p = .004]. In the multiple regression analysis, patients employment, goal ownership, diabetes self - efficacy were entered together with the dichotomous variable allocated to intervention or control group and gender. The results of this regression analysis revealed that goal ownership was the only significant predictor of attrition [or = .138, 95% ci (.038-.510, p = .003] (see table 2). Employment.031.028.264 goal ownership2.005.659.002 goal planning.430.629.494 goal support1.080.871.215 diabetes self - efficacy.204.353.564 multiple logistic regression analysis of 6 month drop - out the results of this study indicate that study non - completers are best characterized on the basis of their s - r cognitions . Compared to study completers, drop - outs had lower levels of goal ownership and goal support, and surprisingly, higher levels of goal planning . In a regression analysis, low autonomous regulation or low goal ownership appeared the best predictor of drop - out over a 6-month time period . Patients with stronger intrinsic motivation to lose weight are thus less likely to drop out . Goal ownership has been shown to be associated with lifestyle changes, medication adherence, and disease management outcomes [13, 21, 22]. Furthermore, lack of goal ownership has been associated with goal disengagement . However, to the best of our knowledge, goal ownership has not yet been linked to drop - out from a (diabetes) weight loss intervention . The importance of goal support has been frequently demonstrated by its associations with both psychological and physiological outcomes in diabetes [24, 25] as well as other conditions . In a recent meta - analysis on the effect of diabetes weight loss interventions on weight and hba1c goal support of a partner or relative appeared an important moderator on weight loss outcomes . Interventions that targeted goal support by including partners or relatives generated better weight loss results than interventions that did not include partners or relatives . Contrary to our expectations, drop - outs reported higher levels of goal planning than study completers . This surprising finding might be interpreted in the light of phases / stages of self - regulation . Study completers who were actively involved in the goal of losing weight, probably had moved beyond the phase of planning and rather focused on behaviors to actively work on goal pursuit . Drop - outs, however, might have not been able to move towards an active stage of goal pursuit and might have remained in cognitive planning activities to plan how to achieve their goal . Contrary to previous research findings, our study did not detect self - efficacy as a predictor of study drop - out . Differences in self - efficacy between study completers and drop - outs were found, but 6-month drop - out could not be predicted by lack of self - efficacy . The absence of this anticipated effect might be due to the specificity of the self - efficacy measure that was used in this study . Self - efficacy was assessed in the specific context of diabetes and weight loss and not in a more general, trait manner, which might be more comparable to personality characteristics . Given the growing awareness of the importance of personality in predicting drop - out, assessing general self - efficacy might be better able to predict drop - out than context specific self - efficacy . In summary, self - regulatory cognitions next to hours of employment, self - regulation cognitions could best distinguish study completers from study drop - outs . Although these outcomes are promising for attrition research in the weight loss field, this study also yielded some limitations . A first limitation concerned the fact that treatment satisfaction was assessed after completion of the group meetings in the intervention and control groups, but not at baseline . Therefore, comparison of study completers and drop - outs in treatment satisfaction was not possible . Of course, drop - out was anticipated and was tried to prevent by drawing firm criteria for study inclusion, discussing realistic treatment expectations, and being flexible in study appointments . By these means, we hoped that the patients chances of agenda conflicts would be low and that the patients with high medical comorbidity and unrealistic treatment expectations would not be included in the study . However, we speculate that the randomized controlled character of this study has increased the number of study drop - outs . Because of the randomized controlled design, it was not possible to take into account patients preferences for treatment in either intervention or control group meetings . Patients satisfaction with the proposed treatment might therefore have decreased and chances of drop - out have increased . With regard to the practical implications of this study, it can be suggested that assessment of goal ownership prior to a weight loss intervention could identify patients who are sufficiently motivated to take part in the intervention . Patients who score low on goal ownership may be offered pre - treatment interventions, based on motivational interviewing and autonomy support to increase their personal motivation and commitment to treatment . Perceived autonomy supportiveness from diabetes care providers proved to increase patients autonomous motivation and perceived competence, resulting in significant reductions in their hba1c values over 12 months . In addition, techniques to increase goal ownership in overweight women with non - insulin - dependent diabetes have been proven successful in increasing session attendance and improving glycemic control . Due to the small sample size of this study, it is hard to generalize the findings . More research is needed to confirm the importance of self - regulation cognitions and skills as predictors of drop - out . Our findings point however at least at an important avenue, which merits to be explored further in future studies.
Pancreatic ductal adenocarcinoma is the most lethal cancer, and more than 60% of pda patients die within a few months . Pda patients have the best five - year survival if surgery, chemotherapy, and radiation are performed in the first or second pre - metastatic stage of development . When pda spreads to other organs, as in stage iv, the five - year survival rate is below 2% . Approximately 270,000 new pda cases are diagnosed worldwide, and 260,000 patients die each year . In 2013 there were more than 45,000 pda cases reported, and almost 38,500 people died from pda in the united states [2, 3]. Pancreatic ductal adenocarcinoma is associated with diabetes mellitus type 2 (t2 dm), and pda risk is higher in the first year after confirmation of diabetes . Diabetes mellitus type 2 is associated with a two - fold increase in the risk of liver, pancreatic, and endometrial cancers . In diabetes mellitus type 2, the patient s cancer risk is increased almost two - fold for breast, colorectal, kidney, and bladder cancer . Pancreatic ductal adenocarcinoma and biliary tract cancers (btc), with similar clinical presentation, are diagnosed mostly at incurable stage iii and iv . In the united kingdom (uk) pancreatic cancer is ninth on the list of leading cancers, amounting to 8000 cases of pda and 2000 cases of btc each year . Obesity is a risk factor for pancreatic cancer as well as increased risk for cardiovascular diseases . Obesity is associated with increased incidence and prevalence of pancreatic cancer . In the usa, obesity concerns mostly women as well as non - hispanic and afro - american populations . Despite being unsolved and poorly understood, some risk factors are mentioned in recent papers, including demographic factors like old age, ethnic origin, and the highest mortality seen in the afro - american population . Concerning europe, the incidence of pda between 2005 and 2007 was highest in men from the baltic countries, followed by the czech republic, hungary, slovakia, and slovenia . Northern european countries like finland and denmark also had higher incidence rates in comparison to the average incidence in europe . The lowest mortality rates in the world (caused by pda / btc) were noted in latin america . The pda / btc mortality rates in women were the highest in some central / eastern and northern european countries, and the lowest were in latin america . In 2007 norwegian pda / btc mortality was higher in women than in men (8), and the incidence of pancreatic cancer over the last two decades has levelled off . Familial cancer conditions cannot be ruled out as a possible risk factor of pad / btc . Genetic predisposition has been found for liver cancer, as well as for pancreatic cancer . The main problem with pda / btc is diagnosis being made too late, because most patients visit their doctors at advanced stages (iii, iv) of pda / btc . However, even for those who are in the first two stages with the possibility of surgery, prognosis is also poor [9, 11]. In 2012, there were 211,500 cases of pda / btc worldwide, and despite progress in its pathophysiology, morbidity and mortality due to pda / btc have not changed much in recent years . In japan, where mortality and morbidity due to pda / btc is the lowest in the world, the incidence of pda / btc has increased in the past four decades and is the fifth leading cause of death in men and the sixth in women . Smoking is the most considering and well - documented risk factor for pancreatic cancer, being responsible for about 25% cases of pda / btc, commonly in western countries, and is still rising in developing countries . Cancer mortality in europe is highest in russia, romania, and in eastern europe, due to their greatest consumption of tobacco . In others parts of western europe with improved smoking cessation processes, in many countries, mostly in the western hemisphere, the populations are living longer than ever, with the longest recorded life span in japan, creating concerns of rising incidence of many kinds of cancer, including pda . Aging correlates well with the incidence of pda / btc, and it is the most recognised risk factor; pda / btc is most often diagnosed between 60 and 80 years of age . Pda / btc is rare below age 45 years . During the almost 40 years since the second world war, the incidence of pc tripled [16, 17]. In poland, after cardio - vascular diseases, cancers are the second overall cause of death, with the highest mortality before the age of 65 years . In poland, in 2010, most cancer - related deaths were caused by the following cancers: as smoking is one of the main cancer risk factors, and more than 30% of the polish population smokes cigarettes, it will be difficult to diminish the incidence rate of cancer in poland [14, 18]. Although pda accounts 95% of all pancreatic cancers, screening of the general population for pda is still not validated . That is why risk factors for pda are an important issue . It appears, from descriptive and retrospective studies, that tobacco smoking and aging are seriously involved in the development of pancreatic cancer [1, 2, 6, 7, 14, 18, 19]. It was reported that smoking is responsible for nearly 25% of pda, and that carbon monoxide, benzene, vinyl chloride, and many other compounds play a destructive role not only in the development, but also in the progression of pda . It was proven that tobacco smoking renders cancer cells more metastatic; nicotine stimulates tumour growth and invasion, which causes cancer cells to be more metastatic and resistant to drugs . Patients who smoke decrease their own survival rate because tobacco directly and negatively interferes with chemotherapy . The protective role against cancers played by oestrogens, helping cells to survive hypoxia, caused by side effects of a smoking habit . Demographic factors, as progress in age, mostly at old age, are not modifiable . Ethnicity indicates that the afro - american population is more vulnerable to pda than white populations . African american and ashkenazic jewish races have more significant risk of pda than the general white population [2, 7]. Occupational exposures for chlorinated hydrocarbons and aliphatic solvents, nickel and chromium compounds, polycyclic aromatic hydrocarbons, and organochlorine insecticides are implicated in the development of pancreatic cancer, despite some studies that deny occupation as a major risk factor . It was reported that the risk of pancreatic cancer was associated with genetic predisposition; however, genetics account for only a small proportion of pdas . Mutation of genes in people with a predisposition to genetic mutations significantly increases the risk of pda, causing hereditary pancreatitis (hp) [3, 14]. Other inherited genetic disorders that are thought to create pda cover include: ataxia - telangiectasia, familial atypical multiple mole melanoma, nonpolyposis colorectal cancer, hereditary breast and ovarian cancer, which cause about 10% of all pancreatic cancers . People suffering from genetic predisposition to mutations like familial pancreatic cancer (fpc) with unknown gene but strong relative risk or hereditary pancreatitis (prs51 gene) have a five- to ten - fold increased risk of pda . One of the most convincing risk factors of pancreatic cancer is weight, particularly obesity . It was reported that: body fatness increases pda risk from 20 to 50% as compared to people with normal bmi; obese people are often diagnosed with pda one year earlier than the average population; individuals with abdominal fatness are more susceptible to pda than individuals with fatness of other body parts; obesity increases the risk of mortality not only from pancreatic cancer but also from some other cancers; higher bmi causes more frequent inflammation and hormonal disruption than normal bmi, creating greater chances for carcinogens, because the rise of bmi is also due to reduced physical activity, which is another risk factor for cancer; obesity, associated with insulin resistance, increases insulin concentration in tissues and blood, which can increase tumour growth promotion . Some authors suggest that the increase in adipokines, leptin, and adiponectin in visceral adipose tissue can increase risk of pancreatic cancer . In europe and economically developed countries of the world, people consume about 2600 calories per day, excluding japan, where caloric consumption is lower (not exceeding 1800 calories per day). That is why our weight is one of the most important factors of our health . If we are becoming overweight earlier in life, we have a greater risk of consuming carcinogenic substances, energy imbalance, and inflammation [1, 3, 4, 6, 8, 15, 18]. Type 2 diabetes mellitus (t2 dm) increases the risk of liver, pancreatic, and endometrial cancers by a factor of approximately two, and the risk of breast, colorectal, kidney, and bladder cancer by a factor of less than two . Evidence suggests that overweight / obesity with consequent insulin resistance and hyperinsulinaemia is the main risk of pancreatic cancer . The risk of pda was higher in the first year after t2 dm diagnosis and also higher in the subsequent years of observation in patients with diabetes mellitus, when compare to non - diabetics . Many studies suggest that pancreatic cancer can initiate and promote t2 dm, and diabetes mellitus does not cause pda . There were also suggestions of a higher incidence rate of pancreatic cancer during the first year of t2 dm diagnosis . It is commonly believed that unfavourable dietary habits such as saturated fat, alcohol consumption, and high salt intake can lead to diabetes mellitus [4, 5, 8, 10, 13, 15, 16]. A two - fold increased risk of pancreatic cancer is seen among patients with diabetic type ii, but not with type i diabetes . A three - fold increase in gene mutations was reported in alcohol drinking and smoking persons, in comparison to persons with moderate consumption of alcohol and non - smokers . However, some studies have failed to prove a relation between alcohol consumption and pancreatic cancer, except a relation of chronic drinking with chronic alcoholic pancreatitis . All alcoholic beverages are cancerogenic when consumed in excess and it is advised that people should drink alcoholic beverages in a moderate pattern . Small portions of alcohol (one drink per day) seem unlikely to pose a risk of pda and remain inconclusive as a risk measure . It was reported that people who drink six or more alcoholic drinks per day for many years have a higher risk of pda than people drinking in moderation . Increased risk of pda concerns mostly persons with a previous history of chronic pancreatitis [3, 7, 10, 13, 14, 1618]. Some other risk factors that can play a distinct role in the creation of pda are frequently described in the literature but are still not sufficiently proven in medical research . As long as our data are not conclusive in aetiology and there is a lack of early screening of pancreatic cancer, it is very important to highlight the role of lifestyle factors and education in the prevention of pda . The risk of pda decreases by more than 50% among higher educated people [6, 13]. Physical activity is a statistically significant protective factor for pda, decreasing the risk of pancreatic cancer by 1237% . To diminish the risk of pda, leisure - time activities are more important than occupational activities however, many epidemiological studies concerning the relationship between physical activity and pda are not consistent . Some authors hypothesise that physical activity reduces glucose levels and unhealthy triglycerols, and prevent type 2 diabetes mellitus, which can reduce pda thereafter . It was proven that physical exercise protects against being overweight and obese, and can reduce insulin resistance [1, 6, 10, 13]. In recent decades, a great deal of medical scientific literature has been aimed at establishing a better understanding the aetiology of pda, to reduce the incidence of pancreatic cancer, and to provide screening programmes for persons with relative risk . It is still not well defined who should be under early surveillance . As in western countries, pancreatic cancer is the fourth leading cause of mortality, which shows an increasing trend in morbidity and mortality, some individuals with leading risk factors are invited to participate in screening programs . The most convincing or probable risk factors that should be included in pda screening programs are [2, 3, 10]: chronic pancreatitis, familial atypical mole - multiple, intraductal papillary mucinous neoplasm (ipmn), mucinous cystic neoplasia (mcn), pancreatic cystic neoplasm (pcn), chronic pancreatitis, hereditary conditions peutz - jeghers syndrom, hereditary pancreatitis, body fatness, high bmi, greater than average childhood growth, any individuals with a five - fold or more relative risk . The literature points to many others suggestive, non - conclusive, or potential risk factors predisposing to pda and mostly focused on lifestyle factors like: smoking, lack of physical activity, and poor diet . Several studies show that high bioavailability of statins significantly reduces the risk of pancreatic cancer . The use of statins for more than one year was associated with a 34% reduction in the risk of pda, and use of statins for more than 10 years caused an almost 50% reduction in pda risk . There is also a growing demand for prophylaxis of pda by using pre - emptive pancreatic surgery with a minimally invasive approach . However, total pancreatic surgery is sometimes necessary due to the protective action against malignant transformation and resection of a precancerous lesion . Intraductal papillary mucinous neoplasm and mucinous cystic neoplasia are the most prone to becoming malignant, and preventive surgery is strongly recommended in such cases . Rarely, lesions of the pancreas may be diagnosed as pda by prophylactic measures (ultrasound, magnetic resonance imaging, computerised tomography). Then the pancreas should be removed before spreading to nearby organs, causing pda recovery . Individuals at high or even modest risk of pancreatic cancer with hereditary conditions and other predisposition for pda should be under surveillance or screening [3, 5, 19]. Pancreatic cancers are usually diagnosed in their advanced stages, disseminated to adjacent and distant organs, when surgical treatment is not recommended . Symptoms of pda include: change in bowel habit, any of the above symptoms can bring people to medical facilities due to the probability of diagnosis of pda . The first pda symptoms include abdominal pain and/or back pain, later pda patients report jaundice, when the pancreatic - head tumour is small . Diabetes mellitus is associated with other signs mentioned above . In pancreatic - tail tumours, unfortunately, the pain is usually too late as a warning symptom because it appears when the cancer is spreading to nearby tissues . It is also often seen that very early signs and symptoms of pda occur for a short period of time and stop thereafter, reassuring patients that nothing wrong is happening . Therefore, new guidelines are needed to start investigation of at - risk subjects [5, 7]. Screening for pda males and females over 50 years old, with higher risk factors, substantially benefits predisposed individuals . Although only 56 cancers in men and 58 in women per 100,000 screened persons are diagnosed, it results in 38 cancer deaths averted . Computerised tomography (ct) of abdomen is the first diagnostic procedure followed by endoscopic retrograde cholangiopancreatography . Magnetic resonance imaging (mri) and use of endoscopic ultrasound device if pancreatic cancer is found, further diagnostic procedures should be taken, including fine - needle aspiration (fna) to obtain a tissue for diagnosis and management, or endoscopic ultrasound - guided biopsy to confirm neoplasm histologically . Ct is not always recommended for diagnosis of pda due to radiation exposure, but the use of endoscopic ultrasound (eus) is increasing [3, 7, 9]. Screening individuals with a relative risk of pc can give greater life expectancy than in the general population . Some individuals with little risk should not be screened due to false - positive results and subsequent unnecessary surgery, complications, and deaths from surgical interventions . Surgical resection, radiotherapy, and chemotherapy remain the best options for patients with pancreatic cancer to improve outcome . Due to an asymptomatic or insufficiently symptomatic process for some experts, primary prevention of the pc is not available . For others, modifiable risk factors are crucial enough to establish prophylactic programs in the general population . In our opinion, effective primary prevention of pda should be started with tobacco cessation because cigarette smoking is one of the modifiable pc risk factors . Five years after cigarette smoking withdrawal, the risk for pc is the same as in the general population [1, 3, 7, 8, 10]. Nicotine can stimulate pc tumour growth, making cancer more metastatic and less responsive to therapy, and reducing the survival rate of pda patients . It was reported that cigarette smoking increases the risk of pancreatic cancer by a factor of two . Cancer mortality in recent decades has generally declined in western europe, including pda, mostly due to cessation of cigarette smoking, but has remained constant in eastern europe . Countries like russia, romania, and poland have high rates of tobacco consumption and incidence of pda, which has not declined yet [15, 18]. We also have suggestive and modifiable risk factors of pancreatic cancer other than smoking . However, more studies are needed, particularly randomised studies, to establish links between lifestyle factors and pda . Many of the risk factors other than smoking are reversible, without side effects, for example: maintaining proper bmi, drinking alcohol in a moderate manner, or to improving physical activities . Many authors consider how to correct unhealthy human habits to improve quality of life, extending life expectancy, and avoiding pancreatic cancer . Modifiable pda risk factors [1, 68, 10, 13, 14, 1618] include: obesity / body fatness, adiposity, high bmi, poor diet as: red meat, processed food, saturated fatty acids, high cholesterol diet, fructose, heavy alcohol drinking /alcohol abuse, small consumption of vegetables and fruits, more than four cups of coffee a day, low - fibre diet, high consumption of sodium salts, smoked meat, food preservatives and additives, lack of systematic physical activity, lack of secondary and/or tertiary education . Eating plenty of healthy grains, vegetables, and fish, as well as limiting red meat and sweets, has been shown to lower the risk of pda among people with higher risk of pda . Tomatoes, broccoli, spinach, and blueberries are considered to have a prophylactic effect on cancers . A growing body of research suggests that people who eat more protein from animal sources have four times increased risk of dying from cancer, as compared to people who consume proteins from fish, fowl, and plant sources like whole grains or nuts . We should focus on monounsaturated fats which control insulin level and blood glucose and can be helpful to people suffering from type 2 diabetes . Deep ocean fish, including salmon, mackerel, sardine, and tuna are major sources of long - chain omega-3 fatty acids, which help to maintain anti - inflammatory processes and have been shown to have some anticancer properties . Chemicals known as heterocyclic amines, nitrates, and heme iron, found in foods, are capable of damaging cells and dna, influencing cancerogenic processes, and triggering cancers of the pancreas, prostate, and colon [7, 10, 13, 16, 20]. A healthy diet, when food intake does not exceed 2000 calories daily, and regular physical activity, account for 2/3 of our lifespan . The five - year survival rate of all cancers taken together has increased in the past three decades due to massive screening programs, sophisticated treatment, and common preventive practice . If we are able to stop people smoking, we can reduce by more than 80% the incidence of lung cancer and diminish substantially the incidence of pancreatic cancer [7, 8, 18, 20]. Aging is considered as a non - modifiable risk factor, characterised by a high incidence of pancreatic cancer in the elderly . However, it was reported that healthy lifestyle, including regular physical activity and healthy diet, can help to avoid diabetes type 2 and obesity, and can slow aging processes and increase longevity . Several studies have shown that the reduction in diabetes type 2 incidence after changing lifestyle habits is related to the decrease of incidence of pda [1, 6, 8, 10]. Eating charred, processed foods, drinking alcohol in excess, and accumulating excessive amounts of carcinogenic substances in our body are more important predictors of carcinogenesis than the process of aging . Our physiological / biological age can differ from our calendar age, giving a possibility to live longer without debilitating diseases . In the world, pancreatic cancer is a rare but fatal disease, with few established risk factors, including: smoking, genetic predisposition, and increasing age [3, 6, 10]. Recent literature has found modern advances in pda treatment and prophylaxis . As long as pancreatic cancers are mostly diagnosed too late, the most important task for every individual on the planet is to take a healthy approach and to stave off death in the short term [2, 6, 7]. Lifestyle changes could play a crucial role in reducing the incidence of pancreatic cancer, without negative adverse effects . Lifestyle changes may also allow us to stay healthier throughout our life with better wellbeing . It does not create additional individual cost to be in good shape, slim, physically active, without smoking, with moderate alcohol consumption, with reduced amounts of products preserved with sodium, and lowered consumption of red meat / processed meat, and eating more vegetables and fruit or being vaccinated to protect against diseases that potentially influence carcinogenesis.
Alport syndrome (as) is a hereditary nephropathy characterized by a family history of hematuria, progressive renal failure, sensorineural hearing loss, and ocular abnormalities (1). Progression to renal failure is predictable in men, whereas the course of renal involvement is much more variable in women, remaining mild in the majority of patients . Approximately 80% of as is inherited in an x - linked manner and is caused by mutations in the col4a5 gene . Nearly all male patients will develop end - stage renal disease (esrd), whereas heterozygous females exhibit a wide variability in disease severity (2,3). To date, more than 700 different col4a5 mutations have been identified (4,5). We herein report a novel mutation in a japanese family with an x - linked as mutation in col4a5 . The patient (patient 1) was a 2-year - old japanese girl who was the first child of healthy non - consanguineous parents . Her maternal aunt had an episode of nephritis, and a renal biopsy was performed during childhood, but the findings were uncertain . At 14 months of age, patient 1 was referred to our hospital because of microscopic hematuria that had persisted from the neonatal period . She had no clinically detectable hearing loss or ocular abnormalities . A urine culture revealed no infection . At 15 months of age, enalapril malate was initiated for proteinuria, with a urinary protein / creatinine ratio (p / cr) of 1.3 - 3.6 g / gcr . At 21 months of age a urinalysis showed 2 + proteinuria (p / cr, 1.3 g / gcr) and 3 + hematuria, with the urine sediment containing> 100 red cells per high - power field . Her blood urea nitrogen (bun) level was 17.7 mg / dl, serum creatinine level was 0.22 mg / dl, serum total protein level was 5.9 g / dl, and albumin level was 3.7 g / dl . Serum c3 and c4 levels were 118.2 mg / dl and 24.8 mg / dl, respectively . Her mother (patient 2) also had a history of proteinuria and hematuria without renal dysfunction, deafness, or ocular abnormalities ., she was admitted to our hospital along with her daughter (patient 1) for a renal biopsy . A urinalysis showed 3 + proteinuria (p / cr, 1.7 g / gcr) and 2 + hematuria, with the sediment containing 10 - 19 red cells per high - power field . Her bun level was 20.0 mg / dl, serum creatinine level was 0.59 mg / dl, serum total protein level was 6.8 g / dl, and albumin level was 4.0 g / dl . Serum c3 and c4 levels were 134.4 mg / dl and 34.2 mg / dl, respectively . Unaffected individuals with kidney diseases are denoted by empty squares (men) and circles (women). Affected individuals with x - linked alport syndrome are denoted by blackened circles (patients 1 and 2). Affected individuals with biopsy - unproven kidney disease genetic analyses revealed that both patients had a heterozygous mutation (c. 2,767g> c) in exon 32 . (b) x chromosome inactivation assays for our patients . In methylation - sensitive enzymes, patient 1 and 2 both showed a random pattern with ratios of 77: 23 and 31: 69, respectively . It was impossible to distinguish whether the mutated allele was possessed by an activated or inactivated x chromosome because both had the same number of cag repeats ., 29 glomeruli were observed on light microscopy; the glomerulus, tubules, and interstitium showed no significant alterations . Immunofluorescence (if) staining for alpha 5 chains of type iv collagen showed segmental and mosaic patterns in the glomerular basement membrane (gbm). Electron microscopy (em) demonstrated diffusely thinned - out gbms (139 - 143 nm) with focal lamellation and splitting . In patient 2, 40 glomeruli were observed on light microscopy, two of which were globally sclerotic . If staining for alpha 5 chains of type iv collagen showed segmental and mosaic patterns in the gbm, bowman's capsule, and distal tubular basement membrane (tbm). Em demonstrated diffusely thinned - out gbms (149 - 166 nm) with dense granules and splitting . In both patients, the merged if staining images for alpha 2 and 5 chains of type iv collagen clarified the findings of segmental and mosaic patterns in the gbm . Patient 1 shows (a) a normal pattern of the alpha 2 chain of type iv collagen in the gbm and (b) segmental and mosaic patterns of the alpha 5 chain of type iv collagen in the gbm and the absence of staining in bowman s capsule and the tbm . (c) the merged findings of the alpha 2 and 5 chains of type iv collagen are shown . (d) electron microscopy reveals a split lamina densa (arrows) and thin gbm . Patient 2 shows (e) a normal pattern of the alpha 2 chain of type iv collagen in the gbm and (f) a mosaic pattern of the alpha 5 chain of type iv collagen in the gbm and tbm . (g) the merged findings of the alpha 2 and 5 chains of type iv collagen are shown . Gbm: glomerular basement membrane, tbm: tubular basement membrane a sequence analysis of col4a5 in the index patient and her mother was performed . The study was approved by the institutional review board of kobe university school of medicine, and written informed consent was obtained . Genomic dna was isolated from each patient's peripheral blood leukocytes using the quick gene mini 80 system (kurabo industries, tokyo, japan), according to the manufacturer's instructions . Mutational analyses of col4a5 were performed using polymerase chain reaction (pcr) and direct sequencing of genomic dna of all exons and exon - intron boundaries . The pcr - amplified products were then purified and subjected to direct sequencing using a dye terminator cycle sequencing kit (amersham biosciences, piscataway, usa) with an automatic dna sequencer (abi prism 3130; perkin elmer applied biosystems, foster city, usa). The analysis revealed that both patients had a heterozygous mutation (c.2767g> c) in exon 32 (fig . 1a). To investigate x chromosome inactivation, the human androgen receptor (humara) assay was performed in both patients . Genomic dna was digested by a methylation - sensitive enzyme, hpaii, at 37c for 18 hours followed by pcr using dna with humara primers, as described previously (6). A dna fragment analysis was performed on a 310 genetic analyzer (thermo fisher scientific, waltham, usa). Fragment data analyses were performed using the gene mapper software program (thermo fisher scientific). The humara assay for patients 1 and 2 revealed that the x chromosome inactivation pattern was 77:23 and 31:69, respectively (fig . The index case was a 2-year - old girl with x - linked as who had inherited a col4a5 novel mutation (c.2767g> c) in exon 32 from her mother . Both patients had histories of hematuria and proteinuria without sensorineural hearing loss or ocular abnormalities . Renal biopsy findings indicated hereditary glomerulonephritis, and genetic analyses were useful in making a final diagnosis . More than 700 disease - causing mutations have already been reported in col4a5 (2,5). In men with x - linked as, clinical features can be predicted from the location of col4a5 mutations (3). Pathologically, patient 1 had less signals in bowman's capsule and the tbm compared with patient 2 . It reported that the genotype - phenotype correlations are not observed in women, even among family members (2,7). Although heterozygous female patients with x - linked as have a normal col4a5 allele, differences in x chromosome inactivation may influence the disease severity (2,8). However, the humara assay in our cases did not confirm non - random x chromosome inactivation, so - called skewed x, since it is usually considered that the non - random inactivation pattern is> 80:20 or <20:80 (9). Reported that 90% of x chromosomes with a normal col4a5 allele were inactivated in the kidney of a woman with a severe as phenotype (10). The different patterns of x chromosome inactivation in the gbm may cause the variable phenotypes in women with x - linked as . Therefore, the inactivation of a high proportion of normal x chromosomes in critical tissues could be clinically severe (11). In this study, skewed x was not detected in the peripheral blood cells of both patients 1 and 2 . However, it remains possible that skewed x operates in specific tissue, namely the gbm, more strongly in patient 1 than in patient 2 . The mutation identified in our patient's family was c.2767g> c, resulting in a glycine to arginine substitution at position 923 in exon 32 . Glycine substitutions are likely to alter the folding of the triple helix of collagen; however, solid evidence is absent regarding type iv collagen (12,13). Gross et al . Proposed the following classification to link the phenotype and genotype (14): i. severe as . Genotype alterations in col4a5 include large rearrangement, premature stop codons, frameshift and donor splice site mutations, and mutations involving the nc1 domain . The phenotype is associated with the early onset of end - stage renal failure (esrf) at -20 years of age, with 80% presenting with hearing loss and 40% presenting with ocular lesions . The genotype is characterized by non - glycine - x - y missense, glycine - x - y substitutions involving exons 21 - 47, and in - frame and acceptor splice site mutations . The phenotype is associated with esrf at -26 years of age, with 65% presenting with hearing loss and 30% presenting with ocular lesions . The phenotype is associated with esrf at -30 years of age, with 70% presenting with hearing loss, and 30% presenting with ocular lesions . According to this classification, our patients were classified as having moderate - severe as . Taken together with no detection of skewed x, however, it would be clinically difficult to predict the prognosis of kidney function in women with as due to the lack of genotype - phenotype correlations and the intra - familial heterogeneity of the phenotype (2). Therefore, our patients should be longitudinally followed while paying careful attention to a progressive increase in proteinuria, as previously suggested (2). There is no specific treatment available for as; however, early intervention with angiotensin - converting enzyme inhibitors (acei) significantly delayed esrd progression and improved life expectancy in x - linked as (15,16). In women with x - linked as, treatment with either acei or angiotensin ii receptor blockers (arbs) inhibits progression to esrd (15). Therefore, it is recommended that patients with x - linked as are annually followed by a nephrologist, and treatment with aceis / arbs should be initiated for patients with microalbuminuria, proteinuria, or hypertension (15,17). In our patients, acei (enalapril malate) was administered to patient 1 but not to patient 2, in whom a renal biopsy was performed 3 months after the delivery of her second child . We had to consider the potential high risk of fetal and newborn morbidity and mortality, which may be associated with acei / arb fetopathy in pregnant women (18,19). In conclusion, the identification of mutations and characteristic pathological findings were useful in making the diagnosis of as . For a close long - term follow - up, the early detection and treatment of women with x - linked as are important.
Hydrogenic impurities, including donors and acceptors, have been widely studied in theoretical and experimental approaches . Recently, mahieu et al . Investigated the energy and symmetry of zn and be dopant - induced acceptor states in gaas using cross - sectional scanning tunneling microscopy and spectroscopy at low temperatures . The ground and first excited states were found to have a non - spherical symmetry . In particular, the first excited acceptor state has td symmetry . Bernevig and zhang proposed a spin manipulation technique based entirely on electric fields applied to acceptor states in p - type semiconductors with spin - orbit coupling . While interesting on its own, the technique could also be used to implement fault - resilient holonomic quantum computing . Studied tunneling transport through the depletion layer under a gaas surface with a low temperature scanning tunneling microscope . Their findings suggest that the complex band structure causes the observed anisotropies connected with the zinc blende symmetry . Kundrotas et al . Investigated the optical transitions in be - doped gaas / alas multiple quantum wells with various widths and doping levels . The fractional dimensionality model was extended to describe free - electron acceptor (free hole - donor) transitions in a quantum well (qw). The measured photoluminescence spectra from the samples were interpreted within the framework of this model, and acceptor - impurity induced effects in the photoluminescence line shapes from multiple quantum wells of different widths were demonstrated . Presented results on the ground - state binding energies for donor and acceptor impurities in a deformed quantum well wire (qww). The impurity effective - mass schrdinger equation was reduced to a one - dimensional equation with an effective potential containing both the coulomb interaction and the effects of the wire surface irregularities through the boundary conditions . Studying the ground - state wave functions for different positions of the impurity along the wire axis, they found that there are wire deformation geometries for which the impurity wave function is localized either on the wire deformation or on the impurity, or even on both . Calculated the magnetic - field dependence of low - lying spectra of a single - electron magnetic quantum ring and dot, formed by inhomogeneous magnetic fields using the numerical diagonalization scheme . The effects of on - center acceptor and donor impurities were also considered . In the presence of an acceptor impurity, transitions in the orbital angular momentum were found for both the magnetic quantum ring and the magnetic quantum dot when the magnetic field was varied . Galiev and polupanov calculated the energy levels and oscillator strengths from the ground state to the odd excited states of an acceptor located at the center of a spherical quantum dot (qd) in the effective mass approximation . They also used an infinite potential barrier model . Using variational envelope functions, janiszewski and suffczynski computed the energy levels and oscillator strengths for transitions between the lowest states of an acceptor located at the center of a spherical qd with a finite potential barrier in the effective mass approximation . Calculated the spectrum of a mn ion in a p - type inas quantum disk in a magnetic field as a function of the number of holes described by the luttinger - kohn hamiltonian . For simplicity, we will study the electronic structures and binding energy of a hydrogenic acceptor impurity in semiconductor nano - structures in the framework of effective - mass envelope - function theory . In our calculations, the finite potential barrier and the mixing effects of heavy- and light - holes are all taken into account . Throughout this paper, the units of length and energy are given in terms of the bohr radius and the effective rydberg constant where m0 and 0 are the mass of a free electron and the permittivity of free space . For a hydrogenic acceptor impurity located at in a semiconductor nano - structure, the electron envelope function equation in the framework of the effective - mass approximation is(1) in the above equations, 1,2, and 3are the luttinger parameters and the subscript n = 0, 1, 2,...correspond to the ground-, first excited-, second excited-,...states, respectively . The quantum confinement potential can be written in different forms for various nano - structures . In eq . 1, is 0 when there are no acceptors and 1 when there are acceptors in the nano - structure . The binding energy of the n - order hydrogenic donor impurity state is explicitly calculated by the following equation:(2) we express the wave function of the impurity state as (3) where lxly, and lz are the side lengths of the unit cell in the x, y, and z directions, respectively . Kx = 2 /lx, ky = 2 /ly, kz = 2 /lz, nx {mx,, mx}, ny {my,, my}, and nz {mz,, mz}. The plane wave number is nxyz = nxnynz = (2 mx + 1)(2 my + 1)(2 mz + 1), where mxmy, and mz are positive integers . We take lx = ly = lz = l = wmax + 25 nm, kx = ky = kz = k = 2 /l, and nx = ny = nz = 7 in the following calculation, where wmax is the maximum side length of the nano - structures . If we take larger nxny, and nz, the calculation precision will be increased somewhat . The matrix elements for solving the energy latent root of the impurity states can be found from eqs . 1 and 5 . The electronic structures and binding energy in the nano - structure can be calculated from the matrix elements . In the following sections, we will give some numerical results for the electronic structure and binding energy of a hydrogenic acceptor impurity in several typical gaas / ga1xalxas nano - structures . 1 = 6.98,2 = 2.06, 3 = 2.93 . The band gaps eg (ev) of bulk gaas and al0.35ga0.65as are 1.519 and 2.072 ev, respectively . The valence - band offset is assumed to be 35% of the band gap difference, so v0 = 193.55 mev . We adopt a square potential energy model in the following calculation, i.e., v(r) = 0 inside and v(r) = v0 outside of the nano - structures . Figures 1 and 2 show the first five energy levels and binding energy levels of an impurity in a qw as functions of the qw width w for an acceptor at the qw center . Figure 1 shows that the energy levels monotonically and quickly decrease as the well width increases . It is well known that the donor binding energy has a peak as the qw width increases . However, fig . 2 shows that the changes of the acceptor binding energies are very complex as the qw widthwincreases . This is because the holes have asymmetric effective masses, and there are mixing effects between heavy- and light - hole states . The energy levels of the first five states as functions of the qw width w for an acceptor at the qw center the binding energy levels of the first five states as functions of the qw width w for an acceptor at the qw center figure 3 shows the binding energy levels of the first five states as functions of the donor positionz0for the qw widthw = 10 nm . This figure shows that the binding energies monotonically decrease as the acceptor moves away from the qw center . The binding energy levels of the first five states as functions of the donor position z0 for the qw width w = 10 nm figure 4a and b shows the impurity energy levels of the first five states as functions of the square qww side length l0(a) and the cylindrical qww radius (b) for an acceptor at the qww center . Compared with fig . 1, we find from fig . 4 that the impurity energy levels decrease slowly as the qww size increases . This is because the acceptor is confined in two directions . The impurity energy levels of the first five states as functions of the square qww side length l0(a) and the cylindrical qww radius (b) for an acceptor at the qww center figure 5a and b is the same as fig . 4a and b, respectively, but are for the binding energy levels instead of the impurity energy levels . The binding energy of the acceptor in the qww is larger than that in the qw because the quantum confinement effects in the qww are larger than in the qw . The same as fig . 4 but for the binding energy levels of the first five states figure 6a and b shows the binding energy of the first five states as a function of the impurity position for a square qww with side widthl0 = 10 nm (a) and for a cylindrical qww with radius (b). The positions of o, a, and b in fig . It is easy see that the binding is the weakest for the impurity located at the corner of the square qww . The binding energy of the first five states as functions of the impurity position for the square qww side length l0 = 10 nm (a) and the cylindrical qww radius (b). The positions of o, a, and b in (a) are indicated in the inserted figure figure 7(a), b, and c gives the impurity energy levels as functions of the spherical qd radius r0(a), the square qd side length w(b), and the cylindrical qd radius and height w () (c) for an acceptor at the qd center . Compared with figs . 1 and 4, we find that the impurity energy levels decrease more slowly in the qd than in the qw or the qww . This is because the quantum confinement effect is larger in the qd than in the qw and qww . The impurity energy levels as functions of the spherical qd radius r0(a), the square qd side width w(b), and the cylindrical qd radius and height (c) for an acceptor at the qd center figure 8a, b, and c is the same as fig . 7a, b, and c, respectively, but are for the binding energy levels . From fig . 8(a), we find that there is only one binding energy for which r0 is greater than about 2.2 nm . The first two quantum states are degenerate and correspond to the first energy level, due to the symmetry of the spherical qd . Figure 8(b) shows that there is only one binding energy level when the side length is between 3 and 10.5 nm . If the side length is greater than 10.3 nm, the second binding energy level arises once again . Figure 8(c) shows that the first two binding energy levels diverge quickly, and the other binding energy levels disappear as the qd radius and height become larger than about 2.5 nm . The same as fig . 7 but for the binding energy levels of the first five states figure 9a, b, and c shows the binding energy as a function of the impurity position with a spherical qd radius of r0 = 5 nm (a), with a cubic qd side length of w = 10 nm (b), and a cylindrical qd radius and height w equal to 5 nm (c). 9b and c are indicated on the inserted qd figure, respectively . As the acceptor moves away from the center, the symmetry decreases, figure 9c shows that there are two binding energy levels when the cylindrical qd radius and height w equal 5 nm . The binding energy is the largest when the impurity is at the qd center, and it is least when the impurity is at the corner . The binding energy as a function of the impurity position with the spherical qd radius of r0 = 5 nm (a), with the cubic qd side length w = 10 nm (b), and the cylindrical qd radius and height w equal to 5 nm (c). The impurity positions of o, a, b and c in (b) and (c) are indicated on the inserted qd figure, respectively in summary, we have calculated the electronic structures and binding energy levels of a hydrogenic acceptor impurity in 2, 1, and 0-dimensional semiconductor nano - structures in the framework of effective - mass envelope - function theory . Our method can be widely applied in the calculation of the electronic structures and binding energy levels of a hydrogenic acceptor impurity in semiconductor nano - structures of other shapes and other semiconductor material systems . This work was supported by the national natural science foundation of china under grant nos 60325416, 60521001, and 90301007.
Children born before week 32 with very - low - birth weight (vlbw: birth weight 1500 g) are more likely to need medical treatment during the perinatal period, and their immature nervous and cardiovascular systems render these children prone to focal brain injuries such as intraventricular hemorrhages and periventricular leukomalacia (pvl) (volpe, 2009). Volpe (2009) has suggested that the complex of encephalopathy of prematurity includes both destructive and developmental disturbances, and primary white matter injury could have secondary effects on cortical and gray matter nuclei development . Although perinatal care and medical treatment in the neonatal intensive care unit (nicu) have improved radically during the last decades with reduced incidence of focal brain injury, the immature brain and exposure to the harsh extra - uterine environment in the nicu are still believed to increase the risk of disrupted brain development in very preterm born survivors . The consequences of such developmental disruptions in an extremely sensitive period of brain growth may be profound alterations of subcortical and cortical morphology that may affect brain function . Previous studies have reported abnormal cerebral white matter in vlbw infants as the most common pathological finding, manifested as reduced fractional anisotropy on diffusion tensor images at 2746 weeks of gestation (ball et al ., 2013), in adolescents (skranes et al ., 2007) and in young adults aged 1822 years (eikenes et al ., 2011). However, changes in cortical and subcortical gray matter have also been demonstrated in children with vlbw as reduced brain cortical surface area at term - equivalent age; (ajayi - obe et al ., 2000; 2006), in toddlers 1822 months old (phillips et al ., 2011), in children at the age of 10 years (grunewaldt et al ., 2014), and in adolescents and young adults (frye et al ., 2010; skranes et al ., both regional thinning and thickening of the cerebral cortex have been reported in children 712 years of age (grunewaldt et al ., 2014; mrner - lavanchy et al ., 2014) and in adolescents at the age of 19 (bjuland et al ., 2014). Possible mechanisms underlying these cerebral changes in the vlbw population may include injuries that affect neuronal migration and thereby cortical development (volpe, 2009). The aim of the present study was to investigate cortical thickness and cortical surface area in 510 year old children born preterm with vlbw and term - born controls . To our knowledge, no previous study has explored regional cortical morphology using continuous cortical surface maps in vlbw children as young as 510 years of age . Using continuous maps of cortical thickness and surface area increases both sensitivity and specificity compared to volumetric methods (rimol et al ., 2012). In addition, the present study explores the relationship between regional measures of cortical morphology and full iq as an overall measure of cognitive functioning . Cognitive abilities have been shown to be reduced in the vlbw population (aarnoudse - moens et al ., 2009; anderson et al ., 2004; lohaugen et al ., 2010; nosarti et al ., 2007; 2004), and reduced cognitive performance has been related to reduced cortical volume in 1415 year old vlbw adolescents (nosarti et al ., 2014). Finally, previous studies of vlbw young adults have shown negative correlations between iq and cortical thickness (bjuland et al ., 2013), and positive correlations with surface area (skranes et al ., 2013). However, these vlbw young adults were born in 19861988, and it is unclear whether the same relationships between cortical morphology and cognitive function exist for school aged vlbw children born after year 2000, who have received modern neonatal intensive care . The children born prematurely with very low birth weight (vlbw) (birth weight 1500 g) were recruited based on admittance to the neonatal intensive care unit (nicu) at st . Sixty - three non - cp children were invited and 57 agreed to participate in the study (31 females). One child (a twin sibling to a vlbw child) with birth weight at 2090 g was included in the data analysis, and post - hoc analysis showed similar brain morphology and iq scores for this child as for the vlbw cohort . The control subjects were recruited from the national norwegian mother and child cohort study (moba) managed by the norwegian institute of public health (magnus et al ., 2006), with ages ranging between 4 and 11 years (n = 143, 70 females). The participants included in the current analysis were living in the same geographical area as the vlbw participants (nord- and sr - trndelag) and had normal vision and hearing . The exclusion criteria were a history of injury or disease known to affect the central nervous system (cns) function, including neurological or psychiatric illness and serious head trauma . Furthermore, if the child was under psychiatric treatment, used psychoactive drugs known to affect cns functioning, had a birth weight below 2500 g, or had any known mri contraindications, they were excluded from participation in the current study . Mri data were collected using a 12-channel head coil on a 1.5 t siemens avanto scanner (siemens medical solutions). The pulse sequence used for morphometric analyses was one 3d t1-weighted magnetization prepared rapid acquisition gradient echo (mprage) scan with the following parameters: repetition time (tr), 2400 ms; echo time (te), 3.61 ms; inversion time (ti), 1000 ms; flip angle, 8, fov 240 240 and acquisition duration of 4 min and 18 s. each volume consisted of 160 sagittal slices with voxel sizes of 1.25 1.25 1.20 mm . Raw datasets were de - identified and transferred to linux work - stations for processing . Each mprage was visually inspected and only scans with no or minimal movement artifacts were included in the analyses . Cortical reconstruction was performed with the freesurfer 5.3.0 image analysis suite, which is documented and freely available for download online (http://surfer.nmr.mgh.harvard.edu/). The technical details of these procedures are described in other publications (dale et al ., 1999; dale and sereno, 1993; fischl et al ., 2004a; fischl and dale, 2000; fischl et al ., 2001). Briefly, this includes motion correction and averaging (reuter et al ., 2010) of multiple volumetric t1 weighted images, removal of non - brain tissue using a hybrid watershed / surface deformation procedure (sgonne et al ., 2004), automated talairach transformation, intensity normalization (sled and pike, 1998), tessellation of the gray and white matter boundary, automated topology correction (fischl et al ., 2001; sgonne et al ., 2007), and surface deformation following intensity gradients to optimally place the gray / white and gray / cerebrospinal fluid (csf) borders at the location where the greatest shift in intensity defines the transition to the other tissue class (dale et al ., 1999; dale and sereno, 1993; fischl and dale, 2000). Once the cortical models are complete, a number of deformable procedures can be performed for further data processing and analysis including surface inflation (fischl et al ., 1999), registration to a spherical atlas which is based on individual cortical folding patterns to match cortical geometry across subjects (fischl et al ., 1999), parcellation of the cerebral cortex into units with respect to the gyral and sulcal structures (desikan et al ., 2006; fischl et al ., 2004b), and creation of a variety of surface based data . This method uses both intensity and continuity information from the entire three - dimensional mr volume in the segmentation and deformation procedures to produce representations of cortical thickness, calculated as the closest distance from the gray / white boundary to the gray / csf boundary at each vertex on the tessellated surface (fischl and dale, 2000). The maps are created using spatial intensity gradients across tissue classes and are therefore not simply reliant on absolute signal intensity . The surfaces were smoothed with a full - width - half - maximum gaussian kernel of 30 mm (662 iterations). Each surface consisted of approximately 160,000 vertices arranged in a triangular grid, and estimates of the cortical area were obtained by computing the area of each triangle in the standardized, spherical atlas space surface tessellation when mapped into the individual subject space . Vertex - wise estimates of cortical area were then computed by assigning one - third of the area of each triangle to each of its vertices (rimol et al ., 2012). The cortical surface of each subject was automatically parcellated using defined gyri and sulci as landmarks, and the surface was divided into 34 anatomical regions for each brain hemisphere defined in freesurfer (desikan et al ., 2006; fischl et al ., 2004a), which were used to anatomically identify the affected regions after significance testing . In the vlbw group, analyses were conducted based on mr - images from 37 children (21 females). Of the 57 who were eligible for mr - scanning, 10 children did not want to be scanned and had cognitive assessment only, and 10 images were excluded due to movement artifacts or disrupted scanning . In the control group we were able to attain 104 mprage images of good quality (54 females). A total of 143 children were invited to mr imaging, 22 children did not want to participate and 17 of the images had to be excluded due to movement artifacts or disrupted scanning . The youngest participants (56 years of age) in both groups were most likely to decline mri scanning or be excluded due to movement artifacts . In the vlbw group, children <6 years of age were assessed with the age - appropriate, complete version of the wechsler preschool and primary scale of intelligence, 3rd edition (wppsi - iii) (wechsler, 2002), whereas children 6 years were assessed with wechsler intelligence scale for children, 4th edition (wisc- iv) (wechsler, 2003). Wppsi - iii provides three iq indices: full scale iq, verbal iq and performance iq, while wisc - iv comprises four indices: verbal comprehension index, perceptual reasoning index, working memory index and processing speed index, and full scale iq . Cognitive abilities in the controls who were 6.5 years of age were assessed with the wechsler abbreviated scale of intelligence (wasi) (wechsler, 1999). The wasi is a validated screening test that is used to assess the following aspects of intelligence: verbal knowledge, visual information processing, spatial and nonverbal reasoning, and general intelligence . Three iq scores can be extracted using the wasi: a verbal iq (viq) score (subtests: vocabulary and similarities) and a performance iq (piq) score (subtests: block design and matrices), which when combined provide an estimated full - scale iq (fsiq) score . The controls who were younger than 6.5 years of age completed a short form of the wechsler preschool and primary scale of intelligence, 3rd edition (wppsi - iii) (wechsler, 2003), including similar subtests: vocabulary, similarities, block design and matrices, and verbal iq (viq), performance iq (piq) and full - scale iq (fsiq) were calculated . Ibm spss statistics 19 edition was used for the analysis of the clinical and cognitive measurements by independent samples t - tests and non - parametric tests . Matlab 2011b was used for statistical analyses of morphometry data . To examine group differences, a general linear model was fitted with cortical surface area or cortical thickness as dependent variable and group, sex and age at mri scan as independent variables in each vertex across the cortical surface . The regression of iq on cortical morphology was tested with the same glm with full iq as an added continuous predictor . The hemispheres were analyzed separately, and effect size and p - maps were generated . Effect size is reported as cohen's d for group comparisons and r = f / (f + df) for the continuous predictors (iq and birth weight). The p - maps were thresholded and multiple comparisons were corrected for with a 5% false discovery rate (fdr) that was applied co - jointly across the hemispheres . Significance tests were performed to investigate differences in cortical morphology between the groups . For the clinical variables birth weight, gestational age, and days on ventilator general linear models were fitted in each vertex across the surface, with cortical surface area or cortical thickness as the dependent variable and one of the clinical variables as a covariate, and adjusted for sex and age at mri scan . Missing data in the independent variables (full iq and birth weight) were dealt with by multiple imputations . Pattern analysis was performed, showing that we had below 5% missing data and that we could assume that data were missing at random . Seven full iq data and two birth weights were imputed and pooled imputations were used in further analyses . Hollingshead's (1975) two factor index of social position based on education and occupation of one parent or the mean index of both was used to calculate socio - economic status (ses). The regional committee for medical research ethics approved the study protocol (project number: 2010/2359), and written informed consent was obtained from the parent / guardian of all participants . Demographic and clinical characteristics of the study groups are shown in table 1 . In the vlbw group, mean birth weight was 1048 g and mean gestational age was 28.5 weeks . There was no significant group difference in age at examination; however, the controls had higher mean socioeconomic status (ses) than the vlbw group . The vlbw group had significantly lower scores than controls on full iq, also after adjusting for socioeconomic status (n = 34/85). There were significant differences in cortical surface area between the vlbw and the control group . The vlbw group showed bilateral reduction in cortical surface area in the frontal, temporal, and parietal lobes (fig . The effect size of the group difference ranged from d = 0.4 to 0.8 in most cortical regions (see appendix fig . Table 2 lists all cortical regions with significant group differences in surface area as determined by the glm (after 5% fdr correction), where 20 out of 35 regions had 90% reduction of cortical surface area in the vlbw group compared with controls . In the control group, there were widespread cortical regions in both hemispheres showing a significant relationship between full iq scores and cortical surface area (see appendix fig . The relationship between full iq scores and cortical surface area did not reach statistical significance . However, the correlation coefficients were 0.20.4 in several cortical regions in both groups, and as high as 0.6 in some regions in the vlbw group (fig . The lack of statistical significance in these analyses is readily explained by loss of statistical power due to the smaller sample size . The vlbw group showed significantly thicker cortex in the frontal (medial orbitofrontal gyrus, rostral anterior cingulate, frontal pole) and occipital regions (pericalcarine sulcus) bilaterally, and a thinner cortex in the right posterior parietal lobe compared with controls (fig . 3). Moderate to large effect sizes (d = 0.60.8) were observed in the frontal and occipital regions (appendix fig . A3). Proportion (%) of cortical regions with significant differences in thickness between the vlbw and the control groups is displayed in table a3 (appendix) with> 90% involvement of the frontal poles, medial orbitofrontal gyri, left rostral anterior cingulate and right pericalcarine sulcus . A4 (appendix) demonstrates the degree of spatial overlap between the observed between - group differences in cortical surface area and cortical thickness . There were no significant correlations between cortical thickness and full iq in either group . Demonstrate a trend - level negative relationship between cortical thickness and full iq in both groups in widespread cortical regions, i.e. The thinner the cortex, the higher the iq scores (fig . Some temporal and parietal regions showed a positive relationship to full iq in the vlbw group, i.e. Thinner cortex was related to lower iq scores (fig . There were no significant associations between birth weight or gestational age and cortical area or cortical thickness in the vlbw group . There was, however, a significant effect of days on ventilator on surface area bilaterally in the dorsal frontal regions, including the superior and medial frontal gyrus, precentral gyrus, and orbitofrontal cortex, as well as the left supramarginal and posterior superior temporal gyrus, and the right precuneus and superior parietal gyrus . There were two subjects with extreme scores on days on ventilator, i.e. More than 3 weeks on ventilator (35 and 47 days), and these subjects also had low gestational age (23.5, 26 weeks). Region - of - interest based examination of cortical surface area and cortical thickness showed that the child with 47 days on ventilator was an outlier on 5 of 36 cortical parcellations (> 2 sd from the mean in the control group), and the child with 35 days on ventilator was an outlier in one parcellation . Excluding these two subjects from the analysis of group differences between vlbw and controls did not affect the results . Finally, in order to check the effect of prolonged exposure to the ventilator, we excluded children who had spent more than 10 days on ventilator . Since this reduces the statistical power to detect, because the sample is smaller than that in the full analysis, we compared maps of effect size (cohen's d). We report significant reduction of cortical surface area in 510 year old vlbw children, relative to a term - born control group . Cortical area was reduced in frontal, temporal, parietal, and occipital regions, and cortical thickness was increased in the medial frontal and occipital lobes, in the vlbw group . Moreover, there were medium sized to large correlations between reduced surface area and thicker cortex and poorer iq scores, in both the vlbw and the control group . However, only in the control group did correlations between reduced surface area and iq reach statistical significance . The vlbw children showed significantly reduced cortical surface area in frontal, temporal, posterior parietal and medial occipital regions, as well as the right anterior cingulate (see fig . 1). Cortical surface area expands significantly during preschool years and into adolescence in normally developing children, with the greatest changes occurring in higher order regions such as the prefrontal cortex and temporal association cortex (brown and jernigan, 2012). However, by the age of 10, the occipital and superior parietal lobes start to show a decrease in surface area, most probably due to pruning (brown and jernigan, 2012). Hence, the reduced surface area observed in the vlbw children could reflect altered maturation of cortical surface area . Reduced cortical surface area has previously been reported in extremely low birth weight (elbw) children at the age of 10 (grunewaldt et al ., 2014), vlbw adolescents at 1516 years of age (frye et al ., 2010), and vlbw late adolescents at 19 years of age (skranes et al ., 2013). The magnitudes of reduction, and precisely which gyri / sulci are affected, differ somewhat between these studies . However, skranes et al . (2013) reported reductions in cortical surface area similar to the reductions observed in the present study, both in terms of magnitude and localization of the affected regions . The fact that similar cortical regions are affected in cohorts aged 510 and 1820, suggests that the morphological abnormalities observed in the present study may not simply reflect delayed maturation but rather aberrant development leading to permanently altered cortical architecture . These results also demonstrate that similar cortical changes are found both in vlbw survivors born in the late 80s and after year 2000, in spite of the advances in perinatal medicine . We speculate whether the explanation for this has to do with prenatal factors, such as fetal growth restriction, or that immature birth exposes the neonate to environmental factors such as inflammation that exert an epigenetic influence on the genes controlling normal cortical development . Performing the analyses of group differences in cortical surface area without the two children who had extreme scores on days on ventilator (32 and 45 days), did not affect the results . Excluding children who had been on ventilator for more than 10 days, in order to exclude a possible effect of prolonged respiratory support on cortical surface area, we observed similar group differences in cortical morphometry as for the full sample, albeit with reduced effect sizes in most regions . However, excluding children with more than 10 days on ventilator implies excluding many of the most immature and sickest individuals, and leaves us with a sample that is not representative of the premature birth population . Nonetheless, it is worth noting that even when subjects with prolonged respiratory support were excluded, a number of cortical regions still showed group differences . One could speculate whether this may reflect adverse prenatal factors, since these regions show cortical deviations even in the individuals who require the least amount of neonatal care . The vlbw children showed thicker cortex in the frontal and occipital lobes bilaterally, which is consistent with previous studies reporting increased cortical thickness in both children and adolescents born prematurely (bjuland et al ., 2005; mrner - lavanchy et al ., 2014; phillips et al ., 2011). With normal development of the cerebral cortex, thickness will increase during early childhood due to late arriving interneurons then decrease, due to pruning, as neural connectivity improves (raznahan et al . Sowell et al . (2004) reported that the pattern of progressive cortical thinning varies across development, in a longitudinal study of normally developing children 511 years old, observing a significant cortical thinning in the dorsolateral frontal regions and bilateral parietal occipital regions, and cortical thickening in perisylvian regions of the ventral frontal lobe and superior temporal lobe with increasing age . Children develop at varying paces and one possible explanation for the group differences in cortical thickness in our study is delayed maturation in the vlbw group . This would be consistent with mrner - lavancy's (2014) study of vlbw children and term - born controls, 712 years old, which reported thicker frontal and parietal cortices in the youngest vlbw children compared to controls but no such group difference in the oldest children . (2014) found cortical thickness differences exclusively in the occipital lobe at 10 years of age in a cohort of elbw . On the other hand, bjuland et al . (2013) found increased cortical thickness in frontal and occipital regions, but also thinner cortex in frontal, parietal and temporal regions, in 19 year old vlbw adolescents . Thus, it is unclear whether the differences observed in the present study reflect aberrant development and permanent cortical changes or, rather, divergent developmental cortical trajectories that converge with increasing age . Longitudinal studies are needed to answer such questions, in order to conclusively determine whether vlbw children born after 2000 have permanent changes in cortical thickness similar to what has been reported for children born in the late 80s . 2009) demonstrated that cortical area and cortical thickness reflect at least two distinct sources of genetic influence, consistent with the developmental origin of cortical architecture described by the radial unit hypothesis (rakic, 1988), and other studies have suggested independent and divergent developmental trajectories for area and thickness (raznahan et al ., 2011; shaw et al ., we found that regions displaying group differences in surface area and cortical thickness overlapped to a limited extent (as shown in fig . A4). Regions displaying overlapping effects were mainly located on the mesial aspect of the hemispheres; anteriorly in the anterior portion of the sfg, medial orbitofrontal cortex, and anterior cingulate, and posteriorly in the pericalcarine sulcus and cuneus . The vlbw children in the present study were born between 23 and 35 weeks of gestation, which is a particularly sensitive period of neural migration and rapid cortical development . Disorders of migration are more likely to occur in the second trimester (zhang et al ., 2013) by either under - migration or over - migration of neurons, and preterm birth may affect processes like neuronal migration, synaptogenesis and apoptosis late in the 2nd and early 3rd trimesters (tau and peterson, 2010) resulting in the kind of deviant cortical thickness and reduced surface area observed here . The migration of neuroprogenitor cells may be hindered in preterm children by germinal matrix hemorrhages that can destroy neuronal precursors, or by injury to guiding glial cells (volpe, 2009). A reduced pool of neuroprogenitor cells and deficient migration can lead to a reduced number of founder cells in the ventricular zone and number of cerebral columns, which may result in decreased surface area (rakic, 1995). However, in our study only three out of 37 vlbw children had intraventricular hemorrhages and none had focal pvl, suggesting that focal perinatal brain injury is probably not the cause of the cortical deviations seen in our vlbw group . Within 2832 weeks of gestation a fast emergence of short - range connectivity, in addition to the long - range association pathways, is observed (takahashi et al ., 2012). In the vlbw population, reduced fractional anisotropy has been reported in the inferior longitudinal and the longitudinal occipito - frontal fascicles (eikenes et al ., 2011; skranes et al ., 2007). Whether diffuse white matter injury causing disrupted connectivity and cortical reorganization leads to reductions in surface area and increased cortical thickness in the vlbw is not known, but cannot be excluded as an explanation for the presently observed cortical changes . The frontal, parietal and occipital regions with deviant cortical surface area and/or increased thickness in the vlbw children are all regions involved in networks receiving long - range association tracts . We therefore speculate that the deviations seen in cortical morphology in the vlbw group may be both primary changes due to cortical maldevelopment as well as secondary to altered white matter microstructure and connectivity . A positive association between cortical surface area and iq was observed in both vlbw and control subjects, albeit as a non - significant trend in the vlbw group . However, the magnitude of the effect was larger in frontal, temporal and medial parietal regions in the vlbw group than that in the control group, although these structure function associations survived significance testing only in the control group due to its larger sample size . The frontal regions in which surface area was related with iq included the caudal middle frontal gyrus, lateral orbitofrontal gyrus, medial orbitofrontal gyrus, pars orbitalis, rostral anterior cingulate, frontal pole and insula . These are regions where the vlbw children have significantly reduced surface area in comparison with controls, and are believed to be important for cognitive functions such as decision making, executive functions, semantics, attention, and working memory . Previous studies have consistently shown poorer executive abilities in individuals born with vlbw than term - born peers, as well as problems with attention and working memory (aarnoudse - moens et al ., 2009; bayless and stevenson, 2007; anderson, 2014; lohaugen et al ., 2010;anderson et al ., 2004), and in the present study, the vlbw group had lower iq scores than those of the controls . Our results indicate that a larger surface area is positively correlated to higher iq, consistent with skranes et al . (2013), and it is tempting to speculate that reduced cognitive function in the vlbw group may be caused, at least in part, by the observed reduction in surface area . There was a negative association, albeit not significant, between iq and cortical thickness in both groups . In the vlbw group, the regions with the strongest negative associations between iq and cortical thickness were also the regions where the vlbw children displayed thicker cortex than controls . This is consistent with previous studies of the relationship between cortical thickness and cognitive functions in normally developing 511 year old children, where cortical thinning in the left dorsal frontal and parietal lobes was correlated with improved verbal performance (sowell et al ., 2004). Moreover, in vlbw adolescents with low iq, full iq and cortical thickness were negatively correlated in the frontal and positively correlated in the parietal lobes (bjuland et al ., 2013). Taken together, our findings suggest that altered cortical development in vlbw children seems to affect their cognitive abilities; however, longitudinal studies are needed to determine whether these deviations persist during further brain maturation throughout school age, adolescence and into early adulthood also for these recent year cohorts of vlbw children . The present study demonstrates altered development of cortical surface and cortical thickness in vlbw children born in 20032007, and these deviations are associated with poorer cognitive abilities . The present brain morphological deviations are evident even in vlbw children without cerebral palsy who have received state of the art medical treatment in the perinatal period, and who did not present with focal brain injuries on neonatal ultrasonography.
The protozoan parasite trypanosoma cruzi is the causative agent of chagas disease, which affects approximately 15 million people in south and central america [1, 2]. It is estimated that about 30% of infected individuals will develop severe chronic forms of the disease, especially the often fatal chagas disease cardiomyopathy (ccc) [14]. Intracellular protozoan parasites are potent stimulators of innate and cell - mediated immunity . The induction of macrophage proinflammatory cytokines by ligands of innate immunity receptors of t. cruzi is considered important in the control of infection and outcome of chagas disease [5, 6]. It has been extensively described that glycosylphosphatidylinositol - anchored mucins - like glycoproteins from trypanosoma cruzi trypomastigotes (tgpi - mucins) activate murine macrophages in vitro to produce the proinflammatory cytokines tumor necrosis factor (tnf-) and interleukin- (il-) 12 as well as nitric oxide (no) [7, 8]. The bulk of evidence establishes that il-12 and il-12 driven th1 cytokines, the ones involved in delayed - type hypersensitivity, are induced during acute infection with t. cruzi in mice, playing an obligatory role in parasite clearance and host survival [912]. T. cruzi tgpi - mucins were shown to initiate the inflammatory response through an activation of toll - like receptors tlr2 [7, 13]. Different components from this parasite are capable of activating tlrs in dendritic cells and macrophages, like the unmethylated cpg motifs present in t. cruzi genome, were identified as a tlr9 agonist . T. cruzi chronically infected chagas disease patients display a th1 cytokine profile which is even more pronounced among ccc patients [16, 17]. It has been described that certain infectious agents, like mycobacterium tuberculosis, possess molecules stimulating innate immunity that can shift the systemic cytokine environment and modify clinical immune profiles . Our group and others have previously reported that heart - infiltrating t cells predominantly produce ifn- and tnf-, suggesting that such th1 t cells play an important pathogenetic role in heart tissue damage in ccc [16, 1922]. Even though acute t. cruzi infection induces il-12 production in mice, little is known about whether t. cruzi or tgpi - mucins exert a similar action in humans . We have previously described the isolation of live t. cruzi trypomastigotes outgrowing from a heart biopsy fragment from a ccc patient, routinely cultured for the study of outgrowing heart - infiltrating t cells [16, 24]. In order to study whether t. cruzi and tgpi - mucins could directly induce the production of the th1-inducing cytokine il-12 in human cells, we studied the cytokine profile in naturally infected supernatants of heart - infiltrating mononuclear cells . We also assessed the effect of cocultivation of t. cruzi and tgpi - mucins with peripheral blood mononuclear cells and purified monocytes on il-12 production . Finally, we assessed the role of ifn- and cd40l signaling on t. cruzi and tgpi mucin - induced il-12 production . The y strain of t. cruzi was maintained in fibroblast cultures and was used as parasite source for purification of tgpi - mucins . For the trypomastigote culture, l-929 fibroblasts were initially infected with blood trypomastigotes in a ratio of one parasite per cell . The infected cultures were maintained in dulbecco's modified eagle's medium (dmem) containing 5% fetal calf serum (fcs) at 33c in 5% co2 . After 4 or 5 days of culture, the parasites were collected daily and centrifuged at 40 g at 4c for 10 min for cellular debris separation, followed by another centrifugation at 700 g at 4c for 10 min . The gpi - mucins were isolated from t. cruzi trypomastigotes as described previously [7, 8] using sequential organic extraction followed by hydrophobic - interaction chromatography in an octyl - sepharose column (amersham pharmacia biotech, uppsala, sweden) and elution with a propan-1-ol gradient (560%). Briefly, biopsy tissue was minced and seeded on to 96-well flat bottom plates in the presence of il-2 and irradiated peripheral blood mononuclear cells (pbmc) until lymphoblast outgrowth was observed; t cell lines were expanded by restimulation every two weeks with 5 g phytohemagglutinin (pha) and irradiated pbmc . Pbmc were obtained from blood of healthy donors and separated by density gradient centrifugation with ficoll - hypaque . All cells were cultured in dulbecco's modified eagle's medium supplemented with 2 mm l - glutamine, 1 mm sodium pyruvate, mem's nonessential amino acids and mem's vitamins (all from gibco, grand island, ny, usa), 50 g / ml gentamicin, 10 mm hepes buffer, and 10% normal human serum (complete medium). This protocol has been approved by the institutional review board of the university of so paulo school of medicine and all subjects provided informed consent . Ten to 12 days after the last pha stimulation, heart - infiltrating t cell lines (from four different individuals, in separate experiments) were stimulated in the presence of irradiated pbmc (5 10/well) plus 5 g / ml pha and supernatants were obtained after 48 h incubation . In another set of experiments, culture conditions included variable components: irradiated pbmc, heart - infiltrating t cell lines (from four different individuals), 5 g / ml pha, 5 10 y strain t. cruzi trypomastigotes obtained from llc - mk2 monolayer cell culture, or 10 pmol / ml of t. cruzi tgpi - mucins . Blocking / neutralizing monoclonal antibodies against cd28, cd40, and ifn- (pharmingen, la jolla, ca) were employed in selected experiments . Human monocytes were obtained by leukapheresis of normal volunteers at the blood bank of national institutes of health (bethesda, md). After density sedimentation of the mononuclear cells with lymphocyte separation medium (organon, teknika, durham, nc), the monocytes were purified by counterflow centrifugal elutriation, as described previously, except that pyrogen - free pbs was used in the elutriation procedure . Monocytes were enriched> 90% as determined by morphology, non - specific esterase staining, and flow cytometry . The purification procedure did not activate the monocytes, as shown by the fact that, after overnight incubation at 37c in suspension, 4% of the cells were il-12r positive or spontaneously secreted any of the cytokines measured . After purification, monocytes were left at 4c overnight and then transferred to 5 ml polystyrene falcon tubes (becton dickson labware, lincoln park, nj) and cultured for 24 h in the presence or absence of 10 pmol / ml tgpi - mucins, in the presence or absence of 100 units / ml of human ifn- (genetech), as indicated . Cytokines ifn-, il-4, il-2, il-10, il-12, and tnf- were measured by double sandwich elisa using the anti - human cytokine antibody pairs (r&d systems, minneapolis). Groups were compared by a nonparametrical test (mann - whitney rank sum test) with graphpad instat software (version 5.0; graphpad). We routinely cultured t cell lines from endomyocardial biopsies from ccc patients for the isolation of t cell lines . In one of these biopsy explants, highly motile t. cruzi trypomastigotes were observed in some of the seeded wells, indicating that the tissue fragments in those wells probably contained a t. cruzi pseudocyst . We therefore compared pha - stimulated cytokine production in the supernatant from the t cell line established from wells containing live t. cruzi parasites (with t. cruzi trypomastigote growth) with the cell line derived from wells of the same biopsy devoid of t. cruzi (no t. cruzi trypomastigote growth). Figures 1(a) and 1(b) depict the cytokine profile of the t cell line obtained from the t. cruzi - positive wells as compared to the t cell line of the same sample, obtained from wells where no t. cruzi trypomastigotes were observed . As can be seen, t. cruzi trypomastigotes outgrowth induced the production of il-12, tnf-, and ifn-, with undetectable levels of il4 . The presence of t. cruzi strongly reduced the levels of il-2 and mildly reduced il-10 levels . To further investigate the phenomenon observed in the endomyocardial biopsies wells we assayed cytokine production in supernatants of human pbmc in the presence of living t. cruzi and/or pha - activated t cells . As shown in figure 2, t. cruzi trypomastigotes can induce moderate production of il-12 directly on irradiated pbmc or in cocultures of pbmc and t cells . However, coculture with pha - activated t cell lines induced a 10- to 100-fold increase in il-12 production by irradiated pbmc . We also tested if purified tgpi - mucins could activate isolated pbmc - derived monocytes in vitro to produce il-12 . As shown in figure 3, tgpi - mucins induce significant production of il-12 by human monocytes, which is further potentiated after ifn priming of cells . In an attempt to study the mechanisms underlying t. cruzi - induced potentiation of il-12 production by human monocytes, we cocultured these cells with pha - activated t cell lines, 5 10 t. cruzi y strain living trypomastigotes, or tgpi - mucins and added neutralizing / blocking antibodies to human ifn-, cd40, and cd28 . Results indicated that blocking ifn- or cd40 individually caused approximately 50% and 35% of inhibition of il-12 production, respectively, while anti - cd28 showed negligible inhibition . The combined effect of the three antibodies induced 85% of inhibition, suggesting that most of the il-12-inducing effects of pha - activated t cell lines are due to ifn- production and cd40-cd40l interactions (figure 4(a)). Similar results were obtained when tgpi mucin was used as stimulus (figure 4(b)) suggesting that these molecules may be the effectors in the t. cruzi - induced il-12 production in humans, as has been previously described in mice . In this paper, we observed that both living trypomastigotes and tgpi - mucins are potent inducers of il-12 production in human monocytes and that this effect depends on cd40-cd40l interaction and ifn- signaling . The finding that spontaneous outgrowth of parasites in culture cells derived from chronically infected myocardium induced the production of t1-type proinflammatory cytokines, like il-12, tnf, and ifn, is in accordance with data from murine models from other studies [2630]. These cytokines, which are induced during acute infection with t. cruzi in mice, play an obligatory role in parasite clearance and host survival [26, 29]. However, the same immunological pattern may participate in mechanisms of tissue damage in chagas disease, indicating that protective and pathological responses must share important characteristics in this context . When parasites were deliberately added to cocultures of irradiated pbmc and activated t cell lines, we observed again high levels of il-12 expression in pbmc, although the t. cruzi stimulus itself was capable of inducing some il-12 expression by pbmc in the absence of activated t cells . This corroborates the findings obtained with cultures with spontaneous outgrowth of t. cruzi trypomastigotes, where we had pha - activated cell lines and t. cruzi trypomastigotes . Our results indicate that pbmc - derived monocytes are the cell population responding to tgpi - mucins with in vitro il-12 production . Although we already observed induction of il-12 production by monocytes using tgpi - mucins as a first signal (microbial stimulus via tlr-2), maximal levels of il-12 are reached only after a second signal through the presence of ifn-, as it has been reported by other studies [32, 33]. The alkylacylglycerolipid component of tgpi - mucins is capable of triggering toll - like receptors-2 at subnanomolar concentrations . Moreover, macrophages derived from tlr2 or myd88 mice are less responsive to tgpi - mucins, further confirming the possible role of the tlr pathway in this process . Our findings that anti - ifn- and anti - cd40l neutralizing antibodies were able to significantly reduce il-12 production indicate this phenomenon is mediated by ifn- and cd40-cd40l interactions . This can be explained by the fact that, in this context, t cells are likely to be the major source of ifn- and membrane cd40l, activators of macrophages involved in many aspects of parasite control [11, 35]. As previously described by chaussabel et al . Cd40 ligation in t. cruzi - infected mice has a protective effect because it is related to upregulation of il-12 as well as no by a direct stimulation of inf- activated macrophages . Previous studies also showed that the cd40-cd40l signaling pathway mediated protective effect with other pathogens such as leishmania, schistosoma mansoni, cryptococcus neoformans, cryptosporidium parvum, and pneumocystis carinii . The enhanced production of ifn-, tnf-, and nitric oxide associated with cd40/cd40l signaling is thought to be responsible for this protective effect . It was shown that ifn stimulus also upregulates the transcription factor t - bet, which in turn maintains il-12r chain expression, possibly resulting in a positive feedback loop that, consequently, keeps the shift towards a th1 response in chagas disease . In summary, our data suggest that the t1-type cytokine profile found in the peripheral blood and among heart - infiltrating t cells is related to previous or ongoing encounters with il-12 generated as a response to t. cruzi gpi - anchored mucin - like glycoproteins.
Deceased donors (dds) with the brain death due to head injury are the major source of organs for transplantation . Recent studies mention incidence of post - head injury disseminated intravascular coagulation (dic) ranging from 24% to 50% . Many centers hesitate in accepting organs from donors with dic due to increased risk of primary graft non - function and/or high chances of morbidity / mortality . In india, about 61% of stage v chronic kidney disease (ckd) patients are not on any form of renal replacement therapy and only 2% are prepared for renal transplantation . There is a wide gap between the demand and supply of organs for patients with end organ failure and potential chances of getting a deceased donor (dd) organ in the present scenario are dismal . To reduce this disparity, an additional approach that could be considered is accepting dd with head injury with disseminated intravascular coagulation (dic) for organ donation since, dd with traumatic head injury are the most common source of dd organ transplantation . Whether to consider a dd with dic is controversial since some studies have shown increased incidence of primary non - function in transplanted kidneys while others have shown good long - term outcome, but initial delayed graft function . We present two successful renal transplants (rtx) from a brain - dead dd with head injury with extensive dic and deranged renal function . A 19-year - old male patient with head injury following a road traffic accident was brought in emergency to a private trauma care hospital . On admission, he was found to be comatose, had fractured right tibia and left femur . On day of admission, his renal function and liver function tests were normal with serum creatinine (scr), 1.40 mg / dl . However, he had deranged coagulation profile with activated partial thromboplastin time: 32.7 s, (control: 27.9 s), prothrombin time: 20 s (control: 10.6 s) and international normalized ratio was 1.89 . His hemoglobin was 7.21 g / dl, total leukocyte count 1.52 10/l and platelet count was 2.08 10/ l . He had hypotension and fall in hemoglobin level; hence, he was transfused 4 units of whole blood and started on vasopressors dopamine at 10 g / kg / min and noradrenaline at 10 g/ min . Blood pressure was maintained around 110 - 120 systolic and 80 - 90 mm of hg diastolic . On 2 day, he developed extensive petechiae and purpura all over the body and oozing of blood through wounds hence 6 units of fresh frozen plasma were transfused . However, he further deteriorated with increase in purpuric spots, further derangement in coagulation profile, decreasing platelet count and hemoglobin level, low serum fibrinogen level and d - dimer> 4000 ng / ml suggestive of extensive dic . Patient was declared brain - dead by neurophysician and neurosurgeon at the interval of 6 h. ultrasonography of the abdomen showed normal sized kidneys . The relatives were explained about organ donation . Since, they agreed he was shifted to our institute . His renal function was deranged with scr, 3.57 mg / dl and blood urea 92 mg / dl . His hemoglobin had dropped to 7.3 g/ dl and platelet count was 5.5 10/l . Both kidneys were harvested and transplanted in two recipients with favorable complement - dependent lymphocytotoxicity cross - match . Laboratory parameters of donor recipient 1-was a 25-year - old male on maintenance hemodialysis for, 55 months . He received induction therapy with rabbit anti - thymocyte globulin (r - atg) 1.5 mg / kg and 3 doses of methyl prednisolone 500 mg/ day . His hematological, coagulation and renal function profile remained within normal range throughout the post - operative period and follow - up period of 1 month . Scr normalized on 5 post - operative day to 1.22 mg/ dl and he was discharged on 7 post - operative day with scr, 1.18 mg / dl on maintenance immunosuppression of tacrolimus 0.08 mg / day, mycophenolate sodium 720 mg twice daily and prednisolone, 20 mg / day . There was no evidence of graft dysfunction throughout the follow - up period of 1 month . She received the same induction and maintenance immunosuppression therapy as recipient 1 . On first post - operative day however, it recovered gradually and normalized on 4 post - operative day without any treatment . Coagulation profile performed performed on 1 and 4 post - operative day was normal and there was no evidence of bleeding diathesis . She was discharged on 12 post - operative day with scr of 0.87 mg / dl with same maintenance immunosuppression as that of recipient 1 . Demographic profile and laboratory parameters of both recipients tacrolimus level of both transplant recipients in immediate post - transplant period and throughout the follow - up period of 1 month was maintained in the range of 7 - 10 ng / ml . Dic is common in dd with head injury with incidence of 59% in open head trauma, 43% in combined open and closed head trauma and 37% in closed head trauma . Mechanisms responsible for dic are activation of the coagulation cascade through release of brain tissue thromboplastin, inflammatory cytokines, tissue factor activation and exposure of phospholipids to circulating blood . Dic causes occlusion of small and medium sized vessels due to the formation of fibrin thrombi . Many centers still hesitate to accept organs from dd with dic due to reported incidence of primary non - function . In one study, fibrin thrombi present in renal biopsy at 1 h post - transplant was not present in biopsy done at 7 days and 6months post - transplant . Recipients of grafts with donor thrombi were more likely to exhibit delayed graft function; however, graft function and survival at 1 and 2 years post - transplant was good suggesting that the presence of donor microvascular thrombosis does nt nt portend poor outcome in rtx . Some studies suggest that the presence of good renal function and renal biopsy is necessary prior to transplantation to exclude renal cortical necrosis due to microvascular thrombosis in dic, but other reports negate this view . Pre - transplant renal biopsy is not a good method to guide organ allocation in cases of donor dic because of false positive and false negative results and also inability of renal biopsy to predict reversibility of fibrin thrombi by glomerular fibrinolytic system after transplantation . One study reported increased incidence of post - transplant thrombocytopenia in recipients of kidney from dic positive donors . It also noted that there was increased incidence of delayed graft function in recipients who had post - transplant thrombocytopenia, but long - term graft survival was good . Recipient thrombocytopenia may be an early sign of intrarenal clotting due to dic; thus, may contribute to delayed graft function or slow graft function . There are some case reports, which showed that even presence of severe dic with thrombotic microangiopathy on renal biopsy and renal function impairment in dd is not a reason for excluding organ from dic positive donor . We selected this donor for organ donation because of his young age and since, there was no any major illness in the past; also he had good urine output prior to transplant . One of our recipients developed transient thrombocytopenia, but there was no clinical or laboratory evidence of dic and also there was no evidence of delayed graft function or slow graft functions in either recipient . To conclude, a carefully selected dd with head injury and dic even with deranged renal function may not be a contraindication for organ donation if other risk factors for primary non - function are excluded.
Intracranial foreign bodies such as needle, wooden and bullets are generally due to penetrating injuries through the orbita, ear or cranial bones or rarely forgotten surgical objects in the brain during surgery . Most of the cases were diagnosed incidentally and/or during evaluation for symptoms such as headache, epilepsy or altered behavior . Patients, who presented with seizure due to intracranial needles, were rarely reported previously . We report a 14-year - old boy with epilepsy resulting from the presence of sewing needle located in the brain . A 14-year - old boy was admitted to the pediatric neurology outpatient department with a history of generalized tonic - clonic (gtc) seizures . There were no previous history of epileptic seizures, head trauma or injury and his family history was unremarkable . On admission his neurological examination was normal, there were no localizing neurological findings or lateralized weakness . After his second seizure sodium valproate treatment (20 mg / kg / day) was started . To determine etiology of seizures cranial magnetic resonance imaging was obtained and magnetic susceptibility artifact in right frontal region was seen (figure 1). To reveal the nature of the object computed tomography (ct) was obtained, which showed a linear density compatible with a sewing needle surrounded with a local encephalomalacic area and calcification (figure 2). Also, skull radiography demonstrated the presence of metallic opacity of a sewing needle, located in a cranio - caudal direction on the right frontal area (figure 3). Based on the needle s location, we thought that it might have been inserted through the anterior fontanel during infancy . The needle was encrusted with an irregular gliotic area (figure 4). During the postoperative 8 months he was seizure - free with valproic acid treatment . Two months after discontinuing valproic acid treatment his seizure reoccurred and a spike wave activity in the right frontal region was determined in his eeg . In the two there are reports of sewing needles and other foreign objects retained in the brain for long periods of time without any symptoms . Since its first description in 1914, approximately forty cases have been reported all over the world, which were reviewed by struiale et al . In this review, about one third of patients with intracranial needles were asymptomatic and had been discovered incidentally . The most seen complaint was long time history of slight headache and secondly was seizure . Initial complaints such as fever, hemiparesis, extrapyramidal signs, cranial nerve palsy, hemi - chorea, brain abscess, nausea, vomiting, lethargy, hemorrhage, meningitis and rarely hypothalamic syndrome had also been reported in this review . The underlying pathophysiologic mechanism of seizures linked to intracranial foreign bodies is not clearly understood . Reported that persistent epilepsy after penetrating brain injuries is as high as 50% at 15 years after the injury, 85% in cases with longer follow - up . Hypothesized that the needle can function as an electric dipole and can be the main reason for epileptogenesis . Although we could not identify any abnormality in his first awake / sleep eeg, his postoperative eeg showed spike wave activity in the same area with the needle in our case . Therefore we thought that the area of gliosis around the needle might be a possible reason for seizures then the needle . The most widely accepted approach is to follow - up without surgical removal when the patients have no clinical signs or symptoms and the diagnosis is purely incidental . The excision of the cortical scar tissue at the sewing needle entrance is highly recommended, especially if there are preoperative eeg abnormalities . We preferred to remove the needle surgically because of the early presentation of the patient with seizure . Most frequently, the patients and their relatives have no idea about how and when the needle was inserted . In most cases, as in ours, the needle presumably was introduced in infancy before the closure of the fontanels; therefore the patient cannot remember anything . Although it is thought that the needle inserted accidently, the conditions such as introduction of the needle by another child without the awareness of parents or other family members and child abuse should be strongly considered in these cases . In conclusion, although there are no clear - cut guidelines regarding the management of these foreign bodies, follow - up with antiepileptic treatment before surgery might be a reasonable approach to the patients with seizure.
The fgf family consists of 18 receptor - binding members that regulate a broad spectrum of cellular activities 1 . The fgf elicits its regulatory signals via activating the fgfr tyrosine kinases encoded by four highly homologous genes . Frs2, also called snt1 for suc13-associating neurotropic factor target 1, is a broadly expressed membrane - anchored adaptor protein that is required for the fgf to activate the map and pi3 kinase pathways, the two major pathways in the fgf signaling cascade 2 - 4 . The frs2 family has two highly homologous members, frs2 and frs2. Frs2 is broadly expressed in adult and fetal tissues, whereas frs2 is more restrictively expressed 5 - 7 . Frs2 null mouse embryos die between embryonic (e) 7.0 - 7.5 days 5 . Although frs2 can compensate for the loss of frs2 with respect to mapk activation in mouse embryonic fibroblast (mef) cells 8, recent reports demonstrate that frs2 and frs2 do not always mediate the same signals 9 - 12 . Frs2 and frs2 share similar structure domains, which include the n - terminal myristylation site that anchors frs2 to the cell membrane, the phosphotyrosine - binding (ptb) domain required for binding to the fgfr, and the c - terminal sequence that contains multiple tyrosine and serine / threonine phosphorylation sites . The ptb - binding domain of the fgfr does not include a phosphorylated tyrosine residue, which is different from the ptb - binding sites of other growth factor receptors 13 . The ptb domain of frs2 also binds cks1, a molecule that triggers degradation of cell cycle regulatory protein p27 during the g1/s transition in the cell cycle 14 . Although not essential 15, a vt (valine - threonine) motif encoded by alternatively spliced sequences in the intracellular juxtamembrane domain of fgfr1 and fgfr2 is important for association with the ptb domain 3,13,16 - 18 . Interestingly, the binding of fgfr1 to frs2, but not frs2, is enhanced by receptor autophosphorylation 15 . Frs2 has six tyrosine phosphorylation sites . Among them, tyr196, tyr306, tyr349, and tyr392 are grb2-binding sites that have been shown to be important for transmitting the signals to the pi3k / akt pathway . Tyr436 and tyr471 are shp2-binding sites that have been shown important for transmitting the signals to the map kinase pathway 19 - 21 . Mice expressing a frs2 mutant that lacks the shp2-binding sites exhibit severe developmental defects; those that lack the grb2-binding sites have less severe defects 19,22 . Fgf stimulation also causes phosphorylations on multiple serine / threonine residues of frs2, which provides a negative feedback for the fgf signaling activity 23 . Frs2 phosphorylation appears to be fgfr isoform - specific, which may result in differential recruitments of downstream signaling molecules and, thus, contribute to signaling specificity of the fgfr 24,25 . We reported previously that the four grb2-binding, but not the two shp2-binding sites, are essential for fgfr1 to activate the fgf - inducible response element (fire) of the mouse syndecan 1 gene 15, which is an enhancer required for the fgf to promote syndecan 1 expression and has been widely used as a reporter for the fgf signaling pathway 15,24,26 . Process for degrading and recycling various cellular constituents, such as long - lived proteins and entire organelles . Autophagy initiates with the formation of autophagosomes in which the isolation membrane engulfs cellular constituents 27 . Autophagosomes then fuse with lysosomes to form autolysosomes where the contents are degraded by acidic lysosomal hydrolases . As a self - digestion system, autophagy may influence cell survival, proliferation, and differentiation by accelerating turnover of old protein or organelles 28 . However, whether autophagy can be regulated by the fgf signaling axis is currently unknown . Here, we show that frs2 is required for the fgf signaling axis to activate the mtor pathway and to suppress the autophagy activity in mefs . Ablation of frs2, as well as inhibition of fgfr or pi3k, but not erk, kinase activity suppressed fgf to activate the mtor pathway and to inhibit autophagy . Thus, the results, for the first time, demonstrate that the frs2-mediated mtor pathway is required for the fgf signaling axis to regulate autophagy, and suggest a new mechanism by which the fgf elicits its regulatory signals . All animals were housed in the program for animal resources of the institute of biosciences and technology, and handled in accordance with the principles and procedures of the guide for the care and use of laboratory animals . The embryos were harvested at embryonic day 14.5 for establishing mef cultures as described 15 . Briefly, e14.5 embryos carrying homozygous frs2 were minced in 3 ml ice - cold 0.25% trypsin - edta solution (sigma - aldrich, st . Louis, mo), incubated at 4 c overnight, and then 37 c for 30 minutes in the same solution . The isolated cells were propagated at a ratio of 1:3 in 6-well plates and maintained in 5% fetal bovine serum / dmem until being used . The mefs were then immortalized by transfection with prsv - tag plasmid that carried the sv40 t antigens . Ad5-cmv - cre - gfp and ad5-cmv - gfp viruses from the center for cell and gene therapy (houston, tx) were used to delete the floxed fragment . Overnight cultured mefs (1x10 cells in 6-well plates) were transfected with 5 g of the indicated frs2 mutants in pcdnazeo plasmids (invitrogen corporation, la jolla, ca) and 5 l lipofectamine (invitrogen corporation). (camarillo, ca), and pi3k (ly294002) was from cell signaling (beverly, ma). The cells were incubated at 37 c for 24 hours, and the culture media were then changed to a serum - free dmem containing 10 g / ml heparin for serum starvation . After incubation at 37 c for 12 hours, the cells were stimulated with fgf2 at the indicated concentrations for the indicated times before being lysed with 200 l ripa buffer containing the protease - phosphatase inhibitor cocktail (sigma - aldrich, inc . The cell lysates equivalent to 50 g total proteins were separated on sds - pages and blotted onto pvdf membranes for western analyses with the indicated antibodies . The sources and dilution of the antibodies are: phosphorylated frs2 tyr196 (1:1000), phosphorylated erk1/2 (1:1000), phosphorylated akt thr308 (1:1000), phosphorylated akt ser473 (1:1000), and phosphorylated s6k1 thr389 (1:1000) from cell signaling technology inc . (danvers, ma); human lc3 (1:500) from novus biologicals, llc (littleton, co); and -actin (1: 5000) from santa cruz biotechnology, inc ., (santa cruz, ca). The specifically bound primary antibodies were detected with the horseradish peroxidase conjugated secondary antibodies and visualized with the ecl - plus chemoluminescent reagents . We previously reported that the mef cells derived from frs2 floxed embryos are able to respond to fgf signals 15 . Since the frs2-mediated pi3k / akt pathway is an upstream regulator of the mtor pathway, we investigated whether fgf activated mtor . Mef cells were treated with fgf2 at the final concentration of 5 ng / ml, and lysed at various time points . Activation of the fgf signaling axis and mtor pathway was analyzed by western blot . Treating with akt and erk1/2, the major downstream molecules of the fgf signaling axis, were also strongly phosphorylated within 5 minutes, which were then gradually reduced . S6k1, the major substrate of the mtor c1 complex, was also strongly phosphorylated, although the phosphorylation was slightly delayed . Consistently, phosphorylation of mtor s2448, an s6k1 phosphorylation site 30, was increased at a lagging mode . Together, the results demonstrate that the fgf signaling axis induces activation of the mtor pathway . Frs2 is an adaptor protein in the fgf signaling pathway, which recruits multiple downstream pathways, including the erk1/2 and pi3k / akt pathways, to the fgfr kinase . To investigate whether frs2 was required for fgf2 to activate the mtor pathway, mefs bearing homozygous frs2 floxed alleles were treated with adenovirus - cre / gfp to delete the floxed fragment unlike mefs bearing floxed frs2 alleles that exhibited strong activation of erk1/2, akt, and s6k1 by fgf2, mefs bearing frs2 null alleles failed to respond to fgf2 with respect to erk1/2, akt, and s6k1 phosphorylation (fig . 2b). The weak activation of erk activation in frs2 null mefs were likely due to frs2-independent pathways . The results indicated that frs2 was required for the fgf signaling axis to activate the mtor pathway . Interestingly, the background phosphorylation of akt s473, an mtor - rictor phosphorylation site 31, was significantly increased in frs2 null mef . This is not surprising since frs2 has been shown to play important roles in cell signaling feedback regulation 32 . To confirm that the results were not associated with the cloning procedure, primary mefs were isolated from frs2 floxed embryos and infected either with adenovirus - gfp or adenovirus - cre / gfp to repeat the experiments . The results demonstrated that ablation of the frs2 alleles significantly impaired activation of akt, erk, and mtor by fgf2 (fig . 2c), which confirmed that frs2 was required for fgf2 to activate the mtor pathway . In addition to the frs2-independent pathway, incomplete disruption of frs2 in primary mefs might also contribute to compromised phosphorylation of erk1/2 induced by fgf2 . Frs2 has 6 tyrosine phosphorylation sites of which 4 are grab2 binding that are important for pi3k / akt activation and 2 are shp2 binding that are important for map kinase activation 19 . Both pi3k / akt and map kinase pathways can be linked to the mtor pathway 33 . To determine which pathway is required for fgf2 to activate mtor in mefs, inhibitor specific for either pi3k or mek1/2 inhibition of pi3k, but not mek1/2, abolished the phosphorylation of s6k1, indicating that only the pi3k / akt pathway was required for fgf2 to activate the mtor pathway (fig . 3). Interestingly, inhibition of the map kinase pathway significantly increased the phosphorylation of akt t308 and s473, which were catalyzed by the pi3k / pdpk1 (phosphoinositide dependent kinase 1) and mtor c2, respectively . Thus, the results suggested that inhibition of the map kinase pathway sustained or promoted activation of the akt - mtor signaling axis . The results are in line with the reports that sustained activation of the map kinase provides a feedback control mechanism of the fgf signaling axis 23 . The mtor pathway is a major signaling pathway that inhibits autophagy 34 . To investigate whether fgf signaling regulated autophagy, we then assessed the influence of fgf2 on lc3 ii abundance . Lc3, also called microtubule associated light chain 3, is a broadly used autophagy indicator . The full - length prolc3 is processed to its cytosolic form, lc3 i, which is activated by conversion to its lipidated form, lc3 ii . The lipidated lc3 ii is translocated to preautophagosomes and autophagosomes, which are then degraded after fusing with lysosomes . Thus, the increased abundance of lc3 ii reflects enhanced autophagic activity or reduced autophagosome turnover 35,36 . To determine whether the fgf signaling axis regulated autophagy, the mefs with either frs2 floxed or null alleles were treated with fgf2, and the abundance of lc3 was assessed by western blot . Treating the frs2 floxed mefs with fgf2 enhanced s6k1 phosphorylation and reduced the abundance of the lc3 ii isoform, suggesting that fgf2 regulated autophagy, likely through the mtor pathway . Consistent with the result that fgf2 treatment did not induce s6k1 phosphorylation in frs2 null mefs (fig . 2b), fgf2 treatment also did not reduce lc3 ii abundance in frs2 null mefs (fig . 4a), indicating that fgf2 regulated the autophagy activity through frs2-mediated pathway(s). As the abundance of the lc3 ii isoform was dynamically controlled, the variation of lc3 ii abundance alone may reflect either an inhibition of lc3 i to lc3 ii conversion or an activation of lc3 ii turnover . Atg1 and atg13 are substrates of the mtor c1 kinase that is activated by the pi3k / akt pathway . Phosphorylation of atg1 and atg13 by mtor c1 changes the conformation of the atg1 complex to an conformation, resulting in suppression of autophagy 34 . As s6k1 is a substrate of the mtor c1 kinase, which is often used as readout for mtor c1 activity, the data of enhanced s6k1 phosphorylation in fgf2 treated mefs inclines toward the possibility that autophagic activity is repressed by increased mtor c1 activity . To clarify how fgf2 regulated autophagy, the mefs were treated with bafilomycin a1 although blocking the degradation of lc3 ii by suppression of fusions between autophagosomes and lysosomes increased the abundance of lc3 ii either in the presence or absence of fgf2, treating the cells with fgf2 still significantly reduced the abundance of the lc3 ii isoform, indicating less lc3 ii formation (fig . 4b). Therefore, the results indicate fgf2 suppresses lc3 i to lc3 ii conversion, and thus, the autophagy initiation in cells . To determine whether the mtor pathway was required for fgf2 to suppress autophagy, rapamycin, an mtor c1 inhibitor, was added to the mef cultures . Treating with rapamycin suppressed the phosphorylation of s6k1 and increased the abundance of the lc3 ii in fgf2 treated mefs (fig . 4c), although it did not affect akt and erk1/2 phosphorylation (data not shown). Separate experiments revealed that treating with rapamycin alone did not increase lc3 ii abundance in mefs (fig . The results suggest that activation of mtor c1 is required for fgf2 to suppress the autophagy activity . Further experiments with other autophagy markers, including expression of p62, atg5, atg12, etc . Reinstatement of full length frs2 expression in frs2 null mefs by transfection restored regulation of fgf2 on the autophagy activity (fig . 5). To determine whether the grb2- or shp2-binding sites were required for regulating autophagy, mutants lacking the four grb2- or two shp2-binding sites were expressed in frs2 null mefs as described 15 . To better demonstrate the difference in lc3 ii abundance, only lightly exposed films were shown . Consistent with the chemical inhibition experiments demonstrating that the pi3k / akt, but not erk, pathway was required for fgf2 to suppress autophagy, expression of the frs2 mutant lacking the grb2-binding sites (y196, y306, y349, and y392) failed to restore the activity, whereas expression of frs2 lacking the shp2 binding sites (y436 and y471) restored the response to fgf2 with respect to autophagy inhibition . The results again demonstrate that the fgf signaling pathway regulates autophagy through the grb2-mediated pi3k / akt pathway . There are four grb2-binding sites on frs2. To further investigate which grb2-binding phosphorylation sites were required for frs2 to mediate the autophagy regulation signals, mutant frs2 carrying an individual grb2-binding site mutation were expressed in frs2 null mefs . Expression of mutants with a substitution of either y196 or y306 with phenylalanine rescued the fgf activity to inhibit autophagy activity, whereas expression of the mutant with a substitution of either y349 or y392 with phenylalanine failed to rescue such defects (fig . The results indicate that y349 and y392, but not y196 and y306, are required for the fgf to suppress autophagy, and imply that each grb2-binding site may mediate a subset of fgf signals . Activation of pi3-kinase is mediated by assembly of gab1 to the grb2/frs2 complex, which enables tyrosine phosphorylation of gab1 by fgfr to generate binding sites for p85, the regulatory subunit of pi3-kinase, thus resulting in recruitment and activation of the pi3-kinase 38 . Although all four grb2-binding sites are involved in activation of the pi3k / akt pathway in 3t3 cells 2, only two grb2-binding sites are involved in autophagy regulation in mefs . The molecular mechanism underlying the results suggest that the autophagy - suppressing signals of the fgf signaling axis are different from the fire - activating signals that are mediated by the four grb2-binding sites additively, since substitution of each grb2-binding site only partially reduces fire activation activity 15 . The fire consists of a 170-bp array of five dna motifs that bind two fgf - inducible fos - jun heterodimers, one inducible ap-2-related protein, one constitutively expressed upstream stimulatory factor, and one constitutive 46-kda transcription factor . It has been shown that fire is selectively activated by the fgf, but not by other tyrosine kinase receptor - activating growth factors 26 . Furthermore, the fgf activates fire independent of its mitogenic activity in prostate cancer cells 24 . Here we showed that the signals of fgf to regulate the mtor / autophagy pathway was different from those for fire activation, and further demonstrate mechanistic diversity of the fgf signaling axis . Furthermore, it has been reported that the lc3 antibody exhibits different detection sensitivities toward lc3 i and lc3 ii 40 . Therefore, future efforts to monitor expression of other autophagic markers, such as p62, atg5, and atg12, etc ., will be needed to confirm the findings and to elucidate the molecular mechanism underlying how fgf signaling regulates autophagic activity . Autophagy is a major cellular pathway to degrade bulky subcellular organelles and macromolecules, and plays important roles in development, metabolism, tumorigenesis, and diseases 41 . However, how autophagy contributes to development is not understood, although it has been proposed that autophagy may influence cell differentiation either by impairing new protein or organelle formation or by accelerating turnover of old proteins or organelles . In this study, we provided the first in vitro evidence that autophagy can be negatively regulated by the fgf signaling axis . Recently, we also discovered that the fgf regulates cardiac progenitor cell and cardiomyocyte differentiation via controlling the mtor pathway - regulated autophagy, and that the frs2-mediated pathways are required for the fgf to suppress premature differentiation of heart progenitor cells . Inhibition of autophagic activity suppresses cardiomyocyte differentiation both in the second heart field progenitors and in embryoid body (eb) cultures (42). In summary, the fgf signaling axis activated the mtor kinase and repressed autophagy by activating the frs2-mediated pi3k / akt pathway . Suppression of mtor activation abolished the repression activity of fgf2 on autophagy, indicating that fgf regulated autophagy via the mtor pathway . This is the first report that the fgf signaling axis plays a crucial role in autophagy regulation, thus, sheding new light on cell signaling mechanisms . We previously reported that the mef cells derived from frs2 floxed embryos are able to respond to fgf signals 15 . Since the frs2-mediated pi3k / akt pathway is an upstream regulator of the mtor pathway, we investigated whether fgf activated mtor . Mef cells were treated with fgf2 at the final concentration of 5 ng / ml, and lysed at various time points . Activation of the fgf signaling axis and mtor pathway was analyzed by western blot . Treating with akt and erk1/2, the major downstream molecules of the fgf signaling axis, were also strongly phosphorylated within 5 minutes, which were then gradually reduced . S6k1, the major substrate of the mtor c1 complex, was also strongly phosphorylated, although the phosphorylation was slightly delayed . Consistently, phosphorylation of mtor s2448, an s6k1 phosphorylation site 30, was increased at a lagging mode . Together, the results demonstrate that the fgf signaling axis induces activation of the mtor pathway . Frs2 is an adaptor protein in the fgf signaling pathway, which recruits multiple downstream pathways, including the erk1/2 and pi3k / akt pathways, to the fgfr kinase . To investigate whether frs2 was required for fgf2 to activate the mtor pathway, mefs bearing homozygous frs2 floxed alleles were treated with adenovirus - cre / gfp to delete the floxed fragment . 2a). Unlike mefs bearing floxed frs2 alleles that exhibited strong activation of erk1/2, akt, and s6k1 by fgf2, mefs bearing frs2 null alleles failed to respond to fgf2 with respect to erk1/2, akt, and s6k1 phosphorylation (fig . 2b). The weak activation of erk activation in frs2 null mefs were likely due to frs2-independent pathways . The results indicated that frs2 was required for the fgf signaling axis to activate the mtor pathway . Interestingly, the background phosphorylation of akt s473, an mtor - rictor phosphorylation site 31, was significantly increased in frs2 null mef . This is not surprising since frs2 has been shown to play important roles in cell signaling feedback regulation 32 . To confirm that the results were not associated with the cloning procedure, primary mefs were isolated from frs2 floxed embryos and infected either with adenovirus - gfp or adenovirus - cre / gfp to repeat the experiments . The results demonstrated that ablation of the frs2 alleles significantly impaired activation of akt, erk, and mtor by fgf2 (fig . 2c), which confirmed that frs2 was required for fgf2 to activate the mtor pathway . In addition to the frs2-independent pathway, incomplete disruption of frs2 in primary mefs might also contribute to compromised phosphorylation of erk1/2 induced by fgf2 . Frs2 has 6 tyrosine phosphorylation sites of which 4 are grab2 binding that are important for pi3k / akt activation and 2 are shp2 binding that are important for map kinase activation 19 . Both pi3k / akt and map kinase pathways can be linked to the mtor pathway 33 . To determine which pathway is required for fgf2 to activate mtor in mefs, inhibitor specific for either pi3k or mek1/2 inhibition of pi3k, but not mek1/2, abolished the phosphorylation of s6k1, indicating that only the pi3k / akt pathway was required for fgf2 to activate the mtor pathway (fig . 3). Interestingly, inhibition of the map kinase pathway significantly increased the phosphorylation of akt t308 and s473, which were catalyzed by the pi3k / pdpk1 (phosphoinositide dependent kinase 1) and mtor c2, respectively . Thus, the results suggested that inhibition of the map kinase pathway sustained or promoted activation of the akt - mtor signaling axis . The results are in line with the reports that sustained activation of the map kinase provides a feedback control mechanism of the fgf signaling axis 23 . The mtor pathway is a major signaling pathway that inhibits autophagy 34 . To investigate whether fgf signaling regulated autophagy, we then assessed the influence of fgf2 on lc3 ii abundance . Lc3, also called microtubule associated light chain 3, is a broadly used autophagy indicator . The full - length prolc3 is processed to its cytosolic form, lc3 i, which is activated by conversion to its lipidated form, lc3 ii . The lipidated lc3 ii is translocated to preautophagosomes and autophagosomes, which are then degraded after fusing with lysosomes . Thus, the increased abundance of lc3 ii reflects enhanced autophagic activity or reduced autophagosome turnover 35,36 . To determine whether the fgf signaling axis regulated autophagy, the mefs with either frs2 floxed or null alleles were treated with fgf2, and the abundance of lc3 was assessed by western blot . Treating the frs2 floxed mefs with fgf2 enhanced s6k1 phosphorylation and reduced the abundance of the lc3 ii isoform, suggesting that fgf2 regulated autophagy, likely through the mtor pathway . Consistent with the result that fgf2 treatment did not induce s6k1 phosphorylation in frs2 null mefs (fig . 2b), fgf2 treatment also did not reduce lc3 ii abundance in frs2 null mefs (fig . 4a), indicating that fgf2 regulated the autophagy activity through frs2-mediated pathway(s). As the abundance of the lc3 ii isoform was dynamically controlled, the variation of lc3 ii abundance alone may reflect either an inhibition of lc3 i to lc3 ii conversion or an activation of lc3 ii turnover . Atg1 and atg13 are substrates of the mtor c1 kinase that is activated by the pi3k / akt pathway . Phosphorylation of atg1 and atg13 by mtor c1 changes the conformation of the atg1 complex to an conformation, resulting in suppression of autophagy 34 . As s6k1 is a substrate of the mtor c1 kinase, which is often used as readout for mtor c1 activity, the data of enhanced s6k1 phosphorylation in fgf2 treated mefs inclines toward the possibility that autophagic activity is repressed by increased mtor c1 activity . To clarify how fgf2 regulated autophagy, the mefs were treated with bafilomycin a1 to suppress the fusion between autophagosomes and lysosomes 37 . Although blocking the degradation of lc3 ii by suppression of fusions between autophagosomes and lysosomes increased the abundance of lc3 ii either in the presence or absence of fgf2, treating the cells with fgf2 still significantly reduced the abundance of the lc3 ii isoform, indicating less lc3 ii formation (fig . 4b). Therefore, the results indicate fgf2 suppresses lc3 i to lc3 ii conversion, and thus, the autophagy initiation in cells . To determine whether the mtor pathway was required for fgf2 to suppress autophagy, rapamycin, an mtor c1 inhibitor, was added to the mef cultures . Treating with rapamycin suppressed the phosphorylation of s6k1 and increased the abundance of the lc3 ii in fgf2 treated mefs (fig . 4c), although it did not affect akt and erk1/2 phosphorylation (data not shown). Separate experiments revealed that treating with rapamycin alone did not increase lc3 ii abundance in mefs (fig . The results suggest that activation of mtor c1 is required for fgf2 to suppress the autophagy activity . Further experiments with other autophagy markers, including expression of p62, atg5, atg12, etc . Reinstatement of full length frs2 expression in frs2 null mefs by transfection restored regulation of fgf2 on the autophagy activity (fig . 5). To determine whether the grb2- or shp2-binding sites were required for regulating autophagy, mutants lacking the four grb2- or two shp2-binding sites were expressed in frs2 null mefs as described 15 . To better demonstrate the difference in lc3 ii abundance, only lightly exposed films were shown . Therefore, lc3 i bands in this figure are too weak to be seen . Consistent with the chemical inhibition experiments demonstrating that the pi3k / akt, but not erk, pathway was required for fgf2 to suppress autophagy, expression of the frs2 mutant lacking the grb2-binding sites (y196, y306, y349, and y392) failed to restore the activity, whereas expression of frs2 lacking the shp2 binding sites (y436 and y471) restored the response to fgf2 with respect to autophagy inhibition . The results again demonstrate that the fgf signaling pathway regulates autophagy through the grb2-mediated pi3k / akt pathway . There are four grb2-binding sites on frs2. To further investigate which grb2-binding phosphorylation sites were required for frs2 to mediate the autophagy regulation signals, mutant frs2 carrying an individual grb2-binding site mutation were expressed in frs2 null mefs . Expression of mutants with a substitution of either y196 or y306 with phenylalanine rescued the fgf activity to inhibit autophagy activity, whereas expression of the mutant with a substitution of either y349 or y392 with phenylalanine failed to rescue such defects (fig . The results indicate that y349 and y392, but not y196 and y306, are required for the fgf to suppress autophagy, and imply that each grb2-binding site may mediate a subset of fgf signals . Activation of pi3-kinase is mediated by assembly of gab1 to the grb2/frs2 complex, which enables tyrosine phosphorylation of gab1 by fgfr to generate binding sites for p85, the regulatory subunit of pi3-kinase, thus resulting in recruitment and activation of the pi3-kinase 38 . Although all four grb2-binding sites are involved in activation of the pi3k / akt pathway in 3t3 cells 2, only two grb2-binding sites are involved in autophagy regulation in mefs . The molecular mechanism underlying the results suggest that the autophagy - suppressing signals of the fgf signaling axis are different from the fire - activating signals that are mediated by the four grb2-binding sites additively, since substitution of each grb2-binding site only partially reduces fire activation activity 15 . The fire consists of a 170-bp array of five dna motifs that bind two fgf - inducible fos - jun heterodimers, one inducible ap-2-related protein, one constitutively expressed upstream stimulatory factor, and one constitutive 46-kda transcription factor . It has been shown that fire is selectively activated by the fgf, but not by other tyrosine kinase receptor - activating growth factors 26 . Furthermore, the fgf activates fire independent of its mitogenic activity in prostate cancer cells 24 . Here we showed that the signals of fgf to regulate the mtor / autophagy pathway was different from those for fire activation, and further demonstrate mechanistic diversity of the fgf signaling axis . Although widely used, lc3 lipidation is not a perfect marker for autophagy 39 . Furthermore, it has been reported that the lc3 antibody exhibits different detection sensitivities toward lc3 i and lc3 ii 40 . Therefore, future efforts to monitor expression of other autophagic markers, such as p62, atg5, and atg12, etc ., will be needed to confirm the findings and to elucidate the molecular mechanism underlying how fgf signaling regulates autophagic activity . Autophagy is a major cellular pathway to degrade bulky subcellular organelles and macromolecules, and plays important roles in development, metabolism, tumorigenesis, and diseases 41 . However, how autophagy contributes to development is not understood, although it has been proposed that autophagy may influence cell differentiation either by impairing new protein or organelle formation or by accelerating turnover of old proteins or organelles . In this study, we provided the first in vitro evidence that autophagy can be negatively regulated by the fgf signaling axis . Recently, we also discovered that the fgf regulates cardiac progenitor cell and cardiomyocyte differentiation via controlling the mtor pathway - regulated autophagy, and that the frs2-mediated pathways are required for the fgf to suppress premature differentiation of heart progenitor cells . Inhibition of autophagic activity suppresses cardiomyocyte differentiation both in the second heart field progenitors and in embryoid body (eb) cultures (42). In summary, the fgf signaling axis activated the mtor kinase and repressed autophagy by activating the frs2-mediated pi3k / akt pathway . Suppression of mtor activation abolished the repression activity of fgf2 on autophagy, indicating that fgf regulated autophagy via the mtor pathway . This is the first report that the fgf signaling axis plays a crucial role in autophagy regulation, thus, sheding new light on cell signaling mechanisms.
We have performed 350 spk transplants over the past 18 years in type 1 diabetic patients with end - stage renal disease . Type 1 diabetes diagnosis is routinely verified by lack of detectable c - peptide after a sustacal challenge . Pancreata and kidneys from the tested donors were transplanted into type 1 diabetic patients between 1998 and 2005 . We measured aab to glutamic acid decarboxylase (gad - aab), the tyrosine - phosphatase - like protein ia2 (ia2-aab), and insulin (insulin - aab) using standard radioimmunoassays . Aab levels are expressed as index levels calculated from the counts per minute of the test sample and the positive and negative control samples . Receiver operating curves identified assay cutoffs of 11.44, 3.72, and 6.85 for the gad, ia2, and insulin aab assays, respectively . Our laboratory participated in the diabetes autoantibody standardization program of the immunology of diabetes society and centers for disease control in 2000, 2002, 2003, and 2005 (8). The institutional review board of the university of miami school of medicine approved the study . Four of 135 (2.96%) donors were aab positive: three donors had gad - aab, and one donor had insulin - aab . Donors with gad - aab had low aab levels . Tables 1 and 2 show the characteristics of the aab - positive donors and corresponding recipients . Two donors with gad - aab were homozygous for the hla - dr4 or -dr3 susceptibility alleles; the remaining gad - aab positive donor carried a presumably protective hla - dr2 . Our spk recipients had a mean sd follow - up of 5 2.1 years . All patients transplanted with a pancreas from a single aab - positive donor became insulin independent; three - fourths of the patients transplanted with a pancreas from an aab - positive donor had normal, insulin - producing grafts 35.8 years after transplant (table 2). The recipient of the pancreas from gad - aab positive donor 1 had a pancreas transplant biopsy 3.2 years after transplantation showing no -cell loss, insulitis, or other abnormalities . This recipient had elevated gad - aab levels preceding the transplant that persisted essentially unchanged during follow - up . The recipient of the pancreas from insulin - aab positive donor 4 developed chronic rejection following discontinuation of immunosuppression 3.3 years after transplant . At that time, the patient returned to insulin dependency despite maintaining residual c - peptide secretion for up to 2.2 years after developing chronic rejection . The patient's last c - peptide level was 2.3 ng / ml . Loss of graft function did not differ among recipients of aab - positive and aab - negative donors (1 of 4 vs. 12 of 131; p = 0.33). There is interest in screening pancreas donors for autoantibodies to identify pre - diabetic donor pancreata that may not be suitable for transplantation and could be made available for research (5). The juvenile diabetes research foundation is supporting large - scale screening to identify aab - positive pancreas donors for research (www.jdrfnpod.org). A recent analysis of pancreas donors aged 2560 years from the general population showed that single aab positivity is not commonly associated with insulitis and -cell loss, via analyzing 0.5 cm bioptic fragments of pancreata that were used for islet cell iso - lation (6). Insulitis was found in only two donors who were positive for 3 aab and not in 59 donors positive for 12 aab . We identified four donors with a single aab, consistent with the reported frequency in organ donors (5). Our data include subjects younger than those in previous studies (5,6): of our donors, 55% were aged <25 years, an age - group with higher type 1 diabetes incidence . Our analysis is unique in providing transplant outcome data from patients who received a pancreas from a single aab - positive donor . All patients became insulin independent on follow - up . In a patient who continued to be euglycemic, a biopsy performed 3 years after transplantation did not evidence islet damage . The recipient of the insulin - aab positive donor pancreas lost transplant function due to chronic rejection related to noncompliance . Overall, our outcome data are consistent with biopsy data from previous studies showing that single aab positivity may not always be associated with clinically significant autoimmunity and -cell damage in organ donors (5,6). The findings are consistent with the low diabetes risk associated with single aab positivity in the general population (9,10). Relevant to clinical pancreas transplantation, our data suggest that single autoantibody positivity is unlikely to affect pancreas transplant outcome and may help to refine strategies for ongoing pancreas donor aab - screening initiatives, of which we remain strong supporters . Limited access to human pancreata with ongoing autoimmunity remains a major obstacle to the advancement of our understanding of human type 1 diabetes.
Cancer of the exocrine pancreas represents the fifth leading cause of cancer death in the western population with a five - year survival rate under 5% . Because of the few treatment options, understanding of the molecular pathology is prerequisite to identify potential molecular targets for drug therapy . The panin (pancreatic intraepithelial neoplasia) classification describes various changes in the pancreatic duct system distinguishing three panin grades (panin 1 panin 3) according to the degree of structural dysplasia and cytological atypia present in the lesions . Microdissection techniques revealed genetic alteration in cancer - causing genes in the putative premalignant lesions similar to pancreatic carcinomas (see table 1). The combination of morphological and genetic observations leads to a tumor progression model for pancreatic carcinoma, comparable to the adenoma - carcinoma sequence in colorectal carcinomas . The sequential acquisition of mutations in the proto - oncogene k - ras and the tumor suppressors ink4a, tp53 and dpc4/smad4 leads to a profound disturbance in cell cycle regulation, a hallmark of pancreatic cancer . Mutations in k - ras, ink4a, tp53 and dpc4/smad4 are frequent, whereas mutations in the tumor suppressor brca2, mismatch repair genes and the serine - threonine kinases akt2 and lkb1/stk11 are rare genetic events . Table 1 summarizes the reported frequencies of major genetic alterations in the pancreatic tumor progression model . Pancreatic cancer has the highest incidence of ras mutations in human tumors identified to date . The mutations of the k - ras gene, h - ras and n - ras are not affected, are generally found in codon 12 . Dependent on the used technique the frequencies of codon 12 mutations reported range from 20 to 100% and occur early in the tumor progression model . The ras family proteins encode small gtp - binding cytoplasmic proteins that mediate pleiotropic effects including cell proliferation, survival and migration . Mutation of codon 12 in k - ras results in a gain of function, because the ras protein remains trapped in the activated state . In contrast primary murine fibroblasts can be efficiently transformed by mutated ras in concert with a second oncogene or loss of a tumor suppressor, like p53 or p16 . The sole expression of oncogenic ras in primary rodent and human cells results in a permanent g1 arrest accompanied by accumulation of p53, p16and p21 . This senescence is thought to be a defense mechanism against oncogenic stress . Whether the observed overexpression of p21, whose frequency parallels that of k - ras mutation in the pancreatic tumor progression model, is part of this defense mechanism or directly linked to the cell cycle by working as an assembly factor for the cyclin d1/cdk4 complex, is not known . Placing p21 in a defence program is speculative but attractive, because it could explain in part the observation that oncogenic k - ras mutations are not specific for malignancy, being present in benign diseases of the pancreas and in early clonal lesions . Moreover, the risk of progression to malignancy is low in the absence of co - operating genetic events [11 - 14]. Despite the high mutation frequency in human pancreatic carcinoma, mice which harbor a latent allele of k - ras g12d capable of spontaneous activation in vivo, develop multiple early onset lung tumors but not pancreatic cancer, further demonstrating the species differences of ras function . The complexity of ras function is amplified through recent data suggesting tumor suppressor properties of ras . Transfection of wildtype ras into rat fibroblasts inhibits anchorage - independent growth and colony formation, induced by the oncogenic ras gene . Loss of the wildtype k - ras allele was observed in some pancreatic carcinoma cell lines with mutation in k - ras (aspc1, capan1 and miapaca) and there was underexpression of the mutanted allele in comparison to the wildtype allele in two other cell lines (su8686 and panc1). A further species difference affects the signaling pathway utilized by oncogenic ras . Whereas in rodent cells the raf / mapk and the pi3k are thought to mediate many effects of oncogenic ras, there are hints that in human cells the guanine nucleotide exchange factor ral is sufficient for ras transformation . Therefore, the net outcome of ras activation in a specific setting is not easy to predict and further studies, including primary cultures of epithelial pancreas cells, are needed . Homozygous deletion of p16/p14locus is a characteristic genetic alteration observed in 80% 95% of human pancreatic cancer and usually occurs in later stages of the tumor progression model [19 - 21]. This locus on chromosom 9q21 encodes the two related tumor suppressor genes ink4a and arf, who's coding sequence partially overlap . Are generated by the use of a different first exon and an alternative reading frame in exon 2 . Whereas ink4a regulates cell cycle progression as an inhibitor of the cyclin d / cdk 4/6 kinase complex, arf directly interacts with mdm 2/hdm 2 to block the interaction with p53 by localizing mdm 2/hdm 2 to the nucleolus and by inhibiting directly mdm 2/hdm 2's e3 ubiquitin ligase activity, contributing to p53 activation (figure 1). Gene deficient mice for p19, the mouse homologue of human p14, strongly suggest that arf is the major tumor suppressor in mice . The specific mutation of the ink4a gene in mice also etablishes p16as a tumor suppressor in mice . Germline mutations in the exon 1 of ink4a are associated with the familial atypical mole - malignant melanoma syndrome, implicating ink4a in human tumor susceptibility . This mutation also predispose to pancreatic cancer, but in contrast to very high penetrance and early onset of melanoma, the penetrance of pancreatic cancer is very low and displays latency similar to the sporadic disease [28 - 30]. In humans ink4a seems to be the more important tumor suppressor for pancreatic cancer development, because germline and sporadic mutations have been identified that target ink4a, but omit arf . Ink4a is inactivated by homozygous deletions and intragenic mutation and in the remaining cases the ink4a gene is turned off through promoter methylation . The ink4a / arf locus . The two products of the ink4a / arf locus encodes for p16and p14(p19 in mice). The tp53 tumor suppressor gene is mutated, especially by missense mutations in sequences coding for the dna binding domain, in greater than 50% of pancreatic adenocarcinomas . The mutations are often accompanied by loss of the wildtyp allele and occur late in the progression model [21,35 - 39]. The transcription factor p53 regulates an essential growth checkpoint that both protects against genomic rearrangement or the accumulation of mutations, and suppresses cellular transformation caused by oncogene activation or the loss of tumor suppressor pathways . P53 is stabilized and activated by extracellular stress including irradiation and intracellular stress such as deregulation of cellular oncogenes . Loss of p53 is associated with aneuploidy, an outstanding feature of pancreatic cancer, indicating that p53 function maintains genomic stability . Germline mutations in tp53 have been described as li - fraumeni syndrome, predisposing to several neoplasms, but pancreatic carcinomas are rare findings . Smad4, which was initially named dpc4, deleted in pancreatic carcinoma, was originally identified as a candidate tumor suppressor that is frequently inactivated in pancreatic tumors . The transcription factor smad4 is an important regulator of the transforming growth factor (tgf-) signaling pathway . Upon receptor activation smad proteins get phosphorylated and heterodimerize with smad4 to transmit upstream signals to the nucleus and transactivate transcription of specific target genes . The smad4 gene is deleted or mutated in over 50% of pancreatic carcinoma, an event occurring late in the tumor progression model . The most prominent biological activity of tgf- is its potent inhibition of cell growth, mediated by a cell cycle g1 arrest, in a wide variety of cells . It is assumed that the growth - inhibitory function of tgf- is important for smad4 tumor suppressor activity . Restoration of smad4 in human pancreatic carcinoma cells suppressed tumor formation in vivo and did not restore tgf- sensitivity . Furthermore a decrease in pro - angiogenic vegf expression and an increase in the angiogenesis inhibitor tsp-1 was observed, so that regulation of an angiogenic switch might contribute to the tumor suppressor function of smad4 . Epigenetic inactivation of the tgf-/smad4 pathway occurs in the presence of activated ras, whereby explaining the reduced selection pressure for loh at the smad4 locus . Brca2 on chromosome 13q encodes for a protein that is needed for the maintenance of genomic stability by regulating dna repair processes . Normal cells, deficient for brca2, accumulate lethal chromosomal aberrations . Inherited brca2 mutation predispose to early onset familiar breast and ovarian cancer . Albeit with lower penetrance and equal age of onset, inherited brca2 mutations also increase the risk for pancreatic cancer . In sporadic pancreatic cancer brca2 is inactivated in 7 to 10% and lately the biallelic inactivation in a high - grade duct lesion was demonstrated, so that it is assumed that brca2 mutation occur late in the neoplastic progression in the pancreas . The autosomal dominant inherited peutz - jeghers syndrome, caused by mutation of the serine - threonine kinase lkb1/stk11 that maps to chromosom 19p13, is associated with an increased incidence of pancreatic carcinoma [57 - 60]. The signaling pathway of lkb1/stk1 is so far unknown, but this gene was shown to be inactivated in 5% of sporadic pancreatic cancer, suggesting a possible role in tumor suppression . The serine - treonine kinase akt2 is a candidate oncogene for human pancreatic cancer and was found to be amplified and overexpressed in pancreatic adenocarcinoma and cell lines in up to 20% [63 - 65]. Akt2 is a downstream effector of the pi3 kinase and can be activated by epidermal growth factor, platelet - derived growth factor and basic fibroblast growth factor, all known to be overexpressed in pancreatic carcinoma . Recently akt signaling was linked to enforced insulin - like growth factor i receptor expression, promoting invasiveness of pancreatic cancer cells . Autosomal dominant inherited lynch syndrome is characterized by an increased risk of developing colorectal, endometrial, ovarian and breast cancers, transitional carcinoma of the ureter and renal pelvis . Mutations in the dna mismatch repair genes, including hmlh1, hmsh2 and hmsh6, cause this syndrome . Pancreatic cancer is included in the tumor spectrum, however, it seems to be a rare finding . Pancreatic cancers occurring in context of the lynch syndrome are different compared to sporadic pancreatic carcinomas in terms of the superior clinical course, histopathology and distinct molecular genetic profiles, including retention of wildtyp ras [72 - 74]. Furthermore, microsattellite instability is unlikely to participate in the oncogenesis of spontaneous pancreatic cancer [75 - 77]. Molecular investigations of pancreatic cancer are complicated by the restricted accessibility of the organ for biopsies . The findings in molecular research on pancreatic carcinoma of the last years is now integrated in a pancreatic tumor progression model, with genetically, epigenetically and morphological defined precursor lesions . Pancreatic cancer is a genetic disease, but the transition between cancer and non - cancer is not regulated by a simple switch activated by a single gene . In fact, multiple mutations must accumulate in a single cell, including overexpression of receptor - ligand systems, oncogene activation and loss of tumor suppressor genes, to develop pancreatic carcinoma . Deregulated cell cycle is the hallmark of many human tumors, including pancreatic carcinoma, and therefore the cell cycle could be placed into the center of pancreatic oncogenesis . As figure 2 illustrates, each of the major genetic alteration mentioned above is involved in cell cycle regulation and leads together to the acceleration of the cell cycle progression and continuous growth . All four major genetic alteration, k - ras, ink4a, tp53 and the tgf-/smad4 tumor suppressor pathway, observed in pancreatic carcinoma, regulate directly or indirectly g1 progression, leading to e2f dependent s phase entry . Further analysis of the underlying molecular mechanism will offer new diagnostic and therapeutic options and, hopefully improve the outcome of this dismal disease in the future.
This disorder is characterized by symmetrical mutilating palmoplantar keratoderma along with periorificial hyperkeratosis (mouth, nose, eyes, genital, anal). One of our patients (case 1), had woolly hair and flexion contracture of a digit, in addition to usual features, while the other had pseudoainhum . A 13-year - old girl presented to our department with curly hair from birth and thickened skin with painful fissures over palms and soles since 5 years of age [figures 1 and 2]. She first developed fissuring over soles at the age of 5 years and later over the palms . Subsequently, warty skin lesions appeared over elbows, knees, dorsa of hands and buttocks . From birth, diffuse keratoderma with blackening and foul smell was noticed over her soles . In palms, along with keratoderma, flexion contracture of right little finger was noticed . She had keratotic papules over dorsa of hands, extensor aspect of knees, elbows and buttocks . She also had perioral erythema, hyperkeratosis and angular chelitis, in addition to perianal hyperkeratosis . Skin biopsy from a palmar lesion revealed massive hyperkeratosis, acanthosis and inflammatory infiltrate in the dermis . Case 1 showing woolly hair, keratoderma of palms and flexion contracture of right little finger shows woolly hair, scanty eyebrows and perioral erythema and scaling a 10-year - old girl presented with thick palms and soles since few years of birth . She had difficulty in walking and grasping objects because of thick and fissured soles and palms . Her parents and siblings were normal . On examination, she had sharply marginated, yellowish, hyperkeratotic, perioral plaque, and transgredient, diffuse palmoplantar keratoderma [figures 3 and 4]. Histopathology of a keratotic papule showed acanthosis, suprabasal cleft and inflammatory infiltrate in dermis . Case 2 showing perioral hyperkeratosis keratoderma extending over dorsal aspect of hands with keratotic papules and constriction of right little finger a 13-year - old girl presented to our department with curly hair from birth and thickened skin with painful fissures over palms and soles since 5 years of age [figures 1 and 2]. She first developed fissuring over soles at the age of 5 years and later over the palms . Subsequently, warty skin lesions appeared over elbows, knees, dorsa of hands and buttocks . From birth, diffuse keratoderma with blackening and foul smell was noticed over her soles . In palms, along with keratoderma, flexion contracture of right little finger was noticed . She had keratotic papules over dorsa of hands, extensor aspect of knees, elbows and buttocks . She also had perioral erythema, hyperkeratosis and angular chelitis, in addition to perianal hyperkeratosis . Skin biopsy from a palmar lesion revealed massive hyperkeratosis, acanthosis and inflammatory infiltrate in the dermis . Case 1 showing woolly hair, keratoderma of palms and flexion contracture of right little finger shows woolly hair, scanty eyebrows and perioral erythema and scaling a 10-year - old girl presented with thick palms and soles since few years of birth . She had difficulty in walking and grasping objects because of thick and fissured soles and palms . Her parents and siblings were normal . On examination, she had sharply marginated, yellowish, hyperkeratotic, perioral plaque, and transgredient, diffuse palmoplantar keratoderma [figures 3 and 4]. Histopathology of a keratotic papule showed acanthosis, suprabasal cleft and inflammatory infiltrate in dermis . Case 2 showing perioral hyperkeratosis keratoderma extending over dorsal aspect of hands with keratotic papules and constriction of right little finger a 13-year - old girl presented to our department with curly hair from birth and thickened skin with painful fissures over palms and soles since 5 years of age [figures 1 and 2]. She first developed fissuring over soles at the age of 5 years and later over the palms . Subsequently, warty skin lesions appeared over elbows, knees, dorsa of hands and buttocks . From birth, diffuse keratoderma with blackening and foul smell was noticed over her soles . In palms, along with keratoderma, flexion contracture of right little finger was noticed . She had keratotic papules over dorsa of hands, extensor aspect of knees, elbows and buttocks . She also had perioral erythema, hyperkeratosis and angular chelitis, in addition to perianal hyperkeratosis . Skin biopsy from a palmar lesion revealed massive hyperkeratosis, acanthosis and inflammatory infiltrate in the dermis . Case 1 showing woolly hair, keratoderma of palms and flexion contracture of right little finger shows woolly hair, scanty eyebrows and perioral erythema and scaling a 10-year - old girl presented with thick palms and soles since few years of birth . She had difficulty in walking and grasping objects because of thick and fissured soles and palms . Her parents and siblings were normal . On examination, she had sharply marginated, yellowish, hyperkeratotic, perioral plaque, and transgredient, diffuse palmoplantar keratoderma [figures 3 and 4]. Histopathology of a keratotic papule showed acanthosis, suprabasal cleft and inflammatory infiltrate in dermis . Case 2 showing perioral hyperkeratosis keratoderma extending over dorsal aspect of hands with keratotic papules and constriction of right little finger olmsted syndrome was first described by olmsted in the year 1927 in a 5-year - old boy with palmoplantar keratoderma and periorificial keratosis . Most cases reported to date have been sporadic apart from few patients who had affected family members . Although both our cases were females, according to literature, males are predominantly affected . Keratotic papules around orifices develop later and similar papules and plaques may appear over neck, axilla, cubital fossa, inguinal region and gluteal region . In addition, these patients may exhibit universal alopecia, exaggerated keratosis pilaris, palmoplantar hyperhidrosis, oral leukokeratosis, corneal dystrophy, tooth anomaly, nail dystrophy, chronic paronychia, suppurative dactrocystitis, joint laxity, deaf mutism, primary sclerosing cholangitis, short stature and hemangioma . In some patients, the condition slowly progresses, leading to mutilating flexion contractures of digits and autoamputation . In longstanding cases, progression to squamous cell carcinoma and malignant melanoma has been reported . The differential diagnosis in the early stage of periorificial erythema could be acrodermatitis enteropathica . In cases of palmoplantar keratoderma, vohwinkle keratoderma and mal de meleda form the differential diagnoses . Since one of our patients (case 1) had woolly hair and palmoplantar keratoderma, initially naxo's syndrome was thought of . The other differential diagnoses that were thought of include erythrokeratoderma periorificialis type, a condition in which affected patient has periorificial and acral keratoderma . As our patient had symmetrical, diffuse, transgredient palmoplantar keratoderma, periorificial keratosis, keratotic papules and plaques over extensor aspect of elbows, knees, gluteal region and dorsa of hands, along with woolly hair, a diagnosis of olmsted syndrome was made . Histopathology of the plaque reveals massive acanthosis, parakeratosis, papillomatosis and slight superficial perivascular infiltrate . Histochemical study done in a patient showed more basal and suprabasal keratinocytes of epidermis with immunoreactivity for k167 marker . Electron microscopic finding of a nucleated cell having keratohyaline granules within the stratum corneum has also been reported . All these findings support the notion that olmsted syndrome is a hyperproliferative disorder of epidermis . Treatment options include topical emollients, topical calcipotriol, keratolytics like salicylic acid and urea . Application of hydrocolloid dressing may alleviate pain in cases of fissures over palms and soles . Debulking surgery followed by skin grafting has been tried in some patients with flexion contractures but recurrence has been noted . Our patients were started on systemic retinoids with mild improvement of keratoderma and significant improvement of periorificial keratotic plaques after 3 months of therapy.
The present analysis was prespecified within the simvastatin ezetimibe in aortic stenosis (seas) study analysis plan . Study design, baseline characteristics, and main outcome results of the seas study have previously been published . In short, 1873 men and women aged 45 to 85 years with asymptomatic mild - to - moderate as having a peak aortic jet velocity between 2.5 and 4.0 m / s by echocardiography were randomized to placebo or to combination treatment with simvastatin 40 mg and ezetimibe 10 mg daily . Patients with known coronary heart disease, heart failure, diabetes mellitus, history of stroke or peripheral vascular disease, clinically significant mitral valve disease, severe or predominant aortic regurgitation, rheumatic valvular disease, aortic valve prosthesis, or renal insufficiency and patients already on lipid - lowering therapy or with a guideline indication for lipid - lowering therapy were not included in the seas study . Core laboratory readings of peak aortic jet velocity and lv mass were available from baseline and at least 1 follow - up echocardiogram in 1656 patients (88% of the study population), who comprise the present study population . Hypertension was defined as history of hypertension, use of antihypertensive drug treatment, or elevated blood pressure at the baseline clinic visits . All patients gave written informed consent, and the study was approved by ethics committees in all participating countries . Echocardiograms were performed at baseline, annually and before planned aortic valve surgery following a standardized protocol at 173 study centers in 7 european countries . Echocardiographic images were stored on videotapes, compact discs, or magnetic optical discs and forwarded for blinded interpretation at the seas echocardiography core laboratory at haukeland university hospital, bergen, norway, as previously published . Lv mass was measured by an autopsy - validated method and indexed to body height in the allometric power of 2.7 . Lv hypertrophy was defined using the prognostically validated cutoff values lv mass index> 46.7 g / m in women and relative wall thickness was assessed from 2lv posterior wall thickness / lv end - diastolic diameter ratio and considered increased if> 0.43 (concentric lv geometry). Pressure recovery was assessed at the aortic sinotubular junction and used for calculation of energy loss index as prognostically validated . The prespecified primary end point of seas was major cardiovascular events, a composite end point, including aortic valve related events (combined aortic valve replacement, hospitalization for heart failure because of aortic stenosis, and death from cardiovascular causes) and ischemic cardiovascular events (combined death from cardiovascular causes, nonfatal myocardial infarction, hospitalization for unstable angina, coronary revascularization, and nonhemorrhagic stroke). All end points were classified by an independent end point classification committee whose members were unaware of study group assignments . We also assessed the post hoc defined composite end point of total mortality and hospitalization for heart failure because of aortic stenosis . Statistical analysis was performed using ibm spss version 22.0 (ibm corporation, armonk, ny). Comparison between groups was performed by paired and unpaired t test, test, and general linear model with post hoc test and bonferroni adjustment as appropriate . Cumulative incidences of cardiovascular events during follow - up were estimated by kaplan meier . Meier plots were used to compare event - free survival in groups of patients with and without lv hypertrophy at baseline . Correlates of the prespecified primary and secondary composite end points were identified by cox regression analysis in univariable and multivariable models and presented as hazard ratio and 95% confidence intervals . In the primary analyses, lv mass index was used as the continuous variable . In secondary models, lv hypertrophy as a dichotomous variable was used . Age, sex, body mass index, peak aortic jet velocity, lv ejection fraction, concentric lv geometry, hypertension, and valvuloarterial impedance were included as covariates in all multivariable models . Aortic valve replacement was included as a time - varying covariate in models assessing cardiovascular death and total mortality . In subsequent models, concentric lv geometry was replaced by stress - corrected midwall shortening, and the presence of aortic regurgitation was added . To take the progressive increase in lv mass during progression of as into account, time - varying cox regression analysis was used . Two - tailed p<0.05 was regarded as statistically significant both in univariable and multivariable analyses . The present analysis was prespecified within the simvastatin ezetimibe in aortic stenosis (seas) study analysis plan . Study design, baseline characteristics, and main outcome results of the seas study have previously been published . In short, 1873 men and women aged 45 to 85 years with asymptomatic mild - to - moderate as having a peak aortic jet velocity between 2.5 and 4.0 m / s by echocardiography were randomized to placebo or to combination treatment with simvastatin 40 mg and ezetimibe 10 mg daily . Patients with known coronary heart disease, heart failure, diabetes mellitus, history of stroke or peripheral vascular disease, clinically significant mitral valve disease, severe or predominant aortic regurgitation, rheumatic valvular disease, aortic valve prosthesis, or renal insufficiency and patients already on lipid - lowering therapy or with a guideline indication for lipid - lowering therapy were not included in the seas study . Core laboratory readings of peak aortic jet velocity and lv mass were available from baseline and at least 1 follow - up echocardiogram in 1656 patients (88% of the study population), who comprise the present study population . Hypertension was defined as history of hypertension, use of antihypertensive drug treatment, or elevated blood pressure at the baseline clinic visits . All patients gave written informed consent, and the study was approved by ethics committees in all participating countries . Echocardiograms were performed at baseline, annually and before planned aortic valve surgery following a standardized protocol at 173 study centers in 7 european countries . Echocardiographic images were stored on videotapes, compact discs, or magnetic optical discs and forwarded for blinded interpretation at the seas echocardiography core laboratory at haukeland university hospital, bergen, norway, as previously published . Lv mass was measured by an autopsy - validated method and indexed to body height in the allometric power of 2.7 . Relative wall thickness was assessed from 2lv posterior wall thickness / lv end - diastolic diameter ratio and considered increased if> 0.43 (concentric lv geometry). Pressure recovery was assessed at the aortic sinotubular junction and used for calculation of energy loss index as prognostically validated . The prespecified primary end point of seas was major cardiovascular events, a composite end point, including aortic valve related events (combined aortic valve replacement, hospitalization for heart failure because of aortic stenosis, and death from cardiovascular causes) and ischemic cardiovascular events (combined death from cardiovascular causes, nonfatal myocardial infarction, hospitalization for unstable angina, coronary revascularization, and nonhemorrhagic stroke). All end points were classified by an independent end point classification committee whose members were unaware of study group assignments . We also assessed the post hoc defined composite end point of total mortality and hospitalization for heart failure because of aortic stenosis . Statistical analysis was performed using ibm spss version 22.0 (ibm corporation, armonk, ny). Comparison between groups was performed by paired and unpaired t test, test, and general linear model with post hoc test and bonferroni adjustment as appropriate . Cumulative incidences of cardiovascular events during follow - up were estimated by kaplan meier . Meier plots were used to compare event - free survival in groups of patients with and without lv hypertrophy at baseline . Correlates of the prespecified primary and secondary composite end points were identified by cox regression analysis in univariable and multivariable models and presented as hazard ratio and 95% confidence intervals . In the primary analyses, lv mass index was used as the continuous variable . In secondary models, lv hypertrophy as a dichotomous variable was used . Age, sex, body mass index, peak aortic jet velocity, lv ejection fraction, concentric lv geometry, hypertension, and valvuloarterial impedance were included as covariates in all multivariable models . Aortic valve replacement was included as a time - varying covariate in models assessing cardiovascular death and total mortality . In subsequent models, concentric lv geometry was replaced by stress - corrected midwall shortening, and the presence of aortic regurgitation was added . To take the progressive increase in lv mass during progression of as into account, time - varying cox regression analysis was used . Two - tailed p<0.05 was regarded as statistically significant both in univariable and multivariable analyses . Compared with patients with normal lv mass index at baseline, the group with lv hypertrophy was older, had higher body mass index, lower lv midwall function, and included more patients with hypertension (all p<0.01; tables 1 and 2). During a median of 4.3-year follow - up, lv mass indexed to height (lv mass index) and concentricity increased, whereas lv endocardial and myocardial function declined (all p<0.001). The prevalence of lv hypertrophy increased from 36% at baseline to 60% at the last study visit (p<0.01). The annual as progression rate did not differ between groups of patients with and without lv hypertrophy at baseline, whether calculated based on change in peak aortic jet velocity (0.210.39 versus 0.200.27 m / s per year), mean gradient (47 versus 45 mm hg / y), or aortic valve area (0.030.25 versus 0.030.29 cm / y, all p>0.3). The average time between the baseline and the last follow - up study was 3.61.2 years . The average time between the follow - up study and an aortic valve event, an ischemic cardiovascular event, and death from any cause was on average 0.590.03, 0.940.06, and 0.800.55 years, respectively . Clinical patient characteristics in the total population and in patients with or without lv hypertrophy at baseline echocardiographic findings in the total study population and in patients with or without lv hypertrophy at baseline during follow - up, each sd higher unindexed lv mass, lv mass / height, and lv mass / body surface area was associated with comparable 21%, 23%, and 25% higher rates of the primary study end point, respectively, in univariable analyses (all p<0.001). The rates of aortic valve events, ischemic cardiovascular events, cardiovascular death, and combined death from any cause and hospitalization for heart failure because of progression of as all increased progressively with increasing quartile of baseline lv mass index and were 1.5, 1.8, 3.2, and 2.5 times higher in the upper lv mass index quartile than in the lowest quartile (figure 1). In multivariable cox regression, higher lv mass index was associated with higher rates of aortic valve events, ischemic cardiovascular events, cardiovascular death, and combined death from any cause and hospitalization for heart failure when adjusted for known prognosticators in as patients like age, sex, body mass index, as severity, lv ejection fraction, concentric lv geometry, and concomitant hypertension (table 3). Similar results were found in a second model, replacing concentric geometry by stress - corrected midwall shortening (hazard ratio, 1.13 for primary end point per 1 sd higher lv mass index [95% confidence interval, 1.021.24]; p=0.017). Adding the presence of aortic regurgitation or type of antihypertensive drug among the covariates did not influence results . Impact of baseline left ventricular mass index (per 1 sd [15 g / m2.7] higher) on the rates of the primary and secondary study end points, hospitalization for heart failure, cardiovascular death, all - cause death, and combined all - cause death and hospitalization for heart failure during> 4.3 years of follow - up in patients with initially asymptomatic as cumulative incidences of aortic valve events (ave), ischemic cardiovascular (cv) events (ice), cv death (cvd), and combined death from any cause and hospitalization for heart failure because of progression of aortic stenosis (death&chf) during> 4.3 years of follow - up in relation to quartile of baseline left ventricular (lv) mass index in mild - to - moderate asymptomatic aortic stenosis . In a secondary set of models, having lv hypertrophy on the baseline echocardiogram was associated with higher rates of the primary and secondary composite study end points and combined all - cause death and hospitalization for heart failure, consistent with the outcome association demonstrated for lv mass index (table 4; figure 2). Impact of baseline left ventricular hypertrophy on the rates of the primary and secondary study end points, hospitalization for heart failure, cardiovascular death, all - cause death, and combined all - cause death and hospitalization for heart failure during> 4.3 years of follow - up in patients with initial asymptomatic as survival free from major cardiovascular (cv) events (a), aortic valve events (b), ischemic cv events (c), and combined death from any cause and hospitalization for heart failure because of progression of aortic stenosis (as; d) in groups of patients with () and without (-) left ventricular hypertrophy (lvh) on the baseline echocardiogram . In multivariable linear regression higher lv mass / height at the last study echocardiogram was associated with male sex (=0.06), and higher mean aortic gradient (=0.15), systolic blood pressure (=0.04), body mass index (=0.14), initial lv mass / height (=0.54), and presence of normal midwall shortening (=0.05, all p<0.05) at the baseline echocardiogram . To take into account the progressive increase in lv mass during progression of as, higher lv mass index during follow - up was associated with a 16% higher rate of the primary study end point, 13% higher rate of aortic valve events, 25% higher rate of ischemic cardiovascular events, 63% higher cardiovascular mortality, and 44% higher combined death from any cause and hospitalization for heart failure (all p<0.01; table 5). In subsequent models replacing energy loss index by peak aortic jet velocity, mean aortic valve gradient, or aortic valve area as measure of as severity, or lv ejection fraction by midwall shortening or stress - corrected midwall shortening, the results did not change (data not shown). Impact of in - study left ventricular mass index (per 1 sd [15 g / m2.7] higher) on the rates of study end points during> 4.3 years of follow - up in patients with initial asymptomatic aortic stenosis this is the first large prospective study to assess the prognostic impact of lv mass and hypertrophy assessed by echocardiography in patients with asymptomatic mild - to - moderate as without known coronary heart disease or diabetes mellitus . As demonstrated by our results, higher lv mass at baseline or during follow - up was associated with higher rates of both the primary and secondary prespecified composite study end points in the seas study, resulting in a considerably increased overall cardiovascular morbidity and mortality . These findings were also independent of documented prognosticators in asymptomatic as, including as severity, hypertension, body mass index, sex, lv ejection fraction, and concentric lv geometry . Of note, patients in the highest versus lowest quartile of lv mass index at baseline had a 13% higher 4.3-year cumulative incidence of aortic valve events and a 11% higher incidence of combined death from any cause and hospitalization for heart failure, corresponding to absolute differences of 3.0% and 2.6% per year, respectively . Traditionally, development of lv hypertrophy in as has been considered a physiological, compensatory process taking advantage of laplace s law to sustain normal systolic function during chronically elevated systolic stress . However, as recently demonstrated, concomitant hypertension, obesity, and the presence of the metabolic syndrome have been associated with increased lv mass in patients with asymptomatic nonsevere as, suggesting that development of lv hypertrophy is multifactorial also in patients with as . The relatively larger impact of hypertension on lv wall volume in mild - to - moderate as than of as itself has also been demonstrated in experimental simulation models by garcia et al . However, having increased lv mass on the baseline echocardiogram was associated with increased event rates in the present study independent of the prognostic impact of concomitant hypertension and increased body mass index previously demonstrated in asymptomatic mild - to - moderate as . Risk prediction in asymptomatic as remains a challenge, including identification of as patient with high risk for development of congestive heart failure, the most prognostically severe complication of as . Both american and european guidelines recommend aortic valve replacement in patients with severe as irrespective of symptoms if lv dysfunction defined as lv ejection fraction <50% is present . Population - based studies have demonstrated that increased lv mass was associated with incident heart failure independent of lv ejection fraction and independent of incident myocardial infarction . The present findings expands this knowledge by demonstrating that also in patients with mild - to - moderate as, increased lv mass index is associated with higher rate of combined death and heart failure independent of lv systolic function . Current european guidelines suggest excessive lv hypertrophy unless because of hypertension among indications for aortic valve replacement in as, as this has been associated with increased perioperative morbidity and mortality and may be less reversible after delayed surgery, precluding an optimal long - term prognosis . The present results from the large seas study document the association of increased lv mass with increased cardiovascular morbidity and mortality also in patients with asymptomatic mild - to - moderate as independent of the presence of concomitant hypertension . Our findings contrast with previous reports from smaller studies in patients with moderate to - severe as . Stewart et al following 183 patients with initially asymptomatic moderate or severe as for a median of 31 months found that neither lv mass nor tissue doppler measures of lv systolic and diastolic function predicted outcome independent of as severity . Similar findings were reported by monin et al in a study of 107 patients with moderate - to - severe as, who therefore did not include lv mass in the suggested risk assessment score for asymptomatic patients with moderate - to - severe as based on their findings . Electrocardiographic lv strain pattern was recently suggested as a strong correlate of mortality and hospitalization for heart failure by greve et al in a seas substudy . Of note, electrocardiographic strain pattern was not significantly associated with either cardiovascular death or all - cause mortality when mean aortic gradient was included as covariate in their multivariable models, in contrast to the strong independent association with echocardiographic lv mass and hypertrophy reported in the present article . However, shah et al documented that electrocardiographic strain pattern as a highly specific marker of midwall myocardial fibrosis, reflecting more advanced myocardial injury, lv decompensation, and impaired prognosis . Concentric lv geometric patterns have been demonstrated to carry individual risk of cardiovascular morbidity and mortality in hypertension . Furthermore, an association with reduced coronary flow reserve as a substrate for reduced myocardial function in hypertensive subjects with lv concentric geometry free from coronary artery disease has been reported by galderisi et al . In patients operated for as, both concentric lv geometry and excessive lv hypertrophy have been associated with higher postoperative mortality . Cioffi et al previously demonstrated that excessive lv hypertrophy was the strongest correlate of combined death, congestive heart failure, and nonfatal myocardial infarction in 218 patients with asymptomatic severe as . Of note, these findings were independent of patient age, extent of aortic valve calcification, renal dysfunction, or the presence of concomitant diabetes mellitus, all factors that have been associated with worsened prognosis in previous studies in asymptomatic severe as . The present results expand this knowledge by demonstrating the independent prognostic importance of higher lv mass in a large prospective study of patients with initially asymptomatic mild - to - moderate as . Patients with known coronary heart disease, heart failure, diabetes mellitus, history of stroke or peripheral vascular disease, other clinically significant valve disease, rheumatic valve disease, or renal insufficiency and patients with a guideline indication for lipid - lowering therapy were not included in the seas study . Thus, projection of study results to these patient groups should be done with caution . In the seas study, referral for aortic valve replacement was left to the decision of the attending cardiologist at the 173 participating centers, and the basis for referral of individual patients for surgery was not captured in the study database . We cannot exclude that presence of extreme lv hypertrophy may have influenced the decision to refer for surgery in individual cases . However, an independent impact of higher lv mass index was also found with the more objective end points cardiovascular and total mortality . Several studies have found speckle strain imaging, in particular 2-dimensional (2d) global longitudinal strain, useful for detecting asymptomatic as patients with more advanced lv injury despite normal lv ejection fraction . Lower global longitudinal strain in these patients has been associated with higher lv mass, concentric lv geometry, more severe as, concomitant hypertension, and with impaired prognosis . Recently, nagata et al reported the superior performance of 3d compared with 2d global longitudinal strain for risk prediction in such patients, also when adjusting for lv mass in multivariate analysis . However, speckle tracking echocardiography was not included in the large seas study, where the majority of echocardiogram were recorded on video tapes during the period 2002 to 2008 . Marchaux et al have demonstrated the usefulness of exercise stress echocardiography for risk stratification in asymptomatic patients with severe as, and current european guidelines include exercise testing for additional risk assessment in patients with asymptomatic severe as . However, exercise testing was not included in the large seas study, which was undertaken in 173 study centers during the years 2002 to 2008 . In patients with asymptomatic as, higher lv mass index patients with known coronary heart disease, heart failure, diabetes mellitus, history of stroke or peripheral vascular disease, other clinically significant valve disease, rheumatic valve disease, or renal insufficiency and patients with a guideline indication for lipid - lowering therapy were not included in the seas study . Thus, projection of study results to these patient groups should be done with caution . In the seas study, referral for aortic valve replacement was left to the decision of the attending cardiologist at the 173 participating centers, and the basis for referral of individual patients for surgery was not captured in the study database . We cannot exclude that presence of extreme lv hypertrophy may have influenced the decision to refer for surgery in individual cases . However, an independent impact of higher lv mass index was also found with the more objective end points cardiovascular and total mortality . Several studies have found speckle strain imaging, in particular 2-dimensional (2d) global longitudinal strain, useful for detecting asymptomatic as patients with more advanced lv injury despite normal lv ejection fraction . Lower global longitudinal strain in these patients has been associated with higher lv mass, concentric lv geometry, more severe as, concomitant hypertension, and with impaired prognosis . Recently, nagata et al reported the superior performance of 3d compared with 2d global longitudinal strain for risk prediction in such patients, also when adjusting for lv mass in multivariate analysis . However, speckle tracking echocardiography was not included in the large seas study, where the majority of echocardiogram were recorded on video tapes during the period 2002 to 2008 . Marchaux et al have demonstrated the usefulness of exercise stress echocardiography for risk stratification in asymptomatic patients with severe as, and current european guidelines include exercise testing for additional risk assessment in patients with asymptomatic severe as . However, exercise testing was not included in the large seas study, which was undertaken in 173 study centers during the years 2002 to 2008 . In patients with asymptomatic as, higher lv mass index is independently associated with increased cardiovascular morbidity and mortality during progression of valve stenosis . The simvastatin ezetimibe in aortic stenosis (seas) echocardiography core laboratory was supported by the msp singapore company, llc, singapore, a partnership between merck & co., inc . And the schering - plough corporation . Drs pedersen, rosseb, and gerdts were members of the scientific steering committee for the simvastatin ezetimibe in aortic stenosis (seas) study in the years 2002 to 2008 and received honoraria for this work . It is well known that the presence of left ventricular (lv) hypertrophy by echocardiography predicts increased cardiovascular morbidity and mortality both in general and hypertensive populations . In patients with aortic valve stenosis (as), lv hypertrophy has traditionally been considered as an adaptive response that keeps lv wall stress close to normal, offsetting the hemodynamic load . Recent publications have demonstrated that the presence of concomitant hypertension, obesity, and metabolic syndrome significantly modulates lv mass and geometry in patients with asymptomatic as independent of as severity . Furthermore, excessive lv hypertrophy in severe as has been associated with incident heart failure and increased mortality . The present study is the first to demonstrate the prognostic impact of lv mass and hypertrophy in a large, prospective study in asymptomatic mild - to - moderate as . Higher lv mass at baseline or during follow - up was associated with considerable increased overall cardiovascular morbidity and mortality . Patients in the highest versus the lowest quartile of baseline lv mass index had 2.6% higher incidence per year of death and hospitalization for heart failure . Emerging data suggest that speckle strain echocardiography may be used for further identification of as patients with more advanced lv injury . Whether asymptomatic as patients with lv hypertrophy however, lv hypertrophy in asymptomatic as should not be regarded as purely compensatory, and referral to a heart valve center for further evaluation may be indicated.
Pharmacists are gradually extending their professional roles, slowly shifting their attention from the passive dispensing of medications to actively caring for their patients . This shift of focus has occurred over many years with different degrees of success and intensity . The impact of the ideals of pharmaceutical care on the change of pharmacy and on its new ethical paradigm cannot be underestimated.1 however, the publication in 2000 of the report to err is human: building a safer health system and others such as an organization with a memory and a spoonful of sugar contributed significantly to drawing attention to the problems associated with pharmacotherapy and stimulated research and discussion about patient safety issues.24 a key role of pharmacists in ensuring the safety of medications prescribed and dispensed to patients emerged.1 at around the same period of time, johnson and bootman published a landmark article in which they estimated the annual cost of adverse effects of ambulatory drug use in the united states to be us$76 billion, equaling the annual cost for procuring drugs . The medical protection society in the uk estimates that medication errors account for approximately 20% of all clinical negligence claims against doctors in both primary and secondary care.5 these significant events helped to bring both the issue of patient safety and the potential role of the pharmacist in enhancing patient safety to the forefront.6 clinicians are not good at following recommendations for best practice and implementing guidelines resulting in underuse, overuse, and misuse of drugs.7 pharmacists are placed in an excellent position to promote rational use of medicines (ie, prescribing, dispensing, and use). Rational use of medicines requires that patients receive medications appropriate to their clinical needs, in doses that meet their individual requirements, for an adequate period of time, and at the lowest cost to them and/or their community.8 the positioning of pharmacists at crucial stages in the drug use process (be it in an outpatient dispensary, a community pharmacy, or beside the patient s bed in a hospital after the drug has been prescribed) allow them to play a vital role in rationalizing drug use through identifying, preventing, and resolving drug - related problems (drps). Medication errors (defined as any preventable event that may cause or lead to inappropriate medication use or to patient harm) are the most prevalent form of drps, and prescribing errors are the most important source of medication errors.9 since the 1970s, medication error research started to extend to nonacute care settings, including nursing homes, outpatient pharmacies, and special patient populations . The term medication misadventuring was introduced by manasse who made a strong argument for increased public policy attention to this problem.10 research that addresses prescribing errors has the potential to influence the perception of, and attitude towards, medication errors to produce fundamental changes within the pharmacy, nursing, medical staff, and in the environment they work within . The literature on prescribing errors is gaining momentum, and the data so far suggests that the problem is not limited to any specific health care environment or defined practice setting . For example, a uk community pharmacy study reported 0.7% prescribing errors identified, and around 28% of the identified problems could have resulted in patient harm.9 in the hospital setting, pharmacists interventions were demonstrated in initiating changes to hospitalized patient management which led to cost savings relating to length of stay, readmission, drugs, medical procedures, and laboratory monitoring.11 technology has a promising role in reducing adverse drug events . It has been reported that computerized decision support systems (cdss) reduced the incidence of over dosage, adverse reactions, and the length of hospital stay.12 cdss were also effective in changing the class of drug prescribed, increasing generic prescribing, and improving activities related to medication management . Other prescribing error reduction strategies include electronic prescribing (ep) and pda - based documentation and use . These proved successful in collecting data on drps and documenting pharmacist interventions.13 electronic prescribing minimized and sometimes eliminated errors of omission (incomplete scripts), such as missing important patient or prescriber s information . These tools also minimized the occurrence of drug - drug interactions, dosage problems, and inconveniences caused by prescribing a medication that was out of stock . In hospitals, information technology (it) has proved cost - effective and even reduced mortality rates.14 interventions that are most effective for influencing prescribing practice include feedback, reminders, educational outreach visits, and patient - mediated interventions.15 grindrod and colleagues suggested that for pharmacists to positively impact prescribing practices they should focus on these strategies rather than relying primarily on passive didactics or dissemination of guidelines.16 despite the evidence published so far on prescribing errors, there is still a paucity of research reporting the role of pharmacists in identifying these errors and the prevalence of near - miss incidents in the prescribing process . Indeed, we could find no previously published work to document and bench - mark the problem of prescribing errors or the role played by pharmacists in identifying these errors in primary health care in qatar, or other countries of the arabian gulf region . We believe it is of paramount importance that all pharmacists positioned at the frontline of the drug - use process promote effective reporting and information sharing on the number, types, causes, and consequences of prescribing errors . This could facilitate research that assists in better understanding the root cause of prescribing errors and the development of appropriate process control measures to minimize them . The aim of this study was therefore to characterize and analyze interventions documented by pharmacists in outpatient pharmacies of a primary health care service in qatar . This prospective, descriptive research project was conducted in four primary health care clinics (coded a, b, c, and d for the purpose of this study) within a primary health care service in the capital city of the state of qatar, in the period from january to march 2008 . Each of these clinics is supported by an embedded pharmacy team composed of a senior pharmacist with wide administrative duties and a number of pharmacists and pharmacy assistants (table 1). Prior to data collection, pharmacists in the four clinics attended two orientation workshops covering the concepts of drps, pharmacy interventions, categorizing intervention, and the documentation process . Pharmacists used online integrated health care software (trakcare; intersystems, cambridge, ma, usa) to document all interventions made . Documented information included: patient s age and gender, drug therapy details, the intervention details, its category, and its outcome (at prescriber level; at patient level; at drug level). Each intervention made was communicated to the respective prescriber by the intervening pharmacist in person or by phone . Intervention data and their outcomes were retrieved weekly from the respective clinics software and entered by a research team member in a dedicated excel spreadsheet (microsoft corp ., the data was then exported to spss software (version 17; spss inc ., were reviewed later by two members from the research team (abdullah adam, and nadir kheir), who also categorized the intervention as per the pharmaceutical care network of europe (pcne) classifications in broad drp classes . The outcome of the intervention (on prescriber or patient level) was recorded in the pharmacy s software (as intervention; a) approved and treatment changed, b) approved and no treatment was changed, c) rejected, information only . Medication counseling, referral made, written information provided was also noted). Each intervention was considered a potential drp and was categorized using an adaptation of the pharmaceutical care network europe classification of drps (revised 01 - 05 - 06 v5.01) (table 2).17 means and percentages were calculated for the numbers and outcomes of interventions, including other variables where applicable . Correlations were expressed as pearson s correlation coefficient (r) and statistical significance was set at p <0.05 . The data were exported from the clinics integrated health care software into the study excel spreadsheet and then to the spss software for analysis . The study looked at interventions made without reference to the identities of specific patient, doctor, or pharmacist . The study was approved by the department of medical services at the primary health care facility in doha, no direct patient involvement (eg, interviews) were involved and, no personal details were published . Our working definition for a pharmacy intervention was: any contact made by a pharmacist during the dispensing process with a prescriber or a patient and that was aimed at rationalizing drug prescribing or use. We adopted the pcne s definition of drps which states that a drp is an event or circumstance involving drug therapy that actually or potentially interferes with desired health outcomes.17 furthermore, we considered a prescribing error as any prescribing decision which results, or had the potential to result in, an unintentional significant reduction in the probability of treatment being timely and effective, or an increase in the risk of patient harm . Each intervention was considered a potential drp and was categorized using an adaptation of the pharmaceutical care network europe classification of drps (revised 01 - 05 - 06 v5.01) (table 2).17 means and percentages were calculated for the numbers and outcomes of interventions, including other variables where applicable . Correlations were expressed as pearson s correlation coefficient (r) and statistical significance was set at p <0.05 . The data were exported from the clinics integrated health care software into the study excel spreadsheet and then to the spss software for analysis . The study looked at interventions made without reference to the identities of specific patient, doctor, or pharmacist . The study was approved by the department of medical services at the primary health care facility in doha, no direct patient involvement (eg, interviews) were involved and, no personal details were published . Our working definition for a pharmacy intervention was: any contact made by a pharmacist during the dispensing process with a prescriber or a patient and that was aimed at rationalizing drug prescribing or use. We adopted the pcne s definition of drps which states that a drp is an event or circumstance involving drug therapy that actually or potentially interferes with desired health outcomes.17 furthermore, we considered a prescribing error as any prescribing decision which results, or had the potential to result in, an unintentional significant reduction in the probability of treatment being timely and effective, or an increase in the risk of patient harm . Of a total of 82,800 patients presenting with prescriptions in a three - month period, there were 594 patients (table 3) whose prescriptions were intercepted for suspected errors (0.72%). The total number of drp - related interventions made in the study period was 890 interventions . Table 4 provides the distribution of all interventions across clinics (total prescriptions dispensed, number and percentage of intervention per clinic and in total). 10.8% of the total prescriptions intercepted were for children who were aged five years or younger and 3.5% were for children who were aged between six to 12 years . Prescriptions for patients who were 60 years or older were 3% of the total prescriptions intercepted, and the majority (83% of the total) was for patients whose ages lie between 13 and 59 years . Overall, the percentage of errors intercepted ranged from 0.4 to 1.9% of the total prescriptions dispensed . The mean across all clinics was 1.7% . Figure 1 shows the classes of interventions made based based upon the pcne classification of drps . Over half of all errors were related to drug choice problems, followed by drug safety problems . When drug choice problems were further analyzed (figure 2), prescribing inappropriate drug therapy contributed 39% of the total, followed closely by duplicate therapy (32%). In interventions classified as safety problems, 51% of the interventions (ie, 188 interventions from a total of 363) were related to dosing errors (figure 3). The outcome of the interventions that were accepted by the prescriber at the drug level are summarized in figure 4 . In 35% of the interventions, a dose was changed, and in 20% the drug was changed . At the patient level, written information was provided to the patient in over 60% of the interventions made, and medication counseling (over and above the routine instructions given at the dispensing window) took place in 24% of all interventions in this category (figure 5). At the drug level, safe patient care requires safety - conscious individuals at the frontline to promote effective reporting and learning systems, to develop systems that facilitate information sharing about the number, types, causes, and consequences of errors, facilitate research in order to have a better understanding of the extent and causes of possible patient harm, and to develop appropriate solutions.17 prescribing errors are a universal problem with similar potential impact on patients safety and quality of care . To be most effective and better influence prescribing, pharmacists require clinical knowledge and a set of technical and social skills to underwrite competency so as to negotiate issues relating to best - practice in prescribing with clinicians . Our anecdotal experience suggests that pharmacists need to embed themselves authoritatively within the clinical team in order to develop a mutual respect . In such a relationship they might develop an understanding of the complex cognitive processes and emotions associated with clinician prescribing . In the clinical setting involved in this study, the senior clinical pharmacists complete the dual authoritative roles of medical pharmacy advisor (mpa) to the regional primary health care team (phct) and as the chairperson of the drugs and therapeutics sub - committee of the corporate clinical governance committee . We believe that pharmacists might achieve better acceptance of drug interventions by clinicians if they reference these interventions to previous, joint professional development sessions and to published literature that is normally resourced by clinicians such as american family physicians steps collection (freely accessible at: http://www.aafp.org/afp/steps). This study provides an insight into the potential role of pharmacists in promoting rational drug use in qatar . It also provided evidence of the magnitude of avoidable prescribing errors that pharmacists could intercept in outpatient pharmacies . The calculated figure of 1.7% prescriptions with an error is more than double that reported in the uk,13 but it is significantly smaller than figures reported elsewhere (sayer and colleagues,18 and leemans and colleagues19) who reported 12.4% and 4.1% prescribing errors, respectively . However, several reasons for these differences in prevalence could be noticed, not the least in importance was the location of the studies (community / private pharmacies versus pharmacies embedded within primary health care setting) and the fact that transcription errors, prescription legal issues and formulary issues were eliminated from our study through the full use of a cpoe, a factor which must have had huge impact on the number of intercepted errors . Overall, drug choice problems and drug safety problems featured high in the list of prescribing errors identified . Considering the daily workload and the reality of the pharmacists priorities (ie, serving patients comes before documenting for the purpose of the study), we think the outcome in terms of documented interventions reported in these three months, while realistic, might be underestimating the real numbers of drps in the clinics involved . This could be due to documentation fatigue among the study pharmacists despite an effort to continuously keep motivating them to document each intervention they made . We have included interventions on the level of the patients in this analysis to have a feeling of the role that could be played by the pharmacist engaging in patient education when the need to do so was identified . In most of the cases involving patients, these strategies are expected to improve health outcomes and enhance patient compliance with drug therapy . Interventions that were more likely to be accepted by the prescribing physicians were those involving dosage errors, duplicate therapy, and drug choice problems, in that order . We have not attempted to trace the fate of rejected interventions in this study . In the absence of a structured validation process however, such an understanding is critical to the success of drp intervention programs . Considering the large proportion of rejected interventions, it might be reasonable to assume that a percentage of those interventions made and rejected were actually correct and well - founded . If this is the case, then it might suggest an inability of the pharmacists concerned to engage into constructive dialogue with the prescribing physicians, despite having evidence that should have supported changing the therapy . Dispensing a prescription that could have an error raises significant ethical issues relating to the pharmacist s hippocratical obligation towards beneficence and nonmalfeasance (the responsibility to do good and the duty to do no harm). In general terms, dispensing a prescription that is suspected to be erroneous violates these basic ethical principles . We found no association between the pharmacists characteristics (ie, gender, age, and years of experience) and the number of interventions made or documented . This should not be surprising, since many other important factors might contribute to the ability of pharmacists to identify errors . These factors, such as the individual pharmacist s motivation, interest, and clinical knowledge, are difficult to assess especially as they fall beyond the scope of the current project, while they could have significantly more influence than other demographics . We were unable to assess the association between errors identified (or accepted / rejected) with the clinician s characteristics for logistical and ethical reasons . The findings of this study should stimulate further research into the root cause for prescribing and medication errors . The results of such research should promote the development of focused continuing education and training material that would specifically address gaps and safety risks in the prescribing process . This material should be delivered in ways that have been shown to improve prescribing behavior and should be extended to both physicians and pharmacists in a multidisciplinary environment . Finally, it should be pointed out that no retrospective validation process was carried out by an independent reviewer on the interventions made or those rejected to ascertain grounds for rejection in this study . While accepted interventions carry an inherent validation by both the intervening pharmacist and the prescribing physician, the absence of validation targeting those interventions which were rejected by the prescribers remains a limitation in this study . Additionally, we have not made any attempt to classify the interventions beyond the pcne classification system into broad drp types . No classification based on potential harm or seriousness of the prevented drps was made . Documenting and analyzing interventions should be a routine activity in pharmacy practice of primary health care services . Progressive pharmacists are recognizing that this is only the first step in the process of improving patient safety . The real challenge is to work with clinicians to develop multiple channel interventions to better translate the exposure of clinicians to incontrovertible evidence into effect.20
Obesity is a growing epidemic worldwide; its prevalence has been rising tremendously over the last 30 years (who, 2013). Excess adiposity is an established risk factor for metabolic diseases including insulin resistance, type 2 diabetes (t2d), hypertension, nonalcoholic fatty liver disease (nafld), polycystic ovarian diseases, and several types of cancer . Obesity is a proinflammatory condition in which hypertrophied adipocytes and adipose tissue - resident immune cells (primarily lymphocytes and macrophages) both contribute to increased circulating levels of proinflammatory cytokines . Metabolic inflammation, is considered a focal point in the pathogenesis of insulin resistance and t2d in humans and rodent animal models [25]. Although liver and muscle show obesity - induced mild inflammatory responses, white adipose tissue (wat) is the key site mediating systemic inflammation . Adipose tissue primary function is to store excess nutrients as triacylglycerols and to release free fatty acids during fasting . A major step forward to the recognition of the major secretory and endocrine role of wat occurred in the 1990's with the demonstration that adipocytes synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (tnf-) and the hormone leptin which regulates appetite and energy balance . Evidence shows that the adipose tissue secretes more than 50 hormones and signaling molecules, collectively called adipokines, which exert their biological roles in an autocrine, paracrine, or systemic manner and influence several physiological processes concerning energy, glucose metabolism, and immunity . More specifically, adipokines can exhibit either proinflammatory or anti - inflammatory properties, thereby contributing to insulin resistance . Adipose tissue from lean individuals preferentially secretes anti - inflammatory adipokines such as adiponectin, transforming growth factor beta (tgf), interleukin (il)-10, il-4, il-13, il-1 receptor antagonist (il-1ra), and apelin . In contrast, obese adipose tissue mainly releases proinflammatory cytokines among which are tnf-, il-6, leptin, visfatin, resistin, angiotensin ii, and plasminogen activator inhibitor 1 . In lean individuals, anti - inflammatory adipokines mediate physiological functions, whilst in states of metabolic diseases, the proinflammatory adipokines modulate insulin resistance either directly by affecting the insulin signaling pathway or indirectly via stimulation of inflammatory pathways . Indeed, serine phosphorylation of insulin receptor substrate (irs) by various adipokines directly or via inflammatory pathways including the c - jun n - terminal kinase (jnk) pathway and i - kappa b kinase (ikk)/nfb pathway disrupts the insulin signaling pathways, possibly giving rise to insulin resistance . Adipokines enlisted in regulation of insulin resistance are adiponectin, leptin, resistin, visfatin, chemerin, tnf-, il-1, il-6, il-8, il-10, plasminogen activator inhibitor 1, monocyte chemoattractant protein-1, and retinol binding protein-4 (tables 1 and 2). Because this topic has been the subject of recent reviews [12, 13] it will not be discussed in detail . We will rather focus on the prototypical adipokines (tnf-, il-6, leptin, adiponectin, and resistin) highlighting their roles in the development of insulin resistance as well as in immunity and inflammation . Tnf- is a potent proinflammatory cytokine, primarily secreted from myeloid cells via activation of mapk and nfb signaling pathways, resulting in the release of other inflammatory cytokines, such as il-1 and il-6 . It was the first wat - derived inflammatory cytokine reported to be implicated in the initiation and progression of insulin resistance [8, 15]. Although originally thought to be mainly secreted by adipocytes, it is now admitted that the majority of tnf- is secreted by adipose tissue - resident macrophages . In rodents tnf- is overexpressed in adipose tissue from obese animals, and obese mice lacking either tnf- or its receptor show protection against the development of insulin resistance . In humans tnf- levels are higher in plasma and adipose tissue of obese individuals, and circulating levels reduce with weight loss . Tnf- levels were also found to be positively correlated with other markers of insulin resistance; nonetheless, acute treatment with tnf- inhibitor in obese subjects with type 2 diabetes reduced other systemic inflammatory markers without reducing insulin resistance, fueling lingering uncertainty about the biological relevance of this pathway in human insulin resistant states . More recently, the long - term assessment of anti - tnf- inhibitor treatment to subjects diagnosed with metabolic syndrome has been shown to improve fasting blood glucose and to increase adiponectin levels, confirming a role for tnf- in obesity - related insulin resistance in humans . A key mechanism by which tnf- induces insulin resistance involved phosphorylation of irs-1 . Beside its direct negative interference with the insulin signaling pathway, tnf- is known to promote lipolysis and the secretion of free fatty acids, which contribute to an increase in hepatic glucose production . Moreover, tnf- inhibits the conversion of preadipocytes to mature adipocytes notably through downregulating adipogenic genes such as peroxisome proliferator - activated receptor gamma (ppar) and ccaat / enhancer binding protein (c / ebp)allowing further recruitment of uncommitted cells and thus possible expansion of adipose tissue mass . Tnf--activated nf-b suppressed genes involved in lipid uptake and storage as well as many adipocyte - specific genes . Tnf- also downregulates the mrna levels of adiponectin, an adipocyte - derived hormone which contributes to the maintenance of peripheral glucose and lipid homeostasis . Nevertheless, the influence of tnf- on immune response mostly results from its enhancing effect on the production of other cytokines, such as il-6, rather than from a direct effect . Il-6 is a multifaceted, pleiotropic cytokine that is a central player in the regulation of inflammation, hematopoiesis, immune responses, and host defense mechanisms . Il-6 is secreted by wat, skeletal muscle, and liver [16, 29]. Because one - third of circulating il-6 in healthy individuals is estimated to originate from adipose tissue, il-6 is considered an adipokine . In wat, only a fraction of il-6 is secreted by adipocytes, the other part being produced by other cells, particularly macrophages . Similarly to tnf-, wat and plasma il-6 expression correlate with increased body mass, waist circumference, and free fatty acid levels, with reduction in circulating il-6 following weight loss . Il-6 has been implicated as a marker for visceral adiposity because visceral adipose tissue releases more il-6 than subcutaneous adipose tissue . Nevertheless, data regarding the role of il-6 in both obesity and insulin resistance are controversial and unresolved . While several studies indicate that increased il-6 levels correlate with adiposity and fat mass, and not necessarily with insulin action or responsiveness [30, 33], another study has pointed to higher il-6 levels in patients with obesity - related insulin resistance . It can be inferred that relentless increase in systemic levels of il-6 may lead to insulin resistance, whereas a transient increase in il-6 may assist in normal glucose homeostasis . In fact, il-6 appears to have dual functions depending on the tissue and metabolic state . During exercise, il-6 increases glucose uptake in the skeletal muscle, leading to muscle hypertrophy and myogenesis and ampk - mediated fatty acid oxidation, as well as having an anti - inflammatory effect . In adipose tissue and liver, however, il-6 will exert proinflammatory activities, increasing insulin resistance by upregulating socs3 (suppressor of cytokine signaling 3) which, in turn, impairs insulin - induced insulin receptor and irs1 phosphorylation . Il-6 may promote dysregulation of fatty acid metabolism in wat as it enhanced mesenchymal stem cell proliferation, maintaining the cells in an undifferentiated state and inhibiting adipogenesis additionally, il-6 was recently shown to stimulate insulin secretion via enhanced glp-1 (glucagon - like peptide-1) expression in pancreatic cells . Thus, obesity - induced il-6 secretion may reflect a mechanism to increase insulin production in the obese insulin resistant state . However, while elevated il-6 secretion from wat and liver is unfavorable, the opposite is true for skeletal muscle . On the other hand, a number of in vitro and in vivo studies demonstrate that il-6 is capable of inducing insulin resistance . In cultured murine adipocytes, il-6 production is strongly increased by tnf- and induces insulin resistance by inhibiting glucose uptake and impairing insulin signaling and action . Whether or not il-6 impairs insulin action in adipose tissue in vivo has yet to be clearly determined . Like tnf-, il-6 can directly affect lipid metabolism and activate pathways to promote increased energy turnover . Il-6 stimulates lipolysis in humans, increases free fatty acid (ffa) concentrations and whole body fat oxidation . Notably, il-6 can decrease the expression and secretion of adiponectin in human adipocytes, as well as other markers of adipocyte differentiation . Therefore, understanding and clarifying its role in the regulation of metabolism is of utmost importance . As stated above, leptin was one of the first proteins shown to be secreted from adipose tissue, through the identification and sequencing of the ob gene from the ob / ob mouse . Leptin is primarily secreted by adipocytes proportionally to fat cell mass and is well known for its key contribution to energy metabolism . Leptin exerts its effect on energy balance mainly by acting on the brain, either directly or indirectly by activating specific centers in the hypothalamus to decrease food intake, to increase energy expenditure, to influence glucose and lipid metabolism, or to alter neuroendocrine function . Daily injection of leptin in ob / ob mice resulted in a rapid reduction in food intake, body mass, and percentage of body fat but maintained lean muscle mass, increased energy expenditure, and restored euglycemia, confirming its important role in energy homeostasis and storage . However, leptin levels are increased in obese subjects, with little or no impact to regulate energy homeostasis, which coined the well - established phrase leptin resistance in obesity . Indeed, preclinical and clinical experiments showed that obese rodents and humans displayed leptin resistance that may directly contribute to the reduction of lipid oxidation in insulin - sensitive organs, leading to accumulation of lipids and insulin resistance [44, 45]. Recently, it has been proposed that socs3 could be involved in negative regulation of leptin - induced intracellular signal transduction in the brain . Moreover, neuronal deletion as well as whole - body knock - out of protein tyrosine phosphatase 1b (ptp1b) increased leptin and insulin sensitivity, preventing body weight gain in a diet - induced obesity animal model [47, 48], hence suggesting that, likewise socs3, ptp1b also orchestrates leptin resistance control . On the other hand, the role of leptin on insulin resistance is still not fully understood . Leptin is decreased in low insulin states, such as experimentally induced diabetes, and increases after insulin treatment . In humans, insulin resistance is associated with elevated plasma leptin levels independently of body fat mass . However, in patients with lipodystrophy, a condition characterized by almost complete lack of adipose tissue, leptin levels are very low and correlate significantly with markers of insulin resistance . Leptin therapy in lipodystrophic patients improves their metabolic state with remarkable improvements in insulin sensitivity, suggesting that leptin acts as a signal that contributes to regulation of total body sensitivity to insulin . Leptin has proinflammatory functions: it stimulates t - cell proliferative responses, polarized nave cd4 t - cell proliferation towards the th1 phenotype, promotes a marked increase in th1-type cytokine production, induces the expression of proinflammatory cytokines by macrophages and monocytes, and acts directly on hepatocytes to promote c - reactive protein expression . The proinflammatory nature of leptin has been noted in several studies, with intravenous injection of endotoxin inducing a sudden rise in leptin levels, as well as endotoxin - induced fever and anorexia in rats, again inducing an increase in leptin levels as part of the inflammatory response . The importance of leptin in immunity was confirmed in obese mice with homozygous mutation in leptin (ob / ob mice) or leptin receptor (db / db mice), in which high levels of lymphocyte atrophy and significant reduced thymus cortex were evidenced . Replacement of leptin in the ob / ob mice or in congenital leptin - deficient children is able to restore normal thymic function, to increase the number of cd4/cd8 t - cells, to promote th1 differentiation, and to reduce thymic apoptosis . We also reported impaired functionality of t - lymphocytes, dendritic cells, and macrophages in ob / ob and high - fat (hf) diet - fed mice [59, 60]. More recently, leptin has also been shown to activate human b lymphocytes to secrete tnf-, il-6, and il-10 via the jak2, stat3, p38mapk, and erk signaling pathways . Besides acting on adaptive immunity, leptin also regulates innate immune cells such as polymorphonuclear neutrophils, monocytes, and natural killer (nk) cells . Leptin can induce chemotaxis of neutrophils, is involved in the development and maintenance of a functional nk (natural killer) pool, and induces the production of il-6 and tnf- from macrophages . Unlike leptin, the circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are decreased in obesity . Adiponectin has important insulin - sensitizing effect: adiponectin - deficient transgenic mouse showed improved insulin sensitivity and association studies have consistently linked plasma adiponectin levels to insulin sensitivity in rodent models and in humans . Among the three major adiponectin isoforms, high - molecular weight (hmw) adiponectin is the most biologically active form and best reflective of the reduction in total adiponectin levels associated with obesity . Indeed, hmw adiponectin levels have been identified as an independent risk factor for insulin resistance . Adiponectin can suppress the production of tnf- and ifn (interferon gamma) and is a negative regulator of t cells, notably through its effect on the t - cell presenting function of dendritic cells . Adiponectin maintains a mutual antagonistic action to tnf-: as mentioned above tnf- inhibits the expression of adiponectin, and conversely adiponectin suppresses lipopolysaccharide- (lps-) induced tnf- production . Resistin is another unique adipocyte - derived signaling cysteine - rich molecule that was first identified in obese mice, deriving its name because of its resistance to the action of insulin . In rodents, resistin is secreted primarily from adipose tissue, whereas in humans resistin can be detected in other tissues like placenta, skeletal muscle, small intestine, spleen, stomach, thymus, thyroid gland, and uterus, being predominantly expressed in macrophages . In rodents, demonstrated that circulating resistin levels are elevated and positively concordant with rising levels of insulin, glucose, and lipids in ob / ob mice and that leptin administration improved insulin sensitivity associated with a decrease in resistin gene expression . Moreover, transgenic mice overexpressing a dominant negative form of resistin showed increased adiposity, possibly owing to enhanced adipose tissue differentiation and adipocyte hypertrophy . Resistin appears to interfere with normal insulin signaling by decreasing insulin receptor and insulin receptor substrate (irs1 and 2) protein expression and phosphorylation level in preadipose 3t3-l1 cells . In addition, resistin has been showed to decrease ampk activation which is known to be implicated as a potential insulin sensitizing molecule . However, the role of resistin in the development of insulin resistance in humans is not as clear as in rodents . Since resistin is preferentially expressed by macrophages in humans, it suggests a proinflammatory role of resistin rather than a role in regulating glucose metabolism . Resistin mrna expression level is higher in obese subjects, likely resulting from increased infiltration of macrophages in the adipose tissue . Several studies have reported positive correlations between resistin levels and insulin resistance in vivo and in vitro . Moreover, genetic studies showed that two single nucleotide polymorphisms (snps: 537a> c and 420c> g) were associated with increased resistin levels in diabetic patients, but not in control subjects . Recently, associations have been reported between resistin and metabolic syndrome components on one hand and early atherosclerosis in obese children on the other hand . Finally, resistin has been demonstrated to stimulate the secretion of several inflammatory factors (e.g., tnf-, il-6, il-8, and mcp-1) known to play a role in the induction of insulin resistance . Therefore, resistin may have an indirect effect on insulin resistance in humans through exacerbating inflammation, which has been shown to disturb insulin sensitivity . During the past decades, il-7 has been identified as the major homeostatic cytokine supporting the survival of and t cells, nkt cells, innate lymphoid cells, and regulatory t cells (tregs). Il-7 is predominantly produced by stromal and vascular endothelial cells, with very low levels of il7 transcripts detectable in adult animals, consistent with the concept that under basal states there are limited amounts of il-7 available for lymphocytes in vivo . In a homeostatic animal, il-7 amount is thought to be constant yet stroma - derived il-7 production can be induced by overt inflammation . Il-7 receptor (il-7r) is composed of the private il-7r chain (cd127) combined with the common gamma (c; cd132) chain and is expressed mainly by t lymphocytes but also by nk cells, macrophages, dendritic cells, lymphoid tissue inducer cells, and certain subsets of b cells . One central characteristic of il-7r expression is its dynamic regulation by cytokines and by the overall metabolic and differentiation state of the cells . For example, tnf- has been reported to upregulate il-7r expression and il-6 to be a critical effector of il-7r signaling . Without il-7 the lymphoid system cannot be built and maintained . Interestingly, the role of il-7 on lymphocyte homeostasis was shown to partly rely on its control of basal lymphocyte glucose metabolism through the expression of the glucose transporter glut-1, which promotes glucose uptake and increases metabolic activity as well as cell size . Recently, we and others identified il-7 as a new secretory product of the adipose tissue, mostly produced by cells of the stromal vascular fraction [82, 83]. Furthermore, we reported that il-7 also contributes to body weight regulation via both hypothalamic and adipose tissue control . Regarding the latter, we showed that il-7 modulates the adipose tissue through acting on its mass and function . In fact, a single administration of il-7 was sufficient to decrease adipose tissue inflammation and to protect mice from obesity in three different models of experimentally induced obesity (i.e., monosodium glutamate - induced hypothalamic obesity, gold thioglucose - induced hypothalamic obesity (wolowczuk i, unpublished data), and hf diet- (hfd-) induced obesity). Strikingly, we showed that il-7 overexpressing mice presented a lipodystrophy - like phenotype: reduced wat mass is associated with impaired adipocyte differentiation and intolerance to glucose and insulin resistance, these traits being commonly associated with lipodystrophy in both animals and humans . The first part of our review showed that the apparent metabolic simplicity of the adipose tissue is illusory; this is also true regarding its cellular composition . Besides lipid - filled mature adipocytes, the tissue is also composed of various stromal cells, including preadipocytes, endothelial cells, fibroblasts, and immune cells . During the progression of obesity, both the adipocyte and the stroma vascular fractions are changed: adipocytes grow larger, secrete predominantly proinflammatory cytokines, and are insulin resistant; coincidently, the nature of wat immune cells is also modified . Notably, proinflammatory macrophage infiltration and inflammation - related gene expression precede the development of insulin resistance and appear to be a cardinal feature of obesity in rodents and humans . Adipose tissue macrophages (atms) accumulate in both the subcutaneous and visceral expanding fat depots, even though macrophage infiltration appears to be more prominent in the latter . Apart from increasing in numbers, adipose tissue macrophages are also phenotypically changed during obesity: while anti - inflammatory m2 macrophages reside in wat of lean mice, obese wat predominantly contains proinflammatory m1 macrophages . Activated m1 atms are a prominent source of proinflammatory cytokines such as tnf- and il-6, which can block insulin action in adipocytes via autocrine / paracrine signaling causing systemic insulin resistance via endocrine signaling (cf . Thus, both recruitment and proinflammatory polarization of atms are required for the development of insulin resistance . In both humans and rodents, atms content positively correlates with inflammation and insulin resistance . Despite its importance in adipose tissue inflammatory responses and systemic insulin sensitivity the recent discovery of micrornas (mirnas) provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function . Mirnas, which correspond to a group of highly conserved, small (i.e., approximately 22 nucleotides in length) noncoding rnas, can trigger either a block in translation and/or mrna degradation [88, 89]. Numerous studies have provided compelling evidence that mirnas are key regulators of cell fate determination and significantly contribute to the pathogenesis of complex diseases, including obesity - associated metabolic diseases [9092]. Recently identified mirna-223 (mir-223) as a potent regulator of macrophage polarization and provided strong evidence supporting the functional significance of this new pathway in metabolic homeostasis . The authors showed a suppressive effect of mir-223 on macrophage proinflammatory activation (m1) and a stimulatory effect on anti - inflammatory activation (m2): high - fat diet - fed mir-223-deficient mice displayed increased adipose tissue inflammation and were more insulin resistant . At the molecular level, a major target of mir-223 in macrophages is pknox1, which itself favors the proinflammatory activation pathway . However, a key question still unanswered by now is how the mir-223/pknox1 pathway interacts with known regulatory pathways that control macrophage activation . The identification of mechanisms underlying functional polarization of macrophages into m1 or m2 might provide new insights into a basis for macrophage - centered therapeutic strategies for metabolic diseases . Similarly to any immune and inflammatory response, macrophage infiltration in the obese adipose tissue results from blood monocyte influx, mainly attracted by the chemokine monocyte chemoattractant protein-1 (mcp-1) which is secreted by hypertrophic adipocytes . It has been reported that mcp-1 secretion is markedly enhanced locally and in plasma of obese rodents and humans . Overexpression, deficiency, or mutation - induced dysfunction of mcp-1 in different mouse models were shown to interfere with atms accumulation, along with insulin - resistance development [95, 96]. However, the role of mcp-1 in promoting atm recruitment and insulin resistance has recently been challenged by the absence of noticeable impact on macrophage accumulation and glucose intolerance resulting from mcp-1 genetic disruption . Furthermore, hfd - fed mcp-1 receptor i.e., ccr2-deficient mice (namely, ccr2 mice) do not normalize atm content and insulin resistance to the levels of lean animals, suggesting that atm recruitment and insulin resistance are also regulated by mcp-1/ccr2 independent signaling pathways . Recently identified and characterized a critical role for ccr5, another c - c motif chemokine receptor, in the regulation of obesity - induced wat inflammatory response and insulin resistance ., ccr5 mice were protected from insulin resistance induced by hf feeding through both reduction in atm accumulation and induction of anti - inflammatory m2 shift in those cells . Additionally, a bone marrow transplantation study revealed that lack of ccr5 expression in macrophages alone could protect mice from the hfd - induced insulin resistance, this being associated with a significant reduction in atm infiltration . In humans, recent studies have also shown upregulation of ccr5 in the visceral fat of morbidly obese individuals in whom macrophage infiltration has been confirmed . However, further studies are needed to evaluate whether ccr5 inhibitor treatment (e.g., maraviroc) affects macrophage activation and other aspects of adipose tissue biology in obese patients . Also, it remains to be established whether the two c - c chemokine receptors, ccr2 and ccr5, play common or unique roles in obesity - induced adipose tissue inflammation and insulin resistance . Alterations in atm content and polarization state occur fairly late in the progression of obesity and probably are not initiating events of inflammation and development of sustained insulin resistance . Evidence has accumulated showing that other changes in adipose - resident immune cells may precede these events . Under this scenario, atms will be effectors of a coordinated inflammatory response that includes the accumulation of proinflammatory t cells (cd8 and th1 cd4 t cells) and the loss of anti - inflammatory regulatory t cells (tregs), as well as the appearance of b cells, nk cells, nkt cells, eosinophils, neutrophils, and mast cells . Adipocytes in lean adipose tissue produce factors such as il-4 and il-13 that induce m2 activation of macrophages and th2 activation of cd4 t cells and maintain treg cell and eosinophil numbers . In obesity, the progressive accumulation of adipose tissue is accompanied by early increased infiltration of proinflammatory cd8 t cells and a shift towards a higher cd8/cd4 ratio . Cd8 infiltration appears to be a key event preceding the depletion of adipose tregs and the increased cd4 th1 cell activation observed in murine models of diet - induced obesity [101, 102]. Increased adipose - resident cd8 t - cell activation also potentiates adipocyte expression of il-6 and tnf- in mice, while cd8 t - cell depletion reverses this effect . Neutrophils are known to play a role in the early stages of inflammatory responses, and it has been recently reported a sustained increased in adipose tissue neutrophil content in hfd - induced obesity with neutrophil secreted elastase being a key effector in this process . The enhanced release of il-8, a factor involved in neutrophil chemotaxis, by hypertrophic adipocytes, may partly explain neutrophil recruitment . Adipose tissue neutrophils produce chemokines and cytokines, facilitating macrophage infiltration, which could contribute to development of insulin resistance . In the 1980s, a new cell population known as natural suppressor cells, distinct from t and nk cells, was described in the bone marrow and spleen of tumor - bearing mice [104, 105]. Myeloid - derived suppressor cells (mdsc) because of their myeloid origin and their ability to suppress immune responses . In fact, mdscs represent a heterogeneous and metabolically plastic population of immature myeloid cells in different stages of differentiation, having in common the capacity to inhibit effector immune responses and to accumulate under conditions of inflammation . The term plasticity here refers to the ability of mdscs to change both their expression of various mediators of suppression (e.g., inos, arginase i) in response to environmental influences (e.g., local il-4/13 or ifn concentration) and also their differentiation state (e.g., becoming more / less neutrophil or myeloid cells). These cells are also defined by their immature state of macrocytic / monocytic, granulocytic / neutrophilic, and dendritic cell precursors and are characterized by the increased production of extracellular degradative enzymes, cytokines, and reactive oxygen and nitrogen species . In mice, since there are several subpopulations within gr-1cd11b cells, several groups further subcategorized mdsc into monocytic mdsc (cd11bly6gly6c) and granulocytic / neutrophil - like mdsc (cd11bly6gly6c), based on the expression of ly6c and gr-1/ly6 g . There is no human marker equivalent to mouse gr-1, human mdsc being typically defined as cd11bcd33cd34cd14hla - dr cells . In addition to heterogeneity, discrepancies exist in cell surface expression of certain activation / maturation markers, such as mhc ii and costimulatory molecules, and of lineage markers (e.g., f4/80) between mdsc . This heterogeneity supports the notion that mdsc include multiple subpopulations of myeloid - derived cells that are at various stages of maturity . In the steady state, mdscs are predominantly present in the bone marrow and participate in the normal process of myelopoiesis . However, under various pathological inflammatory conditions such as cancer, infection, sepsis, graft - versus - host disease, and bone marrow transplantation, a variety of cytokines and soluble factors released induce rapid expansion of mdsc that will accumulate in peripheral lymphoid organs and blood, as well as in tumors, where they have been described to block cd4 and cd8 t - cell responses thus favoring cancer development . In cancer, one key factor controlling mdsc expansion and tumor progression is ppar . Vascular endothelial growth factor (vegf), macrophage colony - stimulating factor (m - csf), or il-6 it has been suggested that mdscs contribute to tumor progression by both facilitating neoangiogenesis and metastasis and by inhibiting antitumor responses . In addition to their recognized role in tumor tolerance, mdscs may also be involved in the induction and maintenance of transplant tolerance . Recently, an exciting observation has been described by xia et al ., showing that mdscs and m2 macrophage induction may be a physiological response to promotion of insulin sensitivity . These authors showed that obese ob / ob mice, as well as wild - type mice fed on high - fat diet, have marked accumulation of anti - inflammatory mdscs and m2 macrophages in adipose tissue . Furthermore, the increase in mdscs and m2 macrophage number was associated with higher response to insulin . Adoptive transfer of mdscs (e.g., gr-1 cells) into high - fat diet - fed mice improved the response of the recipient mice to insulin while, in contrast, mdscs depletion (after treatment with anti - gr-1 antibody) increased their susceptibility to obesity and further worsened their resistance to insulin . Have also described the ability of mdscs to delay onset of type 1 diabetes and insulin resistance, through inducing expansion of antigen - specific tregs and suppressing t - cell proliferation . The mechanisms by which obesity - associated chronic inflammation induces expansion / accumulation of mdscs are arguably stepwise . However, the initial proinflammatory state created in early obesity may induce the accumulation of mdsc in an attempt to curtail overt inflammation, as described in other well - described models of inflammation, and may improve insulin sensitivity . In addition, we recently showed that the mechanistic target of rapamycin (mtor) signaling pathway might be involved in the expansion of mdscs (makki, ms submitted), as well as in myelopoiesis . Although mechanisms by which mdscs enhance insulin sensitivity are unknown, it has been proposed that upregulation of insulin growth factor-1 (igf-1) in the setting of insulin resistance may lead to the accumulation of mdscs or m2 macrophages . Suggestions have been made that not only does insulin resistance induce physiological response for mdsc and m2 macrophage expansion, but insulin may also modulate direct gene transcriptional control of these cells . Therefore, pharmacological enhancement of insulin sensitivity in obese individuals may preemptively hinder the development of mdscs . The complex alterations in adipose tissue secretion of cytokines, adipokines, and chemokines and immune cell composition observed in adipose tissue - related pathologies such as obesity (figure 1) have been, and still are, an active research area . As the proportion of overweight and obese (even among the youngest) continues to rise worldwide, understanding the role of adipose tissue in the pathogenesis of obesity and its metabolic and immune - based complications will be critical to optimize long - term health outcomes . As summarized in the present review, there might be a potential therapeutic value of targeting certain immune resident cells (such as m2, tregs, or mdsc) and/or certain cytokines, adipokines, or chemokines (such as mcp-1/ccr2 or ccr5) to improve insulin resistance and restrain organ damage in type 2 diabetic obese patients by limiting the proinflammatory milieu.
Hyperekplexia is a rare movement disorder characterized by an exaggerated response to trivial stimuli that are mostly acoustic and tactile . Hereditary hyperekplexia is characterized by excessive startle response beginning soon after birth, with the associated generalized hypertonia and exacerbated body stiffness following the startle response . However acquired cases may not have the typical characteristics so described, therefore, the case of a 10-year - old boy who developed an acquired hyperekplexia following an episode of cerebral malaria is reported . This communication is that of a 10-year - old boy who suddenly developed involuntary jerking movements when exposed to loud sound, he had several episodes in a day; this was initially noticed when exposed to loud conversations at home; they had difficulty travelling to the hospital because the jerky movement was stimulated by sound of moving cars and car horn . He could speak but avoided speaking because he felt it could induce the jerking; he could also hear and see and there was no history of loss of consciousness . He was discharged from hospital 2-week before onset of this illness and was treated for cerebral malaria - he had status epilepticus during that period but he completely recovered before discharge . His newborn period was not adversely eventful, and he had normal developmental milestones and scholastic achievement before onset of illness; and there was no behavioral or psychiatric complaint . He had no cranial nerve palsy or signs of meningeal irritation; normal tone globally; he walked with an ataxic gait [video 1]. When he was stimulated [videos 2a and b] he responded with excessive startling . The cerebrospinal spinal fluid analysis, electroencephalogram (eeg) and magnetic resonant imaging of the brain were not remarkable . He was commenced on slow release carbamazepine and 3-week into treatment the excessive startling subsided [video 3] with improved ambulation [video 4]. He is currently being followed - up in the pediatric neurology clinic . Hyperekplexia was first described by kirstein and silfverskiold, in 1958; when they described a family with drop seizures. In 1962, drs kok and bruyn further reported on this hereditary syndrome which was then unknown, and they simply described it as hypertonia in infants . Evaluated members of a dutch pedigree with excessive startle reflexes and called the disorder hyperekplexia . Hyperekplexia has been classified into major and minor, and our patient fulfilled the criteria for hyperekplexia minor . Hereditary hyperekplexia has been associated with mutation in several of the glycine receptor genes and has an established genetic - phenotypic correlation . However, this may not explain the mechanism in all cases because serotonin has also been implicated and sechi et al . In their report documented the beneficial effect of fluoxetine, further substantiating the serotonergic hypothesis . Neuropsychiatric startle syndromes (culture - specific syndromes), reflex and startle epilepsy could easily be confused with hyperekplexia, but the absence of neuropsychiatric and intellectual handicaps in the index case, coupled with normal eeg and following critical review of the video clips made the diagnosis easy . Though this disorder is predominantly hereditary, although cerebral malaria has been associated with neurologic complications but no mention of hyperekplexia has been reported before now; the exact mechanism is not completely understood, however cerebral hypoxia during periods of repeated seizures might be responsible . Vigevano maneuver, which consist of forced flexion of the head and legs toward the trunk may relieve attacks, especially in the newborn period . Clonazepam is the drug of choice in treating hyperekplexia, while levetiracetem has shown promises in managing this disorder . However, other sedative - hypnotics like carbamazepine, phenytoin, diazepam, valproate, and phenobarbital have been used . Due to nonavailability of the first - line drugs we opted for carbamazepine and he responded with complete resolution of the startling . Acquired hyperekplexia is a rare movement disorder; even rarer is its association with cerebral malaria.
Well validated antibodies are crucial to enable scientists to make progress in a wide range of life science disciplines ranging from neuroscience to tissue engineering to plant science . Evidence of validation is important as it allows scientists to choose antibodies that are fit for their experiments and avoid wasting time optimising antibodies that are unsuitable . Validation data also provides reviewers a guide as to whether the antibodies used in a manuscript are likely to give reliable results, something which helps to ensure experimental reproducibility, a topical issue in today s life sciences . Validating an antibody is a complicated process that can involve many different approaches (bordeaux et al ., 2010; howat et al ., 2014) historically antibody validation commonly involved the now controversial antigen pre - adsorption test (holmseth et al ., 2012), while current studies may make use of knockout or knockdown tissue to demonstrate specificity . There are also large scale approaches, capable of validating many antibodies simultaneously (holm et al ., 2012). However, there is no simple experiment that can validate an antibody for all possible applications and samples . For example, validating an antibody for western blotting using a human cell line, does not guarantee the antibody will be suitable in immunohistochemistry using tissue from a rat . Instead antibody validation is a gradual process which involves testing the antibody for specific applications and species / tissues of interest, ideally using a number of approaches . Our aim is to encourage publishing of all independent studies, both positive and negative, which increase understanding of how antibodies perform . These can range from large studies involving hundreds of antibodies, or the use of many tissues or cell lines, to small single figure studies focusing on an individual antibody in a specific setting . The main criteria are; the research group performing the validation should be independent from the company who supply the antibody, the experiments have been sufficiently repeated and the results are accurately and fully reported . It is also crucial that the materials and methods provide enough detail to allow the experiments to be reproduced, something which is often not the case with studies using antibodies (helsby et al ., 2013). A key part of ensuring reproducibility is to make sure the antibodies can be identified by including their supplying company name and code and a resource identifier issued by the research identification initiative (http://scicrunch.com/resources). The instructions to authors (box 1) and guidelines for reviewers have been tailored to facilitate the aim of encouraging a broad range of papers, with a focus on reproducibility and accurate reporting, rather than perceived impact extract from the instructions to authors the antibody validation article collection aims to provide a platform for antibody validation studies and enhance the reliability and reproducibility of antibodies in scientific research . Referees reviewing validation studies will not focus on novelty and impact, but rather on whether the study is scientifically sound and provides all relevant information . F1000research accepts a variety of validation studies, which will be published as research notes: new antibodies; either against a new target or a new antibody raised against an existing target . New applications for existing antibodies; either in a new biological system or a new application tested within an existing / previously tested biological system . Existing antibody applied to a new biological system; new organism / tissue / cell type . Validations of previously tested antibodies that are carried out in more depth than before, in one or more applications . The antibody validation article collection aims to provide a platform for antibody validation studies and enhance the reliability and reproducibility of antibodies in scientific research . Referees reviewing validation studies will not focus on novelty and impact, but rather on whether the study is scientifically sound and provides all relevant information . F1000research accepts a variety of validation studies, which will be published as research notes: new antibodies; either against a new target or a new antibody raised against an existing target . New applications for existing antibodies; either in a new biological system or a new application tested within an existing / previously tested biological system . Existing antibody applied to a new biological system; new organism / tissue / cell type . Validations of previously tested antibodies that are carried out in more depth than before, in one or more applications . This broad approach should encourage a wide range of studies, many of which may never be published without this initiative and we hope that as the collection grows it will become a valuable resource for the thousands of researchers who use antibodies.
Degeneration of the dopaminergic system is still considered to be the pathological hallmark of parkinson s disease (pd), although the serotonergic, cholinergic, noradrenergic and gamma - amino - butyric acid systems are affected as well (hirsch et al . 2003). Importantly, when compared to the dopaminergic circuitry, these systems may actually be involved to a larger degree in the early stages of pd (braak et al . Abnormalities in non - dopaminergic systems have now been implicated in the diversity of part of the motor and non - motor symptomatology in pd (chaudhuri 2006; de rijk et al . In particular, due to the widespread serotonergic innervations, serotonin (5-ht) may play a role in regulating other neurotransmitter activities (fink et al . Post - mortem neurochemical studies in pd have detected up to a 50% 5-ht loss in both the cortex and the basal ganglia brain regions (birkmayer et al . . Moreover, neuronal loss and lewy body formation not only affect the dopaminergic neurons (forno 1996) but also the serotonergic system (jellinger 1987). Whereas, loss of 5-ht in post - mortem pd studies is well established, its degeneration in vivo and particularly its potential role in affecting clinical pd presentation still awaits elucidation . Various lines of evidence have suggested that 5-ht may be involved in the etiology of resting tremor in pd . Loss of striatal dopamine transporter (dat) binding has been shown to correlate with rigidity / bradykinesia, but not with tremor (spiegel et al . 2006) and, unlike rigidity and bradykinesia the response of resting tremor to levodopa therapy is variable at best (koller et al . These findings would argue against a pure dopaminergic etiology of resting tremor . Since the late 1960s there has been continuous debate over the genesis of tremor . Lesion studies in animal models of parkinsonian tremor have mainly targeted midbrain and cerebellar areas (wilms et al . 1999), thereby mainly lesioning ascending serotonergic projections to the forebrain . Tremor induced by harmaline in rats and chickens can be effectively antagonized by 5-ht supplementation (bowman et al . It has been hypothesized that this tremorgenic compound may act indirectly by inhibiting the inhibitory serotonergic projections arising from the raphe nuclei (headley et al . Finally, decrease in midbrain 5ht-1a receptors in pd along with an association with resting tremor has been recently reported in a c - way 100635 pet study (doder et al . Selective neurodegeneration within the thalamus may well contribute to the motor and non - motor symptoms of pd (henderson et al . 2000a, b). Noteworthy is thalamic involvement in the extensive cerebral oscillatory network that is postulated to determine resting tremor in pd, as reported in various magnetoencephalografic studies (schnitzler et al . 2006; timmermann et al . 2003; volkmann et al . Based on primate studies, experimental tremor generated by a thalamo - cortical mechanism has been defined as a parkinson - like tremor, whereas, the olivo - cerebellar system was assessed to be responsible for the faster physiological tremor (delong 1978; lamarre et al . 1975). In the 1980s deep brain stimulation (dbs) was first applied to the thalamus in patients with severe tremor (benabid et al . A multicenter european study reported thalamic dbs to be effective on pd tremor, while the other parkinsonian hallmarks did not receive consistent benefits (limousin et al . Indeed, thalamic dbs is recommended for patients disabled only by tremor (limousin - dowsey et al . Several post - mortem studies as well as biochemical studies of breakdown products of 5-ht in the cerebral spinal fluid (csf) have shown reduced 5-ht activity in the brain of pd patients (birkmayer et al . Moreover, there is a functional polymorphism in the 5-ht transporter (5-htt) synthesis, and the short allele (which synthesizes less 5-htt) is associated with pd depression (mossner et al . Treatment with selective 5-ht reuptake inhibitors (ssri), e.g., citalopram, improves pd depressive symptoms (antonini et al . 2006), although a role for dopaminergic drugs has also been suggested (barone et al . 2006). Interestingly, a [i]-cit spect study showed the capability of citalopram to block thalamic 5-htt in depressed patients (pirker et al . 1995), thus demonstrating the thalamus to be an anatomic site of action of an ssri . In vivo displacement studies in primates have shown striatal [i]-cit binding to be predominantly associated with dat, whereas, binding in the diencephalon was mainly associated with 5-htt (innis et al . [i]-cit has been shown to be useful in monitoring the dopaminergic degeneration of the nigrostriatal pathway in pd (innis 1994; tissingh et al . 1998), but it may be also used to assess the integrity of thalamic 5-htt (de win et al . 2005). In the light of these data we chose to investigate 5-htt binding in the thalamic area by means of [i]-cit spect . The primary aim of this study was to assess whether in pd thalamic 5-htt binding is decreased in vivo . Consequently, we investigated thalamic 5-htt binding as well as striatal dat binding in 32 unrelated drug - nave early pd patients and in 13 healthy subjects using [i]-cit spect . Additionally, we hypothesized that 5-htt binding declines as disease progresses . In order to verify this hypothesis we repeated scans on 26 patients in an average of 17 months later . We postulated that 5-htt loss may be associated with the presence of tremor and depression at disease onset . We studied 32 drug - nave early - stage pd patients as well as 13 healthy volunteers . Diagnosis of pd was made according to uk brain bank criteria (hughes et al . 1992). Patients were recruited regardless of their phenotype, i.e., presence of tremor or depression, as the primary aim of the study was to investigate whether 5-htt binding has decreased in vivo in an early drug - nave pd population . A [i]-cit follow - up scan was performed on 26 patients out of the initial group over a period (mean sd) of 17 9 months . At baseline, all patients were drug - nave for dopaminergic treatment and two patients used benzodiazepines, whereas, at follow - up, they had initiated dopaminergic medication (levodopa or a d2 agonist). Pd patients with dementia were not included: a mini - mental state examination (mmse) score below 26 was used as an exclusion criterion . At baseline, pd severity was assessed using the motor part of the unified parkinson s disease rating scale (updrs - iii). Based on updrs tremor scores (total score on item 20), we categorized patients in two subgroups: pd patients with moderate / severe tremor at onset (pdt subgroup; tremor score 2 in at least one limb) and pd patients without tremor at onset (pdwt subset; tremor score of 0). Note that all patients with tremor had a score equal to zero on item 21: they revealed resting but no action tremor during clinical examination . The two groups displayed no significant difference in rigidity and bradykinesia updrs scores (items 18, 19, 22, 27, 28, 29, 30 and 31). In contrast, patients (n = 12) who had a updrs tremor score of 1 were not included in any of the subset . At the time of the first scan, the beck depression inventory scale (bdi) was used to assess depressive symptoms (visser et al . See table 1 for details on demographic and clinical data of the participants . Table 1demographic and clinical data of the participantsbaseline17 months follow - upco (n = 13)pd (n = 32)pdt (n = 8)pdwt (n = 12)pd (n = 26)pdt (n = 6)pd wt (n = 11)sex (male)822551745age (years, mean sd)53 854 1055 956 1056 954 958 7disease duration (years, mean sd)2 12 12 14 14 14 1updrs motor score18 716 619 8updrs tremor score (item 20)1 13 10updrs bradykinesia / rigidity score (items:18 + 19 + 22 + 27 + 28 + 29 + 30 + 31)13 69 613 6bdi score8 67 78 6pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation data not available demographic and clinical data of the participants pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation data not available patients were recruited at the vu university medical center (vumc), movement disorders clinic . The hospital ethics committee approved the study and all patients signed informed consent forms . Spect studies were performed using a brain - dedicated system, the strichmann medical equipment 810. the strichmann camera system consists of 12 individual crystals, each equipped with a focusing collimator . The transaxial resolution is 7.6 mm full - width at half - maximum (fwhm) (vermeulen et al . [i]-cit (specific activity of> 185 mbq / nmol; radiochemical purity> 99%) was injected intravenously as a bolus, at a dose of approximately 110 mbq . [i] labeling of -cit was performed as described earlier (tissingh et al . Spect image acquisition was performed 24 h after injection, a time point at which [i]-cit binding to striatal dat and thalamic 5-htt is in an equilibrium state (pirker et al . Slices were acquired during 300 s periods from the orbitomeatal line to the vertex with an interslice distance of 10 mm . Data acquisition took place in a 128 128 matrix . Attenuation correction and reconstruction of the images analysis of the [i]-cit binding was performed on two contiguous transverse slices representing the most intense striatal and thalamic binding . A standard region of interest (roi) template was manually drawn with the aid of a stereotactic atlas . In order to distinguish the forebrain from the brainstem, we defined the striatal inferior level to be the anatomic limit between the midbrain and the thalamic region . Specific striatal [i]-cit binding to dat in whole striatum, putamen and caudate nucleus and specific thalamic [i]-cit binding to 5-htt were calculated using the formula: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\left [{{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi}}} \right)} - {\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}} \right]}} \mathord{\left/ {\vphantom {{{\left [{{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi}}} \right)} - {\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}} \right]}} {{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}}}} \right . \kern-\nulldelimiterspace} {{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}} $$\end{document}this formula is referred as the specific to non - specific [i]-cit binding ratio (sns binding ratio). The occipital region was chosen as reference region because of negligible density for both dat and 5-htt (laruelle et al . Difference in mean [i]-cit binding ratio between the various groups was expressed as a percentage and calculated as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\left [{{\left ({{\text{groupx}}\,{\text{sns}} - {\text{groupy}}\,{\text{sns}}} \right)} - {\text{groupx}}\,{\text{sns}}} \right]} \times 100\% . $$\end{document} the investigator performing roi analysis was blind to the subjects diagnosis as well as demographics . An independent sample t test with equal variances not assumed was used to investigate the variation in mean [i]-cit binding values between pd patients and healthy control subjects . We compared [i]-cit binding values between pdt and pdwt cohorts using one - way analysis of variance (anova). A paired simple two tailed t test was used to examine the change between the same group imaging results analyzed at baseline and at follow - up (i.e., pd patients, pdt and pdwt subgroup). The kolmogrov smirnov and the levene s test were applied to screen for normality and equal variance, respectively . Additionally, to increase statistical power, we attempted to normalize pd putamen and pdwt striatum binding values with an algorithmic [log10(x + 1)] and an inverse (1/x) transformation, respectively . Conversely, putamen and thalamic binding values of the pdt cohort at follow - up could not be normalized using these transformations, thus were compared to the respective values of the pdt cohort at baseline using the mann whitney test . The latter was also used to compare putamen and thalamic binding values of the pdt and pdwt subgroup at follow - up . Pearson bivariate correlation was used to correlate bdi scores with thalamic [i]-cit binding values . One - way anova was applied to examine variations in mean bdi score between pdt and pdwt subgroups . Differences between groups were considered significant at a level of p 0.05 . We studied 32 drug - nave early - stage pd patients as well as 13 healthy volunteers . Diagnosis of pd was made according to uk brain bank criteria (hughes et al . 1992). Patients were recruited regardless of their phenotype, i.e., presence of tremor or depression, as the primary aim of the study was to investigate whether 5-htt binding has decreased in vivo in an early drug - nave pd population . A [i]-cit follow - up scan was performed on 26 patients out of the initial group over a period (mean sd) of 17 9 months . At baseline, all patients were drug - nave for dopaminergic treatment and two patients used benzodiazepines, whereas, at follow - up, they had initiated dopaminergic medication (levodopa or a d2 agonist). Pd patients with dementia were not included: a mini - mental state examination (mmse) score below 26 was used as an exclusion criterion . At baseline, pd severity was assessed using the motor part of the unified parkinson s disease rating scale (updrs - iii). Based on updrs tremor scores (total score on item 20), we categorized patients in two subgroups: pd patients with moderate / severe tremor at onset (pdt subgroup; tremor score 2 in at least one limb) and pd patients without tremor at onset (pdwt subset; tremor score of 0). Note that all patients with tremor had a score equal to zero on item 21: they revealed resting but no action tremor during clinical examination . The two groups displayed no significant difference in rigidity and bradykinesia updrs scores (items 18, 19, 22, 27, 28, 29, 30 and 31). In contrast, patients (n = 12) who had a updrs tremor score of 1 were not included in any of the subset . At the time of the first scan, the beck depression inventory scale (bdi) was used to assess depressive symptoms (visser et al . See table 1 for details on demographic and clinical data of the participants . Table 1demographic and clinical data of the participantsbaseline17 months follow - upco (n = 13)pd (n = 32)pdt (n = 8)pdwt (n = 12)pd (n = 26)pdt (n = 6)pd wt (n = 11)sex (male)822551745age (years, mean sd)53 854 1055 956 1056 954 958 7disease duration (years, mean sd)2 12 12 14 14 14 1updrs motor score18 716 619 8updrs tremor score (item 20)1 13 10updrs bradykinesia / rigidity score (items:18 + 19 + 22 + 27 + 28 + 29 + 30 + 31)13 69 613 6bdi score8 67 78 6pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation data not available demographic and clinical data of the participants pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation data not available patients were recruited at the vu university medical center (vumc), movement disorders clinic . The hospital ethics committee approved the study and all patients signed informed consent forms . Spect studies were performed using a brain - dedicated system, the strichmann medical equipment 810. the strichmann camera system consists of 12 individual crystals, each equipped with a focusing collimator . The transaxial resolution is 7.6 mm full - width at half - maximum (fwhm) (vermeulen et al . [i]-cit (specific activity of> 185 mbq / nmol; radiochemical purity> 99%) was injected intravenously as a bolus, at a dose of approximately 110 mbq . [i] labeling of -cit was performed as described earlier (tissingh et al . Spect image acquisition was performed 24 h after injection, a time point at which [i]-cit binding to striatal dat and thalamic 5-htt is in an equilibrium state (pirker et al . Slices were acquired during 300 s periods from the orbitomeatal line to the vertex with an interslice distance of 10 mm . Data acquisition took place in a 128 128 matrix . Attenuation correction and reconstruction of the images analysis of the [i]-cit binding was performed on two contiguous transverse slices representing the most intense striatal and thalamic binding . A standard region of interest (roi) template was manually drawn with the aid of a stereotactic atlas . In order to distinguish the forebrain from the brainstem, we defined the striatal inferior level to be the anatomic limit between the midbrain and the thalamic region . Specific striatal [i]-cit binding to dat in whole striatum, putamen and caudate nucleus and specific thalamic [i]-cit binding to 5-htt were calculated using the formula: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${{\left [{{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi}}} \right)} - {\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}} \right]}} \mathord{\left/ {\vphantom {{{\left [{{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{specific}}\,{\text{roi}}} \right)} - {\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}} \right]}} {{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}}}} \right . \kern-\nulldelimiterspace} {{\left ({{\text{mean}}\,{\text{counts}}\,{\text{in}}\,{\text{non}} - {\text{specific}}\,{\text{roi}}} \right)}} $$\end{document}this formula is referred as the specific to non - specific [i]-cit binding ratio (sns binding ratio). The occipital region was chosen as reference region because of negligible density for both dat and 5-htt (laruelle et al . Difference in mean [i]-cit binding ratio between the various groups was expressed as a percentage and calculated as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $${\left [{{\left ({{\text{groupx}}\,{\text{sns}} - {\text{groupy}}\,{\text{sns}}} \right)} - {\text{groupx}}\,{\text{sns}}} \right]} \times 100\% . $$\end{document} the investigator performing roi analysis was blind to the subjects diagnosis as well as demographics . Statistical analysis was performed using the spss 13.0 software package . An independent sample t test with equal variances not assumed was used to investigate the variation in mean [i]-cit binding values between pd patients and healthy control subjects . We compared [i]-cit binding values between pdt and pdwt cohorts using one - way analysis of variance (anova). A paired simple two tailed t test was used to examine the change between the same group imaging results analyzed at baseline and at follow - up (i.e., pd patients, pdt and pdwt subgroup). The kolmogrov smirnov and the levene s test were applied to screen for normality and equal variance, respectively . Additionally, to increase statistical power, we attempted to normalize pd putamen and pdwt striatum binding values with an algorithmic [log10(x + 1)] and an inverse (1/x) transformation, respectively . Conversely, putamen and thalamic binding values of the pdt cohort at follow - up could not be normalized using these transformations, thus were compared to the respective values of the pdt cohort at baseline using the mann whitney test . The latter was also used to compare putamen and thalamic binding values of the pdt and pdwt subgroup at follow - up . Pearson bivariate correlation was used to correlate bdi scores with thalamic [i]-cit binding values . One - way anova was applied to examine variations in mean bdi score between pdt and pdwt subgroups . Differences between groups were considered significant at a level of p 0.05 . There was no difference with regard to demographic features (age and disease duration) between pd patients and healthy controls and between the pdt and pdwt cohorts (table 1). Pd patients had an average of 61% lower putaminal sns [i]-cit binding ratios than healthy controls . In addition, average caudate nucleus and whole striatum sns binding ratios were also significantly lower in pd patients compared to healthy controls (table 2). We found a significant reduction of 22% in mean thalamic sns [i]-cit binding ratio in pd patients versus healthy controls (table 2). Interestingly, mean sns thalamic binding ratio was significantly lower in the pdt as compared to the pdwt cohort by 19% (table 2, fig . 2). Conversely, there was no significant difference in whole striatum, putamen and caudate nucleus average sns binding ratios between pdt and pdwt (table 2). Table 2mean specific to non - specific [i]-cit binding ratios (mean sd) assessed at baselineregion of interestbaselinecontrols (n = 13)pd patients (n = 32)pdt (n = 8)pdwt (n = 12)striatum, whole8.65 2.614.39 1.10 * 4.38 0.954.21 1.45caudate, whole9.43 2.556.34 1.55 * 5.60 0.986.93 1.93putamen, whole7.72 2.653.02 0.85 * 3.08 0.792.87 1.10thalamus, whole1.98 0.561.55 0.34 * 1.37 0.371.70 0.33 * * significant difference between controls and pd patients (* p 0.02) and between pdt and pdwt patients (p = 0.05)pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation mean specific to non - specific [i]-cit binding ratios (mean sd) assessed at baseline * significant difference between controls and pd patients (* p 0.02) and between pdt and pdwt patients (p = 0.05) pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation average putaminal sns [i]-cit ratio in repeated scans showed a significant decline of 17% from the baseline (table 3). Notably, we found that thalamic sns [i]-cit binding ratio was significantly further decreased at follow - up as compared to the pd baseline measurement by 29% (table 3; fig . 1). Note that there was no significant difference in thalamic sns binding ratios between pdt and pdwt subgroups at the time of the second [i]-cit spect (fig . 2). Whereas, there was no significant difference in putaminal and thalamic radiotracer binding between pdt cohort at baseline, and at follow - up putaminal and thalamic [i]-cit binding was significantly decreased in the pdwt subgroup at the time of the second as compared to the first scan (19 and 39%, respectively) (table 3; fig . 2). Table 3mean specific to non - specific [i]-cit binding ratio (mean sd) at baseline and at follow - up (17 9 months later)region of interestbaseline pd (n = 26)follow - up pd (n = 26)baseline pdt (n = 6)follow - up pdt (n = 6)baseline pdwt (n = 12)follow - up pdwt (n = 11)striatum, whole4.17 0.953.86 0.754.16 0.983.79 0.423.91 1.13.64 0.73caudate, whole6.13 1.475.60 1.365.88 1.365.65 0.685.76 1.585.52 1.16putamen, whole2.84 0.712.36 0.51 * 2.83 0.522.37 0.292.74 0.932.22 0.40thalamus, whole1.50 0.351.07 0.39 1.26 0.341.16 0.291.65 0.311.00 0.45 * * significant difference between pd patients at baseline and at follow - up (p <0.01) and between pdwt subgroup at baseline and at follow - up (p <0.05)pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviationfig . 1scatterplot showing the rate of change in thalamic specific to non - specific [i]-cit binding ratio in healthy controls and in pd patients at baseline and at follow - up . At the time of the first imaging series, mean thalamic [i]-cit binding ratio was significantly lower in pd patients as compared to controls (p = 0.02). Moreover, mean thalamic ratio obtained in pd patients was significantly lower at the follow - up as compared to the baseline (p <0.01). Pd baseline and pd follow - up indicate pd patients at baseline and at the follow - up, respectivelyfig . 2scatterplot showing the rate of change in thalamic specific to non - specific [i]-cit binding ratio in the pdwt as compared to the pdt subgroup over time . Mean thalamic [i]-cit binding ratio was significantly lower in the pdt versus the pdwt subgroup at baseline (p = 0.05). No difference in mean thalamic ratio between the pdt and the pdwt subset was detected at the time of the second imaging series . Moreover, mean thalamic ratio in the pdwt subgroup was significantly lower at follow - up than at baseline (p <0.01). Pdt and pdwt indicate pd patients subgroups with moderate / severe tremor and without tremor at onset, respectively mean specific to non - specific [i]-cit binding ratio (mean sd) at baseline and at follow - up (17 9 months later) * significant difference between pd patients at baseline and at follow - up (p <0.01) and between pdwt subgroup at baseline and at follow - up (p <0.05) pd parkinson s disease, pdt pd patients subgroup with moderate / severe tremor at onset, pdwt pd patients subgroup without tremor at onset, sd standard deviation scatterplot showing the rate of change in thalamic specific to non - specific [i]-cit binding ratio in healthy controls and in pd patients at baseline and at follow - up . At the time of the first imaging series, mean thalamic [i]-cit binding ratio was significantly lower in pd patients as compared to controls (p = 0.02). Moreover, mean thalamic ratio obtained in pd patients was significantly lower at the follow - up as compared to the baseline (p <0.01). Pd baseline and pd follow - up indicate pd patients at baseline and at the follow - up, respectively scatterplot showing the rate of change in thalamic specific to non - specific [i]-cit binding ratio in the pdwt as compared to the pdt subgroup over time . Mean thalamic [i]-cit binding ratio was significantly lower in the pdt versus the pdwt subgroup at baseline (p = 0.05). No difference in mean thalamic ratio between the pdt and the pdwt subset was detected at the time of the second imaging series . Moreover, mean thalamic ratio in the pdwt subgroup was significantly lower at follow - up than at baseline (p <0.01). Conversely no difference was found in the pdt subset . Pdt and pdwt indicate pd patients subgroups with moderate / severe tremor and without tremor at onset, respectively thalamic sns binding ratios did not correlate with bdi scores and there was no difference in bdi score between pdt and pdwt (table 1). We found that mean [i]-cit spect putaminal binding was reduced in all early pd patients at baseline as well as at follow - up . Moreover, average thalamic 5-htt binding was decreased in early drug - nave pd patients compared to controls and declined as the disease proceeded . The pdt cohort displayed a lower 5-htt thalamic binding when compared to the pdwt cohort at baseline, while no difference could be detected during follow - up . Finally, in the pdwt subgroup a further decline in both putaminal and thalamic binding was detected at follow - up, which was not true for the pdt subgroup . To our knowledge, this is the first longitudinal study to investigate 5-htt loss in pd . Van dyck et al . (2000) reported a mean 4.2% per decade age - related decline of 5-htt in the diencephalon of healthy humans by means of [i]-cit spect . However, this decay rate is much lower that the one we presently reported in pd patients (29% over 17 months). Therefore, whereas 5-htt loss may occur with age on a physiological basis, consistent with our results its decay appears more severe in pd patients . These data confirm the first two hypotheses we stated, according to our results 5-htt is decreased in pd and further declines along with the disease . Noteworthy, it has been argued that [i]-cit radiotracer in the thalamus may bind to monoaminergic transporters other than 5-htt . In particular, in a monkey pet study (farde et al . 1994) citalopram as well as desipramine, a norepinephrine transporter (net) blocker, was able to displace thalamic [i]-cit binding by 50 and 40%, respectively . (1993) demonstrated that the selective net inhibitor maprotiline failed to affect [i]-cit binding in monkey diencephalon . (2005) validated [i]-cit to assess in vivo 5-htt in the thalamic area . Importantly, in this study, a double - blind, placebo - controlled, crossover design with citalopram (the most selective 5-htt blocker) was performed . Moreover, it has been demonstrated that citalopram blocks approximately 50% of thalamic [i]-cit binding in depressed patients (pirker et al . 2007), venlafaxine (a 5-htt and net inhibitor) proved to decrease [i]-cit spect binding by 54%, an outcome similar to the one obtained by pirker et al . Therefore, it may be argued that in vivo only a small part of the [i]-cit binding to human thalamus is due to net binding . Finally, it must be taken into account that in the human brain 5-htt is much higher than net thalamic concentration (tong et al . 2007), while the affinity of [i]-cit (or rti-55) for net is lower than for 5-htt (scheffel et al . 1997). To summarize, although we cannot exclude that in vivo [i]-cit binding in the human thalamus, may be partly due to net binding; the largest part likely represents 5-htt binding . Nonetheless, further studies with selective radiotracers for 5-htt are needed to reproduce our findings . In this context, two previous pet studies with selective 5-htt tracers found no significant thalamic 5-htt decrease in pd patients when compared to controls (guttman et al . In particular, kerenyi et al . Describe a trend for lower thalamic 5-htt binding with a decrease of approximately 25%, a range loss similar to the one we presently report (22%). Importantly, the small number of participants investigated in the pet studies mentioned above (9 and 13, respectively) may have played a role for a non - significant thalamic decreased binding outcome . This may be relevant to the present study where a thalamic 5-htt decline over time was demonstrated in a larger cohort of 26 pd patients . At baseline, we found lower thalamic 5-htt density in pd patients with mild to severe tremor compared to patients without tremor . The two cohorts (pdt and pdwt) differed in terms of updrs tremor scores but were comparable in terms of rigidity and bradykinesia . In fact, pdt had a updrs resting tremor score 2 in at least one limb, whereas, the pdwt presented no tremor at all . Resting tremor was the only one out of the cardinal motor features that clinically characterize pd (tremor, bradykinesia, rigidity and postural instability) to differentiate the two patient subsets . Moreover, the absence of significant differences in striatal dat binding between the two subgroups confirms the assumption we made based on previous studies: bradykinesia and rigidity but not tremor correlates with striatal dat binding (spiegel et al . 2006). These data suggest that 5-ht may play a role in the etiology of resting tremor in early pd stages . In this context, haapaniemi et al . Reported a lower thalamic [i]-cit binding in pd compared to controls, although, in contrast with our results, no correlation to tremor scores was detected (haapaniemi et al . We obtained data 24 h post - injection, whereas they acquired scans at 4 h post - injection . Although there is still considerable debate as to the best timing to acquire spect images (brucke et al . 1994b), scans obtained between 20 and 24 h showed to be closer to a state of transient equilibrium (pirker et al . 2000). Recently, doder et al . (2003) reported a reduction of in vivo raphe 5-ht1a binding in pd patients and an association with the severity of updrs tremor scores using c - way 100635 pet . The authors suggested that this reduction expresses loss of 5-ht cell bodies, possibly due to lewy body degeneration . Our present finding indicating thalamic [i]-cit binding decline is in agreement with this hypothesis, since the 5-htt is located exclusively on the 5-ht neuronal membrane . Alternatively, 5-ht and dopaminergic activity may decline in parallel in the striatum and in the midbrain of parkinsonian patients, particularly in patients with tremor onset . Reduced serotonergic function would result in less inhibition and in turn facilitate striatal dopamine release (de et al . We investigated pd patients at onset, and did not investigate radiotracer binding within raphe nuclei; whereas doder et al . Examined participants at an advanced stage and did not analyze the thalamic area . Note that unlike their study, we chose not to correlate thalamic binding to updrs total tremor scores . In fact, patients we enrolled were at an early stage and thus presented a very low total tremor score (mean items 20 total score sd = 1 1). Due to such a low variance in total tremor score, a correlation between these values and sns thalamic binding could not have been evaluated adequately . Also note that both studies did not take into account 5-htt levels in striatal and cortical regions, which express low to moderate 5-htt concentrations . According to braak et al . (2003) midbrain stem is the first component of the somato - motor and emotional motor system to be affected, whereas, the degeneration of cells in the substantia nigra, thalamic nuclei and neocortical areas follows . In the light of this evidence, a comparison between 5-htt availability among brain areas presenting 5-htt in subsequent pd stages by means of selective 5-htt tracers is warranted to better assess the role of 5-ht in pd . In fact, although in pd alterations of striatal and cortical 5-htt may be associated with tremor, in the present study we were not able to assess striatal 5-htt binding, as [i]-cit binds predominantly to dat in the striatum (laruelle et al . Additionally, although we provided some evidence that [i]-cit spect may be used to measure 5-htt in 5-htt - low cortical areas, these measurements must be interpreted with caution (de win et al . Unlike baseline imaging, the repeated scans showed no statistical difference in thalamic 5-htt binding between the pdwt and pdt group . Furthermore, the pdwt subgroup revealed a faster 5-htt thalamic decline as compared to the pdt group . It has been demonstrated that pd patients, who manifest symptoms at a relatively [i]-cit high density, progress substantially faster than those who manifest symptoms at a lower transporter density (seibyl et al . 1999). While we do not have an explanation for this phenomenon, a progressive loss of surviving neurons could be postulated . Both lewy body degeneration and reduced 5-htt inhibiting activity over the striatum might have a major effect in those patients still presenting with a high transporter density . Additionally, we were not able to assess whether tremor had eventually revealed in the pdwt subgroup at the time of the second scan, as patients were under dopaminergic treatment . Indeed the lack of follow - up updrs clinical examinations represents a limitation of our study . 5-ht neurotransmission is impaired in pd, but its causal relationship with tremor remains controversial and it is unclear what the clinical relevance of a 22% thalamic 5-htt loss is . Due to the design of the present study, our findings do not allow a definitive answer on pd tremor but certainly supports the role of 5-htt function . As of yet, [i]-cit spect imaging correlating 5-htt binding decline to depressive symptoms in pd has revealed inconsistent results . Kim et al . (1999) reported no significant correlation between hypothalamic / midbrain 5-htt in pd and updrs - i (depression items). A further study showed no relationship between 5-htt binding and updrs rating of mood in thalamus, but detected a significant correlation in dorsal midbrain (murai et al . Finally, haapianemi and colleagues demonstrated that 5-htt frontal region binding in pd correlates to updrs - i (haapaniemi et al ., we found no correlation between bdi scores and thalamic 5-htt and, even more importantly, there was no difference in bdi between the pdt and pdwt subgroups suggesting no contribution of depressive symptoms to these findings . Whereas, we used bdi, a reliable self - assessment scale to assess depression in pd (visser et al . 2006), other studies used updrs rating for mood, which is a less specific screening tool . We did not assess bdi at the follow - up, therefore depressive symptoms may not have been revealed until a more advanced stage, along with thalamic 5-htt decline . In conclusion, in this study we show loss of thalamic 5-htt binding in pd and its progressive loss over an average of 17 months . At baseline, a decreased thalamic 5-htt density in patients presenting tremor at onset was found, while no correlation could be detected between thalamic [i]-cit binding ratios and bdi scores . In this context, further studies with selective 5-htt tracers are needed to reproduce our findings in early pd . Additionally, new research is warranted to gain a better insight into the causal relation that links 5-ht dysfunction and tremor and depression in pd . Finally, pharmacological challenges with serotonergic agents may be performed to test their potential capacity in counteracting pd tremor.
Pancreatic cancer is well known to be an aggressive and highly malignant condition with varied ways of presentation . Pancreatic cystic neoplasms (pcns) are very uncommon causes of pancreatic malignancy and are often ignored or missed, especially in the early stages . They can be considered as confusing diagnostic problems that are being encountered with greater frequency, especially with modern and advanced imagining technology [1, 2]. Pcns account for <10% of all pancreatic malignancies, but for up to 30% of pancreatic resections performed, and they encompass a spectrum of benign, premalignant (borderline) and malignant lesions [1, 2, 3]. Although relatively rare, it is important to distinguish cystic neoplasms from the far more prevalent entity of benign pseudocysts, which are usually associated with a clinical history and radiographic evidence of pancreatitis . Cystic neoplasms of the pancreas can be classified into four major categories as discussed below . These categories possess varying potentials for malignancy and prognostic outcomes, hence the need for proper diagnosis . We bring to light our experience with a 49-year - old male with no past medical history who was initially diagnosed with acute pancreatitis without any obvious cause at an outside facility after he presented with sudden epigastric pain . He was initially treated conservatively and subsequently discharged home . However, following discharge, his symptoms persisted . Two weeks later, he was readmitted to the outside facility where a laparoscopic cholecystectomy was performed for cholelithiasis that was presumed to be the cause of his persistent symptoms . At the same time he was also found to have a pancreatic pseudocyst on ct of the abdomen / pelvis (fig . 1, fig he was subsequently seen by gastroenterology at our hospital, where he had a eus - fna biopsy of the pancreatic pseudocyst, and was discharged home . However, on discharge, he started experiencing unremitting daily fevers (101104f), nausea and diffuse abdominal pain . At this time he presented to the emergency department as his symptoms continued to worsen . On presentation, he was found to be in painful distress . Physical examination revealed a moderately distended and tender abdomen with guarding in the epigastrium and left lower quadrant . Labs showed a hemoglobin of 11.4 g / dl, a white blood cell count of 10.2 10/l, ast of 85 u / l, alt of 119 u / l and alkaline phosphate of 136 u / l . He was admitted and treated conservatively for presumptive persistent pancreatitis . A ct scan of the abdomen / pelvis with contrast revealed sequela of pancreatitis with complex peri - pancreatic fluid and likely necrosis involving the distal pancreatic body and tail . A fluid level was also identified, raising concern for debris / necrotic tissue versus blood products secondary to recent bleeding into these collections . Multiple ill - defined hypoattenuating lesions scattered throughout the hepatic parenchyma were also identified, although they were considered too small to characterize (fig . The patient was placed on vancomycin and zosyn from admission without any relief of his symptoms . Given these worsening symptoms, he was taken to the operating room for an exploratory laparotomy and possible pancreatic necrosectomy . Upon exploration of the abdomen, on pathological examination, the tumor was found to be composed of highly pleomorphic, discohesive cells with eosinophilic cytoplasm and frequent multinucleated giant cells (fig . The tumor was also positive for epithelial membrane antigen, confirming the epithelial nature of the malignant cells . They range from inflammatory pseudocysts that arise from acute or recurrent pancreatitis to pcns that possess varying malignant potential . Cystic neoplasms are a broad group with various subtypes that are responsible for about 1% of pancreatic cancers . Presenting symptoms and signs are nonspecific and are no basis for diagnosis . In most cases, they remain asymptomatic until they are incidentally discovered or up to very late stages, especially in malignant subtypes . They are typically benign, multicystic lesions formed by glycogen - rich, periodic acid - schiff - positive epithelial cells with a central scar that can be found throughout the pancreas . Abdominal discomfort is the major presenting feature and is mainly secondary to expanding tumor size . Mucinous cyst adenomas are a second subtype and just like their serous counterparts are common among middle - aged females, with a higher potential for malignancy . On imaging they may be seen as one or more cysts with thick, irregular walls along the body and/or tail of the pancreas . Cystic fluid analysis of mucinous cystadenomas reveals a positive mucin stain with an elevated cea level . Intrapapillary mucinous neoplasia is a third subtype and unlike the subtypes discussed earlier, it is more common in middle - aged and older males . Lastly, papillary cystic neoplasms are a rare form of cystic neoplasms found in young females . They have a malignant potential, are locally aggressive and can be found along the body and/or tail of the gland . The most important factor to keep in mind with cystic neoplasms this will help assess the potential for malignancy and hence guide appropriate therapy . According to grobmyer et al . The risk of malignancy is directly proportional to the size of the cysts, especially in mucinous subtypes . Asymptomatic cysts that are <5 cm at their widest diameter without concerning features such as nodularity and/or calcifications can be serially monitored at least yearly with imaging . Eus - fna is usually added to obtain cystic fluid for analysis and cytology, thereby enhancing diagnostic accuracy . Molecular markers and cystic dna are other methods that can be employed in proper diagnosis . In a prospective, multicenter study, khalid et al . [7, 8] and jones et al . Concluded that elevated amounts of pancreatic fluid dna, high - amplitude mutations and specific mutation acquisition sequences are indicators of malignancy . Lesions with a high risk of malignancy and caught early are usually managed by surgery . Lesions in the pancreatic head are resected by a pancreaticoduodenectomy while those in the distal body and tail are resected by a pancreatectomy . Serous cyst adenomas that are asymptomatic and do not raise concern of malignancy do not need to be resected . Cysts that are suspicious for malignancy in patients who are not surgical candidates can be managed with eus - guided ethanol lavage with paclitaxel injection . Cysts with widespread metastases have a very poor prognosis and palliative options may need to be explored . Our experience shows the potential extent of malignant pcns if left untreated or undiscovered . In a matter of 12 weeks, our patient went from being diagnosed with what was initially thought to be acute pancreatitis to metastatic malignant mucinous cystic pancreatic neoplasm that eventually cost him his life . It is our hope that our experience and this article bring to light the importance of proper diagnosis of pcns.
Heart failure can be considered as the last stage of heart diseases and as a significant cause of mortality and morbidity throughout the world.1 a limited effort tolerance is frequently both the first and most important clinical characteristic of heart failure, reflecting the reduction in cardiac function and the alteration in reflex, metabolic, vascular and muscular function.1,2 the maximal exercise cardiopulmonary test is performed to evaluate the functional capacity, therapeutic response and prognosis in heart failure patients.3,4 the six - minute walk test is proposed to be a submaximal exam that could reproduce a patient s daily physical activities and evaluate the prognosis of heart failure patients.5 however, if the original version is followed, the six - minute walk test could demand a higher exercise intensity than that demanded during the patient s daily activities,68 as expressed by a respiratory quotient (rq)>1.0.6 the centers for disease control and prevention recognize submaximal activities as 3 to 6 mets, which corresponds to a moderate intensity for most young to middle - aged adults.6 to make sure that all patients undergo a submaximal test, a careful standardization was proposed using the borg scale (submaximal is defined as between relatively easy and slightly tiring, which is between 11 and 13 on the scale).6 the reproducibility of this new standardization is unknown . The aim of this study was to test the reproducibility of the six - minute treadmill cardiopulmonary walking test (6cwt) using the borg scale in patients with stable heart failure and to make sure that all patients undergo a submaximal test . Twenty - three male heart failure patients (509 years) were included in the study . Patients with any of the following were excluded from the study: non - optimized drug therapy, atrial fibrillation, cachexia, interrupted walk test prior to the sixth minute and noncardiovascular functional limitations, such as osteoarthritis and chronic obstructive pulmonary disease . Female patients were not included in this study due to the reduced number of females available during the study period . All patients were in stable clinical condition, without changes in medication for three months . All patients underwent the exercise test on a programmable treadmill (series 2000; marquette electronics; milwaukee, wi, usa) in a temperature - controlled room (2123 c) with continuous electrocardiography monitoring (max 1; marquette electronics; milwaukee, wi, usa), blood pressure monitoring (auscultation method) and ventilation and gas exchange monitoring (computerized system, vmax 229 model, sensormedics, yorba linda, ca, usa). The 6cwt using the borg scale was performed on a treadmill with zero inclination and patient - controlled speed7 . All patients were advised to keep walking during the test at a pace between relatively easy and slightly tiring (between 11 and 13 on the borg scale). Encouragement was standardized with phrases like if you can walk faster, increase the speed, you are doing very well and if it is tiring, you can reduce the speed . The electrocardiography, ventilatory, hemodynamic and gas exchange variables were continuously evaluated during the test but only collected at the sixth minute . The protocol was approved by the ethical committee of heart institute of so paulo s medical school . Data were analyzed using spss statistical software for windows version 11.5 (spss inc ., chicago, il, usa). The descriptive analysis was presented as mean and standard deviation . Intraclass correlation coefficients (ri), with a 95% confidence interval, were calculated using analysis of variance (anova)24 for the following variables: ventilation (ve, l / min), rq, oxygen consumption (vo2, ml / kg / min), ve / vco2 slope, heart rate (hr, bpm), systolic blood pressure (sbp, mmhg), diastolic (dbp, mmhg) blood pressure and distance (miles). All patients were well - adapted to the six - minute walk test using the borg scale and completed the protocol without interference . Results are listed in table 2 and the linear regression is presented in figure 1 . The distance was highly reproducible between the two tests (0.20 0.03 vs 0.20 0.04 miles or 321 meters; ri=0.88, p<0.0001), as was vo2 (11 2 vs 11 2 ml / kg / min; ri=0.92, p<0.0001). The bland - altman plots for the six - minute walk test using the borg scale are shown in figures 2 and 3 . In this study, we tested the reproducibility of the 6cwt using the borg scale proposed by guimares et al ., 2002.6 the six - minute walk test s guideline does not recommend a treadmill walk test because patients are unable to pace themselves.8 in our study, all patients were well - adapted to the method and were able to pace themselves . Studies with chronic obstructive pulmonary disease showed that the distance walked on a treadmill was less than the distance in a corridor.9,10 following the [current?] Guidelines, the patient s instruction for the six - minute walk test was: walk as far as you can.11 a previous study demonstrated that the six - minute walk test could demand a higher exercise intensity than that achieved while performing daily activities (rq>1.0) when the guideline s standard recommendations are followed.6 the borg scale is a valid measure of relative intensity, showing good correlation with heart and exercise rate.11 some studies require the patient to become familiar with the six - minute walk test.1214 a learning effect of 6% was reported in a cardiac rehabilitation population that completed the test on non - consecutive days.12 in our study, patients did not undergo familiarization, and it was not important for the reproducibility of the principal variables . There is no data available regarding the reproducibility of the six - minute - walk test using the borg scale . In our study, heart rate, it is known that heart rate correlates with exercise intensity14, and we expected this reproducibility because exercise intensity has been standardized by the borg scale . The reproducibility of the maximum cardiopulmonary test was studied previously24 and showed good reproducibility for both heart rate and ventilation at an anaerobic threshold and during a peak exercise test . In the present study, no data are available about the reproducibility of ventilation, heart rate, systolic or diastolic blood pressure during the six - minute walking test . All of our patients showed good rq reproducibility, which remained less than 1.0 during the entire six - minute cardiopulmonary walking test . An rq above 1.0 characterizes a tester near their maximum effort.13 in this study, our methodology assured a submaximal walking test . There are no available studies with six - minute cardiopulmonary walking tests showing rq values . The american thoracic society guidelines for the six - minute walk test11 recommend that patients should walk as far as possible for 6 minutes and recognize that patients will probably get out of breath or become exhausted, suggesting that a maximum exercise test can occur . In our study, the ve / vco2 slope showed good reproducibility . Previous studies showed that the ve / vco2 slope predicts mortality and morbidity in heart failure patients who underwent a maximal cardiopulmonary test.1416 in the 6cwt, this variable is correlated with distance and also predicts mortality and morbidity in patients with primary pulmonary hypertension.14 there is no data available about the ve / vco2 slope during the six - minute walk test in heart failure patients . Reproducibility of the ve / vco2 slope at an anaerobic threshold was investigated in a maximal cardiopulmonary test and showed a good correlation.15 in our study, vo2 at the sixth minute showed good reproducibility . This result could be important in meta - analysis studies because vo2 could be accessed without the influence of a physical therapist s encouragement . Peak oxygen consumption is the standard measurement to evaluate exercise capacity and is a powerful predictor of mortality in heart failure patients.1618 the six - minute walk test distance in heart failure patients correlated with peak o2 consumption and could predict peak vo2 in advanced heart failure patients.30 the centers for disease control and prevention recognize submaximal activities as 3 to 6 mets, which corresponds to moderate intensity for most young to middle - age adults.9 in our study, the mets average was less than 3.2 . The reproducibility of the peak vo2 at anaerobic threshold and during a maximum cardiopulmonary test was investigated, and a good correlation was found.24 walking distance showed good reproducibility in our study . This could be important to evaluate the walking distance in meta - analysis studies without the influence of a physical therapist s encouragement . Previous studies showed that the distance walked during the six - minute walk test strongly predicts mortality and morbidity.16,16,17 a walked distance of less than 300 meters predicts death or hospital admission for inotropic or mechanical bridging to transplantation in a period of six months.18 excess encouragement significantly increased the walked distance19, which is why we standardized it with the borg scale . A study evaluated heart failure patients (n=233) three times: two times within an interval of 30 minutes and another one day after . The tests were performed in a hospital corridor (34 meters of length) with previous familiarization, and all of the exams were very reproducible.15 another study evaluated the distance covered during the six - minute walk test one year after the original test in 1,077 heart failure patients using a 15-meter corridor . The distance correlated with self - perceived symptom changes, as evaluated by a questionnaire . The authors concluded that the distance is sensitive to changes in self - perceived symptoms of heart failure; when the self - perception does not change, the distance walked is reproducible.16 this study was limited by both the number and gender of available patients, although our data showed reproducibility of the most important variables for the 6cwt . Prognosis based on distance should have a different cutoff point than the previously mentioned 300-m cutoff point . More studies with a greater number of heart failure patients (including females) are necessary for a better comprehension of the 6cwt using the borg scale . The 6cwt using the borg scale in heart failure patients was reproducible and assured a submaximal walking test . These results suggest that our methodology may be appropriate for evaluating functional capacity in heart failure patients
Vertebral artery injury is a rare but potentially catastrophic complication of cervical spinal surgery with a reported incidence of 0.07% . Those injuries that have been reported have most commonly involved an anterior approach or following lateral mass screw placement to the cervical spine . Of the 1.8% that have occurred following a posterior approach, all have involved the axial cervical spine . To date, there have been no reports of injury to the subaxial vertebral artery following a routine foraminotomy of the subaxial cervical spine [13]. We describe a case of a patient who developed a delayed left - sided vertebral artery dissection following an uncomplicated left c5/6 foraminotomy . A 46-year - old female presented with a 5-month history of progressive left - sided c6 radiculopathy that had not responded to medical management . Clinical examination confirmed paraesthesia in the left c6 dermatome and mild weakness of the left biceps muscle . A magnetic resonance imaging scan revealed a left - sided c5/6 foraminal stenosis secondary to a focal disc herniation . The initial decompression was performed using a standard high - speed drill with a matchstick attachment and manual irrigation . Following surgical exposure, a successful decompression was achieved and at the time there was no evidence of arterial injury . Throughout the procedure there were no episodes of excessive flexion, extension or lateral rotation at any stage of the procedure . She awoke from the anaesthetic neurologically intact; however, 7 hours later she developed an acute onset of right homonymous hemianopia, expressive dysphasia and became drowsy . We elected to not thrombolyse her due to her surgery but commenced aspirin . Following further neurology review by a stroke consultant on the following morning, we noted that she demonstrated a right homonymous hemianopia without macular sparing, bilateral gaze - evoked and rebound nystagmus, thalamic dysphasia, multifactorial gait difficulty and memory impairment . A ct arteriogram demonstrated an occlusion to the left vertebral artery distal to c7/t1 with reconstitution at the inferior endplate of c5 . This was followed by an area of attenuation and a return to normal calibre at c4 . In addition to this, there was an occlusion of the left distal p3 segment of the posterior cerebral artery . She had acute infarcts involving the left mesial occipital lobe, left posterolateral thalamus, posterior limb of the left internal capsule and left hippocampal tail . In addition, she has bilateral superior cerebellar infarcts with the right side affected more so than the left . She was referred to stroke rehabilitation where she underwent occupational therapy support and visual field testing . As far as we are aware this is the first reported case of delayed vertebral artery dissection following a posterior foraminotomy and rhizolysis [13]. The precise pathophysiology of this complication is difficult to determine given that there was no evidence of injury to the vertebral artery at the time of surgery and there had been no abnormal movements of the neck . Spontaneous dissection of the vertebral artery has been reported in the setting of genetic changes such as low levels of alpha-1 antitrypsin, connective tissue diseases, various genetic polymorphisms, gene mutations, hyperhomocystienemia, the presence of migraines and vessel abnormalities . However, our patient had none of these risk factors and given that the dissection occurred precisely at the surgical site it is difficult to attribute the injury to anything other than either direct or indirect surgical trauma . The absence of any significant haemorrhage at the time of surgery would seem to make indirect trauma more likely and a possible mechanism may be damage induced by use of the high - speed drill . This may occur due to thermonecrosis if the area is not cooled appropriately or due to vibrations transmitted through the bone . Animal studies have demonstrated that repetitive vibration can induce arterial constriction increasing shear stress and subsequent thrombosis . There is also an increase in oxidative activity and an increased sensitivity to alpha-2c - adrenoreceptor mediated vasoconstriction . It has also been demonstrated that vibration between 60 and 800 hz, in the arteries of rat tails, increased discontinuities in the internal elastic membrane with patches of missing tunica intima . In this particular case, it may be that vibrational shear stress contributed to vessel wall injury and subsequent thrombosis; however, this remains to be established . The use of a high - speed surgical drill could have transmitted the vibration through the bone and subsequently caused damage to the tunica intima of the vessel causing a dissection.
Dna sequences coding for the huj591 variable regions were kindly provided by dr . The mb (80 kda) is a bivalent homodimer with each monomer consisting of a single - chain variable fragment (scfv) linked to a human igg1 hinge and ch3 domain . The mb exists as a stable dimer due to the natural association between the ch3 domains as well as the formation of disulfide bonds within the hinge regions . The cys - db (50 kda) is a bivalent homodimer formed from two cross - paired scfv fragments . Each chain consists of the variable regions linked together by a gly - ser linker . A cys tail, consisting of the sequence gly - gly - cys, is fused to the c - terminus to enable the stabilization of the diabody complex by formation of a covalent disulfide bond . Purified huj591 mb and cys - db proteins were provided by imaginab, inc . (inglewood, ca). A 3-fold equivalence of dfo - bz - scn (macrocylics, inc .) In dmso (<5% total volume) was added to a solution of mb or cys - db . Huj591 was reacted with dfo - bz - scn with a 1:5 mab: chelate ratio . All reactions were incubated at 37 c for 45 min with occasional mild stirring . Subsequent purification using a pd10 size exclusion column removed any unreacted dfo - bz - scn and dmso with 0.9% saline as the mobile phase . The purified product was concentrated to a volume of 500 l using a vivaspin 500 (mwco: 10 kda) centrifugal filter . Sds gel electrophoresis was performed to analyze purified dfo conjugates of the mb and cys - db (see the supporting information and si figure 1 in the supporting information). Zr - oxalate (37 mbq) was produced as previously described (see the supporting information). The ph of zr was adjusted to 7.07.2 with 1 m na2co3 . To each separate reaction, approximately 125140 g of mb / cys - db - dfo (mb - dfo, 1.561.75 nmol; cys - db - dfo, 2.52.8 nmol) or 250 g (1.7 nmol) of huj591-dfo was added and incubated at room temperature with intermittent mild shaking . After 11.5 h, the reaction was quenched with 10 l of 50 mm dtpa (ph 7) to remove any nonspecifically bound zr . Crude radiolabeling yields were determined to be> 95% using itlc with the zr - labeled proteins remaining close to the origin at rf = 0.30 while free zr-89 is found near the solvent front at rf = 0.65 . Purification of zr - proteins was performed using a pd10 size exclusion column with saline as the eluting buffer . The final radiochemical purity was> 99% based on itlc analysis . To address retained affinity for the antigen, both radiolabeled constructs zr - mb and zr - cys - db lncap (psma(+)) and pc3 (psma()) human prostate cancer (pc) cell lines (american type culture collection) were cultured in a sterile environment with 5% co2 at 37 c and grown as described . All animal experiments were conducted in accordance with the guidelines set by mskcc animal care and use committee and research animal resource center . For imaging experiments, male athymic nude (nu / nu) mice (68 week old, taconic) lncap cells (3 10) in 1:1 medium: matrigel (bd sciences) were implanted on the left shoulder . Pc3 cells (3 10 in 1:1 medium: matrigel) were injected on the right shoulder . Tumor growth was monitored weekly and measured using vernier calipers with the volume calculated using the formula length width height 0.52 . Internalization of zr - mb, zr - cys - db, and fully human zr - huj591 was investigated on lncap and pc3 cells . Approximately 1 10 cells were seeded in a 12-well plate and incubated overnight . A volume of 2 ml of radiolabeled protein (37 kbq / ml) was added to each well . The plates were incubated at 37 and 4 c for 0.524 h. following each incubation period, the medium was collected and the cells were rinsed with 1 ml of phosphate buffered saline (pbs) twice . Surface - bound activity was collected by washing the cells in 1 ml of 100 mm acetic acid + 100 mm glycine (1:1, ph 3.5) at 4 c . The adherent cells were then lysed with 1 ml of 1 m naoh . Each wash was collected and counted for activity . The% internalized activity was calculated as the ratio of the activity of the lysate and the total activity from the medium, pbs, acid, and base washes . In 12-well plates, 1 (100 g, 0.67 nmol) was either coadministered with the radioactive probes or preincubated for 1 h at 37 c . After addition of zr - mb (12 g, 12.525 pmol) and zr - cys - db (12 g, 2040 pmol) in separate wells, the cells were incubated at 37 c for 1 h and then carefully washed with medium to remove any excess unbound activity . The level of bound radioligands was calculated as% bound, normalized to the amount of activity added . To determine the dissociation constant (kd) of each of the zr - mabs, saturation binding studies briefly, varying concentrations of the zr - mabs (0.033 g / ml, 0.260 pmol / ml) were added to 5 10 cells in pbs containing 1% bovine serum albumin . The same experiment was repeated with additions of cold mabs (140 g / ml for the mb (1.75 nmol / ml) and cys - db (2.8 nmol / ml) and 300 g / ml for huj591 (2 nmol / ml)) to determine nonspecific binding (nsb). After 1 h incubation at room temperature, the cells were washed twice with 1 pbs . Specific binding (sb, nm), derived by subtracting nsb from total bound activity, was plotted against the amount of zr - mabs added . The dissociation constant, kd, was calculated by nonlinear regression fitting of the resulting plot using graphpad prism v. 6.02 . Mice (n = 34) bearing lncap xenografts were administered intravenously (iv) with 7.410.2 mbq of either zr - mb (2838 g, 0.350.48 nmol) or zr - cys - db (2534 g, 0.500.68 nmol) in saline . Small - animal pet studies were conducted using micropet - r4 and focus 120 scanners (concorde microsystems). Using asipro vm software (concorde microsystems), volumes of interest (vois) were measured on various planar sections of the acquired image by manually drawing on the tumor site and on select organs . The mean voi was calculated and expressed as% injected dose per gram of tumor tissue (% id / g). Single tumor bearing mice were administered intravenously with 370555 kbq of either zr - mb (12 g, 12.525 pmol) or zr - cys - db (12 g, 2040 pmol). Competitive inhibition studies were performed with coadministration of 200500 g (2.510 nmol) of nonradioactive mb or cys - db in lncap tumor - bearing mice (n = 35). In a separate cohort of mice (n = 4) bearing the psma(+) tumor, the parent huj591 (500 g, 3.3 nmol) was administered 36 h prior to dosing with zr - mb . Mice were euthanized by co2 asphyxiation after 1, 4, 12, and 24 h p.i . (n = 45 per group). Select tissues including the tumor were harvested and weighed with bound activity measured using a gamma counter (perkinelmer). The tissue uptake (% id / g) was calculated against the total net activity injected . For autoradiographic studies, separate animals were administered with zr - mb, zr - cys - db, and zr - huj591 (7.410.2 mbq) as described above, 24 h before sacrifice . Five minutes prior to sacrifice, animals were injected with hoechst 33342 (1 mg / ml pbs, iv). Immediately following sacrifice, tumors were removed and frozen in oct mounting medium (sakura, europe) and a series of 10 m tissue sections obtained . Autoradiographic distribution of zr at 25 m pixel resolution was obtained by exposing the sections to a bas ms2025 phosphor plate (fujifilm), followed by reading on a ge typhoon 7000ip plate reader . The same sections were then used to obtain high - magnification whole - mount images of hoechst 33342 distribution and h&e staining as previously described . Following autoradiography, sections were fixed in 4% paraformaldehyde and stained for psma expression using rabbit anti - psma antibody (clone epr6253, abcam, 1:100 dilution) overnight at 4 c, followed by goat anti - rabbit - alexa-568 (invitrogen, 1:100) for 1 h at room temperature . Finally, sections were stained with h&e, and digital microsopic images obtained in the same manner . Statistical analysis was performed using a one - way anova test followed by dunnett s multiple comparison test when applicable for biodistribution experiments . A two - tail student s t test was employed in in vitro assays and tumor uptake comparison . Following production and purification of the mb and cys - db, the proteins were analyzed to confirm their purity and identity prior to conjugation . The mb and cys - db migrated primarily as covalent dimers of 80 kda and 50 kda, respectively, under nonreducing sds page conditions, but migrated as monomers of 40 kda and 25 kda, respectively, under reducing sds a low level of product - related impurities (likely partially clipped fragments) of the mb was also detected under the denaturing sds tof - ms confirmed the identity of the mb and cys - db by molecular mass . Facile dfo - bz - scn conjugation and zr - radiolabeling of mb, cys - db, and huj591 were achieved . Sds page gel confirmed purity of both dfo - attached fragments compared to unmodified proteins (si figure 1 in the supporting information). Table 1 summarizes radiolabeling yield, purity, and specific activities of zr - mb and zr - cys - db, which compared favorably to the previously reported values for zr - huj591 . Both zr - mb and zr - cys - db demonstrated excellent radiolabeling yields (> 70%) and radiochemical purities (> 99%). Specific activities of 222296 mbq / mg (17.823.7 mbq/mol) and 259333 mbq / mg (12.916.6 mbq/mol) were attained for zr - mb (n = 8) and zr - cys - db (n = 5) respectively . Both radiolabeled fragments sufficiently retained their affinity for psma with immunoreactivities established at 72.4 1.7% for zr - mb and 72.8 1.4% for zr - cys - db (n = 3 for each). With reports showing psma possessing a putative endocytic functionality, we conducted in vitro internalization studies using lncap cells to compare the rates of internalization of zr - mb, zr - cys - db, and zr - huj591 . The internalization patterns of all three radiotracers appear to be rapid, however, initial slow uptake was displayed by zr - mb at 4 h at 37 c with lower fractions internalized (3.7 0.5%, p = 0.002) (figure 1a, left panel). This was then followed by rapid internalization, peaking at 12 h with 15.1 1.0% (p = 0.027), which reached a plateau at 24 h (16.6 1.0%). In figure 1a (middle panel), zr - cys - db showed consistent linear internalization, spanning all time points (1.0 0.1% at 0.5 h, 3.5 0.2% at 1 h, 4.5 0.5% at 4 h, 8.9 0.7% at 12 h, and 11.9 0.1% at 24 h) at 37 c . At 37 c, zr - huj591 demonstrated internalization with 1.4 0.1% at 0.5 h, 1.8 0.8% at 1 h, 7.3 0.6% at 4 h, 12.1 1.3% at 12 h, and 10.1 1.3% at 24 h (figure 1a, right panel). Minimal internalization was demonstrated by all three imaging probes at 4 c, evidence of an endocytic uptake process . In vitro assays in lncap prostate cancer cells . Both antibody fragment radiotracers were added to lncap prostate cancer cells and incubated at 37 and 4 c at 0.5, 1, 4, 12, and 24 . (a) the internalization rates of zr - mb (left) and zr - huj591 (right) follow a nonlinear trend with slow uptake rates after 4 h of incubation; a subsequent increase in intracellular radioactivity with higher internalized fractions was observed for zr - mb after 12 h. zr - cys - db (middle) displays a linear rate of internalization . All three radiotracers display minimal intracellular accumulation at all time periods at 4 c . (b) in lncap prostate cancer cells, 1 h preincubation or coaddition of huj591 (100 g) with the radiotracer diminished binding of zr - mb (12 g, left) and zr - cys - db (12 g, right). The specificities of both zr - mb and zr - cys - db to psma were evaluated by competitive inhibition with nonradioactive huj591 . In figure 1b (left panel), zr - mb demonstrated as much as 4.96 0.73% binding to lncap cells with no added huj591 . This level of bound mb was satisfactorily blocked as much as 10-fold with both preincubated (0.55 0.19%, p <0.0001) and coadded (0.56 0.3%, p <similar outcomes were observed with zr - cys - db (figure 1b, right panel) where, in the absence of huj591, the radiotracer displayed 4.77 0.21% psma - binding activity; this uptake was significantly diminished by preincubation (0.16 0.08%, p <0.0001) and coaddition (0.15 0.07%, p <0.0001) of huj591 . Zr - huj591, zr - mb, and zr - cys - db demonstrated specific binding and high affinities for psma localized on the surface of lncap cells . The saturation binding studies showed apparent kd values that are similar for all three radiotracers: zr - huj591 (kd = 2.31 0.27 nm), zr - mb (kd = 2.18 0.50 nm), and zr - cys - db (kd = 2.59 0.41 nm). Tissue distributions of zr - mb were analyzed in mice with single implants of psma(+) lncap (figure 2a; si table 1 in the supporting information) or psma() pc3 tumors (si table 2 in the supporting information). Tumor uptake of zr - mb at 1 h (2.3 0.5% id / g) was observed to steadily increase at 4 h p.i . Accumulation of the probe in the tumor increased at 12 h p.i . With 6.2 2.5% id / g and at 24 h p.i . (12.1 3.6% id / g). To establish the specificity of zr - mb for psma in vivo, we performed blocking assays with various doses of nonradioactive mb (si table 3 in the supporting information). At 12 h p.i ., the uptake of zr - mb was moderately decreased by 2-fold at 200 g of coinjected cold mb with 3.73 1.27% id / g (p = 0.21). Increasing the nonradioactive dose to 500 g did not significantly block the uptake either with 3.82 0.70% id / g (p = 0.19). A 500 g huj591 dose was administered iv 36 h prior to injection of zr - mb with the mice (n = 4) euthanized 24 h p.i . Of the radiotracer, which mitigated tumor uptake (5.0 2.4% id / g, p = 0.025). The in vivo specificity of this probe was further strengthened with differences between zr - mb accumulation in psma() pc3 tumors against lncap tumors in (figure 2a, inset). The pc3 tumor sustained a constant accretion of 2.3 0.4% id / g at 1 h (p = 0.28), 2.1 0.3% id / g at 4 h (p = 0.03), 1.7 0.5% id / g at 12 h (p = 0.04), and 2.4 0.3% id / g at 24 h. this nonspecific tumor uptake is attributed to the tumor s leaky vasculature . Ex vivo tissue distribution studies of zr - mb and zr - cys - db administered intravenously in lncap (psma(+)) and pc3 (psma()) prostate tumor - bearing mice . (a) biodistribution of zr - mb at different time points (124 h) in select tissues including the tumor; a blocking dose of 500 g of huj591 36 h prior to intravenous injection of zr - mb effectively mitigated tumor uptake at 24 h p.i . (a, inset) direct comparison between lncap and pc3 tumors displays cumulative zr - mb uptake in the psma(+) xenograft, which was significantly higher as early as 4 h p.i ., whereas nonspecific binding in the negative tumor is observed . (b) zr - cys - db exhibits selective tumor tissue targeting, which peaks at 12 h p.i . A blocking dose of 200 g of nonradioactive cys - db decreases the radiotracer tumor uptake at 12 h p.i . (b, inset) higher uptake of zr - cys - db in lncap tumors compared to pc3 implants at 12 and 24 h p.i . Assessment of zr - cys - db localization in the psma(+) tumor demonstrated uptake with 4.1 1.0% id / g at 1 h, 7.0 3.0% id / g at 4 h, and 12.3 2.5% id / g at 12 h (figure 2b; si table 4 in the supporting information). A decrease in tumor accretion at 24 h (6.5 1.0% id / g) was observed . Blocking with 200 g of cold cys - db at 12 h p.i . Effectively lowered the tumor accumulation to 5.64 1.75% id / g (p = 0.044). Pc3 tumor uptake (figure 2b inset; si table 5 in the supporting information) displayed lower accretion across all time points with 2.36 0.48% id / g (1 h, p = 0.068), 3.65 1.54% id / g (4 h, p = 0.12), 2.75 0.51% id / g (12 h, p = 0.007), and 3.44 0.85% id / g (24 h, p = 0.0011). We performed in vivo pet imaging in mice bearing psma(+) lncap tumors from 1 to 24 h on all three radiotracers (si figure 2 in the supporting information). In figure 3a, planar sections of acquired images revealed accumulation of zr - mb at 12 and 24 h p.i . Maximal intensity projection (mip) at 12 h showed tumor delineation with nonspecific binding observed in the liver and kidneys . Similar results were achieved with zr - cys - db where localized delivery of the radiotracer to the tumor provided high contrasts at earlier time points (figure 3b). In contrast to zr - huj591 (figure 3c), very minimal to no activity is present in the lung and heart in both zr - antibody fragments, proof that the circulating probes were eliminated faster than the intact mab . Quantification of tumor uptake is presented in figure 4a (si table 6 in the supporting information), which displays an increasing trend in tumor uptake for all three radiotracers . At 12 h p.i, the accumulation of both zr - mb (6.85 0.87% id / g) and zr - cys - db (9.84 2.54% id / g) plateaued, whereas zr - huj591 (15.84 1.79% id / g) steadily increased at 12 h p.i . After 24 h p.i ., the tumor uptake of the intact mab was 3-fold higher compared to the scfv proteins . This lower tumor delivery of the engineered mab fragments most likely stemmed from low availability of both fragments in the blood pool . As shown in the vois drawn on the heart (figure 4b; si table 7 in the supporting information), a sharp decreasing trend in radiotracer activity was displayed by zr - cys - db followed by zr - mb across all time points such that, at 12 h, only 2.73 0.61% id / g zr - cys - db and 5.06 2.84% id / g zr - mb remained . At 24 h, residual cardiac activity from both tracers was determined (i.e., 2.00 0.18% id / g for zr - cys - db and 3.26 1.20% id / g for zr - mb). Zr - huj591 heart activity remained consistently higher at all time points (i.e., 16.36 1.57% id / g at 12 h and 12.13 1.26% id / g at 24 h). Representative maximum intensity projections (mips) and planar pet images of (a) zr - mb, (b) zr - cys - db, and (c) zr - huj591 acquired in male athymic nude mice bearing lncap (left shoulder, t) prostate tumors xenografts at 12 and 24 h postinjection . Kidney (k) and liver (l) accumulations were observed for both zr - mb and zr - cys - db and rationalized as routes of excretion . All three probes localize in the tumor, however, only zr - huj591 remains in circulation between 12 and 24 h. time activity curves of zr - mb, zr - cys - db, and zr - huj591 demonstrating (a) tumor volumes of interest (vois), (b) blood clearance through heart vois, (c) tumor - to - muscle ratios, and (d) tumor - to - heart ratios from 1 to 24 h. scrutiny of tumor - to - muscle (t / m) ratios from the pet scans disclosed no significant differences among all three radiotracers at all time points (p> 0.05) (figure 4c; si table 8 in the supporting information). Tumor - to - heart (t / h) ratios (figure 4d; si table 9 in the supporting information) showed no distinctions between zr - mb and zr - huj591; however, analysis of t / h ratios for zr - cys - db provided notably higher values at all time points against the intact mab, for example, 3.58 0.19 vs 0.98 0.19 (p = 0.0002) at 12 h and 4.91 0.26 vs 2.22 0.46 (p = 0.014) at 24 h. distribution of zr - mb (figure 5a), zr - cys - db (figure 5b), and zr - huj591 (figure 5c) at the microscopic level was evaluated using combined digital autoradiography (right panels) and whole - mount brightfield microscopy . The tumor tissues were viable with minimal necrosis observed as shown in the h&e stain (si figure 3a c in the supporting information). Psma expression (red stain, left panels) appeared to be essentially uniform in all lncap tumor sections examined . Binding was generally absent from regions of stromal infiltration . Slightly increased immunoreactivity was observed in tumor necrotic regions, but was assumed to represent nonspecific antibody binding . For each construct, focal uptake of zr was observed in central tumor regions, with seemingly less around the tumor periphery . Regions of high zr uptake were generally centered on areas of high vascular perfusion (as indicated by hoechst 33342 staining in blue, left panels). However, not all perfused regions, in particular those observed around the tumor rim, were associated with zr uptake . It is possible that vascular integrity or leakiness may play a role in determining the distribution of the zr - labeled constructs in this tumor model . This data may serve to indicate that the relative tumor distribution of each of the zr constructs appears to be equivalent over the time course of these experiments . Ex vivo autoradiography and histology . Registered whole - mount micrographs of lncap tumor sections with psma staining (red) coregistered with hoechst 33342 (blue) for perfusion (left) and digital autoradiography (right) of (a) zr - mb, (b) zr - cys - db, and (c) zr - huj591 . Here in this study, we developed psma - targeting pet imaging probes using antibody fragments of huj591 as carriers . We rationalize that these smaller derivatives are necessary to enable lower dose exposures and faster imaging, which can potentially decrease the burden on prostate cancer patients . We opted to utilize zr as the radionuclide of choice for labeling these fragments to provide a systematic and parallel comparison to huj591-pet . These results demonstrated significantly improved pk attributes of both zr - labeled antibody fragments with retained tumor targeting properties and shorter tracer clearance, thus rendering both probes as excellent markers of psma(+) cancer with the advantage of same - day imaging . Facile conjugation of dfo - bz - scn and zr - radiolabeling of mb and cys - db were achieved with high radiochemical yields and purities and with minimal loss of immunoreactivities . Consistently high specific activities for both zr - constructs were produced after numerous labeling experiments . For uniformity, huj591 was also conjugated with dfo - bz - scn, which offers the benefit of shorter and more straightforward conjugation compared to an earlier protocol used by our group . All three probes were shown to internalize albeit with varying kinetics underpinning the critical use of radiometals in labeling internalizing proteins to capitalize on longer retention within the tumor for better imaging contrasts . We further demonstrated the specificity of both zr - mb and zr - cys - db by coaddition or prior incubation of cold huj591 in lncap cells . We next asked whether the binding affinities of zr - mb and zr - cys - db are comparable to zr - huj591 . From our saturation binding assays, all radiolabeled proteins displayed comparable kds, supporting the hypothesis that smaller antibody platforms offer stable association to psma in a similar fashion to the parent mab . Our observed affinities for all zr - mabs are within the range reported for huj591 labeled with i (kd = 1.83 1.21 nm) and in (kd = 3 nm). Two reports of new anti - psma antibody fragments were recently published . Of particular interest is a diabody developed from the same parent huj591 and labeled with tc (tc - j591cdia) with a kd of 5.0 0.5 nm . Two chimeric scfv fragments derived form the parent murine mab 3/f11 (kd 4.0 nm) established kds of 8.3 nm for d7-fc and 9.6 nm for d7-ch3 . Moreover, a previous study investigating a single - chain variable fragment of another psma - targeting mab, d2b, reported a 4-fold lower affinity (8.6 nm) via biacore analysis, thus rendering our antibody fragments potentially superior . Pet imaging visualized tumor uptake and rapid whole body clearance of the zr - mb and zr - cys - db in murine xenografts . Tissue distribution studies showed minimal nonspecific binding of both radiotracers in healthy tissues except in the hepatic and renal organs, which are routes of elimination . This nonspecific binding observed for both liver and kidneys is significantly lower when compared to, for example, tc - j591cdia with> 13 1.8% id / g and> 29.2 3.5% id / g respectively, albeit at 8 h p.i . However, the uptake of our fragments, in particular, zr - cys - db, in the liver and the kidneys is significantly lower . Retention of activity in the kidneys is likely due to a combination of clearance of the antibody fragments and radiolabeled metabolites, in addition to antigen - specific binding, as the proximal renal tubules have been reported to express psma; only the latter would be reduced by blocking studies . Specific tumor delivery of both probes zr - cys - db and zr - mb to psma was validated by addition of excess cold cys - db and huj591 respectively . The competitive inhibition of zr - mb, however, was not straightforward with excess doses of mb inefficiently attenuated . A plausible explanation can be attributed to the nonspecific clearance kinetics of each fragment, whereby competitive inhibition of the mb should be examined at longer time periods (i.e., 24 h p.i .) When tumor uptake has stabilized . The results from autoradiography and histology (figure 5b) reinforce this rationale as zr - mb was found to colocalize with areas of vasculature even at 24 h p.i . ; this, however, does not definitively discriminate between the vascular contributions of each construct . Stronger direct evidence is found in the blood activity at 12 and 24 h obtained from the ex vivo tissue distribution wherein accretion of zr - mb is 2-fold higher than that of the smaller zr - cys - db . May likely stem from the fact that the internalization of psma is critically enhanced by ligand or antibody binding to the antigen . As we have shown in our internalization assays, the kinetics vary with the minibody internalizing at a higher rate after 12 h p.i . With the psma recycling to the cell surface, more binding sites are available for zr - mb delivery with 3% id / g circulating in the bloodpool . In our study, engineered smaller fragments are postulated as viable imaging surrogates of therapeutic antibodies within the context of curtailing wait times between administration and imaging . Clear distinction between the three imaging probes is attained upon analysis of each tracer s in vivo pharmacokinetics due primarily to varying molecular size . Blood pool activity of all radiotracers demonstrated obvious disparities (figure 4b) in clearance kinetics with zr - cys - db clearly showing faster elimination during earlier time points . Zr - mb showed parallel blood residencies at 12 and 24 h with zr - cys - db while zr - huj591 still remained in circulation at higher activities after 24 h. moreover, the measured tumor uptake from the pet images demonstrated significantly higher radiolocalization of zr - huj591 at later time points (i.e., 24 h). We ascribe this observation as a factor of systemic elimination of the fragments rather than loss of affinity . Intact mabs possess innate prolonged blood residencies, significantly increasing its availability for tumor delivery . This extended circulation may benefit immunotherapies, but for diagnostic purposes, faster clearance with retained tumor localization is key to perform imaging within the same day of dosing . Faster blood pool clearance considerably enhances the image quality and resolution, which puts these zr - engineered mabs at a critical advantage . Moreover, the lower tumor delivery should not be taken as a major drawback as contrast or tumor - to - background ratios is considered one of the critical parameters not just in nuclear imaging but across all modalities . From the tumor - to - muscle ratios (figure 4c), no significant disparities in contrast were observed between the antibody fragments and intact huj591, supporting the notion that these intermediate - sized antibody fragments provide similar tumor delineating properties at the time points studied . One of the benefits of using engineered fragments is arguably the lower dose toxicity due to shortened blood residencies and minimized nonspecific healthy tissue accumulation . Blood activities of both antibody fragments are far shorter than that of the intact huj591 with residencies decreasing by 2-fold within 12 h postadministration . Thus, we surmise that, with such rapid kinetics, nontrivial absorbed whole body radioactive doses especially in hematopoietic tissues will be minimized proportionally . Certainly, with smaller antibody fragments, a radioisotope with a half - life matching the pk of the fragment (i.e., cu, t1/2 = 12 h) will afford better dosimetry profiles, especially if the probes are envisioned for repeat monitoring of treatment response in the same patient . Although immunopet potentially holds a vital role in molecular and functional diagnosis, the lengthened circulation of intact antibodies and their nonspecific accumulation in normal tissues can impact radiotoxicity in normal organs, for example, the liver, the spleen, and the rest of the hematopoietic system, upon repeated pet scans . We believe that the use of engineered fragments derived from the intact antibody provides such benefit and advantage with observed terminal half - lives of 311 h in mice and with maintained radiotracer target specificity . Recognizing that imaging tools require rapid clearance of the probe at shorter time periods for optimum contrast and in order to minimize patient burden, the smaller cys - db and mb fragments of huj591 for pet imaging are obvious choices as companion diagnostics of psma - targeted therapy . In summary, small engineered antibody fragments of huj591 have high potential as alternative noninvasive imaging probes for the detection and staging of psma - positive prostate tumors . Both zr - mb and zr - cys - db offer rapid tumor delineation and background clearance, essentially establishing these scfv - based antibody fragments as faster alternatives to the intact mab.
Identified in the 1950s, caveolae are 5080 nm diameter plasma membrane invaginations that are morphologically distinct from clathrin - coated pits . Caveolae are cholesterol- and sphingolipid - rich and considered a subdomain of plasma membrane microdomains or lipid rafts . Small (10200 nm) heterogeneous membrane domains enriched in sterol and sphingolipids that are involved in the compartmentalization of various cellular processes. Multiple studies have described the role of caveolae and rafts in the endocytosis of various ligands (for reviews see refs . Several raft - dependent pathways have been described and raft ligands are quite liberal in their selectivity for a particular route of entry into the cell . The extent and nature of raft - dependent endocytosis is regulated by various cellular components that include caveolin-1 (cav1), cholesterol and dynamin as well as regulators of the actin cytoskeleton . Cav1 is the major component of caveolae and its expression is essential for the formation of caveolar vesicles . An absence of caveolae is noted in cells that do not express cav1 and its reintroduction into these cells induces caveolae formation at the plasma membrane . Caveolin-2 (cav2) facilitates but is not essential for caveolae formation [79]. Caveolae are highly immobile at the plasma membrane and cav1 has been proposed to be a negative regulator of raft - dependent endocytosis [13, 14]. However, upon activation by sv40, cav1 mobility at the cell surface is greatly increased . There is also evidence for raft - dependent endocytic pathways independent of cav1 that are mediated by distinct carrier vesicles [1619]. In this review, we will characterize the various raft - dependent endocytic pathways and discuss their regulation . Various other pathways, commonly referred to as clathrin - independent, have been identified and are under intense investigation . Some of these pathways are cholesterol - sensitive and therefore considered to be raft - mediated . It is important to recognize that clathrin - mediated endocytosis is also sensitive to acute depletion of cholesterol [21, 22] and that raft recruitment has been shown to precede clathrin - dependent endocytosis for egfr, bcr and anthrax toxin [2325]. In addition, macropinocytosis, involving rac1-dependent membrane ruffling at the plasma membrane, can be cholesterol - sensitive, potentially defining another dynamin - independent raft pathway [26, 27]. However, macropinocytosis has been shown to be dynamin - dependent in nih-3t3 and huvec cells [28, 29]. The dynamin- and raft - dependence of macropinocytotic pathways may be cell type and cargo - specific . For the purposes of this review, raft - dependent endocytic pathways will be defined by their clathrin - independence and cholesterol - sensitivity and will not include macropinocytosis . A characteristic of some of these raft - dependent pathways is their dependence on dynamin, a molecule involved in vesicular fission from the plasma membrane [30, 31]. The formation of dynamin - dependent smooth plasma membrane vesicles, or caveolar invaginations can occur both in the presence or absence of caveolins . Similarities between the caveolae and non - caveolin dynamin - dependent raft endocytic pathways led us to refer to them inclusively as caveolae / raft - dependent endocy - tosis . Dynamin - independent raft pathways have been described that are caveolin - independent and invoke tubular intermediates [16, 17]. While the heterogeneity of raft domains is certainly indicative of higher orders of complexity and regulation of their endocytosis, to a large extent, and at least for now, raft - dependent endocytic pathways can be classified based on their caveolin- and dynamin - dependence (fig . This classification is based on mechanistic similarities of the raft - dependent internalization of select ligands at the plasma membrane . Indeed, different cargoes that use similar raft endocytic mechanisms may be internalized via distinct raft domains and targeted to different intracellular sites [14, 33]. (a) several endocytic pathways are characterized as raft - dependent and mediate the uptake of various ligands, including but not limited to those indicated . These include dynamin - dependent pathways that invoke caveolae or non - caveolin vesicular intermediates and that can be referred to as caveolae / raft - dependent endocytosis . While similar mechanisms control the uptake of the indicated raft - dependent ligands, they are not necessarily internalized by the same raft domains or follow similar intracellular targeting routes . (b) cav1 may negatively regulate uptake via the dynamin - dependent, non - caveolin pathway by either stabilizing raft invaginations at the cell surface (1) or by sequestering key components, including cholesterol, dynamin and others, required for raft - dependent uptake (2). Cholesterol is not shown in the flat portion of the membrane to simplify the diagram . Laccer: lactosylceramide; ct - b: cholera toxin b subunit; gpi - ap: glycosylphosphatidylinositol - anchored proteins; amf: autocrine motility factor; il-2: interleukin-2; sv40:simian virus 40 . The simian virus sv40 follows a dynamin - dependent, caveolae - mediated pathway that targets a caveolin - positive endosome, the caveosome, before being delivered to the smooth endoplasmic reticulum . When stimulated by sv40, caveolin, dynamin and actin are recruited sequentially to the caveolae . The raft - dependent endocytic pathway of cholera toxin b - subunit (ct - b) has also been characterized as a dynamin - dependent, caveolar pathway [19, 3537]. Albumin is internalized via a dynamin - dependent pathway that requires cave - olin . In lymphocytes lacking cav1, endocytosis of the interleukin-2 receptor occurs via a clathrin - independent, cholesterol - sensitive pathway that requires dynamin and is regulated by the rhoa gtpase . In nih-3t3 cells, the autocrine motility factor receptor is localized to caveolae and internalization of its ligand, amf, is cholesterol and dynamin - dependent and negatively regulated by cav1 expression [13, 40]. A raft - dependent, dynamin - independent pathway has also been described for ct - b and sv40 [16, 17] that exhibits similarity to a cdc42-dependent pathway followed by gpi - anchored proteins (gpi - ap) and fluid phase markers . In fibroblasts from cav1 knockout mice, sv40 exploits an alternate, cav1-independent pathway that is cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin-2 and arf6 . A similar pathway has also been described for ct - b in cav1 fibrob - lasts where it is arf6-dependent . This pathway invokes not caveolar invaginations but the formation of uncoated tubular endocytic structures and an intracellular dynamin - dependent step for delivery to endosomes and the golgi apparatus . Internalization of ct - b has also been shown to occur via a dynamin - independent pathway defined not by caveolin but by flotillin, another raft component . Ct - b therefore provides an example of an endo - cytic ligand internalized by several pathways including clathrin - coated pits and both dynamin - dependent and independent raft pathways . A recent study showed that 50% of ct - b enters the cell via clathrin - coated pits with the remainder internalized via dynamin - independent, caveolin - independent uncoated tubules . In the same study, the authors showed that about only 2% of the total pool of cav1 positive caveolae contributes to the internalization of ct - b, suggesting that internalization of ct - b via caveolae represents only a minor contribution . However, ct - b internalization was found to be deficient in immortalized cav1 mef - derived cell lines contrasting with the demonstration that primary cav1 mefs show no difference in ct - b uptake compared to wild - type mefs . In hela cells, depletion of flotillin by sirna prevents its uptake via a dynamin - independent route and switches it to a dynamin - dependent route . Variable cell surface expression of the ct - b receptor, gm1 ganglioside, impacts on the extent of its raft - dependent endocytosis . In addition, ct - b concentrations used vary significantly (from 0.05 to 10 g / ml) between studies from different laboratories [17, 42, 45, 46]. Interestingly, in studies defining the dynamin - independent raft pathway, both ct - b and dextran concentrations were relatively low [17, 18]. Variable factors, ranging from expression of ligand receptors to raft components, may impact not only on the extent of ct - b uptake but also on its route of entry into different cells or clonal populations of the same cell type . Raft - dependent endocytosis is therefore a highly complex process in which the same cargo can follow various entry routes and in which different cargo can use similar entry routes with different molecular reg - ulation . This complexity should not preclude efforts to classify these pathways based on common denomi - nators, as proposed in figure 1a . Further characterization of the cargo - specificity and molecular regulation of raft - dependent pathways will lead to a better understanding of what are clearly intricate mechanisms regulated by multiple factors . Fluorescence recovery after photobleaching (frap) experiments have shown that movement of cav1 at the cell surface is restricted by cortical actin as well as through interaction with the actin - binding protein filamin [12, 47]. Caveolar stability at the plasma membrane suggests that rapid, constitutive internalization and turnover of caveolae is unlikely to occur . Rapid, reversible budding of caveolae, or potocyto - sis, was originally suggested to regulate folate internalization . More recently, tirf microscopy was used to show that reversible caveolae budding is limited to the subplasma membrane region by the underlying actin cytoskeleton . Disruption of the actin cytoskeleton induces rapid internalization of caveolar vesicles [49, 50]. Recruitment of sv40 to caveolae induces the transient, localized breakdown of the actin cytoskeleton . Actin depolymerization also induces internalization of tight junction proteins via a caveolae - dependent pathway . However, earlier work showed that disruption of the actin cytoskeleton by cytochalasin d in a431 cells inhibited alkaline phosphatase uptake via caveolae . The submembrane actin cytoskeleton would therefore appear to be a critical regulator of the endocytic potential of caveolae . Gpi - anchored proteins have been shown to be internalized via a cdc42-regulated pathway that is independent of rho and rac . Rhoa regulates il-2 receptor internalization and ct - b endocytosis to the golgi apparatus in cos-1 cells is dependent on rhog . The menkes disease atpase (atp7a) uptake can be inhibited by a rac-1 dominant negative mutant . Differential expression and local activation of rho family gtpases may be a key determinant of the cell type specificity of raft - dependent endocytosis . Threshold levels of cav1 and cholesterol regulate caveolae formation [56, 57]. Various cholesterol modulating agents, including methyl--cyclodextrin, nys - tatin and filipin, have been shown to inhibit both caveolae expression and raft - dependent endocytosis [13, 16, 5760]. Caveolar endocytosis of various lig - ands can be significantly increased by addition of cholesterol or glycosphingolipid to human fibroblasts . Using heterokaryons expressing both cav1-gfp and cav1-rfp, it has been shown that cholesterol depletion increased exchange between otherwise stable cav1 positive structures . Cav1 interacts directly with cholesterol [61, 62] and cholesterol levels in lipid raft fractions obtained from cav1 expressing cells were 34-fold higher than in matched cells lacking cav1 . It is possible that cav1 regulation of raft endocytosis is linked to its ability to sequester cholesterol in raft domains (fig . When in excess and therefore free from interaction with phospholipids, cholesterol shows a higher chemical activity . Cav1 may therefore regulate cholesterol - dependent processes, such as raft endo - cytosis, through sequestration of active cholesterol . Expression of the k44a dynamin mutant increases the number of caveolae in caveolin - expressing nih-3t3 cells as well as the formation of morphologically similar invaginations in ras and abl - transformed nih-3t3 cells expressing little caveolin . Several studies have shown that overexpression of cav1 is associated with reduction, even inhibition of raft - dependent endocytosis [13, 17, 38, 46]. Cav1 overexpression was also found to inhibit the non - caveolar, dynamin - independent endocytosis of ct - b . Reduction of cav1 levels in mammary tumor - derived cell lines is associated with both increased plasma membrane mobility and raft - dependent uptake of ct - b to the golgi apparatus . Interestingly, regulation of ct - b mobility and endocytosis in these cells occurred at cav1 levels below the threshold for caveolae formation (lajoie, nim and nabi, unpublished). This suggests that cav1 may act indirectly to regulate raft - dependent endocytosis by impacting on the composition and endocytic potential of non - caveolar raft domains (fig . Indeed, the idea of dynamic exchange between raft domains is consistent with the ability of raft components, such as cav1 or flotillin, to impact on the raft - dependent endocytosis of select ligands by modulating the endocytic potential of distinct raft domains . Treatment of cells with tyrosine kinase inhibitors blocks caveolae endocytosis while addition of the phosphatase inhibitor okadaic acid triggers endocytosis [17, 37, 51, 70]. Indeed, the use of the non - specific tyrosine kinase inhibitor genistein is generally recognized as a selective inhibitor of raft - dependent endocytic pathways . Cav1 is phosphorylated by src kinase at tyrosine 14; however, the role of cav1 phosphorylation in raft endocytosis is still unclear . Redistribution of tyrosine phosphorylated cav1 from focal adhesions to caveolae upon cell detachment from the extracellular matrix triggers raft - dependent endocy - tosis and plasma membrane depletion of rac . Activation of v - src in rat-1 cells is responsible for cav1 phosphorylation and is associated with loss of plasma membrane caveolae . In addition, cav1 phosphorylation on tyrosine 14 is associated with flattening, aggregation and fusion of caveolae vesicles . However, in pancreatic cancer cells, egf stimulation of src - mediated cav1 phosphorylation leads to a marked increase in the number of assembled caveolae at the cell surface . Src kinase regulation of transcytosis of albumin across the endothelial cell monolayer is associated with cav1 phosphorylation . Src kinase activity is also required for stimulation of caveolae internalization by glycosphingolipids and cholesterol . However, whether cav1 tyrosine phosphorylation is a critical regulator of caveolae internalization remains to be determined . Tyrosine phosphorylation inhibitors do not prevent sv40 recruitment to caveo - lae but do prevent recruitment of dynamin to caveolae suggesting that tyrosine phosphorylation is crucialfor dynamin - dependent caveolae budding . Similarly, the src - dependent internalization of albumin via a gi - coupled pathway requires interaction of its receptor, gp60, with cav1 [38, 76]. Dominant negative src reduces phosphorylation of dynamin-2 and dynamin-2 association with cav1 resulting in reduced albumin uptake . However, the requirement for tyrosine kinases in the raft - dependent uptake of amf in cancer cells expressing low levels of cav1 and in the dynamin - independent raft uptake of sv40 in cav1 cells is indicative of further complexity for the role of tyrosine phosphorylation in raft - dependent endocytosis . A sirna screening approach of kinase inhibitors identified a large group of 208 human kinases as regulators of sv40 entry and 39 of them were involved in caveolae / raft trafficking . Application of a similar approach to other raft ligands may identify common and, potentially, distinct kinases that control raft - dependent endocytosis of various raft ligands . Cav1 has a well - established scaffolding function implicated in the sequestration of cytokine receptors and lipid - anchored signaling intermediates as well as cholesterol [80, 81]. Sequestration of egfr and tgf r to caveolae and interaction with cav1 is associated with inhibition of signaling capacity [76, 8284]. These studies were later confirmed when it was shown that cav1 was able to induce sequestration of the receptor and to directly bind egfr [86, 87]. Moreover, the second cysteine region of egfr contains sequences that target the receptor to caveolae / raft domains . Upon stimulation with egf, egfr is no longer localized in low density raft fractions, consistent with its migration from caveolae to clathrin coat pits upon stimulation . Alternatively, cav1 may indirectly regulate egfr signaling through regulation of the cholesterol content of lipid rafts . When stimulated with a high egf dose, egfr is internalized via a caveolae / raft - dependent pathway associated with ubiquitination of the receptor . Similarly, clathrin - dependent uptake of tgf r is associated with subsequent signaling events via smad2 phosphorylation in eea1-positive endo - somes while its caveolae / raft - dependent is associated with receptor degradation through binding to the smad7-smurf2 complex . Raft - dependent endo - cytosis is therefore both regulated by and impacts on cell signaling . Raft - dependent endocytosis includes various cholesterol - sensitive endocytic routes, distinct from clathrin - mediated endocytosis, which can be classified based on their dependence on cav1 and dynamin . These pathways share sensitivity to cholesterol depletion as well as to other more selective regulators whose cell - specific expression may impact on the endocytic pathway followed by multiple raft - dependent ligands . By impacting indirectly on raft domain organization, various raft components, including cholesterol, cav1 and flotillin, cav1 acts as a determinant of raft - dependent endocytosis by stabilizing rafts at the cell surface, via receptor recruitment or through sequestration of cholesterol and other critical determinants of raft - dependent endocytosis . Further study of raft - dependent endocytosis should lead to the further classification and identification of specific regulators of the endocytic potential of these varied pathways . Open questions that remain relate to the molecular regulation of raft - dependent endocytosis and how the heterogeneous composition of raft domains, including but not limited to cargo, impacts and determines their endocytic potential, mechanism of internalization and intracellular targeting.
Antrodia cinnamomea, a taiwan - specific mushroom, has been reported to have numerous biological activities including hepatoprotection, anti - inflammation, antihepatitis c virus activity, and anticancer activity . Liver cancer ranks fourth in cancer - related mortality around the world (who 2008) and is the second leading cause of cancer deaths in taiwan . Twenty years ago, the major cause of liver cancer in taiwan was hepatitis virus infection, such as hepatitis b virus (hbv) infection . The incidence of hbv infection is very low now, mostly due to the hbv vaccine policy of taiwan government . However, the mortality of liver cancer is still high, which may be due to social culture and unhealthy living habits such as drugs abuse, drinking, and overworking . There are three common types of cell death: apoptosis, autophagy, and necrosis . Apoptosis, also called type i programmed cell death, involves dna fragmentation, caspase induction, and phosphatidylserine translocation from the inner side of the cell membrane to its outer side; autophagy, also recognized as type ii programmed cell death, forms autophagosome as the major phenomenon; and necrosis would cause inflammation and disrupt organelles . There is no study on the determination of major mechanism of antcin k - induced cell death in human liver cancer cells . Proteins must go through a series of post - translational modifications and be fully folded in order to be transported from the endoplasmic reticulum (er). Proteins with incomplete or incorrect folding will remain in the er or be degraded by the proteasome in the cytoplasm . Many physiological and pathological circumstances such as hypoxia, oxidative damage, deficiency in er calcium ion, and viral infection may cause disorders of protein folding in the er, in which proteins with incomplete folding will accumulate on the er surface and cause er stress . In order to resist er stress, cells will start a series of reactions to adapt to the circumstance and regulate the er stress, which include promotion of protein degradation, inhibition of protein translation, and activation of relevant genes . However, if the er stress persists, apoptosis or autophagy would be induced, which will eventually lead to cell death . The unfolded protein response is primarily a survival response, acting to resolve dysregulation of protein - folding pathways . If the normal luminal environment cannot be restored, the response to er stress would be switched from survival to apoptosis . Perk, ire1, and atf-6 are all involved in the induction of proapoptotic as well as prosurvival pathways . Both perk and atf-6 induce expression of the transcription factor c / ebp homologous protein (chop), which in turn leads to reduced expression of b - cell lymphoma 2 (bcl - xl) and increased expression of a number of proapoptotic proteins, including bh3-only protein bim and its downstream protein b - cell lymphoma extra - large (bax)/bak.9, 10, 11 furthermore, bcl-2 family proteins at the er also regulate apoptosis through both direct modulation of signaling pathways leading to caspase activation and modulation of ca signaling of er.12, 13 calcium is a common metal ion, which plays a dominant role in cell death signaling transduction . It has been reported that the concentration of calcium ion can regulate the stability of organelles such as mitochondria and er . Here, we will demonstrate how antcin k modulates cell death through the change of calcium distribution in human liver cancer cells ., it would induce reactive oxygen species (ros) production, and the ros may attack some organelles such as mitochondria . Next, the permeability transition pore of mitochondria would be opened by ros or other stimulation, and then by some proapoptotic protein bax / bak overexpression, which could release cytochrome c into the cytoplasm . Finally, cytochrome c would increase the cleavage caspase-9 protein expression, which triggers caspase-3 activation . There are other caspase - independent death pathways in mitochondria, such as htra2/omi, endonuclease g (endo g), and apoptotic - induced factor (aif) released from mitochondria.15, 16 basswood cultivated a. cinnamomea (bcrc930103) mycelial powder was supplied by po - zo co., ltd (taipei, taiwan). Ethyl acetate (ea) was added to 500 g dry power of a. cinnamomea to a total volume of 4 l and stirred for 3 days . The extract was decanted, and the solvent was removed using a rotary evaporator at 50c three times for each sample . The column was consecutively eluted with 10%, 15%, 20%, 30%, 50%, 70%, and 100% ea / hexane . The fraction with 100% ea / hexane contained the highest amount of antcin k. it was further purified by high - performance liquid chromatography to obtain antcin k with> 90% purity (fig . Antimycotic, 2,7-dihydrodichlorofluorescein diacetate, dulbecco's modified eagle's medium, fetal bovine serum, fluo-3-acetoxymethyl ester, nonessential amino acids, rhod-2-acetoxymethyl ester, and 3,3-dihexyloxacarbocyanine iodide were purchased from invitrogen (carlsbad, ca, usa). Anti--actin antibody, anti - aif antibody, anti - bcl - xl antibody, anti - bax antibody, anti - bak (d4e4) rabbit mab antibody, anti - caspase-9 antibody, anticleaved caspase-3 rabbit mab (asp175)(5a1e) antibody, anti - chop (l63f7) mouse mab antibody, anti - cytochrome c antibody, anti - endo g antibody, anti - htra2/omi antibody, anti - parp antibody, antirabbit igg hrp - linked antibody, and antimouse igg hrp - linked antibody were obtained from cell signaling technology (beverly, ma, usa). Caspase-3 assay kit and annexin v - fitc apoptosis detection kit were purchased from bd biosciences (san jose, ca, usa). Anti - lc3b antibody was purchased from genetex (irvine, ca, usa). Adenosine diphosphate / adenosine triphosphate (adp / atp) ratio assay kit and lactate dehydrogenase (ldh) cytotoxicity assay kit were purchased from biochain institute (hayward, ca, usa). All other chemicals were of analytical or reagent grade and obtained from sigma - aldrich (st louis, mo, usa). Human hepatoma hep 3b cell line was a kind gift from professor ming - shi shiao (medical research and education department, taipei veterans general hospital, taipei, taiwan). Hep 3b cells were cultured in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum, 1.5 g / l sodium bicarbonate, 1% nonessential amino acids, and 1% antibiotic antcin k was diluted in dimethyl sulfoxide (dmso) prior to being added to cultures . Liver cancer cells at a concentration of 5 10 cells / well were seeded in 96-well plates and incubated for 24 hours, followed by treatment with 0m (0.3% dmso), 80m, 100m, and 125m antcin k and incubated further for 24 hours and 48 hours . At the end of the stipulated period, 100 l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) solution (0.5 mg / ml) was added, and the cells were incubated at 37c for 4 hours . The resulting mtt formazan was dissolved in 100 l dmso and the absorbance recorded at 570 nm using a powerwave ht microplate spectrophotometer (bio - tek, winooski, vt, usa). Hep3b cells (1 10 cells / well) were seeded in 96-well plates for 24 hours, followed by treatment with 200 l medium containing 0m, 80m, 100m, and 125m antcin k and lysis solution (as a positive control), and incubated for another 48 hours . The plates were then centrifuged at 250 g for 10 minutes, and 100 l of supernatant was transferred to corresponding cells of a new 96-well plate . Following this, 45 l of assay mixture containing lactate, nicotinamide adenine dinucleotide, iodonitrotetrazolium, and diaphorase was added to each well, protected from light, and incubated for 60 minutes . The absorbance was recorded at 490 nm using a bio - tek powerwave ht microplate spectrophotometer . The cells (1.75 10 cells / well) were seeded in a four - well plate for 24 hours . For the 4,6-diamidini-2-phenylindole (dapi) staining, after 24 hours of incubation, the cells were treated with 350 l medium containing 0m, 80m, 100m, and 125m antcin k for 24 hours . After 24 hours of treating, the cells were washed and fixed by 4% paraformaldehyde for 30 minutes, and then washed twice . After washing, the cells were blocked for 1 hour by 5% bovine serum albumin and 0.1% triton x-100 . After washing, the slides were mounted and examined under fluorescence microscopy (olympus ix51; olympus, tokyo, japan). For the mitochondria calcium staining, after 24 hours of incubation, the cells were treated with 350 l medium containing 0m, 80m, 100m, and 125m antcin k for 30 minutes . After 30 minutes of treatment, the cells were incubated with 350 l medium containing 5m rhod-2-acetoxymethyl ester for 1 hour and protected from light . After washing, the cells were incubated with 5% bovine serum albumin at 37c for 30 minutes . The slides were mounted and examined under a leica tcs sp5 ii confocal fluorescence microscope (leica, solms, germany). The cells (5 10 cells / well) were seeded in 96-well plates for 24 hours . After 24 hours of incubation, the cells were treated with 100 l medium containing 0m, 80m, 100m, and 125m antcin k for 24 hours . After 24 hours of treatment, 90 l lysis solution, which contains luciferin and luciferase, was added to each well and incubated for 10 minutes . The luminescence was integrated for 5 seconds using a beckman coulter dtx-880 microplate reader (beckman coulter, brea, ca, usa). The cells (3 10 cells / dish) were seeded in 6 cm dishes for 24 h hours . After 24 hours of incubation, the cells were treated with 6 ml medium containing 0m, 80m, 100m, and 125m antcin k for 30 minutes to detect ca, 24 hours to detect ros, 48 hours to detect autophagosome, 48 hours to determine mitochondrial membrane potential, and 24 hours to determine the percentage of cells undergoing apoptosis, using an annexin v - fitc / propidium iodide (pi) assay kit (bd biosciences). At the end of the stipulated period, the cells were harvested and washed . For the detection of ca, ros, and mitochondrial membrane potential, the cells were separately stained by 4m fluo-3-acetoxymethyl ester, 5m dihydrodichlorofluorescein diacetate, and 4m 3,3-dihexyloxacarbocyanine iodide at 37c for 30 minutes . For the detection of autophagosome, the cells were permeabilized with 0.25 mg / ml digitonin for 5 minutes . Following this, the cells were washed twice, pelleted by centrifugation at 1000 g, and incubated with anti - lc3-ii antibody (1:2000) for 30 minutes . After two washes, the cells were stained by alexa fluor 488 anti - rabbit igg antibody (1:500) for 1 hours and protected from light . All these cells were washed and filtered prior to being analyzed by a becton - dickinson facscan flow cytometer (becton - dickinson, hercules, ca, usa). A total of 10,000 cells per sample were collected, and the mean fluorescence intensity and percentage of mitochondrial membrane potential detected by fl1-h (530 15 nm) were analyzed using winmdi 2.8 software . For the detection of the percentage of apoptosis cells, the cells were resuspended in binding buffer [10 mmol / l hepes / naoh (ph 7.4), 140 mmol / l nacl, 2.5 mmol / l cacl2] and stained with annexin v - fitc and pi at room temperature for 15 minutes in the dark . Cells were washed and filtered prior to being analyzed by a beckman coulter fc500 flow cytometer (beckman coulter, pembroke pines, fl, usa). A total of 10,000 cells per sample were collected, and the apoptotic cells were defined as annexin v - fitc - positive and pi - negative cells.19, 21, 22 for western blotting, the cells (5 10 cells/10 ml / dish) were seeded in 10 cm dishes for 24 hours . After 24 hours of incubation, the cells were treated with 10 ml medium containing 0m, 80m, 100m, and 125m antcin k for 48 hours . After 48 hours of treating, total cell extracts were prepared in protein extraction solution, which contained 1 mm phenylmethanesulfonylfluoride, 1 mm edta, 1m pepstatin a, 1m leupeptin, and 0.1m aprotinin . The cell lysates were sonicated and cleared by centrifugation, and the protein concentration in the lysates was measured by lowry's method . Polyacrylamide gel electrophoresis gels, transferred to polyvinylidene fluoride membranes, blotted with specific primary antibodies, and then labeled by horseradish peroxidase - conjugated secondary antibody according to the manufacturer's instructions . The membranes were performed using the enhanced chemiluminescence and the ec3 300 corning uvp biospectrum ac system (corning, ny, usa), and the relative density of each band after normalization for -actin was analyzed using image j 1.45 software . For the comet assay, the cells (5 10 cells/10 ml / dish) were seeded in 6 cm dishes for 24 hours . After 24 hours of incubation, the cells were treated with 6 ml medium containing 0m, 80m, 100m, and 125m antcin k for 24 hours . After 24 hours of treatment, cells were examined for dna damage using the comet assay described previously . All results are reported as mean standard deviation (sd), and the differences between the antcin k - treated group and the control group were analyzed by one - way analysis of variance (anova) and duncan's multiple comparison tests (sas institute inc ., cary, nc, usa) to determine significant differences among treatments (p <0.05). After treating hep 3b cells with 0m, 80m, 100m, and 125m antcin k for 24 hours or 48 hours, cell viability was analyzed by mtt assay . 1b shows that after being treated with 80m, 100m, and 125m antcin k for 24 hours, compared to the negative control (0.2% dmso, cell survival rate set at 100%), the cell survival rates were 85.7 5.0%, 63.8 5.8%, and 53.8 5.3%, respectively; after 48 hours of treatment, cell survival rates decreased to 76.7 2.1%, 65.0 8.6%, and 46.0 4.2%, respectively . After treating hep 3b cells with 0m, 80m, 100m, and 125m antcin k, for 48 hours, the autophagosome formation was analyzed by flow cytometry . 1c shows that after being treated with 80m, 100m, and 125m antcin k, the lc3-ii contents were significantly decreased, and not increased . After treating hep 3b cells with 0m, 80m, 100m, and 125m antcin k and lysis solution for 48 hours 1d shows that after being treated with 80m, 100m, and 125m antcin k for 48 hours, compared with the positive control (lysis solution, lactate dehydrogenase leakage rate set at 100%) and negative control (0.2% dmso, lactate dehydrogenase leakage rate set at 0%), the lactate dehydrogenase leakage rates were 8.56 5.24%, 1.46 7.85%, and 7.21 7.48%, respectively, which were all low . We assessed the effect of antcin k on the induction of dna aggregation in hep 3b cells by dapi staining . The immunofluorescence at 24 hours showed that 80m, 100m, and 125m antcin k treatment resulted in the formation of chromatin condensation in hep 3b cells . The nuclei of control cells were round and dim, whereas those became condensed and bright after antcin k treatment (fig . Higher concentrations of antcin k led to a longer dna migration smear (comet tail) (fig . A quantitative evaluation was sought using annexin v - fitc dye to detect the translocation of phosphatidylserine from the inner (cytoplasmic) leaflet of the plasma membrane to the outer side (cell surface). Compared with negative control cells, 80m, 100m, and 125m antcin k induced, respectively, 11.25 0.51%, 33.28 4.67%, and 39.87 3.83% of apoptotic cells in hep 3b cells in 24 hours (fig . 3a). After treating hep 3b cells with 0m, 80m, 100m, and 125m antcin k for 24 hours, ros generation was analyzed by flow cytometry; in addition, after treating hep 3b cells with the same concentrations of antcin k for 48 hours, the adp / atp ratio was analyzed with bioluminescence . 3b shows that as the antcin k dose increased, ros generation in cells increased significantly (the maximum value being 1.49 times greater than the minimum) and had a dose - dependent effect . 3d shows that as the antcin k dose increased, the adp / atp ratio in cells increased significantly . The adp / atp ratios were 0.9 0.9%, 5.9 1.5%, 14.7 2.8%, and 16.1 2.0%, respectively, for 0m, 80m, 100m, and 125m antcin k treatment . Using fluo-3-acetoxymethyl ester, a cell - permeable ca indicator, we assessed the effect of antcin k treatment on cytosolic ca . The level of cytosolic ca induced significantly by 100m and 125m antcin k at 30 minutes . In addition, treatment with 125m antcin k similarly induced a 1.74-fold increase in ca level (fig . Mitochondrial free calcium levels were quantitated in cells that were still viable with the cell - permeability acetoxymethyl ester of the fluorescent marker rhod-2 . Using rhod-2-acetoxymethyl ester, a selective indicator for mitochondrial ca, the distribution of calcium in 0m and 125m antcin k - treated hep 3b cells was revealed by confocal immunofluorescence microscopy (fig . Hep 3b cells were treated with 0m, 80m, 100m, and 125m antcin k for 48 hours, after which the mitochondrial membrane potential was analyzed and quantified by flow cytometry . 3c shows that as the antcin k dose increased, the mitochondrial membrane potential in cells decreased significantly (the maximum potential being 0.34 times greater than the minimum) and had a dose - dependent effect . After treating hep 3b cells with 0m, 80m, 100m, and 125m antcin k for 48 hours, unfolded protein response - related protein expression 4a shows that as the concentration of antcin k increased, the chop expressions increased in dose - dependent manners, compared with the negative control (0.2% dmso). As bcl-2 family proteins play an important regulatory role in calcium flux and mitochondrial apoptosis, we next studied the effect of antcin k on the protein expressions of bcl - xl, bax, and bak in hep 3b cells . The results showed that the bcl - xl expressions were decreased in dose - dependent manners and the protein expressions of bax and bak were increased in dose - dependent manners compared with the negative control, after treatment with various concentrations of antcin k in 48 hours . After treating hep 3b cells with 0m, 80m, 100m, and 125m antcin k for 48 hours, caspase - independent and caspase - dependent apoptosis - related protein expression was analyzed by western blotting . 4b shows that antcin k significantly increased the protein expression of cytochrome c in a dose - dependent manner, and the protein expressions of cleaved caspase-9, parp, and caspase-3 were also increased . In addition, 4c shows that antcin k truly increased the caspase - independent apoptosis - related protein expressions of aif, htra2, and endo g at 48 hours in hep 3b cells . Currently known active ingredients of a. cinnamomea include polysaccharides, benzenoids, triterpenoids, and steroids . Among them, triterpenoids have received more attention due to their potent anticancer effects . Out of three artificial cultivation methods for a. cinnamomea, basswood cultivation is most valuable and yields the highest content of triterpenoids, followed by solid culture, and liquid fermentation material, which is almost free of triterpenoids . Antcin k is a triterpenoid from fruiting bodies of basswood cultivated a. cinnamomea and accounts for 0.5% of a. cinnamomea . Furthermore, it has previously been reported that antcin k can inhibit ros generation in human neutrophils to attain anti - inflammatory effects and inhibit na / k - atpase . Our current study demonstrated that antcin k inhibited the proliferation of human liver cancer cells . Treatment of hep 3b cells with antcin k caused the cells to undergo apoptotic cell death through the mitochondrial and er stress signaling pathway . Er and mitochondria interact both physiologically and functionally, and one of the most critical aspects of this interaction is calcium signaling between the two organelles . As evident from ros production and the results of atp level analysis (fig . 3b and d), antcin k can increase ros generation in human hepatoma hep 3b cells and also reduce the atp level of the cells, suggesting that er may be damaged by oxidation and in a hypoxic state . 4a), when er stress was induced, the protein expression of chop increased, downregulated bcl - xl, and activated bax / bak channel to promote intracellular calcium ion release from the er then transfer into the mitochondria . We studied the interaction of er and the mitochondrial pathway in our ongoing efforts to determine the apoptotic mechanism of antcin k against hep 3b cells . Apoptosis occurs upon the perturbation of cellular ca homeostasis, such as cytosolic ca overload, er ca depletion, and mitochondrial ca increase.33, 34 ca overload can induce mitochondrial depolarization and, subsequently, the release of apoptosis - inducing factors (such as cytochrome c), which leads to the sequential activation of caspase-9 and caspase-3.35, 36 as evident from the results of the calcium ion signal analysis (fig . 2c and d), mitochondrial calcium ion signals were increased obviously after antcin k treatment; antcin k might cause release of calcium ions from the er into the cytoplasm, leading to the mitochondrial uptake of these ions . In addition, as shown by the results of the mitochondrial membrane potential (fig . 3c), antcin k can affect the cytosolic ca homeostasis and induce mitochondrial depolarization . 4b and c), caspase - independent and caspase - dependent apoptosis - related proteins were released, including htra2, aif, endo g, and cytochrome c; cytochrome c activated caspase-9 and caspase-3, and cut downstream protein parp, ultimately leading to cell apoptosis . Su et al have shown that eburicoic acid, a triterpenoid from a. cinnamomea, induced autophagy in human hepatoma hep 3b cells of the er stress pathway and increased the calcium ion concentration as well as the protein expressions of bcl-2 and beclin-1 . This study reported that antcin k could cause er stress in hep 3b cells and also increase calcium ion concentration in the cytoplasm, which were consistent with the findings of the previous studies conducted in our laboratory . However, the most interesting issue is that antcin k could not induce autophagy in hep 3b cells; instead, it caused apoptosis, decreased the protein expression of bcl - xl, and changed mitochondrial membrane potential . Why did eburicoic acid induce autophagy whereas antcin k cause apoptosis in hep 3b cells, although both these active components (eburicoic acid and antcin k) were from a. cinnamomea? The reason for different cell death modes may be the different protein expressions of bcl-2 and bcl - xl . Persisting er pressure will cause apoptosis or autophagy, and eventually lead to cell death; the cell death mode, on the other hand, depends on factors such as cell types, growth conditions, and drug structures . After comparing the protein expressions of apoptosis and autophagy, we observed that apoptosis would inhibit bcl-2 and bcl - xl, while autophagy could activate bcl-2 and bcl - xl, which happened to be opposite effects . Previous studies revealed that bcl-2 and bcl - xl exert two separate functions, depending on their subcellular localization . On the one hand, they induce apoptosis by (directly or indirectly) changing mitochondrial membrane potential . On the other hand, they can activate autophagy by liberating beclin-1 from its inhibition by bcl-2 or bcl - xl, allowing it to activate the lipid kinase activity of vps34 at the level of the er . However, the cell death mechanism of triterpenoids from a. cinnamomea was not the same . It may be caused by the different chemical structures, cell lines, environments, bioactivities, and bioavailabilities; even some pure components had the effect of coordination . Although we have limited references to clarify the cell death mechanism of the triterpenoids from a. cinnamomea, future studies will certainly reveal the anticancer mechanism of a. cinnamomea . Several studies have demonstrated that a. cinnamomea has a good inhibitory effect on cancer.3, 41, 42 this study is the first of its kind to show that antcin k has an antiproliferative effect in liver cancer cells . Antcin k treatment induced hep 3b cell death via promotion of intracellular ros generation and atp depletion, leading to er stress . Moreover, the detail mechanism are followed by antcin k treatment induced er stress and mitochondria membrane permeability change through the induction of ca release, chop and bax / bak protein expression but downregulation of bcl - xl . In addition, it also induced caspase - dependent apoptosis by activating caspase-9 and caspase-3, and caspase - independent apoptosis by inducing protein expressions of aif, endo g, and htra2.
Grip strength indicates the ability of hand muscles to grip, and has been used for risk stratification to predict future health problems in individuals1, to assess upper limb impairment, or to develop a suitable treatment plan2 . A decrease in grip strength makes it difficult for subjects to use their hands for many daily activities, and recovery from this muscle weakness is a major goal of rehabilitation3 . According to biomechanical principles, gripping force is produced by the complex activation of forearm muscles, with co - contraction of forearm flexors and extensors4 . In particular, it has been reported that the strength of wrist extensors is highly correlated with grip strength3,4,5 . During grasping, the wrist should be stabilized by wrist extensors at a fixed position of approximately 30 of wrist extension3 and nearly 5 of ulnar deviation5 . In a study by shimose et al.4, wrist extension training was performed and led to an increase in gripping force and increased activation of wrist extensors after training . Adhesive taping has been used as a therapeutic modality, and is clinically effective in reducing pain, improving joint movement, increasing muscle strength, performing fascial and mechanical correction, enhancing blood and lymphatic circulation, and stimulating sensory mechanisms2, 5,6,7,8,9 . Kinesio tape (kt) with elastic properties was developed by kenzo kase in 19735, 9, 10, and the taping method originated from the hypothesis that an external component could aid the functions of muscles and other tissues10 . The stretch applied on the tape provides a pulling force on the skin and creates more space by lifting the fascia and soft tissue9, which improves communication with mechanoreceptors and increases the number of motor units recruited10 . Non - elastic tape (nt) was first introduced in 1984 by jenny mcconnell11, and includes the application of a protective undertape, followed by a rigid overtape which is used to apply tension12 . It has been reported to enhance muscle activity, improve joint alignment, affect proprioception and neuromuscular control, and restore functional movement12,13,14,15 . Subsequently, kase reported that one of the effects of kinesio tape is to increase muscle strength9, and several studies have been conducted with respect to tape application methods . However, the true effect of taping on muscle strength remains unclear2, 8 . Moreover, most previous studies compared application techniques using elastic taping, but studies that evaluated muscle strength using nt are limited . Moreover, none of the studies have applied nt and kt in the same way to assess grip strength in normal subjects . The purpose of this study was to evaluate the immediate effect of forearm kinesio taping and non - elastic taping application on maximal grip strength in healthy adults . We hypothesized that application of both tapes can improve muscle strength by facilitating contraction . A total of 20 healthy adults participated in this study, and were divided into two groups (kt and nt). Inclusion criteria were absence of any known cardiopulmonary conditions and previous surgery on the tested upper extremity, and absence of any active joint pain or other related symptoms in the prior 6 months8 . Exclusion criteria were very hairy or very fragile skin5, central or peripheral neurological deficits, or consumption of alcoholic beverages or pharmaceutical substances 24 hours prior to the start of this study10 . All subjects were informed about and understood the details of the research procedure, and provided signed, informed consent prior to participation, in accordance with the ethical standards of the declaration of helsinki . The present study was conducted in a single - blind fashion so that the subjects did not realize the purpose of the experiment . Participants were seated in a chair without leaning, with the shoulder in slight abduction (15), the elbows in 90 flexion, and forearms neutral in supination / pronation1, 3 . In all subjects, maximal grip strength was first measured with a jamar digital hand dynamometer (sammons preston, usa), and the reliability test for the hand dynamometer indicated an intraclass correlation coefficient of 0.9739 . The subjects were asked to grip the handle of the dynamometer with maximum effort for 3 s with a one - minute rest break between trials, and the values of the 3 trials were recorded2, 3, 9; this was considered the initial maximal grip strength5 . After the measurement of maximal grip strength, one hand of each subject was taped and subjects were immediately asked to perform the hand grip movement with maximum strength in the same standardized manner . The tape was applied on the dominant hand based on the edinburgh handedness inventory, and the principal investigator applied the tape for all subjects2, 8 . For most of the basic applications, the muscle and tissue were placed in a stretched position, and all subjects were asked to perform elbow joint extension with wrist joint flexion while the tape was being applied . Based on a study by kouhzad mohammadi et al.2, the length of the tape was measured from 2 cm inferior to the lateral epicondyle of the humerus to the styloid process of the radial wrist joint line before application . The area to be taped was cleaned with an alcohol swab16, and the i - shaped kinesio tex tape was applied over the common wrist extensor muscles from their origin to insertion with 50% stretch tension in order to facilitate muscle function2, 5, 9 . Using the kt methods, the nt was applied in the same position and direction . A protective hypoallergenic tape (endura fix tape, endura - tape pty . Ltd ., australia) was carefully applied over the skin without creating tension, and a rigid non - elastic tape (endura sports tape, endura - tape pty . Australia) was applied directly over the undertape with enough tension to create crimping or puckering of the skin12 . Spss software was used to examine the change in maximal grip strength after forearm taping . The shapiro - wilk test was used to check the normal distribution of data before analysis2 . Because the collected data did not meet the normality assumption (p<0.05), two non - parametric tests were used . For comparisons between the two independent groups (kt and nt), the mann - whitney u test was applied . For comparisons between two dependent quantitative signs as non - parametric tests were used, median values replaced mean values for comparison11 . No significant difference was found in the maximum grip strength between the two groups (z=1.066, asymp . However, in the kt group, the maximal grip strength was significantly increased compared to the initial value (z=2.519, asymp . (2-tailed) = 0.012); in the nt group, there was no significant difference in the maximal grip strength (z=0.354, asymp . Table 1table 1.changes in maximal grip strength (kg) just before (pre) and immediately after (post) tapingpre - testpost - testm sdm sdkt group31.6 7.133.1 8.4nt group36.1 11.635.9 11.7median values were used for comparison . * values are means sd . Shows the maximal grip strength just before and immediately after application of the tape . A recent study showed that there is a strong correlation between grip strength and wrist extension strength3, with the tape applied over the forearm extensor muscles to determine the change in the maximal grip strength . The use of adhesive tape is gradually increasing in the clinical field, and several studies have examined it as a therapeutic intervention that may lower costs and improve outcomes6 . It has been suggested that both rigid and elastic taping techniques alter muscle force, neuromuscular control, and proprioception17 . This study examined the effects of two tapes with different elastic properties on maximal grip strength . First, significant increases in maximal grip strength were observed immediately after kinesio taping over the extensor region of the forearm . Previous studies reported the same results2, 5, 10; in particular, the study by lemos et al.10 showed that the increase in grip strength was maintained for 48 h after the application of kt . It is thought that the effect of kinesio taping on muscles is due to the reflex mechanism of the nervous system . Most studies have reported that taping over the skin constantly stimulates cutaneous mechanoreceptors, thus providing more sensory signals to the central nervous system for information integration2, 5, 9, 11, 18 . In addition, reduction of motor neuron threshold induced by cutaneous stimulation would influence the recruitment of the motor unit, which can facilitate muscle contraction, and ultimately improve muscle strength5 . Kouhzad mohammadi et al.2 also suggested that kinesio taping increases sensory feedback of the taped region through skin stretching, thus facilitating contraction of inactive muscles10 . Although the working mechanism of nt has been shown to be similar to that of kt13, 16, no effective change in maximal grip strength was noted immediately following the application of nt . As in our study, donec et al.5 applied 5-cm wide nt medical tape (mefix) in a placebo group and compared it with a kt taping group, and found a significant difference in maximal grip strength in only the kt group . Alexander et al.12, 15 applied nt on the skin overlying the lower fibers of the trapezius and gastrocnemius in healthy subjects, and motor - neuron pool excitability was assessed using the hoffmann reflex (h reflex). The result was surprising in view of the fact that taping is thought to facilitate muscle fibers; however, taping was found to inhibit the h reflex . We assume that these results considered the muscle length using rigid nt application methods . Unlike kt, nt was applied with enough tension to create crimping or puckering of the skin . Alexander et al.12, 15 suggested that if the muscle is held in a shortened position by the tension of the tape, there may be reduction in tonic muscle spindle activity . The reason grip strength was not increased in another study should be discussed8, 9, 19, 20 . Different taping application techniques with different tensions and forms can provide different tactile stimuli19 . While we applied the tape in the direction of muscle origin to insertion with 50% of its maximal length tensed in the stretched position, they applied 75% tension . In another study by chang et al.9, kt was applied over the common wrist flexor muscles from their insertion to origin with 1520% tension in healthy subjects, and there was no statistically significant difference . In contrast, the study by kouhzad mohammadi et al.2 showed that there was a significant association between grip strength and taping region, and that taping of the extensor region increased the maximal grip strength significantly, compared to taping of the flexor region . In our study, nt did not increase muscle strength, but park and kim21 and lee et al.13 showed increased grip strength using electromyography in patients with lateral epicondylalgia, with the tape applied in a spiral or cuff form around the elbow joint . Moreover, the study by chou et al.22, in which the tape was applied from the distal end of the lateral malleolus, wrapping around the posterior lower leg in subjects with chronic ankle instability, found a significant increase in h: m ratio in the soleus muscle . Although taping techniques are used in clinical practice in order to achieve various therapeutic effects, there is limited evidence regarding the effects on muscle strength . Two types of tapes were applied in the same way, and the results showed that the kt group had a statistically significant difference in muscle strength; however, in the nt group, there was no difference in muscle strength . We believe our research will provide guidelines for clinical practice for use in subjects who need to increase their muscle strength with use of the tape.
In chronic obstructive pulmonary disease (copd), many different patient groups are represented . Patients include those with chronic bronchitis to the emphysematous, with overweight or with nutritional depletion, and from irreversible obstruction to having a reversible component besides persistent obstruction . By gold definition,1 and in daily practice, all these groups are termed copd . In most recent (therapeutic) copd trials,2,3 however, a strong entry selection occurred to ensure population homogeneity, thereby diminishing external validity.4 from large prospective studies2,3,5,6 it has become clear that the progressive lung function loss in copd cannot be altered by inhaled corticosteroid therapy . However, these randomized clinical trials strictly excluded patients with any form of reversibility for methodological reasons.7 in practice, the diagnostic prednisolone test has been used widely to identify the patients responding to oral steroids, thus foreshadowing the presumable efficacy of inhaled corticosteroid therapy . It is disputed what proportion of copd patients suffer from persistent obstruction with a reversible component . Estimates differ from 10% to 30% depending on clinical setting.79 it is estimated that 20%30% of patients with copd may experience a significant improvement in fev1 from short - term corticosteroid use.7,1012 in case of persistent obstruction with a significant reversible component, a diagnostic prednisolone test can be performed, although the validity of this test is questioned and different cut - off points for response are advised according to several international guidelines.1,1315 as a consequence, the utility and predictive value of responders within the copd population is vigorously debated.7,9 small, short - term studies in selected patient groups have described different regimes of prednisolone testing, and as a result the more or less accepted current form (14 days of 30 mg prednisolone) has been part of the diagnostic work - up of copd - patients.1619 however, the prednisolone test was never validated prospectively in a primary care population.20 in severe disease without reversibility it has recently been shown not to be useful by american thoracic society (ats) criteria.21 as a result, it was concluded that a short course of oral corticosteroids is a poor predictor of the long - term response to inhaled corticosteroids in copd . The aim of this study was therefore to determine the predictive value and usefulness of the prednisolone test; to what extent is the clinical efficacy of inhaled corticosteroids (fluticasone propionate 500 g bid) versus placebo related to a positive test response in a primary care population during three years of follow - up . The coopt trial22 is a double - blind, double - dummy, randomized placebo - controlled clinical trial with a three - year follow - up undertaken in the netherlands between 1998 and 2004 . General practitioner (gp)-diagnosed patients with chronic bronchitis and copd from 44 general practices participated in the study, when postbronchodilator forced expiratory volume in one second (fev1) was between 40%90% predicted, and fev1/forced vital capacity (fvc) was below 88% (males) or 89% (females) according to former european respiratory society (ers) criteria.13 a clear history of asthma, allergic rhinitis, or atopic eczema was an exclusion criterion, while reversibility to bronchodilators was not . Outcome measures were health status, as measured with the chronic respiratory disease questionnaire (crq),23 exacerbation frequency and postbronchodilator fev1 . An exacerbation was defined as an episode with one or more subsequent unscheduled contacts with either a gp or a pulmonologist due to worsening of respiratory symptoms . In this randomized clinical trial a three - leg design was used, with fluticasone propionate and n - acetylcysteine as intervention groups, the third leg as placebo . An independent statistician generated a randomization list based on a block size of three for treatment allocation to balance the three treatment arms by study center . Neither investigators nor patients were aware of the group assignment . In this analysis we compared the fluticasone propionate 500 ug twice daily administered as dry powder inhalation by diskus inhaler versus placebo legs . A wash - out period of three months preceded the study, allowing any effects of stopping inhaled steroids or n - acetylcysteine to subside . During this wash - out phase, 12% of original study candidates withdrew . In effect, the study group can be described as patients clinically diagnosed as having copd by their gp, but who did not get worse if inhaled steroids were stopped for three months . Before randomization took place, all patients underwent the prednisolone test, before and after which lung function measurements were taken . The diagnostic prednisolone test is generally defined as fev1 measurements before and after 14 days of 30 mg prednisolone, but cut - off points for a positive response differ among the various international guidelines . The ats14 considered an increase of> 12% and 200 ml of baseline fev1 as a positive response, while the british thoracic society (bts)15 stated an increase of> 15% of baseline fev1 as a positive response . By contrast, the ers13 used to recommend a 10% increase of fev1 predicted in their guidelines, but this recommendation has been left out in the 2004 ers / ats guidelines.24 specific criteria for positive response have consequently also been deleted from the british nice guidelines25 and the executive summary of the gold guidelines.1 differences in longitudinal scores on health status, exacerbation frequency, and postbronchodilator fev1 were tested on an intention to treat basis, by using statistical techniques for repeated measurements . The placebo group has been taken as representing the natural course, relative to the intervention groups . For statistical testing of differences in exacerbation frequency between placebo and intervention groups during the study period we used a correlated time - event model26 (genmod - procedure in sas; poisson distribution, compound symmetry correlation structure). To analyze the effects on health status, a longitudinal analysis was performed on the crq - total score, while decline analyses for repeated fev1 measurements were used for statistical testing of differences in annual lung function . The regression model for these outcomes accounted for correlation between repeated measurements27 (proc mixed in sas, compound symmetry correlation structure). The number of responders differs from 25 (9%, ers) to 44 (16%, ats) depending on guideline used . Responders show a significant higher proportion of females, almost equaling men, in comparison with nonresponders across all guidelines . Age, smoking behavior, pack - years, degree of obstruction, and reversibility to bronchodilator do not differ significantly . The long - term effectiveness of fluticasone propionate relative to placebo in responders versus nonresponders is shown according to different guideline criteria . Outcome variables are exacerbation rate, health status, and postbronchodilator fev1 . On average, clinically relevant differences in health status were not reached . Responders according to ers experienced a borderline significant effect of inhaled fluticasone on health status (0.29 points / year, p = 0.05), and to a lesser extent in bts responders (0.26 points / year, p = 0.06). Ats - responders did not experience any significant benefit from long - term fluticasone use . Possible clinically relevant reductions in exacerbation rate (rate ratio 0.67) and fev1 decline (39 ml / year) occurred in bts responders, but did not reach statistical significance . These effects were similar (rate ratio 0.68, fev1 decline 30 ml / year) in ers - responders, while much smaller in ats responders (rate ratio 0.78, fev1 decline 8 ml / year). To our knowledge, this study for the first time prospectively relates the responses to long - term inhaled corticosteroid therapy to different cut - off points for prednisolone testing in primary care . Between 9% to 16% of the copd population was classified as a responder depending on criteria used . On average, clinically relevant differences in health status responders according to the former ers guideline (fev1 increase of 10% predicted) experienced a borderline significant effect of inhaled fluticasone on health status (0.29 points / year, p = 0.05) during three years of treatment . A similar, but not significant effect on health status (0.26 points / year, p = 0.06) was seen using the former bts criteria, while ats responders showed less effect (0.20 points / year). Possible clinically relevant reductions in exacerbation rate (rate ratio 0.67) and fev1 decline (39 ml / year) occurred in responders according to bts, but did not reach statistical significance . Similar results were seen in the ers group (rate ratio 0.68 and + 30 ml, respectively) but were less similar when the ats criteria was applied (rate ratio 0.77 and + 8 ml, respectively). Interestingly, the only other long - term prospective study21 that assessed prednisolone testing on these outcomes, but not in a primary care population, concluded on the basis of using ats criteria that there was no relationship between the short term response to prednisolone and the rate of decline in fev1 or health status. In fact, our results are in line with that widely cited study, since we only found any possibly meaningful results using the ers and bts criteria . In addition, the isolde researchers looked at the so - called callahan criteria (fev1 increase of 20% of baseline), which were derived from a meta - analysis12 looking at oral corticosteroid therapy, not prednisolone testing . In fact, this cut - off indeed also showed a significant effect, but this was deemed by the isolde researchers to be the result of confounding . Thus, the criteria used appears to matter and we cannot recommend the use of the former ats criteria when performing prednisolone trials, based on our results and on those of the isolde researchers . In our study, the proportion of primary care copd patients meaningfully labeled as responder ranges from 9% (ers) to 12% (bts), which is considerably lower than expected . We therefore fear that our study was underpowered, since estimates in literature ranged from 20%30% responders . However, it is also possible that we underestimated the number of potential responders in the population, since for ethical reasons our study design provided a wash - out period to exclude all patients that experienced more than two exacerbations when inhaled steroids were taken in the three months preceding entry to the study.28 in our population, this means that steroid - dependent patients were not allowed to enter the study . Contrary to the isolde researchers, we found a significant gender difference in prednisolone responsiveness across all criteria groups . Women appear particularly prone to copd when exposed to similar amounts of tobacco29 and interestingly, the proportion of female copd patients responding to prednisolone was consistently higher than among nonresponders at baseline . No other significant differences were seen between responders and nonresponders in terms of age, lung function, current smoking, or pack - years of smoking . This apparent gender difference in prednisolone response therefore deserves further study, since earlier studies probably did not include sufficient numbers of females with copd, whose prevalence has been seen to rise only in recent years . In this study, a clear history of asthma, allergic rhinitis or atopic eczema was an exclusion criterion, while reversibility to bronchodilators was not . However, we found no relationship between prednisolone response and bronchodilator reversibility, which was very similar across all criteria groups (table 1). We consider it highly relevant to clinical practice that reversibility to bronchodilators apparently does not correspond to prednisolone responsibility, since these terms are sometimes interchangingly used as parameters of the same phenomenon . These study data indeed suggest that the response to oral steroids may describe a different underlying inflammatory process than the response to bronchodilation, which is of a fundamentally different origin . Our results suggest a borderline significant effect on health status in ers responders, but is it clinically meaningful? The minimum clinical difference on the crq is 0.5 points, which is clearly higher than the 0.29 points reached on average in our study, which renders this result marginal . However, the rate ratio for exacerbations (32% less than placebo) and even the fev1 decline (30 ml / year less than placebo) point in the same direction in both the ers and bts groups (33% less exacerbations and 39 ml / year less fev1 decline, respectively), which may indicate possible clinical relevance . The systematical difference in effects on all three outcomes compared to the nonresponders (and indeed the total group of copd patients) suggests that this rather small group of responders to prednisolone could be a limited subgroup in primary care, which would need different medical treatment . Since the number of responders (using the ers or bts criteria) now can be expected to be around 10% in a primary care population like ours, we think these results would probably need replication in a larger primary care study . This small but possibly relevant proportion is identical to the 10% that was found in the earlier meta - analysis on the benefits of oral corticosteroid therapy for copd patients.12 as has been stated before,30 it is important to keep an open mind about the possibility that there may be responder and nonresponder subgroups and to continue to seek ways to identify and characterize them, especially in primary care where heterogeneity is common.20 oral steroid testing distinguishes a limited proportion of copd patients, but does not reveal clinically relevant benefit from inhaled steroid treatment on health status.
Risk benefit analysis with or without concern for legal issues has always been the mainstay of clinical practice . Beneficence, insofar as it is a principle in medicine, has to a greater or lesser degree always been based on this issue . Even under the accusation (and hopefully this argument is resolved and exhausted now), whatever decisions doctors used to take was based on what in their opinion was in the patient s best interests and for the patient s good . Leaving patients ignorant of their disease and outside the realm of decision - making was considered a therapeutic privilege and done in order to protect the patient from news which he or she did not or should not know in order to keep them from grief which could cause them more harm than good . The term values is used in order to emphasize that it is not merely a question of relativism or simply because we understand human nature better . It was indeed the philosophical debate which contributed considerably to increasing the principle (in the kantian sense) of respecting autonomy and trumping it over beneficence, until beauchamp and childress1 came around to give us (or rather, lay down what was in the air) the four principles . These evolved into an ethical framework and indeed doctors and health care providers were invoked to use these principles in their decision - making, calling them mid - level principles . Principlism came under attack2 and the defenders went on to invoke methods of using principles, such as specifying and balancing . Specification and balancing were meant to address the particular situation, yet steering away from situational ethics . Assessing there is overlap also with the principle of justice because these decisions invariably also involve the use of resources that can be allocated elsewhere (without, of course, bowing to utilitarianism, as indeed even a deontologist must consider her duty to society). In the meantime, the field of gerontology and geriatric medicine has taken on new life, and rightly so; we are not only concerned with the elderly as a specific group, needing special needs with special attention to differences in medical care, just as children need a different approach to adults, but also because our populations, and perhaps cultures, are aging.3 making decisions concerning risk benefit for the elderly invariably therefore takes on a completely different dimension than for other adults, not only because where the elderly are concerned there is a different physiology and an aging body, but also because we are dealing with perhaps an aging culture, and more persistently so because we have to face the bottom line question: is it worth treating this patient at this stage of life? These may be blunt questions, but it is a reality from which discussions of end - of - life decision - making ensue . The issues of cardiopulmonary resuscitation, advance directives, and research into longevity have arisen . Because we are discussing an ethical concept, and not merely a clinical algorithm, what we have at base is our attitude, in philosophical terms, towards our elderly populations . The first discusses the reality of principles as an ethical framework and whether in essence it is our values towards the above questions which define outcomes . Secondly, it discusses three real - world situations, the third of which discusses how we view elderly in this postmodern society . Decisions are usually based on inherent cultural beliefs from which emanate virtues and general principles . Inherently we are what we are because we have learned basic concepts throughout life which set the scene for our principles . But both the use of and the foundation of these principles are inherently attached to our concept of what is or should be right and wrong . A difference at this level, and you invoke a different reasoning on how the same principles are used to arrive at another different answer . Indeed when we deliberate a moral problem we start with an empathic feeling of what is right and what is wrong . Rational thought about a situation may allow us to change our course of action and perhaps the final decision, but our initial value system remains essentially unchanged . Indeed, it is probable that we have conflict amongst principles because we have different value systems . Gregory tillett perhaps best explains this conflict.4 tillett, in his analyses of resolving conflict distinguishes three concepts, or categories, of differences between ideas, humans, relationships, and between internal (within oneself) issues, ie, problems, disputes, and conflicts . A problem can be resolved by management, ie, by the agreement on how something can or should be done . We can easily conceive of a difficult clinical situation in which one consultant or family practitioner asks the advice of another, and doctors within a team discuss courses of action between themselves and with patients, especially within a context where there is refusal of a particular kind of treatment . Tillett points out that these problems can indeed become disputes and can generate conflict.4 a dispute occurs when two (or more) people (or groups) perceive that their interests, needs, or goals are incompatible and they seek to maximize fulfilment of their own interests or needs, or achievement of their own goals (often at the expense of others). Consider, for example, the situation of an elderly woman who is being taken care of at her daughter s home . This daughter has three brothers, one of whom had distanced himself from his sister due to a dispute with her husband . The main contention was that this brother convinced the others that their mother should be put in a home because he refused to visit her at their sister s home . Of course, prima facie, the best interests of the patient were for her to remain at home . However, it transpired that the woman was very depressed because this son, who was evidently her favorite, was not visiting her . The undue pressure was indeed coercive and the woman conceded to go to the home in order to be able to see all her children, not at all a voluntary choice . In the meantime after eight months in hospital and no remedy in finding a place at an institution, the decision had to be reversed to take into consideration again her best interests, ie, avoidance of a hospital infection . Because the daughter was still willing to take her back, this course of action was taken, and the necessary legal procurements arranged . At each stage however a value - laden decision was taken, which brings us to the third category, ie, conflict . Conflict, again as defined by tillett, arises when two (or more) people (or groups) perceive that their values or needs are incompatible whether or not they propose, at present or in the future, to take any action on the basis of those values.4 whilst problems and disputes arise within specific situations, conflicts do not need one . Two parties can be in conflict because of beliefs or because of the values they uphold, and hence the heated debates on such issues as abortion and euthanasia . But, as in the case described here, we may find that superficially, at least, the people involved had the same values in that they all loved their mother, but when it came to taking a decision which was in the best interest of their mother, it was rather a decision in their own best interests which had become evident, until a situation arose which gave enough strength to override these different values . The difference in value was between one in which x would rather have his mother in a home and be able to see her (and perhaps that she would be able to enjoy him), and the same person admitting that notwithstanding that his mother would not be able to see him, his sister was in a better position to take care of her and that given the state of existing homes, she may be better off . The conflict was that between x and the husband of x s sister, and not the situation . Admittedly there can be a hazy line between a dispute and a conflict, as in this case . One has to delve into the narrative of each participant in order to reveal a conflict and understand that the matter is not simply one of disputes . In this case, the brothers agreed that the mother be allowed to go to their sister s home on the condition that the sister use her pension only and not the mother s savings in order to take care of her, ie, if she needed a new bed or a commode, they were not willing to contribute to either . This coercion reveals the underlying conflict between the daughter s husband and the dominant brother, notwithstanding a resolution of the conflict . It seems that the needs or values of the children trumped the needs of the mother or the value to respect and meet her needs . Certainly this was not a dispute any more but a conflict, which unfortunately the medical practitioner or team may not be able to handle, unless by enforcing legal processes which may not be in the interests of all parties and not so conducive to conflict resolution . Justice is conflict.5 doctors make decisions in the light of beneficence and at the same time doing justice to the person(s) and the situation at hand . By doing justice, we obviously do not merely mean that the situation is handled well and correctly (within, that is, the boundaries of standards of care), but we seek that justice is done morally . Deciding whose justice and which rationality is certainly not easy in the modern sense, especially in dealing with end - of - life situations, whether to treat or not to treat, whether to leave elderly for acute treatment at their home, or incur a more expensive, perhaps better treatment, but in a hospital setting . Certainly there can be hardly a better exposition of this philosophical reality than that put forward by macintyre.5 whether he does justice to the topic is probable but whether he resolves the dilemma is uncertain . Certainly he speaks extensively of tradition, which is an argument extended through time, in that it can be a medical tradition or a religious tradition or simply a tradition of horse - riding . Solomon6 believes that morality cannot be reduced to any strict kantian system of principles . The very fact that the four principles of health care require further specification and balancing shows that a deeper sense of the real situation is needed, and what this usually boils down to is the narrative of the parties involved who bring forth their values and needs . Solomon does not particularly like macintyre s use of traditions, in the sense that it still leaves open the nostalgia for religion and a sense of community, but goes on to admit that aristotle did invoke a sense of community when speaking about virtues . Neither does he associate virtue with feminist ethics, values not being supposed to be held captive to gender, even though feminist ethics may have a point in contrasting male principles which are hard, oppressive, and impersonal, with the female virtues of warmth and caring . Colloquially, by having principles, we are actually talking about the values a person holds as well . If one has a so - called value for life and is not in favor of euthanasia, one invokes principles to lead one to this conclusion . Conversely, if one s value system of life includes terminating suffering even by allowing physician - assisted suicide, then one will use the same four principles to reach the opposite goal . Having summarized the philosophical foundations on which risk benefit analysis is based, various relevant clinical situations faced in health care are grouped . Jonsen et al7 classify three forms of disease and goals of medicine that are summarized here with particular reference to care and decisions for the elderly . These authors say that in the first, the patient suffers from an acute illness that, once diagnosed, can be readily treated and cured . In the second, the patient experiences a process that causes serious disabilities and which, while some relief can be provided, will progress despite treatment and eventually case death . In the third, the patient suffers a chronic disease that can be effectively treated so as to relieve many of the most debilitating effects . For simplicity they use the acronyms acure (acute, critical, unexpected, responsive, and easily diagnosed and treated), care (critical, active, recalcitrant, and eventual), and cope (chronic, outpatient, palliative, and efficacious). Perhaps using simply acute, critical, and chronic would have been quite an accurate substitute, but these acronyms serve the purpose just as well and are applied here . These authors point out that physicians habitually approach medical problems by attempting to determine the indications for or against medical intervention . In fact, assessing risk and benefit takes both medical intervention and ethical dilemmas to task, especially when considering elderly patients and when one asks oneself whether one should treat or not in a particular case . Medicine remains a science of uncertainty; even these acronyms are understood as being probability statements rather than absolute designations . Indeed, uncertainty makes for good decision - making in the face of risk benefit analysis, and not simply one of clinical competence but also one which is value - laden . These goals apply to acute and relatively straightforward conditions, especially once a diagnosis has been made . The situations may be critical and unexpected, such as meningitis, reversible and easily treated (by easy one of course does not mean easy in the strict sense, but that one knows what to do and has an immediate plan of action based on evidence - based medicine and/or standards of care). Ethical issues in this situation may range from simply refusal of treatment to families not wishing the elderly patient to know . Recent literature suggests a more culturally sensitive approach in these clinical situations,810 respecting wishes which may be culturally sensitive . In japan for example, it may be considered disrespectful to let an elderly person know of the medical condition, such that the sons and daughters take on the responsibility . The second category (care) describes patients with active, progressive, and deleterious conditions . One can easily see many elderly people in such situations, although this is by no means restricted to this section of the population . Under this category lie most of the ethical dilemmas facing end - of - life decisions, such as withdrawing of life support, decisions not to intubate, decisions not to resuscitate, and irreversible coma / brain death . So are the mainline ethical discourses of ordinary versus extraordinary treatment, killing versus allowing to die, treating versus tender loving care, etc . What is in fact ordinary and extraordinary treatment? Standards of care weigh in heavily . Even in invoking the principle of double effect by using high doses of morphine, for example, to treat palliatively, knowing that there is a risk that the patient may die of the dose of morphine and that this was a foreseen but unintended consequence, one must understand the nature of increasing doses of morphine and not be caught in a dispute of hastening death by going about the principle of double effect . Indeed, even though the principle has four basic rules to it, it is heavily burdened on the moral agent to ascertain that it is morally and correctly invoked (just as it takes a moral agent, for example, not to abuse the abortion act in the uk). There is always a standard of care in increasing dose strengths, although this is a patient - sensitive decision and hence can be externally seen as subjective . There is no duty to treat when treatment is judged to be useless, nor a duty to treat when treatment is deemed extraordinary . Although today proportionate and disproportionate are terms which may be used instead of ordinary versus extraordinary, the latter are not only still the most commonly used terms, but they are probably the best guideline to make a judgment on risk benefit . However, one must clearly understand the meaning of extraordinary to make best use of the term, and for this one must look at its original catholic origins . Extraordinary means of preserving life are all medicine, treatments, and operations, which cannot be obtained or used without excessive expense, pain or other inconvenience for the patient or for others, or which, if used, would not offer reasonable hope of benefit to the patient.11 a closer look at this definition shows how this covers most, if not all, legal issues that may arise when making moral choices on risk benefit . When assessing risk, we are indeed facing the particular and singular clinical picture of this patient, including fitness for surgery, side effects and interaction of drugs, wishes of the patient, and physical state of the patient . The definition clearly does not take into account the state - of - the - art of medicine and technology, and something that is considered extraordinary today may by no means remain so tomorrow . Conversely, something that is quite ordinary treatment in one country, either because of culture or economic status, may indeed be extraordinary in another . Family need not be put to excessive burden, especially if they are required to pay or to go through extraordinary measures to meet the patient s needs . Clearly, where insurance and state cannot or will not pay, the family cannot morally be held responsible to take all measures necessary (in malta, it has almost become a societal pressure to go to the uk for care when one finds no hope on the island, even if consultants say that no further treatment can be found there). Of particular note is that even medicines and excessive expense are mentioned in the definition, along with pain and inconvenience to the patient or family . Hence a treatment which cannot be afforded or which will cause prolonged agony need not be given . These issues are mostly found in outpatient and community settings of family practice, and deal with the chronic and palliative . Although jonsen et al lament that the ethical issues here may be less evident, they in fact may represent the same issues discussed above . Cope is a good acronym because in fact we are working with the patient to although the same goals of preserving life, preserving function, and reducing pain and suffering are used, they take on a more patient - centered approach and reach compromises with the patient that allow him or her to participate in the treatment . Whilst there may be no drama as in life - and - death situations, there is a quality - of - life perspective and also end - of - life decision - making to face . The american college of family physicians have recently paid much attention to helping families cope with end - of - life and also address the cultural aspects of individuals and families.12 this departs considerably from the earlier days of bioethics when autonomy meant revealing everything to the patient and almost burdening the patient with information, giving details in order to allow them to feel they were making a choice, or preferably making the choice themselves . This renewed cultural sensitivity brings back the onus on the practitioner to be truly patient - centered and to share the burden with the patient who usually indeed seeks the doctors advice . The bigger burden lies with difficult patients, ie, those who are noncompliant and those who pose problems such as never being fully satisfied . In these situations, the doctor may be impelled to transfer the care to someone else, because a breakdown of the relationship is perceived to have occurred . Yet patient relationship as well as compromise, and this may in turn translate into breakdown occurring less often . Legal issues may arise with the family, who may not be satisfied with the care imparted to their parents . However, we need to acknowledge in this regard that the involvement of the family as a community - based approach to treating the elderly is still compatible with the patient - centered approach.13 of course the concept of aging has changed over time . Today, with discourse on aging, one may find less difficulty with, eg, an elderly man seeking help for erectile dysfunction . Yet many bioethics committees recommend against women having fertility treatment beyond their menopausal age . With advancing technology, the right to treatments, perhaps with aging populations, will probably give ground to the yearning of the elderly spirit to continue enjoying life beyond the social boundaries thus far accepted . Leon kass, former chairman of the us council on bioethics, found considerable opposition to his general wariness about reproductive technology and efforts to forestall aging.14 therefore, risk benefit analysis cannot look at the social concept of aging in a postmodern and perhaps posthuman society . The concern for us now lies in making the same analysis of legality in risk - benefit not only with regard to medical care but also in terms of research, this being imperative to the advancement of medicine . We have to decide which research is moral and which not to finance with public funds . There are strong arguments for the moral imperative to research elderly subjects further, even though research in this realm is both ethically required and ethically suspect.15 do we carry out research in order to increase longevity, to curb the aging process, or even perhaps to stop aging all together? Whereas better cures and medical advances have already brought this out, it was never really a medical imperative . It was, at most, a welcome, although perhaps foreseen consequence of treatment and care . Yet the line is quickly drawing a distinction between a rise in the elderly population due to better survival and a deliberate search for survivorship and longevity, although such research into the genome and molecular cell biology is rapidly becoming hazy . In discussing aging and the sociology of embodiment, featherstone and hepworth16 give an interesting account worth reflecting upon . At the end of the day, the body is the bottom line, when we survive to advanced old age.17 elias, in his old age acknowledged, with an air almost of awe, that there is a simple reality of a finite life.18 therefore, the body in decline, for featherstone and hepworth,19 is a central issue for contemporary society to come to terms with . They assert that this does not mean that historically, when life expectancy was much shorter, people did not strive to prolong their life . Fear of death, intensity of bereavement, and the longing for earthly pleasures are all issues to be faced . It is precisely in the struggle to reconstruct this cultural inheritance of pessimism that the element of difference between past and present attitudes towards aging through the later period of the life course may be found.19,20 this reconstruction, according to these social authors, lies in moving away from the pessimistic and melancholic processes of aging to one of optimism and a period enriched with distinctive creative possibilities.16 only in this way can the structure of feeling and attitudes towards aging be changed, especially with regard to the aging body . This social reconstruction (or better, change in social acceptance), will then perhaps illuminate our attitude towards caring for our elderly in a new way and not simply as seeing them one of the vulnerable populations, even though they may be . We may see new light in research into the elderly and perhaps even in some form of longevity and prevention of body decline, which would bring with it a continuation of activity, be this sexual, educational, or occupational . We are already seeing countries prolong the retirement age due to improved standards of living and quality of life, and this not merely because elderly populations are on the rise and we need to continue collecting taxes from them . In understanding our ethical and legal attitudes to the risk and benefits of treatment, we must indeed be wary therefore of any desire by groups, such as practitioners of geriatric medicine, to make claims of a specialized form of knowledge and to legitimize the imposition of controls over aging members of the population.21 one should not take this to mean that geriatric medicine should not exist, but to entail a change in concept even of the discipline itself ., much as family doctors are advocates for seeing the family more consistently in the biopsychosocial dimension, acting as family counselors and advocates for patient rights . Notwithstanding the discipline of geriatrics and the vulnerability experienced by an increased percentage of this sector of the population, we cannot continue to see old age as a form of pathology and the body in decline as a medicalized entity, which according to katz, is an accusation often leveled at medicine . Perhaps we need to understand that the stages model of life is a cultural clich, as are expressions like ticking of the clock.22 the implications for the sociology of ageing are clear: it is no longer possible to make adequate generalizations about the aging process that are grounded on biological assumptions about the ages of life . Nor is it particularly useful to adopt schemata of the life course based upon loosely conceptualized models of unspecified processes of interaction between the ontologically distinctive entities, body, self and society . As a consequence contemporary models of ageing into old age must be increasingly post - modern, by which we mean they must anticipate even more advanced forms of biocultural destabilization.16 these authors eloquently argue how our struggle is not really with biologic decline, but with the social construction of aging that has been the center of debate . Hormone replacement therapy, even if it had an element of success, was more about remaining young and active, hidden behind the excuse of better heart and skin outcomes, until the reality of the cancer issue came to light, ie, something which many were very skeptical about from the outset . The social pressure to take hormone replacement therapy cannot be denied, in that many women may have taken it seeking a better skin complexion and less wrinkling, or at least have been encouraged to take the medication for these reasons . It is interesting that postmodern feminist studies of the interpretation of aging were associated with wisdom . Before patriarchal societies, the female crone was seen as a naturalistic and matriarchal part of life, with wrinkles being badges of honor.23 history has brought about a separation of the self from the body and society, and the seeking of youth is found at the base of this force . Whether this is the result of patriarchal influence which feminism blames is beyond the scope of this paper . The profound reality is that it is an existential issue and an entity to be reckoned with . This dualism, ie, the separation of the self from the body, lies in the very nature of the fear of aging in terms of a declining body . Is an expression that perhaps was less heard in primitive society, which adorned their elders with a wisdom and respect much less seen today . This lies at the heart and concept of the recent change towards end - of - life decision - making . Western culture is learning yet again from populations which lagged behind the materialistic taint and preserve many of their traditional values, and such is the case for eastern countries . Featherstone and hepworth continue to make the argument that whilst accepting the limits of the body and the aging process as having its own rewards, they adopt a negative attitude to technologic advances whereby medical intervention, such as hormone replacement therapy, is a possibility . Nowadays, in ethical circles, words like thus, the main question would be whether hormone replacement therapy is unnatural and a threat to womanhood? Indeed, there is much sense in not seeing technology as an external factor to human nature, but as a relationship between nature and the human bodily nature (the creator of the technology itself), which is a culturally dynamic process . We have stopped seeing technology as part of our nature, because it is human nature which creates it, just as a monkey uses a stick, and perhaps moral discourse is more based on a fear of progress rather than a balancing of moral choices . When seen in this light, we are be forced to accept a change in balancing risk as opposed to benefit . Whilst medicine has been attacked for being paternalistic and indeed domineering, in a foucaultian sense it is the world in general that has strived for technology and improvement of the human condition . Medicine has certainly taken advantage of new technologies for better cure rates, but research is often not carried out by medicine as such, but by corporations who strive to satisfy the thirst of society, and profit of course in the process . There is a delicate, and sometimes controversial, balance with cultural and religious values . There is nostalgia, ie, a longing to remain attached to certain roots.24 the reality of the new field of bioethics, as opposed to the centuries old hippocratic tradition, emerged at a time when technology was also blooming . Therefore, we may still be tied to concepts of not treating the elderly because of their age . The balance between cultural nontelling, and indeed giving a treatment adequate to what the body can withstand (it would be unwise to opt for surgery, but certainly a long - term course of tamoxifen, even if palliation is not needed) is not that difficult to conceive . However, accepting cultural criteria, such as the children of this woman, indeed in their late 60s themselves, of not giving her the bad news, even if she had never explicitly expressed a wish not to know, is the morally (and perhaps legally) correct thing to do . What is probably wrong in our approach to the elderly is to see the changing body as a pathologic process . Even if we can come to accept change in body parts, better appearance, etc, as socially acceptable, there will always be a time of reckoning with death and the human condition . Decline begins very early on in life, perhaps immediately after peak physical growth is reached . There are certainly problems that affect the elderly more, as there are problems that affect children more . Two years for a 70-year - old is a larger proportion of one s remaining lifespan than the same number of years for a 40-year - old . Certainly balancing risks and benefits may not work out in favor of the elderly person . A new social awareness towards aging helps cultivate an attitude that old age is worthwhile for what it is rather than for what the elderly do or what they have . Whilst looking at the risk and benefit of treatment, we cannot ignore the larger risk benefit picture of what research and medicine hold in the future . Advanced research must be respected as part of the human need to ask, invent, and discover . However, a balance must be struck so as not to uproot people from their cultures because this is what gives people their identities . Simone weil warns against uprooting people, cautioning that uprootedness leads to misery and spiritual lethargy on one hand and to violent efforts to adapt and uproot those not already uprooted.
Leptin, a 167amino acid hormone, was discovered in 1994 and is secreted mainly by adipocytes . Plasma leptin levels are significantly correlated with body mass index (bmi) and the total amount of body fat . A recent study reported that total fat mass is the strongest predictor of circulating leptin . The discovery of leptin made it clear that adipose tissue is not only a regulator of body weight but also an endocrine organ with feedback loops between the brain and peripheral tissues ., the gastric mucosa is the only tissue secreting leptin in an exocrine rather than an endocrine fashion . Plasma leptin levels decrease during fasting or energy restriction and increase during refeeding, overfeeding, and surgical stress . Insulin, glucocorticoids, serotonin, and estrogen have been reported to stimulate leptin secretion. [1417] in this paper, we focus on the regulation of leptin secretion by insulin . Plasma leptin was associated with bmi in obese subjects and with fasting plasma insulin levels . In humans, plasma leptin levels exhibited a pulsatile and circadian pattern, peaking at night and reaching its nadir in the morning. [1820] sinha et al, reported that the circadian rhythm of leptin levels is not associated with insulin levels or food intake . In contrast, schoeller et al, suggested that diurnal leptin levels are altered by meal timing . Many studies on the relationship between postprandial increases in insulin and leptin have been conducted; however, conclusions about the effect of insulin on leptin are controversial . Dagogo - jack et al, reported that plasma leptin levels did not change post - prandially and concluded that, at least in the short term, insulin does not increase leptin secretion in humans . Some investigators have reported similar results. [2224] the results of studies using the glucose clamp technique support the finding that insulin is not a short - term regulator of leptin secretion . Physiological and supraphysiological euglycemic - hyperinsulinemic clamps did not change plasma leptin levels in response to insulin for up to 120 or 200 min, regardless of the insulin - sensitive or insulin - resistant status of the subjects, and the plasma leptin levels increased only after more than 4hr. [2729] similar findings have been reported in patients with type 2 diabetes mellitus . Vidal et al, reported that neither a caloric restriction nor a 3hr euglycemic - hyperinsulinemic clamp changed the level of leptin mrna in abdominal subcutaneous fat tissue, despite changes in metabolic parameters such as decreased insulinemia, glycemia, and resting metabolic rate, and increased plasma ketone bodies . They suggested that leptin gene expression is either not acutely regulated or not regulated by fasting - related metabolic factors . On the other hand, similar results were reported by saad et al, who used a glucose clamp in humans . The authors suggested that several previous studies apparently overlooked decreases in leptin levels due to saline infusion and therefore could not detect the acute effect of insulin on leptin . Carlson et al, reported that postprandial leptin increases correspond with insulin levels at 15 and 30 min . Otukonyong et al, stated that leptin secretion was influenced by consumption of foods high in fat, thereby increasing the insulin for up to 200 min after food intake . Koopmans et al, reported that pharmacological insulin infusion stimulated leptin increase in 2 h, although 4 h are required to observe a rise in plasma leptin levels after physiological insulin infusion in rodents . Pagano et al, also reported that insulin had an acute effect on leptin secretion . Furthermore, insulin is important for inducing an acute increase in plasma leptin levels in rats with streptozotocin - induced diabetes . In addition to euglycemic - hyperinsulinemic clamps, a hypoglycemic - hyperinsulinemic clamp has been reported . During hyperinsulinemic euglycemia however, the leptin profile observed when a hypoglycemic clamp was used differed from the leptin profile in euglycemic conditions: the increase was smaller and it was delayed . Further, wellhoener et al, showed a smaller increase in serum leptin levels during hypoglycemic conditions than during euglycemic conditions, despite the identical rates of insulin infusion; the total amount of dextrose infused during the clamp was significantly related to the changes in serum leptin levels . The reduced leptin secretion during fasting may be, directly or indirectly, due to falling glucose levels . The attenuating effect of prolonged hypoglycemia on hyperinsulinemia - induced leptin secretion may be caused by the response to hypoglycemia rather than the hypoglycemia itself . Whether insulin regulation of leptin secretion in humans and rodents is acute, continuing from minutes to several hours is controversial . On the other hand, in vitro studies have revealed that insulin does not affect leptin mrna levels for several hours . Leptin secretion insulin stimulates leptin secretion through a posttranscriptional mechanism that is mainly mediated by the pi3k - pkbmtor pathway, or other unknown pathways . It has been suggested that the chronic effect of insulin is mediated by glucose metabolism . Irs: insulin receptor substrate, pi3k: phosphoinositide 3-kinase, pkb: protein kinase b, mtor: mammalian target of rapamycin, er: endoplasmic reticulum moreno - aliaga et al, demonstrated, in 3t3-l1 cells, that leptin mrna was increased after 48 h of treatment with insulin and was inhibited by 2-deoxy - d - glucose (2-dg), a competitive inhibitor of glucose transport and phosphorylation . They concluded that insulin - stimulated glucose metabolism, and not insulin per se, mediates the effects of insulin to increase leptin mrna . Researchers frequently use 3t3-l1 adipocytes to study adipogenesis, fatty acid metabolism, and insulin - regulated trafficking . When the standard isobutylmethylxanthine / dexamethasone / insulin (ibmx / dex / ins) protocol is applied, 3t3-l1 fibroblasts differentiate into mature adipocytes, but leptin expression is very limited . Zeigerer et al, modified the standard protocol to better define the molecular mechanisms underlying leptin secretion of adipocytes . They added a peroxisome proliferator activated receptor (ppar) gamma agonist to the ibmx / dex / ins differentiation cocktail, which caused a five - fold increase in the leptin mrna levels . Under these conditions, insulin stimulation for 15 min induced a two - fold increase in leptin secretion without new protein synthesis . The effect of insulin on leptin exocytosis was blocked by brefeldin a, but not by the phosphoinositide 3-kinase (pi3k) inhibitor wortmannin or the protein synthesis inhibitor cycloheximide . This suggests that leptin is targeted to a regulatory secretory compartment in 3t3-l1 adipocytes, where its release is stimulated by insulin via a pi3k - independent mechanism . Leptin mrna was detectable in mature 3t3 - 442a cells, but not premature cells . These findings suggest that leptin mrna expression depends on cell culture lines or maturity of cells and some important factors may be missing ex vivo . When rat epididymal fat was incubated with or without insulin for 4hr in vitro, leptin secretion increased by about 80% at all - time points studied . After 10 min of insulin treatment, the amount of tissue - associated leptin had decreased, presumably because of increased secretion . Later, both tissue - associated leptin and total leptin production had increased in insulin - treated fat tissue . Before insulin treatment after insulin treatment, leptin staining in many cells became fainter and was restricted to a narrow band near the plasma membrane . These results suggest that insulin increases both secretion and production of leptin and stimulates the transport of leptin from the endoplasmic reticulum . Mueller et al, reported that the insulin - regulated increase of leptin secretion was more closely related to the amount of glucose taken up by adipocytes than to insulin concentration . Two inhibitors of glucose transport, phloretin and cytochalasin - b, and 2 inhibitors of glycolysis, iodoacetate and sodium fluoride, inhibited leptin secretion as well . In addition, they revealed that metformin and vanadium, antidiabetes agents that increase glucose uptake by peripheral tissues, increased glucose uptake and inhibited leptin secretion by cultured adipocytes . The inhibition of leptin secretion by metformin was related to an increase in the metabolism of glucose to lactate, so the effect of increasing leptin by glucose utilization involves the metabolism of glucose to a fate other than anaerobic lactate production . They concluded that glucose transport and metabolism are important factors in the regulation of leptin expression and secretion . The precise intracellular compartmentalization and trafficking pathways leading to the secretion of leptin and the molecular components that mediate the transport of leptin are still poorly understood . Cammisotto et al, found that a portion of leptin was localized in the endoplasmic reticulum and golgi apparatus, and also in small intracellular vesicles . Although incubation of isolated adipocytes with insulin did not increase leptin mrna levels for several hours, insulin did increase leptin concentration . However, the cellular content and secretion of leptin increased in parallel and were significantly different from basal secretion only 45 mins after insulin stimulation . These stimulating effects were abolished by cycloheximide and brefeldin a. in contrast, transcriptional inhibitor actinomycin d did not have any effect before or after insulin stimulation . Golgi secretory vesicle pathway and that short - term leptin secretion does not involve changes in mrna levels . Supporting this interpretation, one study found that actinomycin d did not block insulin - stimulated leptin secretion . In this study, the pi3k inhibitor ly294002, the map / erk kinase inhibitor pd98059, and the immunosuppressant rapamycin these agents had no effect on basal levels of leptin secretion; however, all 3 inhibitors markedly decreased both insulin- and dexamethasone - stimulated leptin secretions . These findings suggest a complex set of signaling pathways involved in mediating insulin- and dexamethasone - stimulated leptin synthesis and secretion . Reported that incubation of isolated rat adipocytes with insulin for 60 mins rapidly increased leptin synthesis, with little or no leptin secretion . Over 60 mins monensin, an inhibitor of protein translocation, had no effect on leptin synthesis, but it blocked the insulin - mediated secretion of leptin . It has been suggested that insulin promotes leptin secretion by increasing leptin synthesis, rather than by promoting the secretion of a preexisting cytosolic pool of leptin . Saladin et al, showed that leptin mrna levels in rats increased with food intake and insulin injection and decreased with fasting; additionally, the insulin increased leptin mrna expression in adipocytes . Zheng et al, reported that abdominal fat (epididymal and perirenal fat pads) had higher leptin mrna levels than subcutaneous fat . Leptin mrna levels increased after a 2.5-hr infusion of insulin into fasted rats in the abdominal fat, however, the level of leptin mrna did not change in the subcutaneous fat . Lee et al, showed the mechanisms of increased serum leptin in response to feeding by using metabolic labeling to directly assess leptin biosynthesis, secretion, and turnover . Starvation decreased serum leptin, adipose tissue leptin content, and leptin secretion during 3 h of incubation . Insulin did not acutely increase leptin biosynthesis in vitro, but pulse - chase studies showed that in adipose tissue from fed rats, insulin accelerated the secretion of leptin after 30 and 60 min of chase . The researchers conducting these studies concluded that feeding, rather than starvation, influenced leptin production at multiple posttranscriptional levels: synthesis, tissue storage, turnover, and secretion . Kolaczynski et al, reported that insulin indirectly regulates leptin production in human adipose tissue . They investigated whether leptin mrna changes in response to insulin in vitro and in vivo under euglycemic and hyperglycemic conditions . Healthy lean, obese, and type 2 diabetes mellitus subjects were infused with insulin for 5hr in a euglycemic clamp and for 6472 hrs in a hyperglycemic clamp . Short - term euglycemic - hyperinsulinemia had no effect on the levels of circulating leptin . During the prolonged hyperglycemic clamp, a rise in leptin was observed at least 40 hrs later . In the presence of insulin in vitro, leptin mrna increased at 72 hrs, followed by an increase in leptin secreted into the medium . They concluded that insulin does not acutely stimulate leptin production; however, a long - term effect of insulin on leptin production could be demonstrated both in vivo and in vitro . Russell et al, investigated in vitro regulation of leptin expression in adipose tissue of severely obese women and men before and after culture with insulin and/or dexamethasone . Leptin mrna levels and leptin secretion were greater in subcutaneous versus omental adipose tissue before culture . Dexamethasone transiently increased leptin mrna in both depots after one day of culture, but leptin secretion only increased in omental adipose tissue . Insulin did not increase leptin mrna in either depot but increased leptin secretion in subcutaneous tissue throughout the seven days of culture . The combination of insulin and dexamethasone increased leptin mrna and leptin secretion in both depots at day one and maintained leptin expression throughout seven days of culture . Insulin and glucocorticoid had depot - specific effects and functioned synergistically as long - term regulators of leptin expression in omental and subcutaneous adipose tissue from obese subjects . Wabitsch et al, also concluded that both insulin and cortisol are physiological regulators of leptin expression in human adipose tissue . A partially contradictory study was reported by casabiell et al, they reported that insulin has a dual action in leptin regulation: an early (less than 48 hrs) inhibitory action, followed (4896 hrs later) by stimulation . While the inhibitory phase was observed at every glucose concentration tested (range, 1mm25 mm), the stimulatory phase required the presence of physiological or supraphysiological glucose concentrations . This dual effect of insulin was not due to modification of leptin mrna levels, suggesting that it depends entirely on posttranslational mechanisms . They concluded that insulin - related inhibition and stimulation are due to the metabolic changes triggered by the insulin - induced increase in glucose uptake . Bado reported that gastric mucosa secretes leptin, and both feeding and administration of cholecystokinin-8 increase plasma leptin . Cammisotto et al, showed that the gastric mucosa largely contributes to levels of circulating leptin, particularly levels measured at the time of food intake . Nevertheless, very little is known about insulin - regulated secretion of leptin by gastric mucosa . However, insulin regulates the long - term leptin secretion of adipose cells by a transcriptional or posttranscriptional mechanism . The regulation may be mediated by glucose metabolism, but the mechanism is not yet fully understood . Few studies during the last few years have investigated insulin - regulated leptin secretion by adipose cells . This could be because of the very low levels of leptin mrna expressed by the traditional 3t3-l1 adipocyte cell line or because the cell lines that closely mimic the in vivo state are absent or because a cell strain highly sensitive to hormonal signals is not available . Understanding the short - term and long - term insulin - regulated mechanisms of leptin secretion could lead to the development of new treatments for obesity and its comorbidities, which are serious public health concerns.
Extubation failure (the need for re - intubation) is associated with increased intensive care unit and hospital mortality, increased length of stay in the intensive care unit and hospital, greater need for tracheostomy and for long - term acute care, and increased costs . Underlying severity of illness, premorbid health status, and complications directly associated with re - intubation fail to explain the adverse outcomes seen with extubation failure . Clinical deterioration between the time of extubation and the re - establishment of ventilatory support the study reported by lee and coworkers is one of a series of recent investigations examining whether corticosteroids can prevent postextubation upper airway obstruction (uao), which is a common cause of extubation failure . Intubation and the endotracheal tube (ett) may cause laryngotracheal injury, resulting in inflammation, mucosal ulceration, edema, or granuloma formation . This can lead to glottic or subglottic narrowing, which manifests as stridor, respiratory distress, or respiratory failure after removal of the ett . Factors associated with increased risk for postextubation uao include female sex (probably resulting from small airway size), trauma patient, age above 80 years, excessively mobile or overly large ett size, ratio of ett size to laryngeal diameter above 45%, ratio of patient height to tube diameter, duration of intubation, tracheal infection, absence of cough, absence of sedation, low glasgow coma scale score, or excess cuff pressure [2 - 5]. Research into detection of uao, with the ett in place, has recently focused on using the quantitative cuff leak test . During this maneuver the patient breathes on assist control ventilation, the endotracheal cuff is deflated, and the difference between inspired and expired tidal volume is compared . An obstructed upper airway results in similar inspiratory and expiratory volumes, whereas a patent airway results in a substantial difference as a large volume of gas escapes around the tube . This quantitative cuff leak can be reported as either a percentage of inspired tidal volume or as an absolute cuff leak volume (clv). Previous investigators have found that the risk for postextubation stridor is increased when clv is less than approximately 12% to 25% of inspired volume or an absolute value of less than 110 to 130 ml [2,6 - 9]. Although previous studies conducted in pediatric patients found that corticosteroids reduce the prevalence of postextubation uao by nearly 40% and may reduce the need for re - intubation, earlier controlled trials in mechanically ventilated adults did not corroborate those findings . Francois and colleagues recently compared 20 mg methylprednisolone (given every 4 hours for 12 hours before extubation) with placebo in nearly 700 adults patients who had been intubated for at least 36 hours . Corticosteroid pretreatment was associated with decreased risk for postextubation uao (3% versus 22%), need for re - intubation (4% versus 8%), and need for re - intubation secondary to uao (0.3% versus 4%). The number needed to treat (nnt) was eight to prevent one case of stridor and 26 to prevent one case of re - intubation . Cheng and colleagues used a reduced clv (24% of inspired tidal volume) to define and study patients at high risk for postextubation uao . Patients randomly assigned to methylprednisolone (40 mg every 6 hours for four doses) were less likely either to experience stridor (7% versus 30%) or to require re - intubation (7% versus 19%) than were those receiving placebo . The study reported in this issue of critical care also targeted high - risk patients by examining those ventilated for at least 48 hours and with a clv below 110 ml . Patients were randomly assigned to receive placebo or dexamethasone 5 mg every 6 hours for 24 hours, and were then extubated 24 hours later . The dexamethasone group was less likely to develop postextubation stridor (10% versus 27.5%; nnt = 5.7) without a difference in need for re - intubation (2.5% versus 5%; nnt = 40). An important observation is that dexamethasone led to a significant increase in clv that persisted for 24 hours after the last dose (for example, at the time of extubation). Given that no further improvement in clv occurred after the last dose of dexamethasone, one could argue for immediate extubation at that time rather than waiting an additional 24 hours . The study by lee and coworkers also revealed that 14 out of 285 in the non - randomized cohort (4.9%), who had a clv above 110 ml, developed stridor . Examining these patients and those randomly assigned to placebo, only 20% with postextubation uao required re - intubation, possibly a result of the effective use of inhaled racemic adrenaline (epinephrine) and noninvasive ventilation . Among placebo patients, 73% did not develop postextubation uao, despite a clv below 110 ml; similar findings have been noted by other investigators . A falsely low clv may result from secretions adherent to or pooled around the ett . Alternatively, with the cuff deflated, the patient may breathe additional tidal volume around the tube (in addition to machine delivered volume), leading to a falsely low measurement of inspired tidal volume . This phenomenon can be overcome by delivering the machine breath with the cuff inflated and then deflating the cuff just before expiration . The evidence is now mounting that corticosteroids can prevent postextubation uao, and possibly the need for re - intubation, but should all patients be intubated for longer than 36 to 48 hours receive such therapy? Although a short course of corticosteroids may be relatively safe, further study is warranted . This author believes the focus should continue to be on targeting patients at greatest risk . Although the study by francois and coworkers examined an' unselected' cohort, the 22% incidence of postextubation uao suggests a high risk group . Using the clv can help to identify a cohort at greater risk, but the sensitivity and specificity of the test is suboptimal . Using this test alone to determine need for prophylactic corticosteroids will result in an unnecessary 12 to 24 hour prolongation of intubation for three out of every four such patients . Another approach is to identify first a high - risk cohort based on clinical factors (for instance, age, sex, tube size, and so on) and then to apply clv to determine which patients should receive corticosteroids before extubation . Clv = cuff leak volume; ett = endotracheal tube; nnt = number needed to treat; uao = upper airway obstruction.
Overactive bladder (oab) is a common and vexing problem in children, and despite great advancement in therapies, a certain percentage of patients remains resistant to treatment . We believe that children whose symptoms do not resolve with timed voiding, laxatives, anticholinergic medications, and biofeedback physical therapy do so because of an undiagnosed and inadequately treated megarectum and that therapy directed specifically at the dilated rectum will resolve oab symptoms most efficaciously . In this study, we evaluated the efficacy of daily enemas for the treatment of oab in children . Exclusion criteria included neurogenic cause of bladder dysfunction, urinary tract infection, prior lower - urinary - tract surgery, and any diagnosed anatomical abnormalities of the urinary tract that could influence voiding function, such as posterior urethral valves . Oab was defined as uncontrolled daytime urge incontinence, and bladder function was measured using the pediatric voiding dysfunction symptom score (dvss). The 40 control patients were treated with traditional therapies, including timed voiding, osmotic laxative peg3350 (regardless of bowel history to maintain daily, soft bowel movements), and in select cases, anticholinergic medications and/or biofeedback therapy . The 20 remaining patients were prescribed only a daily enema (liquid glycerin suppository for ages 2 to 5, pediatric fleet enema for ages 6 to 11) and enough osmotic laxative to maintain soft spontaneous bowel movements, with no other therapy or voiding schedule . If the voiding symptoms resolved while on the daily enemas, patients were instructed to taper off the daily enemas over a 2-month time period (an enema every other day for a month, and then an enema twice weekly for a month). All patients were evaluated on each visit with complete history and physical, urinalysis, bristol stool scale (bss), rome iii criteria, kub x - ray, and pediatric voiding dysfunction questionnaire . Data analysis was performed using spss statistics version 23 (ibm corp, armonk, ny). For the nonparametric variables dvsss, bss scores, and rome iii scores comparisons were made using mann - whitney u tests . For the continuous variable maximum rectal diameter on kub a student t test was used . Comparisons were made both within groups for the pretreatment and posttreatment phases as well as between groups . A total of 60 children (20 experimental and 40 controls) were included in this study . Table 1 demonstrates the mean dvss, bss, rome iii, and rectal diameters prior to treatment . There was no significant difference on any of these measurements between the control and treatment groups . Table 2 demonstrates the mean posttreatment measures and the mean change in each metric after the treatment period . Patients who underwent enema had significantly more improvement in dvsss and significantly greater change in maximum rectal diameters than control patients (figures 1 and 2). Of note, both control and treatment groups demonstrated significant improvement in all measured variables after the treatment period . Abbreviations: dvss, pediatric voiding dysfunction symptom score; bss, bristol stool scale . There were no variables that were significantly different between the control and treatment groups prior to enema . Abbreviations: dvss, pediatric voiding dysfunction symptom score; bss, bristol stool scale . Participants who underwent enemas had a significantly greater improvement in dvsss and maximum rectal diameters . Pretreatment and posttreatment pediatric voiding dysfunction symptom score (dvsss): the treatment group showed a significantly greater improvement in dvss when compared with the control group . Pretreatment and posttreatment maximum rectal diameters: the treatment group also showed a significantly greater improvement in maximum rectal diameter on kub when compared with the control group . The historical teaching regarding oab of childhood was that a congenital obstruction interrupted urine flow and led to the development of detrusor hypertrophy and hyperactivity, and the accepted therapy was serial and repeated dilation of this obstruction . As time progressed, this anatomical obstruction was found to be the result of a willful dyssynergic contraction of the pelvic floor during voiding, and the treatment was changed to biofeedback physical therapy . This nonphsyiological contraction of the urethral sphincter was no medical curiosity but a disease process so severe that it could influence the natural history of many childhood disorders, such as vesicoureteral reflux and nocturnal enuresis, and in extreme cases induce renal failure . There is some debate among scientists as to whether this dyssynergic sphincter contraction is a learned or inborn condition . It is the author s opinion that in children with an intact nervous system, uninhibited voiding dominates the infantile period of voiding prior to toilet training . This is most clearly represented by the progressive bladder growth in pre toilet - trained children, with increasing compliance . This is mirrored in children with cerebral palsy, who demonstrate progressive bladder growth when they maintain an uninhibited, infantile voiding pattern . The teaching of bladder overactivity as the natural progression of an obstructed voiding pattern evolved from pathological models of voiding dysfunction such as posterior urethral valves and the neurogenic bladder, which develops in myelodysplastic patients with discoordinated sphincters . This led to the development of the modern model of dysfunctional elimination, an acquired condition where children paradoxically fail to relax their pelvic floor during elimination, resulting in bowel and bladder pathology . This study sought to investigate an alternative theory regarding the origins of dysfunctional elimination, with a therapy directed toward that cause to examine its benefits . This was first noticed in the 1960s, largely because of the presence of urinary symptoms in children with hirschsprung s disease . In his seminal work, shopfner noted that the distention of the colon, especially the rectum, could have profound effects on bladder function . Oregan furthered this work with several groundbreaking studies linking rectal distention to bladder overactivity, with excellent success in treating nocturnal enuresis, urinary tract infections, and vesicoureteral reflux simply by alleviating this rectal distention . What has unfortunately hampered the development of this work has been the lack of a uniform definition of constipation . Oregan defined constipation not as functional constipation, but mainly as the presence of fecal soiling, incomplete rectal emptying, and/or grossly decreased level of perception to balloon insufflation on anorectal manometry . The interesting discovery that oregan made was that often children with oab symptoms presented with no functional signs of constipation, yet had markedly abnormal anorectal manometry studies . In other words, they often volitionally delayed defecation until the rectum distended to fill the anatomical pelvis and then would reach a new, abnormal homeostasis where stools would evacuate at regular intervals and with surprisingly normal appearance; yet the rectal tone would be so diminished as to have abnormal manometry studies . Put another way, these children were changing the rectum from a sensing organ (which in normal circumstances provides cues on the need to defecate), to a storage organ, with decreased sensation, and often resultant fecal soiling, but more often than not normal stooling patterns . Oregan early on discovered what we have also demonstrated in this study that parental reporting of their children s bowel habits by bss or rome iii criteria is often inaccurate, and even when accurate, often not helpful in diagnosing rectal distention in children with oabs . Not only that, but in children with completely normal bowel habits, rectal distention can be the main or sole cause of urinary symptoms, leading to compression of the bladder, uninhibited contractions, and often urethral obstruction, all of which has been proved years ago . The ability of rectal stool to induce uninhibited bladder contractions is well understood and was described as early as the 1980s; this work has been supported by numerous studies, although the exact mechanisms have yet to be defined . The uncanny ability of children to be cured of nocturnal enuresis by simply restoring normal rectal tone is a great testament to this relationship . What oregan proposed was that in chronically constipated children; the rectum is never empty, necessitating the repetition or maintenance of rectal sphincter complex contraction to maintain fecal continence . In other words, the pelvic floor contractions during voiding are not a willful process that can be unlearned, but a physiological response to stool withholding . And this could be reversed by directing therapy specifically at rectal dilation, in other words daily enemas, with excellent results . So what if the modern theory of dysfunctional elimination and the resultant oab is wrong? We have proved that simply emptying the rectum repeatedly with the goal of restoring normal rectal tone resolves oab in children more efficaciously than the standard of care . In fact, if the current model of voiding dysfunction were accurate, it should be impossible for our treatment to have been beneficial at all because we made no effort to influence pelvic floor function . Some would argue that our therapy would make pelvic floor contractions worse . Yet, in almost all children, the bladder symptoms resolved . And in children whose symptoms a daily enema regimen specifically targeted at restoring normal rectal tone is more effective than the standard of care for the treatment of oab in children . Sjh contributed to the conception and design; contributed to the acquisition, analysis, and interpretation of data; drafted the manuscript; gave final approval; and agrees to be accountable for all aspects of work ensuring integrity and accuracy.
Ameloblastoma is a unique neoplasm of the jaws arising from odontogenic epithelium presenting in a variety of clinico - radiological and histological forms . Although histologically benign, ameloblastoma represents a locally aggressive lesion with potency to cause extensive destruction of jaws with infiltration into the surrounding soft tissues . Very rarely, they may present in temporal / infratemporal fossa, orbit, anterior skull base and with intracranial extensions . A 32-year - old indian female reported to our institution in october 2010 with a painless swelling in the left temporal region . She had undergone a fine needle aspiration cytology (fnac) which was reported elsewhere as she gave a history of prior surgery (2005) for the lower jaw swelling . Clinical examination showed a ~8 cm 7 cm 5 cm, soft, fluctuant, non - pulsatile, and non - tender swelling in left temporal fossa along with a 6 cm scar in the submandibular region . Oral cavity examination was normal [figure 1a and b]. A computed tomography (ct) showed a well - defined, 8 cm 6 cm, uniform, oval, cystic, homogenous lesion medial to the left zygomatic arch, infiltrating the pterygoid and temporalis muscles, abutting the deep lobe of the parotid gland, and causing erosion of the left zygomatic arch [figure 2a and b]. (a and b) frontal and worm's eye views show swelling over the left side of face arising from temporal region . Furthermore, note the submandibular scar from the previous surgery and the cosmetic deformity due to surgery and the swelling (a and b) radiological appearance of the lesion in left temporal fossa on computed tomography (detail description in text) on fnac, amber colored, non - purulent fluid showed inflammatory infiltrate of macrophages, occasional giant cells, atypical squamous cells with enlarged nuclei and keratinous cytoplasm . A core biopsy showed fibroadipose tissue with tiny focus of a tumor with squamoid differentiation infiltrating skeletal muscle . Squamous cell carcinoma was reported . However, clinical course and radiological features of the lesion were pointing toward a benign pathology . After thorough work - up, patient underwent excision of left temporal mass with infratemporal fossa clearance via a combined temporal and cervical approaches under general anesthesia [figure 3a]. Reconstruction was carried out using the free anterolateral thigh flap to provide for oral cavity lining and bulk in the temporal area . Intraoperative exposure of the tumor and post - operative frontal view after 1 year (a) h and e stained sections showing ameloblastoma with solid and cystic patterns . (a) papanicolaou stain (x400) (b) giemsa stain (x400) it is commonly seen in adults between 30 years and 50 years without gender predilection . It runs an indolent course and therefore remains asymptomatic for a long time and can attain massive sizes . Clinically, ameloblastomas are classified as (1) solid / multicystic (2) unicystic, (3) extraosseous / peripheral . The follicular type is further divided into acanthomatous, desmoplastic, granular cell, basal cell, and clear cell and mixed variety . Reichart et al ., found the solid variant to be the most common (92%) while the unicystic (6%) and peripheral variants (2%) were rare . Enucleation, cauterization or local curettage, are conservative approaches while segmental resection is referred to as radical . Due to benign histology, it is likely that a conservative surgical approach may be favored . However, conservative treatment modalities have very high recurrence rates (90% for mandibular tumors, 100% for maxillary tumors). Time for recurrence is highly variable, (range 1 - 30 years, average 5 years). Recurrence is attributed to inadequate removal, seeding, aggressive histology and spread along the muscle attachment . Treatment of recurrence often mandates extensive ablative and reconstructive surgery with inherent morbidity, even in expert hands . We hypothesize that recurrence in the temporal region probably occurred due to retraction of temporalis muscle already infiltrated by tumor with / without a small fractured fragment of coronoid process (weakened due to existing pathology and fracturing during the surgical manipulation) [figure 5]. Schematic representation showing large ameloblastoma (grey shaded area) involving ramus coronoid and condyle of mandible histopathology is the gold standard to confirm the diagnosis of recurrences at such rare sites . In this case, the histopathological diagnosis (fnac, biopsy) of squamous cell carcinoma was perplexing . Fnac of the lesion showed a few scattered epithelial cells resembling squamous cells with mild nuclear atypia, along with a few foamy macrophages [figure 4 (b and c)]. It is difficult to differentiate ameloblastic epithelial cells from squamous cells in fnac due to overlapping morphological features . Small clusters of ameloblastic epithelial cells in core biopsy can resemble squamous cells . A scanty focus of ameloblastoma in core biopsy, hence can lead to suspicion of oral squamous cell carcinoma . Ameloblastoma recurrences can occur at non - conventional sites such as temporal and infratemporal fossa . Fnac or core biopsy of these recurrent lesions may be inconclusive or even misleadingly suggestive of malignancy . Recurrences are due to incomplete removal at primary surgery, rather than de novo lesions . Strict adherence to the principles of ablative surgery, which include adequate surgical exposure, en bloc removal with adequate bony (1 - 1.5 cm) and soft - tissue margins, is mandatory . The policy of including the next uninvolved anatomic structure for adequate clear margins must be followed . Extensive bone destruction of the vertical ramus, coronoid, and condylar process are high - risk features and should be evaluated using computerised tomography / magnetic resonance imaging (preferred) to look for infiltration of attached muscles . Similarly, maxillary ameloblastomas with erosion of bony walls are high risk tumors as they may infiltrate pterygoid muscles posteriorly or extend superiorly into orbital floor, paranasal sinuses . Clinico - radiological follow - up
Accurate diagnosis based on disease - related characteristics is a prerequisite for successful treatment and improves patients prognosis . To determine the diagnostic value and future potential of a certain diagnostic tool the process from cartilage matrix damage to generalized degeneration represents a disease continuum in which the reversibility of the inflicted damage varies depending on the stage of the disease . This process is thought to be initiated by changes in nutritional status due to sclerosis of the underlying bone and/or by microdamage as a consequence of biomechanical (over)load . Related to this, repetitive low - impact injuries and single - event high - impact injuries during sports accelerate the development of damage to the articular cartilage matrix, putting active young adults at risk for early onset of cartilage degeneration and eventually osteoarthritis at middle age . During the early stages following cartilage damage, the loss of proteoglycans and a disruption of the collagen network lead to impaired matrix biomechanics as characterized by tissue softening . Softened articular cartilage has a reduced capacity to resist and conduct impact forces during physiological loading, giving rise to surface fibrillation and fissures . Continued loading of this damaged cartilage matrix has a negative influence on disease progression, eventually leading to generalized cartilage degeneration as occurs during osteoarthritis . Over the past decades, the spectrum of cartilage diagnostics has provided several options to recognize, visualize, quantify, and analyze the events involved in the progression from a focal cartilage defect to generalized disease . Clinical signs and symptoms, radiographic analysis, arthroscopy and magnetic resonance imaging (mri), and newer techniques, such as ultrasound, delayed gadolinium- enhanced mri of cartilage (dgemric), optical coherence tomography, and genetic profiling, address different aspects of cartilage morphology and function . This article aims to provide an update and insight into cartilage diagnostics for clinical and research purposes, from early matrix damage and degeneration to generalized intraarticular disease with a focus on reliability, clinical value, current status, and possible applications . The most severe cartilage degeneration is usually found in osteoarthritis . Presently, the most frequent clinically applied diagnostic modality for osteoarthritis is signs and symptoms as presented by the patient . Interestingly, the clinical symptoms of osteoarthritis are not related to cartilage degeneration but to other pathological events in osteoarthritis . Pains, stiffness, functional impairment, crepitus, swelling, and restricted movement are the clinical key characteristics of osteoarthritis . Of these although the exact mechanisms by which pain in osteoarthritis is generated remains unknown nociceptive fibers are found only in the subchondral bone and joint capsule but not in cartilage it is believed that intraarticular factors released from bone or synovium cause hypersensitivity of related structures such as the periosteum, subchondral bone, or marrow bone . Concomitant intraarticular hypertension and ischemia due to synovitis could be other sources of joint pain . Also, subchondral venous obstruction, resulting in raised intraosseous pressure, is associated with severe degenerative changes in the joint and could be a source of pain at the end stage of the disease . Another typical symptom in osteoarthritis is morning stiffness, which lasts less than 30 minutes, in contrast to inflammatory arthropathy, as defined by the diagnostic criteria of osteoarthritis by the american college of rheumatology . Osteophyte formation, subchondral bone remodeling, and capsular thickening are biological changes that result in functional impairment and difficulties with activities of daily living . Clinical characteristics of osteoarthritis are evaluated by several classifications and questionnaires, focusing on symptoms, daily functioning, and quality of life, and help to report series of cases or describe the success rate of an intervention . This is indicated by the very low sensitivity (20%-49%) of major clinical signs, such as pain and morning stiffness, when compared to radiographic scoring systems . In general notwithstanding, in current daily practice, they are the most important reason for a surgeon to decide for arthroplasty once radiographic osteoarthritis has been proven . The combination of clinical and radiographic disease characteristics to diagnose end - stage cartilage degeneration is commonly used in daily clinical practice . Although cartilage itself is invisible on plain radiography, it can be used to identify some disease - related characteristics . They described several radiographic features, such as osteophytes, periarticular ossicles, joint space narrowing, subchondral pseudocystic sclerotic areas, and altered shape of bony ends . Nowadays, these radiographic changes are generally accepted to be hallmarks of severe cartilage degeneration and a representation of osteoarthritis . Despite the large inter- and intraobserver error (8%-31% observer bias), the kellgren and lawrence scale is frequently applied for the individual assessment of a patient s disease progression or effect measurement in a clinical trial . More recently developed scoring systems for radiographic cartilage damage, by altman and gold and nagaosa and colleagues, provide a further subcategorization of these individual radiographic features and show good intra- and interobserver reproducibility . Despite this, several studies show a poor to moderate correlation between the radiographic characteristics of degenerative cartilage and the actual degree of cartilage damage as determined by arthroscopy . Novel developments for the radiographic evaluation of ongoing cartilage degeneration based on computerized measurements of the generally accepted radiographic features might help to standardize measurement of these features and thus form a valuable tool to monitor disease progression or treatment effect in clinical trials . These computerized measurements show a good inter- and intraclass reliability and correlation (correlation scores varying from 0.50 - 0.99) to radiographic scoring systems . However, the position of the patient influences the shooting angle of the radiographic image and thus the computerized measurements of the radiographic features . Therefore, these analytical algorithms may entail practical problems during the follow - up of patients . Thus, although the assessment of radiographic characteristics for the diagnosis of osteoarthritis is still frequently applied in daily practice, the actual extent of cartilage degeneration shows a poor correlation with these parameters . Although clinical signs and radiography will only indirectly suggest cartilage damage and degeneration, arthroscopy introduced the advantage of direct visualization of the actual cartilage damage . Macroscopic signs of matrix damage, fibrillation, and softening can be assessed easily during arthroscopy by surface evaluation and cartilage probing . A disadvantage, however, is the subjective character of these observations . In an attempt to quantify and standardize the arthroscopic evaluation of cartilage damage, several scoring systems (e.g., the french society of arthroscopy scoring system [sfa] and outerbridge scales), based on size, grade, and localization of cartilage damage, have been developed . When tested for accuracy, these systems show average to good interobserver reproducibility (0.52 - 0.62) and intraobserver reliability (0.66 - 0.80) but tend to have higher agreement (81% intraobserver agreement) for severe degenerative lesions compared to intermediate and lower graded lesions (65% intraobserver agreement). This suggests that arthroscopic grading may not be suitable for quantitative assessment of early cartilage damage . Alternative macroscopic scoring systems, such as the international cartilage repair society (icrs) and oswestry arthroscopy score (oas) score, have also been developed to provide a macroscopic evaluation of regenerative cartilage repair . These systems showed good inter- and intraobserver reliability (0.62 and 0.56 icrs and 0.73 and 0.65 oas, respectively) and can therefore be applied as an outcome measure in clinical trials on cartilage regeneration . Because macroscopic damage as visualized by arthroscopic evaluation will most likely be irreversible, arthroscopy seems to be a good method of grading severe focal cartilage lesions but has inferior sensitivity for the diagnostic workup of early matrix - related cartilage damage . Ultrasonic evaluation of the articular cartilage is primarily based on the speed of sound in cartilage . The thickness of articular cartilage, as a representation of the tissue status, has been calculated from the speed and the so - called time of flight . However, the reported discrepancy between the speed of sound in healthy (1658 - 1760 m / s) and degenerated (1567 - 1600 m / s) cartilage highly influences the thickness measurement . In an attempt to provide a biomechanical quantification of the cartilage status, ultrasound measurements were combined with indentation tests . However, given the variation of the speed of sound in cartilage according to the state of the tissue, resulting in large measurement errors on thickness and biomechanical moduli, the possible clinical application of this mechano - acoustic quantification of articular cartilage can be debated . As articular cartilage matrix constituents influence the attenuation and (sub)surface reflections of high - frequency ultrasound waves, more detailed evaluation of ultrasound reflex echoes has been performed to describe the pathological changes during cartilage matrix damage and degeneration . However, the defined quantitative ultrasound parameters showed weak correlations to biochemical scoring and were only able to distinguish healthy from severely degenerated samples . Analogous to ultrasound, optical coherence tomography (oct) departs from reflections of near infrared light instead of sound waves . In addition, cross - sectional images can be derived from up to 2 mm deep into the tissue . The histological fibrillation index, a measure of surface fibrillation, was shown to correlate well to the oct - derived fibrillation index . However, even though oct is able to show structural changes in (sub)surface collagen orientation and disorganization, proteoglycan loss as part of early (traumatic) matrix damage is not likely to be detected . Another more practical limitation is the requirement of the oct probe to be placed exactly perpendicular to the cartilage surface . Thus, both ultrasound and oct allow for more objective measurements of cartilage quality than simple probing, but the discriminative quality in the detection of various stages of de- or regeneration should be tested to really determine their additional clinical value . The broad spectrum of clinically available and recently developed mri techniques, scoring systems, and sequences allows for a sensitive analysis of cartilage from focal lesions to generalized disease . The fat - suppressed spoiled gradient - echo sequence (spgr) sequence produces a high cartilage signal and low signal from the adjacent joint fluid and is currently the standard for quantitative morphological imaging of cartilage . Semiquantitative measurements of cartilage volume, thickness, and surface area, derived from various scanning sequences, show excellent inter- and intraobserver reliability and long - term precision errors ranging from 1.4% to 3.9%, which make these parameters attractive for longitudinal studies, patient follow - up, and diagnostic procedures . The availability of higher field strengths, up to 3 t, makes these measurements even more accurate, with accuracy errors for 1.5 t field strengths ranging from 11% to 17% and from 3% to 7% for 3.0 t field strengths . Several semiquantitative mri scoring systems for osteoarthritis have been developed focusing on size and location of the lesions and subchondral, cartilaginous, bone, and meniscal abnormalities . The knee osteoarthritis scoring system (koss) has a good overall reproducibility (intraclass correlation [icc] 0.77) but a limited reproducibility for cartilaginous and subchondral tissue, with an icc of 0.64 and 0.63, respectively . The interobserver agreements of the whole organ magnetic resonance imaging score (worms) are good, with an icc for cartilage loss that is even greater than 0.90 . Overall, these scoring systems provide good quality for evaluation of the osteoarthritic status of the joint . However, evaluation of a single case will take approximately 45 minutes and therefore limits the clinical implementation . The scoring system developed by marlovits et al . Showed good interobserver reliability (icc> 0.80 for 8 of 9 features) and significant correlations to subscales of the knee injury and osteoarthritis outcome score (koos), although the number of included patients was limited . Although current mri sequences and scoring systems allow for good diagnostic accuracy for moderate to severe cartilage degeneration when compared to radiography, newer techniques have been developed to focus on imaging of cartilage constituents as possible tools in the detection of early cartilage damage . Degradation of the collagen matrix in cartilage enhances the mobility of water protons, which can be sensitively detected in vivo by quantitative mri t2 relaxation time . Water proton mobility (measured by quantitative t2 mapping) also seems to reflect collagen architecture and density of articular cartilage . An mri - based technique that enables quantification of proteoglycans is dgemric, which is based on the negative proteoglycan - related charge (also called fixed charged density [fcd]) in cartilage . Intravenously administered diethylenetraminepentaacetic acid (gd - dtpa) is distributed at high concentrations in cartilage areas with low proteoglycan content and vice versa and therefore allows for mapping of proteoglycan distribution in articular cartilage (fig . 1). This technique shows good in vivo reproducibility and good correlations (correlation scores 0.95 - 0.96) to the biochemically determined proteoglycan content in vitro . A decrease of dgemric signal has been observed after posterior cruciate ligament rupture when compared to the pretrauma signal, indicating a disturbance of the cartilage matrix after knee trauma . In addition, significant correlations have also been described between the proteoglycan content in synovial fluid and t1gd signal in the acute phase after anterior cruciate ligament rupture . Conventional magnetic resonance imaging (left) and dgemric scan (right) of articular cartilage . In addition to dgemric, the t1 mri technique also provides a quantitative map of the proteoglycan distribution in articular cartilage . The advantage of the t1 mri technique over dgemric is that it does not need intravenous administration of contrast agents . In vitro studies show a strong correlation (correlation scores 0.92 - 0.98) between proteoglycan content and changes in t1 relaxation times . Another technique that also uses the fcd to visualize proteoglycan loss from articular cartilage is sodium mri . The loss of negatively charged proteoglycans results in a lower fcd and induces a loss of positively charged sodium ions from the tissue, which can be visualized by quantitative namri . This technique can only be performed at higher field strengths (3 t) but is promising in detecting early proteoglycan loss from articular cartilage . Although conventional mri sequences and scoring systems offer a good analysis of all the structures within the joints, they are only able to detect an articular cartilage defect when it is actually present, making them less suitable for the detection cartilage matrix disturbances as a disease stage preceding focal lesions . Newer experimental mri techniques, such as dgemric, t1 mri, and sodium mri, do provide a validated quantitative measurement of specific articular cartilage matrix constituents, making them promising tools for the evaluation of early damage to articular cartilage . Besides the development of imaging techniques and arthroscopic devices to quantify cartilage matrix damage and degeneration at different stages of the disease, molecular markers of damaged and degenerating articular cartilage have been studied in serum, urine, and synovial fluid to provide for more sensitive hallmarks of degenerative cartilage disease . The irreversibility of articular cartilage damage is hypothesized to coincide with a phenotypic shift of articular chondrocytes . This shift may result in inappropriate expression of genes encoding for matrix constituents and eventually to decreased matrix stability . Elevated levels of keratan sulfate (ks) and cartilage ogliomeric matrix protein (comp) were found not only in serum of patients with radiographic osteoarthritis (oa) but also in serum of patients with recent joint trauma, such as anterior cruciate ligament rupture or medial meniscectomy . In joint trauma patients, collagen neoepitopes have mainly been used as oa markers . In patients with knee pain, urine and serum levels of various collagen neoepitopes generated by protease cleavage, among which are c - telopeptide of collagen type ii, collagen type ii cleavage neoepitope, and collagen type i and ii cleavage neoepitopes c1,2c, have been shown to correlate with the severity of radiographic oa . Inversely, high serum levels of propeptide collagen type ii were inversely correlated to oa . Concentrations of mmp-1, mmp-3, procollagen type i c - peptide (picp), tissue inhibitor of metalloproteinase-1 (timp), proteoglycans (pgs), and deoxypyridinoline (dpd) showed a typical decrease in synovial fluid during the first year after autologous chondrocyte transplantation (act) surgery, suggesting an inhibition of the degenerative process upon treatment . In addition to the extracellular matrix genes and degrading proteases, genes closely related to chondrocyte differentiation and chemokine and endothelin pathways have been related to early degenerative changes in human chondrocytes . Also, various cytokines have been implicated to be involved in cartilage degeneration during oa . However, synovial fluid levels of most inflammatory cytokines are low or undetectable, and it is not known to what extent serum cytokine levels are affected, which renders them unsuitable as diagnostic markers . In early joint degeneration, levels of interleukin-15 (il-15) were found to be increased in the synovial fluids from patients with meniscus tears and cartilage thinning . This area of research still needs expansion . For a more extensive update on the ever growing field of molecular markers in cartilage disease the various techniques available for the diagnosis of cartilage disease are based on imaging, biochemical, and biomechanical characteristics of articular cartilage . Technical improvement and increasing knowledge of disease initiation and progression can be expected to positively influence current diagnostic modalities and form a basis for the development of new procedures . It has to be kept in mind, however, that the capacity for sensitive diagnosis of cartilage status in itself will not improve treatment of early cartilage disease, and even if new treatments can be developed, they may not be applied as long as a patient does not have any clinical signs and articular cartilage appears normal at regular arthroscopy . These aspects are important to consider in future development of diagnostic and therapeutic strategies in clinical practice.
Dementia, a syndrome usually resulting from a chronic or progressive brain disorder such as alzheimer s disease, currently affects 18 million people worldwide . Dementia is characterized by a decline of cognitive functions such as memory, language, recognition, reasoning, and judgment . The risk of developing dementia increases with age; thus, the number of patients with dementia will increase worldwide over the coming decades along with the demographic aging of the population . A considerable number of patients suffering from dementia at moderate to advanced stages are institutionalized . Factors contributing to institutionalization vary and can include behavioral and psychological symptoms of dementia [2, 3] as well as physical performance or level of independence in activities of daily living (adl). Indeed, dependence in adl is a critical risk factor for institutionalization [4, 5]. Furthermore, dependence for adl causes direct and indirect costs related to the care of this population to increase in parallel . Recent data about the relationship between cognitive functioning and physical performance suggest that regular physical activity could provide cognitive benefits for patients with alzheimer s disease [7, 8]. A high - intensity physical activity program may delay adl decline in older nursing home residents with severe cognitive impairment . However, sound evidence on which form of physical activity has the highest impact is still lacking . Knowing what can be done, in which form, at what time, how long, and at what stage of the illness is of crucial importance to caregivers, to healthcare professionals, and to public health decision makers this is especially important at moderate to severe stages of the illness when adl ability decreases . The objectives of this review are (1) to describe the different types of physical activity programs designed for patients with moderate to severe dementia and (2) to identify the impact of these activities on functional independence in adl . A computerized search strategy was applied using the following mesh terms: (motor activity [mesh] or exercise [mesh] or physical activity) and dementia [mesh] and (activities of daily living [mesh] or functional independence). The consulted databases were medline via pubmed, cinahl, cochrane library, isi social sciences citation index, otseeker, and pedro . We completed our computerized search by checking the reference lists of the included articles, of five systematic reviews [7, 1013], and one meta - analysis . Studies were included if they investigated physical activity interventions applied to moderately to severely demented persons (defined as an mini - mental state examination (mmse) score of 17 or less) [15, 16]. Additional inclusion criteria were the presence of outcome measures for adl, the randomized controlled trial design, and the language of the article; only articles written in english, french, german, or italian were considered . Quality assessment was carried out using the scale developed by downs and black which is appropriate for assessing randomized and non - randomized studies . We adapted the last criterion of this scale, i.e., the power of the study, and attributed one point if the authors did a prior sample size calculation . Two authors (eb, nk) independently assessed the quality of the included papers after having completed the training of the scale application on one study which had not been included in the review . Disagreement was resolved through discussion, and if necessary, the judgment of a third person (avg) was retained . We summarized the characteristics of each study sample, the physical activity interventions and their modalities of application, the measured adl outcomes, the assessment tools used to measure adl capacities, and the effect on the patients capacities to perform adl . For missing data, the original author of the trial was contacted in order to receive the required information . Three meta - analyses were conducted in order to combine the results of the different studies included in our review . The first analysis compared the pre- and post - treatment values of the experimental group, the second analysis compared the pre- and post - treatment values of the control group, and the last analysis compared the post - treatment difference of both groups . Since the number of included studies was small and since different exercise programs were proposed across these studies, the use of additional meta - analyses for the comparison of the different intervention modes was not feasible . Published results were first converted into a common effect size measure: the standardized mean difference which is defined as the mean difference divided by the corresponding standard deviation . Bias was removed using hedges g method, and the homogeneity between studies was evaluated by the q test . Here, the homogeneity was rejected for all analyses, meaning that the true effect size in each study can be considered as similar, but not as identical . The solution was then to compute the overall effect size under the assumption of a random effect model . In practice, each study was weighted by the inverse of a variance composed of two terms: the variance of the study and a constant computed from q and representing the variability of the effect size across studies [21, 22]. The study by steinberg et al . Could not be included in the meta - analyses because the results were in a form incompatible with the computation of standardized mean differences . For similar reasons, the study by stevens and killeen so, to be coherent between the analyses, we decided to perform twice each of the first two analyses: with and without the stevens and killeen s study . Since the results with only three studies included did not significantly differ from those with the four studies included, we reported results for the latter case only . Results were reported as forest plots including the effect sizes and the 95% confidence intervals . We summarized the characteristics of each study sample, the physical activity interventions and their modalities of application, the measured adl outcomes, the assessment tools used to measure adl capacities, and the effect on the patients capacities to perform adl . For missing data, the original author of the trial was contacted in order to receive the required information . Three meta - analyses were conducted in order to combine the results of the different studies included in our review . The first analysis compared the pre- and post - treatment values of the experimental group, the second analysis compared the pre- and post - treatment values of the control group, and the last analysis compared the post - treatment difference of both groups . Since the number of included studies was small and since different exercise programs were proposed across these studies, the use of additional meta - analyses for the comparison of the different intervention modes was not feasible . Published results were first converted into a common effect size measure: the standardized mean difference which is defined as the mean difference divided by the corresponding standard deviation . Bias was removed using hedges g method, and the homogeneity between studies was evaluated by the q test . Here, the homogeneity was rejected for all analyses, meaning that the true effect size in each study can be considered as similar, but not as identical . The solution was then to compute the overall effect size under the assumption of a random effect model . In practice, each study was weighted by the inverse of a variance composed of two terms: the variance of the study and a constant computed from q and representing the variability of the effect size across studies [21, 22]. The study by steinberg et al . Could not be included in the meta - analyses because the results were in a form incompatible with the computation of standardized mean differences . For similar reasons, the study by stevens and killeen could not be included in the third meta - analysis . So, to be coherent between the analyses, we decided to perform twice each of the first two analyses: with and without the stevens and killeen s study . Since the results with only three studies included did not significantly differ from those with the four studies included, we reported results for the latter case only . Results were reported as forest plots including the effect sizes and the 95% confidence intervals . Further reasons for exclusion comprised other or missing interventions (125), other outcomes (49), other languages (12), other populations (51), and other designs (16). After reading the full text, we excluded two additional studies with samples whose mmse score was higher than 17 (fig . 1). The results of the quality assessment (table 1) showed that only one of the five randomized clinical trials included demonstrated a high - quality score (25 of 27).fig . The first column represents the number of articles identified on the consulted databases (medline, cinahl, pedro, isi social sciences citation index, cochrane, otseeker) and those found by hand search . The second column refers both to the articles excluded after reading the title or the abstract and to the reason of exclusion . The last column contains the number of the articles that were assessedtable 1results of the quality assessmentitemsdescriptionscorereportingkwak et al . Hypothesisis the hypothesis / aim / objective of the study clearly described?11111main outcomesare the main outcomes to be measured clearly described in the introduction or methods section?01111included patients characteristicsare the characteristics of the patients included in the study clearly described?01100interventions of interestare the interventions of interest clearly described?11100distributionare the distributions of principal confounders in each group of subjects to be compared clearly described?12211main findingsare the main findings of the study clearly described?11111random variabilitydoes the study provide estimates of the random variability in the data for the main outcomes?11101adverse eventshave all important adverse events that may be a consequence of the intervention been reported?01100characteristics of patients lost to follow - uphave the characteristics of patients lost to follow - up been described?00101probability valueshave actual probability values been reported (e.g. 0.035 rather than <0.05) for the main outcomes except where the probability value is less than 0.001?01111external validityrepresentativity of the participantswere the subjects asked to participate in the study representative of the entire population from which they were recruited?00111representativity of the prepared subjectswere those subjects who were prepared to participate representative of the entire population from which they were recruited?00000representativity of the staff, places and facilitieswere the staff, places, and facilities where the patients were treated, representative of the treatment the majority of patients receive?00100internal validity biasblind study subjectswas an attempt made to blind study subjects to the intervention they have received?00000blind staffwas an attempt made to blind those measuring the main outcomes of the intervention?01100data dredgingif any of the results of the study were based on data dredging, was this made clear?11101adjustment of the analysesin trials and cohort studies, do the analyses adjust for different lengths of follow - up of patients, or in case control studies, is the time period between the intervention and outcome the same for cases and controls?11111statistical testswere the statistical tests used to assess the main outcomes appropriate?11110compliancewas compliance which the intervention / s reliable?01100validity and reliability of the main outcomeswere the main outcome measures used accurate (valid and reliable)?01001internal validity confoundingsubjects recruited from the same populationwere the patients in different intervention groups (trials and cohort studies) or were the cases and controls (case control studies) recruited from the same population?01111subjects recruited over the same period of timewere the patients in different intervention groups (trials and cohort studies) or were the cases and controls (case control studies) recruited over the same period of time?00111randomizationwere study subjects randomized to intervention groups?11111randomized intervention assignmentwas the randomized intervention assignment concealed from both patients and health care staff until was complete and irrevocable?00100adequate adjustment for confoundingwas there adequate adjustment for confounding in the analyses from which the main findings were drawn?11100losses of patients to follow - upwere losses of patients to follow - up taken into account?00100powerclinically important effectdid the study have sufficient power to detect a clinically important effect where the probability value for a difference being due to chance is less than 5%?00110total score1019251214 flowchart of identified and included studies . The first column represents the number of articles identified on the consulted databases (medline, cinahl, pedro, isi social sciences citation index, cochrane, otseeker) and those found by hand search . The second column refers both to the articles excluded after reading the title or the abstract and to the reason of exclusion . The last column contains the number of the articles that were assessed results of the quality assessment the main weaknesses of the included articles were the lack of information concerning the following three criteria: external validity, description of randomization, and description of the patients lost to follow - up . Blinding of therapists and participants is not feasible when applying physical activity interventions, and hence, item 14 was not retained as a quality criterion . The agreement between the two raters when applying the downs and black scale was good with a chance corrected kappa coefficient of 0.67 . We checked the internal validity of the included studies by applying the cochrane criteria that are specific for randomized trials . The results confirmed that none of the studies was free of bias . According to the cochrane criteria, the main weaknesses consisted of (1) the unclear process of randomization for four [23, 24, 26, 27] out of the five included studies, (2) the unequal treatment time in two studies [25, 26], (3) the lack of blinding of the outcome assessors in three studies [24, 26, 27], and (4) the missing report of withdrawals in three studies [24, 26, 27]. The extracted information is summarized in tables 2 and 3.table 2main characteristics of the selected studiesauthorsdesignpopulationprogram referenceintervention: experimental groupintervention: control groupparticipants ratedropoutkwak et al . Experimental group: age 79.7 (6.6) years, mmse 14.5 (5.3). Exercise intensity was gradually increased from 30% to 60% of expected maximal oxygen consumptionno informationno information0/30rolland et al . Experimental group: age 82.8 (7.8) years, mmse 9.7 (6.8). Control group: age 83.1 (7.0) years, mmse 7.9 (6.4)experience and based on two other references [6, 7]aerobic, strength (lower extremity), balance training, fast walking, flexibility, program accompanied by music . Intensity: at the beginning light intensity and was gradually increased over the first month . No restriction in nursing, physiotherapy, medical care, advice, or any other healthcare support134/42911/67 (eg)13/67 (cg)steinberg et al . Randomized controlled trial stratified for gender and age over 7527 community dwelling persons with alzheimer disease . Experimental group: age 74 (8.1) years, mmse 15.5 (5.4). Control group: age 76.5 (3.9) years, mmse 20.1 (5.1)graduate program at the johns hopkins bloomberg school of public healthaerobic fitness: brisk walking, strength training, balance and flexibility training, intensity: compliance measurehome visit, with recommendations27/300/27stevens and killeen randomized controlled trial120 nursing home residents from 6 different nursing homes . Control group 2: 80.5 years, mmse scores> 9 <23program was designed based on knowledge concerning physiological adaptation in older frail people and in consultation with the school of sport and exercise science, southern cross university, lismoreaerobic by moving joint and large muscle groups . Control group 2: social visits equivalent in duration and frequency as those undertaking the exercise program in the experimental groupno information45/120francese et al . Experimental design12 severely demented residents of a medicare nursing facilityprogram was based on previous interventions for frail or impaired residentsactivities such as catching, throwing, and kicking balls, leg weight exercises, parachute reaches, program accompanied by music, intensity: gentle aerobic exertionsing - along video12/300/6 (eg)1/6 (cg)mean age (in years) and mean mini - mental state examination scores and the corresponding standard deviation are reported for the experimental and the control group . (data not available), and stevens and killeen reported a significant improvement of adl scores . . Showed significant delay of adl deterioration, only after 12 months of program duration . Post - treatment adl scores of the control group of stevens and killeen s study (data not available) decreased significantly compared to baseline assessment . Adl scores of both groups deteriorated significantly in the study of rolland et al . Compared to baseline assessmenteg experimental group, cg control groupindication of sd of mean age as well as mean mmse scores and the corresponding sd for each group is missinginformation related to age and mmse scores is missingtable 3intervention modalities of the selected studiesauthorsprogram duration (months)session duration (minutes)frequency per weekexercise leadergroup size participants (number)outcomeskwak et al . 3no information6 (aerobic), 4 (strength, balance and flexibility training)exercise physiologistindividualhand activity relevant for adlstevens and killeen 3303researchersno informationadlfrancese et al . (data not available), and stevens and killeen reported a significant improvement of adl scores . Rolland et al . Showed significant delay of adl deterioration, only after 12 months of program duration . Post - treatment adl scores of the control group of stevens and killeen s study (data not available) decreased significantly compared to baseline assessment . Adl scores of both groups deteriorated significantly in the study of rolland et al . Compared to baseline assessment main characteristics of the selected studies mean age (in years) and mean mini - mental state examination scores and the corresponding standard deviation are reported for the experimental and the control group . (data not available), and stevens and killeen reported a significant improvement of adl scores . . Showed significant delay of adl deterioration, only after 12 months of program duration . Post - treatment adl scores of the control group of stevens and killeen s study (data not available) decreased significantly compared to baseline assessment . Adl scores of both groups deteriorated significantly in the study of rolland et al . Compared to baseline assessment eg experimental group, cg control group indication of sd of mean age as well as mean mmse scores and the corresponding sd for each group is missing information related to age and mmse scores is missing intervention modalities of the selected studies kwak et al ., (data not available), and stevens and killeen reported a significant improvement of adl scores . . Showed significant delay of adl deterioration, only after 12 months of program duration . Post - treatment adl scores of the control group of stevens and killeen s study (data not available) decreased significantly compared to baseline assessment . Adl scores of both groups deteriorated significantly in the study of rolland et al . Compared to baseline assessment the included studies were conducted between 2006 and 2009, except for one publication dating back to 1997 . Three studies [24, 25, 27] included nursing home residents and two studies [23, 26] community - dwelling people . The participants were 75 years or older and, according to the mmse scores or population description, moderately to severely demented . The physical activity programs differed in each study, and in one study, references for the activity programs were missing . The most frequently mentioned interventions were strengthening exercises, balance, and gait training [23, 25, 26] as well as endurance training [2325]. The frequency varied between biweekly and daily and the duration of each session between 20 and 75 min . The intensity of exercises was inconsistently reported with few information: oxygen consumption, aerobic exertion [24, 25, 27], and compliance measures [23, 25]. Each study used a different assessment tool . Among them, the katz index was the most common adl assessment tool . One article indicated the applied assessment method and the considered adl items . In three studies [23, 24, 26], the physical activity program significantly improved adl performance in the experimental group by the end of the intervention . It was a small but clinically meaningful difference in the experimental group compared to the control group after 12 months of intervention; the intermediate result after 6 months was not significant . Physical activity practice showed a significant effect already after 6 months, although modalities of the physical activity program were quite similar between these two studies . One very small study including only six participants in the experimental group did not show any significant effect on adl performance . Among the controls, adl performance deteriorated over the short observational period (table 4).table 4outcomes: mean score and standard deviation of adl assessmentsbaseline scoretimepost - intervention scorep valueegcgegcge / creferenceassessment toolmax scoremeansdmeansdmonthsmeansdmeansdadl deteriorationkwak et al . Acsm24617.535.4612.074.52change scoreassessment toolmax scoretimedifferences pre-/post - interventionp valuemonthsegcgadlcgadle / cstevens and killeen self - help skill2434.2922.80.524ns*jebsen total time test assesses hand function which is relevant for adl performance . American college of sports medicine method assesses adl according to the seven following categories: clothing, eating, moving, urinating, bathing, controlling feces, and washing hands . (data not available), and stevens and killeen reported a significant improvement of adl scores . . Showed significant delay of adl deterioration, only after 12 months of program duration . Post - treatment adl scores of the control group of stevens and killeen s study (data not available) decreased significantly compared to baseline assessment . Adl scores of both groups deteriorated significantly in the study of rolland et al . Compared to baseline assessmentbi barthel index, self - help skill is an adl item of the revised elderly persons disability scale, jtt jebsen total time test, cads changes in advanced dementia scale, acsm american college of sports medicine, na data non - available, time time since baseline assessment, adl decline of adl performance, max score maximum score, eg experimental group, cg control group, e / c in - between group comparison, ns not significantp <0.05; * * p <0.01control group 1 = no intervention; control group 2 = social intervention outcomes: mean score and standard deviation of adl assessments jebsen total time test assesses hand function which is relevant for adl performance . American college of sports medicine method assesses adl according to the seven following categories: clothing, eating, moving, urinating, bathing, controlling feces, and washing hands . (data not available), and stevens and killeen reported a significant improvement of adl scores . . Showed significant delay of adl deterioration, only after 12 months of program duration . Post - treatment adl scores of the control group of stevens and killeen s study (data not available) decreased significantly compared to baseline assessment . Adl scores of both groups deteriorated significantly in the study of rolland et al . Compared to baseline assessment bi barthel index, self - help skill is an adl item of the revised elderly persons disability scale, jtt jebsen total time test, cads changes in advanced dementia scale, acsm american college of sports medicine, na data non - available, time time since baseline assessment, adl decline of adl performance, max score maximum score, eg experimental group, cg control group, e / c in - between group comparison, ns not significant * p <0.05; * * p <0.01 control group 1 = no intervention; control group 2 = social intervention figures 2, 3, and 4 summarized the main results of each meta - analysis . Figure 2 showed that the adl status did not significantly change between the pre- and post - measurements among treated patients (aggregate effect size 0.30, 95% confidence interval [0.49; 1.09]). We examined the effect of including or not including stevens and killeen s study in the meta - analysis, and it did not change our conclusion . When performed on the control group (fig . 3), the same analysis indicated a significant decline of the adl functioning between the pre- and post - measurements (0.63 [1.02; 0.23]). 4) did not show any real difference in post - treatment values of both groups (0.43 [0.63; 1.50]).fig . 2meta - analysis conducted for the comparison of the pre- and post - treatment values in the experimental groupfig . 3meta - analysis conducted for the comparison of the pre- and post - treatment values in the control groupfig . 4meta - analysis conducted for the comparison of the pre- and post - treatment values in the both groups meta - analysis conducted for the comparison of the pre- and post - treatment values in the experimental group meta - analysis conducted for the comparison of the pre- and post - treatment values in the control group meta - analysis conducted for the comparison of the pre- and post - treatment values in the both groups thus, the decline in adl performance in demented subjects may be due not only to disease progression but also to physical inactivity . One study showed that the effect on adl performance disappeared after cessation of the physical activity programs . This observation may explain why three [23, 24, 26] out of five trials showed a significant effect on adl performance, but only one reported clinically meaningful results despite differences in their physical activity programs . The negative evolution of adl performance in all control groups except one tends to support the relationship between physical activity and functional abilities . The most severe adl degradation was observed in the study which included the patients with the lowest mmse scores . Delaying adl deterioration in nursing home residents with moderate or severe cognitive impairment through a physical activity program may be both clinically and economically relevant as adl care in this patient group accounts for two thirds of the total care time . The descriptive results were supported by our meta - analyses and allowed us to conclude as forbes et al . Did that even if the proposed treatments have not proven their efficiency in improving the adl status of the patients, they could nevertheless limit the decline in adl functioning . However, the physical activity programs varied among the included studies . In line with yu and kolanowski as well as taylor et al ., we confirm that there are no clinical practice guidelines for aerobic exercises for persons with dementia . Reasons for the lack of both studies on the matter and practice guidelines may include the individual variability of the aging process and the limitation of physical activity practice due to the disabilities of the very old . Nonetheless, endurance can positively influence the physiological aging process of the cardiovascular system at a central and peripheral level . Aging per se causes loss of muscle strength, and regular strengthening exercises can counteract to some degree this loss in the very old . Based on this knowledge, the content of the physical activity programs covering endurance, gait, and strength training proposed in the included studies seemed to be appropriate . Questions related to the duration and intensity of the physical activity program, the duration of each session, or their frequency remain unanswered . One high - quality study applied a physical activity program with moderate to high intensity and showed a significant delay of adl decline only after 12 months duration, but not after 6 months . A high intensity strengthening program during 3 months achieved a similar result in dementia nursing home residents . Paterson and warburton suggest that the intensity of physical activity should be at least moderate in order to improve adl performance in the elderly . The literature on the topic does not either give clear indications on how long an ideal activity program should last . Yu and kolanowski, based on their summary of current knowledge concerning the prevention of alzheimer s disease, suggest an ideal program duration of 2 months and the ideal session frequency of three times per week for an aerobic exercise program designed for a medically stable population with dementia and aimed at improving their adl performance . The most important parameter was regularity of exercising with high exercise adherence significantly preventing adl decrease . The result of the study of littbrand et al . Including residents with less severe dementia showed no lasting effect of physical activity training on adl . Demented people are likely to need some encouragement to stay physically active and to slow the decrease of their adl performance especially in those with moderate to severe dementia . Overall, the optimal frequency and intensity of physical activities which provide a satisfactory long - term effect are still unknown and their determination highly relevant . Physical activity programs were accompanied by music in only three studies [24, 25, 27], although there is a growing body of evidence that music in advanced stages of dementia could help improve the performance of patients [33, 34]. According to a qualitative study, demented patients claimed that programs should respond to their psychological and social needs, whereas their caregivers considered maintenance of functional independence as the most important goal . Thus, physical activity in groups accompanied by music could more easily respond to the expectations of demented patients and increase their adherence to a physical activity program . In addition, a further criterion to respect is the meaningfulness of the proposed activities . The reduced communication skills, the frailty of this population, and the increased risk of falling may require limited group sizes . An individual approach, as opposed to group sessions, allows meeting a patient s needs more specifically as community - dwelling participants may prefer exercising at home . The need for individual adaptation of physical activity programs has been stressed by yu and kolanowski as patients with moderate to severe dementia do not express reliably exertion and need guidance to exercise at the targeted level . Variability in content and modalities of physical activity programs (session frequency and duration) impede any precise recommendation for clinical practice . The variety of applied adl assessment tools hampered comparison of the program effect on the adl performance across all studies . Any recommendation to use objective measurement tools for moderately to severely demented subjects was altogether missing from the literature . However, it has little or no sensitivity to small changes and is therefore inappropriate for longitudinal studies . Only one study applied a population specific measure, i.e., the changes in advanced dementia scale, to assess adl performance in demented persons . Assessing mobility is crucial as it is a key capacity among elderly with moderate to severe dementia . Mobile patients decrease caregiver burden, and their quality of life may be better . The majority of items in the changes in advanced dementia scale deal with cognitive abilities to perform adl and not physical abilities . However, while physical activity programs act primarily on motor tasks, they may also have an impact on cognition . Given the preponderance of items related to cognition, physical components of adl capacities may be underestimated when applying the changes in advanced dementia scale . The preceding considerations may explain the clinicians and researchers difficulties in selecting the appropriate adl assessment tool . Higher levels of physical activity were beneficial and decreased the number of falls in a mildly to moderately demented population . No study reported serious adverse events related to physical activity, but steinberg et al . Found trends of poorer quality of life and increased depression in their exercise group and discussed the possibility that physical activity may cause distress . However, the finding of higher depression scores was not corroborated by other studies [43, 44]. . The methodological quality of the majority of the included studies is low, and information regarding the reliability, validity, and sensitivity to change (i.e., the clinically minimal important change) of the various assessment tools applied to a moderate or severely demented population is missing . The scores obtained for external validity were low which limits the generalizability of the results and the formulation of recommendations . Biases were due to inappropriate randomization methods in four out of the five included studies [23, 24, 26, 27], to the lack of blinding of the outcome assessors in three studies [24, 26, 27] and two studies [23, 26] used adl assessment tools which were not validated for the moderately and severely demented . The clinical importance of the results was threatened by the insufficient power of three out of the five included studies [23, 26, 27]. The absence of interventions for the control group in two trials [25, 26] represented a further weakness and prevented interpretation of the impact of physical activity programs on adl in these studies . The small number of studies corresponding to the selection criteria of our review represents an important limitation of this paper . Although we tried to identify all significant studies, we do not pretend to have conducted a comprehensive review . Defining dementia severity using mean mmse scores was a pragmatic decision as mmse scores are the most frequently reported severity measures . However, although populations were described as being moderately to severely demented in the included studies, not all authors reported dementia severity scores [24, 27]. We are aware of the confounding education levels by interpreting mmse scores as well as the questionability of the cutoff scores which define moderate and severe dementia . While a uniform definition of severity is still lacking [47, 48], the relevant mmse scores are still debated . Evidence for efficacy of physical activity programs on adl performance in the elderly with moderate to severe dementia remains very limited . One high - quality study showed a small but statistically significant and clinically meaningful effect . Further investigations determining ideal physical activity program content as well as appropriate session duration and frequency are warranted . The variable program content between studies is of concern since it provides little guidance to clinicians as to what protocols may be the most suitable in patient care . Gaining a more complete picture of the impact and limits of physical activity programs in this domain is urgent . While the number of people with dementia is growing, reducing adl dependence contributes to a better control of costs and alleviates family and caregiver burdens . In this context, the issue of the most effective dose for physical activity programs (nature, frequency, duration) is of crucial importance to healthcare quality and costs.
Patients with functional bowel disorders (fbds) manifest variable combinations of intestinal symptoms without structural and/or biochemical abnormalities . The latter concept has been challenged by growing evidence showing low - grade inflammatory changes in the gut and altered gut - brain axis signaling.1,2 according to the rome iii classification, fbds include the irritable bowel syndrome (ibs), functional bloating (fb), functional constipation, functional diarrhea, and unspecified fbd, and they are attributed to abnormalities likely originating from the small bowel, colon, and rectum.3,4 since fbds lack objective biomarkers, their diagnosis is based on the clinical symptoms reported by patients, physical examination, and the exclusion of alarm symptoms / signs (eg, blood in stools, anemia, weight loss, and others). Although fbds are not regarded as life threatening, these conditions can significantly worsen the patient s quality of life . Indeed, fbds are responsible for prolonged absenteeism from work as well as for suboptimal performance in the workplace with relevant social costs.5,6 amongst fbds, ibs is certainly the most common clinical entity affecting up to 20% of the general population.7 classically, an ibs diagnosis revolves around abdominal pain / discomfort in conjunction with altered bowel habits . The clinical phenotypes include ibs with constipation, with diarrhea (ibs - d), alternating bowel or mixed (the most frequent pattern in western industrialized countries), and unsubtyped according to stool frequency and consistency.3,8,9 the pathogenesis underlying ibs is only partly understood and notoriously referred to as multifactorial being attributable to dysfunction of the gut - brain axis . In this context, recognized mechanisms in ibs span a wide spectrum including gut dysmotility, low - grade inflammation, visceral hypersensitivity, changes of gut microbiome, infections, altered gut barrier function, and genetic and psychosocial factors.1014 the role of dietary factors in ibs pathogenesis is a topic of great interest.1517 indeed, more than 60% of patients with ibs relate the occurrence of bloating and abdominal pain to the ingestion of certain foods . The majority of these patients report worsening of symptoms between 15 minutes to a few hours after meal intake.18 however, only recent animal and human studies have focused on the key role of specific foods in altering gut physiology . The aim of the present review is to provide an overview highlighting the major aspects of the complex interplay existing between foods and gut function with relevance to ibs . Specifically, the reader will have an update on the role of gluten / wheat sensitivity as potential dietary triggers evoking gut dysfunction and symptoms in ibs . The pathophysiology of ibs is still not well understood, limiting the capacity to effectively treat the disorder.19 enteric infections are the strongest environmental triggers for ibs, constituting the well - characterized subgroup of post - infective ibs,20 which is associated with dysbiosis, low - grade inflammation and altered intestinal permeability.21 these mechanisms have also been proposed in the general ibs population, but results are not as consistent as in post - infective ibs.22 in addition to enteric infection, other environmental and psychosocial triggers have been linked with ibs . Interestingly, many of these triggers induce visceral hypersensitivity, changes in gut microbiota, and altered levels of enteric hormones and neurotransmitters which may explain symptom generation.19,22,23 alterations in gastrointestinal transit, which may be caused by stress,24 have also been reported in ibs patients . Although ibs is considered to affect mainly the colon, several studies have reported motility alterations also in the esophagus, stomach and small intestine, which often correlate with patients symptoms.25 several studies and a recent meta - analysis2628 have demonstrated bile acid (ba) malabsorption, at least in a sub - population of ibs - d . In a study of 119 patients with ibs,28 32% had abnormal colonic transit measured by scintigraphy at 24 or 48 hours, with accelerated transit in 48% of ibs - d patients; the causes of abnormal transit are unclear . Ba sequestrants have been proposed as a possible therapeutic option, however, the mechanism of action of these ba sequestrants to ameliorate bowel function is not yet clearly demonstrated.29 of all the possible non - infectious environmental triggers, food is a likely candidate that can affect a variety of physiologic parameters important in ibs such as motility, visceral perception, brain - gut interactions, microbiota composition, permeability, immune activation, and neuro - endocrine function.30 among the foods reported to associate with ibs symptoms, those rich in carbohydrates,19 gluten and wheat31,32 are common . However, food sensitivity or allergy cannot be always confirmed in patients that recognize a specific food, as a trigger of their symptoms.31 therefore, a better understanding of the dietary factors involved in ibs and their underlying mechanisms is key to determine the real benefit of exclusion diets in ibs . Ingestion of a meal activates an array of complex mechanisms enabling the digestive system to perform the complex task of digestion and absorption of nutrients as well as expulsion of waste . Under normal circumstances, our gut has adapted to respond with a tightly regulated system of neuro - immune interactions necessary to maintain proper gut function and homeostasis . Dietary factors, however, can be harmful in certain circumstances and can cause intolerance, allergy, or hypersensitivity via a number of different mechanisms . Food intolerance, such as lactose intolerance, classically relates to a reaction to food components based on some metabolic deficiency.3335 food allergy and hypersensitivity instead refer to undesirable immune - based reactions that cause symptoms . The most common food sensitivities have been related to proteins in nuts, wheat, and milk.36 some of these reactions are typically ige mediated (allergy), while others recognize non - ige immune pathways . Patients with ibs often report worsening of symptoms by a wheat containing diet.37 gluten is a group of immunogenic proteins in wheat, which causes celiac disease, an inflammatory and autoimmune disease, in genetically susceptible people.38 this led to the hypothesis that a subgroup of ibs patients could develop mild immune or functional alterations in the absence of celiac disease.39 gluten proteins are indeed insufficiently degraded by gut proteases, leaving undigested peptides that could trigger innate immune mechanisms that may be of importance in ibs . A mouse study determined that gluten sensitization causes altered smooth muscle contractility and altered barrier function, which are mechanisms commonly associated with ibs . It is important to stress that other proteins in wheat, such as -amylase / trypsin inhibitors (atis)40 and wheat lectin agglutinin,41 have recently shown to induce innate immune pathways . Atis have been shown to trigger a toll - like - receptor (tlr)-4 mediated activation40 and it remains to be determined whether this is associated with gut functional changes . In addition to protein fractions, wheat contains a group of carbohydrates, called fructans . These are members of fermentable oligosaccharide, disaccharide, mono - saccharides, and polyols (fodmaps) that are poorly absorbed in the small intestine.37,40,42 these substrates are very important for gut health, supporting microbiota diversity and short chain fatty acid production43 but under certain circumstances could cause, through excessive bacterial colonic fermentation, symptoms such as bloating and altered bowel habits.19,44 regardless of the offending component, the withdrawal of wheat from the diet in a subgroup of patients with ibs has indeed been shown to improve symptoms.45 although unselected ibs patients display clinical improvement after wheat withdrawal, only a proportion may truly reflect underlying gluten sensitivity (table 1).4656 the absence of specific biomarkers and the great proportion of patients with placebo effect observed in ibs trials, are 2 barriers for identifying the specific role of wheat components in generating ibs symptoms . Therefore, double - blind - placebo - controlled - crossover (dbpcc) trials are a reliable way to establish whether wheat and its fractions are involved in ibs symptom induction.50,51,53,56 different mechanisms have been proposed to explain how gluten may trigger gastrointestinal symptoms in the absence of celiac disease (figure). In vitro studies have demonstrated that digests of gliadin increase the expression of co - stimulatory molecules and the production of proinflammatory cytokines in monocytes and dendritic cells.40,57,58 certain toxic (that only stimulates the innate immune response) gliadin - derived peptides such as the 3143mer, may evoke epithelial cell dysfunction, increased il-15 production and enterocyte apoptosis.59 recent studies have demonstrated increased expression of tlr-2 in the intestinal mucosa of non - celiac compared to celiac patients, suggesting a role of the innate immune system in the pathogenesis of non - celiac reactions to gluten or other wheat components.49 other studies have shown that monocytes from hla - dq2 + non - celiac individuals spontaneously release 23 fold more il-8 than monocytes from hla - dq2 negative patients . This suggests that patients without celiac disease (no enteropathy and negative specific serology), but with positive hla - dq2 status, may represent a subpopulation reacting mildly to gluten.60 in terms of gut dysfunction, gluten sensitization in mice has been shown to induce acetylcholine release, one of the main excitatory neurotransmitters in the gut, from the myenteric plexus.57 this correlates with increased smooth muscle contractility and a hypersecretory status with increased ion transport and water movements.57 these functional effects induced by gluten were not accompanied by mucosal atrophy, and were not observed after sensitization with non - gluten proteins . Interestingly gluten - induced gut dysfunction was particularly notable in mice transgenic for the human celiac gene hla - dq8.57 atis, a group of wheat proteins that confer resistance of the grain to pests, are strong inducers of innate immune responses via tlr4 and via the myeloid differentiation factor 88-dependent and -independent pathway.40 this activation occurs both in vitro and in vivo after oral ingestion of purified atis or gluten, while gluten - free cereals display no or minimal activities.61 the role of atis in ibs is not yet known, however there is clear description of a mechanism that could be involved in the generation of gut dysfunction and symptoms . These mechanisms are different from those proposed for gluten and thus it is conceivable that they could co - exist in given patients or have a synergistic effect . Ibs and celiac disease are common conditions, and they may overlap by chance . However, celiac disease is 34 times more common in ibs patients compared to controls suggesting that both disorders could be mechanistically associated.62 active screening by celiac serology and biopsy reveals that 4% of patients labeled as ibs have underlying celiac disease.6265 this overlap between the 2 conditions is different from the issue of wheat components, including gluten, causing functional symptoms in patients without celiac disease.39 clinical studies have revealed that a subgroup of ibs patients in whom celiac disease and wheat allergy were ruled out, displayed intestinal and extra - intestinal symptoms after wheat ingestion . Three consensus conferences defined this new syndrome as non - celiac gluten sensitivity (ncgs) or, alternatively, non - celiac wheat sensitivity (ncws) given the possibility for immune reactions or intolerances to other wheat components as explained above.6668 it is important to bear in mind the complexity of food hypersensitivities and intolerances, many of which could coexist in the same patient . This constitutes a confounder that we must be aware of for the design and interpretation of clinical trials.69 the prevalence of ncgs / ncws is still not clearly established mainly due to the hitch in standardizing international diagnostic criteria . In united states this condition seems to be identified more frequently in tertiary referral centers than in primary care . Data obtained in primary care practice, from the national health and nutrition examination survey (nhanes), indicate that the estimated prevalence of ncgs / ncws was 0.6% over 7762 subjects.70 on the other hand, data published from the celiac disease center in baltimore (university of maryland), showed a prevalence of suspected ncgs / ncws of 6% over 5896 subjects.66 moreover, an italian multicenter study prospectively evaluated the prevalence of ncgs / ncws and celiac disease in pediatric and adult centers for gluten related disorders.71 among 12 255 subjects consecutively investigated during a one - year survey, 391 (3.2%) cases of suspected ncgs / ncws and 340 (2.8%) celiac patients were identified . Based on these results, it is possible to estimate a ratio between ncgs / ncws and celiac disease of 1.15:1, data in line with the nhanes survey . Although ncgs / ncws can occur at any age, this condition seems to occur more frequently in adulthood with a mean age at diagnosis of 40 years . The female gender, similarly to ibs, is more affected by this sensitivity with a ratio up to 5:1.71 some clinical trials have attempted to investigate underlying functional and immune abnormalities in patients with a clinical picture of ncgs / ncws . Initial results showed that there is increased expression of tlr2 in the small intestine of patients with ncgs/ ncws.49 this was in contrast with the normal expression of il-17a, il-6, ifn-, il-17, and il-21 in the duodenal mucosa of these patients, which seemed to rule out the contribution of adaptive immunity.72 recent work has suggested increased ifn- levels in small intestinal biopsies of ncgs / ncws patients following a short - term gluten challenge.73 although this cytokine is a key mediator of t - helper 1 adaptive immune responses, it could also be produced by innate cells such as intraepithelial lymphocytes (iels).73 another relevant pathogenic aspect that has been investigated in ncgs / ncws pertains to possible functional changes, such as the increase of intestinal permeability . Using the lactulose / mannitol test, ibs patients with self - reported sensitivity to gluten did not exhibit significant alterations in sugar permeability.49 this may be related to the population involved or technical difficulties in the interpretation of clinical permeability tests . However, in contrast to this study, vazquez - roque et al,52 using the same method, found increased intestinal permeability in a subgroup of hla - dq2/dq8 + ncgs/ ncws patients with ibs - d, once again raising the possibility of a particularly vulnerable subpopulation . This underscores the importance of careful patient phenotyping in studies involving functional symptoms and adverse reactions to food components . It also raises the concept of a subgroup of genetically predisposed individuals carrying celiac markers, but without active celiac disease, that may be more sensitive to low - grade inflammation or functional changes induced by gluten . An initial study enrolling ibs patients fulfilling criteria for ncgs / ncws showed symptom recurrence following gluten reintroduction.48 in a second trial from the same group,51 ibs patients that responded to a gluten - free diet were challenged with low dose of gluten (2 g / day), high dose of gluten (16 g / day) or whey protein (16 g / day) after 2 weeks of low dose fodmaps diet . It is worthwhile to note that patients increased their symptoms with all challenges, including placebo, and that gluten challenge did not show a dose response . Carroccio et al50 demonstrated that ibs patients randomized to receive whole wheat vs placebo, had a significant worsening of their intestinal and extra - intestinal symptoms after wheat ingestion . A recent dbpcc trial used pure gluten vs rice starch (control)-containing capsules to clarify the exact role played by gluten in symptom generation in patients with highly suspected ncgs / ncws.54 the results showed that pure gluten ingestion induced recurrence of a variety of symptoms including, bloating, abdominal pain, foggy mind, aphthous stomatitis, headache and depression, but not in all of ncgs patients . Two recent dbpcc trials55,56 performed in gluten / wheat sensitive strongly suggested that gluten / wheat was responsible for symptom generation in up to one - third of ibs patients . Overall the results indicated that gluten and other wheat proteins cause symptoms in the absence of celiac disease in a subset of the ibs population (table 2). The dilemma is that a gluten - free diet may also be low in fodmaps . Some studies have assessed the efficacy of low - fodmaps diet in ibs with variable results (table 3). However, none of these trials included a placebo group, thus introducing a possible bias hampering the actual value of a low - fodmaps diet both in terms of diagnosis and as a therapeutic intervention . A recent dbpc trial (without crossover) comparing a low - fodmap diet with traditional dietary advice in ibs, showed no difference among the 2 strategies, thus renewing controversy of the specificity of therapeutic effect of fodmaps exclusion in the general ibs population.74 moreover, this study indicated that patients are more likely to react positively to fodmaps restriction, when already reducing fodmaps from their diet before initiation of the trial . Finally, the data on the effect of fodmaps in ibs were pooled in three systematic reviews, and one of them included a meta - analysis . In the systematic reviews from rao et al75 and moayyedi et al76 the authors showed that the data could not be combined for meta - analysis and concluded that low - fodmaps is of uncertain benefit in ibs . In a third review,77 with less rigorous inclusion criteria, the authors were able to pool the data and found that low fodmap diet was effective in ibs . However, the results from this meta - analysis should be taken cautiously due to a great heterogeneity in population included and the comparator for the intervention (table 3). Ncgs / ncws is often considered a self - diagnosis, usually reported by the patient . For the physician, it is based on the thorough evaluation of the clinical features according to the indications proposed by the consensus conferences on this syndrome.6668 identification of biomarkers that allow us to diagnose ncgs / ncws with more specificity and differentiate it from the unselected ibs population will be key to define and manage this condition.78,79 the clinical picture of patients with ncgs / ncws is characterized by symptoms occurring shortly after consumption of wheat/ gluten containing meals and disappearing or recurring in a few hours / days after specific withdrawal or challenge.6668 symptoms that characterize ibs, such as bloating, abdominal discomfort / pain, altered bowel habits and tiredness, are present in ncgs / ncws . It has been suggested that a clinical distinction can be made between general ibs and ncgs / ncws because of more extra - intestinal manifestations involving the central and/or peripheral nervous system, joint / muscle (fibromyalgia - like) and skin manifestations in the latter.71 due to the lack of biomarkers, the diagnosis of ncgs/ ncws remains highly presumptive being based only on clinical and exclusion criteria.80,81 the improvement of symptoms after a gluten - free diet, which is regarded as the major diagnostic criteria for ncgs / ncws, might be due to a placebo effect which often follows the elimination of some foods from the diet.82,83 in this context it is important to stress the negative potential influence (nocebo effect) generated by media on the deleterious effect of wheat consumption . There is no scientific evidence to support that gluten/ wheat consumption is deleterious to the overall population and such sensitivity seems to be limited to a subpopulation of patients that present with ibs symptoms . Despite some disproportionate press on one hand, and some healthy skepticism on the other, there is no doubt that awareness and interest in ncgs / ncws continues to grow . Antibodies to native gliadin (aga) have been suggested as a potential diagnostic marker for ncgs / ncws diagnosis, in the absence of specific celiac serology such as tissue transglutaminase, endomysial and deamidated gliadin antibodies.84 aga have been detected in the sera of about half of ncgs / ncws patients being predominantly of the igg class . Although aga are not specific for ncgs / ncws being detectable in various conditions, ie, autoimmune disorders, connective tissue diseases and even in healthy controls, their positivity, especially at high titers, in patients with a clinical picture suggestive of gluten / wheat sensitivity may be regarded as a diagnostic adjunct . In parallel to symptom resolution, aga normalized in almost all patients with ncgs / ncws within 6 months of gluten - free diet.85 in cases with suspected ncgs / ncws, the most important clinical issue is to rule out celiac disease while the patient is on a gluten - containing diet . Patients with ncgs / ncws have normal duodenal mucosal histology, although an increased number of iels ranging from 25 to 40/100 epithelial cells are found in at least 40% of cases, suggesting an accompanying low - grade inflammation.71 there is no increase of t - cell receptor / iels in biopsies of patients with ncgs / ncws . Some studies found that hla - dq2 and/or hla - dq8 genes were present in ~50% of ncgs / ncws patients, which is slightly higher than in the general population.84 on the other hand, some studies have suggested that ibs patients who carry celiac susceptibility genes are more likely to respond to the gluten - free diet.52 this apparent controversy may be explained by the fact that overall the ncgs / ncws population may be heterogeneous and responsive to multiple stimuli . It could be speculated that those with celiac susceptibility genes may be more prone to develop mild immune responses and gut dysfunction to gluten . Up to 20% of ncgs / ncws show mild laboratory abnormalities, such as low levels of ferritin, folic acid, vitamin d and b12, most likely related to a minimal inflammatory state in the intestinal mucosa.86 evidence of osteopenia detected by bone densitometry has been found in about 50% of ncgs / ncws.87 finally, recent data reported a high prevalence of serum autoantibodies (antinuclear antibodies, ana) and a frequent association with autoimmune disorders (ie, hashimoto s thyroiditis) in patients with ncgs / ncws.88,89 no specific guidelines are yet available for the treatment of ibs patients with ncgs / ncws . Gluten is a common ingredient of many food items and its complete removal is almost impossible to achieve . Exposure to as little as 1050 mg of gluten (a breadcrumb) can cause intestinal lesions and symptoms in celiac patients, although the threshold for gluten tolerance is unknown in the ncgs / ncws population . Moreover, there seems to be an individual level of tolerance in ncgs / ncws.6668 experts in the field recommend that investigations to rule out celiac disease and wheat allergy should be performed in people who consider themselves as gluten / wheat sensitive before starting a gluten - free diet.66 patients should be warned that an inappropriate dietary restriction can cause nutritional deficiencies . Because gluten - rich grains are important sources of nutrients in the general diet, their exclusion could potentially have major effects on nutritional status . Lower caloric9093 and fiber intake,90 but a higher intake of total and saturated fat91,92 was observed in the diet of celiac patients compared to healthy control subjects on a gluten containing diet . Lower levels of folate, niacin, vitamin b12, vitamin e, vitamin a, phosphorus, calcium, zinc, and selenium were described in the diet of celiac individuals compared to control subjects.9093 currently, it is still difficult to draw a conclusion on the nutritional adequacy of a gluten - free diet because of discordant results noticed from the studies regarding macro- and micro - nutrient intake . However, the evidence suggests that following a gluten - free diet may be detrimental if not properly evaluated and medically indicated . Dietary restriction has been shown to affect the richness and composition of small intestinal and fecal microbiota, reducing beneficial bacterial groups such as firmicutes.94,95 it is necessary to consider that after any restriction diet, adaptations in gut physiology and microbial metabolism could increase sensitivity to the subsequent re - introduction of gluten / wheat or fodmaps . The persistence of symptoms after a period of gluten - free diet (at least 6 weeks) in patients with suspected ncgs / ncws suggests that other food sensitivities / intolerances may be responsible for symptom generation.37 in this respect, the patient may benefit from a low - fodmaps trial excluding rapidly absorbable carbohydrates such as those naturally contained in legumes, onions, honey, pears, water melon, dry fruits, fennel, and dairy products . Foods can be triggers of gastrointestinal and extra - intestinal symptoms in a proportion of ibs patients . Inside the spectrum of the so - called food hypersensitivity, ncgs / ncws has been recognized as a newly identified gluten - related disorder which presents clinically with overlapping symptoms of ibs.6668 along with gluten, other wheat and food components, have emerged as functional digestive symptom triggers . Within wheat, a potential culprit of symptom generation is ati, a still poorly investigated soluble protein fraction of wheat.40 a role for fodmaps, detectable not only in gluten - containing cereals (wheat, rye, and barley), but also in milk, honey, and legumes, has been proposed, although a recent dbpc trial did not find this restriction more efficient than traditional dietary advice for ibs patients.74 we have discussed the evidence for some specific dietary components, such as gluten and other wheat components, to cause functional digestive symptoms and extra - intestinal manifestations matching the current criteria for ibs . As with overall ibs, the mechanisms underlying symptom generation in the subgroup of patients with ncgs / ncws are still poorly understood . Our review focused on clinical features with the intent to expand current knowledge in this area of gastroenterology . A better understanding of food hypersensitivity and intolerances as well as the development of biomarkers will enable physicians to design tailored dietary approaches to treat patients with food - related functional bowel disorders.
Medical research has increased greatly in many developing countries during the recent decade, motivated by the need to improve health in these countries . Since medical research involves human participants, such research needs to be guided by fundamental ethical principles to ensure the protection of their rights and welfare . Furthermore, international standards mandate the review of research by research ethics committees (recs) [2, 3]. However, research regulations do not exist in many developing countries, and commentators have expressed concerns regarding the extent of individual and institutional research ethics capacity, including the existence of functioning ethics review systems [4, 5]. Accordingly, several studies have demonstrated that research ethics review is not optimal in the developing world, including the middle east . For example, abou - zeid and colleagues found that 28% of researchers in the middle east region did not obtain ethical clearance for their research proposals submitted for funding [6, 7]. In many of these proposals these investigators also showed that many basic elements of informed consent were omitted from the submitted informed consent forms . Concerns have also been expressed regarding the capacity building efforts for recs as well as the challenges that prevent the optimal functioning of recs [1014]. For example, sleem and colleagues showed that barriers to the effective functioning of recs in egypt include insufficient training of members, lack of diverse membership, and limited resources . Less than optimal functioning recs have been highlighted by recent research - related scandals with occasionally tragic consequences [15, 16]. These results demonstrating less than optimal individual and institutional research ethics capacity may be explained by the relative novelty of research ethics regulations and the recent requirement of ethics review of research in the developing world, including the middle east . As such, little is also known regarding academics' attitudes towards recs, their practices in research ethics (e.g., informed consent), and training opportunities in research ethics . Recently, asem and colleagues assessed the knowledge and attitudes of the faculty at cairo university towards research informed consent . Their results showed that many academics lacked training in research ethics and that their attitudes towards several practices in the research setting were not optimal . However, these investigators also showed acceptance of the faculty towards the establishment of recs and a desire for educational programs in research ethics . The field of dentistry is committed to ongoing research investigating the causes and treatment of dental diseases and adheres to the same ethical standards embraced by the fields of medicine . However, little research has investigated the attitudes of dental faculty towards concepts of research ethics, including the acceptability of recs and their desire for training in research ethics . Recently, commentators have expressed concerns in dental research related to aspects of scientific misconduct [19, 20]. Accordingly, our objectives were to assess the knowledge, awareness, and attitudes of dental faculty regarding recs and training in research ethics, as well as potential independent variables associated with our findings . Our results will help institutional officials understand better how well recs are accepted in their institutions and also help them develop relevant educational programs in research ethics directed towards dental faculty . We conducted a cross - sectional survey study performed during the period between april and june 2007 . We recruited members of the dental faculty (demonstrators, assistant lecturers, lecturers, assistant professors, associate professors, and professors) at king abdulaziz university (kau), jeddah, saudi arabia and at ain shams university (asu), cairo, egypt . Defined as faculty with a status of demonstrators or assistant lecturers at asu and those with a status of demonstrators or lecturers at kau . Demonstrators at both asu and kau cared for patients under supervision, worked on their research projects for their master's theses, and assisted upper level faculty members in students' clinical sessions . Assistant lecturers at asu and lecturers at kau held similar academic roles (the rank of assistant lecturers did not exist at kau): both had obtained their master's degree, worked on their research projects for their phds, and attended all students' clinical sessions . Defined as faculty with a status of lecturers and assistant professors at asu and a status of assistant and associate professors at kau . Lecturers at asu and assistant professors at kau held similar academic roles: both faculty types had obtained their phds, were cosupervisors for students' theses, worked on their research projects for their promotions, and gave lectures to undergraduate students . Assistant professors at asu and associate professors at kau held similar academic roles (the rank of associate professor did not exist at asu): both worked on their research projects for their promotions, supervised theses, and gave lectures to both under- and postgraduate students . Defined as faculty who achieved the status of professors . At both universities, professors conducted and supervised research projects / theses and gave lectures to both under- and postgraduate students . Ain shams university and king abdulaziz university are among the most important universities in their respective countries . King abdulaziz university was founded in 1967 and its faculty of dentistry and its four departments (oral basic and clinical sciences, oral / maxillofacial rehabilitation, preventive dental sciences, and conservative dental sciences) were established in 1985 . At king abdulaziz university, one of the coauthors h. f. el - dessouky, distributed the surveys by placing them into the mailboxes of the faculty in the dental school . At ain shams university, another coauthor r. a. fadl, faculty was asked to return the surveys back anonymously by placing it in a general mailbox in the department's office . We developed a questionnaire based on our study objectives, taking guidance from the previous literature regarding research ethics in the developing world . The first part collected demographic information of the participants: age, gender, academic position, prior participation in human subjects research (e.g., research involving human subjects and/or human biological samples), number of research projects involved in, and prior training in research ethics (e.g., having attended a course or a workshop in research ethics). We did not ask for any details regarding any courses or workshops the faculty had attended . The second part of the survey assessed the participants' self - awareness of research ethics principles and functions of research ethics committees . Are you familiar with ethical principles that govern conducting research involving human subjects? Are you fully aware of the functions of ethics committees? The first part of the knowledge section consisted of several case scenarios involving the ethics of clinical research in dentistry and asking the respondents to answer questions based on these cases . Case 1 (informed consent describing risks and benefits) thirty patients from the outpatient clinic of the faculty of dentistry were enrolled in a study that aimed to evaluate the flexible denture base material as compared with conventional denture base . One of the most serious disadvantages of the resilient denture liners is colonization and infection of the material surface by candida albicans . An oral consent has been taken from the patients without full description of the risks and benefits . Which of the following best describes obligations of informed consent?the investigators can conduct the research without any ethical responsibility.a written consent with a brief description of the procedures must be taken.a full description of the risks and benefits should be stated in the informed consent.there is no need for informed consent, as the patients were enrolled from the outpatient clinic . Thirty patients from the outpatient clinic of the faculty of dentistry were enrolled in a study that aimed to evaluate the flexible denture base material as compared with conventional denture base . One of the most serious disadvantages of the resilient denture liners is colonization and infection of the material surface by candida albicans . An oral consent has been taken from the patients without full description of the risks and benefits . Which of the following best describes obligations of informed consent?the investigators can conduct the research without any ethical responsibility.a written consent with a brief description of the procedures must be taken.a full description of the risks and benefits should be stated in the informed consent.there is no need for informed consent, as the patients were enrolled from the outpatient clinic . The investigators can conduct the research without any ethical responsibility . A written consent with a brief description of the procedures must be taken . A full description of the risks and benefits should be stated in the informed consent . There is no need for informed consent, as the patients were enrolled from the outpatient clinic . Case 2 (research involving children)one hundred children of both sexes, age range from 6 to 15 years, were randomly selected from the outpatient clinic of the faculty of dentistry . One group will have their extensively carious teeth extracted, while the other group will go through pulpotomy in an attempt to keep the tooth as long as possible in their mouth.a clear description of the procedure should be explained to the child's parent / guardian.an assent (oral approval) should be taken from the child.an assent should be taken from the child as well as a written informed consent from the child's parent / guardian.no need to have an assent or consent as the children were already enrolled in the outpatient clinic and ready to receive any type of treatment . One hundred children of both sexes, age range from 6 to 15 years, were randomly selected from the outpatient clinic of the faculty of dentistry . One group will have their extensively carious teeth extracted, while the other group will go through pulpotomy in an attempt to keep the tooth as long as possible in their mouth.a clear description of the procedure should be explained to the child's parent / guardian.an assent (oral approval) should be taken from the child.an assent should be taken from the child as well as a written informed consent from the child's parent / guardian.no need to have an assent or consent as the children were already enrolled in the outpatient clinic and ready to receive any type of treatment . A clear description of the procedure should be explained to the child's parent / guardian . An assent should be taken from the child as well as a written informed consent from the child's parent / guardian . No need to have an assent or consent as the children were already enrolled in the outpatient clinic and ready to receive any type of treatment . Case 3 (retrospective research on stored samples originally collected for clinical purposes)fifty patients from the outpatient clinic of the faculty of dentistry were diagnosed as having lichen planus . Biopsies were taken from the patients after their approval to confirm the clinical diagnosis (patients were not charged any money). A month later, a research on lichen planus is planned by the faculty involving all biopsies that were previously obtained from the patients . This research cannot be done without the approval of the patients.the biopsies belong to the faculty of dentistry, so no patient approval is needed.it is for the researchers to decide whether to take the patient's consent or not.it is up to the dean or head of department to decide what to do with the biopsies without patient's interference . Fifty patients from the outpatient clinic of the faculty of dentistry were diagnosed as having lichen planus . Biopsies were taken from the patients after their approval to confirm the clinical diagnosis (patients were not charged any money). A month later, a research on lichen planus is planned by the faculty involving all biopsies that were previously obtained from the patients . This research cannot be done without the approval of the patients.the biopsies belong to the faculty of dentistry, so no patient approval is needed.it is for the researchers to decide whether to take the patient's consent or not.it is up to the dean or head of department to decide what to do with the biopsies without patient's interference . The biopsies belong to the faculty of dentistry, so no patient approval is needed . It is for the researchers to decide whether to take the patient's consent or not . It is up to the dean or head of department to decide what to do with the biopsies without patient's interference . Case 4 (confidentiality in medical research)eighty patients from the outpatient clinic were enrolled in a research . The aim of the research was to differentiate between two different treatment modalities in the management of periodontal intraosseous bony defects . Patients' research files should be coded to ensure patients' confidentiality.no need for confidentiality as the procedures are common in dental practice.it is left to the investigator to decide whether to keep the research data confidential or not.the dean or head of the department is the one to decide regarding the provisions of confidentiality . The correct answer for cases 1 and 2 is c, while for cases 3 and 4 the correct answer is a.these answers were based on concepts of research ethics drawn from research ethics guidelines . The second part of the knowledge section consisted of the following two questions . Which of the following are considered guidelines in research ethics? Nuremberg code, declaration of helsinki, belmont report, council of the international organizations of the medical sciences (cioms), orall of the above . Review the ethical aspects of the research, determine whether informed consent is needed, review the scientific design of the research, protect the welfare and rights of the subjects in the research, ormake research more difficult to perform, other . 1 and either (a, b, c, d) or (a, b, and d) for question no . 2, because both universities in this study have scientific committees that review the scientific design of the research, and therefore, it is conceivable that faculty might have thought that their rec does not review the scientific design of the research . The cioms guidelines recognize this possibility, as they state in their guidelines that a research ethics committee must either carry out or arrange for a proper scientific review or verify that a competent expert body has determined that the research is scientifically sound . Respondents were required to choose from a 5-point likert scale ranging from 1 to 5 (1-strongly agree, 2-agree, 3-not sure, 4-disagree and 5-strongly disagree). The fifth part of the questionnaire assessed respondents' attitudes towards certain practices in the conduct of research . These practices included those involved with informed consent, enrolment of vulnerable individuals, confidentiality, and responsible conduct of research . Respondents were required to answer yes, no, or uncertain . The aim of the research was to differentiate between two different treatment modalities in the management of periodontal intraosseous bony defects . Patients' research files should be coded to ensure patients' confidentiality.no need for confidentiality as the procedures are common in dental practice.it is left to the investigator to decide whether to keep the research data confidential or not.the dean or head of the department is the one to decide regarding the provisions of confidentiality . Patients' research files should be coded to ensure patients' confidentiality . No need for confidentiality as the procedures are common in dental practice . It is left to the investigator to decide whether to keep the research data confidential or not . The dean or head of the department is the one to decide regarding the provisions of confidentiality . The correct answer for cases 1 and 2 is c, while for cases 3 and 4 the correct answer is a. these answers were based on concepts of research ethics drawn from research ethics guidelines . The second part of the knowledge section consisted of the following two questions . Which of the following are considered guidelines in research ethics? Nuremberg code, declaration of helsinki, belmont report, council of the international organizations of the medical sciences (cioms), orall of the above . What do you think is the role of a research ethics committee? Review the ethical aspects of the research, determine whether informed consent is needed, review the scientific design of the research, protect the welfare and rights of the subjects in the research, ormake research more difficult to perform, other . Which of the following are considered guidelines in research ethics? Nuremberg code, declaration of helsinki, belmont report, council of the international organizations of the medical sciences (cioms), orall of the above . Declaration of helsinki, council of the international organizations of the medical sciences (cioms), or what do you think is the role of a research ethics committee? Review the ethical aspects of the research, determine whether informed consent is needed, review the scientific design of the research, protect the welfare and rights of the subjects in the research, ormake research more difficult to perform, other . Review the ethical aspects of the research, determine whether informed consent is needed, review the scientific design of the research, protect the welfare and rights of the subjects in the research, or make research more difficult to perform, correct answers were e for question no . 1 and either (a, b, c, d) or (a, b, and d) for question no . 2, because both universities in this study have scientific committees that review the scientific design of the research, and therefore, it is conceivable that faculty might have thought that their rec does not review the scientific design of the research . The cioms guidelines recognize this possibility, as they state in their guidelines that a research ethics committee must either carry out or arrange for a proper scientific review or verify that a competent expert body has determined that the research is scientifically sound . . Respondents were required to choose from a 5-point likert scale ranging from 1 to 5 (1-strongly agree, 2-agree, 3-not sure, 4-disagree and 5-strongly disagree). The fifth part of the questionnaire assessed respondents' attitudes towards certain practices in the conduct of research . These practices included those involved with informed consent, enrolment of vulnerable individuals, confidentiality, and responsible conduct of research . We entered data from completed questionnaires into microsoft excel and then converted it to spss version 13.0 (statistical package for social sciences, 2009). For purposes of analysis, we collapsed the categories of strongly agree and agree . We report in percentages the positive responses of the available choices (yes as opposed to no and do not know and the strongly agree and agree responses for those questions employing a likert scale). We used chi - square tests to determine, in bivariate analyses, the association of each of the independent variables (gender, academic position, prior ethics training, and prior involvement with research) with each of the main outcome of interest (dependent responses involving knowledge, awareness, and attitudes). We used covariates that were significant in a multivariate logistic regression analysis to determine independent predictors (covariates) of the dependent responses . We report in the text the odds ratios and confidence intervals for those associations that were significant . This study was approved by the recs at king abdulaziz university, ain shams university, and the university of maryland . Potential participants received a cover letter attached to the survey tool that included the following elements of informed consent: the purpose of the research study, potential benefits and risks, and that participation was voluntary and refusal to participate would not be associated with any academic penalty . To ensure anonymity, the recs waived the requirement of signed written consent; completion of the survey implied participants' provision of informed consent . Of the 100 questionnaires distributed at each university (total of 200), we received responses from 125 individuals . There were 75 surveys from king abdul aziz university (kau) and 50 from ain shams university (asu), representing response rates of 50% and 75%, respectively, and a total response rate of 62.5% . Table 1(a) shows the demographic data of the faculty from the universities, both separately and combined . There were no significant differences between the two universities regarding the demographic data . The combined results showed that there were slightly more women faculty compared to men (55.2% versus 43.2%), and there was an almost even distribution among the different faculty types . Regarding prior research experience, almost three quarters of the respondents from both universities (71.2%) had performed research involving human subjects or human tissue samples or both . Regarding prior ethics training, a minority (36.8%) of the respondents had prior training in ethics, whereas a majority (63.2%) had no such prior training . Of those faculty who received ethics training, 16 (35%) had attended both a course and a workshop, whereas 30 (65%) had attended either a course or a workshop, but not both (data not shown in table). Table 1(b) shows the association between gender and academic position with prior research experience (percentage of faculty performing research and number of projects / faculty) and prior ethics training . Research experience was higher with men compared with women, both in terms of the percentage of faculty (85.2% versus 61.2%, p <.01) and in the mean number of research projects per faculty (7.56 versus 3.31, p <.01). Regarding research experience, while the percentage of faculty performing human subjects research was similar among the different faculty types, the mean number of research projects per faculty was higher for the more senior faculty . Faculty at asu and kau performed similar mean number of projects per faculty (5.34 versus 5.20 projects / faculty, p = ns; data not shown in table). Regarding prior ethics training, men and women showed similar percentages, while there was a significantly higher percentage of mid - level faculty who had prior ethics training (57.5% versus 28.2% and 26.2% for professors and juniors, the percentage of faculty with prior ethics training was similar between those with and without prior research experience involving human subjects (37.1% versus 32.4%, resp . Table 2 shows the respondents' responses regarding their awareness of research ethics principles and the functions of recs . Less than half of the respondents stated that they were familiar with research ethical principles, and less than a third stated that they were familiar with the functions of recs . A higher percentage of faculty at kau compared with those at asu stated their familiarity with research ethics principles (p <.05). Table 2 also shows the association between the responses and demographic (independent) variables . Professors were significantly more likely to state that they were familiar with research ethics principles (p <.01); mid - level faculty was significantly more likely to state they were familiar with the functions of an rec (p <.01). Were significantly more likely to state they were familiar with research ethics principles and with the functions of recs compared with those who had no such prior training (both p <.01). There was a tendency for faculty with prior research experience involving human subjects to more likely state they were familiar with research ethics principles and functions of recs compared with faculty without such research experience, but these differences did not reach statistical significance . We performed a multiple logistic regression analysis to determine which independent variables were the strongest predictors of the responses in table 2 . Our analysis showed that prior ethics training was a strong predictor for stating a familiarity with research ethics principles (p <.001, or 10.10; 95% ci, 2.4541.67) and for stating a familiarity with the functions of recs (p <.001; or 5.95; 95% ci 2.4114.68). 14) and questions (items no . 5 and 6) that assess respondents' knowledge in research ethics . More than half of the respondents gave the correct answer for the first case involving an informed consent issue, while less than half of the respondents gave correct answers for the other three case scenarios . A small number of respondents (12.0%) knew the guidelines in research ethics (item no . 5) and less than a third knew the roles of recs (item no . The average score was 40.2%; 15.2% of the respondents gave correct answers to at least five of the questions, while approximately half (56.0%) knew the correct answers to at most two of the questions . Table 3 also shows the association between the responses and the demographic (independent) variables . Faculty at kau were significantly more likely to give the correct response to the 4th and 6th items (both p <.05). Mid - level faculty were significantly more likely to give the correct answers to several of the knowledge questions (2nd, 3rd, and 6th items; p <.01, p <.01, and p <.05, resp .) Compared with the other faculty types . There was a tendency for faculty with any prior ethics training or prior research experience to more likely give correct answers to all of the questions compared with those without prior ethics training or prior research experience; but these differences only reached statistical significance for item no . 5 for faculty with prior ethics training (p <.01) we performed a multiple logistic regression analysis to determine which independent variables were the strongest predictors of the responses in table 3 . Our analysis showed that mid - level faculty was a strong predictor of knowing the correct responses to the 2nd and 3rd cases; (both p <.001; or 8.62; 95% ci 2.8725.64; or 10.75; 95% ci 3.1637.03; resp . ); and that prior ethics training was a strong predictor for knowing the guidelines in research ethics (item no . 5; p <.001; or 10.55; 95% ci, 2.6042.86). Regarding the overall score, faculty at kau compared with those at asu achieved a higher average score (47.0% versus 37.0%, p <.01), whereas mid - level faculty had a higher average score compared with that obtained by the professor and junior faculty (55.8% versus 33.8% and 32.2%, resp ., p <.01). Kau faculty, mid - level faculty, and those with prior ethics training were significantly more likely to give correct answers to at least five of the questions (p <.01, p <.01, and p <.05, resp . ). Logistic regression revealed that kau faculty and mid - level faculty were strong predictors for knowing the correct answers to at least five of the questions (p <.05; or 4.69; 95% ci, 1.1319.53; p <.01; or 9.52; 95% ci, 1.6655.56, resp . ). Table 4 shows the respondents' attitudes to recs and research ethics education . Greater than 90% of the respondents agreed that an rec would be helpful, there is a need for an rec in each institution, and that human subject research must be reviewed by an rec . Furthermore, less than 20% believed that ethical review is only necessary for international collaborative research and less than 10% thought that the presence of scientific committees made the existence of an rec unnecessary . However, almost half (44%) thought that recs would delay research and would make research harder to perform . A large majority of the respondents (greater than 90%) were in favor of research ethics education for postgraduates, investigators, and members of recs . Table 4 also shows the association between these attitudes and the demographic variables . Of note, those without any prior ethics training were significantly more likely to think that an rec would be helpful (p <.01). Were significantly more likely to believe that an rec would delay research (p <.05). Professors compared with the other faculty were significantly more likely to agree that ethical review of research is only necessary for international research (p <.01). Multiple logistic regression analysis showed that none of the independent variables were strong predictors for any of these attitudes . A significant majority of the respondents (> 90%) believed in the need for confidentiality protections of research participants' data (question no.1). A large majority (> 85%) also held strong opinions regarding the importance of informed consent, as indicated by their responses to questions no . Less than 10% believed that patients should not be told about the risks of research because they may not enroll in the study . However, almost a third of the respondents thought it was not necessary to obtain research informed consent for blood samples that were obtained for clinical tests (question no . 7). Almost 40% of the respondents thought that vulnerable groups such as children and the mentally ill could provide informed consent (question no . 8). A small minority (<10%) of the respondents thought that if surrogates are not available to give informed consent for vulnerable individuals, it would still be proper to enroll such individuals in research (item no . Slightly more than 10% of the respondents thought it is proper to fabricate data to improve the outcome of the research if such an act did not cause harms to patients (question no . Table 5 also shows the association between these attitudes and the demographic variables . Of note, men and those with prior research experience were significantly more likely to agree that vulnerable groups could provide informed consent (both p <.01). Research informed consent is not necessary for blood samples obtained for clinical test and that it is okay to fabricate data prior research experience was a strong predictor for agreeing that vulnerable groups could provide informed consent (p <.01; or 4.55; 95% ci 1.5413.41). This survey study showed several key findings that should be of interest to educators and policy makers . First, the surveyed dental faculty indicated a high endorsement for the existence of recs, as most thought that such committees should review research, that they would be helpful, and that they should exist in universities . Previous studies observed similar results regarding the acceptance of recs among academics in sudan and egypt [17, 23]. However, while the faculty in this study endorsed the existence of recs, almost half of them held the opinion that such committees would delay research and make it more difficult to perform research . Commentators from western countries, where recs have been in existence for more than twenty years, have recently written about concerns with excessive bureaucratic details that cause costly delays in research approval [2426]. In our study, respondents who are more likely to harbor the belief regarding delays in research by recs were mid - level faculty and those with prior ethics training . First, faculty, in general, might not understand the extent of the processes needed for an adequate review of research . Indeed, less than a third of all of the respondents stated their familiarity with the functions of recs, underscoring that efforts are needed to enhance faculty awareness of the operations of the recs . Second, the finding that faculty with prior ethics training were more likely to believe that recs would delay research raises the question as to whether the prior training gave a mistaken impression about recs by emphasizing the many processes of recs without stressing the benefits of rec review . Third, that mid - level faculty was more likely to believe that recs would delay research can be explained by them having had more unfavorable experiences with their recs . Indeed, mid - level faculty was shown to have conducted more human subjects research projects than the junior faculty and might have had more interactions with recs compared with professors, who probably did the majority of their research when recs were not in existence . Finally, it is possible that these results regarding the associations between mid - level faculty and prior ethics training with the belief that recs delay research might represent false positives, since these independent variables were not significant on multiple logistic testing . Future qualitative research involving in - depth interviews is warranted to further explore the basis of faculty attitudes regarding the process of research review by recs . Our survey also yielded interesting results regarding the attitudes of the dental faculty towards certain research ethics practices . A large majority of the faculty appears to be aware of the accepted practices regarding confidentiality protections and several aspects regarding the informed consent process . These results contrast with the concerns mentioned regarding informed consent practices by commentators working in egypt . However, approximately a third of the respondents held the attitude that research performed on blood samples obtained for clinical purposes do not require informed consent . Professors were more likely to agree with this practice compared with the other faculty . Asem and colleagues observed similar results regarding this issue among the faculty at cairo university . Another concern we discovered regarding informed consent is that almost 40% of the faculty believed that certain vulnerable subjects (e.g., children and the mentally ill) could provide informed consent to participate in research . This result raises the underlying issue of how informed is informed consent for subjects who might lack decision making capacity . This result is made more significant by the findings of another study showing that research participants who participated in studies on oral health in nigeria had poor understanding of several key elements of the informed consent process . Accordingly, we suggest future training efforts for dental faculty to be focused on informed consent issues, including those issues involved with concepts of understanding and vulnerability . A small but significant minority (approximately 10%) thought it is permissible to fabricate data and professors were more likely to hold this opinion compared with the other faculty types . For example, eastwood and colleagues found that approximately 2% of respondents were willing to fabricate data for a grant application or a paper, whereas about 27% would be willing to select or omit data to improve the results . Similarly, a study of biomedical trainees showed that 15% admitted of personal misconduct, and they were willing to fabricate, select, or omit data for publishing a paper or obtaining a research grant . Finally, a recent meta - analysis of surveys involving scientists' self - report of research misconduct yielded a pooled weighted estimate of almost 2% of the scientists replying that they had fabricated, falsified, or modified data at least once, with a range from 0.3% to 4.9% . These numbers reflecting self - report are lower than those in the previously mentioned studies reporting what individuals would be willing to do, thus highlighting the difference between perception and actual practice . Finally, martinson and colleagues also showed that scientists late in their careers admitted to fabrication at rate higher than those early in their careers . Reasons that may account for differences between the different faculty levels regarding fabrication include (a) senior faculty have had more opportunities to engage in misconduct, (b) perceptions of being caught might change during one's career, and (c) senior faculty received their education and work habits at a time when there were different behavior standards . Specifically, less than half (36.8%) of the 125 respondents from both universities received prior training in research ethics . Furthermore, the overall average score achieved on the knowledge questions was only 40.2%, and only about 15% of the faculty was able to give correct answers to at least five of the six questions . Thus, lack of training and knowledge gaps on research ethics exist among the academic dental faculty . Interestingly, while the variable prior ethics training was shown to be an independent predictor for awareness of ethical principles and the functions of recs, it was not an independent predictor for achieving a high score on the knowledge - type questions . These results raise the issue that current training programs in research ethics might be insufficient . Interestingly, mid - level faculty compared with the other faculty types were more likely to score well on the knowledge - type questions . Several reasons can be offered to explain these results regarding the mid - level faculty . First, as shown in table 1(b), mid - level faculty was more likely to have received prior ethics training compared with the other faculty types . However, as explained previously, prior ethics training was not an independent predictor for knowledge . But, ethics training coupled with ample research experience might have led these faculty to obtain the amount of theoretical and practical knowledge necessary to answer the questions on our survey . Indeed, mid - level faculty had a combination of ethics training and research experience (defined by number of projects / faculty) that together was greater than observed for the other faculty types . Alternatively, mid - level faculty might have had more recent opportunities to travel abroad for their academic education and thus had more exposures with research ethics issues from their experiences at these other universities . Further research should probe for the factors that account for any differences in knowledge gaps between the different faculty types . Although knowledge gaps exist in research ethics, our findings showed that all faculty levels were favorable towards research ethics training for postgraduates, investigators, and rec members . Previous studies have had mixed results regarding the effects of ethics education on knowledge . A 3-day workshop in research ethics involving clinicians and scientists in a nigerian university improved participants' knowledge and application of the principles of research ethics, international guidelines and regulations, and operations of recs . Other investigators have shown similar findings regarding the effects of ethics training [3436]. However, other studies have demonstrated the lack of an effect of prior ethics instruction on the extent of research ethics knowledge [37, 38]. Also, previous studies have yielded inconclusive results regarding positive relationships between ethics education and moral conduct [29, 30, 3941]. These findings do not necessarily call into question the value of ethics education, but rather the quality of the educational experiences, as well as the long - range effects of short training in research ethics . Further research is needed to determine the teaching methods (e.g., face - to - face or distance learning) that are most effective in addressing the existing knowledge gaps in research ethics . First, our study was based on convenience sampling, thus the professionals who completed the survey may not reflect the awareness, knowledge, and attitudes of the entire membership of the dental faculty of the two universities . Second, this study involved one university in each of two countries in the middle east, thus further limiting the generalizability of our results . Future studies are warranted to investigate the knowledge, awareness, and attitudes of academics from other faculties in other universities . Third, the knowledge type questions we used in our study do not reflect the broad range of topics in research ethics . However, the questions on the survey represent basic information that academics should be expected to know in the area of research ethics . Finally, we did not obtain detailed information regarding the types of courses and workshops that the faculty had attended, as well as the methods of instructions used in their training . Despite these limitations second, there seems to be an acceptance of the need for education in research ethics among all faculty levels . Since, as revealed in our study, prior research ethics training was not an independent predictor for the extent of knowledge in research ethics, attention should be directed towards the type and extent of training needed to further enhance faculty knowledge on research ethics issues . We therefore recommend further development of educational instruction in research ethics for all university faculty, with special emphasis on vulnerable participants, responsible conduct of research, and the roles and functions of recs . Recently, educational initiatives have been organized in many regions in the developing world [42, 43]. Such efforts can lead to enhanced knowledge and acceptance of research ethics principles among investigators . Finally, we recommend qualitative studies to further explore the attitudes of faculty towards recs and certain practices in research ethics.
The ir spectra were collected on a perkin - elmer spectrum 100 instrument with universal atr . Nmr data were collected on a jeol eca-600 spectrometer operating at 600.17 mhz for h, 150.9 mhz for c, and 60.8 mhz for n (instrument reference set to liquid nh3). The edited - ghsqc was optimized for 140 hz, and the long - range jh, c (ghmbc and band selective ghmbc) were optimized for 8 hz . Chemical shifts are referenced to solvent (dmso - d6 h observed at 2.50 ppm and c observed at 39.5 ppm). Chemical shifts for n were referenced to liquid nh3 with long - range jh, n optimized for 6 hz . The esims spectra were measured on a finnigan ltq mass spectrometer located at hboi . The hresims spectra were measured using a kratos ms50tc mass spectrometer at the university of california, riverside . Medium - pressure liquid chromatography was conducted on an isco - teledyne combi - flash rf4x system . Hplc was performed using a hitachi lachrom quaternary gradient hplc system equipped with diode array detector (l-7455) monitoring at 230 nm and elsd detection . Preparative fractionation was conducted using mass - directed collection on a waters autopurification system equipped with a waters 2535 quaternary gradient module, a waters 2998 pda detector, a waters 515 hplc pump, a waters sfo system fluidics organizer, a waters 2767 sample manager, and a waters 3100 mass detector operating in positive and negative ion esi mode . The sponge (hboi specimen number 22-x-00 - 4 - 010) was collected using the johnson - sea - link human - occupied submersible at a depth of 131 m off ocean cay, bahamas, approximately 20 nautical miles south of bimini (latitude: 25 24.21 n; longitude: 79 14.21 w). It is an unidentified species of asteropus (phylum porifera, class demospongiae, subclass heteroscleromorpha, order tetractinellida, suborder astrophorina, family ancorinidae). The sponge is most closely related to asteropus niger hajdu and van soest, 1992 . The sample was massive - spherical in shape with clusters of oscules distributed over the surface . Its ectosome and choanosome were black inside both in situ and in air; it is dark brown in etoh . While the spicule complement is similar to that described for a. niger, that species is characterized by two size categories of megasclere oxeas, one of which is less than 500 m long . The smaller category of megasclere oxeas in the material studied is rare, and the dimensions are much longer and thicker than those reported for a. niger . A taxonomic voucher specimen preserved in etoh is deposited in the harbor branch oceanographic museum (catalog number 003:01087). The frozen sponge asteropus (211 g) was diced and extracted exhaustively by macerating with etoh (3 200 ml) using a waring blender . The combined filtered extracts were concentrated by distillation under reduced pressure to yield 11.10 g of crude residue . The residue was partitioned between etoac (300 ml) and h2o (3 200 ml) to give after removal of solvent 1.44 g of etoac extract . The aqueous phase was further partitioned with n - butanol (3 200 ml) to afford, after removal of solvent, 7.11 and 1.94 g of aqueous and n - butanol extracts, respectively . The n - butanol extract was fractionated using a combi - flash rfx4 system equipped with a 15.5 g redi - sep rp-18 rf gold isco column operating at 200 psi with a flow rate of 30 ml / min . Fractions were eluted using a linear gradient of a [h2o / ch3cn (95:5 v / v + 0.1% tfa)] and b [ch3cn (+ 0.1% tfa)]; t = 0 min a: b (95:5 v / v), t = 23 min a: b 0:100 v / v, hold for 2 min . Further purification of a fraction eluting at 10 column volumes (approximately 4 min) (200 mg) was carried out on a waters autopure lcms mass - directed preparative purification system using a vydac 218tp protein and peptide c-18 preparative column (150 22 mm, 10 m particle size) eluted with a linear gradient of a [h2o / ch3cn (95:5 v / v + 0.1% tfa)] and b [ch3cn (+ 0.1% tfa)], t = 0 min a: b (95:5 v / v), t = 23 min a: b (0:100 v / v), hold for 2 min, and selecting mass collection at m / z 367 and 447 . This led to the isolation of 1 (44.1 mg, 1.9 10% of wet weight) and 2 (5.9 mg, 2.7 10% of wet weight). Dark blue solid; uv (meoh) max (log) 209 (3.82), 270 (3.89), and 295 (3.63); ir (film) 3399, 3206, 1611, 1568 cm; h nmr (dmso - d6) see tables 1 and s5; c nmr (dmso - d6) see tables 1 and s5; hresims m / z [m + h] 367.0081 (calcd for c18h12brn2o2, 367.0082). H and c nmr data were measured at 600.2 and 150.9 mhz, respectively . Congested areas of the spectrum; assignments were made on the basis of a band - selective h c ghmbc experiment . Two decimal places are shown to distinguish resonances of very similar chemical shifts, but that could be clearly resolved in the band - selective experiment . Dark brown solid; uv (meoh) max (log) 202 (3.44) and 268 (3.39); ir (film) 3390, 2924, 1587, 1471, 1438 cm; h nmr (dmso - d6) see tables 1 and s20; c nmr (dmso - d6) see tables 1 and s20; hresims m / z [m h] 444.9489 (calcd for c18h10brn2o5 s, 444.9499). The minimum inhibitory concentrations (mics) were determined for c. albicans and methicillin - resistant s. aureus as described by chen et al . Compounds 1 and 2 were evaluated for their cytotoxicity toward the panc1 human pancreatic cancer (atcc no . Crl1469) and nci / adr - res ovarian adenocarcinoma cell lines using a method previously described in the literature . The ir spectra were collected on a perkin - elmer spectrum 100 instrument with universal atr . Nmr data were collected on a jeol eca-600 spectrometer operating at 600.17 mhz for h, 150.9 mhz for c, and 60.8 mhz for n (instrument reference set to liquid nh3). The edited - ghsqc was optimized for 140 hz, and the long - range jh, c (ghmbc and band selective ghmbc) were optimized for 8 hz . Chemical shifts are referenced to solvent (dmso - d6 h observed at 2.50 ppm and c observed at 39.5 ppm). Chemical shifts for n were referenced to liquid nh3 with long - range jh, n optimized for 6 hz . The esims spectra were measured on a finnigan ltq mass spectrometer located at hboi . The hresims spectra were measured using a kratos ms50tc mass spectrometer at the university of california, riverside . Medium - pressure liquid chromatography was conducted on an isco - teledyne combi - flash rf4x system . Hplc was performed using a hitachi lachrom quaternary gradient hplc system equipped with diode array detector (l-7455) monitoring at 230 nm and elsd detection . Preparative fractionation was conducted using mass - directed collection on a waters autopurification system equipped with a waters 2535 quaternary gradient module, a waters 2998 pda detector, a waters 515 hplc pump, a waters sfo system fluidics organizer, a waters 2767 sample manager, and a waters 3100 mass detector operating in positive and negative ion esi mode . The sponge (hboi specimen number 22-x-00 - 4 - 010) was collected using the johnson - sea - link human - occupied submersible at a depth of 131 m off ocean cay, bahamas, approximately 20 nautical miles south of bimini (latitude: 25 24.21 n; longitude: 79 14.21 w). It is an unidentified species of asteropus (phylum porifera, class demospongiae, subclass heteroscleromorpha, order tetractinellida, suborder astrophorina, family ancorinidae). The sponge is most closely related to asteropus niger hajdu and van soest, 1992 . The sample was massive - spherical in shape with clusters of oscules distributed over the surface . Its ectosome and choanosome were black inside both in situ and in air; it is dark brown in etoh . While the spicule complement is similar to that described for a. niger, that species is characterized by two size categories of megasclere oxeas, one of which is less than 500 m long . The smaller category of megasclere oxeas in the material studied is rare, and the dimensions are much longer and thicker than those reported for a. niger . A taxonomic voucher specimen preserved in etoh is deposited in the harbor branch oceanographic museum (catalog number 003:01087). The frozen sponge asteropus (211 g) was diced and extracted exhaustively by macerating with etoh (3 200 ml) using a waring blender . The combined filtered extracts were concentrated by distillation under reduced pressure to yield 11.10 g of crude residue . The residue was partitioned between etoac (300 ml) and h2o (3 200 ml) to give after removal of solvent 1.44 g of etoac extract . The aqueous phase was further partitioned with n - butanol (3 200 ml) to afford, after removal of solvent, 7.11 and 1.94 g of aqueous and n - butanol extracts, respectively . The n - butanol extract was fractionated using a combi - flash rfx4 system equipped with a 15.5 g redi - sep rp-18 rf gold isco column operating at 200 psi with a flow rate of 30 ml / min . Fractions were eluted using a linear gradient of a [h2o / ch3cn (95:5 v / v + 0.1% tfa)] and b [ch3cn (+ 0.1% tfa)]; t = 0 min a: b (95:5 v / v), t = 23 min a: b 0:100 v / v, hold for 2 min . Further purification of a fraction eluting at 10 column volumes (approximately 4 min) (200 mg) was carried out on a waters autopure lcms mass - directed preparative purification system using a vydac 218tp protein and peptide c-18 preparative column (150 22 mm, 10 m particle size) eluted with a linear gradient of a [h2o / ch3cn (95:5 v / v + 0.1% tfa)] and b [ch3cn (+ 0.1% tfa)], t = 0 min a: b (95:5 v / v), t = 23 min a: b (0:100 v / v), hold for 2 min, and selecting mass collection at m / z 367 and 447 . This led to the isolation of 1 (44.1 mg, 1.9 10% of wet weight) and 2 (5.9 mg, 2.7 10% of wet weight). Dark blue solid; uv (meoh) max (log) 209 (3.82), 270 (3.89), and 295 (3.63); ir (film) 3399, 3206, 1611, 1568 cm; h nmr (dmso - d6) see tables 1 and s5; c nmr (dmso - d6) see tables 1 and s5; hresims m / z [m + h] 367.0081 (calcd for c18h12brn2o2, 367.0082). H and c nmr data were measured at 600.2 and 150.9 mhz, respectively . Congested areas of the spectrum; assignments were made on the basis of a band - selective h c ghmbc experiment . Two decimal places are shown to distinguish resonances of very similar chemical shifts, but that could be clearly resolved in the band - selective experiment . ; uv (meoh) max (log) 202 (3.44) and 268 (3.39); ir (film) 3390, 2924, 1587, 1471, 1438 cm; h nmr (dmso - d6) see tables 1 and s20; c nmr (dmso - d6) see tables 1 and s20; hresims m / z [m h] 444.9489 (calcd for c18h10brn2o5 s, 444.9499). The minimum inhibitory concentrations (mics) were determined for c. albicans and methicillin - resistant s. aureus as described by chen et al . Compounds 1 and 2 were evaluated for their cytotoxicity toward the panc1 human pancreatic cancer (atcc no . Crl1469) and nci / adr - res ovarian adenocarcinoma cell lines using a method previously described in the literature.
While the beneficial effects of exercise for physical and mental health are well recognized, average physical activity is below recommended levels, and it remains a challenge to support the adoption of active behaviours . Virtual reality environments are increasingly being used to encourage individuals to exercise more regularly, for the prevention of sedentary lifestyles among relatively healthy individuals, including, and as part of treatment, in those with mental health or neurological disorders . When exercising in virtual environments for the benefit of mental health, there is a need to consider the theoretical sense of presence . Sense of presence is defined as a perceived feeling of immersion when exposed to a virtual environment; it is perfected when one is completely unaware of their real surroundings [47]. Beyond subjective assessments to explore a sense of presence, there is increasing reliance on more objective physiological measurements, for example, heart rate monitoring . Only a few studies have investigated the neural responses that underlie the sense of presence perception . There has been a particular focus on the neural activation patterns that accompany and may regulate the sense of presence during the process of habituation to virtual environments [9, 10]. Recently, frontal brain regions (i.e., dorsolateral prefrontal cortex) were identified to form a key node for a sense of presence network (spn;). Participants who are highly engaged and most attentive during imagination and movement representation studies show strong spn activation . Electroencephalography (eeg) frequency analyses have also been used to explore the neural activation patterns associated with sense of presence perception . Alterations in alpha oscillations, particularly over the frontoparietal brain regions, have been associated with perceived sense of presence . Notwithstanding continuous debates on inconsistent findings, alpha and beta oscillations are also associated with mental state, for example, physical and psychological strain [10, 13]. It has been shown that sense of presence perception depends largely on the characteristics of the virtual environment and differs with screen size, duration of exposure, and the realism of the presentation [10, 1416]. Whether the addition of a real exercise (e.g., cycling on an ergometer) within an immersive virtual environment alters sense of presence perception, or the accompanying changes in neural activation patterns or mental state, is not known . In healthy participants, the beneficial effects of exercise on mental state and mental well - being are reflected by neurophysiological and behavioural adaptations [17, 18]. These effects of exercise on mental well - being are suggested to be largely dependent on the volume or dose of exercise [19, 20] and are also influenced by a preference bias towards self - paced (moderate - intensity) exercise . Studies of brain activity and the alterations in alpha and beta oscillations with exercise suggest the presence of a general model of cortical arousal (mca;). According to this, a reduction in alpha activity and an increase in beta activity reflect cortical arousal; and the reverse is associated with relaxed cortical states that may be recorded immediately after self - paced (moderate - intensity) exercise . The relationship between exercise - induced changes in mental state and neural activation patterns may also be described on the basis of the transient hypofrontality theory (tht;). Transient hypofrontality would suggest that the eeg changes that accompany exercise reflect redistribution of limited cortical resources away from less required brain regions (e.g., decreased activity in frontal brain regions) towards more required brain regions (e.g., increased activity in motor regions). Thus, based upon the dominant role of frontal brain regions in mental state, this exercise - induced redistribution is in favour of well - being (i.e., decreased activity in frontal brain regions as a refreshment of mental state capacity). Transient hypofrontality is particularly evident in response to self - paced (moderate - intensity) exercise, and similar responses are seen across a diverse range of participants including younger and older participants and those with and without a mental impairment [2426]. With this, combining traditional interpretations of alterations in eeg frequency bands (i.e., mca) with the latest understanding of cortical redistribution (i.e., tht) seems reasonable . It is not known whether these same neural responses occur during exercise or movement representation in a virtual environment, and this is important to establish if virtual environments are to be used to facilitate exercise as a therapy for mental well - being . Therefore, the objective of this study was to investigate the interactive effects of virtual environment exposure and exercise on physiological and perceptual responses in healthy adults . Specifically, we aimed to examine the influence of moderate - intensity exercise (i.e., self - paced cycling), compared with movement representation, within three levels of virtual environment exposure (a complex three - screen mode, a simple one - screen mode, and a no - screen control) on cortical neural oscillations (i.e., frontal alpha and beta activity), perceived sense of presence, and mental state (i.e., perceived physical state, motivational state, and psychological strain). Participants were eighteen healthy volunteers (7 females, 11 males) with no known history of neurological or musculoskeletal disorders (age: 28.78 5.19 years; height: 177.50 10.15 cm; weight: 75.44 13.23 kg). Participants considered themselves recreationally active with no particular experience of being exposed to virtual environments . This study was approved by the institutional human research ethics committee and was conducted in accordance with the declaration of helsinki . Participants attended a single experimental test session, following familiarisation with the cycle ergometer and the test environment on the same day . Experimental procedures were arranged in a randomized and controlled study design . After baseline measurements, exercise or no - exercise trials were conducted within three levels of virtual environment exposure . Each trial was 5 minutes in duration and was followed by posttrial assessments of heart rate, perceived sense of presence, and eeg as well as mental state (figure 1). An immersion square system (bonn - rhein - sieg university of applied sciences;) back projection (8 gb ram, amd radeon hd 5800 series) on three screens (260.0 cm width, 195.0 cm height) provided 4200 1050 pixel resolution (each 1400 1050 pixels). Screens were arranged in a square surrounding the participant's visual field (figure 2). Screen configuration allowed for three levels of virtual environment exposure: (1) one - screen mode (osm) with only the front screen switched on, (2) three - screen mode (tsm) with all three screens switched on, and (3) control with all screens switched off . During each trial, participants were seated on a fivis bicycle simulator (bonn - rhein - sieg university of applied sciences;). During both the osm and tsm trials, 3d content was identical and comprised an endless two - lane road (straight) in an urban setting . The 3d video display was synchronised with the actions of the bicycle, including steering (i.e., handlebar movement), velocity (i.e., pedal cadence and torque), and braking (i.e., backpedal or handbrake). With respect to dose [19, 20] and preference, exercise trials were performed at a self - paced moderate intensity that was defined by consistent heart rate monitoring and verbal instructions prior to each exercise trial (i.e., please pace your pedalling consistently to meet your perceived moderate intensity). During no - exercise trials, participants sat on the cycle ergometer without moving and the virtual bicycle (3d video) travelled the course at a fixed virtual speed of 22.0 km / h . Heart rate was monitored using a polar rcx5 portable heart rate monitor (polar electro oy, finland). Average heart rate (bpm) at rest and during the period immediately after each trial was used for analysis . Perceived sense of presence was assessed immediately after each trial using a simple verbal query / response - questionnaire [28, 29]. Participants were asked, without hesitating, how strongly do you feel connected to the virtual environment at this moment? Responses were anchored to an 11-step scale from 0 (not at all) to 10 (totally). The bodyfinder module is sensitive to short - term mood alterations and has been developed to determine current perceived physical state (peps) based on the subdomains of physical energy, physical fitness, physical flexibility, and physical health . This module has been used and validated in various biomedical settings, including exercise physiology and internal medicine studies . The feelfinder module comprises a shortened version of the ez - scale (eigenzustandsskala;). In comparison to other psychological adjective scales (e.g., poms) the ez - scale assesses motivational state (mot), in addition to the commonly assessed psychological strain (psych). This study used a 16-item ez - scale, comprising eight subdomains to generate assessments of mot (self - confidence, willingness to seek contact, social acceptance, and readiness for strain) and psych (relaxation, positive mood, calmness, and recovery). For this study, the moodmeter was configured with 32 adjectives (16 peps, 8 mot, and 8 psych) that were stored and presented to participants on a handheld axim x50 pocket pc (dell, usa) in a quasi - random sequence . Upon the presentation of each adjective, participants were asked to indicate how well that adjective described their current physical or mental state by selecting one of six options from 0 (not at all) to 5 (totally). The response time for each adjective was limited to five seconds so as to discourage rational deliberation, and this time was shown with a progress bar at the bottom of the screen . With this, the moodmeter was specifically designed to detect short - term alterations that are of particular relevance for exercise - related research . On each occasion, completion of the mood assessment (all 32 adjectives) took less than two minutes . For further details on the development and operation of the moodmeter, please refer to kleinert . To record cortical neural oscillations, brainvision recorder 1.20.0701 together with a portable acticap system was used (brainproducts gmbh, germany). A permeable - to - air eeg - cap that is adapted to individual head size the eeg - cap consisted of 64 ag / agcl electrodes, arranged in the international 10:20 system: fp1, fp2, af7, af3, af4, af8, f7, f5, f3, f1, fz, f2, f4, f6, f8, ft9, ft7, fc5, fc3, fc1, fc2, fc4, fc6, ft8, ft10, t7, c5, c3, c1, cz, c2, c4, c6, t8, tp9, tp7, cp5, cp3, cp1, cpz, cp2, cp4, cp6, tp8, tp10, p7, p5, p3, p1, pz, p2, p4, p6, p8, po7, po3, poz, po4, po8, o1, oz, o2, and po10 . Fcz (reference) and afz (ground) were added and po9 served as horizontal electrooculogram (eog) to detect eye movements . Supervisc electrode gel (easycap gmbh, germany) was added to each electrode to optimize conductivity . Distances between electrodes were> 25 mm to avoid bridging (though not measured, perspiration was not noticeably profuse in any participant). A 60-second resting eeg was recorded (sampling rate of 500 hz) with eyes closed, while seated on the cycle ergometer before (rest) and after each trial . Analogue eeg data were amplified and digitally converted for analyses using brainvision analyser 2.1.0.327 (brainproducts gmbh, germany). Low and high cutoff filter frequencies ranged between 7.5 and 45.0 hz (time constant 0.02 seconds, 48 db / octave). Based on the eog, gratton's standard ocular correction was performed to reduce eye - moving artifacts . After segmentation and an automatic artifact rejection (gradient <35 v, min / max amplitudes 100 v), a minimum of twelve 4-second segments remained for fast fourier transformation (spectral analysis: resolution at 0.24 hz, hanning window of 10%). Averaged segments were pooled into frontal (fp1, fp2, af7, af3, af4, af8, f7, f5, f3, f1, fz, f2, f4, f6, f8, ft9, ft7, fc5, fc3, fc1, fc2, fc4, fc6, ft8, and ft10), central (c5, c3, c1, cz, c2, c4, c6, cp5, cp3, cp1, cpz, cp2, cp4, and cp6), parietal (p7, p5, p3, p1, pz, p2, p4, p6, and p8), and occipital (po7, po3, poz, po4, po8, o1, oz, o2, and po10) electrode sites before exporting prominent frequency bands alpha (7.512.5 hz) and beta (12.535.0 hz) as mean activity in v . For statistical comparisons, frontal, central, parietal, and occipital pools were each referenced to a global pool (export of all respective other electrode sites). All statistical analyses were performed using the software statistica 7.1 (statsoft, tulsa, usa). Repeated - measures analysis of variance (anova) was used to detect condition effects and interactions between exercise (exercise, no - exercise) and virtual environment exposure (control, osm, and tsm) for heart rate, cortical neural oscillations (eeg: alpha, beta), and mental state (peps, mot, and psych). Fisher's least significant difference (lsd) was applied post hoc where interactions and main effects were identified . Friedman's anova followed by wilcoxon paired samples test was used to identify changes in the perceived sense of presence . Possible correlations between cortical neural oscillations (eeg: alpha, beta), mental state (peps, mot, and psych), sense of presence, and heart rate in both no - exercise and exercise trials were determined using pearson's correlation coefficient . Data (n = 18) in figures are presented as mean confidence interval (0.95) and as mean standard error of mean in the text and tables . Heart rates showed significant increases from rest to no - exercise and exercise trials (f(3,51) = 39.63, p <0.05), and these changes were consistent across the three levels of virtual environment exposure (table 1). Ancova revealed no significant effect of gender on heart rate (= 0.58, f(7,9) = 0.92, p> 0.05). Sense of presence increased from no - exercise to exercise trials, with increasing levels of virtual environment exposure from control to osm and tsm (chi(18,5) = 83.98, p <0.05; table 2). Ancova revealed no significant gender effect for perceived sense of presence (= 0.52, f(6,10) = 1.52, p> 0.05). There were significant main effects where mental state differed between exercise and no - exercise trials and between the different levels of virtual environment exposure (f(4,68) = 3.37, p <0.05). Post hoc analysis revealed increases in peps and psych from control to osm and tsm during the exercise trial, whereas mot increased from control to osm and tsm in the no - exercise trial (table 3). There was no significant gender effect (ancova: = 0.52, f(15,1) = 1.22, p> 0.05). Frontal alpha activity showed significant differences between no - exercise and exercise trials across the various virtual environment exposures (f(2,34) = 5.90, p <0.05). Post hoc analysis revealed frontal alpha activity increased from control to tsm during the no - exercise condition, whereas the respective global alpha activity did not change with levels in virtual environment exposure . The reverse was shown during the exercise trial where the respective global alpha activity increased from control to tsm, whereas frontal alpha activity did not change significantly with levels in virtual environment exposure (figure 3). Ancova revealed no significant effect of gender for alpha activity (= 0.12, f(12,4) = 2.45, p> 0.05). Central (f(2,34) = 1.60, p> 0.05), parietal (f(2,34) = 2.16, p> 0.05), and occipital (f(2,34) = 2.86, p> 0.05) alpha activity revealed no significant changes compared to their respective global alpha activity . Frontal beta activity showed significant differences between no - exercise and exercise trials during the different levels of virtual environment exposure (f(2,34) = 5.11, p <0.05). Post hoc analysis revealed that, during the no - exercise trial, frontal beta activity increased from control to tsm by trend, whereas the respective global beta activity did not change significantly . These findings were reversed during the exercise trial, where the respective global beta activity increased from control to tsm by trend, and frontal beta activity did not change significantly (figure 4). There was no significant gender effect (ancova: = 0.15, f(12,4) = 1.92, p> 0.05). Central (f(2,34) = 1.59, p> 0.05), parietal (f(2,34) = 0.78, p> 0.05), and occipital (f(2,34) = 1.55, p> 0.05) beta activity revealed no significant changes compared to their respective global beta activity . For the no - exercise trial, positive correlations could be obtained between cortical neural oscillations and mental state as well as between cortical neural oscillations and heart rate (table 4). For the exercise trial, a positive correlation could be obtained between mental state and heart rate (table 5). This study aimed to investigate the interactive effects of virtual environment exposure and exercise on neurophysiological and perceptual responses in healthy adults . We verified that exposure to increasing levels of virtual environment exposure led to increases in perceived sense of presence, and a key finding was that this change in perception was further enhanced by the inclusion of exercise . Measures of mental state responded to increasing levels of virtual environment exposure, and the addition of exercise led to increases in physical state (peps) and psychological strain (psych) and a reduction in motivational state (mot). The addition of exercise to the virtual environment had a significant influence on the eeg responses, with a shift from strong frontal alpha and beta activity responses during the no - exercise condition to strong global alpha and beta responses when exercise was added to the virtual environment exposure . Perceived sense of presence increased with increasing levels of virtual environment exposure, from control to one - screen mode (osm) and three - screen mode (tsm), and in each condition sense of presence was further amplified with the addition of exercise . Though not correlated, these changes in sense of presence were underlined by changes in heart rate . The increases in heart rate during the no - exercise condition are consistent with previous studies, and collectively these findings support the use of heart rate monitoring as an objective physiological measure that may reflect sense of presence during exposure to virtual environments . There was a significant increase in peps and psych during exercise in the virtual environments compared with the no - exercise condition . The increase in peps (perceived physical state) reflects the increased heart rate and effort during exercise, and such a response is well documented in the literature [1720]. The strong psych (psychological strain) response to exercise in the virtual environments appears to be at odds with evidence of the beneficial and calming effects of exercise, although exercise alone in the present study (during control) did in fact lead to a slight (not significant) reduction in psych . The increase in psych during exercise in the virtual environment was accompanied by a reduction in motivation (mot). Though surprising, it seems reasonable that exercising in virtual environments may lead to less motivation, relative to an increasing virtual environment exposure (i.e., from control to osm to tsm) that is coexistent with an increase in psych (e.g., increased discomfort). Also, it has previously been suggested that virtual reality increases the psychological demands of an environment, and these demands increase with increased duration of exposure . Our findings suggest that the addition of exercise increases the demands of a virtual environment and this results in a pronounced psychological strain . In contrast, it appears that less demanding virtual environments (i.e., without exercise) result in a stronger motivational state, thus, in line with the above suggested less motivation while exercising in the virtual environment . The increase in global beta activity after exercise in the virtual environment is consistent with the hypothesis of a generalised model of cortical arousal (mca). According to the mca, the increase in beta activity is consistent with cortical arousal and has previously been associated with the incidence of cybersickness, akin to motion sickness, and this corresponds with the elevated psychological strain and the decreased motivation during this trial . Rather than a corresponding decrease in alpha activity, as would be expected according to the mca, global alpha activity also increased during exercise in the virtual environment . This is suggestive of a relaxed cortical state, which is consistent with the low motivational state in the current trial, but at odds with the elevated psychological strain . Assessment of cortical current density allows for the localisation of neural changes to specific regions of the brain . The transient hypofrontality theory (tht) reflects redistribution of cortical activity away from the frontal regions [23, 25], thus allowing for greater cortical resources to maintain mental state . While there were significant increases in frontal cortical neural oscillations in the virtual environment during the no - exercise condition, the addition of exercise led to reductions in both alpha and beta frontal activity, relative to global activity . This is consistent with the tht and presumably is driven largely by the increase in physical and psychological strain . From a sense of presence perspective, the neural activation patterns are in line with previous neuroimaging studies, suggesting that frontal brain regions form a key sense of presence node . Though documented in a nonexercise fmri study (i.e., rollercoaster scenarios), baumgartner et al . Suggest lesser cortical activation in the dorsolateral prefrontal cortex accompanied by a stronger sense of presence perception . In addition, it is well accepted that frontoparietal brain regions are strongly involved in movement control . Even the imagination of movements or motor representation is likely to shape the sense of presence perception in virtual environments . With this and previous evidence that postural changes enhance sense of presence in virtual environments, it seems reasonable that the inclusion of real exercise (i.e., self - paced cycling on an ergometer) similarly fosters and possibly intensifies sense of presence perception compared to motor representation (i.e., automatic drive). This is underlined by increased sense of presence perception from no - exercise to exercise trials as well as from control to osm and tsm . Despite plausible findings, we are aware that the present study is limited by a rather small number of participants . Additionally, lack of data pertaining to exercise intensity (e.g., watt / rpm, vo2) makes it difficult to verify the standardisation of exercise conditions; however, the heart rate responded as would be expected to further support the moderate exercise definition, which is mainly owing to available infrastructure . The primary aim of this study was to examine cortical neural oscillations and related alterations in mental state in response to exercise in immersive virtual environments compared to movement representation . Changes in psychological strain and physical state were generally mirrored by neural activation patterns of frontal and global alpha and beta activity . Furthermore, these changes are likely to indicate, based on the model of cortical arousal and, in particular, the transient hypofrontality theory, that exercise augments the demands of virtual environment exposures and this possibly contributes to enhanced sense of presence.
Hypomethylation of the imprinted h19/igf2 locus in 11p15 and matupd7 are two major epigenetic etiologies in srs . Matupd7 was first reported by spence et al in a cystic fibrosis patient with srs like phenotype (short stature and slight asymmetry). After this paper, other cases affected by pre and postnatal growth retardation but not srs phenotype with matupd7 were published[46]. The incidence of srs is approximately 1/3000 and more than 60 srs patients with matupd7 have been reported till the end of 2002[216] and matupd7 has been found in approximately 5 - 10% of srs patients with unexplained etiology . After description of matupd7 srs cases, both arms of chromosome 7 were searched for candidate genes . Although 7p aberrations of four patients with a srs phenotype have been described, no pathogenic mutations have been identified in several studies focusing on the search for point mutations in 7p - encoded genes so far that might be functionally related to the srs phenotype . On the other hand, two papers published by hannula et al and reboul et al described srs cases with a segmental maternal upd 7q showing the importance of human imprinted genes in 7q for human growth and srs . However, screening studies on mutations in the imprinted genes and transcripts on 7q chromosome region failed to detect any pathogenic variants[2024]. Ring chromosome 15, deletion of 15q, deletion of 8q11e13, translocations associated with a breakpoint in 17q25, deletion of csh1, duplications of 11p15, and epimutations of the imprinting center region 1 (icr1) on 11p15 have been reported as the other reasons of srs . Loss of methylation of icr1 gene on 11p15, resulting in a down - regulation of igf - ii and h19 genes which were found in 38 - 64% of cases with srs, have been reported as the most frequent reason in the etiology of srs as mentioned before[32, 33]. Hypomethylation of matupd7 has not been studied in turkish population up to date so far . Therefore, to ascertain the frequency of matupd7 in the cause of srs in turkish population, we decided to screen matupd7 to perform genotype and phenotype correlation . A total of 13 srs patients aged between 2 - 9 years, from two genetic subdivisions of the pediatric departments of two university medical school hospitals / turkey were included in this study . Diagnosis of srs was established in the patients according to the previously described in literature . Chromosomal analysis and subtelomeric fluorescent in situ hybridization (fish) (telvysion probes) (vysis / abbott) were performed to identify microscopic and submicroscopic imbalances on all patients . The study was approved by the faculty ethics committee of the university . Dna preparation: dna was extracted from 2 ml venous blood taken from all patients and their parents using (dna isolation blood mini kit, invitek, germany) standard procedures . Genotyping for matupd7 in 13 srs patients was performed on all patients with different chromosome tetra and dinucleotide repeat microsatellite markers (d7s460, d7s821, d7s1808, d7s1818, d7s1804, d7s2195 and d7s2446) by polymerase chain reaction (pcr) (abi pcr system 9700). Pcrs were performed in 25l reaction containing 50ng dna, 1xdynazyme ii buffer, 1.5 mm mgcl2, 300 um of each dntp, 0.3 um of each primer, 1% dimethyl sulfoxide, and 1.25 u taq dna polymerase (fermantase, leon - rot, germany). Amplification was performed with an initial denaturation of 5 minutes at 94c, followed by 30 to 35 cycles each of 30 s at 94c, 75 s at 55c, and 60 s at 72c with a final extension at 72c for 10 minutes . Cases of matupd7 were genotyped for additional chromosome by 7 microsatellite markers, to verify matupd7 by pcr (fig . Examples of str genotyping showing maternal uniparental heterodisomy 7 in srs patient . A: allelic patterns of the marker d7s821 . Chromosomal analysis and subtelomeric fluorescent in situ hybridization (fish) analysis were found to be normal in all patients . Clinical features of 13 srs patients including mat upd7 patient are given in table 1 . There were no significant differences between clinical features of mat upd7 case and other srs cases except congenital heart defects . Echocardiographic studies revealed ventricular septal defect (vsd), patent ductus arteriosus (pda), and patent foramen ovale (pfo) in the patient with matupd7 . Clinical features of silver - russell syndrome patients iugr: intrauterin growth retardation / pngr: postnatal growth retardation / chd: congenital heart defect the matupd7 case was a 2-year - old girl, the first child of non - consanguineous parents, born 5 weeks before term (33 weeks). There was a history of fetal karyotyping because of intrauterine growth retardation which was found to be normal . On physical examination, her weight was 7500 gr (<3 percentile), height 73 cm (3 - 10 percentile), and head circumference 48.5 cm (50 - 75 percentile). She had relative macrocephaly, triangular face, broad prominent forehead, low set ears, downslanted corners of the mouth, micrognathia, clinodactyly of the 5th digit and bracydactyly as dysmorphological features (fig . Facial features including relative macrocephaly, triangular face, broad prominent forehead, down slanted corners of the mouth, micrognathia and hemihypertrophy of the left side of the body are seen . Developmental milestones were as follows: sitting without support at age of 8 months, walking alone and speaking several words at age of 14 months . It has been reported that matupd7 cases cover approximately 10% of all srs patients[8, 9]. In the study presented matupd7 was detected in 7.6% of srs patients, which is slightly lower than that in previous reports and this could be caused by the low number of srs patients in our study . Srs has variable characteristics, however it is easy to recognize with typical symptoms, but diagnosis may be difficult in milder cases . Our matupd7 patient presented with mild srs characteristics as the other matupd7 cases previously described (table 2). Matupd7 has been considered to be a subgroup among srs patients with mild dysmorphic features, severe feeding difficulties and delayed speech . Our patients represented the spectrum of dysmorphic features and developmental difficulties seen typically in patients with srs (table 1). The dysmorphic features in matupd7 patients have been reported to be mild in both srs[8, 16, 34] and patients without srs, and these patients have also been defined as having intrauterine growth retardation (iugr) and postnatal growth retardation (pngr) with slight dysmorphic features . It is often difficult to establish a diagnosis of a child with iugr, growth failure and dysmorphic features . Although growth retardation of unknown cause has been associated with upd, hannula et al showed that it is not worthwhile to screen those patients for matupd7 unless they show characteristic features of srs and severe growth retardation . The matupd7 patient described here shows the fulfilling of diagnostic criteria for srs and supports the suggestion that matupd7 is not a general cause for growth retardation . Interestingly matupd7 was also reported in three patients with cystic fibrosis (mim 219700) and unexpectedly severe short stature[3, 5, 12]. Among these three cases one had growth hormone (gh) deficiency . Screening for matupd7 is therefore advisable if abnormally short stature occurs with cystic fibrosis or other recessive disorders mapped to chromosome . Although matupd and icr1 hypomethylation patients have overlapping features, asymmetry, fifth finger clinodactyly and congenital anomalies are more commonly seen in patient with icr1 hypomethylation, whereas learning difficulties and referral for speech therapy are more likely with matupd7 . Our matupd7 patient had mild dysmorphic features of srs that was similar to the patients previously described[8, 9, 11]. Peg1/mest [37, 38] and cop35 both at 7q32, and grb1036,37 at 7p11.2-p12,3 are the imprinted genes in srs that have been identified so far . Paternal mutation or a deletion in the related imprinting gene or genes presents with similar symptoms as in matupd7 patients . On the other hand, fetal growth regulator genes egfr (epidermal growth factor receptor), igfbp1, and igfbp3 (insulin - like growth factor binding protein 1 and 3) are the other candidate imprinting genes that were thought to be responsible in srs etiology . Congenital heart defects observed in this patient which has not been reported in matupd7 cases could be the result of one of these or other imprinted gene dysfunctions . Imprinting genes on chromosome 7 may be responsible for this additional anomaly or there may be coincidence . Methylation analysis indicated that in maternal but not paternal alleles, cpg islands are completely methylated in the promotor region, however, a recent study showed a de novo deletion in 7q32 affecting the paternal imprinted mest / peg1 gene copy . It is generally diffucult to exclude mosaicism in an infant with upd in srs but it is identified in a patient related with relatively mild srs - like phenotype . In addition, iugr and prenatal abnormalities are associated with confined placental mocaicism (cpm). Cpm is occasionally found with upd, where after a meiotic error leads to trisomy, followed by trisomic rescue leads to a upd cell line in the fetus . Therefore, in cases of upd originating from cpm, it is difficult to ascertain whether the growth retardation is caused by upd in the fetus, or the upd cell line or trisomic cell line in the placenta . We coincidently ruled out cpm, by observing the normal karyotype after chorionic villus sampling with the indication of advanced maternal age . One of the limitations in our study is the lack of evaluation of the other responsible etiologies, such as hypomethylation of the imprinted h19/igf2 locus, submicroscopic genomic alterations and mutations in the candidate genes . Matupd7 was detected in 7.6% of srs patients firstly in turkish population, which is slightly lower than that in previous reports . It is also worth to emphasize that congenital cardiopathy of the matupd7 case has not been described in the literature to date . We suggest testing all patients with intrauterine and severe postnatal growth retardation with only slight signs of srs.
Neuroblastoma, a pediatric malignancy of the developing sympathetic nervous system, is a multifaceted disease with biological and clinical courses ranging from relentless progression to spontaneous regression or differentiation into benign ganglioneuroma . Given these different phenotypes, therapeutic regimens vary between wait - and - see approaches to the most intense multimodal treatment . Accurate prediction of the natural clinical course of each individual patient at the time of diagnosis is therefore an essential prerequisite for therapeutic decision - making . Clinical variables such as stage of the disease and age of the patient at diagnosis are well established predictors of neuroblastoma outcome . In addition, non - random cytogenetic aberrations have been shown to be associated with clinical courses in neuroblastoma and are increasingly used in risk stratification systems (reviewed in [1 - 3]). Whereas amplification of the oncogene mycn and several other genomic alterations, such as loss of the chromosomal regions 1p and 11q or gain of 17q, have been shown to be strong markers of poor outcome, hyper - diploidy of the tumor cells is associated with a favorable clinical phenotype . However, whereas current risk estimation systems for neuroblastoma mostly succeed in discriminating patients with divergent outcomes, further improvements are required to prevent fatal events in low - risk and intermediate - risk groups and to avoid unnecessary cytotoxic treatment of patients in whom spontaneous regression will occur . The advent of microarray - based comparative genomic hybridization (array - cgh) has facilitated the analysis of chromosomal alterations in the cancer genome, providing pangenomic alteration profiles with exceptional spatial resolution in a single experiment . Initial array - cgh studies of primary neuroblastomas confirmed the clinical significance of known copy number variations and narrowed down breakpoint regions of non - random chromosome aberrations . In a recent survey, caren et al . Investigated 165 primary neuroblastomas using affymetrix 250k single nucleotide polymorphism arrays and compared the survival of patient subgroups defined by genomic alterations . Patients with only numerical chromosomal aberrations and no other alteration had a favorable long - term outcome . In contrast, the survival of patients characterized by mycn amplification, loss of 11q or gain of 17q was considerably worse, whereas no death or disease was observed in patients with tumors harboring segmental chromosome alterations other than those previously mentioned . These findings support results from previous studies indicating that a limited number of predictive genomic alterations are sufficient for risk assessment of neuroblastoma patients (reviewed in)., however, indicated that global genomic profiles may add significant prognostic information to current neuroblastoma risk estimation . In this study, the prognostic significance of overall genomic alterations was investigated in a cohort of 493 primary neuroblastomas by bacterial artificial chromosome array - cgh . Whereas patients with tumors showing only numerical chromosome aberrations had an excellent survival, those with tumors harboring segmental genomic alterations showed a high risk of relapse and a poor outcome . Amplification of mycn was confirmed to be a strong predictor of adverse outcome, but other single genomic alterations, such as loss of 11q or gain of 17q, were overridden by the presence of any kind of segmental alterations in multivariate analyses . Another significant difference between these two studies was noticed in the fraction of tumors with only numerical chromosome alterations . In the work of janoueix - lerosey this subgroup comprised 47% of the tumors, whereas it accounted for 28% of the cases in the study of caren et al . . Similar to the latter findings, this subgroup constituted 21% of the cases in a preliminary analysis of our array - cgh data . These differences might in part be attributed to distinct compositions of the cohorts under investigation . However, they may also result from the lower spatial resolution of the microarrays used in the study of janoueix - lerosey et al . Than in the other surveys, which might have resulted in the detection of a smaller fraction of tumors with small gains or deletions and in the classification of fewer patients into subgroups with segmental aberrations . Taken together, although the results of these two comprehensive studies are promising with respect to prognostic classification of neuroblastoma using array - cgh, the clinical significance of global genomic alterations needs to be further evaluated in independent studies and compared with current risk estimation strategies . An inherent disadvantage of array - cgh analysis is its propensity to disregard low - level copy number losses or gains in samples with a high proportion of contaminating stromal cells . This potential bias has been taken into account by janoueix - lerosey et al . By analyzing only samples with a tumor content of at least 60%, whereas the tumor content was not specified in the study of caren et al . . This discrepancy in the experimental set - up may have resulted in a higher fraction of flat genomic profiles (that is, with no alterations) in the latter study (19%) as compared with the former study (4%). This suggestion is supported by the finding of only 2% flat genomic profiles in another study in which a tumor content of 60% had been used for sample selection . Because of the rare occurrence of neuroblastomas without any chromosomal alterations, the clinical outcome of these patients has so far remained elusive . Nevertheless, the routine application of array - cgh in clinical practice might be considerably limited by the issue of contaminating stromal cells, because defined thresholds of tumor content will a priori exclude a substantial fraction of samples from the analysis . In addition, genomic heterogeneity within a single tumor might be missed by array - cgh analysis . Although the frequency and the clinical consequences of genomic heterogeneity in neuroblastoma need to be clarified, it might be advisable to complement array - cgh analyses of neuroblastoma samples with methods for detecting chromosomal aberrations on the single cell level, such as fluorescence in situ hybridization, to evaluate the concordance of the results and to validate the clinical implications in large patient cohorts . As an alternative to the overall genomic pattern as a prognostic marker, several reports have provided compelling evidence that specific gene - expression patterns can predict the natural courses of neuroblastoma patients with unprecedented accuracy [11 - 15]. These studies have shown that gene - expression - based classifiers can distinguish patients with contrasting clinical courses in almost all prognostic subgroups, including those defined by prognostic genomic makers such as mycn amplification or loss of 11q . A systematic comparison of global genomic and transcriptomic classification results is still lacking, however . The routine application of expression - based prognostic markers in clinical practice might be limited by the instability of mrna in comparison with dna, which will require strict adherence to elaborated standard operating procedures in the processing of tumor samples . In addition, similar to array - cgh approaches, classification results of gene - expression - based predictors might be influenced by the relative amounts of stromal cells in the samples . In contrast to classifications based on genomic alterations, however, the prognostic significance of gene - expression profiles might be conferred by the stromal cells themselves, as has been described in other cancer entities, such as lymphoma or breast cancer . Re - evaluation of the gene functions from existing gene - expression classifiers and validation of the predictive accuracy in neuroblastoma cohorts with low tumor contents will reveal the contribution of non - tumorous cells to the prognostic validity of gene - expression - based classifiers in neuroblastoma . Because of the strong association of numerical and segmental cytogenetic alterations with patient outcome, it has been suggested that neuroblastoma comprises two distinct clinico - genetic classes . The first type corresponds to patients with favorable outcome and is characterized by mitotic dysfunction leading to whole chromosome gains or losses, whereas the second type corresponds to aggressive disease and is characterized by defects in maintaining genomic stability leading to segmental chromosome alterations . Given the prevalence of mycn amplification and loss of 11q in unfavorable neuroblastoma, and the inverse correlation between these aberrations in high - risk neuroblastoma, it has been furthermore hypothesized that the natural behavior of high - risk tumors is mainly conferred by these two aberrations . In the work of caren, this suggestion was substantiated by the finding that patients with mycn amplification and those with loss of 11q differed significantly in both their age at diagnosis and their median survival time . However, whereas the influence of mycn amplification on aggressive growth in neuroblastoma has been mostly proven, the effect of 11q loss on neuroblastoma biology is less clear . In a recent integrative genomics analysis of primary neuroblastoma, it was demonstrated that tumors with loss of 11q make up two distinct biological subgroups that differ in their clinical phenotype as well as in their gene - expression patterns . These results suggest that 11q loss is not a primary determinant of neuroblastoma tumor behavior, indicating that the biology of neuroblastoma is more complex than the association of genomic alterations with patient outcome might suggest . We expect that the emerging application of next - generation sequencing will unravel novel genomic alterations that contribute to the programming of the various neuroblastoma phenotypes, which will lead to a refined molecular classification of this malignancy . The prognostic significance of specific single genomic markers is well established in neuroblastoma, and has led to their implementation in current risk assessment . Recent studies suggested that overall genomic profiles may further improve neuroblastoma risk estimation . Before routine use in clinical practice, the prognostic impact of global genomic alterations needs to be validated prospectively and compared with current stratification systems . In addition, it needs to be evaluated whether analysis of overall genomic profiles, gene - expression - based classifiers, or the combination of both will contribute most to an improved risk estimation of children with neuroblastoma . In any case, such analysis will require elaborate standard operating procedures to avoid technical pitfalls and defined interpretation guidelines to ensure reliable treatment stratification of each individual patient in future clinical trials . This work was supported by grants from the bundesministerium fr bildung und forschung (bmbf) through the national genome research network plus (ngfnplus, grant 01gs0895) and the frdergesellschaft kinderkrebs - neuroblastom - forschung e.v.
Teeth are shown to be distinctive organs made of the most persistent mineralized tissues in the human body . Teeth are known to be resistant to mechanical, chemical, physical, and thermal types of devastations . Therefore, teeth play a vital role in identification of skeletal remains, chiefly in cases where there is a poor preservation of skeletal remains, the identification is not possible by standard methods.1 - 4 dental profile comprises a group of specific individual characteristics related to the teeth and surrounding tissues . These characteristics help in the estimation of age, sex, race, socio - economic status, personal habits, oral and systemic health, occupation and dietary status of the person . The variations in tooth form are a common occurrence in permanent dentition, and these variations have an ethnic, forensic, and anthropological significance.1,3 - 6 the study of tooth form is achieved by measurements and out of the two proportions - widths and length, the former is considered more important . Any measurement on teeth unaccompanied by age, race, and sex must be treated with great reserve . Based on the tooth crown metric traits, tooth morphology is studied from an interdisciplinary perspective (biology, anthropology, dentistry, paleopathology, archeology, forensic science) because teeth can be used in the assessment of biological relationships between population . This is can achieved by comparatively analyzing past and present human groups in an attempt to explain the historical, cultural, and biological macro and micro - evolutionary processes that lead to an understanding of the origin, formation, contacts, displacements, migrations pathways, and isolates that have led to the populating of the planet and ethnic variation of humanity.7 in general, population have been grouped as microdontic, mesodontic, and megadontic . Hence, measurements of dental crown size, provide greater objectivity and provide the most comprehensive and discriminatory description of human dentitions . But metric variations of the human dentition have not been utilized to their full possibility by anthropologists concerned with patterns of human biological variation in indian population.6,8,9 hence, this study forms part of a larger investigation aimed at using dental crown features - mesiodistal (md) and buccolingual crown dimensions between four ethnic groups, to develop a probabilistic model to distinguish individuals from specific human population, particularly for forensic purposes . Highlighting patterns of tooth size between these groups and consider the findings in relation to genetic and environmental influences . The study samples consisted of 400 individuals from four different ethnic groups including hindu, islam, christian, and iranians . Dental casts were obtained from all the participants using alginate impressions that were poured up with high - quality dental stone . Measurements were obtained from all the permanent maxillary and mandibular central incisors, lateral incisors, canines, first and second premolars, first and second molars using digital vernier caliper (mitutoyo corp - japan) (figure 1). All the third molars were excluded because impressions were taken before eruption of these teeth and the teeth that were not fully erupted, had carious lesions or restorations, or were crowded and/or exhibited any evidence of tooth wear or model damage were excluded from this study . Digital vernier caliper (mitutoyo - japan corp .) Used to measure buccolingual and mesiodistal tooth dimensions . (2005) as being the maximum distance between the mesial and distal proximal surfaces of the tooth crown (figure 2). The maximum labiolingual or buccolingual (bl) crown dimension was defined as the greatest distance between buccal and lingual surfaces of the crown perpendicular to and bisecting the line defining the md dimension (brook et al ., 1999; each tooth was measured on two separate occasions, and the mean value of the measurements was used . Different recording sheets were used on each occasion to ensure no access to the previous measurements . If there was a discrepancy> 0.4 mm between the recordings, the measurements were discarded . The mean values of the four groups were compared pair - wise using the spss statistical software package for analysis of variance . Reliability testing across the four ethnic groups showed similar results, indicating that each set of data was reliable to 0.1 mm and that valid comparisons between the groups could be made . The mean md dimensions of all the maxillary and mandibular teeth together with descriptive statistics are presented in tables 1 and 2 and also buccolingual dimensions of all the teeth are presented in tables 3 and 4 . Comparison of mean values of mesiodistal tooth dimensions of maxillary teeth between four different ethnic groups . Comparison of mean values of mesiodistal tooth dimensions of mandibular teeth between four different ethnic groups . Comparison of mean values of buccolingual tooth dimensions of maxillary teeth between four different ethnic groups . Comparison of mean values of buccolingual tooth dimensions of mandibular teeth between four different ethnic groups . The combined md and buccolingual crown dimensions for the christian sample were largest overall compared with the other three groups while those of the iranians sample were the smallest . There was also a great range of dimensions in all the groups and the difference observed between these groups was seen to be statistically significant . Comparison of coefficients of variation between the first and second teeth of each tooth type, e.g., upper central incisor versus upper lateral incisor, showed the later forming teeth usually demonstrated greater variation . Teeth can provide evidence about the nature and extent of diversity between human population and variations in dental crown size have been reported between different population.1,2 numerous factors can contribute to variation in tooth size, and these may be described broadly as genetic, epigenetic, and environmental influences.3 - 5 previous studies have confirmed the presence of sexual dimorphism within the human dentition8 - 10 and examples of ethnic differences and geographic variability in tooth size have been documented.2 in the present study, we found significant differences in tooth size between the four ethnic groups studied, with christians having generally larger md crown dimensions . The variations in crown dimensions observed in the four groups are likely to reflect genetic and environmental differences between this group and the other three considered here . A synthesis of data on dental dimensions from different population worldwide has indicated that western eurasian population tend to have the smallest teeth, with indigenous australians, melanesians, micronesians, sub - saharan africans, and native americans tending to have large teeth . East and southeast asian population were found to be intermediate in tooth size between these groups.11 the data presented here for the four modern population match this pattern . Hanihara and ishida have suggested that the distribution of tooth sizes observed in their study may be due to the impact of agriculture on the operation of natural selection on tooth size, with the use of agriculture reducing the effects of natural selection.11 this hypothesis is not supported by the data for the romano - british population, which showed smaller md dimensions than were observed in any of the modern population . If the smaller tooth size in western eurasian population was due to a longer history of agriculture in these population, then it would be expected that the romano - british population would have larger teeth than both the modern british and north american population . Instead, it is possible that genetic differences may be the contributing factor to the differences observed.11 - 13 we propose that the systematically varied md and buccolingual tooth width seen in these population groups is associated with specific genetic and epigenetic factors . Although only young individuals were included in this study, it is possible that a limited amount of tooth wear may have occurred even in these young individuals . Hillson identified a series of factors affecting tooth wear.14 these include masticator forces, non - chewing parafunctional activities, use of teeth as tools, and the nature of the diet . A tough fibrous diet requiring prolonged mastication, and the abrasivity of food consumed, could potentially contribute to tooth wear, as seen in older individuals.14 - 20 patterns can also be detected within the dentition between the four population . Although the christian population has the largest md dimensions for most of the dentition, there are some exceptions to this trend . These included the maxillary central incisor, mandibular central and lateral incisors and mandibular canine, which are largest in the muslim population, and the maxillary first and second molar, which are largest in the hindu population . The extent of the differences in tooth dimensions varied from tooth to tooth, as shown in tables 1 - 4 . The overall pattern is seen to follow the morphogenetic field concept as recently revised by townsend et al . With later - forming teeth in each tooth type being smaller and more variable.3,4,8,10 the values of coefficients of variation (tables 1 - 4) also showed that these later - forming teeth tended to be more variable in md and buccolingual dimensions . Hence, we propose that a there might be a strong genetic contribution to variation in tooth size but environmental factors may also play a role . For example, low birth weight has been linked to a reduction in the md width of deciduous teeth.21,22 alvesalo has shown that there is sexual dimorphism displayed in the dentition, with males tending to have larger teeth than females, reflecting x chromosome linkage with the y chromosome also having an impact . For example, both 47, xxy males and 47, xyy males have larger teeth than 46, xy males.8 - 10 by using a standardized methodology, significant differences in md and buccolingual crown dimensions have been demonstrated between ethnic groups . There were varying patterns of tooth size between the groups and the later - forming teeth in each tooth type were smaller and showed greater variation . These differences reflect different contributions of genetic and environmental influences to tooth size variability within and between human population.
Internet addiction disorder (iad) is defined as one s inability to control his internet using, which could lead to physical, psychological, and social difficulties (1). Owing to web - based technologies and increased internet access in latin america and asia, use of internet has dramatically increased across the world, with the global number of internet users reaching more than 2.3 billion in 2011 (2). Internet provides many positive benefits to users as it allows them to obtain new information, knowledge, and even entertainment . Its explosive growth in the last decade had a huge impact on communication and interpersonal behavior . Internet was originally designed to facilitate communication and research activities, but the dramatic increase in its use in recent years has led to pathological use (3, 4). Today clinical specialists have reported cases of this disorder observed in their clinics . As a new form of addiction in recent years, it has attracted the attention of researchers in psychology, psychiatry, sociology, and other scientific fields (5). In his research, yang found that internet addiction has seven main reasons: insecurity, financial problems, marital discontent, work stress, anxiety, family disputes, and limited social life (6). Griffiths defined internet addiction as a non - chemical addiction originating from interactions between human and machines and added that technological addiction is either passive (television) or active (computer games), prompting and reinforcing the characteristics facilitating addictive tendencies (4). Internet addiction is accompanied by several psychological problems, including self - doubt, anxiety, and depression (7). Perceived social support is defined as a mental sense of belonging, acceptance, and being loved by others; thus, it can foster secure relationships for a person . Theorists believe that perceived social support, as a comparative source, can decrease the harmful effects of stressful factors in one s environment (8). It is believed that the social support of friends, family, and others decreases psychological stress in people . Shaikhalizade et al . (9), cohen and sayme (1985), vex (1998), and sarasoon (1990) referred to the major point that decreasing social support increases the vulnerability of physical and mental structures . Concurrent with many studies worldwide, a few researches are conducted in iran (10). It was found that because of internet addiction, students had weaker social relations and weaker mental health; they preferred using internet to having social relationship with their friends, family, spouse and other people . The current research aimed to investigate comprehensive social support, self - esteem, and internet addiction among university students . In the current descriptive research, the statistical sample consisted of 101 female students of al - zahra university (an all - female university), tehran, iran . Sample size was computed with = 0.05, = 0.10, r = 0.29 using equation 1: cluster sampling was employed to select participants; subjects completed the multidimensional scale of perceived social support, rosenberg self - esteem scale, and yang internet addiction tests . Perceived social support (mspss) is a measurement with 12 items provided by smith et al . To evaluate perceived social support from three resources: family, friends, and effective persons around us . This scale measures perceived social support by examining these three areas . It has three subscales: family (items 3, 4, 8, and 11), friends (items 6, 7, 9, and 12), and others (items 1, 2, 5, and 10). The total alpha coefficient of test is 0.91, and the alpha coefficient of its subscales ranges from 0.90 to 0.95 . This scale has good construct validity, because it has no correlation with the social goodness scale of marlow - kron . A high score in this scale shows that there is high level of perceived social support (11). In the iranian subjects, perceived social support values for family, friends, and others were 89%, 86%, and 82%, respectively (12). The internet addiction test (iat) developed by kimberly young is a reliable and valid measure of addictive use of internet . It consists of 20 items that measure mild, moderate, and severe levels of internet addiction . The total score for each item was calculated; the higher the score, the greater the level of addiction . Scores are interpreted as follows: 20 - 49 points indicates an average online user . The user may surf the web a bit too long at times, but has control over usage; 50 - 79 points indicates the user is experiencing occasional or frequent problems because of internet, and its full impact on the user s life should be considered; 80 - 100 points indicates the user s internet usage is causing significant problems in his / her life . Its impact should be alleviated, and the problem caused by internet usage should be directly addressed (13). The rosenberg self - esteem scale is a 10-item, self - report measure of global self - esteem . It consists of 10 statements related to overall feelings of self - worth or self - acceptance . The items are ranked on four - point scales ranging from strongly agree to strongly disagree . Among the iranian subjects, the coefficient of internal consistency in the whole subjects was 0.84 (14). After completion of the questionnaires, the data were analyzed by correlation and multiple regression analysis tests using spss version 21 . Perceived social support (mspss) is a measurement with 12 items provided by smith et al . To evaluate perceived social support from three resources: family, friends, and effective persons around us . It has three subscales: family (items 3, 4, 8, and 11), friends (items 6, 7, 9, and 12), and others (items 1, 2, 5, and 10). The total alpha coefficient of test is 0.91, and the alpha coefficient of its subscales ranges from 0.90 to 0.95 . This scale has good construct validity, because it has no correlation with the social goodness scale of marlow - kron . A high score in this scale shows that there is high level of perceived social support (11). In the iranian subjects, perceived social support values for family, friends, and others were 89%, 86%, and 82%, respectively (12). The internet addiction test (iat) developed by kimberly young is a reliable and valid measure of addictive use of internet . It consists of 20 items that measure mild, moderate, and severe levels of internet addiction . The total score for each item was calculated; the higher the score, the greater the level of addiction . Scores are interpreted as follows: 20 - 49 points indicates an average online user . The user may surf the web a bit too long at times, but has control over usage; 50 - 79 points indicates the user is experiencing occasional or frequent problems because of internet, and its full impact on the user s life should be considered; 80 - 100 points indicates the user s internet usage is causing significant problems in his / her life . Its impact should be alleviated, and the problem caused by internet usage should be directly addressed (13). The rosenberg self - esteem scale is a 10-item, self - report measure of global self - esteem . It consists of 10 statements related to overall feelings of self - worth or self - acceptance . The items are ranked on four - point scales ranging from strongly agree to strongly disagree . Among the iranian subjects, the coefficient of internal consistency in the whole subjects was 0.84 (14). After completion of the questionnaires, the data were analyzed by correlation and multiple regression analysis tests using spss version 21 . According to the descriptive statistics, about 22.8% and 77.2% of the subjects had high low levels of self - esteem, respectively . Results of the descriptive statistics based on table 1 and figure 1 showed that, based on answers to question 1, 29.7%, 9.9%, and 13.9% of the subjects said that they frequently, often, and always used internet more than they intended, respectively . Results of the descriptive statistics showed that, based on answers to question four concerning effective support by family or friends, 29.7% believe that they enjoyed family support, and 23.8% enjoyed friends support (figure 2). Results of the stepwise regression test showed that the scale of internet addiction and the subscale of family were predicative variables for self - esteem (r = 0.137, p <0.005, f2, 96 = 77.7). The regression model showed that it could be possible to predict one s self - esteem score by the internet addiction scale and family subscale (y = 3.66 - 0.085 ait + 0.734 family). Scale of perceived social support includes the subscales of family, friends, and others (tables 2 and 3). Scale of perceived social support includes the subscales of family, friends, and others . The current research investigated perceived social support, self - esteem, and internet addiction among university students . The findings demonstrated that persons with low self - esteem were more vulnerable to internet addiction than the ones with high self - esteem . This result conforms to those of the previous studies (7, 13). According to the previous studies, addicted users had low self - esteem (yang and tony; kaplan, great and benyard). To explain these results, davis s theoretical model (15) can be used . Based on his model, low self - esteem as a psychological damage could predispose a person to becoming an internet - addicted user . Results of the current study showed a significant correlation between addiction to internet and perceived social support, the subscales of family, and others . A study found that addicted students had low social relationships; internet addiction may lead to weaker social, personal, familial, and friendly relationships in university students . (17) indicated that strengthening familial relationships and promoting youth positive development could prevent internet addiction in adolescents . Patients with internet addiction have more anxieties as well as prominent disruption in familial relationships (18). Kim et al . (16) in his studies found that the levels of depression and suicide were the highest in the internet - addicted people . (19) indicated a high prevalence of internet addiction among chinese adolescent internet users . The study highlighted the importance of stressors in interpersonal and school - related problems, which are mainly mediated with a negative coping style, as risk factors for internet addiction (19). Kaplan s studies showed that internet - addicted persons have low levels of social relationships compared with others (20). (13) showed that internet usage, in spite of creating a figurative space for relationships with others, is unable to create relationships with friends and family . Davis theoretical model can explain these results . Based on this model, loneliness due to social isolationism or lack of social support, the findings of several studies on internet addiction also showed that persons who have low self - esteem or social skills are exposed to internet addiction more than others; they look for their identity or ranking in the figurative space of internet . However, it cannot be concluded that people with low self - esteem have the inclination to use internet or that internet abuse may led to weaker social relationships and create loneliness in a person, eventually causing depression or low self - esteem . Based on the previously discussed problems concerning internet usage addiction, it is necessary to provide instruction for proper use of this technology, especially to universities and students as national assets for development . Due to the availability of wireless technology at al - zahra university, study subjects were selected from the students of this university . Unfortunately, since it is an all - female university, all participants were female . It is recommended that future studies compare the internet addiction among females and males . Furthermore, it is recommended to study the relationships between internet addiction, depression factors, perceived social support, and self - esteem in males . Due to the availability of wireless technology at al - zahra university, study subjects were selected from the students of this university . Unfortunately, since it is an all - female university, all participants were female . Furthermore, it is recommended to study the relationships between internet addiction, depression factors, perceived social support, and self - esteem in males.
A 41-year - old, male farm worker presented to our hospital with a 3-month history of recurrent right - upper - quadrant pain . Computed tomography demonstrated a 7.41-cm cyst in the right lobe of the liver (figure 1). After the diagnosis, medical treatment with albendazole 400 mg per day was initiated 4 weeks before surgery . The cyst was approached laparoscopically by using the same hydatid asepsis techniques as in open surgery . A veress needle was introduced and hypertonic saline (20% naci) was injected to surround the cyst . Afterwards, we punctured the cyst, and hypertonic saline (20% naci) was injected into it . The saline solution was allowed to remain for 5 minutes and changed 4 to 5 times . The germinative layer and hydatid daughters were removed with care and placed in extraction bags (figure 2). Therapy consists of inactivation of the cyst with scolicide (hypertonic saline), removal of the cyst contents without contaminating the patient, followed by appropriate management of any remaining cavity . The cyst is approached laparoscopically by using the same hydatid aseptic techniques as in open surgery . The procedure is contraindicated in patients with secondarily infected cysts, or suspected biliary communication (bile - stained aspirate), owing to the increased risk of complications . In these cases the laparoscopic technique is safe and simple and has the advantages of other abdominal laparoscopic operations . It also fulfills the prerequisite of open surgery of hydatid cyst of the liver, namely, the prevention of intraperitoneal spillage of cyst contents.
Scleroderma or systemic sclerosis (ssc) is a complex autoimmune disease affecting 1/100,000 individuals among the caucasian population . Higher rates have been reported in usa, australia, and eastern europe and lower rates have been reported in northern europe and japan . Even though current clinical and diagnostic utilities have led to a better understanding of the disease scleroderma is a heterogeneous disease with a wide range of clinical manifestations ranging from mild skin fibrosis with minimal internal organ disease to severe skin and organ involvement . The three main pathological events that are involved in scleroderma pathogenesis are mainly endothelial damage, fibrosis, and autoimmune dysregulation . Etiopathogenesis of scleroderma is characterized by fibroproliferative alterations, cellular and humoral immune abnormalities resulting in a severe and often progressive fibrotic process . Scleroderma can also be subdivided according to different criteria, such as involvement of organs and the presence of specific antibodies which are hallmarks of the disease . These autoantibodies are disease - specific and usually mutually exclusive and correlate with the extent of skin involvement and associated disease manifestations . The most common are dna topoisomerase (anti - scl70), anti - centromere antibodies (cenp a and/or b protein). These autoantibodies are marker antibodies for relatively distinct clinical phenotypes of ssc where anti - scl70 antibodies are a marker for dcssc and ssc patients with clinically significant pulmonary fibrosis with a poor prognosis whereas anti - centromere antibodies typically are associated with lcssc, uncommon pulmonary fibrosis, and late onset of pulmonary hypertension but generally are associated with an overall good prognosis [3, 4]. Geoepidemiologically it has been noted that clinical features and presence of these disease - specific autoantibodies vary across the globe and ethnicities . The low incidence of ssc and the clinical variability result in difficulties in understanding the disease pathogenesis . There is an unmet need for validated biomarkers for scleroderma disease diagnosis, classification, and future therapeutic approach for management of scleroderma patients . This study was designed to look at differences in the clinical features among subset of scleroderma patients with an emphasis on autoantibodies in scleroderma patients from mumbai, western india . This prospective study was conducted in 110 scleroderma patients from rheumatology department of king edward memorial hospital, mumbai, india, and national institute of immunohaematology, mumbai, india, over the period of 3 years (20102012). This study was carried out after obtaining the requisite ethics committee approval and a written consent from patients . Scleroderma patients were classified according to american college of rheumatology / european league against rheumatism (acr / eular) criteria [6, 7]. Ssc patients were classified into two clinical subgroups based on the extent of skin involvement, limited cutaneous ssc (lcssc), and diffuse cutaneous ssc (dcssc) that are associated with different clinical complications and prognoses . Diffused cutaneous (dcssc) patients had delayed raynaud's phenomenon, severe constitutional symptoms, arthralgias, carpal tunnel, puffy hands and legs, palpated tendon friction rubs, skin thickening progressing from fingers to trunk rapidly, potentially severe pulmonary fibrosis, and cardiac and renal involvement . Lcssc patients had raynaud's phenomenon alone for years, rate constitutional symptoms, minimal arthralgias, puffy fingers, telangiectasias and late calcinosis, skin thickening limited to hands and face, and mild pulmonary fibrosis . Severe organ system involvement noted like musculoskeletal involved joints / tendons of fingertip to palm distance 4.0 + cm and severe proximal muscle weakness on physical examination . Severe renal manifestations involved renal crisis with serum creatinine 3.0 + mg / dl at any time . Pulmonary arterial hypertension was diagnosed by right - sided heart catheterization according to standard definitions . Pulmonary fibrosis was seen on chest radiography and raynaud's phenomenon was self - reported or reported in patients with at least a 2-phase colour change in finger(s) and often toes consisting of pallor, cyanosis, and/or reactive hyperemia in response to cold exposure . Pregnant and postmenopausal women, smokers, patients with diabetes, and patients with significant hyperlipemia were excluded . Standard investigations like cbc, esr, routine biochemical tests (renal and liver function tests and electrolytes), chest x ray, and ecg were carried for all patients . Anti - nuclear antibodies (ana) (biorad, usa), anti - endothelial cell antibodies (aeca) (euroimmune, germany), and anti - keratinocyte antibodies (aka) (euroimmune, germany) were tested by indirect immunofluorescence test (iif). Anti - scl70 antibodies (anti - i), anti - centromere antibodies, and anti - cyclic citrullinated peptide (ccp) antibodies were tested by elisa using commercially available kits (euroimmune, germany). Continuous variables were expressed as mean sd . Pairs of groups were compared using student's test for normally distributed continuous distribution . A total of 110 patients with scleroderma having mean age of 34.7 10.7 years at evaluation and a mean disease duration of 43.7 35.4 years were included in the study (table 1). There were 100 females (91%) and 10 males (9%) included in this study . The female to male ratio was 10: 1 . At evaluation women were slightly older than males with mean sd of 35.6 10.5 as compared to males (30.7 5.7). The disease duration ranged between 6 and 120 months with a mean sd of 43.7 35.4 . It was observed that 45 patients (40.9%) had dcssc lesions and 32 patients (29.1%) had lcssc lesions . The remaining 33 patients (30%) had other autoimmune overlaps wherein patients had features of scleroderma combined with features of a second connective tissue disease at the time evaluation . Clinical presentation revealed that 108 patients (98.2%) had cutaneous manifestations, 85 patients (77.3%) had pulmonary manifestations, 12 patients (10.9%) had renal involvement, 43 patients (39.1%) had musculoskeletal involvement, 8 patients (7.3%) had gastrointestinal involvement, and 15 patients (13.5%) had cardiovascular involvement . As shown in table 2, the overall frequency of ana in ssc patients studied was 85.5% . It was observed that 60 patients (63.9%) had speckled pattern, 17 patients (18.1%) had nucleolar pattern, 7 patients (7.4%) had centromere pattern, 2 patients (2.1%) had rim / peripheral pattern, and the remaining 2 patients (2.1%) had speckled and nucleolar pattern . The frequency of anti - scl70 antibodies, anti - centromere antibodies, aeca, and aka was 62.7%, 22.7%, 30%, and 40.9%, respectively . For anti - scl70 antibodies among dcssc compared with lcssc there was a statistically significant difference (p <0.0115, or = 3.5030, and 95% ci = 1.32569.2570). When anti - centromere antibodies were compared in both the groups, there was a statistically significant difference noted (p <0.0044, or = 0.1626, and 95% ci = 0.04650.5684). When dcssc and lcssc patients were compared statistically, there was no statistically significant difference for ana, aeca, and aka (p> 0.05). When dcssc and lcssc patients together were compared with overlap patients, geoepidemiology studies suggest that systemic sclerosis (ssc) is more common, occurs only at a younger age, and is more severe in african americans than caucasians . The major differences noted were mainly in their clinical and serological phenotypes . These differences can be further related to different environmental exposure as well as immunogenetic makeup of these patients . The studies on clinical and serological profile of ssc patients have important implications for both clinical interventions and future pathogenic studies . De souza mller et al . Had documented 65.62%, 26.04%, and 8.33% frequency for dcssc, lcssc, and overlap among their total ssc patients from brazil . Indian data on ssc patients revealed that, among north and south indian ssc patients, the clinical presentation varies [10, 11]. Though there are a few published records from western india there is no information available from the eastern part of the country giving details of clinical profile of indian ssc patients . Demographic and serological similarities / differences in indian scleroderma patients in various regions across the country are as shown in table 4 [1015]. Ssc - related autoantibodies among scleroderma patients were compared in different ethnicities like caucasian americans, african americans, and latin americans by krzyszczak et al . . The reported frequency of anti - scl70 and anti - centromere antibodies was 15%, 17%, respectively, in caucasian american, 35%, 0% in african american, and 20%, 40% among latin american ssc patients . Had reported an incidence of 94.2% ana positivity among german ssc patients and among brazilian ssc patients, 92.4% ana positivity had been reported [4, 16]. Johnson et al . Had reported a much lower incidence (17%) for anti - scl70 autoantibodies among canadian ssc patients, as compared to 62.7% in the present study whereas the incidence for anti - centromere antibodies was 29% which was similar to the present study (22.7%). 19%, 21% anti - scl70 antibodies and anti - centromere antibodies from pittsburg, usa, 17%, 16% from toronto, 35%, 27% from madrid, and 22%, 28% from berlin ssc cohort had been documented . Among brazilian ssc patients anti - scl70 frequency reported was 17.8% which was mainly associated with dcssc (p <0.015) whereas anti - centromere antibodies (33.3%) were commonly associated with lcssc subtypes . Had reported a total incidence of 30.1% for anti - scl70 antibodies where dcssc subtype was strongly associated with anti - scl70 autoantibodies (p <0.0001). Frequency of anti - centromere antibodies reported by the same group was 35.9% and a positive correlation was found in lcssc patients with anti - centromere antibodies (p <0.0001). De souza mller et al . Also had reported a positive correlation of anti - centromere antibodies with lcssc form (p <0.01) which are similar to the present study . Previous studies had shown that anti - centromere antibodies were strongly associated with renal dysfunction in lcssc patients and cenp - b was a major target antigen reported for aeca in lcssc patients indicating the association between anti - centromere antibodies and aeca autoantibodies leading to aeca mediated endothelial dysfunction due to an underlying autoimmune mechanism [1821]. Further endothelial dysfunction may be associated with high incidence of cerebrocardiovascular diseases which needs to be studied in ssc patients . This study throws light on a need for some biomarker antibodies and discovery of new target antigens among scleroderma patients and their immunodiagnostic potential . These disease - specific antibodies along with disease phenotype variation across the country will help in understanding the geoepidemiological picture of scleroderma in different geographical regions in india . This could possibly represent new diagnostic and/or prognostic markers of scleroderma in the near future.
Adrenocortical carcinoma is a rare malignancy comprising only about 0.2% of all cases of childhood cancer . This rare malignancy is associated with a high rate of recurrence and mortality with a reported 5-year event - free survival estimate of 54.2% in children . Pediatric adrenocortical tumors (acts) occur most commonly in females and in children less than four years . Eighty to ninety percent of patients have functional tumors with endocrine manifestations at diagnosis and the majority present with virilization, alone or in combination with overproduction of other adrenal hormones . We report an infant with cushing syndrome without the clinical features of androgen or mineralocorticoid excess, consequently diagnosed with adrenocortical carcinoma . The patient is a caucasian female born at term with a birth weight of 3.06 kg; pregnancy and delivery were uncomplicated . At six months of age her linear growth apparently had been arrested between three and six months of age, although she gained weight at an accelerated rate (figure 1). Prior to referral, laboratory evaluation by her primary care physician revealed serum cortisol of 240 mcg / dl (normal, 4.522.7 mcg / dl); total testosterone of 185 ng / dl (normal, 677ng / dl), and dhea - s of 401 mcg / dl (normal, 1696 mcg / dl). Serum acth was less than 5 pg / ml (normal, 1060 pg / ml). Her weight at initial evaluation in our clinic at six months of age was 7.52 kg (6075th percentile) and length was 59.1 cm (<5th percentile). Physical examination showed cushingoid facies, buffalo hump, facial acne, and poor muscle tone (figure 2). An mri of the abdomen showed a heterogeneous right adrenal mass measuring 3.6 5.4 3.8 cm; this mass was found to extend as a tumor thrombus into the inferior vena cava . The tumor thrombus appeared to extend to approximately 2 cm from the right atrium . A doppler ultrasound showed minimal, but present flow around the ivc tumor thrombus which measured 2.5 0.9 1 cm . Laboratory evaluation showed serum cortisol 70.8 mcg / dl (normal, 4.522.7 mcg / dl); aldosterone 4.2 ng / dl (normal 6.586.0 ng / dl); testosterone 59 ng / dl (normal, 677 ng / dl); acth <5 (normal, 1060 pg / ml). She was prescribed ketoconozole prior to surgery due to moderately elevated blood pressure . However, surgical resection was undertaken and intraoperative findings showed a large, well - circumscribed mass in the right adrenal gland measuring 5.2 4.0 3.5 cm mass which did not invade the liver, kidney or other surrounding soft tissues . On intraoperative transesophageal echocardiography, there appeared to be thickening of the wall of the superior vena cava of unknown significance . The intracaval clot did not extend to the right atrium and cardiopulmonary bypass was not required . Blood flow was visualized around the caval clot with no evidence of adherence to the caval wall . The adrenal mass and thrombus were removed en bloc (figure 3(a)). The mass showed five to ten percent necrosis of the mass with spotty calcifications (figures 3(b) and 3(c)). She received stress dosing with hydrocortisone during and after surgery; she was reduced to a physiologic replacement dose by 15 days postoperatively . She was considered to have stage 1 adrenocortical carcinoma based on the relatively small tumor size, complete surgical resection, lack of metastases, and normalization of hormonal markers postoperatively . The tumor cells demonstrated a low mitotic count (<1 to 2 per 50 high power fields and no atypical mitoses). In addition, the mib-1 index was low at <1% to 12% with regional variability . Tumor immunohistochemistry for p53 protein showed weak staining with no accumulation in the nucleus; when p53 amplification is present, the defective proteins accumulate within the nucleus due to abnormal cellular processing . To evaluate for a germline p53 mutation in the patient, whole gene sequencing was performed for the tp53 gene and did not reveal any known mutation . Quarterly pet scanning to monitor for recurrence is underway, and has been negative for 24 months post - operatively . Two months after surgery, laboratory evaluation showed total testosterone level of <7.0 ng / dl (normal, 677 ng / dl); dhea <1.0ng / ml (normal, <2.9 ng / ml); dhea - s <15.0 ng / dl (normal, 1696 mcg / dl). Acth was 7.6 pg / ml (normal, 1060 pg / ml). Growth velocity began to increase within two months after surgery, and five months after surgery was 25 cm / year consistent with catch - up growth . Our patient demonstrated classic features of glucocorticoid excess including growth failure, hypertension, acne, plethora and a buffalo hump with excessive weight gain . The differential diagnosis of cushing syndrome in an infant includes adrenocortical tumors (carcinoma or adenomas), as well as multinodular adrenal hyperplasia . In addition, cushing disease, ectopic acth production, and iatrogenic cushing syndrome should be considered . Miller et al ., demonstrated that of 60 infants with cushing syndrome, 48 had adrenal tumors . Two had ectopic acth production, five had nodular adrenal hyperplasia, four had undefined adrenal hyperplasia and one had an acth producing tumor . Therefore, first an adrenocortical tumor should be excluded in an infant with cushing syndrome as long as there is no history for exposure to exogenous glucocorticoids . In our case, the unilateral adrenal mass in the mri indicated an adrenal tumor and histopathological findings confirmed an adrenocortical carcinoma . Pediatric adrenocortical tumors have distinct clinical and biological features in comparison to those which occur in adults . It is estimated that there are 1920 new cases of adrenocortical carcinoma in children and adolescents per year in the united states according to the surveillance, epidemiology and end results program . Most of these patients are either toddlers, early school age children or in their late teens as pediatric adrenocortical tumors have a bimodal age distribution . There is a peak in incidence before four years of age (0.4 cases per million) which then declines during the subsequent ten years . Interestingly, the majority (60%) of pediatric adrenocortical tumors occur in children less than five years which is similar to the distribution seen with tumors of embryonic origin . Although our patient fits the common age distribution for an adrenocortical tumor, her presentation with cushingoid features alone is unusual especially at such a young age . Although biochemically she demonstrated elevations in both androgens and cortisol, she had no evidence of virilization on physical examination . Michalkiewicz et al ., found in a registry of 254 pediatric patients with acts that 55% presented with virilization alone . Only 5.5% percent of this group presented with isolated cushing syndrome, and this tended to occur in older children (median age, 12.6 years). In addition, lefevre et al ., showed similar results in their analysis of 42 children with acts in france . Therefore, our patient is remarkable in that she presented with cushingoid features alone during infancy . The tendency for adrenocortical tumors to occur in younger age groups and to be predominantly androgen - producing could be linked to their relationship to the fetal adrenal cortex . The fetal adrenal cortex is composed of two distinct zones: the outer definitive zone which is steroidogenically quiescent until late gestation and an inner zone which appears to produce steroid hormones throughout gestation . The inner fetal zone makes up 8590% of the total fetal adrenal cortex at birth from which time it begins to undergo apoptosis . The histopathologic features of acts in young children suggest that they arise from the fetal zone which has a tendency towards androgen production due to differential expression of steroidogenic enzymes at different times in development . The primary steroid product of the fetal adrenal cortex is dehydroepiandrosterone sulfate (dhea - s) which is the precursor of placental estrogen . Thus, the derivation of adrenocortical tumors of infants, and young children from the fetal zone of the adrenal cortex could help to explain their preferential elaboration of androgens . Our patient's tumor was negative for p53 mutation which is estimated to occur in 8090% of all pediatric adrenocortical tumors . Interestingly, a distinct germline p53 mutation, tp53 r337h, has been described in brazilian patients with adrenocortical tumors [5, 6]. This mutation leads to an abnormal folding of the tp53 protein and accumulation in the nucleus; loss of heterozygosity at this locus is though to be involved in tumorigenesis . In a study evaluating penetrance of this mutation, our patient has a favorable prognosis given complete surgical resection, small tumor size, lack of metastasis and young age . From the international pediatric adrenocortical tumor registry report including data from 254 patients, those with completely resected tumors weighing less than 200 gm and without metastasis, had a five year event free survival rate of 91% . This study also showed that age less than four years was independently associated with better prognosis; multivariate analysis showed adjusted odds ratio of 2.6 for age less than four years . As mentioned earlier, her follow - up pet scans and hormonal markers have remained negative for 24 months postoperatively . In conclusion, although rare, acts should be considered in the differential diagnosis of cushing syndrome in the pediatric age group . While virilizing features are typically dominant, acts may present with isolated cushing syndrome . Early diagnosis, adequate perioperative management with specific emphasis on postoperative glucocorticoid replacement, complete excision of tumor and close follow - up for recurrence and metastases are crucial to improve survival.
Microarray technology is now routinely used for genome - wide mrna expression and epigenetic profiling . As a consequence, this may improve the interpretation of new experimental studies through comparison with data already publicly available, and allow for large - scale meta - analysis . Use of data in the repositories gives increased statistical power, and facilitates confirmation of hypotheses coming from one study with data from another study, identification of artifacts and separation of primary effects from secondary (1). For example, meta - analysis of microarray data was useful for identification of common transcriptional profiles of neoplastic transformation and progression (2), discovery of genes disproportionately overexpressed in specific cancer types (3), construction of robust high - resolution gene coexpression networks (4), and identification of rhythmically expressed genes in drosophila (5). There are many hurdles to be taken when integrating data from different studies, such as the necessity to map the probes on different arrays to transcripts, the necessity to account for different experimental designs and analysis algorithms, and to retrieve the relevant datasets from microarray data repositories . The latter is addressed by mare, the tool that we developed . Currently, there are no public software tools, which provide combined and interactive access to the main microarray data repositories . Microarray retriever (mare) facilitates meta - analysis by enabling searching and batch data retrieval from the two major public microarray repositories: gene expression omnibus (geo, national center for biotechnology information; 6,7) and arrayexpress (ae, european bioinformatics institute; 8,9). Mare allows the user to search these repositories for experiments with accession numbers, authors, species, date of submission, array platform and keyword search terms . In addition, users can first search pubmed on authors and keywords for relevant literature and then search geo and arrayexpress for experimental data associated with this literature . Alternatively, a custom list of pubmed ids can be uploaded to obtain data for specific articles known in advance . After the search is complete, mare enables downloading of selected results in one step . This avoids the time - consuming procedure of manual and sequential downloading from the web or ftp sites of the repositories . Are performed remotely by sending queries to ncbi e - utilities (10). A 3-s delay between the file is downloaded from the ebi during a search if the previous version of the file is older than 1 h (http://www.ebi.ac.uk/arrayexpress/q-aer/). The mare web interface contains three boxes for input of the query terms: accessions (a), authors / keywords (b) and species / date / platform (c). Box a accepts accession numbers of geo experiments (series and datasets), accession numbers of arrayexpress experiments and pubmed ids of papers potentially associated with experimental data in either of the repositories . Box b specifies authors and keywords to be searched for in the meta - data present in the microarray repositories and/or pubmed . The queries can contain logical operators, brackets and quotes (a detailed description is provided in the online help file). Box c enables searching on or limiting searches on specific species (for example, mus musculus and rattus norvegicusto search for experiments where both species were examined), date of submission to the repository (can be an interval) and platforms (using platform accession numbers or platform keywords). In the keywords field (box b) and the platform keywords field (box c), entrez limits can be added to the input terms, e.g. P53[title]. The limits are only used for searches in geo and pubmed . Query logics is used to define how the boxes a, b and c should be combined to generate the complete query . Search options contains the following options: to search for experiments and the associated platforms or to search for platforms only;to search in geo and/or in arrayexpress;to retrieve series and/or datasets from geo;to retrieve all experiments from arrayexpress or only those, which do not duplicate data already retrieved in the geo search;to retrieve raw data for experiments or to retrieve only the processed data . An email address should be entered in the start search box before the search can be started . Mare will send a notification with the url of the data archive to this email address . To search for experiments and the associated platforms or to search for platforms only; to search in geo and/or in arrayexpress; to retrieve series and/or datasets from geo; to retrieve all experiments from arrayexpress or only those, which do not duplicate data already retrieved in the geo search; to retrieve raw data for experiments or to retrieve only the processed data . Subsequently, the results are combined according to the user - defined logics and redundant hits are removed . Search on each of the underlined input terms is performed independently, after which the results are combined . After obtaining series on datasets and datasets on series for geo and filtering out experiments already found in geo for ae search on each of the underlined input terms is performed independently, after which the results are combined . After obtaining series on datasets and datasets on series for geo and filtering out experiments already found in geo for ae when searching with geo accession numbers, the user can select retrieve gse and gds. Doing so, series associated with the found datasets and datasets associated with the found series are automatically retrieved . This is similar to the search logics implemented in geo entrez . If retrieve only gse or retrieve only gds is chosen, entries which meet the query themselves are retrieved (series or datasets, respectively). When searching arrayexpress, the user can select to retrieve only data not retrieved from geo. In that case, ae experiments that duplicate geo entries already found in the search will not be displayed on the results page . Primary accession numbers and secondary accession numbers of ae experiments are used to distinguish those that are also found in geo . E - geod - number, where the number indicates the corresponding gse, secondary accession numbers coincide with the gse accession numbers in geo . When platform terms are entered in an experiments and platforms search, the platforms are found first . Subsequently, experiments associated with them are retrieved and combined with experiments found for boxes a and b. platforms in the results page are those which correspond to the found experiments . Advantages of using platform terms in a search for experiments and platforms are that the experimental data retrieved will be limited to a specific platform, and that only those platforms will be displayed on which experiments have been performed (function not available in geo). When a search for platforms only is performed, the repositories are searched for platforms, which meet the platform terms and all platforms, including those with no associated experiments, are displayed in the results page . As soon as some of the search results are selected for downloading and the downloading job is started, an email message is sent to inform the user that the job has been started . Processed data from geo are downloaded in geo soft format (6). Processed data from the arrayexpress ftp site are downloaded in all available formats (8). If the user has chosen to download raw data, these data are downloaded as well . Mare keeps the load on the geo and arrayexpress servers minimal by downloading all data on a per file basis instead of downloading all files at once . While downloading, files are retrieved from multiple directories on the ftp servers of the repositories and organized locally so that files for the same experiment are placed in the same folder . After the downloading job is complete, the downloaded files are zipped into a single archive . Finally, an email is sent to the user with the url to the archive on the mare server . Are performed remotely by sending queries to ncbi e - utilities (10). A 3-s delay between the file is downloaded from the ebi during a search if the previous version of the file is older than 1 h (http://www.ebi.ac.uk/arrayexpress/q-aer/). The mare web interface contains three boxes for input of the query terms: accessions (a), authors / keywords (b) and species / date / platform (c). Box a accepts accession numbers of geo experiments (series and datasets), accession numbers of arrayexpress experiments and pubmed ids of papers potentially associated with experimental data in either of the repositories . Box b specifies authors and keywords to be searched for in the meta - data present in the microarray repositories and/or pubmed . The queries can contain logical operators, brackets and quotes (a detailed description is provided in the online help file). Box c enables searching on or limiting searches on specific species (for example, mus musculus and rattus norvegicusto search for experiments where both species were examined), date of submission to the repository (can be an interval) and platforms (using platform accession numbers or platform keywords). In the keywords field (box b) and the platform keywords field (box c), entrez limits can be added to the input terms, e.g. Query logics is used to define how the boxes a, b and c should be combined to generate the complete query . Search options contains the following options: to search for experiments and the associated platforms or to search for platforms only;to search in geo and/or in arrayexpress;to retrieve series and/or datasets from geo;to retrieve all experiments from arrayexpress or only those, which do not duplicate data already retrieved in the geo search;to retrieve raw data for experiments or to retrieve only the processed data . An email address should be entered in the start search box before the search can be started . Mare will send a notification with the url of the data archive to this email address . To search for experiments and the associated platforms or to search for platforms only; to search in geo and/or in arrayexpress; to retrieve series and/or datasets from geo; to retrieve all experiments from arrayexpress or only those, which do not duplicate data already retrieved in the geo search; to retrieve raw data for experiments or to retrieve only the processed data . Subsequently, the results are combined according to the user - defined logics and redundant hits are removed . Search on each of the underlined input terms is performed independently, after which the results are combined . After obtaining series on datasets and datasets on series for geo and filtering out experiments already found in geo for ae search on each of the underlined input terms is performed independently, after which the results are combined . After obtaining series on datasets and datasets on series for geo and filtering out experiments already found in geo for ae when searching with geo accession numbers, the user can select retrieve gse and gds. Doing so, series associated with the found datasets and datasets associated with the found series are automatically retrieved . This is similar to the search logics implemented in geo entrez . If retrieve only gse or retrieve only gds is chosen, entries which meet the query themselves are retrieved (series or datasets, respectively). When searching arrayexpress, the user can select to retrieve only data not retrieved from geo. In that case, ae experiments that duplicate geo entries already found in the search will not be displayed on the results page . Primary accession numbers and secondary accession numbers of ae experiments are used to distinguish those that are also found in geo . E - geod - number, where the number indicates the corresponding gse, secondary accession numbers coincide with the gse accession numbers in geo . When platform terms are entered in an experiments and platforms search, the platforms are found first . Subsequently, experiments associated with them are retrieved and combined with experiments found for boxes a and b. platforms in the results page are those which correspond to the found experiments . Advantages of using platform terms in a search for experiments and platforms are that the experimental data retrieved will be limited to a specific platform, and that only those platforms will be displayed on which experiments have been performed (function not available in geo). When a search for platforms only is performed, the repositories are searched for platforms, which meet the platform terms and all platforms, including those with no associated experiments, are displayed in the results page . As soon as some of the search results are selected for downloading and the downloading job is started, an email message is sent to inform the user that the job has been started . Processed data from geo are downloaded in geo soft format (6). Processed data from the arrayexpress ftp site are downloaded in all available formats (8). If the user has chosen to download raw data, these data are downloaded as well . Mare keeps the load on the geo and arrayexpress servers minimal by downloading all data on a per file basis instead of downloading all files at once . While downloading, files are retrieved from multiple directories on the ftp servers of the repositories and organized locally so that files for the same experiment are placed in the same folder . After the downloading job is complete, the downloaded files are zipped into a single archive . Finally, an email is sent to the user with the url to the archive on the mare server . The layout of the results page is compact and gives a good overview with hyperlinks to the entries in geo or ae . Search parameters are configured on the first page, entries to download are chosen on the next page after the search is complete, i d of the accepted downloading job is finally reported . Search parameters are configured on the first page, entries to download are chosen on the next page after the search is complete, i d of the accepted downloading job is finally reported . The red boxes and text indicate the different sections of the results page . To illustrate the performance of our application, we retrieved all data from geo and arrayexpress connected with a particular area of research ovarian cancer . A query on keyword ovarian[title] and (cancer[title] or tumour[title] or tumor[title]) (box b) limited to the species homo sapiens (box c) in the annotations of the two repositories and in pubmed resulted in 16 geo series (29 platforms) and 13 arrayexpress experiments (19 platforms). Processed data for all experiments and complete descriptions of all associated platforms were selected for download . Downloading all data to the mare server took 2 h and 50 min . Retrieving the complete archive from the mare server using the size of the archive was 2.5 gb, while the size of the unzipped data comprised 5.5 gb . As stated earlier, keyword searches in geo and arrayexpress can be performed in the meta - data of the repositories and/or pubmed . Search in pubmed can result in relevant entries not found by an equivalent search in the geo annotation fields . P53[title] or tp53[title] yielded 30 geo series (as on 12 january 2008). Searching for geo series with four of these 11 experiments (gse2155, gse3072, gse7678 and gse8023) contain neither of the terms p53 or tp53 in their titles nor in the geo annotation (except for the citation field which is not queried via geo entrez). Nevertheless, all of these experiments were manually checked to be relevant for p53 research . In this way, searching in pubmed increases the recall of the search . Microarray retriever provides the following options, which are not available in geo and arrayexpress: simultaneous access to both repositories;search in the repositories via pubmed;selection of platforms on which experiments have actually been done;batch downloading of entries . Besides mare, there are several other tools which have an option of downloading microarray data from public sources: geoquery (11), seqexpress (12) and arrayquest (13). All of them are limited to geo, mare being the only tool which combines both of the major public microarray repositories . Mare is also the only tool which offers robust searching functions using diverse types of parameters and keywords . Another mare - specific function is search in the microarray repositories via pubmed . Finally, it is currently the only existing tool specifically designed for searching and downloading of public microarray data to a local machine . Mare is therefore a valuable addition to the existing tools, which are primarily designed for data analysis . Simultaneous access to both repositories; search in the repositories via pubmed; selection of platforms on which experiments have actually been done; batch downloading of entries . In the coming years, large - scale meta - analysis of microarray data will become more and more important . Our tool facilitates this type of analysis by querying and automatic downloading of relevant data from the most frequently used microarray data sources in the public domain.
Haemangiomas are the most common benign liver neoplasm and result from ectatic growth of the vasculature . These lesions can grow to a significant size and are referred to as giant haemangiomas once larger than 5 cm . Despite the absence of invasive or metastatic potential, hepatic haemangiomas can result in pain, obstructive symptoms, rupture with life threatening haemoperitoneum or a consumptive coagulopathy known as kasabach merrit syndrome . Consensus agreement on the indications for surgical resection, other than for symptomatic lesions, including for giant haemangioma, is lacking at present . There is a paucity of available literature outlining the behaviour of haemangiomas during pregnancy, however female sex hormones have been implicated in their pathogenesis and growth . The nature and extent of this relationship is yet to be fully elucidated . On the other hand, the diagnosis of a symptomatic giant haemangioma during pregnancy presents a dilemma because the safety of neither conservative nor surgical resection has been rigorously investigated . At our centre, a 30-year - old g2p0m1 female underwent resection of a symptomatic giant hepatic haemangioma during the second trimester without any complication . An otherwise well 30-year - old female presented to her general practitioner complaining of intermittent right upper quadrant discomfort over the preceding months . The patient was referred to hospital and ct imaging confirmed a pedunculated giant hepatic haemangioma (13.8 cm 10.2 cm 7.4 cm) attached to segment 4 of the liver (figs 1 and 2). Two smaller intra - parenchymal haemangiomas in segment 6 and 8 were also identified and measured 0.5 cm and 1.3 cm, respectively . Despite a negative urine beta hcg test at the time of ct scanning, she was subsequently found to be pregnant . False negative results such as this are reported to occur, especially in the early luteal phase when the urine hcg may not have reached the diagnostic threshold . Approximately 1 year prior, she had ceased the combined oral contraceptive after taking it for 10 years and was planning to conceive . Figure 1:coronal ct image in the portal venous phase demonstrating the large exophytic haemangioma . Figure 2:axial ct image in portal venou phase at level slightly inferior to aortic bifurcation demonstrating the peripheral nodular enhancement of the haemangioma . Axial ct image in portal venou phase at level slightly inferior to aortic bifurcation demonstrating the peripheral nodular enhancement of the haemangioma . On first review in our clinic she was counselled by a consultant liver surgeon and obstetrician about the available management options and associated risks . The surgical options discussed included; close observation throughout pregnancy and elective resection post - partum, termination of pregnancy followed by resection or resection during the second trimester of pregnancy . Our patient decided to have the tumour resected whilst pregnant due to the concern of rupture, accepting the risks of surgery to herself and the foetus . The operation was scheduled for the second trimester as this is the period in which subsequent miscarriage or preterm labour are least likely . The resection was performed at 18 weeks gestation under general anaesthesia via a small upper midline incision . Upon entering the peritoneal cavity the exophytic haemangioma (fig . This allowed removal with minimal disruption of liver parenchyma, major vasculature or bile ducts . Minimal blood loss occurred and operating time was approximately 1 h. the smaller intra - parenchymal lesions were not resected as they were not the likely cause of her symptoms and represented a lower risk of rupture . Intraoperative foetal monitoring was not indicated due to the pregnancy still being in the previable phase . Figure 3:intraoperative image demonstrating the exophytic nature of the haemangioma and relevant anatomical structures . Obstetric examination in the immediate post - operative period was consistent with a healthy foetus and the patient was discharged from hospital on the third post - operative day . Histopathological examination of the tumour confirmed a haemangioma weighing 412 g. the patient received outpatient follow - up and did not experience any surgical or obstetric complications . A healthy infant was delivered at term via an elective caesarean section due to maternal request . There is little available literature describing the outcome of liver haemangioma resection during pregnancy . To our knowledge, the only available publication is an isolated case report of a successfully resected giant haemangioma at 16 weeks gestation due to kasabach merit syndrome . The scarcity of data may be representative of how infrequently this operation is performed during pregnancy . Both pregnancy and hormonal therapy, including the oral contraceptive, have been associated with haemangiomas enlargement . Rapid growth and haemorrhagic complications have been reported but a recent small case series (four patients, seven pregnancies) found that these tumours can be safely observed during pregnancy . A larger observational cohort study in non - pregnant individuals found that haemangioma resection can be justified due to minimal morbidity and no mortality in the 13 patients who underwent surgery . However, these authors also found that no tumours under 5 cm increased in size or ruptured in patients who received conservative management . Obviously, extrapolation of these findings to our patient would be misguided as the large peripheral exophytic tumour was> 5 cm and she was pregnant . Spontaneous rupture is the complication most feared because this is associated with a mortality rate approaching 80% . A recent large study has estimated that the rupture rate of giant liver haemangioma is 3.4% with peripherally located and exophytic lesions being at higher risk . Accelerated growth, increased intra - abdominal pressure and direct contact with the gravid uterus are all plausible mechanisms for spontaneous rupture or worsening symptoms during pregnancy . Intrapartum rupture of liver haemangiomas has been reported, supporting the notion that increased intra - abdominal pressure may be a causative factor . Our patient's tumour was assessed as being high risk for complications during pregnancy due to its size, exophytic nature and subumbilical position . Therefore, we felt that the risks associated with surgery were justified in this case as spontaneous or intrapartum rupture could be an unsurvivable event . To our knowledge, this is only the second case report of a successful hepatic haemangioma resection during pregnancy . The natural history of these lesions during pregnancy is incompletely understood and resection should be considered if worsening symptoms or the clinical concern for rupture is significant . An interdisciplinary approach that involves surgical, anaesthetic and obstetric specialists is required to perform this procedure safely, and a good overall result for mother and child is possible.
Angiopoietin-2 (ang2) works in concert with vegf to promote neoangiogenesis, and in the absence of vegf, vessels that have been destabilized by ang2 will undergo apoptosis and regress . Though there has been numerous biochemical data to support this paradigm [17], there is sufficient data to suggest a more complex role for ang2 . For example, at high concentrations ang2 acts as an agonist of tie2/tek, providing a prosurvival signal to endothelial cell (ec), which is a similar function as ang1 . Although all tumor models show an upregulation of ang2, its role in tumor angiogenesis has proven to be quite complex and variable, depending on the tumor model investigated [814]. Ang2 upregulation is seen primarily in ec of small cell lung cancer, hepatocellular carcinoma, neuroblastoma, gastric cancer, colon cancer, and kaposi sarcoma, with ang2 being associated with poor prognosis in many of these tumors [816]. Upregulation of ang2 along with vegf upregulation suggests that vessel destabilization by ang2 is a critical step required to allow for vegf - induced neoangiogenesis . In astrocytomas, ang2 has been found to be upregulated in gbms compared to lgas and nb [11, 1719]. The source of ang2 is mainly reported to be the ec; however, one study and our own unpublished data suggest that ang2 may also be expressed by malignantly transformed astrocytoma cells . A noteworthy observation made by stratmann et al . Is that expression of ang2 appears to be vessel size- or vessel type - dependent . Ang2 expression was confined to ec of smaller vessels and not seen in larger vessels suggesting that ang2 promotes in - situ angiogenesis and is more intimately involved with capillary - like vascular structures in tumors [18, 20]. A more recent study identifies ang2 as a marker of tumor cell invasion in high - grade astrocytomas, with little ang2 expression seen in the center of human gbm compared to the invasive peripheral edge of the tumors where ang2 is expressed by both the vascular and neural elements . They also found that upregulation of ang2 in u87 xenografts had a pronounced invasive phenotype compared to the parental u87 mg xenografts that had no ang2 expression . They propose that ang2 confers a more invasive phenotype to the tumor cells via either activation of mmp-2, independent of tie2/tek receptor activation, or perhaps via activation of integrins . In this study, we have focused on deciphering the distinct contribution of ang2 to gbm angiogenesis and vessel development . Additionally, ang2 induces vascular architectural changes that are pathological and aberrant in comparison to control tumor vessels . This aberrancy in vasculature is not seen in human gbms, which suggests that ang2 is not constantly upregulated in human tumors and alludes to a stage - dependent upregulation of ang2 . Established human u87-mg gbm cells were obtained from american type culture collection (atcc, rockville, md) and u373-mg cell lines were a gift from b. westermark (uppsala, sweden). These gbm lines were chosen as they provide variability in their degree of baseline angiopoietin and vegf - a expression, in addition to variable tumorigenicity potential and differences in genetic aberrations . They were maintained in dulbecco's minimal essential medium (dmem) (cellgro, herndon, va) supplemented with 10% fbs and penicillin - streptomycin antibiotics . Full - length human ang2 cdna (a gift from k. alitalo, helsinki, finland) was subcloned into the psec vector (invitrogen) to allow generation of myc - histidine epitope - tagged constructs . The ang - myc / his sequence was subcloned into the bamh1 and ecor1 sites of the pcagg vector that contained a cmv promoter along with a chicken -actin enhancer element . Stable cell lines expressing ang2, were generated by transfection of the vector pcagg - ang - myc / his - zeocin into u87 and u373 gbm lines using lipofectamine 2000 (gibco / brl) as per the manufacturer's instructions . Twenty stable clones, selected with 1mg / ml of zeocin (invitrogen), were examined for ang2 expression by western blot analysis as described below . Two single clones with highest expression of ang2 above baseline parental levels, as well as one pooled clone of ang2 were selected for each of the three gbm lines (u87:ang2, u373:ang2). Corresponding control stable cell lines were generated using empty - vector transfectants . As described previously, briefly, u87-mg cell lines were transfected with ptet - off (clontech, palo alto) vector and stable clones selected and maintained in 1 mg / ml and 500 g / ml of g418, respectively . Thirty of the tet - off clones were assayed by transfecting with the reporter construct ptre - luc and subsequent examination of luciferase activity with a luciferase assay . The highest tetracycline inducible clone was selected to generate double stable tet - off cell lines (data not shown). Double stable tet - off u87-mg cell lines overexpressing ang2 were generated by cotransfecting u87-mg: tet - off stable cells with ptre - ang2 with the ptk - puromycin vector . Stable clones were selected in 3 mg / ml of puromycin, and twenty clones were tested for induction of ang2 expression by immunoprecipitation followed by western blotting, as described below . For control u87-mgtet - off double stable cell lines, ptre - red vector expressing the ds - red fluorescent protein was used . In vitro testing of tetracycline induction of ang2 expression was determined using varying doses of doxycycline, with the most tightly regulated clones expressing ang2 selected for in vivo experiments . Subcutaneous xenografts were generated by growing u87-mg stable clones overexpressing ang2 in the flanks of nod - scid mice . For each stable clone, seven mice were injected with 10 cells suspended in 300 l of pbs, with five mice injected with control empty vector transfectants . Tumor growth was measured biweekly, using calipers by two observers in a blinded fashion . As per animal protocol, mice were sacrificed by cervical dislocation after 100 mg / kg brdu injection (sigma - aldrich). Tumors were cut in cross sections, with two cross - sections kept in formaldehyde for paraffin blocks and immunohistochemical analysis and the remaining tumor stored in liquid nitrogen . Tet - off regulated human u87-mg: ang2 cells (10) were stereotactically injected 3 mm deep into the frontal cortex of nod - scid mice . Mice were treated with dox in the drinking water with three doses of 0, 1, and 10 mg / ml . These doses were selected based on prior published studies demonstrating that dox crosses the blood - brain barrier efficiently to regulate gene expression in the brain . When animals exhibited symptoms consistent with failure to thrive or raised intracranial pressure, the mice were sacrificed by perfusion fixation after brdu injection and tail vein injection of 2% evans blue solution (2 ml / kg) to determine intraluminal blood flow and vessel permeability . The time interval between the injection of evans blue and the perfusion and killing of the mice was approximately 30 minutes . Four different tumor portions were each cut at 5 m consecutive paraffin sections and stained with the ec specific marker anti - factorviii (dako; 1: 2000), followed by detection with an avidin - biotin complex method-3,3-diaminobenzidine (vectastain elite; vector laboratories) system . Microvessel density (mvd) was calculated by counting the number of hollow lumen vessels in ten high - power fields (hpf:500x) and in five hpf at vascular hot spots . Areas that included abnormal vascular structures, such as glomeruloid bodies, were not included in the mvd count as the functional status of these vascular units in both human and xenograft tumors is not known . All analyses were carried out using the microcomputer image device (mcid - imaging research, inc .) Linked to a color ccd camera (sony dxc 970 md) mounted on a transmitted - light microscope (zeiss axioskop). Ihc for ec and smc staining was performed using factorviii antibody and smooth muscle antigen (sma) staining . Standard hematoxylin and eosin (h&e) staining and immunohistochemical analysis was performed on 5 m tissue sections from paraffin embedded tissue blocks . Primary antibodies used include: factorviii (rabbit polyclonal antibody #a0082; dako; used at 1: 2500) and a polyclonal goat anti - ang2 antibody (1: 200 and 1: 400, santa cruz). Secondary antibody was a goat antimouse antibody (zymed) used at 1: 200, and antigens were detected using the avidin - biotin complex method (vector laboratories) and diaminobenzidine substrate . All analyses were completed using statview 4.1 for the macintosh (abacus concepts, berkeley, ca). All errors were calculated as the standard error of the mean (s.e.m . ). One - tailed student's t - test was used to compare means (two sample, unequal variance) with p <.05 considered statistically significant . Parental u87 mg and u373 mg cells have no detectable ang2 (figures 1(a) and 1(b)). Overexpression of ang2 did not alter the in vitro proliferation rate, morphology, or the vegf expression of the cells compared to parental controls (data not shown). Stable transfectants overexpressing the highest levels of ang2 (a2 - 1) and one pooled (a2-p) clone were selected for subsequent experiments (figures 1(a) and 1(b)). Tet - off regulated ang2 stable clones were also established in u87 mg cells, with the most tightly regulated clones selected for in vivo studies (figure 1(c)). In the u87mg: ang2 tet - off clone, dox at 5000 ng / ml was sufficient to decrease ang2 expression to undetectable levels, as seen in control cell lines (figure 1(c)). We assessed the impact of ang2 on the growth of gbm xenografts in both subcutaneous (s.c). And intracranial (i.c .) Tumor models using stable cell lines of u87 mg and u373 mg overexpressing ang2 (figure 1(b)). Xenografts, ang2 overexpression resulted in a significantly faster tumor growth and larger final tumor size compared to controls (figure 1(b) and table 1). In i.c . Xenografts, ang2 conferred a growth advantage as suggested by a significant decrease in survival and tumor proliferation (figure 1(c) and table 2). The response to ang2 was dose - dependent with respect to survival, tumor proliferation, and vascularity (figure 1(c) and table 2). Mice treated with 0 mg / ml of dox in the drinking water, hence those with xenografts expressing high levels of ang2, had a significantly shorter survival time, and tumor proliferation was increased by 2.2-fold compared to the mice receiving either 1 or 10 mg / ml of dox, which had comparable survival to controls (figure 1(c) and table 2). Ang2 is not expressed endogenously by u87 mg cells (figure 1(a)), therefore, addition of dox can completely turn - off exogenous ang2 and result in similar tumor growth and survival of mice as that of controls . Ang2 upregulation resulted in increased mvd and altered vessel size and ec distribution in both s.c . And xenografts (figure 2, tables 1 and 2). Additionally, in both s.c . And i.c . U87mg: ang2 xenografts, there was an abnormal vascular architecture, characterized by preponderance of small vessels, cord-like distribution of ec and whirling of ec present throughout the tumors, in addition to increased numbers of dilated vessels (figure 2(a)i and ii). The alterations in the microvasculature were dependent on levels of ang2 expression that were regulated by dox in the i.c . U87mg: ang2 models (figure 1(b) and table 1). At high levels of ang2 (0 mg / ml dox), a large number of dilated vessels were present, along with abnormal ec distribution throughout the tumor (figure 2(b)). With dox suppression (10 mg / ml dox) of ang2, ec distribution, vessel size, and the overall microvasculature architecture returned to similar structural patterns as is seen in control u87 mg tumors (figure 2(b) and table 2). These vascular alterations have not been reported previously; though the recent publication by hu et al . And lee et al . Makes the observation of impaired angiogenesis by ang2, astrocytomas angiogenesis is postulated to be highly tumor stage - dependant . At their initial growth phase, they coopt and parasitize existing host vessels in an attempt to support their growth, thus the first phase being independent of the tumor angiogenic process [18, 2224]. The second growth phase begins when the host mounts a defensive response and the parasitized vascular supply regresses resulting in tumor hypoxia and necrosis, which in turn triggers upregulation of ang2 and vegf [23, 24]. Therefore, ang2 appears to play a highly phase - dependent role in the progression of malignant astrocytomas . It plays a pivotal role in the cooption of host vessels in the initial phase; and in supporting in - situ tumor angiogenesis, while in the second phase it allows destabilization of mature vessels, by antagonizing ang1-mediated tie2/tek activation, in order to facilitate mitogenic stimulation of ecs by vegf and promoting tumor neovascularization [22, 24]. Xenografts, led to an increase in growth rate, final volume and proliferation of gbms along with an increase in tumor angiogenesis . Ang2 upregulation resulted in an alteration of vascular structures, marked by abnormal ec distribution, with ec forming cord or capillary - like structures and areas of ec whorling present throughout the tumor, in addition to a high number of dilated vessels . In the model used in this study, there is constant upregulation of ang2 throughout all stages and phase of gbm tumor growth, potentially providing a continual trigger for host vessel cooption and promoting in - situ angiogenesis, thereby increased tumor growth . On the other hand, lee et al . They observe a bimodal expression pattern of ang2 in astrocytomas and support the postulate that ang2 is a vessel destabilize, seen at sites of tumor cell growth, tumor periphery, and around sites of necrosis, presumably to promote neoangiogenesis and support tumor cell growth . However, quite contrary to what one would predict based on this observation, lee et al . Also found that ang2 treatment of u87 mg xenografts did not promote but rather restricted astrocytoma growth . Moreover, at first glance these results appear to be in opposition with our observations; however, on closer analysis, both findings can be seen as corroborative and together explain the complex tumor phase - dependent role of ang2 . Xenografts on day 4 after tumor implantation followed by biweekly injections of ang2, whereas in our model ang2 is upregulated constantly throughout all stages of tumor growth . The difference between the level and stages of ang2 upregulation in the two models supports the postulate that ang2 plays a highly tumor stage - dependent role . Another evidentiary data that ang2 plays a stage dependent role in glioma angiogenesis is the fact that tumor vascular structures observed in our xenograft models are not evident in human gbm specimens, indicating that ang2 is not upregulated throughout all stages of human gbms, and most likely plays a very precise role at specific stages of gbm growth . The abundant cord or capillary - like vessels in the u87mg: ang2 xenografts may be a result of ang2-mediated modulation of ec motility, migration, and invasion . Demonstrate regions of ang2-expressing tumors actively invading the brain, high levels of mmp-2 expression, and increased angiogenesis . Additionally, ang2 is known to influence the fate of new tumor vessels, differentiating them into capillaries versus arteries or venous structures . The most likely explanation is that ang2 presents an inhibitory signal, preventing ang1-mediated maturation by of the newly formed tumor vessels . Taken together our data indicates that increased ang2 promotes angiogenic growth of gbms . Constant upregulation of ang2 throughout all phases of tumor growth results in the abnormal vascular structures seen in our xenografts that are not present in human gbms, suggesting that ang2 upregulation in gbms is very much a tumor stage dependent process and not constant throughout all stages of gbm growth . Future studies are required to decipher the precise temporal role of ang2 and whether the combinatorial impact of other angiogenic cytokines with ang2 can be used for therapeutic targets in treatment of gbms.
The increased prevalence of overweight and obese children is not new, yet it has been viewed more recently as a public health epidemic [13]. According to the centers for disease control and prevention (cdc), approximately 12.5 million, or 17%, of american children are obese . More recent studies show that over 10% of 25-year - olds would be classified as overweight, whereas this number increases to 15% in the adolescent age group . Interestingly, in 2000 the us preventive services task force (uspstf) did not find enough evidence to recommend for or against routine screening for overweight status in either children or adolescents as a means of mitigating further health sequelae, yet now the task force is revisiting this idea as of 2010 . It is known that a higher prevalence of comorbid diseases attributable to obesity is seen in both adults and children, especially modifiable cardiovascular risk factors and sequelae . In the long - standing bogalusa heart study, body mass indexes (bmi) performed in childhood and adolescence, as a measurement for obesity, predicted intima - media thickness in adults . Obesity is not only related to modifiable cardiovascular risk factors, but also to several other health - related conditions that may persist or worsen in adulthood [7, 8]. This includes asthma, orthopedic disorders, depression and anxiety, liver abnormalities, and endocrine related issues including type 2 diabetes, hyperlipidemia, and polycystic ovarian syndrome (pcos). Further studies may reflect resolution in these conditions with increased awareness of the association between obesity and multiple comorbid conditions . Therefore, we sought to examine the incidence of the diverse sequelae of obesity and morbid obesity, as evidenced by its associated comorbidities in a single, large, free - standing, children's hospital . This study was conducted with the approval of the institutional review board at boston children's hospital (irb - p00001304). All inpatient and outpatient visits to boston children's hospital between january 1, 2000, and december 31, 2012, were included in the dataset . These visit were queried through the informatics for integrating biology and the bedside (i2b2) data warehouse platform (i2b2 v. 1.4; usa) implemented at the institution . The i2b2 system allowed investigators to perform queries on an enterprise data repository through its web - based interface . Selected demographics information in epic computer system and information on hospital - billed diagnoses in cerner, in the form of international classification of diseases, 9th revision, clinical modification (icd-9) codes, were available through i2b2 . The data collected was not stratified based on age, gender, nor whether the visit was an inpatient or outpatient visit in nature . Using the i2b2 web client, queries were entered in the form of inclusion or exclusion filter groups based on icd-9 codes and date criteria, with date criteria based on the start date of the encounters . We obtained counts for different patient groups by year based on the respective administrative diagnosis . Temporal constraints were set to treat all groups independently as opposed to have the filter groups occur in the same financial encounter . To account for the children and adolescents in our study population, both adult and pediatric icd-9 criteria were used in determining obesity status, utilizing both the bmi categorization standard as defined by world health organization and the percentile standard as defined by american medical association [12, 13]. Icd-9 code criteria for obesity status and comorbid conditions are further described in table 2 . A single condition was considered met if one or more of its icd-9 criteria were coded . To help limit the number of double - counting, a patient was only counted once in each patient group regardless of how many icd-9 criteria of the same condition the patient has met in that particular year . The main comorbid conditions included diabetes mellitus, hypertension, hyperlipidemia, sleep apnea, degenerative joint disease, asthma, and depression . Overall rates of these related comorbid conditions were calculated for obese and morbidly obese versus nonobese patients, as well as the annual increase for all patients over that same time period . Other obesity - related comorbid conditions that we examined included acanthosis nigricans, nonalcoholic steatohepatitis (nash), pcos, glucose intolerance, insulin resistance, left ventricular hypertrophy (lvh), elevated blood pressure, and pseudotumor cerebri . The incidence rate ratio (irr) was then calculated to see if obese and morbidly obese patients were disproportionately affected by these conditions . In total, a retrospective review of 3,185,658 person - years in nonobese, 26,404 person - years in obese, and 25,819 person - years in the morbidly obese children was conducted . The annual rate of patients coded with obesity increased from 0.86% in 2000 to approximately 2.78% in 2012 at our institution as shown in figure 1 . Figure 1 also illustrates that the annual rate of patients coded with morbid obesity increased from 0.34% in 2000 to 1.51% in 2012 . When comparing differing major comorbid conditions, it appeared that the proportions of patients coded as obese or morbidly obese remained constant at the various time points including 2000, 2005, and 2010 . On the other hand, when comparing the differing preconditions, it appeared that there was a slight increase in the proportions of patients coded as morbidly obese versus obese . This was especially prominent in the cohort of endocrine conditions including acanthosis nigricans, pcos, and insulin resistance . The annual rate of all major comorbidities (diabetes mellitus, hypertension, hyperlipidemia, sleep apnea, degenerative joint disease, asthma, and depression) increased in all pediatric patients over the study period . Other obesity - related comorbid conditions, or preconditions, including acanthosis nigricans, nash, pcos, glucose intolerance, insulin resistance, lvh, elevated blood pressures, hypertension, and pseudotumor cerebri, were also increased among all pediatric patients over the study period . However, when comparing annual rates in obese patients alone, the annual rates for obese patients were higher across all categories as compared to the annual rates calculated in all patients (table 3). All of the obesity - related comorbidities were higher among obese versus non - obese patients over the time period from 2000 to 2012 . All comorbidities were also higher in the morbidly obese versus non - obese patients over the same time period . All of the preconditions were noted to have the same findings; all the preconditions were noted to be higher among obese, as were morbidly obese patients versus non - obese patients from 2000 to 2012 . The or did approximate the irr with respect to all these conditions . These findings are seen in table 3 . This current study illustrates the change in the spectrum of obesity - related comorbid conditions in an overweight, pediatric population at a single institution over the past decade utilizing a hospital database system . As expected, children continue to experience the same comorbidities and preconditions associated with obesity as compared to adults . Among the weaknesses of this study, perhaps the most glaring is that this administrative database may have underestimated the overall incidence of obesity, the overall rate of obesity being only 2 - 3% at our institution . However, in spite of this, the spectrum of comorbidities seen in our pediatric patients and the knowledge of these have significant clinical implications . Both the medical and surgical treatment of adult obesity has led to associated refinement of treatment modalities; therefore, we hope to do the same with these data that identify the vast host of comorbidities that are most injurious to our children . On a national level, may et al . Have recently published results regarding cardiovascular sequelae in the national health and nutrition examination survey (nhanes) from 1999 to 2008 . Again, the major cardiovascular risk factors including diabetes, hypertension, and hyperlipidemia were elevated across all cohorts but specifically increased in the overweight and obese cohorts . Similar to their study, we also continued to see a dose - response increase in reportable comorbid conditions across both our obese and morbidly obese cohorts . Unlike their study, we did not collect individual data, but rather administrative coding of diagnoses across both inpatient and outpatient visits . Accordingly, there could have been biased introduced with the respective coding, but our hope is that this large sample ameliorated some of the associated, inherent reporting bias . Comorbid conditions including asymptomatic cardiovascular risk factors (e.g., hypertension, hyperlipidemia) and symptomatic cardiovascular risk factor (e.g., type 2 diabetes) are also prevalent in both the obese and morbidly obese subgroups . In fact, our study shows that hyperlipidemia and hypertension have the broadest effects with both our obese and morbidly - obese populations having the highest weighted or and irr . These results are similar to results of a population - based sample of quebec children and adolescents, where almost one - third of their obese population had unfavorable risk factors; this was evidenced by elevations in apolipoprotein b, high - density lipoprotein cholesterol, triglycerides, insulin, glucose, c - reactive protein, and systolic blood pressure . Thus, despite increased awareness of obesity and intensive medical interventions, obesity and its associated cardiovascular disease risk factors continue to represent an unabated major healthcare burden to this population as a whole ., it is suggested that these comorbidities with cardiovascular implications lend to an increased rate of premature death in early adulthood after adjustment for other confounders . In juxtaposition to this, report that the increased risk for morbidity and mortality may be overestimated following adjustment for adult bmi; however, one of the limitations of this study includes that it was a meta - analysis of very small studies . When examining our data more closely, it is very concerning the degree to which certain preconditions have arisen, including acanthosis nigricans, nonalcoholic steatohepatitis, glucose intolerance, and insulin resistance . These aforementioned conditions will eventually become life - long diseases; therefore, it is imperative to continue to reduce those comorbidities with cardiovascular implications, while at the same time providing more longitudinal analysis of obesity's long - term effects . Obesity and morbid obesity are not only involved in modifiable cardiovascular risk factors, but are also intimately involved in the respiratory system . The evidence correlating the changes in the reactivity and the nature of the pulmonary disease seen with obesity is not new . For example, the nhanes data continues to show that excess weight has stronger association with nonatopic asthma (or: 2.46, 95% ci: 1.21, 5.21) than atopic asthma (or: 1.31, 95% ci: 0.702.57). This becomes a vicious cycle for these children as they are often unable to perform activities of daily living and are less likely to perform in exercise programs, which continues to compound weight - related issues . Our data shows that, even beyond an expected increase in asthma, there has also been a substantial increase in those documented with sleep apnea . For example, our data suggests that the odds of having sleep apnea was 6.48 (95% ci: 6.12, 6.85) times more likely if a patient was coded as obese and 7.60 (95% ci: 7.07, 8.17) times more likely if a patient was coded as morbidly obese as compared to normal weight counterparts . Similar to cardiovascular risk factors, asthma, sleep apnea, and orthopedic problems are also increased in our obese and morbidly obese cohorts . A common theme among these conditions is that they limit the child or adolescent's ability to participate in social activities, which can further alienate this ever - growing proportion of our society . For example, in adults, increasing bmi and subsequent obesity lend itself to knee pain, disability, loss of work productivity, and diminished quality of life . More recent studies in children suggest that obese children report more frequent and severe joint pain with subsequent loss of function [1821]. It is well known that obesity in children and adolescents results in decreased / reduced health - related quality of life . As referenced by the above orthopedic studies, depression might have been masked as complaints of increased joint pain or inability to perform activities performed by non - obese peers . Further studies are needed to elucidate this connection . Even though our study did not examine health - related costs specifically, according to one study, the actual cost of obesity is related to the direct costs of its associated medical conditions . Continued attention and us dollars have been driven toward using policy and environmental changes to help combat the obesity epidemic; only in the last decade or so had there been a shift towards medical and operative interventions . Whereas it has been shown in adults that patients with reduced comorbid conditions attained through weight - loss surgery live longer, the same conclusions have yet to be applied to the pediatric population . In fact, oyetunji et al . Remarked that despite this increased prevalence of obesity and morbid obesity among children and adolescents, only approximately 2% of morbidly obese children with a major comorbidity underwent a bariatric procedure . This may be due in part to a myriad of factors including necessity for higher volume centers for pediatric bariatric procedures, as well as concern for permanent procedures to be performed in children and adolescents . However, there is still a need for novel and equitable approaches to pediatric obesity . This study had important strengths and weaknesses . In the future, we hope to have same functional database as, for example, the national surgical quality improvement program (nsqip) database, which relies on specifically trained personnel who use well - defined terms for data collection and dissemination . We encountered visits that had no documentation regarding vital data points including height, weight, or bmi, thus contributing to the underestimation of the prevalence of obesity . Despite these aforementioned weaknesses, this study's strengths included both its size as well as the duration of the data collection . Obese patients, as well as morbidly obese patients, had significant increases in comorbid conditions including hypertension, hyperlipidemia, sleep apnea, orthopedic issues, and type 2 diabetes mellitus as evidenced by the elevated irr, or, and tightly controlled confidence intervals . In conclusion, these data show the change in the spectrum of obesity - related comorbid conditions in this pediatric population over the past decade . As expected, the children studied have experienced the same dramatic increases in obesity as seen in the adult population and obesity was independently correlated with major comorbidities . Future research will be needed to implement other treatment options to combat the wide variety of disease sequelae.
Jaw dislocation has been reported in the literature as a rare complication of bronchoscopy, intubation, transesophageal echocardiogram, and otolaryngology and dentistry procedures [1, 2, 3, 4]. However, temporomandibular joint (tmj) dislocation is not commonly recognized by gastroenterologists as a potential complication of esophagogastroduodenoscopy (egd) [5, 6, 7, 8, 9, 10]. In this report, we present a patient with temporomandibular displacement after egd and discuss how to identify this complication, the differential diagnosis of postendoscopy jaw pain, and the methods for prevention . We will also review the literature regarding other cases involving temporomandibular displacement following upper endoscopy to assist in the future identification of this complication . A 48-year - old woman with hypertension, rheumatoid arthritis, seasonal allergies, and long - standing solid food dysphagia presented to our gastroenterology clinic with progressively worsening dysphagia and transient food impaction . The patient reported approximately 7 years of solid food dysphagia, but prior to this, has had no upper gastrointestinal symptoms, trouble swallowing, or dietary modification . She had sought prior gastrointestinal evaluation, and an endoscopy 2 years after symptom onset showed rings and furrows, but biopsies were not obtained and dilation was not performed . Though no specific treatments were initiated, the patient did note an improvement in her ability to swallow following the procedure . One year prior to her presentation, she developed recurrent solid dysphagia which progressively worsened . Solid foods, particularly bread and meat, would get stuck in the upper chest and resulted in transient impactions 23 times per week . In most instances, if she waited, the food would ultimately pass; however, in some cases, she would have to regurgitate the food . She reported eating slowly, taking small bites, chewing thoroughly, and drinking a lot of liquids during meals . Because of suspicion of eosinophilic esophagitis (eoe), she was placed on a proton pump inhibitor (omeprazole 20 mg twice daily), and repeat endoscopy was performed . She was given propofol for sedation, and the endoscopy went smoothly, but it was noted that she was yawning multiple times during the procedure . Immediately after the procedure, the patient experienced severe left - sided facial pain . On examination, there was a spasm over the left masseter muscle, and she was unable to close her mouth . In an attempt to control her pain and relax her muscle spasm, the patient was given 25 g of fentanyl and a 30-mg dose of propofol, but these were not successful . Given the severity of her pain and her inability to close her mouth, she was referred to the emergency department for suspicion of jaw dislocation . In the emergency department, the patient required additional doses of fentanyl for pain control . A maxillofacial ct showed an anterior dislocation of the left tmj without a mandibular fracture (fig . 1, 2). The initial attempt at jaw reduction using a 2-mg intravenous dose of midazolam sedation was unsuccessful . The oral maxillofacial surgery team was consulted and successfully reduced the jaw dislocation following administration of a 100-mg dose of ketamine . She remained in the jaw strap and on a mechanical soft diet for 7 days . She was closely followed over the next several days and rapidly improved to her baseline . Further history was obtained and while she has had no prior jaw dislocation, she did report intermittent jaw pain . Of note, the results of the biopsy from her endoscopy confirmed the diagnosis of eoe, which will require the patient to undergo future endoscopies . Anterior dislocation of the tmj is the most common type of jaw dislocation with multiple potential etiologies . Superior and posterior dislocations are rare and almost exclusively associated with trauma and fracture . While anterior dislocation may occur as a result of a dystonic reaction to drugs and seizures, a majority of anterior dislocations occur with extreme opening of the mouth . These causes may include eating, singing, laughing, vomiting, or even yawning, as was noted for the patient presented here . In a retrospective study of 96 cases of tmj dislocation, extreme mouth opening while yawning was identified as the most common cause of dislocation, representing approximately 46% of reported cases . Several reports discuss tmj dislocation as a complication of bronchoscopies, dental procedures, induction of anesthesia, and transesophageal echocardiograms [1, 2, 3, 4]. Amongst the potential iatrogenic causes, several additional factors have been identified as potential sources for increasing the risk of jaw dislocation in cases outside of gastroenterology . These include prolonged procedure time, increased age, and relaxation of the musculature caused by sedating medications . This is an unusual complication of egd, and we have only been able to identify 6 cases reported within the last 30 years in patients ranging from 23 to 83 years of age [5, 6, 7, 8, 9, 10]. In these cases, the endoscopies were uncomplicated, but immediately following each procedure, the patients complained of facial pain and the inability to close their mouth (similar to our case), with two exceptions . The first was a patient who was intubated prior to the procedure and unable to communicate symptoms; the second was a patient who presented the following day with facial pain and the inability to close her mouth appropriately . Another consistent finding amongst these cases and our patient is that all had anterior dislocations . However, the ability to reduce these displacements varied based on the degree or laterality of dislocation and patient experience with prior displacements . For example, in the report from lacy et al ., a patient who had no prior history of dislocations experienced a bilateral displacement which required additional sedation for successful reduction . However, in a report of a young woman with only a unilateral displacement and a well - documented history of tmj dislocations, the patient was able to reduce the displacement herself with the assistance of her endoscopist requiring no further sedation . While a prior history of dislocation is a risk factor for jaw displacement following egd, the lack of a predisposing history does not exclude patients from the possibility of having a tmj dislocation . Of the 6 reported cases, only 2 patients reported a prior history of tmj displacement [6, 10]. Anesthesia is frequently cited as a risk factor for tmj dislocation because it relaxes the masseter and temporalis muscles, which usually act as stabilizers . While propofol was used in our case, in the other reports of jaw dislocation following egd, a benzodiazepine, midazolam or diazepam, was used for sedation [5, 6, 7, 8, 9, 10]. While most reported cases of tmj displacement from a gastroenterology procedure occur as a complication of egd, 2 cases of jaw dislocation have been reported following endoscopic retrograde cholangiopancreatography, and 1 was noted after percutaneous endoscopic gastrostomy tube placement [13, 14, 15]. While jaw dislocation was felt to be the most likely source of this patient's jaw pain following endoscopy, reduction was not immediately attempted due to concern for other possible causes . When patients suffer from tmj dislocation, they will often be unable to close their mouth and have drooling and garbled speech along with pain . For this reason, acute stroke was part of the differential diagnosis and should always be considered in the correct clinical context . Another factor considered in this case was a pathologic fracture . While the diagnosis of tmj dislocation is made based upon clinical examination, radiographic imaging the disadvantage of delayed reduction is an increased risk of developing muscular spasms, which ultimately makes reducing the dislocation more challenging . . She will require additional upper endoscopies for surveillance of treatment response given her new diagnosis of eoe . However, no specific recommendations exist at this time regarding measures that can prevent jaw dislocation during an endoscopy . Given that most jaw dislocations seem to occur as a result of anesthesia, appropriate surveillance of patients who may be over- or undersedated can be important in preventing complications from endoscopy . While it is important to note if a patient has a prior history of tmj dislocation, the lack of such a history should not diminish monitoring for displacement after the procedure . While jaw dislocation is a very rare complication of upper endoscopy, it is important for gastroenterologists to be aware of this possibility, recognize it in the recovery area, and appreciate the differential diagnosis . Similar to previous recommendations [9, 10], as a part of routine evaluation, it may be beneficial to observe patients during mastication after the procedure.
In april 2007, a 30-year - old man from bahia sought care for a 6-day febrile illness that began 9 days after he found a tick attached to his right wrist while hiking and camping in the chapada diamantina national park in paty valley (124826s, 411953w), a semiarid region in bahia . Primary signs and symptoms were fever (3940c), severe myalgia, and swelling and pain at the site of the tick bite . Two days after onset of illness, the man noticed a scab forming on his right wrist and painful swelling in his right axillary region, followed 2 days later by a generalized rash and painful ulcerative lesions in the mouth . The patient sought medical care, and an outpatient physician prescribed acetaminophen and cefadroxil, which did not reduce symptoms . On day 6 of his illness, the patient sought care from an infectious disease specialist, who noted a 2.5-cm eschar on the patient s wrist (figure 1, panel a); disseminated papular rash on his face, trunk, and upper extremities (figure 1, panel b); and several small erosions on his tongue, buccal mucosa, and lips (figure 1, panels c, d). The mucosal erosions were painful, and some skin papules formed small pustules (figure 1, panel e). In the right axilla was a tender, enlarged, 3-cm lymph node . Results of a hemogram and blood biochemistry were unremarkable except for a high level (425 u / l) of lactic dehydrogenase . A rickettsial disease was considered, and the patient was given doxycycline (100 mg 2/d) for 14 days . The fever and generalized rash resolved within 2 days, and the eschar healed completely within 2 weeks after initiation of therapy . Lesions on day 6 of illness of patient with eschar - associated rickettsial disease, bahia, brazil, 2007 . A) eschar on right wrist; b) papular skin rash on left elbow; c) ulcerated lesion on lower lip; d) erosions on tongue mucosa; e) vesicular papular lesions on trunk . Acute - phase and convalescent - phase serum samples were evaluated by microimmunofluorescence assay for antibodies to spotted fever group rickettsiae (sfgr) (4). Before antimicrobial drug therapy was started, biopsy specimens of the papule and the scab from the eschar were collected, preserved in 10% formol, and evaluated by routine histopathology, immunohistochemical staining, and pcr (4,5). Serum collected on day 6 of the illness was nonreactive with r. rickettsii and r. parkeri antigens (class - specific immunoglobulin g [ig] and igm <32 for both assays, cutoff> 64). Subsequent testing determined igg / igm titers on day 12 to be 128/<32 against r. parkeri and 128/32 against r. rickettsii antigens and on day 19 to be 128/64 and 512/32, respectively . Hematoxylin and eosin stained sections of the papule biopsy specimen demonstrated lymphohistiocytic perivascular inflammatory cell infiltrates in the superficial to middle dermal layers . Immunohistochemical staining for sfgr showed rare antigens in a few small foci of perivascular inflammation . Gq900666) from the papule specimen each had 100% identity to homologous gene sequences of sfgr detected recently in an eschar specimen from a patient from peruibe, so paulo (3). The sequences from both organisms were most related to sfgr strain s previously reported from armenia (6) but were not identical to r. sibirica, r. parkeri, and r. africae (figure 2). Gq853064) had 99% identity to the homologous fragment of r. parkeri (genbank accession no . Genetic relationships of the spotted fever group rickettsiae (sfgr) detected in tissue of patient with eschar - associated rickettsial disease, bahia, brazil, 2007 . The phylogenetic optimal tree was inferred by using the neighbor - joining method, and distances were evaluated by implementing the kimura 2-parameter model of substitution (sum of branch length = 0.58588522). In total, 323 nt sites of glta and 401 nt sites of ompa were concatenated and evaluated; primer sequences and sites containing gaps and deletions were excluded from the analysis . Statistical reliability of the tree is based on 1,000 bootstrap replicates; only bootstrap values> 50 are shown above the branches . The corresponding sequences of reference species and isolates were obtained from the national center for biotechnology information genbank database . Bahia and other previously characterized sfgr; b) expanded tree of relationships among new sfgr to r. africae, r. parkeri, r. sibirica, rickettsia sp . During the past decade, many newly identified tick - borne rickettsiae from south america have been described (1,2), including r. parkeri, r. massiliae, r. amblyommii, r. bellii, and other rickettsia spp . Of unknown pathogenicity . We describe another confirmed case of a novel eschar - associated sfgr disease in brazil . Development of an eschar is a characteristic manifestation of rickettsioses caused by r. parkeri, 364d rickettsia, and r. massiliae (4,7). Possible eschar formation in association with rocky mountain spotted fever has been reported (8), but this manifestation does not seem to be a hallmark of disease caused by r. rickettsii or of other rickettsioses in brazil and south america (2). Bsf has been most often confirmed solely by serologic testing; however, atypical clinical manifestations, including eschar formation and lymphadenopathy, have been described (912). Lymphadenopathy and ulcers on the oral mucosa, as found for this patient, have been found in patients with rickettsiosis caused by r. parkeri and african tick bite fever (caused by r. africae) (4,13) but not in the index case - patient from so paulo (3), who seemed to have less severe clinical manifestations than the patient described in this report . In the scientific literature from brazil, the earliest reference to an eschar in a suspected case of bsf was in 1932 (12). Subsequent eschar - associated cases have been identified in regions where bsf is endemic (e.g., the states of minas gerais, rio de janeiro, and espirito santo) (911) and in regions where it is not endemic (e.g., states of santa catarina, situated along the argentina border, and bahia, where the case reported in this article occurred). Furthermore, clinical descriptions of eschar - associated rickettsioses in brazil have been reported from bsf - endemic areas with large populations of a. dubitatum ticks but no known a. triste ticks, which are recognized vectors of r. parkeri in southern brazil (15). Although a. dubitatum, a human biting tick that is highly prevalent in many bsf - endemic areas (2), is a potential candidate for transmission of r. parkeri to humans in brazil, this tick species and its vertebrate hosts, capybaras, have not yet been described in the paty valley, bahia, where the patient acquired the rickettsial infection . Unfortunately, the ticks causing both cases in so paulo and bahia were not available for identification . The taxonomic status of the etiologic agent of this novel rickettsiosis in brazil cannot be definitively determined until it is isolated . On the basis of the available genetic information presented here and elsewhere (3), the pathogen detected in the cutaneous lesion of the patients from bahia and so paulo is equally distant from r. africae, r. parkeri, and r. sibirica . Each of these 3 sfgr is among species long accepted by international committee of systematics of prokaryotes, and this status is consistent with their long evolutionary divergence and differences in their vectors and geographic distributions . Molecular confirmation can and must therefore be used to identify new rickettsial agents because they cannot be identified by clinical case presentations or serologic analyses . Additional efforts will be required to establish the full genetic diversity and range of tick and animal reservoirs of sfgr in brazil and to determine the prevalence and clinical presentations of different rickettsioses in humans . Clinicians should be alert for tick - borne infectious diseases resulting from ecotourism activities, especially in parks and ecologic reserves in the areas of the atlantic forest and other areas of brazil where many rickettsiae - infected ticks have been identified and most bsf cases have been reported . Since submission of this article, recent investigation in brazil has identified a. ovale ticks as potential vectors for the spotted fever group rickettsia sp . Described here (16).
Pancreatic neuroendocrine tumors (pnets) are a relatively rare and heterogeneous group of neoplasms comprising 12% of all pancreatic neoplasms and 2.6% of pancreatic cancers . Due to the widespread use of high - quality imaging techniques, the incidence of pnets has remarkably increased from 0.17 to 0.43/100,000 over the past three decades in the united states . Furthermore, autopsy studies indicate that the prevalence of pnets may be even higher . Depending on whether clinical symptoms are related to excessive secretion of endocrine hormones, pnets are divided into functional and nonfunctional pnets (nf - pnets). Nf - pnets are defined by the absence of a hormone hypersecretion syndrome and account for approximately 90% of all pnets . Nf tumors are asymptomatic or present with nonspecific symptoms related to local mass effect or metastatic disease and are associated with a poorer prognosis than functional tumors, probably due to delayed diagnosis and higher malignant potential . Compared to pancreatic ductal adenocarcinoma, nf - pnets often have an indolent outcome but postoperative recurrence is not rare . Lymph node metastasis (lnm) regarding nf - pnets, lymph node status is regarded as an important prognostic factor in both the european neuroendocrine tumor society (enets) tnm staging system and the american joint committee on cancer (ajcc) cancer staging system . In current guidelines, the national comprehensive cancer network (nccn) guidelines do not advocate routine lymphadenectomy in tumors <2 cm, while this procedure is recommended for tumors that are 12 cm because of the risk of lnm . Moreover, previous reports have demonstrated that only 3040% of patients with nf - pnets present with lnm at diagnosis, which suggests the importance of preoperative recognition of patients at high - risk of lnm, who may benefit from regional lymphadenectomy . Therefore, the identification of reliable predictors of lnm is important in guiding clinical management decisions and avoiding an unnecessary lymphadenectomy in low - risk patients . The aims of this study were: (1) to evaluate the impact of lnm on postoperative recurrence of patients with surgically treated nf - pnet; (2) to evaluate the feasibility of preoperative prediction of lnm in nf - pnets using preoperatively available variables . This was a mono - institutional retrospective cohort study of the clinical records of 100 patients who underwent pancreatic surgery with curative intent for nf - pnet between january 2004 and december 2014 . All patients who were diagnosed with nf - pnet were included (n = 111), whereas syndromic patients (n = 1), patients lost to the postoperative follow - up (n = 6) and patients with distal metastasis (n = 4), were excluded from this study . Nonfunctioning neoplasms were defined by the lack of any clinical syndrome caused by excess hormonal secretion . In total, information about clinical presentation, demographics, data regarding surgical procedures, postoperative course and complications, pathologic findings, and follow - up was collected . All patients underwent presurgical computed tomography (ct) evaluation of the dimensions, local invasiveness, and the presence of lymph node or distant metastasis . The pathological diameter of neoplasms was defined as the largest diameter of the surgical specimens . Standard or parenchyma - preserving resection was selected for according to tumor size and anatomical location . All patients in this study had at least one lymph node sampled on resected specimens . The extent of regional lymphadenectomy was the same as that performed in cases of pancreatic ductal adenocarcinoma . For tumors located in the pancreatic head, regional nodes consisted of those located along the common bile duct, common hepatic artery, portal vein, superior mesenteric vein, posterior and anterior pancreatic head, and the right lateral wall of the superior mesenteric artery . For tumors located in the pancreatic body or tail, regional nodes included those along the common hepatic artery, celiac axis, splenic artery, and splenic hilum . The surgical specimens of all cases were classified according to the world health organization (who) classification criteria (2010). All pnets were divided according to a grading scheme based on mitotic count or ki67 index into g1 (mitotic count <2/10 high - power fields (hpf) and/or 2% ki67 index), g2 (mitotic count 220/10 hpf and/or 320% ki67 index), and g3 (mitotic count> 20/10 hpf and/or> 20% ki67 index). The enets recommended tnm staging system was used for tumor staging . Primary tumors (t stage) were classified into four categories: t1, tumor limited to the pancreas and size <2 cm; t2, tumor limited to the pancreas and size 24 cm; t3, tumor limited to the pancreas and size> 4 cm or invading the duodenum or bile duct; and t4, tumor invading adjacent organs (stomach, spleen, colon, and adrenal gland) or the wall of large vessels (celiac axis or superior mesenteric artery). All patients enrolled in this study underwent a postoperative clinical and radiological follow - up . All patients underwent a radiological examination by ct scans every 612 months after surgery, and magnetic resonance imaging was performed if necessary . If patients had any symptoms suspected to be associated with tumor progression during follow - up, a radiological examination was performed immediately to rule out recurrence or distant metastasis . Disease - free survival (dfs) was calculated as the months between surgery and 30 jun 2015 or the first documented disease recurrence . An acute postoperative mortality was defined as death which occurred within 30 days after surgery . The data on the operation and postoperative morbidity was collected from the electronic patient records of our institution . Phone interviews were conducted for all patients with a response rate of 95% (105 patients) and data on survival status, date of death, and tumor recurrence were collected . For all living patients, data were expressed as a mean standard deviation for continuous variables and as number and percentage for categorical variables . The comparison between subgroups was performed by the analysis of variance for quantitative variables and by the chi - square test or fisher exact test for categorical variables, when necessary . The cox regression model was used in univariate and multivariate analyses to evaluate the independent predictive factors of postoperative recurrence . Hazard ratios (hr) and 95% confidence intervals (ci) were also calculated . Variables with p 0.05 in univariate analysis were included in the multivariate model . Logistic regression analysis performed in a stepwise fashion with backward selection was used to evaluate the value of clinical factors for predicting lnm and to establish the preoperative predictive model . Receiver operating characteristic (roc) curve analysis was performed to assess the predictive power of the models through calculating the area under the curve (auc). Spss version 20.0 (ibm, usa) was used to perform all the data analysis . This was a mono - institutional retrospective cohort study of the clinical records of 100 patients who underwent pancreatic surgery with curative intent for nf - pnet between january 2004 and december 2014 . All patients who were diagnosed with nf - pnet were included (n = 111), whereas syndromic patients (n = 1), patients lost to the postoperative follow - up (n = 6) and patients with distal metastasis (n = 4), were excluded from this study . Nonfunctioning neoplasms were defined by the lack of any clinical syndrome caused by excess hormonal secretion . In total, information about clinical presentation, demographics, data regarding surgical procedures, postoperative course and complications, pathologic findings, and follow - up was collected . All patients underwent presurgical computed tomography (ct) evaluation of the dimensions, local invasiveness, and the presence of lymph node or distant metastasis . The pathological diameter of neoplasms was defined as the largest diameter of the surgical specimens . Standard or parenchyma - preserving resection was selected for according to tumor size and anatomical location . All patients in this study had at least one lymph node sampled on resected specimens . The extent of regional lymphadenectomy was the same as that performed in cases of pancreatic ductal adenocarcinoma . For tumors located in the pancreatic head, regional nodes consisted of those located along the common bile duct, common hepatic artery, portal vein, superior mesenteric vein, posterior and anterior pancreatic head, and the right lateral wall of the superior mesenteric artery . For tumors located in the pancreatic body or tail, regional nodes included those along the common hepatic artery, celiac axis, splenic artery, and splenic hilum . The surgical specimens of all cases were classified according to the world health organization (who) classification criteria (2010). All pnets were divided according to a grading scheme based on mitotic count or ki67 index into g1 (mitotic count <2/10 high - power fields (hpf) and/or 2% ki67 index), g2 (mitotic count 220/10 hpf and/or 320% ki67 index), and g3 (mitotic count> 20/10 hpf and/or> 20% ki67 index). The enets recommended tnm staging system was used for tumor staging . Primary tumors (t stage) were classified into four categories: t1, tumor limited to the pancreas and size <2 cm; t2, tumor limited to the pancreas and size 24 cm; t3, tumor limited to the pancreas and size> 4 cm or invading the duodenum or bile duct; and t4, tumor invading adjacent organs (stomach, spleen, colon, and adrenal gland) or the wall of large vessels (celiac axis or superior mesenteric artery). All patients enrolled in this study underwent a postoperative clinical and radiological follow - up . All patients underwent a radiological examination by ct scans every 612 months after surgery, and magnetic resonance imaging was performed if necessary . If patients had any symptoms suspected to be associated with tumor progression during follow - up, a radiological examination was performed immediately to rule out recurrence or distant metastasis . Disease - free survival (dfs) was calculated as the months between surgery and 30 jun 2015 or the first documented disease recurrence . An acute postoperative mortality was defined as death which occurred within 30 days after surgery . The data on the operation and postoperative morbidity was collected from the electronic patient records of our institution . Phone interviews were conducted for all patients with a response rate of 95% (105 patients) and data on survival status, date of death, and tumor recurrence were collected . For all living patients, data were expressed as a mean standard deviation for continuous variables and as number and percentage for categorical variables . The comparison between subgroups was performed by the analysis of variance for quantitative variables and by the chi - square test or fisher exact test for categorical variables, when necessary . The cox regression model was used in univariate and multivariate analyses to evaluate the independent predictive factors of postoperative recurrence . Hazard ratios (hr) and 95% confidence intervals (ci) were also calculated . Variables with p 0.05 in univariate analysis were included in the multivariate model . Logistic regression analysis performed in a stepwise fashion with backward selection was used to evaluate the value of clinical factors for predicting lnm and to establish the preoperative predictive model . Receiver operating characteristic (roc) curve analysis was performed to assess the predictive power of the models through calculating the area under the curve (auc). A two - sided p <0.05 was considered to indicate statistical significance . Spss version 20.0 (ibm, usa) was used to perform all the data analysis . The clinical and pathological data of the 100 nf - pnets are shown in table 1 . Among the 100 patients, more than 50% (n = 53) were symptomatic at the time of diagnosis . Overall, 81 patients (81%) underwent standard pancreatic resection, while 19 patients (19%) underwent parenchyma - preserving pancreatic resection . Postsurgical complications occurred in 55 patients (55%), among which, postoperative pancreatic fistula (popf) (50.9%) was the most common . Approximately, 40% of popfs were classified as grade a according to the international study group of pancreatic fistula criteria . Clinical and pathological data of patients with nonfunctioning pnets * who 2010 classification; enets recommended tnm staging system . Pnets: pancreatic neuroendocrine tumors; enets: european neuroendocrine tumor society; tnm: tumor - node - metastasis; who: world health organization . According to the 2010 who classification, among the 100 nf - pnets, 61 patients (61%) were diagnosed as g1 tumors, 24 (24%) patients as g2 tumors, and 15 (15%) as g3 tumors . Among the 85 patients with g1 or g2 tumors, 44 (51.8%) were asymptomatic . In contrast, among the patients with g3 tumors, 12 (80%) had symptoms before surgery . Compared to g1 neoplasms, g2 or g3 neoplasms had larger diameter (g2 vs. g1: 4.4 cm 2.6 cm vs. 2.8 cm 2.1 cm, p = 0.001; g3 vs. g1: 4.6 cm 2.0 cm vs. 2.8 cm 2.1 cm, p = 0.004). Tumor size in ln+ patients was larger than that in ln - patients (4.7 cm 2.6 cm vs. 3.2 cm 2.2 cm, p = 0.01). According to the enets recommended tnm staging system, 29 (29%) patients had t1 tumors, 35 (35%) had t2, 31 (31%) had t3, and 5 (5%) had t4 . Among the 100 patients, 27 patients (27%) had stage i neoplasms, 32 (32%) had stage iia, 22 (22%) had stage iib, and 19 (19%) had stage iiib . Pathological examination revealed the presence of angioinvasion and perineural invasion in 15 patients (15%) and 7 patients (7%), respectively . Malignant behavior was found in 31 (31%) of all nf - pnets and four (8.5%) of 47 patients with radiological tumor size <2.5 cm with lnm identified in 3 (75%) of these patients . The 1, 5, and 8-year dfs was 90.2, 64.147.1%, respectively [figure 1]. During the follow - up period, variables associated with dfs in the univariate analysis are shown in table 2 . In the multivariate analysis, lymph node positive (hr = 3.995, 95% ci: 1.58510.06, p = 0.003), angioinvasion (hr = 4.049, 95% ci: 1.472 - 11.135, p = 0.007), and high tumor grading (g3 vs. g1 + g2: hr = 7.286, 95% ci: 2.779718.980, p = 0.000048) were significantly associated with decreased dfs in patients with resected nf - pnet [table 2]. The 5- and 8-year dfs was 79.4% and 71.4%, respectively, for ln - patients compared to 24.9% and 8.3%, respectively, for patients with ln+ disease (p = 0.000001) [figure 2]. Disease - free survival of patients with nonfunctional pancreatic neuroendocrine tumors . The 1, 5, and 8-year disease - free survival was 90.2%, 64.1% and 47.1%, respectively . Univariate and multivariate analysis of risk factors of dfs * size on resected specimens; who 2010 classification; enets recommended tnm staging system . Dfs: disease - free survival; sd: standard deviation; hr: hazard ratio; 95% ci: 95% confidence interval; ln: lymph node; enets: european neuroendocrine tumor society; tnm: tumor - node - metastasis; who: world health organization . The 5-year disease - free survival was 79.4% for lymph node - patients compared to 24.9% for patients with ln + disease (p = 0.000001), ln: lymph node . Among these 100 patients with nf - pnets, 92 who underwent regional lymphadenectomy variables that could be measured preoperatively were selected for the univariate analysis of the feasibility of preoperative prediction of ln status . In the univariate analysis, factors associated with ln metastasis were radiological tumor diameter> 2.5 cm (odds ratio [or] = 5.667, p = 0.010), elevated ca199 (or = 4.714, p = 0.017), high tumor grading (g2:g1, or = 6.125, p = 0.007; g3:g1, or = 14.000, p = 0.000322), and presence of symptoms (or = 3.545, p = 0.026) [table 3]. In the multivariate analysis, tumor grading (g2 vs. g1: or = 6.287, p = 0.008; g3 vs. g1: or = 12.407, p = 0.001) was an independent predictor of lnm [table 3]. The rate of lnm progressively increased from g1 to g3 (g1, g2 vs. g3: 7.5%, 33.3% vs. 53.3%). Considering the difficulty of preoperative retrievability of tumor grade, we excluded tumor grade from the analysis, and as a result, radiological tumor size> 2.5 cm (or = 5.430, p = 0.013) and presence of symptoms (or = 3.366, p = 0.039) were independently associated with lnm [table 3]. Radiological diameter was consistent with pathological diameter (34.1 mm vs. 34.7 mm, p = 0.237). When tumor size was treated as a continuous variable, the correlation with ln metastasis also reached significance (or = 1.313, p = 0.016). Roc analysis demonstrated radiological tumor diameter was a reliable and feasible predictor of lnm in patients with resectable nf - pnet with an auc of 0.693 [figure 3a]. Compared to neoplasms with radiological size> 2.5 cm (32.1%), tumors 2.5 cm had an obviously lower risk of lnm (7.7%). The various clinicopathologic factors reviewed in this study stratified by a tumor size cut - off of 2.5 cm are summarized in table 4 . Compared to tumors 2.5 cm, tumors> 2.5 cm had higher tumor grade and greater malignant potential . A cut - off of> 2.5 cm was associated with a sensitivity of 85% for the presence of ln+ disease . Other cut - offs were also examined [table 5]. With the purpose of promoting the predictive power, we constructed a preoperative predictive model of ln metastasis based on radiological tumor size combined with symptoms . The auc of this model was 0.747 [figure 3b], and the probability of ln+ for every patient was calculated . For the patients with an ln+ risk of 20%, 89.6% of these patients were, actually, ln - negative . Of the 21 incidentally discovered patients with tumors size 2.5 cm on ct scans, only one (4.8%) had lnm and none presented a postoperative recurrence during follow - up . Univariate and multivariate logistic regression analysis of ln metastasis * who 2010 classification; multivariate analysis excluding tumor grade . Or: odds ratio; 95% ci: 95% confidence interval; ln: lymph node; who: world health organization . The area under the curve of radiological tumor size (a) is 0.693, while the area under the curve of the constructed predictive model (b) is 0.747 . Clinicopathologic factors stratified by radiological tumor size * standard resections include pancreaticoduodenectomy and distal pancreatectomy . Atypical resections include middle pancreatectomy and enucleation; laparoscopic and robotic surgery; who 2010 classification; enets recommended tnm staging system . Sd: standard deviation; ln: lymph node; who: world health organization; enets: european neuroendocrine tumor society; tnm: tumor - node - metastasis . Predictive values of different radiological size cut - offs for ln metastasis in patients with nf - pnets npv: negative predictive value; ppv: positive predictive value; auc: area under the curve; nf - pnets: nonfunctional pancreatic neuroendocrine tumors; ln: lymph node . The clinical and pathological data of the 100 nf - pnets are shown in table 1 . Among the 100 patients, more than 50% (n = 53) were symptomatic at the time of diagnosis . Overall, 81 patients (81%) underwent standard pancreatic resection, while 19 patients (19%) underwent parenchyma - preserving pancreatic resection . Postsurgical complications occurred in 55 patients (55%), among which, postoperative pancreatic fistula (popf) (50.9%) was the most common . Approximately, 40% of popfs were classified as grade a according to the international study group of pancreatic fistula criteria . Clinical and pathological data of patients with nonfunctioning pnets * who 2010 classification; enets recommended tnm staging system . Pnets: pancreatic neuroendocrine tumors; enets: european neuroendocrine tumor society; tnm: tumor - node - metastasis; who: world health organization . According to the 2010 who classification, among the 100 nf - pnets, 61 patients (61%) were diagnosed as g1 tumors, 24 (24%) patients as g2 tumors, and 15 (15%) as g3 tumors . Among the 85 patients with g1 or g2 tumors, 44 (51.8%) were asymptomatic . In contrast, among the patients with g3 tumors, 12 (80%) had symptoms before surgery . Compared to g1 neoplasms, g2 or g3 neoplasms had larger diameter (g2 vs. g1: 4.4 cm 2.6 cm vs. 2.8 cm 2.1 cm, p = 0.001; g3 vs. g1: 4.6 cm 2.0 cm vs. 2.8 cm 2.1 cm, p = 0.004). Tumor size in ln+ patients was larger than that in ln - patients (4.7 cm 2.6 cm vs. 3.2 cm 2.2 cm, p = 0.01). According to the enets recommended tnm staging system, 29 (29%) patients had t1 tumors, 35 (35%) had t2, 31 (31%) had t3, and 5 (5%) had t4 . Among the 100 patients, 27 patients (27%) had stage i neoplasms, 32 (32%) had stage iia, 22 (22%) had stage iib, and 19 (19%) had stage iiib . Pathological examination revealed the presence of angioinvasion and perineural invasion in 15 patients (15%) and 7 patients (7%), respectively . Malignant behavior was found in 31 (31%) of all nf - pnets and four (8.5%) of 47 patients with radiological tumor size <2.5 cm with lnm identified in 3 (75%) of these patients . The 1, 5, and 8-year dfs was 90.2, 64.147.1%, respectively [figure 1]. During the follow - up period, variables associated with dfs in the univariate analysis are shown in table 2 . In the multivariate analysis, lymph node positive (hr = 3.995, 95% ci: 1.58510.06, p = 0.003), angioinvasion (hr = 4.049, 95% ci: 1.472 - 11.135, p = 0.007), and high tumor grading (g3 vs. g1 + g2: hr = 7.286, 95% ci: 2.779718.980, p = 0.000048) were significantly associated with decreased dfs in patients with resected nf - pnet [table 2]. The 5- and 8-year dfs was 79.4% and 71.4%, respectively, for ln - patients compared to 24.9% and 8.3%, respectively, for patients with ln+ disease (p = 0.000001) [figure 2]. Disease - free survival of patients with nonfunctional pancreatic neuroendocrine tumors . The 1, 5, and 8-year disease - free survival was 90.2%, 64.1% and 47.1%, respectively . Univariate and multivariate analysis of risk factors of dfs * size on resected specimens; who 2010 classification; enets recommended tnm staging system . Dfs: disease - free survival; sd: standard deviation; hr: hazard ratio; 95% ci: 95% confidence interval; ln: lymph node; enets: european neuroendocrine tumor society; tnm: tumor - node - metastasis; who: world health organization . Impact of lymph node status on disease - free survival . The 5-year disease - free survival was 79.4% for lymph node - patients compared to 24.9% for patients with ln + disease (p = 0.000001), ln: lymph node . Among these 100 patients with nf - pnets, 92 who underwent regional lymphadenectomy were selected for analysis of predictors of lnm . Of these patients, variables that could be measured preoperatively were selected for the univariate analysis of the feasibility of preoperative prediction of ln status . In the univariate analysis, factors associated with ln metastasis were radiological tumor diameter> 2.5 cm (odds ratio [or] = 5.667, p = 0.010), elevated ca199 (or = 4.714, p = 0.017), high tumor grading (g2:g1, or = 6.125, p = 0.007; g3:g1, or = 14.000, p = 0.000322), and presence of symptoms (or = 3.545, p = 0.026) [table 3]. In the multivariate analysis, tumor grading (g2 vs. g1: or = 6.287, p = 0.008; g3 vs. g1: or = 12.407, p = 0.001) was an independent predictor of lnm [table 3]. The rate of lnm progressively increased from g1 to g3 (g1, g2 vs. g3: 7.5%, 33.3% vs. 53.3%). Considering the difficulty of preoperative retrievability of tumor grade, we excluded tumor grade from the analysis, and as a result, radiological tumor size> 2.5 cm (or = 5.430, p = 0.013) and presence of symptoms (or = 3.366, p = 0.039) were independently associated with lnm [table 3]. Radiological diameter was consistent with pathological diameter (34.1 mm vs. 34.7 mm, p = 0.237). When tumor size was treated as a continuous variable, the correlation with ln metastasis also reached significance (or = 1.313, p = 0.016). Roc analysis demonstrated radiological tumor diameter was a reliable and feasible predictor of lnm in patients with resectable nf - pnet with an auc of 0.693 [figure 3a]. Compared to neoplasms with radiological size> 2.5 cm (32.1%), tumors 2.5 cm had an obviously lower risk of lnm (7.7%). The various clinicopathologic factors reviewed in this study stratified by a tumor size cut - off of 2.5 cm are summarized in table 4 . Compared to tumors 2.5 cm, tumors> 2.5 cm had higher tumor grade and greater malignant potential . A cut - off of> 2.5 cm was associated with a sensitivity of 85% for the presence of ln+ disease . Other cut - offs were also examined [table 5]. With the purpose of promoting the predictive power, we constructed a preoperative predictive model of ln metastasis based on radiological tumor size combined with symptoms . The auc of this model was 0.747 [figure 3b], and the probability of ln+ for every patient was calculated . For the patients with an ln+ risk of 20%, 89.6% of these patients were, actually, ln - negative . Of the 21 incidentally discovered patients with tumors size 2.5 cm on ct scans, only one (4.8%) had lnm and none presented a postoperative recurrence during follow - up . Univariate and multivariate logistic regression analysis of ln metastasis * who 2010 classification; multivariate analysis excluding tumor grade . Or: odds ratio; 95% ci: 95% confidence interval; ln: lymph node; who: world health organization . The area under the curve of radiological tumor size (a) is 0.693, while the area under the curve of the constructed predictive model (b) is 0.747 . Clinicopathologic factors stratified by radiological tumor size * standard resections include pancreaticoduodenectomy and distal pancreatectomy . Atypical resections include middle pancreatectomy and enucleation; laparoscopic and robotic surgery; who 2010 classification; enets recommended tnm staging system . Sd: standard deviation; ln: lymph node; who: world health organization; enets: european neuroendocrine tumor society; tnm: tumor - node - metastasis . Predictive values of different radiological size cut - offs for ln metastasis in patients with nf - pnets npv: negative predictive value; ppv: positive predictive value; auc: area under the curve; nf - pnets: nonfunctional pancreatic neuroendocrine tumors; ln: lymph node . Nf - pnets are relatively rare and heterogeneous pancreatic neoplasms with a remarkably increasing incidence . Compared to functional neoplasms, nf neoplasms show a worse outcome in part due to the delay of diagnosis and higher malignant potential . The optimal management for pnet is still controversial . Given its positive impact on survival, surgical resection has become the treatment choice for most patients with nf - pnets . In recent years, small nf - pnets are increasingly being discovered incidentally in cross - sectional imaging for other purposes and routine regional lymphadenectomy when surgical resection is considered in such cases remains controversial . For these reasons, we conducted a mono - institutional retrospective study of resectable nf - pnets with the purpose of (1) elucidating the clinical significance of lnm and (2) identifying reliable predictors of lnm to help surgeons to make informed treatment decisions . Our clinical data demonstrated that the 5-year dfs of resectable nf - pnets was 64.1% . Lnm is a significant prognostic predictor for most malignant tumors, although the impact of lnm on the survival of patients with nf - pnets remains open to debate . Many previous studies have yielded conflicting evidence regarding the prognostic value of lnm for pnet . The difference in patient selection and low lymph node sampling rates during resection for pnets may account for these inconsistencies . In accordance with our results, several previous studies have demonstrated lnm is significantly associated with a poor prognosis . Both the enets - tnm staging system and the ajcc cancer staging system regard lymph node status as an important prognostic factor . In our study, patients with lnm had a significantly higher risk (nearly 4-fold) of postoperative recurrence compared with those without lnm, which supports the necessity for regional lymphadenectomy in patients with high - risk of lymph node involvement . Since lnm is apparently related to prognosis, the ability to distinguish patients with high - risk of lnm preoperatively is of great importance . Nf - pnets classified, according to the criteria of the 2010 who classifications, were divided into three groups using a grading scheme based on the ki-67 index or mitotic count . Our study confirmed the significant correlation between the 2010 who grading system and postoperative recurrence, which is consistent with recent studies . In addition, lnm occurred more frequently in patients with g2 or g3 nf - pnets than in those with g1 tumors (g1, g2 vs. g3: 7.5%, 33.3%, 53.3%); therefore, it was presumed that these patients would benefit from node clearance and routine lymphadenectomy was recommended . However, classification of tumor grade usually depends on pathological examination and the possibility of a preoperative evaluation of ki-67 is still questionable . Endoscopic ultrasonography (eus) combined with fine - needle aspiration (fna) or tru - cut needle biopsy (tcb) can be used to evaluate tumor pathology preoperatively . Preoperative evaluation of ki-67 index, combined with lesion size and imaging findings may help surgeons to decide on the best therapeutic approach and whether lymphadenectomy should be performed . However, there is a lack of data regarding the accuracy of fna cytology in the assessment of ki-67 value . All of the available reports describe studies with small sample sizes . In a recent study, use of eus tcb needles can overcome many of the problems that reduce the accuracy of fna, but they are not feasible for most patients as they are often performed only at high - volume centers, can be challenging for small masses, and are associated with a risk of pancreatitis and bleeding . Moreover, intratumoral ki-67 heterogeneity limits the accurate preoperative evaluation of ki-67 value by eus fna . Considering the limitations of preoperative evaluation of ki-67 index, can easily be obtained preoperatively by use of radiological techniques, it has been extensively studied to identify the patients with lnm . Increasing tumor size reported that radiological tumor size 4 cm was an independent predictor of nodal metastasis in low and intermediate grade nf - pnets . In a retrospective study of 116 patients undergoing resection for nf - pnet, toste et al . Demonstrated that radiological tumor size 2 cm predicted nodal metastasis with a sensitivity of 93.8% and only two (7.4%) of 27 patients with tumor size <2 cm had lnm . Hashim et al . Reported that patients with tumor diameter> 1.5 cm were 4.7 times more likely to have lnm compared to those with smaller tumor diameters . They also found two (12%) of 17 patients with tumor size 1 cm and five (13%) of 38 patients with tumor size 1.5 cm had lnm . In contrast, some studies indicate that tumor size is not an accurate predictor of lnm . Reported that there was no difference in tumor size for patients with and without nodal metastasis (5.2 cm vs. 4.6 cm). <3 cm had nodal metastasis, while only 1 patient in their series with a tumor <2 cm was ln positive . Reported there was no association between decreasing tumor size and decreased percentage of cases presenting with regional nodal metastasis . Our study suggests radiological tumor size is a sensitive predictor of lymph node status and demonstrates a strong correlation between increasing radiological tumor size and lnm . The incidence of lnm in patients with radiological size> 2.5 cm was more than 4 times greater than that in patients with a tumor size 2.5 cm . A cut - off of> 2.5 cm was associated with a sensitivity of 85% for the presence of positive lns . Three of 39 (7.7%) patients with a tumor size 2.5 cm had nodal metastasis and 2 patients (11.1%) with a tumor size 1.5 cm had nodal metastasis . Given that smaller tumors are associated with low rates of lnm, better histology, and a better outcome than larger tumors, it is unclear whether lymphadenectomy should be avoided for small nf - pnets . In a retrospective study of 1854 patients with nf - pnets 2 cm, gratian et al . Demonstrated that there was no difference in 5-year overall survival in patients undergoing surgical resection between those who underwent lymphadenectomy and those who did not . Interestingly, in this study, 29% of tumors 2 cm and 33% of tumors 0.5 cm presented with regional lnm with a median of eight lymph nodes sampled, which was unexpectedly higher than had been reported previously . However, patient selection bias might overestimate the malignant potential of these tumors and account for the high rate of lnm . Nccn guidelines suggest that lymphadenectomy should not be performed routinely for tumors <2 cm but should be considered in tumors that are 12 cm in size . The benefits of lymphadenectomy in patients with small tumors is still unknown and more clinical data or well - designed clinical trials are needed to resolve this problem; however, such clinical trials are limited by the relative rarity and indolent behavior of small nf - pnets . In our study, patients with small tumors (2.5 cm) has a very low - risk of lnm . In addition, recurrence occurred in only one of the patients with small tumors 84 months after surgery, and no deaths caused by these tumors were reported during the follow - up . Nccn guidelines and current staging systems use the same cut - off of 2 cm as that used in pancreatic ductal adenocarcinoma, although patients with nf - pnets have a much better prognosis than those with pda . A cut - off of 2 cm has a low specificity for estimating the risk of lnm; consequently, many patients undergo unnecessary lymphadenectomy under this criterion . Based on our results, a cut - off of 2.5 cm was shown to be appropriate and safe . Compared to 2 cm, a cut - off of 2.5 cm had a similar sensitivity, but higher specificity and negative predictive value, showing that a cut - off of 2.5 cm is more effective in distinguishing the patients who require ln resection . Given the previously demonstrated strict correlation between tumor diameter and poor survival after resection of nf - pnets, raising the threshold may result in some tumors with malignant potential being considered benign . Preoperative evaluation of ki-67 index, combined with lesion size and imaging findings, may help surgeons to decide the best therapeutic approach . Since low - grade tumors are associated with a very low - risk of lnm and excellent survival, relaxing the indications for lymphadenectomy seems to be feasible . Based on the results of this study, we identified radiological tumor size as a noninvasive and reliable factor to predict lnm in nf - pnets . A radiological diameter 2.5 cm yielded a powerful correlation with the risk of lnm . The presence of symptoms at diagnosis was also shown to be a predictor of lnm . Although the symptoms of patients with nf - pnets are always unspecific, they are thought to be related to local mass effect or metastatic disease; in other words, tumor burden . Incidental detection of tumors is a strong prognostic factor for postoperative progression . In this study, incidentally discovered tumors were slightly smaller in size (3.2 cm vs. 3.7 cm) and associated with a much lower risk of lnm (12.2% vs. 29.2%). Reported only 6% of nf - pnets 2 cm were malignant when discovered incidentally . In our study, incidentally discovered small (2.5 cm) nf - pnets had a very low - risk of lnm (4.2%) and no cases of postoperative recurrence or death due to the disease occurred . Considering the low - risk of ln involvement and the positive outcomes, routine lymphadenectomy is not recommended as an addition to pancreatic resection in cases of asymptomatic small nf - pnets when surgery is considered . In conclusion, preoperative prediction of ln involvement is feasible and radiological tumor size, which can be measured easily and accurately, was shown to be a useful and alternative variable correlated with ln involvement . Our results suggest that parenchyma - sparing resection without regional lymphadenectomy is a reasonable option for selected patients with incidentally discovered nf - pnets 2.5 cm when surgical resection is considered because of the low probability of lnm and a good outcome . In contrast, lymphadenectomy should be performed routinely in patients with nf - pnets> 2.5 cm.
The issue of the early diagnosis of acute kidney injury (aki) has been debated for years . Other limitations are the paucity of available experimental models of aki and the inadequate capability of selected marker molecules to detect the impairment of kidney function in real time . On neutrophil gelatinase - associated lipocalin (ngal) as an early marker of acute kidney injury has partially overcome the above mentioned limitations and seems to demonstrate that diagnosing aki in its early stages is possible and useful . The problem may lie in the inadequacy of the renal replacement therapies that we have applied so far; however, this is questionable and it only applies to the late stages of aki, when the organ function has been lost and replacement by artificial organ support is required . There are many contributions showing that technology has evolved in parallel with the worsening of the clinical conditions of the patients being treated, and it is because of this that the mortality in this condition has not changed over the years . We are now realizing that for a number of non - complicated aki cases, mortality can be significantly reduced especially if an adequate renal replacement therapy is provided . Nevertheless, high mortality rates still pertain to complicated cases associated to multiple organ failure or septic syndromes . Only recently have we discovered that most of the preventive measures for aki which are efficacious in the experimental settings do not show comparable positive results in the clinical setting . This can be explained by the inability to identify the time of injury in the clinical setting . In the experimental models we apply the renal insult at a known time and thus we are able to apply the prevention or protection protocol timely and effectively . In the real clinical presentation it is only seldom and in specific cases (for example, elective cardiac surgery) where we can capture the exact moment of kidney insult and put in place counter measures . In order to make a solid diagnosis of aki in its early phases we have been missing adequate classification and staging criteria until only a few years ago . In 2002, during the second acute dialysis quality initiative (adqi) consensus conference held in vicenza, a new classification of aki called rifle (risk, injury, failure, loss, end - stage renal disease) was proposed based on the level of serum creatinine rise or urine output reduction . The rifle classification has been validated now in many papers and it is the most current and accurate tool to define the level of aki and the level of associated risk of mortality . Now that we have rifle we may speculate that aki can be diagnosed much earlier than in the past identifying a specific category such as risk, injury or failure . Such definitions suggest that in some cases, the aki process has begun much earlier than can be detected by the rise in serum creatinine . Creatinine is a reliable marker of kidney function but its constant and slow production makes its rise small and late in the case of an acute injury . There remains a need for a much earlier marker in order to diagnose aki as soon as possible . Using genomic and protein microarray technology, a series of molecules have been identified as potential markers for aki; among them ngal which is a 25 kda protein, generally expressed in low concentrations, and is greatly increased in the case of epithelial damage . In several papers ngal has been demonstrated to rise significantly in patients with aki but not in the corresponding controls [11 - 13]. Furthermore, this rise in ngal occurs in various studies at 24 to 48 hours before the rise in creatinine is observed . Ngal both in urine and plasma is an excellent early marker of aki with an area under the receiver operator characteristic curve (auc) in the range of 0.9 . The molecule still requires a complete evaluation in different clinical settings but the promise is both fascinating and scientifically sound . Today, there are many studies ongoing to elucidate the nature of the association between ngal and aki in the critical care settings . The same holds true in the setting of different types of cardiac surgery, contrast induced nephropathy, worsening renal function in heart failure (hf) and sepsis patients . The study of zappitelli et al . In critically ill children combines for the first time the new rifle classification of aki with the validation of ngal as an early marker of kidney injury . This innovative approach brings a new hope for a timely diagnosis of aki and thus a timely institution of measures for prevention and protection . Looking to the natural evolution of aki, we can identify different milestones along the timeline of the syndrome . The cells start to produce biomarkers of injury and only subsequently does the clinical picture of the syndrome develop with the typical sign and symptoms . Therefore we could imagine that the molecular and cellular clocks always anticipate the clinical clock, which is always late . The biological clock of biomarkers displays an intermediate time in this progression but it most certainly is reflective of an earlier stage when compared to the clinical clock . We need biomarkers that are sensitive (early appearance) and specific (typical of organ injury). They must be easy to detect and measure (possibly at bedside); they must correlate with severity (offering accurate prognosis), quantitatively describing the level of injury even in the absence of typical clinical signs . Finally they must be adequate to indicate treatment initiation (theranostics) enabling future studies to compare efficiency and efficacy of therapeutic measures and techniques . Thus, in the timeline of the aki syndrome early biomarkers represent a unique possibility for a timely diagnosis and intervention to protect the kidney from further insults and to prevent the tissue damage from the existing risk factors . If we wait for the clinical clock to activate the alarm we will always be late . Adqi = acute dialysis quality initiative; aki = acute kidney injury; auc = area under the receiver operator characteristic curve; hf = heart failure; ngal = neutrophil gelatinase - associated lipocalin; rifle = risk, injury, failure, loss, end - stage renal disease . Cr received a speaker fee from biosite at the meeting of ism in brazil in may 2007.
Keratoconus is a chronic bilateral noninflammatory corneal degeneration, characterized by localized corneal thinning and corneal steepening, leading to irregular astigmatism and impaired visual acuity . Corneal collagen cross - linking has found a broad international application for keratoconus in recent years [2, 3]. The combination of ultraviolet a (uva) light with riboflavin as a photosensitizing agent produces interfibrillar cross - linking between corneal stromal collagen fibers, thus increasing corneal rigidity and halting the ectatic process . Several adjuvant therapies in combination with cxl have been proposed in an effort to develop a technique that can offer patients keratectasia corneal stability together with improved functional vision, including intracorneal rings and phakic intraocular lenses . Topography - guided prk to correct ametropia and irregular astigmatism in form fruste and frank keratoconus was introduced over a decade ago [79] with good results . However, concerns were raised about the long - term complications, being a tissue subtraction technique . The mechanism of topography - guided ablation is the fitting of an ideal corneal shape (usually a sphere) under the present topography map with the ablation of tissue in between . Topography - guided prk flattens not only some of the cone peaks but also an arcuate broader area of the cornea away from the cone, usually in the superior nasal periphery; this ablation pattern will resemble part of a hyperopic treatment and thus will cause some amount of steepening, or elevation adjacent to the cone, effectively normalizing the cornea . Kanellopoulos and binder were the first to combine cxl with topography - guided prk in the management of keratoconus . They introduced a two - step procedure with cxl performed first and topography - guided prk after a 1-year interval . However, the fact that cross - linked corneas may have a different ablation rate from normal corneas which could lead to unpredictable prk results and the hypothesis that the removal of the cross - linked corneal tissue by the prk procedure could decrease the stiffening effects of the cxl treatment in addition to the increased possibility of haze formation after prk were serious limitations . Since then, several studies have evaluated the simultaneous use of topography - guided prk followed immediately by cxl in progressive keratoconus [9, 1113]. The aim of this study is to evaluate the effectiveness of combined corneal collagen cross - linking and topography - guided prk in cases of mild and moderate keratoconus using an advanced ablation software with the quest laser platform (nidek, gamagori, japan). This nonrandomized prospective clinical study was conducted on 20 eyes of 14 caucasian patients in between february 2012 and december 2013 . The study was conducted within the tenets of the declaration of helsinki after the approval of the institutional review board . All cases were performed at eye care center for refractive surgery, cairo, egypt . Inclusion criteria included grade 1 or 2 stable keratoconus (amsler - krumeich classification) documented by topography with a corneal thickness not less than 450 m and above 18 years of age . Exclusion criteria included patients below 18 years of age, advanced (stage 3 or 4 amsler - krumeich classification), bscva worse than 1 logmar, central corneal scars, cases that underwent previous refractive surgery, and patients with history of herpetic eye disease or who were pregnant or lactating . Preoperative examination included complete ocular examination included the following: assessment of uncorrected visual acuity (ucva) and best spectacle corrected visual acuity (bscva), slit - lamp examination, intraocular pressure (iop) measurement using goldman's applanation tonometry, assessment of manifest and cycloplegic refraction, and fundus examination using indirect ophthalmoscopy . Corneal topography was performed using optical path difference (opd scan ii) scanning system (nidek, gamagori, japan). Measurements were repeated at least 3 times, and the best image was chosen for the final analysis (the best one was being defined as the image with the best quality peaks for individual points). Soft contact lenses were stopped one week before topography while rigid gas permeable and hard contact lenses were stopped 2 weeks before topography . Corneal pachymetry was done using pacscan 300p ultrasound pachymeter (sonomed escalon, ny, usa) with map mode to measure 5 points at the cornea (centre, superior, inferior, temporal, and nasal area). Measurement accuracy and repeatability are assured by each scan actually consisting of 256 individual measurements and an automatic measurement algorithm to ensure that only scans with proper probe alignment are accepted . Surgeries were performed by 2 surgeons (y. s. mostafa and a. m. sherif). After topical anaesthesia, the epithelium was mechanically removed within an 8.5 mm diameter using hockey knife . Topography - guided prk was performed with the aim to normalize the cornea, by reducing irregular astigmatism and also treating part of the refractive error . Custom ablation transition zone (catz) ablation profile (quest, nidek, japan) was used . The catz algorithm delivers aspheric treatment zones combined with the treatment of corneal elevation irregularities or corneal wavefront at the surgeon's choice . The treatment was planned using the final fit software version 1.11t3,4 (nidek, co., ltd .) Based on the curvature and elevation maps from the placido disc system of the linked topography device opd scan ii.to ensure minimal tissue removal, the effective optical zone diameter was decreased to 5.5 mm . Correction of up to 70% of cylinder in addition to up to 40% of the spherical component without exceeding a maximal ablation depth limit of 50 m was attempted . An example of the ablation pattern is shown in figure 6.after completion of ablation, 0.02% mitomycin c was applied for 30 seconds, followed by copious irrigation with balanced salt solution . After topical anaesthesia, the epithelium was mechanically removed within an 8.5 mm diameter using hockey knife . Topography - guided prk was performed with the aim to normalize the cornea, by reducing irregular astigmatism and also treating part of the refractive error . Custom ablation transition zone (catz) ablation profile (quest, nidek, japan) was used . The catz algorithm delivers aspheric treatment zones combined with the treatment of corneal elevation irregularities or corneal wavefront at the surgeon's choice . The treatment was planned using the final fit software version 1.11t3,4 (nidek, co., ltd .) Based on the curvature and elevation maps from the placido disc system of the linked topography device opd scan ii . To ensure minimal tissue removal, correction of up to 70% of cylinder in addition to up to 40% of the spherical component without exceeding a maximal ablation depth limit of 50 m was attempted . After completion of ablation, 0.02% mitomycin c was applied for 30 seconds, followed by copious irrigation with balanced salt solution . For the next 20 minutes, 0.1% riboflavin 5-phosphate plus 20% dextran t 500 ophthalmic solution (ribolink isotonic, optos, australia) was applied topically every 2 minutes . The solution appeared to soak into the corneal stroma rapidly, as it was centrally devoid of bowman's layer . Following the initial riboflavin administration, collagen cross - linking was performed by projecting ultraviolet (uv) light at 370 nm wavelength (365 to 375 nm) and 3 mw / cm radiance at 4.5 cm onto the surface of the cornea for 30-minute x link cross - linking system (opto global, australia). The device has an accurate internal power meter and feedback loop control deliver consistent uv irradiation during the entire procedure and eliminates the need for periodic calibration.a bandage contact lens was placed on the cornea at the completion of the procedure and was removed at 35 days following complete reepithelialization.postoperative treatment included the topical antibiotic moxifloxacin (vigamox, alcon) four times a day for the first week and topical nonsteroidal anti - inflammatory drops nepafenac 0.1% (nevanac, alcon research ltd ., fort worth, tx) for 3 to 5 days until complete epithelial healing . This was followed by an antibiotic / steroid combination (tobradex, alcon) to be tapered over 60 days . 1000 mg vitamin c was given orally for 30 days.postoperative examinations were performed by 2 independent observes (m. a. ammar and a. a. osman).patients were assessed 1 month, 3 months, 6 months, and 12 months after surgery . On each visit, the patients were examined for uncorrected visual acuity (ucva), best corrected visual acuity (bcva), epithelial healing, and haze formation according to fantes haze grading system using slit lamp and corneal topography was performed using opd scan ii . Total eye aberrometry was not recorded in all cases and thus was not included in the results . For the next 20 minutes, 0.1% riboflavin 5-phosphate plus 20% dextran t 500 ophthalmic solution (ribolink isotonic, optos, australia) was applied topically every 2 minutes . The solution appeared to soak into the corneal stroma rapidly, as it was centrally devoid of bowman's layer . Following the initial riboflavin administration, collagen cross - linking was performed by projecting ultraviolet (uv) light at 370 nm wavelength (365 to 375 nm) and 3 mw / cm radiance at 4.5 cm onto the surface of the cornea for 30-minute x link cross - linking system (opto global, australia). The device has an accurate internal power meter and feedback loop control deliver consistent uv irradiation during the entire procedure and eliminates the need for periodic calibration . A bandage contact lens was placed on the cornea at the completion of the procedure and was removed at 35 days following complete reepithelialization . Postoperative treatment included the topical antibiotic moxifloxacin (vigamox, alcon) four times a day for the first week and topical nonsteroidal anti - inflammatory drops nepafenac 0.1% (nevanac, alcon research ltd ., fort worth, tx) for 3 to 5 days until complete epithelial healing . This was followed by an antibiotic / steroid combination (tobradex, alcon) to be tapered over 60 days . Postoperative examinations were performed by 2 independent observes (m. a. ammar and a. a. osman). Patients were assessed 1 month, 3 months, 6 months, and 12 months after surgery . On each visit, the patients were examined for uncorrected visual acuity (ucva), best corrected visual acuity (bcva), epithelial healing, and haze formation according to fantes haze grading system using slit lamp and corneal topography was performed using opd scan ii . Total eye aberrometry was not recorded in all cases and thus was not included in the results . Data management and analysis were performed using statistical package for social sciences (spss) version 17 . Data were summarized using means and standard deviations . To examine the changes between the different time periods for normally distributed variables, a one - way repeated measures analysis of variance, the analyses were performed by the friedman test, a nonparametric repeated measures one - way analysis of variance . Pairwise comparisons were done using the wilcoxon signed test after adjusting the p values using the bonferroni corrections . The predictability index was not included because we were not sure about the fine accuracy of the subjective spherical equivalent and because the aim was more of surface regularization rather than spherocylindrical correction . The study included 20 eyes of 14 patients: 11 eyes belonged to males (55%) and 9 eyes belonged to females (45%). The age of patients ranged from 18 to 29 years with a mean of 23.6 3.2 . The mean ablation depth was 48.09 9.6 m (range 23.357.3 m). The mean target cylindrical correction was 1.25 0.85 d (range 03.25 d). The preoperative and 1-, 3-, 6-, and 12-month postoperative sphere, cylindrical, and spherical equivalent data are shown in table 2 . There was a statistically significant difference between the mean preoperative logmar ucva (0.83 0.37) (0.21 0.19 decimals) and 1-month postoperative logmar ucva (0.47 0.23) (0.41 0.27 decimals) (p <0.001). Ucva continued to improve progressively until the end of the 12-month follow - up period (0.25 0.26 logmar) (0.63 0.25 decimals) (p <the preoperative mean logmar bcva (0.27 0.31) (0.62 0.29 decimals) showed an insignificant change (p = 0.99) one month postoperatively (0.27 0.29 logmar) (0.61 0.28 decimal) and continued to improve over the follow - up period, reaching 0.18 0.21 logmar at 3 months (p = 0.108), 0.12 0.19 logmar (0.82 0.29 decimal) at 6 months (p = 0.04), and 0.08 18 logmar (0.89 0.27 decimal) at 12 months (p = 0.02) (as shown in figure 2). Two eyes (20%) showed no improvement in bcva in lines, 10 eyes (50%) gained one line, 10% gained two lines, 10% gained 3 lines, and one eye (5%) gained 4 lines in bcva . One eye (5%) lost one line in bcva at the 12th month follow - up in comparison to preoperative bcva due to grade 3 haze (as shown in figure 3). The efficacy index (postoperative mean decimal udva divided by preoperative mean decimal bcdva) was 0.66 one month after surgery, improved to 0.88 at 6 months, and continued to improve at 12 months, reaching 0.97 . The safety index (postoperative mean decimal bcdva divided by preoperative mean decimal bcdva) showed a progressive improvement from 0.96 at one month, reaching 1.31 at 6 months and settling at 1.39 at 12 months . The mean of planned cylindrical correction was 1.25 0.85 d and the mean achieved cylindrical correction was 1.29 1.08 d at 12 months (as shown in figure 4). The median of the preoperative cylinder was reduced from 2.13 d to 1.25 d at one month (p <0.001). It remained stable over the follow - up period (1 d at 3, 6 months (p <0.001) and 1.13 d at 12 months (p <0.001)). The mean keratometric asymmetry was reduced from 3.01 2.03 d preoperatively to 1.25 1.2 d at 12 months (p <0.001). The improvement in mean kmax and mean kmin was stable over the 12-month follow - up period as shown in table 1 . 30% of cases had mild haze 1 month after surgery that was gradually reduced with topical steroids, reaching 20% at 3 months, 10% at 6 months, and 5% at 12 months . This study evaluated simultaneous topography - guided prk and corneal cxl for treatment of early cases of keratoconus . It has been possible to use topography - guided excimer laser treatments in highly irregular corneas that are beyond the limits of wavefront measuring devices, making this approach more efficient in treating highly irregular astigmatism, such as in keratoconus, as its measurements are based solely on the cornea surface reflection . Sequential cxl - topo - guided prk one year apart was first introduced to address the refractive element of keratoconus using prk and the biomechanical aspect using cxl . However several later studies reported better results with simultaneous topo prk and cxl regarding ucva, bcva, keratometry reduction, and haze [3, 16]. In our study, the preoperative mean ucva was 0.83 0.37 which improved at the 1st month postoperatively to 0.47 0.32 with a p value of <0.001 which was statistically significant . It continued to improve over the following months, reaching 0.26 0.25 at the last follow - up (12th month). These results are better than the results of lin et al . In 2012 and comparable to the results reported by alessio et al . In 2013, mukherjee et al . In 2013, and kanellopoulos in 2009 and slightly worse than the results of kymionis et al . In 2009 . The preoperative mean bcva was 0.27 0.31 which showed a gradual improvement over the follow - up period reaching 0.12 0.19 (p = 0.04) at 6 months . At the last follow - up (12th month) the mean bcva was 0.08 0.18 (p = 0.02). 20% of eyes in our study showed no improvement in bcva in snellen chart lines, 50% of eyes gained one line, 10% gained two lines, 10% gained 3 lines, and one eye (5%) gained 4 lines in bcva . These results are comparable to the results reported by kymionis et al ., kanellopoulos, and stojanovic et al . In 2010 . While one eye (5%) lost one line in bcva at the 12th month postoperative follow - up in comparison to preoperative bcva, lin et al . Reported that 12.5% of eyes lost 1 line and 4% lost> 2 lines of bcva . The reduction in mean kmax in our study was around 3.3 d, similar to the results of kymionis et al ., chan et al ., alessio et al ., and mukherjee et al . Regarding corneal haze, only one eye (5%) had visually significant haze (grade 3). Eight eyes (40%) had trace haze (grade 1) and 55% of eyes had no haze at all at the end of the 12-month follow - up . This concurs with other studies on simultaneous collagen cross - linking and topography - guided prk [16, 18]. Previous studies [3, 1113, 1618] evaluated the technique of simultaneous topography - guided prk with cxl in keratoconus with encouraging results and few postoperative complications . To our knowledge, there are no previously published studies describing the use of the catz software of the quest nidek excimer laser system for topography - guided prk in combination with collagen cross - linking in keratoconus . Our results showed significant progressive improvement of ucva and bcva and low risk of haze throughout the 12-month follow - up period . One of the limitations of the study was that the maximum ablation depth of 50 m was exceeded in two cases (53.5 m and 57.3 m). In addition, rigid gas permeable and hard contact lenses were stopped 2 weeks only before surgery which may be too short for some corneas . In addition, the follow - up period in our study was 12 months and due to the reports of progressive excessive corneal flattening [19, 20] which may lead to hyperopic shift up to several years after cxl, further studies with longer follow - up periods are needed to further evaluate the long - term outcomes of this technique.
Korea provides cash compensation to occupationally injured workers as a part of worker's compensation insurance . Interest in the return - to - work of occupationally injured workers has risen since 2001, and policy to promote their return - to - work has been in full scale since 2005 (1). Supported by various businesses led by the korea workers' compensation & welfare service, the return - to - work rate of occupationally injured workers in korea showed marked increases to 49.9% in 2007 and 70.4% in 2011 (2). Although this rate increased rapidly within a short period, related studies, have been limited, focusing on demographic characteristics, occupational injury - related characteristics, vocational rehabilitation, and correlation with business owners that influence return - to - work (1, 3 - 6). Research in other countries has mostly focused on individual factors, such as the worker's age, educational level, vocational characteristics, and psychosocial factors, or examined return - to - work programs or business owner factors . Most studies investigated impairment severity as a factor influencing return - to - work (7 - 13). However, impairment includes not only simple anatomical or functional problems but also limitations of activities or participation (14). The world health organization considers impairment to be a comprehensive notion including body function and structure, limitations of an individual's participation in society or in an occupation, and environmental factors (15). The perspectives of different countries regarding impairment vary, and people accept even the same impairment differently; thus, each country considers different limitations to occupations and activities according to impairment (16). (17) analyzed impairment assessment and income reduction in california and found that income reduction of the same impairment grade differed according to impairment type . Accordingly, california established standards to differently assess the disability rate according to impairment type . Korea took these standards as a benchmark for assessing the disability rate according to occupation and impairment type (18, 19). Therefore, because impairment type influences occupation and future income, it is believed to influence return - to - work . This article examines the effect of impairment type among occupationally injured workers on their return - to - work . This study was conducted with 109,746 participants with impairment grades who received medical treatment due to occupational injury during the 3 yr from january 1, 2009 to december 31, 2011 . The data on the subjects were acquired from the administrative data of the korea workers' compensation & welfare service (kcomwel). Rehabilitation counselors in 55 kcomwel branches confirmed the status of return - to - work through the employment information acquired from the korea employment information services (keis) every december and from telephone interviews . We excluded from the study 20,655 persons who did not have clearly locatable injuries, 4,898 persons whose return - to - work could not be determined, and 4,072 persons without company information before injuries . We also excluded 140 persons with pneumoconiosis, 87 with oral function impairment, 118 with nasal function impairment, and 82 with bony deformity . Return - to - work was classified for investigation into four types: " return to former work ", " work at a new firm ", " self - employment ", and " unemployment " . The first three categories were defined as completion of return - to - work, and " unemployment " as incompletion of return - to - work . The impairment grades and impairment types were decided by a physician according to the korean workers' compensation act guidelines . The impairment classification has 14 grades, with the severest impairment as grade 1, and the slightest as grade 14 . This article reclassified impairment severity into 4 groups: severe (grades 1 - 3), moderately severe (grades 4 - 7), moderate (grades 8 - 10), and mild (grades 11 - 14). The korean workers' compensation act guidelines classify 26 types of impairment according to affected body parts and physiologic functions; however, this article reclassified the impairment types into 9 groups . Upper limbs, lower limbs, and spine disabilities were included as anatomical and functional disabilities . Arm and finger disabilities were included with upper limb impairments; and leg and foot disabilities were included with lower limb impairments . Eyelid, auricle, and outside nose disabilities and scars were reclassified as figure impairments . We excluded masticatory disorder, speaking disorder, nasal function disorder, body deformity, pneumoconiosis, and thoracoabdominal disorder because they were difficult to combine with other disorders, and because they have low frequency . The pre - injury business size was classified into businesses with fewer than 30 workers, between 30 and 49, between 50 and 99, and over 100 . The pre - injury occupational category was largely classified into white collar, blue collar, and service workers . We performed chi - square tests to examine the factors that influence return - to - work, return - to - work according to impairment type, and characteristics that distinguish impairment grades . To investigate the characteristics of return - to - work according to impairment type, we performed logistic regression adjusted by age, sex, impairment grade, pre - injury business size, and pre - injury occupational category . Logistic regression was used to analyze the association between the level of return - to - work, " return to former work ", " work at a new firm ", and " self - employment ", and impairment type, adjusted by age, sex, impairment severity, pre - injury businesses size, and pre - injury occupational category . Examining for multicolinearity of independent variables, we found the variance inflation factor (vif) to be less than 10, indicating that there was no multicolinearity . This study was approved by the institutional review board of human research of yonsei university (approval no . This study was conducted with 109,746 participants with impairment grades who received medical treatment due to occupational injury during the 3 yr from january 1, 2009 to december 31, 2011 . The data on the subjects were acquired from the administrative data of the korea workers' compensation & welfare service (kcomwel). Rehabilitation counselors in 55 kcomwel branches confirmed the status of return - to - work through the employment information acquired from the korea employment information services (keis) every december and from telephone interviews . We excluded from the study 20,655 persons who did not have clearly locatable injuries, 4,898 persons whose return - to - work could not be determined, and 4,072 persons without company information before injuries . We also excluded 140 persons with pneumoconiosis, 87 with oral function impairment, 118 with nasal function impairment, and 82 with bony deformity . Return - to - work was classified for investigation into four types: " return to former work ", " work at a new firm ", " self - employment ", and " unemployment " . The first three categories were defined as completion of return - to - work, and " unemployment " as incompletion of return - to - work . The impairment grades and impairment types were decided by a physician according to the korean workers' compensation act guidelines . The impairment classification has 14 grades, with the severest impairment as grade 1, and the slightest as grade 14 . This article reclassified impairment severity into 4 groups: severe (grades 1 - 3), moderately severe (grades 4 - 7), moderate (grades 8 - 10), and mild (grades 11 - 14). The korean workers' compensation act guidelines classify 26 types of impairment according to affected body parts and physiologic functions; however, this article reclassified the impairment types into 9 groups . Upper limbs, lower limbs, and spine disabilities were included as anatomical and functional disabilities . Arm and finger disabilities were included with upper limb impairments; and leg and foot disabilities were included with lower limb impairments . Eyelid, auricle, and outside nose disabilities and scars were reclassified as figure impairments . We excluded masticatory disorder, speaking disorder, nasal function disorder, body deformity, pneumoconiosis, and thoracoabdominal disorder because they were difficult to combine with other disorders, and because they have low frequency . The pre - injury business size was classified into businesses with fewer than 30 workers, between 30 and 49, between 50 and 99, and over 100 . The pre - injury occupational category was largely classified into white collar, blue collar, and service workers . We performed chi - square tests to examine the factors that influence return - to - work, return - to - work according to impairment type, and characteristics that distinguish impairment grades . To investigate the characteristics of return - to - work according to impairment type, we performed logistic regression adjusted by age, sex, impairment grade, pre - injury business size, and pre - injury occupational category . Logistic regression was used to analyze the association between the level of return - to - work, " return to former work ", " work at a new firm ", and " self - employment ", and impairment type, adjusted by age, sex, impairment severity, pre - injury businesses size, and pre - injury occupational category . Examining for multicolinearity of independent variables, we found the variance inflation factor (vif) to be less than 10, indicating that there was no multicolinearity . This study was approved by the institutional review board of human research of yonsei university (approval no . 4 - 2013 - 0365). The sample consisted of 67,182 males (84.7%) and 12,146 females (15.3%), and the average age was 47.6 yr (sd, 11.4 yr). The mild impairment group was the largest with 60,128 participants, and the most common impairment type was upper limbs impairment, which affected 31,292 participants (39.5%). In case of the pre - injury business size, the number of participants who returned to work was 55,154 (69.5%); and those who did not were 24,174 (30.5%). With respect to the rate of return - to - work by age, workers in their 30s and 40s had the highest return - to - work rate (79.3%) and those in their 60s or older had the lowest (52.0%). In general, the rate of return - to - work decreased with age (p<0.001). The rate of return - to - work in male was 71.2%, which was higher than the rate in female (60.5%) (p<0.001). For the rate of return - to - work according to impairment type, figure impairments showed the highest rate of 78.0%, and neuropsychiatric impairments showed the lowest at 30.3% (p<0.001). For the rate of return - to - work according to impairment grade, the mild group (grades 11 - 14) had the highest rate at 72.4%; and the severe group (grades 1 - 3) had the lowest at 16.8% (p<0.001). As a whole, pre - injury business size of over 100 workers had 72.8% of return - to - work, while those with less than 30 workers showed 68.2% (p<0.001). Regarding pre - injury occupational category, white collar workers showed the highest rate of return - to - work at 80.1%, and service workers showed the lowest at 64.0% (table 1; p<0.001). For impairments of the upper and lower limbs and eyes, the mild group (grades 11 - 14) showed the highest rate of return - to - work; and the severe group (grades 1 - 3) showed the lowest (p<0.001). For spine injuries, the moderately severe group (grades 4 - 7) showed the highest rate of return - to - work at 72.2%, but the sample size was small . Therefore, except for the group, the mild group showed the highest rate of return - to - work . For injuries of the oral cavity, ears, and figure, there were no statistically significant differences in return - to - work rates between the impairment grades . For neuropsychiatric impairment, the moderate group (grades 8 - 10) showed the highest rate of return - to - work at 46.4%, and the severe group (grades 1 - 3) was the lowest at 16.9% (p<0.001). For upper and lower limb, eyes, and neuropsychiatric impairments, the rate of return - to - work decreased with the severity grade . For injuries to the oral cavity, ears, and figure, there were no statistically significant differences in return - to - work rates between the impairment severity grades (table 2). Logistic regression was used to analyze the association between return to work and impairment type, adjusted by age, sex, impairment severity, pre - injury businesses size, and pre - injury occupational category . Compared to injuries of the upper limbs, the odds ratio of return to work was 0.63 (95% ci, 0.60 - 0.65) for injuries involving the lower limbs, 0.62 (95% ci, 0.59 - 0.66) for the spine, 0.75 (95% ci, 0.66 - 0.86) for the eyes, 0.44 (95% ci, 0.37 - 0.53) for the ears, 0.75 (95% ci, 0.72 - 0.79) for pain, and 0.36 (95% ci, 0.32 - 0.41) for neuropsychiatric impairment which were statistically significant (table 3). Logistic regression was used to analyze the association between the level of return - to - work, " return to former work ", " work at a new firm ", and " self - employment ", and impairment type, adjusted by age, sex, impairment severity, pre - injury businesses size, and pre - injury occupational category . In case of return to former work, compared to injuries of the upper limbs, the odds ratio was 0.65 (95% ci, 0.62 - 0.68) for injuries involving the lower limbs, 0.64 (95% ci, 0.61 - 0.67) for the spine, 0.67 (95% ci, 0.59 - 0.76) for the eyes, 0.67 (95% ci, 0.54 - 0.83) for the oral cavity, 0.29 (95% ci, 0.23 - 0.36) for the ears, 0.76 (95% ci, 0.64 - 0.91) for the figure, 0.62 (95% ci, 0.60 - 0.65) for pain, and 0.38 (95% ci, 0.33 - 0.43) for neuropsychiatric impairment which were statistically significant . In case of work at a new firm, compared to injuries of the upper limbs, the odds ratio was 0.77 (95% ci, 0.65 - 0.91) for involving neuropsychiatric impairment, 1.49 (95% ci, 1.20 - 1.84) for the oral cavity, 1.51 (95% ci, 1.24 - 1.85) for the ears, 1.28 (95% ci, 1.06 - 1.54) for the figure, and 1.26 (95% ci, 1.21 - 1.32) for pain which were statistically significant . However, lower limbs, spine, and eyes impairment had no statistical significance . In case of self - employment, compared to injuries of the upper limbs, the odds ratio was 1.23 (95% ci, 1.11 - 1.35) for injuries involving the lower limbs, 1.38 (95% ci, 1.23 - 1.54) for the spine, 1.40 (95% ci, 1.07 - 1.82) for the eyes, and 1.17 (95% ci, 1.06 - 1.29) for pain which were statistically significant . Oral cavity, ears, figure, and neuropsychiatric impairment had no statistical significance (table 4). Various factors are related to the return - to - work of occupationally injured workers . Among them prior studies on impairment and return - to - work have emphasized impairment in specific parts as well as severity of impairment (7, 21 - 24). (23) found that not only severity of impairment but also the locations and causes of impairment had important effects on return - to - work . However, chang et al . (25) reported that, even if impairment severity are the same in upper limbs, they showed differences in return - to - work according to impairment types . Therefore, it was found that not only severity of impairment but also impairment type play important roles in return - to - work . The study results showed statistical significance relative to upper limb impairment, of the lower ors for involving the spine, eyes, ears, pain, and neuropsychiatric impairment on return - to - work . On the other hand it is believed that general opinions in korea about impairment are reflected in the results . Differing from expert opinion, people in the general public tend to think that upper limb impairment is less serious than lower limb impairment, and that impairments of the eyes, ears, mental ability, intellect, or spine are more serious (26). Experts in impairment evaluation consider arm function to be 60% and leg function to be 40% of the whole body functioning (14, 27). However, korean people commonly believe that leg function equally or more important than arm function (26). In korea, because social conditions such as transportation, road conditions, and building structures are unfavorable for the disabled, impairment related to walking and movement is regarded to be most important . Therefore, we suggest that impairments involving the lower limbs, spine, or eyes lead to the greatest difficulties in finding jobs and returning to work because they are commonly considered to be more serious . When return - to - work is classified into return to the former work, work at a new firm, and self - employment to examine the return - to - work ors according to impairment types, we found that impairments of the lower limbs, spine, and eyes had a lower rate of return to the former work (or 0.64 - 0.67), but a higher rate of self - employment (or 1.23 - 1.40), compared to upper limbs impairment . We believe that this difference shows that the difficulty arises in finding a job in compliance with an individual's personal situation rather than from a problem with return - to - work due to objective functional impairment . In korea, pain disorder had only one impairment grade, mild . The number in this group of injured workers was next highest after the group with upper limb impairment . The return - to - work or for impairment due to pain was lower than that of upper limb impairment, which we believe is not because pain disorder restrict physical function but because of avoidance of return to the original work, based on the worker's subjective judgment . That is, pain disorder, compared to upper limb impairment, showed undesirable to return to the former work (or 0.62) but desirable to work at a new firm (or 1.26) and to have self - employment (or 1.17). Therefore, it appears that pain disorder often activates a subjective avoidance of the former work . In fact, pain disorder is viewed not as clear impairment but as feigned illness (28). In this study, neuropsychiatric impairment had lower or for the return - to - work compared to upper limb impairment . Specifically, we found that return to the former work (or, 0.38) and work at a new firm (or, 0.77) were lower, and self - employment (or, 0.95) was similar, to that of upper limb impairment . In the study cases of neuropsychiatric impairment, impairment grades ranged from severe to moderate, and thus, it is assumed that the reason for not returning to work is health restrictions . In fact, health was the reason given for unemployment by 87.1% of participants with neurological disorder who did not return - to - work . For impairments of the ears, compared to upper limbs impairment, the return - to - work or was low . Specifically, return to the former work was lower (or, 0.29), work at a new firm was higher (or, 1.51), and self - employment (or, 1.18) was similar to that of upper limb impairment . Ears impairment differed from other disabilities in that the exposure period of the harmful factor (noise) increased impairment levels with increasing age . It appears that the work environment of the former work influenced the severity of the impairment . This suggests that these participants preferred to work in another place with less noise exposure . Results showed that participants with impairments of the lower limbs, spine, and eyes and related to moving the body were more likely to become self - employed; workers with impairments of the oral cavity and figure were most likely to work at a new firm; those with pain disorder tended to return - to - work at a new firm or to become self - employed; those with serious neuropsychiatric impairment mainly found self - employment; and those with ears impairment usually returned to work at a new firm . It is significant that return - to - work ors according to these impairment types were statistically significant even after adjusting pre - injury occupational categories . Because the return - to - work rates of blue collar and service workers, who do manual labor, were lower than for white collar workers . The different views of experts versus the general public about impairment, impairment severity, and impairment type are likely to affect return - to - work . This study minimized the effects of selection bias that may have been created by utilizing the national institution's data through overall inspections of the return - to - work of occupationally injured workers . In addition, because this study was not based on a questionnaire about impairment types and levels but was instead based on evaluations by doctors, these have high accuracy . However, because this study concentrated on people who were recognized as occupationally injured workers and given impairment grades, it has the limitation that it did not include the disabled who were not recognized as occupationally injured workers . In conclusion, the study findings indicate that, in addition to individual characteristics and socioeconomic factors, the impairment severity, type, and patterns should be seriously considered in the planning by businesses and others to help injured workers return - to - work, such as via vocational rehabilitation.
Alagille syndrome is clinically defined by neonatal cholestatic jaundice with intrahepatic bile duct hypoplasia associated with additional findings, including a characteristic facies, peripheral pulmonary artery stenosis, butterfly - like vertebral anomalies, and ocular abnormalities . Ocular findings include posterior embryotoxon, iris abnormalities, optic disc anomalies, and fundus changes (irregular pigmentation at the level of the retinal pigment epithelium, diffuse hypopigmentation, and punched out chorioretinal atrophy).1,2 to our knowledge, there are no cases in the literature presenting with alagille syndrome and chorioretinal atrophy involving the macula, as examined by optical coherence tomography (oct) and fundus autofluorescence . Herein, we report oct and fundus autofluorescence changes in a patient with this syndrome . An 11-year - old girl with alagille syndrome was referred to the jichi medical university hospital before a liver biopsy during observation after liver transplantation . Liver transplantation was performed at the age of 20 months, and left pulmonary artery balloon dilation was performed at the age of 8 years . She had an unusual triangular facies characterized by a broad overhanging forehead, deep - set hyperteloric eyes, and a small pointed chin . Best - corrected visual acuity in the right eye was 12/20 with 6.25 d 1.0d 90, and in the left eye was 8/20 with 4.25 d 2.0 d 90. the patient was orthophoric and showed normal ocular movements . On slit lamp examination, funduscopy showed diffuse choroidal hypopigmentation with increased visibility of the choroidal blood vessels and symmetric well circumscribed macular discoloration (figure 1). Decreased retinal thickness was seen on oct (rs-3000, nidek, japan). On the basis of macular etdrs (early treatment diabetic retinopathy study) sectors, thickness of the center (central fovea), inner ring (13 mm from the central fovea) and outer ring (36 mm from the central fovea) were 222 m, 254.8 m, and 241.0 m, respectively, in the right eye . In the left eye, in particular, the outer retinal (nuclear) layer and the photoreceptor inner segment / outer segment junctions were irregular and discontinuous, corresponding to macular discoloration (figures 2 and 3). Foveal architecture was preserved in both eyes with thinning . In high - magnification oct images, the border between the chorioretinal atrophy and the hypopigmented area of the macula was clearly defined . The outer retinal thickness in the chorioretinal atrophic area was thinner than that in the hypopigmented area . Fundus autofluorescence imaging (heidelberg retina angiograph 2, heidelberg, germany) showed hypofluorescent areas in the peripapillary regions extending along the macula corresponding to the chorioretinal atrophy . These had an appearance similar to a sleep mask (figure 4). A mottled pattern of hyperautofluorescent areas was also detected in the macula . Visual field testing with goldmann perimetry only showed enlargement of the blind spots in both eyes . Serum vitamin a and vitamin e levels were 94 (normal range 97316) iu / dl and 0.51 (normal range 0.751.41) mg / dl, respectively . In the largest series of patients with alagille syndrome, hingorani et al1 evaluated 22 patients with the condition . The most common ocular abnormalities in alagille syndrome were posterior embryotoxon (95%), iris abnormalities (45%), diffuse fundus hypopigmentation (57%), speckling of the retinal pigment epithelium (33%), and optic disc anomalies (76%).1 in studying retinal changes in alagille syndrome, hingorani et al1 reported that irregular pigmentation at the level of the retinal pigment epithelium, most often manifested as a speckling or granularity of the retinal pigment epithelium, was distributed diffusely in the mid peripheral / peripheral zone.1 the few reports concerning macular changes in alagille syndrome describe symmetric, well circumscribed, horizontally oval areas of speckled reddish brown macular discoloration,2 pigment clumping in the macula,3 and circumpapillary geographic chorioretinal atrophy with half - moon shapes involving the macular area.4 however, chorioretinal atrophy involving the macular area is rare . In this case, diffuse choroidal hypopigmentation with increased visibility of the choroidal vessels and a sleep mask - like hypofluorescent area corresponding to chorioretinal atrophy as examined by fundus autofluorescence were detected . To our knowledge, there are no similar cases in the literature of alagille syndrome presenting a chorioretinal atrophy involving the macula with a sleep mask appearance as examined by oct and fundus autofluorescence . A possible explanation for chorioretinal atrophy in alagille syndrome is failure of absorption of fat - soluble vitamins . Romanchuk et al5 suggested that failure of fatsoluble vitamin absorption plays a major role in the evolution of retinopathy in alagille syndrome . The retinoid cycle is a vitamin a - linked metabolic circuit which occurs between the outer segment of the photoreceptor and the retinal pigment epithelium to maintain visual function . Low serum vitamin e levels have been well documented in children with malabsorption due to cholestatic liver diseases.6 the role of vitamin e in vision is less clear, but it is believed to have an important protective function as a lipid antioxidant for the extremely high concentrations of polyunsaturated fatty acids found in the outer segments . Increases in the number of lipofuscin granules in the cytoplasm of retinal pigment epithelium in vitamin e - deficient rats were observed.7 the retinal pigment epithelium cells of the peripheral and equatorial zone contain large numbers of lipofuscin granules within their cytoplasm.8 thus, both of these vitamins are believed to be important in maintaining the integrity of the photoreceptor outer segments and the retinal pigment epithelium cells . Fundus autofluorescence has been investigated in various fundus diseases associated with changes in the retinal pigment epithelium . Fundus autofluorescence is presumed to derive from lipofuscin in retinal pigment epithelium cells and to represent metabolic activity of retinal pigment epithelium involving turnover of photoreceptor outer segments . Hypofluorescence is thought to correspond to areas of decreased metabolism resulting from photoreceptor and/or retinal pigment epithelium atrophy, and has been used as a marker of the integrity of the retinal pigment epithelium / photoreceptor complex . The peculiar findings in this patient were abnormalities of the inner segment / outer segment junction and the outer segments of photoreceptors, suggestive of loss of structural integrity of the photoreceptors . Therefore, we suggest that transient hypovitaminosis due to alagille syndrome early in life might contribute to the retinal degeneration seen in this case . Finally, anatomic evidence of retinal degeneration induced by vitamin a or e deficiencies suggests that the structural disruption of photoreceptors is more advanced in cones and most pronounced in the macula, with lesser involvement of the peripheral retina.9 however, it is not clear whether our case represents a congenital or acquired change due to alagille syndrome.
The current trend in industrial tasks is moving towards more time - intensive production with standardized, short cycle times1 and limited completion times2 since an aim of the manufacturing industry is to attain high work productivity . Process standard times, such as the work pace or duty cycle time for a particular task, are determined by a process engineer based on task time analysis . However, because workers must work in their designated work locations and must adhere to predetermined task times3, their capacities and productivity state are often overestimated . Hard production standards generally produce high work productivity compared with low or no production standards4 . In general, tasks become more repetitive in the case of harder production standard times and may expose workers to a higher risk of work - related musculoskeletal disorders (wmsds). The capability of workers performing repetitive tasks and the risk of wmsds can be assessed by energy expenditure measurement7 . Energy expenditure is a physiological measurement used to assess the influence of physical fatigue on work performance among industrial workers7, 8 . Hence, estimation of energy expenditure is important indeed, as it serves as a reference in design of tasks that will not induce fatigue and wmsds among workers . The ability to accurately track energy expenditure (ee) would be beneficial in the prevention of wmsds7, 10 . Energy expenditure rate may vary according to the levels of production standard time assigned to workers . Therefore, the objective of this study was to investigate the effects of energy expenditure rate on work productivity performance at different levels of production standard time in order to identify the maximum capability of the workers . This data could be used to help ensure that tasks assigned to workers will not induce fatigue and to minimize the risk of wmsds . A total of 20 subjects, 10 male and 10 female industrial workers, were recruited for a series of experimental tasks . The subjects were between the ages of 22 and 45 years old (30.97.711). They were first briefed on the experimental task process flow and equipment to be used prior to performing the series of experimental tasks . Each subject was given an information sheet outlining their involvement in the study and its potential risks . The study was approved by the local ethics committee . Written informed consent was obtained from each subject to ensure that they fully agreed to participate in the study . The subjects were then instructed to adopt a comfortable sitting posture with the sitting height adjusted individually to obtain a knee angle of 90. the working height was standardized by placing the work table s surface 5 cm below the position of the wrist when the elbow was flexed at 9011 . The subjects were required to perform the experimental tasks after familiarizing themselves with them for 30 minutes . The subjects were given two types of component, plastic clips and plastic foam rings . The subjects were instructed to connect the foam rings to the plastic clips using a jig, which pushed the foam rings onto the clips . The subjects performed the tasks according to the production standard times assigned to them . The production standard times used in the experimental tasks were 100% normal standard time (psn - normal), 126% normal standard time (psh - hard), and 140% normal standard time (psvh - very hard). The normal standard time was determined from a methods - time measurement (mtm) analysis . Heart rate and energy expenditure rate were recorded using the actiheart monitoring device, and work productivity of the subjects was recorded for every 30 minutes . Work productivity data were recorded in terms of quantity per hour and the percentage of normal standard time achieved . The results for work productivity at different levels of production standard time are summarized in table 1table 1.work productivity at different levels of production standard timeproduction standard (ps)work productivity targetwork productivity (quantity / hour)percentage of normal standard (%) psn100%851118.0psh126%890123.0psvh140%928129.0 . The very hard production standard time resulted in the highest output, followed by the hard production standard time and the normal production standard time . Work productivity data were then analyzed to investigate the effect of production standard times on work productivity . Repeated measures anova was carried out for this purpose, and the results revealed that production standard time had a significant effect on work productivity (wilk s lambda = 0.257, f (2, 18) = 2.8, p <0.001, multivariate partial eta squared = 0.743). It is evident that the average work productivity differed significantly among the three production standard times (work productivity targets). The means and standard deviations of energy expenditure and heart rate are summarized in table 2table 2.mean and standard deviation for energy expenditure (kcal / min) and heart rate (bpm) at different levels of production standard timeproduction standard timeenergy expenditureheart ratemeanstandard deviationmeanstandard deviationpsn1.030.0589.83.05psh1.190.1496.74.69psvh1.360.59102.45.75 . It can be observed that the workers energy expenditure and heart rate were higher for harder production standard times . Repeated measures anova analysis revealed that the energy expenditure increased significantly as the production standard time became harder (wilk s lambda = 0.06, f (3, 17) = 89.036, p <0.005, multivariate partial eta squared = 0.940). The energy expenditure rate for the hard production standard time was higher than that for the normal production standard time, with the percentage difference being 15.5% . The assignment of a very hard production standard time resulted in an increase in energy expenditure relative to the normal and hard production standard times, with the percentage increases being 32.5% and 14.6%, respectively . The results showed that work productivity increases significantly as the production standard time becomes harder . This indicates that the workers were able to achieve higher work productivity in the case of harder production standard times compared with the normal production standard time . This observation agrees well with the findings of shikdar and das12, who showed that work productivity increases in the case of harder production standard times . The work productivity target is attainable with the normal production standard time, but this is not the case for the hard and very hard production standard times . The work productivity target for the hard production standard time was 126% of the normal standard time . The results showed that the workers were only able to achieve 123% of the normal standard time . Similarly, the work productivity target for the very hard production standard time was 140% of the normal standard time, and it was found that the workers were only able to achieve 129% of the normal standard time . There is an increase in job requirement in the case of harder production standard times . In general, workers perform more repetitions of tasks in the case of harder production standard times and are exposed to a higher risk of wmsds . The results agreed well with the results of previous studies, which also reported an association between the risk of contracting wmsds with higher repetition of tasks13, 14 and increases in job requirement15 . The results indicated that workers tend to slow down in the case of harder production standard times due to wmsd risks . The results are consistent with the findings of previous studies, which showed that workers tend to slow down when they are fatigued due to wmsds16 . The findings concerning work productivity can be attributed to the variations in energy expenditure rate at different levels of production standard time . The energy expenditure rate for an activity was found to increase significantly as the production standard time becomes harder . This agrees with the findings of li et al.7, who revealed that energy expenditure increases when the frequency of tasks increases . The percentage change in energy expenditure rate for the very hard level (32.5%) relative to the normal level was twice that of the hard level (15.5%). The results also revealed that the average energy expenditure rates for an activity in the case of the normal (1.03 kcal / min) and hard production standard times (1.19 kcal / min) were less than the maximum value, 1.2 kcal / min, for light repetitive tasks involving both arms in a previous study17 . The energy expenditure value reported by garg et al.17 is used as a reference because it serves as a reliable benchmark in estimating energy expenditure7, 18 . In contrast, the average activity energy expenditure rate obtained in the case of the very hard production standard time (1.36 kcal / min) exceeded the reference value, which suggested that the workers were exposed to a higher risk of wmsds . The heart rate of the workers was also found to be higher in the case of a harder production standard time, and the energy expenditure rate increased with the increment in heart rate . These results agreed well with the findings of a previous study that discovered a linear relationship between energy expenditure, heart rate, and oxygen uptake19 . The dynamic muscle exertions during repetitive tasks require oxygen, and the metabolic demands of the muscles increase as activity increases . Therefore, consumption of oxygen will increase in conjunction with an increase in heart rate in order to circulate more blood, which will carry oxygen to the working muscles . The results of this study indicated that workers are exposed to a higher risk of wmsds when carrying out tasks with a very hard production standard time . This is because the energy expenditure rate exceeds the maximum capability of workers when working with a very hard production standard time . The results are supported by the findings of previous studies that emphasized the need to accurately track the energy expenditure rate of workers in order to prevent wmsds7, 10 . In conclusion, working with an energy expenditure rate that is either equal to or above the maximum energy expenditure rate of the tasks will decrease work productivity performance due to the onset of physical fatigue and increase the risk of wmsds . Therefore, it is important to identify the maximum energy expenditure rate to ensure that the tasks assigned will not result in excessive fatigue in workers, which would lead to a risk of wmsds and a reduction in work productivity performance.
Physical activities can preserve brain function and improve blood flow and oxygen transfer2 as well as slow the loss of hippocampal formational tissue in the aging brain . Because of its unique three - dimensional movement, horseback riding has been applied as a physical therapy to stimulate movement to maintain balance3 . One of its effects is to transfer movement to the pelvis, producing an effect very similar to pelvic tilting4 . In addition, horseback riding exercises parts of the body that are not used by other frequently performed exercises, including agonistic muscles, which are deep muscles that contract and relax to maintain balance5 . Psychological research on horseback riding exercise indicates it decreases depression in teenagers with emotional disorders6 as well as improves teenagers self - esteem and self - control7 . However, there is little information about horseback riding exercise as a physical therapy and even less information about whether such exercise is effective for elderly people or indeed how it may affect them . Therefore, this study investigated the effects of horseback riding exercise on the background electroencephalogram (eeg) of elderly people who performed horseback riding for 8 weeks . All participants or their guardians provided informed consent prior to participation . Before the start of the study, the purpose and methods were explained in detail to the participants . The participants were divided into 2 groups: a horseback riding exercise group and a control group (n = 10 each). Riding was performed for 15 minutes, 3 times per week for 8 weeks . The post - exercise tests were performed over a period of 8 weeks in the same manner as pre - study tests . We investigated the background eegs, specifically the relative alpha power index, in both groups . For the exercise, we used jeju horses from the i rehabilitation center - affiliated horse riding course; these horses are normally used to provide riding experiences to visitors and are therefore healthy, accustomed to their surrounding environment, well trained, tame, and have stable strides . All participants wore protective gear including safety helmets, vests, and boots . At the beginning of the tests, the warm - up exercises were performed at a walking speed, which was the slowest speed (110 m / min). For this study, the slowest walking speed was maintained for the participants safety and comfort . In the exercise, an instructor led a horse by the bridle while another assistant supported a participant by holding him or her by the leg prevent them from falling from the horse and provide assistance as needed . Quantitative electroencephalography can be used to diagnose neurological changes in the brain; it constitutes an electric signal due to an active change in the brain that can capture the electrical flow between neurons through electrodes attached to the surface of the head8 . We collected the eeg data using a computerized polygraph (polyg - i, laxtha inc ., korea) and telescan, which is a real - time analysis program . The eeg electrodes were attached to the surface of the head of the participants as they were seated in a comfortable reclining chair . To reliably capture data, the same researcher attached the electrodes and operated the testing instruments . Body and head movements were controlled as much as possible, and the participant kept their eyes closed for 3 minutes while resting in order to minimize the interruption of waves caused by eye movement . The eeg signals were analyzed for 60120 seconds, excluding the first and last 60 seconds when the test might have been influenced by external environments . The background eeg data were analyzed on the basis of a monopolar derivation from 8 points on the surface of the head; the 8 electrodes were attached to fp1, fp2, f3, f4, t3, t4, p3, and p4 according to the international 1020 electrode system . The saved data were transformed into frequencies using a fast fourier transform, which expresses the quantitative relationship between eeg frequencies and their intensity . Power spectrum analysis was subsequently applied, and the band - to - band power was calculated . The relative alpha power (813 hz/450 hz), which emerges in a state of relaxation and rest, was subdivided into the relative slow alpha power (811 hz/450 hz) and the relative fast alpha power (1113 hz/450 hz) and analyzed . The pre- and post - intervention data were compared within each group and between groups by using paired t - tests independent t - tests, respectively . There were no significant differences in the general characteristics between groups (p> 0.05) (table 1table 1.general characteristics of participants (n = 20)categoryhorseback riding(n=10)control(n=10)age (years)69.5 3.269.7 3.5height (cm)157.6 8.7158.5 6.8weight (kg)58.8 7.959.2 6.7bmi bmi: body mass index after the horseback riding program, the relative slower alpha power was analyzed; it appeared to be active in the t3 and p4 domains but was suppressed in the fp1, fp2, f3, f4, t4, and p3 domains (table 2table 2.relative slow alpha power before and after the interventionpre - interventionpost - interventionhorseback ridingfp10.321 0.1470.308 0.177fp20.337 0.1500.326 0.148f30.339 0.1400.337 0.151f40.341 0.1590.319 0.164t30.288 0.1310.314 0.119t40.254 0.1540.212 0.096p30.369 0.1460.367 0.147p40.345 0.1970.351 0.166controlfp10.243 0.1370.288 0.177fp20.246 0.1350.277 0.176f30.251 0.1410.302 0.181f40.237 0.1530.279 0.201t30.216 0.1160.225 0.131t40.164 0.1410.210 0.155p30.291 0.1200.309 0.150p40.277 0.1510.325 0.180data are moreover, after the horseback riding program, the relative faster alpha power appeared to be active in all domains of the horseback riding exercise group but suppressed in all domains of the control group (table 3table 3.relative fast alpha power before and after the interventionpre - interventionpost - interventionhorseback ridingfp10.055 0.0330.058 0.033fp20.060 0.0340.061 0.034f30.059 0.0280.067 0.038f40.057 0.0250.062 0.039t30.060 0.0250.073 0.031t40.054 0.0190.055 0.029p30.088 0.0720.098 0.059p40.078 0.0570.093 0.049controlfp10.048 0.0260.045 0.028fp20.047 0.0300.045 0.031f30.058 0.0370.050 0.034f40.051 0.0420.046 0.035t30.059 0.0370.050 0.034t40.040 0.0240.039 0.018p30.064 0.0420.054 0.038p40.054 0.0370.048 0.027data are mean sd . . There was a significant difference between the horseback riding exercise and control groups with respect to the f3 domain (p <0.05) (table 4table 4.comparison of relative fast and slow alpha powerhorseback ridingcontrolrsafp10.013 0.1240.025 0.093fp20.020 0.1370.012 0.131f30.006 0.1330.037 0.114f40.020 0.1520.043 0.136t30.013 0.1120.018 0.075t40.040 0.1500.031 0.113p30.000 0.0920.018 0.122p40.026 0.1830.018 0.137rfafp10.000 0.0530.013 0.034fp20.000 0.0530.006 0.025f30.020 0.0410.019 0.040f40.007 0.0590.000 0.036t30.007 0.0250.013 0.050t40.007 0.0450.006 0.044p30.007 0.0250.000 0.051p40.013 0.0350.000 0.051p <0.05, rsa: relative slow alpha power, rfa: relative fast alpha power). P <0.05 * p <0.05, rsa: relative slow alpha power, rfa: relative fast alpha power during acute graded maximal exercise, the alpha wave power is reportedly decreased in the frontal and temporal lobes8, 9 . Alpha wave power is an index of stable emotional status or mental health; in this regard, alpha wave power appears to be more closely associated with mental stress than exercise intensity, which is physically recognized . The present study evaluated the changes in background eeg, specifically alpha wave power, which indicates stability and relaxation10, 11 . The relative slow alpha power was analyzed before and after the horseback riding exercise program; the results show it increased equally in all brain wave domains in the control group . In the horseback riding exercise group, the relative slow alpha power was active in the t3 and p4 domains but suppressed in the fp1, fp2, f3, f4, t4, and p3 domains . After the horseback riding exercise program, the relative fast alpha power was increased in all domains of the horseback riding exercise group . Horseback riding exercise is considered to enhance concentration and comfort by activating the brain waves in all domains . The relative alpha power by the motor learning process is reported to increase in the fz, cz, oz, c3, c4, t3, and t4 domains during learning through exercise imagination but is decreased in those observing and performing behaviors12, 13 . After the learning process, the relative alpha power differs significantly in participants who observe and actually perform behaviors compared to those who perform exercise imagination in the pz, oz, c4, t3, and t4 domains, indicating the brain activation level is higher during exercise learning through behavior observation and actual performance than imagination exercise learning . In the present study, there were small differences in the attachment points of the 8 brain wave channels, background eeg, and evoked eeg . However, the results corroborate the notion that learning through exercises helps increase the relative alpha power . The main limitation of this study is its lack of generalizability owing to the relatively small sample as well as the fact that the daily life habits aside from the horseback riding exercise program were known only through personal interviews . Therefore, individual life habits may have affected the results . Accordingly, further studies with more subjects and better evaluation methods are required to confirm the results . Moreover, further studies should investigate the psychological, cognitive, and physical changes as a result of horseback riding . Finally, comparative studies involving different age groups and patients suffering from differing diseases and conditions should be performed.
Spermatic cord torsion (sct) is one of the true urological emergencies that mainly develop during puberty and the early adolescent period, with a calculated annual incidence of 1 in 4,000 among those under 25 years old . The salvage rate of an affected testis largely depends on the duration and degree of torsion . Although sct can be seen at any age, in fact, there have been few reports of older men with sct . Because of its rarity in the older age group, the lack of suspicion for sct may lead to delayed diagnosis or misdiagnosis . To the best of our knowledge, this seems to be the first reported case of sct in an aged patient older than 60 years in korea . Herein, we report this very unusual case of missed sct occurring in a 63-year - old man . A couple of weeks previously, he had experienced right scrotal pain when he woke up . He was then seen by a primary care physician . However, the physician failed to find any abnormalities other than epididymal enlargement on scrotal ultrasonography . The patient was then referred because his symptoms did not improve despite empirical antibiotics use . He denied having any history of trauma to the testis, strenuous exercise, sexual activity, or febrile illness . He also had no known sexually transmitted diseases . On physical examination, a swollen, edematous, and erythematous change in the right hemiscrotum was noted . The right testis was enlarged, hard, consistent, and somewhat tender on palpation compared with the intact left testis . The results of a complete blood count, chemistry profile, and urinalysis were normal . Gray - scale ultrasonography of the scrotum demonstrated heterogenous echotextures in the right testis that were combined with a thickened scrotal wall and hyperechoic supratesticular mass (fig . Color doppler sonography revealed that blood flow was present in the asymptomatic left testis, but absent in the right testis . An increased blood flow in peritesticular structures adjacent to the nonperfused testis was identified (fig . Although very rare, a few cases of intrascrotal torsion of a testis tumor have been reported . Therefore, we could not absolutely exclude the possibility of a testis tumor and thus planned to perform radical orchiectomy of the symptomatic right testis . The patient was taken for surgical exploration of the right testis through a right inguinal incision . The frozen section examination of the right orchiectomy specimen revealed testicular infarction with suppurative inflammation . Hence, left transscrotal orchiopexy was done to prevent contralateral torsion at a later date . An underlying anatomic abnormality, the bell - clapper deformity, was found in the left testis . In the cut section, the right testis was 4.3 cm3.2 cm4.2 cm in size and had reddened parenchyma rimmed with several scattered lesions of dirty yellow pus - like exudates . Final histopathological examinations revealed a total hemorrhagic infarct of the testis and diffuse chronic inflammation of the peritesticular soft tissues (fig . The causes of acute scrotal pain vary with age, but in all ages, infection and sct are the most common etiology . Differentiating sct from infection in the epididymis or testis is critical . Although intravaginal sct is primarily a condition of puberty and early adolescence, it is not exclusive to that population . The onset of pain in sct is usually acute; however, up to 25% of patients also have a gradual onset . Thus, delayed presentation or diagnosis may result in patients with gradual onset of scrotal pain . In addition, age may be another factor that affects the testicular salvage rate . Because sct is uncommon and is associated with more severe cord twisting in the older aged group, the prognosis of testicular salvage in this age group therefore, a high index of suspicion is the first step to determine the presence of sct even if symptoms are equivocal in older patients . Color doppler ultrasound performed by a trained radiologist proved to be a somewhat more desirable modality because it is performed more rapidly and equal to nuclear scintigraphy with respect to sensitivity and specificity . Patients who are very early in the course of torsion may have only minimal enlargement of the testis with normal or slightly decreased echogenicity . As time goes on, the echogenicity of the testis becomes heterogeneous owing to hemorrhage and necrosis . A swollen epididymis may be also seen because the deferential artery supplying the epididymis is often involved . An abrupt increase in the size and alteration of the spermatic cord may be noted below the point of torsion . This finding of a twisted cord is a highly sensitive and specific sign of sct . In the present case, we suggest some possible reasons for the misdiagnosis during the patient's first visit at the local clinic . In most cases of sct, the epididymal head is only mildly enlarged, whereas the spermatic cord, which passes just superoposteriorly to the epididymal head, is grossly enlarged . Thus, the swollen spermatic cord may be mistaken for an enlarged epididymal head . In cases of missed torsion, . This flow may be mistaken for testicular capsular artery flow, thus causing the diagnosis of torsion to be missed . Less likely, a testis with spontaneous detorsion may be hyperemic, simulating epididymo - orchitis . Because a missed diagnosis is the most frequent medicolegal concern in our litigious society clinical symptoms and physical examination are often not enough for prompt diagnosis, especially in cases with delayed presentation . Because the adult type of sct is thought to be rare, this rarity may result in missed torsion unless special attention is paid to the possibility of sct . The present unusual case reminds us to maintain suspicion of sct in males of any age presenting with acute scrotum.
Titanium - based alloys have high corrosion resistance because they form a thin, stable oxide layer . Nevertheless, fluoride - containing prophylactic agents can cause corrosion and associated discoloration of titanium - based orthodontic wires . Resistance to corrosion, superelastic properties, and permanent shape memory, make the orthodontic treatment more efficient for clinicians by decreasing the number of wire changes required for each patient . Furthermore, ni ti wires, thanks to their pseudoelastic properties, allow the orthodontists to apply light and continuous forces to the teeth, reducing the risk of patient discomfort, tissue hyalinization and undermining resorption . It is important to recommend the regular use of fluoride - containing products, in particular gels and mouthwashes, during the course of orthodontic treatment to help prevent dental caries . Additionally, systemic fluoride may be ingested orally by drinking tea and fluoridated bottled water and also taking dietary supplements . Fluoride mouthwashes, available in 0.05% and 0.2% fluoride concentrations, are frequently prescribed by orthodontists for weekly and even daily use to prevent caries . It has been clearly shown that ni ion release, due to the corrosion, can cause allergenicity, toxicity and carcinogenicity [58]. It can also cause allergic contact dermatitis, the incidence of which is as high as 2030% [911]. The corrosion phenomenon not only may influence the mechanical properties of the metal appliances, but also may affect the body due to leached metal ions [1214]. Because of the ionic, thermal, microbiological, and enzymatic properties of the oral environment, patients are often exposed to corrosion products . Atomic absorption spectrometry has been used to analyze the ni ion release [1618]. The second method is to use electrochemical tests [7,1921] in artificial saliva to assess the electrochemical properties in order to evaluate the corrosion resistance of niti alloys in vitro . There are many published studies on the corrosion resistance of niti alloys in physiological solutions, particularly in both fluoridated and non - fluoridated fusayama . Moreover, niti archwires are significantly more stable and resistant to corrosion than stainless steel archwires [2325]. Furthermore, it has been well documented that fluoride has a negative effect on the corrosion resistance of niti alloys [22, 23]. However, there is little information to interpret and compare the adverse effects of different concentrations of fluoride available in typical mouthwashes . The purpose of the present study was to investigate and compare the electrochemical corrosion characteristics of three different types of niti and one stainless steel archwires in fluoride mouthwashes with two different concentrations . In order to achieve this objective, open circuit potential measurements and potentiodynamic and potentiostatic polarization tests for the comparison purposes, three types of commercially available niti wires and a stainless steel wire were selected . The media for corrosion tests were the artificial saliva used as the control and the artificial saliva containing 0.05wt% and 0.2wt% sodium fluoride . Wires: the selected niti orthodontic wires were divided into three groups: niti-1 rema - titan (dentaurum, germany), niti-2 sentalloy (gac, usa), and niti-3 global (global, canada). The last group consisted of ss archwires (dentaurum, germany). All of the wires were 0.016 inches (0.41 mm) in diameter and shaped as preformed lower arches . Each test sample contained three pieces of wire 6 mm in length (12 specimens for each test solution). Meyer artificial saliva . Table-1 exhibits the composition of the artificial saliva, which closely resembles natural saliva . Contents of the fusayama - meyer artificial saliva all the constituents were provided from merck chemical company, germany . The ph was measured with a ph - meter (wtw, d82362, weilheim, germany). The ph of this reference saliva, corresponding to the first test solution, was 5.35 . To evaluate the effect of fluoride concentration on the corrosion resistance of commercial niti archwires, different amounts of naf were added to the artificial saliva to prepare 0.05% and 0.2% naf concentrations, simulating the fluoride concentrations in commercially available fluoridated mouthwashes . Their covers were made of teflon plates having two openings to allow the insertion of electrodes (azarteb, urmia, iran) and two smaller holes for the tips / ends of the archwires . The jars filled with artificial saliva containing 0.05 wt% and 0.2 wt% naf were stored at 371c in a bain - marie bath (pars azma type 1400, iran). Before each test, the wires were degreased ultrasonically with acetone for 15 minutes at ambient temperature, then washed with distilled water and dried with a hairdryer . A 6 mm portion of each wire was marked and immersed in the media . The reference electrode was ag / agcl, and the auxiliary electrode was made of platinum (azarteb corp ., corrosion resistance analyses: the corrosion resistance analyses comprised of polarization tests (potentiostatic and potentiodynamic), the corrosion potential monitoring and evaluation of the surface topography of wires . The corrosion potential was measured over time by plotting the polarization curves once the potential was stabilized, to determine the corrosion resistance of the titanium alloys in different media . The potential for all the wires in each media was recorded every 10 seconds for a period of 24 hours . To determine the corrosion current density (icorr) in the potentiodynamic polarization tests, the archwires were polarized both anodic and cathodic (300mv to 1500mv) relative to the reference electrode . Each curve represents one of the three experiments of each material in each medium and all the curves were obtained from reproducible individual measurements . The ecorr, icorr values are the mean values of the three experiments with a maximum error of 1015% (figure 1). Depicting various parameters in a polarization plot the changes in corrosion current density were recorded as dependent of time . In the end, the topographic evaluation of working electrodes (the wires) was done using sem (cambridge, s-360, england). The corrosion potential of the different alloys was measured after 24 hours in the four test solutions . As shown in table 2, this finding suggested that the corrosion resistance of archwires diminished by increasing the naf concentration . Corrosion potentials (mv / sce) of the samples in different media after 24 hours the polarization curves were plotted in the potential range of 300 mv to + 1500 mv at a scanning rate of 0.5mv / s . The corrosion current increased and the potential of breakdown decreased (the potential at which the alloys started to corrode) as the fluoride concentration increased (tables 3 and 4). The corrosion current densities (10 6 a / cm 2) of the samples in different media potential of breakdown (mv) of the samples in different media in artificial saliva, there was a little difference between niti-2 and the two other wires in cathodic area . In the anodic area, the plots showed a large range of passivity: beyond this area, a trans - passivity phenomenon occurred . Comparison of niti archwires polarization plots in artificial saliva in contrast, stainless steel wires showed a pitting corrosion registered at 630mv while ni - ti-3 as the representative of niti alloys revealed the pits in a potential roughly over 1100 mv (figure 3). Potentiodynamic polarization plot for stainless steel and niti-3 (global) archwires in artificial saliva since the anodic reactions are responsible for metal dissolution during polarization of an alloy, studying the anodic branch of polarization curves obtained from potentiodynamic experiments is of interest . Since the corrosion current density of stainless steel archwires in potentiostatic tests was far more than that of niti alloys, it was not possible to chart it in figure 4 next to other niti wires . Both the potentiostatic tests and the corrosion potential (ecorr) over time confirmed the passivity of niti wire in neutral environment (figures 4 and 5). Comparison of niti archwires potentiiostatic plots in artificial saliva comparison of niti archwires corrosion potential over time in artificial saliva figure 5 shows the changes in corrosion potential of nitinol and stainless steel archwires in simulated saliva solution . For nitinol archwires, this polarization of anodic reaction is due to the integration of the passive layer on the surface in the form of tio2 . The initial corrosion potential of stainless steel was almost 200 mv more positive than nitinol archwires and it was stable for the course of experiment at about 125 mv . This behavior may be attributed to the stability of passive film; mainly cr2o3, on stainless steel . However variation of corrosion potential for about 60,000 seconds indicated that the alloy showed passivity . Potentiostatic results of nitinol archwires at 200 mv potential above the corrosion potential are shown in figure 4 . As seen, the current densities of all three niti alloys when applied above 200 mv showed a sharp decrease and reached a stable condition . This behavior proves that all alloys are in passive state at 200mv applied anodic potential . The only marginal difference mentionable is the greater tendency of niti-1 (dentaurum) archwire that showed a sharper drop in passive current density for the first 100 seconds of the experiment and a lower stable passive current density at the end of the course of experiment in comparison with the other two niti archwires . However, the passive current density of the three alloys at the end of the experiment was about 50 na / cm2 . This negligible passive current density proves the stability of these alloys in artificial saliva environment due to the formation of a corrosion resistant passive film . An increase in the corrosion current density was observed as the concentration of fluoride increased . The corrosion current densities in the saliva containing 0.05 wt% fluoride were 0.92 a / cm2 for niti-1, 0.13 a / cm2 for niti-2 and 0.26 a / cm2 for niti-3 wires . In 0.2wt% fluoridated saliva, icorr were 1.4 a / cm2, 0.36 a / cm2 and 0.51 a / cm2 for niti-1, niti-2 and niti-3 archwires, respectively (figures 6 and 7). Comparison of niti archwires polarization plots in artificial saliva with 0.05wt% naf comparison of niti archwires polarization plots in artificial saliva with 0.2wt% naf the increase in corrosion currents in response to increased concentration of naf confirmed that the corrosion resistance had a reverse correlation with naf concentration . Topographic assessment by sem showed a pitting corrosion in different shapes in all the wires tested . In both stainless steel and niti-1 wires, the shape of pits was well - defined; whereas, in the other wires, the pits were accompanied by fissures and porous surfaces (figure 8). Sem images of pitting corrosion in a)dentaurum niti-1 archwires b)gac niti-2 archwires c)global niti-3 archwires d)dentaurum stainless steel archwires results of the polarization test in the artificial saliva revealed that all niti wires showed passive behavior, no pitting corrosion and trans - passivity due to water oxidation . Niti-1 group showed the highest resistance while the stainless steel wires exhibited passivity in corrosion potential . As seen in figure 3, steel wires showed pitting corrosion current density in lower potentials . This indicated that the stainless steel passive film was more protective than that of niti in this saliva solution . However, the resistance of this alloy to pitting corrosion was much lower than that of niti due to surface inclusions . Potentiostatic results of nitinol and stainless steel wires at 200mv above the corrosion potential are shown in figure 4 indicating that nitinol wires were passive . Additionally, the passivity current density continuously decreased with time and after 15 minutes, it reached around 500 na / cm2 for the three niti wires . The experiments demonstrated that the corrosion potential of niti alloys experienced a pronounced decline by adding fluoride, which suggested that the corrosion resistance of niti orthodontic archwires decreased in presence of fluoride in an acidic environment . Potentiodynamic tests revealed that niti-2 group showed pitting corrosion in higher anodic potentials (around 1200 mv) by adding fluoride ions to the media in comparison with the other two niti wires . This behavior is due to the local dissolution of tio2 oxide layer in the presence of fluoride ions at higher potentials . In presence of fluoride ions, the corrosion potential decreased, the corrosion current density increased and the passivity current density increased as well . These indicate that the anodic reaction was depolarized . Therefore, dissolution of tio2 increased in presence of fluoride . A similar decrease in corrosion potential of niti alloys created by fluoride has also been reported in the literature [7, 22, 27]. Fluoride ions attacked the protective oxide layer on all the alloys tested . In artificial saliva, it can be concluded from these observations that the tio2 protective layer on the surface of niti wires is much more resistant than the cr2o3 layer on stainless steel wires, confirming the findings of rondelli and schiff [22, 26]. The acceleration in corrosion behavior of nitinol wire in the saliva solution in presence of fluoride can be attributed to either the increase in aggressive ion concentration or the nature of fluoride as an accelerator ion . There are few studies discussing the effect of fluoride ions on the orthodontic niti wires using potentiostatic tests in different anodic potentials and the evolution of the corrosion current density in time as an addition to the traditional potentiodynamic tests . Potentiostatic tests confirmed that the corrosion resistance of niti wires decreased by adding naf to artificial saliva, but the polarization plot of niti-1 group was different from that of the other niti wires as the increase of current density was pronounced and parallel to the x axis . In contrast, the increase of current density in the other niti groups led to a slight curve approximately parallel to y - axis . Sem demonstrated that the shape of corrosion pits was also different for the niti-1 group when compared with the others and its smoother surface contrasted the rough surface of other wires . Probably, the cold working / manufacturing of these wires is responsible for the differences seen in the corrosion behavior and polarization plot of these wires . The pictures show a well - defined saucer - shaped pits with a honeycomb form in depth for niti-1 but fissures and cracks along the archwires length for niti-2 and niti-3 . It may explain the different breakdown potentials of niti-1 and the shape of its corrosion pits in presence of naf . Consequently, it could be pointed out that the pitting potential of niti orthodontic wires in fluoride can be significantly influenced by their surface topography . Although the corrosion resistance decreased in artificial saliva with 0.05wt% naf, the corrosion pits were created in high potentials (approximately + 200mv). In addition, the corrosion potentials in all three groups of niti archwires and stainless steel group were the same in the mentioned electr lytes in spite of their difference in shape of corrosion pits . Though our results may seem to differ from those of lee et al, suggesting that different niti archwires had dissimilar corrosion resistance in acidic fluoride - containing artificial saliva . This controversy may be due to the difference in the concentration of fluoride ions in the two studies . They used the maximum 0.5wt% fluoride in their experiment, which was 10 times more than the concentration used in our study . In the solution containing 0.2wt% naf, our finding is consistent with that of previous studies, which have shown that a niti orthodontic archwire may corrode in commercially available fluoride mouthwashes . Furthermore, our results confirm the studies reporting that the protective role of tio2 formed on ti alloy is compromised by fluoride ions when the naf concentration exceeds 0.1wt% . Aggressive behavior of fluoride ions on titanium alloys in aqueous environment has been attributed to the chemical reaction of fluoride ions with the tio2 passive film and destruction of the tio2 passive film by the formation of soluble tif62-ion (following reaction). This reaction leads to deterioration of passivity by thinning of tio2passive film leading to the depolarization of the anodic branch of polarization curve . It means that higher passive current density can be reached in presence of fluoride due to the depolarization of passive film . The increase of passivity current density may also be attributed to the increase in tio2 oxide layer defects in presence of f - ions and its oxide chemical dissolution by the abovementioned reaction . Therefore, it is wise to use fluoride mouthwashes with lower weight percentage of fluoride when their consumption seems necessary, especially during long - term orthodontic treatments as these incur the risk of poorer physical and electrochemical characteristics of the wires . Moreover, the nickel ions released from alloys attacked by fluoride may cause toxic and allergic reactions . The oral cavity conditions are, however, more complex and some other types of corrosions such as crevice corrosion or galvanic corrosion also need to be considered, as well as food debris accumulation, diet and differences in oxygen concentrations . In the artificial saliva, all the niti archwires were found to be passive, in contrast to the stainless steel wire, which showed pitting corrosion . The addition of fluoride to artificial saliva decreased the corrosion resistance of all the archwires tested . This may be due to the destructive effect of fluoride on the protective oxide coating.
Renal cell carcinoma originates in the renal cortex and usually presents at an advanced stage with a 50% mortality rate after 5 years of diagnosis . It classically presents as hematuria, flank pain and abdominal mass but may also manifest with a variety of paraneoplastic syndromes including anemia, hypercalcemia, erythrocytosis, amyloidosis and thrombocytosis . Notably, more than 90% of metastases in the lung have been detected by chest x - ray (cxr) in asymptomatic patients . Pulmonary embolism involves the establishment of the tumor in the pulmonary vasculature, but renal cell carcinoma also frequently invades the lymphatic system . Isolated lymph node metastases due to renal cell carcinoma are infrequent but have been reported in medical literature [5, 6]. Spread beyond the lymph nodes may occur through the lymphatic system, resulting in lymphangitic carcinomatosis . Lymphangitis carcinomatosa of the lungs is distinct from pulmonary metastases as the tumor emboli do not proliferate or spread locally . They are often seen in collections measuring less than 10 microns and frequently trigger the coagulation cascade and obstruct the flow of blood in pulmonary capillaries . This may result in severe respiratory distress and often becomes the direct cause of death . This case report details the presentation and clinical course of a patient with renal cell carcinoma who was found to have lymphangitic carcinomatosis which rapidly progressed in a time span of two weeks resulting in his demise . He denied night sweats and weight loss, and was a lifelong non - smoker . Hilar lymph nodes (3/3), para - aortic lymph nodes (8/8) and interaortocaval lymph nodes (7/7) tested positive for tumor involvement . The left kidney was found to have a 9.8-cm mass at the lower pole and biopsy showed clear cell type renal cell carcinoma with focal sarcomatoid and rhabdoid cells . He was readmitted two weeks later with right flank pain, dyspnea, fever and cough productive of yellowish sputum . Physical examination was significant for tachycardia, tachypnea, bilateral crepitations in the chest and bilateral 1 + pedal edema . His arterial blood gas showed respiratory alkalosis with an elevated alveolar - arterial (a - a) gradient of 52 . His cbc showed leukocytosis at 19,800 cells/l with 88% neutrophils, hemoglobin of 9.8 g / dl and a platelet count of 588 k/l . His lfts were deranged with an albumin of 2.5 g / dl, alk of 315 and ast / alt of 166/184 . His esr was elevated at 134 and bun / creatinine rapidly rose from 13/1 to 24/1.9 . Notably, his cxr showed marked infiltration with nodular opacities in all lung fields bilaterally . 2b shows his cxr at the second admission which reveals marked changes compared to his initial cxr . He was started on zosyn and vancomycin and was later switched to aztreonam and clindamycin on account of worsening renal function . Ct scan to rule out pulmonary embolism could not be performed due to renal failure and doppler ultrasound of the lower extremities was negative for dvt . Ct scan without contrast showed nodular opacities with extensive septal thickening as depicted in fig . An echocardiogram showed a hyperdynamic left ventricle with ef of 80% . The right ventricle size and function were normal . Bronchoscopy with transbronchial biopsy of the right medial lobe was done and biopsy samples revealed non - small cell cancer with clear cell features consistent with renal cell carcinoma and lymphangectatic invasion . Immunohistochemistry showed that the tissue stained positively for vimentin, cd10 and cd 31 as depicted in fig . His renal function worsened and he was transferred to the medical icu where he developed septic shock, he was made dnr and passed away shortly thereafter . The autopsy report showed florid lymphangectatic invasion with focal necrosis, affecting all lobes of both lungs . Lymphangitic spread of metastatic cancer has been observed in many malignancies including breast, lung, pancreatic, colonic and cervical cancers [7, 8]. Symptoms of tachycardia, tachypnea, hypoxemia and clear lung fields in a patient with malignancy should raise the suspicion of lymphangitic carcinomatosis . Infiltration of the tumor cells into the superficial lymphatic system can be readily detected as lymphedema in some malignancies, especially breast cancer . This may even be the initial manifestation of a malignancy or may portend massive tumor burden due to an internal malignancy . The patient may suffer from intense pain, ulceration and necrosis if the superficial lymphatic system is affected as against a painless gradual course when the deeper lymphatics are involved . Ventilation perfusion scans may be normal or may show a mottled appearance with multiple small, peripheral, subsegmental perfusion defects with normal ventilation . This pattern can be readily distinguished from the pattern seen with thromboemboli . Pulmonary angiography is insensitive and may show subtle defects including subsegmental filling defects, pruning and tortuosity of the third- to fifth - order vessels, or delayed filling of segmental arteries . Fdg - pet similarly may detect large emboli with avid uptake but may not be sensitive to detect smaller collections of tumor cells . The gold standard of diagnosis is lung biopsy with demonstration of microscopic emboli of tumor cells in the pulmonary vasculature and detection of circulating tumor cells in the blood drawn from the pulmonary artery catheter . However, it should be noted that lymphangitic carcinomatosis may not necessarily be blood borne . Pulmonary lymphatics communicate with the lymphatic system of the neck and direct extension of head and neck cancers has been observed to occur through this route . Lymphangitic carcinomatosis may also present in an insidious manner being the first sign of an occult malignancy . It has also been noted to be asymptomatic with the only finding being an abnormal cxr . The presence of vimentin and cd10 noted on immunohistochemistry confirmed the diagnosis of clear cell renal cell carcinoma in this patient . It is interesting to note that the tissue samples of this patient were positive for cd31 which inversely correlates with tnm staging and nuclear grade . The sarcomatoid features noted in some samples excised during his initial surgery may explain the aggressive course of his disease as it has been shown that sarcomatoid renal cell carcinoma is a rapidly progressive disease with poor overall survival [14, 15]. This case report demonstrates the rapid clinical deterioration a patient may experience due to pulmonary lymphangitic carcinomatosis . It is notable that this patient underwent successful resection but developed lymphangitic carcinomatosis within two weeks which likely was the direct cause of his death . Early detection with the help of imaging mediated by tumor - specific antibodies can result in therapeutic interventions which may slow the progression of the disease or may even be life - saving.
Myiasis is defined by zumpt as the infestation of live human and vertebrate animals with dipterous larvae, which, at least for a certain period, feed on the host s dead or living tissue, liquid body - substances, or ingested food . Traumatic myiasis is caused by fly larvae infesting pre - existing traumatic lesions or actively gaining access to tissues . Fly species that cause traumatic myiasis can be divided into obligate or facultative ones according to the nature of the host - parasite association . Obligate traumatic myiasis involves larvae that can only develop on living tissue, whereas in facultative myiasis the larvae can develop on both living and dead tissues . Here, we describe a rare case of traumatic myiasis in a domestic cat (felis catus l., mammalia: felidae) caused by an association of sarcophaga tibialis macquart (diptera: sarcophagidae) and lucilia sericata (meigen) (diptera: calliphoridae). A 10-year old european shorthair cat, missing from home for about 3 days in august 2014, was found near its home in the village of san martino (ferrara province, italy) with a large wound on the rump near the base of its tail, which was almost completely detached (fig . The veterinaries assumed that the injury had been caused by a dog bite and found an extensive traumatic myiasis in the wound caused by a high number of diptera larvae (fig . The veterinaries treating the cat collected 60 larvae (all first or second instar), which were placed in plastic test tubes and immediately brought to the laboratory of urban ecology, department of life sciences and biotechnology, university of ferrara, for identification . The wound was washed with an antiseptic (povidone - iodine), and ivermectin was administered subcutaneously . Afterwards, the cat s tail was surgically amputated and during surgery the cat was drip - fed with physiological solution, a vitamin support, and a corticosteroid . After suturing the wound the cat was treated with broad - spectrum antibiotics (streptomycin and penicillin). In the laboratory of urban ecology the larvae were divided into 2 groups: 15 of them were immediately fixed in 4% formaldehyde and the other 45 were reared in a plastic box containing 90 g of cow liver, at 25c, 50% relative humidity and a 16/8 (l / d) photoperiod . Upon reaching the third instar, the larvae were again divided into 2 groups according to their length (respectively 14.80.7 mm and 11.61.0 mm) and aspect (fig . Morphological investigations were performed with a nikon smz 800 stereomicroscope (nikon instruments europe, amsterdam, the netherlands) and a nikon eclipse 80i optical microscope (nikon instruments europe), both connected to a nikon digital sight ds - fil camera (nikon instruments europe). The identification of s. tibialis was carried out by examination of adult males (fig . 2c) emerged from the first group of larvae, based mainly on aspects of the genitalia (fig . The identification of the second species as l. sericata was carried out using the identification key of szpila and was based on morphology of adult males (fig . 2e) emerged from the second group of larvae, namely the bright yellow basicosta (fig . 2f). All examined larvae and adults are deposited in the laboratory of urban ecology, department of life sciences and biotechnology, university of ferrara, ferrara (italy). Reports of myiasis in cats are uncommon, probably because they usually groom themselves carefully . In the present case, infestation by these 2 fly species was favoured by a deep and extensive wound that had weakened the cat, preventing it from properly grooming and cleaning its fur . Cases of myiasis in humans and animals caused by an association of more than one diptera species have been reported in the literature . The species involved are chrysomya rufifacies (macquart) (diptera: calliphoridae) with chrysomya megacephala (fabricius) (diptera: calliphoridae), sarcophaga ruficornis (fabricius) (diptera: sarcophagidae) with musca domestica (linnaeus) (diptera: muscidae), and c. megacephala and chrysomya bezziana villeneuve (diptera: calliphoridae) with lucilia sp . (diptera: calliphoridae). Myiasis caused by s. tibialis in association with another species had been reported only once, by villeneuve with sarcophaga crassipalpis macquart (diptera: sarcophagidae). L. sericata has been reported to cause myiasis in association with chrysomya albiceps wiedemann (diptera: calliphoridae) and wohlfahrtia magnifica (schiner) (diptera: sarcophagidae). The case we report is the first of myiasis caused by an association of s. tibialis and l. sericata . S. tibialis is a thermophilic, heliophilic, and generally synanthropic species, probably indigenous to the afrotropical region and widespread in africa (including the canary islands, madagascar, and the seychelles), central and southern europe, turkey, the chagos archipelago in the oriental region, and oceania . The species is widespread in italy, including sardinia and sicily . Like most sarcophagidae, eggs laid in groups or together with larvae have been reported not to hatch . As in most flies, abasa reported that at 20 - 22c the development from first larval stage to adult lasts about 20 - 27 days, whereas aspoas stated that at 25c development from first instar to adult lasts 20 - 22 days . Recently, villet et al . Reported that the optimal temperature for development of this species (based on lowest larval mortality) is about 20c . In our study, females of s. tibialis and l. sericata were attracted by the smell released by the wound and subsequently deposited larvae and eggs on it . Adults of s. tibialis are usually attracted by decaying matter such as carcasses and excrements, where larvae may or may not be laid . Larvae of this species have been reared in the laboratory on liver, fish carrion, fresh bird meat, dead snails, and chopped grasshoppers [3,17 - 19]. Only 2 well - documented cases of cutaneous human myiasis caused by s. tibialis were reported in the literature . Both occurred in tripoli (libya) in 1913, the first in may and the second in july . Both cases were connected to poor sanitation and the myiasis was localized to the scalp . The first case involved a bedridden boy affected by peritoneal tuberculosis and tinea capitis, the second occurred in a little girl affected by tinea capitis, pediculosis, and pyoderma . According to these reports, onorato hypothesized a possible connection between tinea capitis and cutanous myiasis by s. tibialis . Besides these 2 cases, there is a hint of another case of human traumatic myiasis in algeria mentioned by villeneuve, but without any further detail . Also, patton and evans stated that s. tibialis is known to cause intestinal myiasis in europe, but they did not report any data on the number of cases and localities of occurrence therefore, the present case is the first record of s. tibialis causing myiasis in italy and is probably the first well - documented case of this kind in europe . On the other hand, l. sericata is a widely distributed agent of myiasis in temperate areas and a probable cosmopolite . In the united kingdom, south africa, and new zealand, l. sericata is a relevant agent of myiasis in sheep . Adults are diurnal and commonly breed on carcasses, dirty or wet sheep fleece, garbage and manure; they are also attracted by open wounds and lesions where conditions are such that the larvae can complete their development, causing myiases such as fly strike in sheep . Females start to lay eggs 5 - 9 days after emerging from the puparium and within 3 weeks they lay 2,000 - 3,000 eggs in 9 - 10 batches . The larval developmental rate is strongly temperature - dependent: between 22c and 29c, development from egg to adult occurs in 13 - 19 days . In southern europe, l. sericata is considered a synanthropic species associated to houses and rural areas undergoing urbanization . Together with calliphora vicina robineau - desvoidy (diptera: calliphoridae) and lucilia illustris meigen (diptera: calliphoridae), l. sericata is among the 3 species characterizing urban and suburban areas in europe . In our report the cat contracted myiasis in san martino, a small country village about 4 km from the city of ferrara (ferrara province, italy). Myiasis caused by l. sericata has been reported in domestic cats in america, europe, and asia and may be traumatic [5,29 - 33], gastrointestinal (anal and rectal), urogenital (vaginal, penile and perineal) [30,35 - 37], or ocular . Our report is the first case of traumatic myiasis by l. sericata in a domestic cat in italy, albeit in association with s. tibialis . The only other case of myiasis by l. sericata in a domestic cat in italy was a rectal one, although cutaneous myiases by this species have been reported in sheep and dogs, and humans . The present results on the first case of myiasis caused by an association of s. tibialis and l. sericata and on the first case of myiasis by s. tibialis in italy were obtained through a close cooperation between veterinaries and entomologists . Studies on the biology, distribution, and modes of infestation of diptera causing myiasis could largely benefit from similar collaborations . Accurate and detailed information on the distribution of species of medical and veterinary interest is limited in italy . Thus, the compilation of an infestation map should be a priority objective for veterinary interventions and for ecological and biological studies.
The imgt / hla database was established to provide a locus - specific database (lsdb) for the allelic sequences of the genes in the hla system, also known as the human major histocompatibility complex (mhc). This complex of over four megabases is located within the 6p21.3 region of the short arm of human chromosome 6 and contains in excess of 220 genes (1). The core genes of interest in the hla system are 21 highly polymorphic hla genes that mediate the host response to infectious disease and influence the outcome of cell and organ transplants . With a nomenclature spanning over 50 genes and 3000 alleles, there is an obvious need for a lsdb to curate these highly polymorphic variants . The sequencing of hla alleles began in the late 1970s predominantly using protein - based techniques to determine the sequences of hla class i allotypes . The first complete hla class i allotype sequence, b7.2 now know as b070201, was published in 1979 (2). The first hla class ii allele defined by dna sequencing, dra0101, followed in 1982 (3). While the first hla antigens were named during the 1960s (4), the first hla dna sequences or alleles were named by the who nomenclature committee for factors of the hla system (5) in 1987 . At that time 12 class two years later, in 1989, the nomenclature committee assigned official allele names to 56 novel class i alleles and 78 class ii alleles (6). Advances and availability of sequencing technology meant that in 2007 the nomenclature committee was able to name over 400 new alleles, with the number of hla - b alleles exceeding 1000 in 2008 . The dissemination of new allele names and sequences is of paramount importance in the clinical setting . The importance of a single recognized source for this data led to the first incarnation of the database, the hla sequence databank (hla - db) (7), which allowed the periodic publication of hla class i (811) and class ii (1217) sequence alignments in a variety of journals . By 1995, the numbers of new alleles being reported warranted the publication of monthly nomenclature updates (18), which continues to this day . That year also saw the first distribution of the hla sequence alignments online through the web pages of the tissue antigen laboratory at the imperial cancer research fund (icrf), london, uk . This work transferred to the anthony nolan research institute (anri) in 1996 where it continues to this day . The latest incarnation, is the imgt / hla database (1922), which began in 1997 as part of a collaboration involving the icrf, anri and the european bioinformatics institute (ebi). The first public release of the imgt / hla database was made on the 16th december 1998 (23). Since then the database has been updated every 3 months, in a total of 40 releases, to include all the publicly available sequences officially named by the who nomenclature committee at the time of release . The imgt / hla database contains entries for all hla alleles, and alleles of some related genes, officially named by the nomenclature committee . These entries are derived from expertly annotated copies of the original embl - bank / genbank / ddbj entries . This means that the imgt / hla database may contain multiple entries for any single allele . These component entries are submitted to the database either by the original author, or by our curators, when sequences of interest have been identified by data - mining but have yet to be submitted to the database . To distinguish each imgt / hla entry from the component embl entries, each new allele the accession numbers follow the format hla00000, where the 00000 represents a numerical code . It must be noted that all sequences within the imgt / hla database should also be available from the more general nucleotide sequence databases: embl - bank (24,25), genbank (26) and the dna database of japan (ddbj) (27,28). The main problem when accessing hla sequences from these databases lies in the definition of the sequence . Despite the work of the members of the who nomenclature committee for factors of the hla system in monitoring hla allele designations and maintaining the sequences, they have no control of how sequences are defined in these generalist databases . Readers should, therefore, be aware that entries in these generalist databases may be incorrectly named, contain unofficial designations or contain known, but uncorrected, sequencing errors . The main access point for the user is the world wide web (www), which allows users to employ a number of search tools and other facilities to retrieve, manipulate and analyse hla data . The first area comprises information and help pages that provide background on the database and provide in - depth help on the tools and data available and documentation of the imgt / hla file formats . These core tools allow the users to perform sequence alignments, allele queries and sequence searches as well as queries more relevant to how the data are used and interpreted in a clinical setting . The third area comprises final pages that provide links to commonly used third - party applications such as the sequence - analysis tools at the ebi, including srs, blast and fasta . As the primary users of the database are members of the clinical hla community involved in transplantation of tissues and organs, the most commonly accessed tools have been written to aid in their common queries . All tools are written in perl as cgi scripts and access restricted views of the underlying oracle relational database . The transplant and tissue typing community have two main queries; either to retrieve information on a particular allele or to view how a number of alleles differ in sequence . To answer these questions, the database provides a detailed report on any allele, as well as an interactive alignment tool to view how allelic sequences differ . The allele search tool provides a simple - to - use interface for retrieving allele information . The output, see figure 1, for each allele includes the official allele designation, previously used designations and the unique imgt / hla accession number . Other information provided includes the date that the allele was named, current status (as some allele designations have been deleted) and information on the individual or cell line from which the sequence was derived . Links to all component embl - bank / genbank / ddbj entries are also included . Recently, information from the hla dictionary (29) has also been added to some entries . The dictionary presents the serological equivalents of hla - a, -b, -c, -drb1, -drb3, -drb4, -drb5 and -dqb1 allotypes . The data summarizes equivalents obtained by the who nomenclature committee for factors of the hla system, the international cell exchange (ucla), the national marrow donor program (nmdp), the 13th international histocompatibility workshop, recent publications and individual laboratories . Any citations are also included with, wherever possible, a link to the pubmed entry for that citation . The pubmed link provides an online version of the abstract as well as links to other citations by the author and to similar papers . The final section of the output details the official nucleotide and protein sequence as well as any genomic sequence for the allele that is available . The report provides cross - references to a text flat - file in srs (hla0001), the omim entry for hla - a, the source entries in embl - bank (aj278305-z93949) and to the seminal citations in pubmed . The full entry also contains the nucleotide sequence at both the cds and genomic level . The report provides cross - references to a text flat - file in srs (hla0001), the omim entry for hla - a, the source entries in embl - bank (aj278305-z93949) and to the seminal citations in pubmed . Other information provided includes links to the source material the full entry also contains the nucleotide sequence at both the cds and genomic level . Hla allele sequences can differ from each other by as little a single nucleotide substitution, within a genomic sequence of 3300 bases . Such nucleotide differences between the alleles of prospective transplant donors and recipients can make the difference between a successful transplant, graft failure and death . This means that the database must be able to quickly and easily display this information to the user . The hla community is interested in seeing the polymorphisms in terms of the changes to the sequence rather than as a list of individual single nucleotide polymorphisms (snps). To this end, we have developed the alignment tool, rather than push the users into producing their own alignments for the sequences of interest or simply just reporting the polymorphic positions . These alignments allow a visual interpretation of sequence similarity so that polymorphic positions and motifs, found in multiple alleles, can easily be identified . The representation of hla sequences in this manner can be useful when designing reagents for hla typing, such as primers or oligonucleotide probes or comparing mismatches when looking at potential donors . The interface provided lets the user define a number of key variables for the alignments, these include the gene(s) to be aligned, the alleles of interest and the reference sequence they are aligned against, as well as the type of sequence: nucleotide coding region, nucleotide genomic and the amino - acid sequence of the protein, to be aligned . The alignment tool uses standard formatting conventions for the display of sequence alignments and alignments adhere to standard conventions for displaying evolutionary events and numbering . An example of alignments specially tailored to the hla transplant community is in the presentation of alleles with an alternative splice site . For most alleles, the nucleotide sequence displayed as a coding sequence (cds) the sequence displayed will contain the spliced exons plus any alternatively spliced segment that lies within the traditional exon framework, when compared to a reference sequence . The otherwise missing sequence is also included and highlighted to emphasize the region of interest, rather than omit it, a feature important for the design of reagents that allow for typing of the alternatively spliced allele . Figure 2 illustrates how an alternatively spliced allele (a0111n) is represented in the sequence alignments . Identity to the a01010101 allele is shown by hyphens (-) and the exon borders are indicated by a pipe (\|). The single nucleotide mutation in the third base of codon 175, introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . It is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest, rather than omit it, this feature is important for the design of reagents to allow for typing of this allele . Identity to the a01010101 allele is shown by hyphens (-) and the exon borders are indicated by a pipe (\|). The single nucleotide mutation in the third base of codon 175, introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . It is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest, rather than omit it, this feature is important for the design of reagents to allow for typing of this allele . The previous text - only versions of the alignments are still requested and as a result, are available from the anri website and in a zipped file in the ftp directory . For users who prefer to use other existing software to produce their own alignments, then the ftp directory contains files in popular formats for them to download and import . Recent developments to the website have seen the addition of a search tool for identifying primer and probe sequences . Many hla typing laboratories who have designed their own reagents for hla typing have spreadsheets detailing probe - hit patterns for different alleles . These are used when typing samples to identify known alleles based on the reaction patterns seen . Each time a new release of the database was made it was necessary to manually update these ever - expanding lists by cross - referencing the primer sequence with the sequence alignments, which with the rapidly increasing numbers of alleles was becoming a slow and laborious task . The new probe & primer search tool allows users to enter a list of primer sequences and the tool will search the known alleles for the presence of these sequences and report any matches in a file format suitable for cutting - and - pasting into existing spreadsheets . The tool is currently limited to coding sequences but as the number of genomic sequences in the database expands it will be modified to search these regions as well . The imgt / hla database is also involved in developing data format standards for hla information exchange between the reference database, hla typing laboratories and commercial typing - kit manufacturers . This work, which is being performed in collaboration with other immuno - informatics groups will provide both an xml output format for the imgt / hla database as well as xml reporting format for tissue typing laboratories . The xml output will contain similar information to that described for the flat files and allele output (30). The rise of high - throughput genome typing has seen the expansion of genome browsers like ensembl (31). These browsers have a different priority in how you view a gene, the alleles and any snps . The imgt / hla database is working with groups like ensembl, embl - bank and uniprot (32) to help define hla references to suit all parties at the different levels through the development of locus reference genomic sequences (lrgs). A current project is to improve cross - referencing of the hla data with that from other systems . The aim is to make sure that when users find an entry referring to an hla allele in a third - party system they can also find a link back to the imgt / hla entry for that allele, which should be considered the primary reference for the sequence . The main access point for the user is the world wide web (www), which allows users to employ a number of search tools and other facilities to retrieve, manipulate and analyse hla data . The first area comprises information and help pages that provide background on the database and provide in - depth help on the tools and data available and documentation of the imgt / hla file formats . These core tools allow the users to perform sequence alignments, allele queries and sequence searches as well as queries more relevant to how the data are used and interpreted in a clinical setting . The third area comprises final pages that provide links to commonly used third - party applications such as the sequence - analysis tools at the ebi, including srs, blast and fasta . As the primary users of the database are members of the clinical hla community involved in transplantation of tissues and organs, the most commonly accessed tools have been written to aid in their common queries . All tools are written in perl as cgi scripts and access restricted views of the underlying oracle relational database . The transplant and tissue typing community have two main queries; either to retrieve information on a particular allele or to view how a number of alleles differ in sequence . To answer these questions, the database provides a detailed report on any allele, as well as an interactive alignment tool to view how allelic sequences differ . The allele search tool provides a simple - to - use interface for retrieving allele information . The output, see figure 1, for each allele includes the official allele designation, previously used designations and the unique imgt / hla accession number . Other information provided includes the date that the allele was named, current status (as some allele designations have been deleted) and information on the individual or cell line from which the sequence was derived . Links to all component embl - bank / genbank / ddbj entries are also included . Recently, information from the hla dictionary (29) has also been added to some entries . The dictionary presents the serological equivalents of hla - a, -b, -c, -drb1, -drb3, -drb4, -drb5 and -dqb1 allotypes . The data summarizes equivalents obtained by the who nomenclature committee for factors of the hla system, the international cell exchange (ucla), the national marrow donor program (nmdp), the 13th international histocompatibility workshop, recent publications and individual laboratories . Any citations are also included with, wherever possible, a link to the pubmed entry for that citation . The pubmed link provides an online version of the abstract as well as links to other citations by the author and to similar papers . The final section of the output details the official nucleotide and protein sequence as well as any genomic sequence for the allele that is available . The report provides cross - references to a text flat - file in srs (hla0001), the omim entry for hla - a, the source entries in embl - bank (aj278305-z93949) and to the seminal citations in pubmed . The full entry also contains the nucleotide sequence at both the cds and genomic level . The report provides cross - references to a text flat - file in srs (hla0001), the omim entry for hla - a, the source entries in embl - bank (aj278305-z93949) and to the seminal citations in pubmed . The full entry also contains the nucleotide sequence at both the cds and genomic level . Hla allele sequences can differ from each other by as little a single nucleotide substitution, within a genomic sequence of 3300 bases . Such nucleotide differences between the alleles of prospective transplant donors and recipients can make the difference between a successful transplant, graft failure and death . This means that the database must be able to quickly and easily display this information to the user . The hla community is interested in seeing the polymorphisms in terms of the changes to the sequence rather than as a list of individual single nucleotide polymorphisms (snps). To this end, we have developed the alignment tool, rather than push the users into producing their own alignments for the sequences of interest or simply just reporting the polymorphic positions . These alignments allow a visual interpretation of sequence similarity so that polymorphic positions and motifs, found in multiple alleles, can easily be identified . The representation of hla sequences in this manner can be useful when designing reagents for hla typing, such as primers or oligonucleotide probes or comparing mismatches when looking at potential donors . The interface provided lets the user define a number of key variables for the alignments, these include the gene(s) to be aligned, the alleles of interest and the reference sequence they are aligned against, as well as the type of sequence: nucleotide coding region, nucleotide genomic and the amino - acid sequence of the protein, to be aligned . The alignment tool uses standard formatting conventions for the display of sequence alignments and alignments adhere to standard conventions for displaying evolutionary events and numbering . An example of alignments specially tailored to the hla transplant community is in the presentation of alleles with an alternative splice site . For most alleles, the nucleotide sequence displayed as a coding sequence (cds) the sequence displayed will contain the spliced exons plus any alternatively spliced segment that lies within the traditional exon framework, when compared to a reference sequence . The otherwise missing sequence is also included and highlighted to emphasize the region of interest, rather than omit it, a feature important for the design of reagents that allow for typing of the alternatively spliced allele . Figure 2 illustrates how an alternatively spliced allele (a0111n) is represented in the sequence alignments . Identity to the a01010101 allele is shown by hyphens (-) and the exon borders are indicated by a pipe (\|). The single nucleotide mutation in the third base of codon 175, introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . It is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest, rather than omit it, this feature is important for the design of reagents to allow for typing of this allele . Identity to the a01010101 allele is shown by hyphens (-) and the exon borders are indicated by a pipe (\|). The single nucleotide mutation in the third base of codon 175, introduces a new splice site and as a result the region shaded in green is spliced out and is not part of the cds . It is helpful to include the otherwise missing sequence and highlight it to emphasize the region of interest, rather than omit it, this feature is important for the design of reagents to allow for typing of this allele . The previous text - only versions of the alignments are still requested and as a result, are available from the anri website and in a zipped file in the ftp directory . For users who prefer to use other existing software to produce their own alignments, then the ftp directory contains files in popular formats for them to download and import . Recent developments to the website have seen the addition of a search tool for identifying primer and probe sequences . Many hla typing laboratories who have designed their own reagents for hla typing have spreadsheets detailing probe - hit patterns for different alleles . These are used when typing samples to identify known alleles based on the reaction patterns seen . Each time a new release of the database was made it was necessary to manually update these ever - expanding lists by cross - referencing the primer sequence with the sequence alignments, which with the rapidly increasing numbers of alleles was becoming a slow and laborious task . The new probe & primer search tool allows users to enter a list of primer sequences and the tool will search the known alleles for the presence of these sequences and report any matches in a file format suitable for cutting - and - pasting into existing spreadsheets . The tool is currently limited to coding sequences but as the number of genomic sequences in the database expands it will be modified to search these regions as well . The imgt / hla database is also involved in developing data format standards for hla information exchange between the reference database, hla typing laboratories and commercial typing - kit manufacturers . This work, which is being performed in collaboration with other immuno - informatics groups will provide both an xml output format for the imgt / hla database as well as xml reporting format for tissue typing laboratories . The xml output will contain similar information to that described for the flat files and allele output (30). The rise of high - throughput genome typing has seen the expansion of genome browsers like ensembl (31). These browsers have a different priority in how you view a gene, the alleles and any snps . The imgt / hla database is working with groups like ensembl, embl - bank and uniprot (32) to help define hla references to suit all parties at the different levels through the development of locus reference genomic sequences (lrgs). A current project is to improve cross - referencing of the hla data with that from other systems . The aim is to make sure that when users find an entry referring to an hla allele in a third - party system they can also find a link back to the imgt / hla entry for that allele, which should be considered the primary reference for the sequence . The imgt / hla database provides a centralized resource for everybody interested, clinically or scientifically, in the hla system . The database and accompanying tools allow the study of all hla alleles from a single site on the world wide web . It should aid in the management and continual expansion of hla nomenclature, providing an ongoing resource for the who nomenclature committee . The earliest version of the imgt / hla database, december 1998, included only 964 alleles, covering 24 genes and was limited to much simpler tools and interfaces . The latest release, july 2008, contained over 3300 alleles for 34 genes, with this number set to grow as the database continues to receive and name over 450 new alleles a year . The expansion of the database content has been reflected in its use, in 1999 the website averaged just over 1500 visitors per month, in 2008 this had increased to over 7500 visitors viewing over 40 000 pages per month . The challenge for the database is to keep up with this increase in sequences, develop new tools for the visualization of the sequences whilst maintaining the high standards set in the presentation and quality of the hla sequences and nomenclature to the research community . This work was supported by histogenetics; abbott laboratories inc . ; the american society for histocompatibility and immunogenetics; the anthony nolan trust; bag healthcare; biotest; the european federation for immunogenetics; innogenetics; invitrogen; the marrow foundation; the national marrow donor program; one lambda inc . ; initial support for the imgt / hla database project was from the imperial cancer research fund (now cancer research uk) and an eu biotech grant (bio4ct960037).
Findings from the disciplines of embryology, anatomy, and physiology of the clitoris should form the basis of the discourse about the biological basis of the female orgasm . The anatomy of the clitoris is described in human anatomy textbooks, but often it is neglected by sexological textbooks, for this reason some researchers have proposal and divulged a new anatomical terminology for the clitoris . This review is a revision of the anatomical terms proposed by helen o'connell, emmanuele jannini, and odile buisson . This is a revision of the helen o'connell and emmanuele jannini articles published by journal of sexual medicine and of odile buisson's article published by gyncologie obsttrique fertilit . The aim of this presentation is to provide a comprehensive overview of anatomy of the distal vagina . This would aid communication between clinicians, researchers, and the nonclinician regarding this anatomy . The other components are the walls of the vagina and its associated exocrine glands, which will focus instead on the distal vagina, the site of the female sexual response and the area where confusion in terminology most exists . For instance in her article nothing is written about the size of the distal vagina and of its gross and microscopic anatomical structure . Moreover, the term distal vagina is not used in human anatomy and there are no exocrine glands in the walls of the vagina; the focus of the female sexual response is not the distal vagina, but the clitoris (with its glans: the female primary erogenous zone, which covers the distal part of the corpora cavernosa of the clitoris from which it is independent). There is general consensus about female sexual anatomy and the standard terminology employed in this respect, such as the anatomical terms used to describe the components of the vulva in all human anatomy textbooks [27]. Wrote: there is no uniformity to the vagina, the distal vagina having distinctly different properties to the proximal vagina, reflecting their different developmental origins from the urogenital sinus and mullerian duct . Fliegner, quoted by o'connell et al ., in 1994 writes: a review of the embryology of vaginal epithelium suggests that it is entirely of urogenital sinus origin . The vagina and cervix develop without the involvement of the mllerians ducts; only just the body of the uterus and uterine tubas are formed by the mllerian ducts, and in males the prostatic utricle is the homologue of the female vagina [2, 5, 6, 10]. For example, the lateral walls are very different from the posterior vaginal wall . For this statement, in o'connell et al . 's article, there are no references and the authors do not clarify if they relate to the gross or microscopic anatomy and they do not write the differences between lateral and posterior walls of the vagina . The distal vagina is a structure that is so interrelated with the clitoris that it is a matter of some debate whether the two are truly separate structures . Deep to the vaginal wall mucosa laterally lies only the clitoris . . The vagina has no anatomical relation with the clitoris (figure 1) [27]. The urethra is separated from the distal vagina by a discrete white layer referred to as the periurethral fascia . The anterior vaginal wall is separated from the posterior urethral wall by the urethrovaginal septum (its thickness is 1012 mm) [27]. Although the literature written for lay readers implies that the urethra is erectile in nature or is surrounded as a tube by vessels, this idea is not supported by anatomical studies . The corpus spongiosum of the female urethra is reported in anatomy textbooks [57] and in anatomical studies: grafenberg, in 1950, writes analogous to the male urethra, the female urethra also seems to be surrounded by erectile tissues like the corpora cavernosa, yang et al . In 2006, write the spongy tissue surrounding the urethral lumen is composed of smooth muscle fibres, with multiple small vessels (figure 10). Some have termed this the corpus spongiosum, as in the male . The labiae, like the clitoris, are derived embryologically from the undifferentiated phallus . Only the corpora cavernosa of the clitoris and the glans are formed from the phallus . Labia minora, vestibule of the vagina, and vestibular bulbs are formed from the pelvic and phallic parts of the urogenital sinus and from the urogenital folds [2, 5, 6, 1315]. Clitoral bulbs is an incorrect term from an embryological and anatomical viewpoint, in fact the bulbs do not develop from the phallus, and they do not belong to the clitoris: clitoral bulbs is not a term used in human anatomy, the correct term is the vestibular bulbs [27, 1317]. The urethral orifice and distal urethra are surrounded by the erectile tissue of the clitoral bulbs . The external urethral orifice is situated on the vaginal tubercle (i.e., carina urethralis) and it is not surrounded by the erectile tissue of the vestibular bulbs; the female urethra is not encircled by the clitoris, and it is not in related with the crura and body of the clitoris [27, 1315]; the female urethra is only 3 - 4 cm long and the authors do not clarify the meaning of distal urethra . The clitoris is composed of the glans, which is its only external manifestation . The clitoris itself, in turn, being covered by the vulva . The whole clitoris (glans, body, roots, or crura) is an external genital organ: the glans and body are visible while the roots are hidden, therefore they are not internal [27, 1315]. The tiny glans clitoris, a component of the clitoris composed primarily of large nerve trunks and sensory receptors, is often referred to as the clitoris . . In o'connell's article there are no references for this assertion, and in the pictures of the vulva in anatomy textbooks always there is the body of the clitoris [27]. This area was emphasized in the descriptions of kobelt, and more recently by arien, sevely, and douglas . . The author arien cited by o'connell et al . Does not exist, and reference 29 is not correct, the authors are not arjen a, turnhout v, hage j, diest p., but van turnhout aa, hage jj, van diest pj . 's article there is an error, the article by yang c. et al ., it is not published in 2005, but in 2006 . The interrelationship between the clitoris, urethra, and vagina has been studied by various modalities including ultrasound, mri ., also this is an incorrect reference, this article does not investigate the interrelation between the clitoris, urethra, and vagina, and mri was not used, georgiadis et al . However, this is the first account of brain regions involved in the experience of clitoral stimulation pet was used because it is more robust to motion artifact than fmri . The clitoral complex, composed of the distal vagina, urethra, and clitoris, is the location of female sexual activity, analogous to the penis in men . This definition has no embryological, anatomical, and physiological support and in the male penis there is no vagina [27, 1316, 19]. Laqueur, in 1990, wrote that how adam, renaldus columbus baptized (amoris dulcedo) in 1559, what he had found in nature: a female penis . To describe the cluster of erectile tissues (i.e., clitoris, vestibular bulbs and pars intermedia, labia minora, and corpus spongiosum of the female urethra) responsible for female orgasm, the correct anatomical term should be the female penis [1315]. In measurement of the thickness of the urethrovaginal space in women with or without vaginal orgasm: a response from the study authors (journal of sexual medicine, 2008), answering to rebuttal of professor vicentini, jannini stated that: female sexuality is still a hidden area of medicine, which needs an honest, expert scientific approach in order to move forward . This is not a very appropriate statement, because there are many scientific mistakes in the article written by him with gravina et al . . Grfenberg described an erogenous zone located in the anterior vaginal wall and subsequent studies have correlated the focus of female sensitivity with the external urethral sphincter . G - spot of ladas, whipple, and perry does not correspond to the external urethral sphincter but to the intraurethral glands: komisaruk et al ., in 2006 write stimulation of the pelvic nerve may also occur with stimulation of the area of the g - spot (the area of the female prostate gland) and may also account for the reports of orgasm and female ejaculation from the urethra experienced by some women (and why in this book the science of orgasm nothing is written about the clitoris?). Furthermore the authors also write: between the thickness of urethrovaginal space, or g - spot, clitoris - urethrovaginal complex, also known as the g - spot: therefore in the same article gravina and jannini et al . Wrote 3 definitions of g - spot and each one of them write that they made an echography of the g - spot, but in their article there is no picture that shows a g - spot! Female genital anatomy and the physiology of female sexual function have been scientifically neglected in the past . The female genital anatomy has been described in human anatomy textbooks and the female genital physiology has been described for the first time in dickinson's textbooks in 1949 and subsequently by masters and johnson [27, 15, 19, 25]. The urethrovaginal space (where the halban's fascia runs) seems critical, being constituted of fibroconnective tissue and large numbers of blood vessels, glands, muscular fibers, and nerve endings . Urethrovaginal space is an incorrect term from a scientific point of view, the anterior vaginal wall is separated from the posterior urethral wall by the urethrovaginal septum [26]. In addition halban's fascia, a layer of dense connective situated in the bladder - vaginal septum, does not correspond to the male corpus spongiosum as some sexologists believe: this assumption has no embryological and anatomical support [5, 6, 26]. The most interesting finding of our study is the evidence that women who experience vaginal orgasm have an urethrovaginal space thicker than those who do not the self - reported nature of presence or absence of vaginal orgasm is another strong limitation of our findings by vaginal orgasm we mean the orgasm experienced after direct stimulation of the anterior vaginal wall by penetration . One vaginal orgasm at least once in the past month, in women that reported at least two acts of sexual intercourse per week, is not a meaningful difference with women without vaginal orgasm, besides authors did not clarify the measures of this space considered as normal, and they do not specify the position of coitus . As there is now evidence that the clitoris is related to the distal third of the urethra in the perivaginal space the close physical proximity of the urethra and the clitoris to the anterior vaginal wall suggests an association between these anatomical structures and sexual function the presence of pseudocavernous tissue (clitoral bulb) in the anterior vaginal mucosa is a frequent but not universal finding (86%). The vagina has not anatomical relation with the clitoris and in the anterior vaginal mucosa there is no clitoral bulb [27]! In fact, the anterior vaginal wall is an active organ, transmitting, during intercourse, the effect of penile thrusting in the vagina to the clitoris, by stretching the two ligaments that insert around its base . Have quoted a current opinion of 200 words: this statement is not corroborated by any anatomical or physiological evidence . However, our data cannot directly demonstrate that the thickness of an anatomical space may generate a mechanism that can be related to the creation of an orgasm but, in conclusion, the results here presented allow us to speculate that there may be a functional correlation between the thickness of urethrovaginal space, or g - spot, and the ability to experience the vaginal orgasm . G - spot is not a term used in human anatomy: grafenberg, in 1950, describes some cases of female and male urethral masturbation and the corpus spongiosum of the female urethra . Grafenberg wrote that the intraurethral glands could only release fluids that are not urine during the orgasm: but he did not report an orgasm of intraurethral glands . There are no ultrasonographic images or anatomical pictures of the g - spot, and the female prostate has no anatomical structure that can cause an orgasm: g - spot does not exist and the hypothetical area named g - spot should not be defined with grafenberg's name [11, 13, 28]. In the g - spot and lack of female sexual medicine (gyncologie obsttrique fertilit 2010), odile buisson writes: g - spot was popularized by sexologist beverly whipple in 1980 in honor of the gynecologist ernst grafenberg, the dynamic study of the clitoris urethra - vaginal complex, the vaginal penetration causes a close contact between the inner clitoris and the distal anterior vaginal wall . G - spot is a hypothesis, and there is no anatomical evidence of the vaginal orgasm which was invented by freud in 1905, without any scientific basis [11, 13, 19, 20, 28, 30]. Clitoris is localized under the urogenital diaphragm, in front the pubic symphysis and in the anterior perineal region and the roots of the clitoris are located in contact with the ischiopubic ramus, covered by the ischiocavernosus muscles: they cannot come into contact with the anterior vaginal wall and the inner clitoris does not exist [27, 13, 30]. The female perineal urethra, which is located in front of the anterior vaginal wall, is about one centimeter in length and the g - spot is located in the pelvic wall of the urethra, 2 - 3 cm into the vagina . The male penis cannot come in contact with the venous plexus of kobelt (situated until the angle of the clitoris) or with the roots of the clitoris (which do not have sensory receptors or erogenous sensitivity) during vaginal intercourse (figure 2) [27, 13, 30]. Buisson stated that the clitoris is composed of two arcs, the first consisting of two corpora cavernosa along the right and left ischiopubic ramus, with a length of 1215 cm; they join on the summit of the vulva to form a bend 90 degrees forward: the raphe; the raphe ends in the glans clitoris, the visible part of the clitoris . The second arc consists of two bulbs that surround the lateral walls of the vagina . This buisson's statement is not corroborated by any embryological, anatomical, or physiological evidence: the clitoris is not composed of two arcs [27, 12, 13]. In the article by o. buisson there are only hypotheses to explain others that are conclusions without scientific basis (and she does not write how many women would have the g - spot): the clitoral stimulation is important to have an orgasm and the clitoris exists in all women (i.e., 100%! ), why not simply stimulate, during intercourse with penetration of the penis, the clitoris with a finger? The meaning of words is important in science, but particularly in the female sexuality: gynecologists, sexual medicine experts, and sexologists should spread certainties for all women not hypotheses or personal opinions, they should use correct scientific terminology: clitoral / vaginal / uterine orgasm, g / a / c / u spot orgasm, female ejaculation, are terms that should not be used by sexologists, women, and mass media; clitoral bulbs, clitoral or clitoris - urethrovaginal complex, urethrovaginal space, periurethral glans, halban's fascia erogenous zone, vaginal anterior fornix erogenous zone, genitosensory component of the vagus nerve, and g - spot, are terms used by some sexologists but they are not accepted or shared by experts in human anatomy . Women have the right to feel sexual pleasure: in all healthy women, orgasm is possible, with effective stimulation [13, 19, 25, 32, 33]. Make sex, make love, do not need necessarily to finish with an intercourse, and the female orgasm can be triggered by various noncoital sex play, that is, foreplay, partner masturbation, and cunnilingus . Sexologists should define have sex, make love, the situation in which the orgasm happens in both partners with or without a vaginal intercourse [3234]. I hope that this review article will give rise to some fruitful discussions on the topic of female sexuality.
To conclude, stereo- and chemodivergent asymmetric reaction pathways are observed upon treatment of alkylarylketenes and chloral with chiral nhcs, giving selectively either -lactones (up to 88:12 dr, up to 94% ee) or -chloroesters (up to 94% ee), with 2-arylsubstitution or -branching within the alkyl chain of the ketene unit leading to the -chlorination pathway . Computational studies on a model system have allowed the structural parameters that lead to selectivity in these reaction processes to be analysed . Current research from this laboratory is directed toward developing alternative uses of nhcs and other lewis bases in asymmetric catalysis . For general experimental details, full characterisation data, nmr spectra and hplc traces, see the supporting information . To a flame dried schlenk flask under an argon atmosphere was added nhc precatalyst (0.10 mmol), base (0.09 mmol) and toluene (6 ml) and the mixture stirred for 15 min . The mixture was then cooled to 0 c in an ice / h2o bath followed by addition of a 0 c solution of the requisite ketene (1.00 mmol) in toluene (12 ml), immediately followed by chloral (1.00 mmol). Toluene (2 ml) was added to wash residual reactants into solution and the reaction was stirred for the stated time at 0 c before opening the flask to the air for 30 min and concentration in vacuo . The resulting crude residue was purified by flash silica chromatography (ether: petrol) to provide either the isolated lactone or chlorinated ester as stated . In instances where ketene dimerization was competitive with lactonisation or chlorination the ketene was added dropwise . To a flame dried schlenk flask under an argon atmosphere was added nhc precatalyst (0.10 mmol), base (0.09 mmol) and toluene (6 ml) and the mixture stirred for 15 min . The mixture was then cooled to 0 c in an ice / h2o bath followed by addition of chloral (1.00 mmol). A 0 c solution of the requisite ketene (1.00 mmol) in toluene (12 ml) was subsequently added over 0.5 h. the reaction was stirred for an additional 3 h at 0 c before opening the flask to the air for 0.5 h and concentration in vacuo . The resulting crude residue with the stated diastereomeric ratio was purified by flash silica chromatography (ether: petrol) to provide either the isolated lactone or chlorinated ester . To a flame dried schlenk flask under an argon atmosphere was added nhc precatalyst (0.10 mmol), base (0.09 mmol) and toluene (6 ml) and the mixture stirred for 15 min . The mixture was then cooled to 0 c in an ice / h2o bath followed by addition of a 0 c solution of the requisite ketene (1.00 mmol) in toluene (12 ml), immediately followed by chloral (1.00 mmol). Toluene (2 ml) was added to wash residual reactants into solution and the reaction was stirred for the stated time at 0 c before opening the flask to the air for 30 min and concentration in vacuo . The resulting crude residue was purified by flash silica chromatography (ether: petrol) to provide either the isolated lactone or chlorinated ester as stated . In instances where ketene dimerization was competitive with lactonisation or chlorination the ketene was added dropwise . To a flame dried schlenk flask under an argon atmosphere was added nhc precatalyst (0.10 mmol), base (0.09 mmol) and toluene (6 ml) and the mixture stirred for 15 min . The mixture was then cooled to 0 c in an ice / h2o bath followed by addition of chloral (1.00 mmol). A 0 c solution of the requisite ketene (1.00 mmol) in toluene (12 ml) was subsequently added over 0.5 h. the reaction was stirred for an additional 3 h at 0 c before opening the flask to the air for 0.5 h and concentration in vacuo . The resulting crude residue with the stated diastereomeric ratio was purified by flash silica chromatography (ether: petrol) to provide either the isolated lactone or chlorinated ester . As a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors
The traditional management of atlantoaxial instability, deemed irreducible after traction is transoral odontoid excision followed by a posterior fixation.1 an intimate knowledge of the microsurgical anatomy of the atlantoaxial region and increased experience with surgical techniques to expose and manipulate the atlantoaxial joint spaces now enable the spinal surgeon to manipulate the atlantoaxial spinal complex and achieve reduction in most cases of atlanto - axial sub - luxation obviating the need for a transoral procedure234 and its associated co - morbidities . We have retrospectively analyzed atlantoaxial instability treated by using a combination of c1-c2 joint release and manipulation of the atlanto - axial complex . 66 cases of atlantoaxial instability were diagnosed and treated at our institute between 2005 and 2010 . All patients presented in casualty with suspected cervical spine injury and concussive head injury were subjected to x - rays of the cervical spine . In cases with pain in cervical area, restricted neck movement and neurological deficit with apparently normal cervical x - ray, active lateral flexion and extension films were used to asses instability indicated by a atlanto - dens interval (adi) of more than 3 mm or a posterior atlanto - dens interval (padi) of less than 19 mm . X - ray open mouth view was used to assess lateral mass overhang and look for overlapping of the facets (winking sign)56 which would indicate a rotatory subluxation . All the patients were subjected to magnetic resonance imaging (mri) of cervical spine . The cases in which c1 lateral mass - c2 transpedicular stabilization was considered, were also subjected to computed tomography (ct) scans to assess odontoid separation, the feasibility of placement of the screws and the proximity to the foramen transversarium.6 ct angiogram was performed in cases where c1-c2 intraarticular fixation or c1 lateral mass - c2 transpedicular screw was used to assess the vertebral artery.78910 the time interval between the injury and presentation varied from 1 to 97 days (mean = 12.8 days). Of these 66 cases, 3 cases in which destruction of c1 lateral mass, c1-c2 joints and posterior elements prevented c1-c2 fixation were subjected to occipito cervical fixation and 39 cases were posttraumatic while 16 were associated with congenital abnormalities and 8 cases were associated with degenerative spine disease . All patients underwent detailed neurologic evaluation on admission and were graded on the american spinal injury association (asia) impairment scale (ais). Following evaluation of radiological images, we divided the atlantoaxial subluxations into three basic types [figures 1a - d]. A line diagram showing normal cranio - cervical junction (adi: atlanto - dens interval, padi: posterior atlanto - dens interval) a line diagram showing type a atlantoaxial subluxation increased adi, decreased padi a line diagram showing type b atlantoaxial subluxation increased odontoid - opiston distance, decreased basion - c1 arch distance, altered power's ratio a line diagram showing type c atlantoaxial subluxation increased atlanto - dens interval (adi), decreased posterior atlanto - dens interval (padi), increased interspinous distance there is a forward translation of c1 over c2 causing an increase in the adi with a reduction of the padi . There is no significant increase in the c1-c2 interspinous distance (isd) though there is a minimal anterior inferior angulation c2 body.10 these constitute the classical posttraumatic atlantoaxial subluxations . There is forward translation of c1 over c2 associated with an odontoid fracture and an intact transverse ligament . In this case however, the dens - opisthion distance is increased and the basion c1 arch distance is decreased altering the power's ratio . There is no significant increase in the c1-c2 interspinous distance [figure 2a - d]. (type b) (b) postoperative ct (sagittal) showing reduced atlantoaxial subluxation . (c) postoperative ct (axial) showing screw positioning in c1 (atlas) (d) postoperative ct (axial) showing screw positioning in c2 (axis) the c2 moves posteriorly and superiorly in the sagittal plane and the c1 arch goes downward and forward causing an increase in the adi and increase in the c1-c2 interspinous distance, both of which may be increased on flexion [figures 3a - d]. (b) preoperative ct (sagittal) showing atlantoaxial subluxation (type c). (c) postoperative ct (sagittal) showing reduced atlantoaxial subluxation (type c) all 63 cases were immobilized preoperatively with a philadelphia cervical collar . No attempt was made preoperatively to achieve reduction using skeletal traction . In two cases, a large bony mass possibly a callus was present between the c1 arch and the odontoid process which would have prevented reduction of the c1-c2 subluxation . Only the c1 arch and callus were excised . While in other case, odontoidectomy was also performed . In both cases, the patient is positioned prone on a radiolucent table and the occipito c1-c2 complex is exposed through a midline incision . The c1-c2 joint capsule was opened widely and the joint space curetted out as completely as possible . The c2 root was cut whenever the surgeon felt it is necessary to provide a better field for decompression of the joint . Once the joint cavity was freed, a thin vertebral spreader and chisel was used to distract the joint and assess the mobility . Manipulation was attempted only after the joint spaces were noted to be adequately free . In type a and b subluxations, the reduction technique we perform is as follows:1112 a 21 g wire loop is passed under the posterior arch of c1 . Under fluoroscopic guidance, the wire loop is used to exert traction on the c1 arch in a posterior direction where the assistant applies pressure on c2 spinous process in an anterior and upward direction . The reduction is achieved after which a fixation and fusion procedure is performed . In cases with type c subluxation, a modified reduction procedure is adopted . In these cases, the interspinous ligaments between c1-c2 and the muscles attached to the c2-c3 posterior elements are dissected to allow movement at c2-c3 interspinous region after which the c1 arch is manipulated downward and backward using the sublaminar wire loop . Simultaneously, a vertebral spreader is placed between c2-c3 laminae and the interspinous space distracted under fluoroscopy till reduction is achieved following which fixation of c1-c2 is performed . On table evaluation of reduction in type a and type c, subluxations was considered optimal if adi was less than 3 mm and near optimal if adi was 3 - 5 mm.13 in cases with type b subluxation, the alignment of odontoid with the c2 body produced a good indicator of reduction . The power's ratio being difficult to measure on fluoroscopy during surgery, we have taken an odontoid displacement distance2 of 0 - 2 mm as optimal reduction and 2 - 4 mm as near optimal reduction.13 in cases where reduction was near optimal, the posterior arch of c1 was excised to provide adequate decompression . Bilateral c1 lateral mass c2 transpedicular screws and in 8 cases unilateral c1 lateral mass c2 transpedicular screws were used as vascular and bony anomalies prevented bilateral fixation.14 of the two cases subjected to a transoral decompression one patient underwent c1 lateral mass c2 transpedicular fixation . In the second case, c1-c2 fixation could not be done as the patient developed hypotension on table hence an intraspinous iliac bone graft was used to distract the c2 spinous process from the subaxial spinous processes to achieve and maintain reduction . Type b cases were also subjected to open mouth x rays . Neurological assessment and grading as per asia impairment scale (ais) was performed . Radiological and neurological evaluation was repeated at 6 weeks, 3 months, 6 months, 1 year and on yearly basis subsequently . The c1-c2 complex was considered stable if there was no movement between the c1 posterior arch and c2 spinous process on lateral flexion extension films . Fusion was considered to be achieved if trabecular continuity existed between the intraarticular spaces and between the c1 posterior arch and c2 in cases subjected to a gallie fusion.11 all patients were mobilized to sitting position on the third postoperative day . Patients with neurological deficit or minimal neurological deficit were ambulated after a week on a halo brace and continued the brace for 6 weeks . Patients who could not be ambulated were managed with a philadelphia collar for 6 weeks . There is a forward translation of c1 over c2 causing an increase in the adi with a reduction of the padi . There is no significant increase in the c1-c2 interspinous distance (isd) though there is a minimal anterior inferior angulation c2 body.10 these constitute the classical posttraumatic atlantoaxial subluxations . There is forward translation of c1 over c2 associated with an odontoid fracture and an intact transverse ligament . In this case however, the dens - opisthion distance is increased and the basion c1 arch distance is decreased altering the power's ratio . There is no significant increase in the c1-c2 interspinous distance [figure 2a - d]. (type b) (b) postoperative ct (sagittal) showing reduced atlantoaxial subluxation . (c) postoperative ct (axial) showing screw positioning in c1 (atlas) (d) postoperative ct (axial) showing screw positioning in c2 (axis) the c2 moves posteriorly and superiorly in the sagittal plane and the c1 arch goes downward and forward causing an increase in the adi and increase in the c1-c2 interspinous distance, both of which may be increased on flexion [figures 3a - d]. (b) preoperative ct (sagittal) showing atlantoaxial subluxation (type c). (c) postoperative ct (sagittal) showing reduced atlantoaxial subluxation (type c) all 63 cases were immobilized preoperatively with a philadelphia cervical collar . No attempt was made preoperatively to achieve reduction using skeletal traction . In two cases, a large bony mass possibly a callus was present between the c1 arch and the odontoid process which would have prevented reduction of the c1-c2 subluxation . Only the c1 arch and callus were excised . While in other case, odontoidectomy was also performed . In both cases, the patient is positioned prone on a radiolucent table and the occipito c1-c2 complex is exposed through a midline incision . The c1-c2 joint capsule was opened widely and the joint space curetted out as completely as possible . The c2 root was cut whenever the surgeon felt it is necessary to provide a better field for decompression of the joint . Once the joint cavity was freed, a thin vertebral spreader and chisel was used to distract the joint and assess the mobility . Manipulation was attempted only after the joint spaces were noted to be adequately free . In type a and b subluxations, the reduction technique we perform is as follows:1112 a 21 g wire loop is passed under the posterior arch of c1 . Under fluoroscopic guidance, the wire loop is used to exert traction on the c1 arch in a posterior direction where the assistant applies pressure on c2 spinous process in an anterior and upward direction . The reduction is achieved after which a fixation and fusion procedure is performed . In cases with type c subluxation, the interspinous ligaments between c1-c2 and the muscles attached to the c2-c3 posterior elements are dissected to allow movement at c2-c3 interspinous region after which the c1 arch is manipulated downward and backward using the sublaminar wire loop . Simultaneously, a vertebral spreader is placed between c2-c3 laminae and the interspinous space distracted under fluoroscopy till reduction is achieved following which fixation of c1-c2 is performed . On table evaluation of reduction in type a and type c, subluxations was considered optimal if adi was less than 3 mm and near optimal if adi was 3 - 5 mm.13 in cases with type b subluxation, the alignment of odontoid with the c2 body produced a good indicator of reduction . The power's ratio being difficult to measure on fluoroscopy during surgery, we have taken an odontoid displacement distance2 of 0 - 2 mm as optimal reduction and 2 - 4 mm as near optimal reduction.13 in cases where reduction was near optimal, the posterior arch of c1 was excised to provide adequate decompression . Bilateral c1 lateral mass c2 transpedicular screws and in 8 cases unilateral c1 lateral mass c2 transpedicular screws were used as vascular and bony anomalies prevented bilateral fixation.14 of the two cases subjected to a transoral decompression one patient underwent c1 lateral mass c2 transpedicular fixation . In the second case, c1-c2 fixation could not be done as the patient developed hypotension on table hence an intraspinous iliac bone graft was used to distract the c2 spinous process from the subaxial spinous processes to achieve and maintain reduction . Type b cases were also subjected to open mouth x rays . Neurological assessment and grading as per asia impairment scale (ais) was performed . Radiological and neurological evaluation was repeated at 6 weeks, 3 months, 6 months, 1 year and on yearly basis subsequently . The c1-c2 complex was considered stable if there was no movement between the c1 posterior arch and c2 spinous process on lateral flexion extension films . Fusion was considered to be achieved if trabecular continuity existed between the intraarticular spaces and between the c1 posterior arch and c2 in cases subjected to a gallie fusion.11 all patients were mobilized to sitting position on the third postoperative day . Patients with neurological deficit or minimal neurological deficit were ambulated after a week on a halo brace and continued the brace for 6 weeks . Patients who could not be ambulated were managed with a philadelphia collar for 6 weeks . Postoperative radiological and neurological evaluation was possible in all 61 cases at 6 months (2 mortalities in the postoperative period). None of our patients showed neurological deterioration following manipulation and fixation procedures postoperatively [table 1]. Neurological evaluation optimum reduction was achieved in 26 cases of type a (78.7%), 19 cases of type b (82.6%) and 5 cases of type c (71.4%). Reduction was near - optimal in 7 cases of type a, 4 cases of type b and 2 cases of type c [table 2]. Reduction was noted to be optimal in 36 of the 45 cases treated with bilateral c1 lateral mass c2 transpedicular fixation, 6 out of the 8 cases treated with c1- c2 trans - articular fixation and 7 of the 8 cases treated with unilateral c1 lateral mass - c2 transpedicular fixation [table 3]. Postoperative radiological evaluation radiological evaluation of various surgical options carried out in this series evaluation at 6 months revealed good stability and fusion in 42 of the 44 cases managed with bilateral c1-c2 lateral mass fixation (one patient expired in the immediate postoperative period and could not be assessed), 7 out of 8 cases treated with trans - articular c1-c2 fixation and all cases treated by unilateral c1 lateral mass c2 transpedicular fixation showed good stability and fusion at 6 months [table 3]. Two cases treated with bilateral c1 lateral mass c2 transpedicular fixation were noted to have failure of fixation on followup as evidenced by backing out of the left c2 screw in one case and both c1 and c2 screws on the right side in another (grade 2 transgression). One case treated with trans - articular c1-c2 screw fixation presented with partial backing out of screws, this patient was noted to have osteoporotic bone . All three cases had achieved near - optimal reduction and showed abnormal mobility on lateral flexion extension films at 6 months . The patients treated by trans - articular fixation underwent reexploration with replacement of screws and augmentation with sub - laminar wires and gallie fusion . There were two mortalities in our series; both were of a poor neurological grade (ais b) preoperatively . One patient who underwent initial transoral decompression developed hemodynamic changes during the posterior instrumentation procedure and expired postoperatively (7 day). Another patient treated with bilateral c1-c2 lateral mass transpedicular screws also had polytrauma including thoracic injuries and expired on the 3 postoperative day . Seven cases developed pneumonitis in the postoperative period; of these, four patients required further tracheostomy and ventilator care . Of the four patients who underwent tracheostomy, two cases had lower cranial nerve dysfunction preoperatively . On table vertebral artery injury noted in one patient treated with bilateral c1 lateral mass c2 transpedicular fixation and two cases where unilateral fixation was done . This needed local compression to achieve hemostasis and no neurological consequences were noted in all these three cases . Graft site infection was noted in three cases and deep venous thrombosis in two cases . The management of atlantoaxial sublulxation traditionally has been based on the ability to achieve reduction using preoperative skeletal traction . Reducible subluxations being managed by posterior fixation procedure and irreducible subluxations being subjected to an internal transoral odontoid excision followed by posterior fixation often in a sub - optimal position.1 while the transoral odontoid excision does remove the main compressive element, the procedure itself can be associated with significant morbidity and in addition the excision of odontoid along with the fact that posterior fixation is often done in the sub - optimal position can result in significant restriction of neck movement . The excision of the odontoid process also presents problems while performing the posterior stabilization procedure . The inability to visualize the odontoid on fluoroscopy makes accurate passage of trans - articular c1 c2 screws difficult . Also, the degree of reduction achieved is difficult to assess on fluoroscopy in patients in whom the odontoid has been excised . The risk of vertebral artery injury is also higher in cases where trans - articular screws are placed in sub - optimally reduced subluxations following transoral odontoid excision.15 in cases who have undergone transoral odontoid excision the length of c1 lateral mass screw is difficult to assess and anterior transgression of screws beyond the lateral mass can result in the carotid artery injury.15 however, the traction effect of the rod - screw construct aids in the reduction of atlantoaxial subluxation unlike in trans - articular fixation.16 as mentioned earlier we have divided atlantoaxial subluxation into three types . Types a, b are essentially translation type of injuries and are more commonly found following trauma . In type a, the transverse ligament is incompetent allowing the odontoid to subluxate back . These cases tend to be amenable to reduction using the manipulation process mentioned earlier.12 in type b subluxations, the transverse ligament is intact however the odontoid is fractured and the fractured segment sub - luxates along with the c1 vertebra over the c2 narrowing the padi unlike in type a subluxations however the adi stays constant . In type b subluxations where reduction is achieved on extension by exertion of transoral pressure on the c1 arch odontoid complex, it is possible to achieve stabilization by placing a trans - odontoid screw in the fractured odontoid process . While this procedure effectively restricts the translation of c1 over c2, there is the possibility of rotational movement; hence, at present we continue to treat type b fractures with posterior reduction and fixation as in type a fractures and utilize trans - odontoid screws only in fractures not associated with instability.1718 type c subluxations are more commonly found associated with degenerative and congenital craniovertebral pathologies . But can be associated with traumatic injuries also . In this type of subluxations, the c2 body is rotated backward in the sagittal plane unhindered by an incompetent transverse ligament . This movement is associated with two important anatomical changes: firstly, odontoid rotates backward and upward and subsequently causes neural compression . In some cases, secondly, there is increased c1-c2 interspinous distance with a decrease in c2-c3 interspinous distance . In some long standing cases, fibrous or osseous union of c2-c3 spinous processes may be noted . In type c subluxation in addition to release of c1-c2 joint spaces, separation of the osseoligamentous attachments between the c2 and c3 posterior elements is needed following which c2-c3 interspinous distraction is used to rock the c2 vertebra anteriorly; consequently, the odontoid will rotate anteriorly and downward which achieves both reduction and relief of neural compression . Goel et al.,1516 have classified basilar invagination into type a and b and have postulated that type a basilar invagination may be associated with a fixed atlantoaxial subluxation possibly posttraumatic in which the odontoid is tilted horizontally with an increased omega angle . Goel has reported excellent reduction of subluxation and basilar invagination following distraction of the atlantoaxial joints.1920 using a combination of atlantoaxial joint release and the manipulation procedures discussed we have been able to achieve complete or near complete reduction in all but two cases treated by us in the last 5 years . In these two cases, the presence of a bony lesion (possibly callus) interposed between the anterior arch of c1 and the odontoid precluding any possibility of a primary posterior reduction . We have found that excision of the c1 arch and the callus alone was enough obviating the need to excise the odontoid which can be manipulated back into normal position during the second posterior fixation procedure . To conclude, c1-c2 joint space dissection combined with manipulation of the c1-c2 complex can be safely used to achieve optimal and near optimal reduction in most cases of atlantoaxial instability . Transoral decompression can be avoided in most cases but may be required in cases where osseous growth between the odontoid and c1 anterior arch prevent reduction by posterior manipulation.
Disturbances within the arachidonic acid (aa) cascade have been proposed as a pathophysiological mechanism in the regulation of mood and suggested to contribute to the underlying biological background for bipolar disorder (bazinet, 2010). This is corroborated by preclinical evidence pointing to the arachidonic cascade as a target of drugs used to manage bipolar disorder (rapoport and bosetti, 2002; bazinet, 2009), indicating that lithium (galimberti et al ., 2014), valproate (kieseppa et al ., 2014), carbamazepine (lee et al ., 2012), and antipsychotics (cheon et al ., 2011 in contrast, the antidepressants imipramine and fluoxetine, which may induce mania, increase brain aa turnover, while bupropion, which may be at lower risk of inducing mania (post et al . These findings have led to the formulation of the arachidonic acid theory of bipolar disorder (bazinet, 2009). Additional evidence of downstream disturbances in the cascade, with increased levels of aa - derived prostaglandins in saliva (ohishi et al ., 1988), cerebrospinal fluid (linnoila et al ., 1983), and peripheral blood (lieb et al . Have supported a role for this pathway in bipolar disorder . In a recent genome - wide association study meta - analysis, one of three novel loci identified to be associated with bipolar disorder was near the ptgfr gene encoding the prostaglandin f receptor (chen et al ., 2013), which is highly expressed in the brain . Arachidonic acid, particularly abundant in the brain, is a polyunsaturated fatty acid present in the cell membrane phospholipids, from which it is freed by cytosolic phospholipase a2 (cpla2; rapoport, 2014). The isoenzyme cpla2 iva is selective for aa hydrolysis and its activity is modulated by lithium and carbamazepine (rapoport et al . The expression of cpla2 iva, as well as of the cyclooxygenase (cox)-2 enzyme, has been found to be altered in the post - mortem brain tissue of bipolar disorder patients (kim et al ., 2011). Aa acts as a precursor in the production of prostaglandin h2 (pgh2), mediated by cox, which, in turn, is converted to prostaglandins, thromboxanes, or prostacyclins (funk, 2001). The conversion of pgh2 to prostaglandin d2 (pgd2) is catalyzed by prostaglandin d synthase (ptgds), encoded by the ptgds gene, and preferentially expressed in the brain . Pgd2 functions as a neuromodulator as well as a trophic factor in the central nervous system (taniguchi et al ., 2007) and reduction of both pgh2 and pgd2 is catalyzed by the aldo - keto reductase family 1 member c3 (akr1c3) enzyme, encoded by the akr1c3 gene, resulting in synthesis of prostaglandin f2 alpha (figure 1). The arachidonic acid cascade and prostaglandin metabolism pathway related to the function of ptgds and akr1c3 . Akr1c3, aldo - keto reductase family 1 member c3; pgd2, prostaglandin d2; pgf2, prostaglandin f2; pgh2, prostaglandin h2; pla2, phospholipase a2; ptgds, prostaglandin d synthase . Evidence of aa cascade and prostaglandin pathway dysregulation on a transcriptional level in bipolar disorder is limited . Two studies by the same group, a case study (n = 1; begemann et al ., 2008) and an extended case series (n = 4; gurvich et al ., 2014) of rapid - cycling bipolar disorder patients identified the ptgds and akr1c3 as differentially regulated between manic and depressive episodes and a case - control study of children and young adults with bipolar disorder (n = 9) and adhd (marn - mndez et al ., 2012) found the ptgds gene was differentially expressed between bipolar disorder patients and patients with adhd . In bipolar disorder, which is characterized by phenotypically distinct, recurrent episodes of various polarities, gene expression alterations have the potential to inform on pathophysiological processes related to illness activity and affective state and to the nature of the illness itself . 2014), gene expression changes longitudinally between affective states have not been investigated, and no studies have included assessment of healthy control subjects of these specific genes . In a recent meta - analysis of 17 studies of gene expression alterations in peripheral blood in bipolar disorder patients, comprising 565 patients and 418 healthy control subjects (munkholm et al ., 2012), we showed that findings were limited overall by lack of replication across studies and limited control for possible confounders of gene expression levels . The present study is the first to investigate repeated measures over time of the gene expression of ptgds and akr1c3 in rapid - cycling bipolar disorder patients in a euthymic or current affective state and in healthy control subjects . We hypothesized that mrna expression of ptgds and akr1c3 was deregulated in patients in a euthymic or current affective state compared with healthy control subjects as well as in bipolar disorder patients between current affective states (depressed, manic / hypomanic, or mixed) compared with those in the euthymic state . A longitudinal, naturalistic design was employed, accommodating assessment of patients during multiple affective states of varying polarity . Patients with a potential diagnosis of rapid - cycling bipolar disorder were recruited through referral by psychiatrists at hospitals or outpatient facilities throughout the region of zealand, denmark, with study recruitment taking place during the period of june 2010 to may 2012 . Inclusion criteria were: adults aged 1870 years; and a diagnostic and statistical manual of mental disorders, 4th version (dsm - iv) diagnosis of rapid - cycling bipolar disorder, defined by the occurrence of at least four mood episodes (mania, hypomania, depression, or mixed) during the preceding year in the context of bipolar disorder . Exclusion criteria were: significant physical illness (ie chronic heart disease, chronic pulmonary disease, inflammatory disease, chronic infectious disease, or neurodegenerative disease); current drug abuse; insufficient danish language skills; and pregnancy . Two bipolar patients declined further examination after one and three month follow - ups, respectively; the remaining bipolar patients were followed for a minimum of six months with a mean (standard deviation [sd]) follow - up period of 11.9 (3.0) months . Upon signs of new affective episodes, patients were evaluated with clinical assessments of mood and collection of blood samples which, when possible, were repeated after return to a subsequent euthymic state or change to an affective episode of opposite polarity . In cases of clinical signs of acute infection, any allergic symptoms, or any other acute medical condition, assessment and biochemical analysis blood samples were, on average, collected from bipolar patients 3.41.7 (range, 110) times during the study . Samples were obtained during euthymia in 34 patients (mean 2.01.3 [06]), major depression in 26 patients (mean 1.71.7 [05]), mania / hypomania in 11 patients (mean 0.71.2 [05]), and in a mixed state in a total of 6 patients (mean 0.20.4 [01]). Forty healthy control subjects were recruited among blood donors affiliated with the blood bank at rigshospitalet, copenhagen . Inclusion criteria were: adults aged 1870 years; and no history of psychiatric disorder in the subjects or their first - degree relatives . Healthy control subjects were evaluated with clinical assessments and collection of blood samples on two separate occasions approximately three months apart . Assessment and biochemical analysis were postponed if there were clinical signs of acute infection, any allergic symptoms, or any other acute medical condition . Mean (sd) follow - up time for the healthy control subjects was 2.9 (0.9) months . The study protocol was approved by the committee on health research ethics of the capital region of denmark (protocol no . All participants were assessed by a specialist in psychiatry (dr munkholm), using standardized semi - structured interviews . The schedules for clinical assessment in neuropsychiatry interview (wing et al ., 1990) was used for diagnostic purposes and was based on available case material, referral reports, the interview with the participant, and the hypomania checklist (angst et al ., 2005), completed by the participant . A dsm - iv diagnosis of rapid - cycling bipolar disorder was established for the patients and comorbid psychiatric illness, if present, was recorded . For healthy control subjects, a clinical diagnosis according to dsm - iv, without applying duration criteria, was established at each study visit concurrently with the collection of samples for laboratory analysis . Severity of depressive symptoms was assessed using the 17-item hamilton depression rating scale (hamd-17; hamilton, 1967), employing a structured interview guide (williams, 1990) translated to danish, and manic symptoms were assessed using the young mania rating scale (ymrs; young et al ., 1978), with a time period of three days applied . Medication, alcohol intake, and smoking habits during the two weeks prior to assessment were recorded . Categories of affective states were based on clinical evaluation according to the schedules for clinical assessment in neuropsychiatry interview combined with the hamd-17 and ymrs rating scales without applying duration criteria: euthymic (hamd-17 and ymrs <8), depressive (hamd-17> 7 and ymrs <8), manic / hypomanic (ymrs> 7 and hamd-17 <8), and mixed state blood samples were obtained in the fasting state between 0830 and 1030 hours, after a minimum period of 15 minutes rest, concurrently with the clinical evaluation . Nine milliliters of blood were drawn by venipuncture into a citrate phosphate dextrose adenine containing vacuum tube (vacuette), which was kept at room temperature before and after the blood draw . Peripheral blood mononuclear cells (pbmc) were collected applying the standard ficoll - paque plus isolation procedure (ge healthcare life sciences), within one hour of blood draw . Total rna was extracted from pbmc by use of trizol reagent (life technologies). Rna quality and quantification was measured spectrophotometrically using a nanodrop (nanodrop technologies) spectrophotometer and software, applying the 260/280 and 260/230 ratio algorithms . Cdna was synthesized from rna with a high capacity ddna reverse transcription kit (life technologies). The cdna was subjected to quantitative real - time polymerase chain reaction (pcr) using the viia 7 real - time pcr system (life technologies) with sybr green pcr master mix (life technologies). Gene - specific sequence oligonucleotide primers (ptgds, akr1c3, gapdh, tbp, and sdha) were purchased from tag copenhagen . A set of three genes, the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene, the tata box binding protein (tbp) gene, and the succinate dehydrogenase complex, subunit a, flavoprotein (sdha) gene, were used as candidate reference genes for normalization . Akr1c3, aldo - keto reductase family 1 member c3; gapdh, glyceraldehyde-3-phosphate dehydrogenase; ptgds, prostaglandin d synthase; sdha, succinate dehydrogenase complex, subunit a, flavoprotein; tbp, tata box binding protein . The stability of candidate reference genes was assessed using the normfinder software (akhondzadeh et al ., 2009). Sdha exhibited the highest stability in comparisons between bipolar disorder patients and healthy control subjects, as well as between affective states in bipolar disorder patients (sdha = 0.139; tbp = 0,322; gapdh = 0.271), and no combination of two genes showed higher stability . Semi - quantitative ptgds and akr1c3 mrna levels, assessed by cycle threshold (ct) were thus expressed relative to sdha . We measured the ct = ct (each gene) - ct (sdha) for each sample . Relative levels of expression were determined using the comparative ct method (ricken et al ., 2013) method, calculated by 2 t. all assays were performed in triplicate with laboratory personnel blinded to the clinical status of participants . In addition, standard clinical chemistry parameters were analyzed, including fasting blood glucose and fasting lipid parameters . Independent t - tests were used to test differences in age between healthy control subjects and bipolar disorder patients, and the chi - squared test was used to examine differences in categorical demographic and clinical variables . For our main analyses we employed a two - level linear mixed effects model, accommodating both variation of the outcome variables within subjects (intra - individual variation) and between subjects (inter - individual variation). Level one represented repeated measures of ptgds and akr1c3 mrna levels and level two represented between - subject variation . We conducted two separate sets of analyses, one on comparisons between bipolar disorder patients and healthy control subjects (set a) and one on comparisons between affective states among bipolar disorder patients (set b). In both sets, unadjusted mixed - model analyses with expression levels of each gene as the dependent variables were firstly conducted (model a-1 and b-1) followed by several a priori models specified within each set of analyses (a and b, models 24). All models included a random intercept to accommodate correlations in the outcome variables over time within each participant . The assumptions of independence of errors, homoscedasticity, and normality were met . To evaluate the correlation between levels of ptgds and akr1c3 expression, a pearson s correlation analysis was performed, using residual values produced by our mixed model (model a-2). Patients with a potential diagnosis of rapid - cycling bipolar disorder were recruited through referral by psychiatrists at hospitals or outpatient facilities throughout the region of zealand, denmark, with study recruitment taking place during the period of june 2010 to may 2012 . Inclusion criteria were: adults aged 1870 years; and a diagnostic and statistical manual of mental disorders, 4th version (dsm - iv) diagnosis of rapid - cycling bipolar disorder, defined by the occurrence of at least four mood episodes (mania, hypomania, depression, or mixed) during the preceding year in the context of bipolar disorder . Exclusion criteria were: significant physical illness (ie chronic heart disease, chronic pulmonary disease, inflammatory disease, chronic infectious disease, or neurodegenerative disease); current drug abuse; insufficient danish language skills; and pregnancy . Two bipolar patients declined further examination after one and three month follow - ups, respectively; the remaining bipolar patients were followed for a minimum of six months with a mean (standard deviation [sd]) follow - up period of 11.9 (3.0) months . Upon signs of new affective episodes, patients were evaluated with clinical assessments of mood and collection of blood samples which, when possible, were repeated after return to a subsequent euthymic state or change to an affective episode of opposite polarity . In cases of clinical signs of acute infection, any allergic symptoms, or any other acute medical condition, assessment and biochemical analysis blood samples were, on average, collected from bipolar patients 3.41.7 (range, 110) times during the study . Samples were obtained during euthymia in 34 patients (mean 2.01.3 [06]), major depression in 26 patients (mean 1.71.7 [05]), mania / hypomania in 11 patients (mean 0.71.2 [05]), and in a mixed state in a total of 6 patients (mean 0.20.4 [01]). Forty healthy control subjects were recruited among blood donors affiliated with the blood bank at rigshospitalet, copenhagen . Inclusion criteria were: adults aged 1870 years; and no history of psychiatric disorder in the subjects or their first - degree relatives . Healthy control subjects were evaluated with clinical assessments and collection of blood samples on two separate occasions approximately three months apart . Assessment and biochemical analysis were postponed if there were clinical signs of acute infection, any allergic symptoms, or any other acute medical condition . Mean (sd) follow - up time for the healthy control subjects was 2.9 (0.9) months . The study protocol was approved by the committee on health research ethics of the capital region of denmark (protocol no . Patients with a potential diagnosis of rapid - cycling bipolar disorder were recruited through referral by psychiatrists at hospitals or outpatient facilities throughout the region of zealand, denmark, with study recruitment taking place during the period of june 2010 to may 2012 . Inclusion criteria were: adults aged 1870 years; and a diagnostic and statistical manual of mental disorders, 4th version (dsm - iv) diagnosis of rapid - cycling bipolar disorder, defined by the occurrence of at least four mood episodes (mania, hypomania, depression, or mixed) during the preceding year in the context of bipolar disorder . Exclusion criteria were: significant physical illness (ie chronic heart disease, chronic pulmonary disease, inflammatory disease, chronic infectious disease, or neurodegenerative disease); current drug abuse; insufficient danish language skills; and pregnancy . Two bipolar patients declined further examination after one and three month follow - ups, respectively; the remaining bipolar patients were followed for a minimum of six months with a mean (standard deviation [sd]) follow - up period of 11.9 (3.0) months . Upon signs of new affective episodes, patients were evaluated with clinical assessments of mood and collection of blood samples which, when possible, were repeated after return to a subsequent euthymic state or change to an affective episode of opposite polarity . In cases of clinical signs of acute infection, any allergic symptoms, or any other acute medical condition, assessment and biochemical analysis blood samples were, on average, collected from bipolar patients 3.41.7 (range, 110) times during the study . Samples were obtained during euthymia in 34 patients (mean 2.01.3 [06]), major depression in 26 patients (mean 1.71.7 [05]), mania / hypomania in 11 patients (mean 0.71.2 [05]), and in a mixed state in a total of 6 patients (mean 0.20.4 [01]). Forty healthy control subjects were recruited among blood donors affiliated with the blood bank at rigshospitalet, copenhagen . Inclusion criteria were: adults aged 1870 years; and no history of psychiatric disorder in the subjects or their first - degree relatives . Healthy control subjects were evaluated with clinical assessments and collection of blood samples on two separate occasions approximately three months apart . Assessment and biochemical analysis were postponed if there were clinical signs of acute infection, any allergic symptoms, or any other acute medical condition . Mean (sd) follow - up time for the healthy control subjects was 2.9 (0.9) months . The study protocol was approved by the committee on health research ethics of the capital region of denmark (protocol no . All participants were assessed by a specialist in psychiatry (dr munkholm), using standardized semi - structured interviews . The schedules for clinical assessment in neuropsychiatry interview (wing et al ., 1990) was used for diagnostic purposes and was based on available case material, referral reports, the interview with the participant, and the hypomania checklist (angst et al a dsm - iv diagnosis of rapid - cycling bipolar disorder was established for the patients and comorbid psychiatric illness, if present, was recorded . For healthy control subjects, a clinical diagnosis according to dsm - iv, without applying duration criteria, was established at each study visit concurrently with the collection of samples for laboratory analysis . Severity of depressive symptoms was assessed using the 17-item hamilton depression rating scale (hamd-17; hamilton, 1967), employing a structured interview guide (williams, 1990) translated to danish, and manic symptoms were assessed using the young mania rating scale (ymrs; young et al ., 1978), with a time period of three days applied . Medication, alcohol intake, and smoking habits during the two weeks prior to assessment were recorded . Categories of affective states were based on clinical evaluation according to the schedules for clinical assessment in neuropsychiatry interview combined with the hamd-17 and ymrs rating scales without applying duration criteria: euthymic (hamd-17 and ymrs <8), depressive (hamd-17> 7 and ymrs <8), manic / hypomanic (ymrs> 7 and hamd-17 <8), and mixed state (hamd-17> 7 and ymrs> 7). Blood samples were obtained in the fasting state between 0830 and 1030 hours, after a minimum period of 15 minutes rest, concurrently with the clinical evaluation . Nine milliliters of blood were drawn by venipuncture into a citrate phosphate dextrose adenine containing vacuum tube (vacuette), which was kept at room temperature before and after the blood draw . Peripheral blood mononuclear cells (pbmc) were collected applying the standard ficoll - paque plus isolation procedure (ge healthcare life sciences), within one hour of blood draw . Total rna was extracted from pbmc by use of trizol reagent (life technologies). Rna quality and quantification was measured spectrophotometrically using a nanodrop (nanodrop technologies) spectrophotometer and software, applying the 260/280 and 260/230 ratio algorithms . Cdna was synthesized from rna with a high capacity ddna reverse transcription kit (life technologies). The cdna was subjected to quantitative real - time polymerase chain reaction (pcr) using the viia 7 real - time pcr system (life technologies) with sybr green pcr master mix (life technologies). Gene - specific sequence oligonucleotide primers (ptgds, akr1c3, gapdh, tbp, and sdha) were purchased from tag copenhagen . A set of three genes, the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene, the tata box binding protein (tbp) gene, and the succinate dehydrogenase complex, subunit a, flavoprotein (sdha) gene, were used as candidate reference genes for normalization . Akr1c3, aldo - keto reductase family 1 member c3; gapdh, glyceraldehyde-3-phosphate dehydrogenase; ptgds, prostaglandin d synthase; sdha, succinate dehydrogenase complex, subunit a, flavoprotein; tbp, tata box binding protein . The stability of candidate reference genes was assessed using the normfinder software (akhondzadeh et al ., 2009). Sdha exhibited the highest stability in comparisons between bipolar disorder patients and healthy control subjects, as well as between affective states in bipolar disorder patients (sdha = 0.139; tbp = 0,322; gapdh = 0.271), and no combination of two genes showed higher stability . Semi - quantitative ptgds and akr1c3 mrna levels, assessed by cycle threshold (ct) were thus expressed relative to sdha . We measured the ct = ct (each gene) - ct (sdha) for each sample . Relative levels of expression were determined using the comparative ct method (ricken et al ., 2013) method, calculated by 2 t. all assays were performed in triplicate with laboratory personnel blinded to the clinical status of participants . In addition, standard clinical chemistry parameters were analyzed, including fasting blood glucose and fasting lipid parameters . Independent t - tests were used to test differences in age between healthy control subjects and bipolar disorder patients, and the chi - squared test was used to examine differences in categorical demographic and clinical variables . For our main analyses we employed a two - level linear mixed effects model, accommodating both variation of the outcome variables within subjects (intra - individual variation) and between subjects (inter - individual variation). Level one represented repeated measures of ptgds and akr1c3 mrna levels and level two represented between - subject variation . We conducted two separate sets of analyses, one on comparisons between bipolar disorder patients and healthy control subjects (set a) and one on comparisons between affective states among bipolar disorder patients (set b). In both sets, unadjusted mixed - model analyses with expression levels of each gene as the dependent variables were firstly conducted (model a-1 and b-1) followed by several a priori models specified within each set of analyses (a and b, models 24). All models included a random intercept to accommodate correlations in the outcome variables over time within each participant . The assumptions of independence of errors, homoscedasticity, and normality were met . To evaluate the correlation between levels of ptgds and akr1c3 expression, a pearson s correlation analysis was performed, using residual values produced by our mixed model (model a-2). There were no significant differences between bipolar disorder patients and healthy control subjects with regard to age, gender, educational level, or bmi . More patients were smokers but alcohol consumption was higher among healthy control subjects (table 2). Data are expressed as mean standard deviation (range) or n (%). Bipolar disorder patients were overall on stable medication for a month before study entry and during the course of the study, with few participants changing medication despite alterations of affective state, owing to the fact that the majority of patients received intensive outpatient treatment and, despite pharmacologically advanced treatment, continually experience affective episodes . Four patients stopped and one patient started selective serotonin reuptake inhibitor treatment, two patients started lithium treatment, two started anticonvulsant treatment, and one started antipsychotic treatment during the study period . One bipolar disorder patient suffered from co - morbid obsessive - compulsive disorder; no participants suffered from comorbid generalized anxiety disorder . Four bipolar disorder patients reported mild, well - controlled hypertension and two reported mild, intermittent reflux esophagitis . One healthy control subject reported intermittent symptoms of allergic rhinitis, but not during the study period . The majority of the patients received specialized treatment at the mood disorders clinic, psychiatric center copenhagen, rigshospitalet, copenhagen, denmark, and all of the patients were outpatients at the time of inclusion . Symptom severity of participants at the time of assessment and sampling are presented in table 3 . Hamd-17, hamilton rating scale, 17 items; ymrs, young mania rating scale . * manic patients, n = 19/hypomanic patients, n = 5 . In an unadjusted analysis (model a-1), lower levels of ptgds mrna expression were observed in all affective states compared with healthy control subjects; however, only between a euthymic state and healthy control subjects was this difference statistically significant (b = -0.060, 95% confidence interval [ci; -0.117; -0.002], p = 0.041). No difference was observed in an unadjusted analysis for akr1c3 mrna expression between bipolar disorder patients in any affective state and healthy control subjects [f(4, 184.86) = 0.343, p = 0.8]. Adjusting for age and gender (model a-2), a statistically significant down - regulation of ptgds mrna expression was present in bipolar disorder patients in both the euthymic state (b = -0.073, 95% ci [-0.130; -0.017], p = 0.012), the depressive state (b = -0.062, 95% ci [-0.120; -0.003], p = 0.038), and the manic / hypomanic state (b = -0.076, 95% ci [-0.144; -0.008], p = 0.028), while the lower levels observed in a mixed state did not reach statistical significance (b = -0.053, 95% ci [-0.143; 0.037], p = 0.2; figure 2a). In an adjusted analysis (model a-2), no difference was observed in akr1c3 mrna expression between bipolar disorder patients in any affective state compared with healthy control subjects [f(4, 184.66) = 0.207, p = 0.9; figure 2b]. Age, but not gender, was weakly but positively associated with both ptgds mrna expression (b = 0.003, 95% ci [0.001; 0.005], p = 0.007) and akr1c3 mrna expression (b = 0.001, 95% ci [0.000; 0.001], p = 0.01). In an exploratory analysis of clinical and demographical variables (bmi and alcohol intake) possibly associated with ptgds and akr1c3 mrna expression (model a-3), where smokers were excluded because of uneven distribution between groups, alcohol intake was not associated with ptgds mrna expression (p = 0.2) or akr1c3 mrna expression (p = 0.5) and bmi was not associated with either ptgds mrna expression (p = 0.8) or akr1c3 expression (p = 0.2). In this exploratory analysis, only among bipolar patients in a manic / hypomanic state was the down - regulated ptgds mrna expression compared with healthy control subjects statistically significant (b = -0.100, 95% ci [-0.0.197; -0.003], p = 0.044) with the non - significant findings for akr1c3 unaltered . Ptgds (a) and akr1c3 (b) mrna expression in rapid - cycling bipolar disorder patients and healthy control subjects . Levels represent back - transformed ct values based on a linear mixed - model analysis adjusted for age and gender (model a-2). Ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic (p = 0.01), depressive (p = 0.04), and manic / hypomanic (p = 0.03) state compared with healthy control subjects; no difference in ptgds mrna expression was observed between affective states . Mrna expression did not differ between affective states or between bipolar disorder patients and healthy control subjects . Akr1c3, aldo - keto reductase family 1 member c3; bd, bipolar disorder; ct, cycle threshold; ns, not significant; ptgds, prostaglandin d synthase . In a subgroup analysis including only bipolar disorder patients in a euthymic state of more than one month (model a-4), thus minimizing a possible effect of the previous episode on gene expression in a euthymic state, ptgds mrna expression remained significantly down - regulated in comparison to healthy control subjects (b = -0.073, 95% ci [-0.145; -0.001], p = 0.048), while there was no difference between groups for akr1c3 (p = 0.6). In an unadjusted analysis (model b-1), there was no overall difference in mrna expression between any current affective state and the euthymic state of either ptgds [f(3, 139.820) = 0.208, p = 0.9] or akr1c3 [f(3, 138.188) = 0.294, p = 0.8]. There was also no difference in either ptgds mrna expression (p = 0.6) or akr1c3 mrna expression (p = 0.4) between the depressed and the manic / hypomanic state . Adjusting for age and gender (model b-2) and subsequently adding the covariates bmi, smoking status (no / yes), alcohol intake, illness duration (10 years/<10 years) and medication (no / yes) in an exploratory analysis (model b-3) did not alter the results . In this analysis, none of the covariates entered into the model, including individual medications, were significantly associated with ptgds or akr1c3 mrna expression levels . Specifically, there was no effect of either lithium (b = -0.014, 95% ci [-0.067; 0.039], p = 0.6), anticonvulsant (b = -0.034, 95% ci [-0.091; 0.023], p = 0.2), antipsychotic (b = -0.001, 95% ci [-0.075; 0.075], p = 0.9), or antidepressant use (b = -0.005, 95% ci [-0.065; 0.056], p = 0.9) on ptgds mrna levels . Non - smoking status compared with smoking status was similarly not associated with ptgds mrna levels (b = 0.065, 95% ci there was no association between hamd-17 scores or ymrs scores and mrna expression levels of ptgds and akr1c3 in depressive and manic / hypomanic bipolar disorder patients, respectively (model b-4). Post hoc subgroup analysis of patients not taking acetylsalicylic acid (n = 35) or statins (n = 33) during the study period revealed no difference in either ptgds or akr1c3 mrna expression between affective states (data not shown). Ptgds and akr1c3 mrna expression levels were weakly correlated [r(239) = 0.299, p <0.001]. There were no significant differences between bipolar disorder patients and healthy control subjects with regard to age, gender, educational level, or bmi . More patients were smokers but alcohol consumption was higher among healthy control subjects (table 2). Data are expressed as mean standard deviation (range) or n (%). Bipolar disorder patients were overall on stable medication for a month before study entry and during the course of the study, with few participants changing medication despite alterations of affective state, owing to the fact that the majority of patients received intensive outpatient treatment and, despite pharmacologically advanced treatment, continually experience affective episodes . Four patients stopped and one patient started selective serotonin reuptake inhibitor treatment, two patients started lithium treatment, two started anticonvulsant treatment, and one started antipsychotic treatment during the study period . One bipolar disorder patient suffered from co - morbid obsessive - compulsive disorder; no participants suffered from comorbid generalized anxiety disorder . Four bipolar disorder patients reported mild, well - controlled hypertension and two reported mild, intermittent reflux esophagitis . One healthy control subject reported intermittent symptoms of allergic rhinitis, but not during the study period . The majority of the patients received specialized treatment at the mood disorders clinic, psychiatric center copenhagen, rigshospitalet, copenhagen, denmark, and all of the patients were outpatients at the time of inclusion . Symptom severity of participants at the time of assessment and sampling are presented in table 3 . Hamd-17, hamilton rating scale, 17 items; ymrs, young mania rating scale . In an unadjusted analysis (model a-1), lower levels of ptgds mrna expression were observed in all affective states compared with healthy control subjects; however, only between a euthymic state and healthy control subjects was this difference statistically significant (b = -0.060, 95% confidence interval [ci; -0.117; -0.002], p = 0.041). No difference was observed in an unadjusted analysis for akr1c3 mrna expression between bipolar disorder patients in any affective state and healthy control subjects [f(4, 184.86) = 0.343, p = 0.8]. Adjusting for age and gender (model a-2), a statistically significant down - regulation of ptgds mrna expression was present in bipolar disorder patients in both the euthymic state (b = -0.073, 95% ci [-0.130; -0.017], p = 0.012), the depressive state (b = -0.062, 95% ci [-0.120; -0.003], p = 0.038), and the manic / hypomanic state (b = -0.076, 95% ci [-0.144; -0.008], p = 0.028), while the lower levels observed in a mixed state did not reach statistical significance (b = -0.053, 95% ci [-0.143; 0.037], p = 0.2; figure 2a). In an adjusted analysis (model a-2), no difference was observed in akr1c3 mrna expression between bipolar disorder patients in any affective state compared with healthy control subjects [f(4, 184.66) = 0.207, p = 0.9; figure 2b]. Age, but not gender, was weakly but positively associated with both ptgds mrna expression (b = 0.003, 95% ci [0.001; 0.005], p = 0.007) and akr1c3 mrna expression (b = 0.001, 95% ci [0.000; 0.001], p = 0.01). In an exploratory analysis of clinical and demographical variables (bmi and alcohol intake) possibly associated with ptgds and akr1c3 mrna expression (model a-3), where smokers were excluded because of uneven distribution between groups, alcohol intake was not associated with ptgds mrna expression (p = 0.2) or akr1c3 mrna expression (p = 0.5) and bmi was not associated with either ptgds mrna expression (p = 0.8) or akr1c3 expression (p = 0.2). In this exploratory analysis, only among bipolar patients in a manic / hypomanic state was the down - regulated ptgds mrna expression compared with healthy control subjects statistically significant (b = -0.100, 95% ci [-0.0.197; -0.003], p = 0.044) with the non - significant findings for akr1c3 unaltered . Ptgds (a) and akr1c3 (b) mrna expression in rapid - cycling bipolar disorder patients and healthy control subjects . Levels represent back - transformed ct values based on a linear mixed - model analysis adjusted for age and gender (model a-2). Ptgds mrna expression was down - regulated in rapid - cycling bipolar disorder patients in a euthymic (p = 0.01), depressive (p = 0.04), and manic / hypomanic (p = 0.03) state compared with healthy control subjects; no difference in ptgds mrna expression was observed between affective states . Mrna expression did not differ between affective states or between bipolar disorder patients and healthy control subjects . Akr1c3, aldo - keto reductase family 1 member c3; bd, bipolar disorder; ct, cycle threshold; ns, not significant; ptgds, prostaglandin d synthase . In a subgroup analysis including only bipolar disorder patients in a euthymic state of more than one month (model a-4), thus minimizing a possible effect of the previous episode on gene expression in a euthymic state, ptgds mrna expression remained significantly down - regulated in comparison to healthy control subjects (b = -0.073, 95% ci [-0.145; -0.001], p = 0.048), while there was no difference between groups for akr1c3 (p = 0.6). In an unadjusted analysis (model b-1), there was no overall difference in mrna expression between any current affective state and the euthymic state of either ptgds [f(3, 139.820) = 0.208, p = 0.9] or akr1c3 [f(3, 138.188) = 0.294, p = 0.8]. There was also no difference in either ptgds mrna expression (p = 0.6) or akr1c3 mrna expression (p = 0.4) between the depressed and the manic / hypomanic state . Adjusting for age and gender (model b-2) and subsequently adding the covariates bmi, smoking status (no / yes), alcohol intake, illness duration (10 years/<10 years) and medication (no / yes) in an exploratory analysis (model b-3) did not alter the results . In this analysis, none of the covariates entered into the model, including individual medications, were significantly associated with ptgds or akr1c3 mrna expression levels . Specifically, there was no effect of either lithium (b = -0.014, 95% ci [-0.067; 0.039], p = 0.6), anticonvulsant (b = -0.034, 95% ci [-0.091; 0.023], p = 0.2), antipsychotic (b = -0.001, 95% ci [-0.075; 0.075], p = 0.9), or antidepressant use (b = -0.005, 95% ci [-0.065; 0.056], p = 0.9) on ptgds mrna levels . Non - smoking status compared with smoking status was similarly not associated with ptgds mrna levels (b = 0.065, 95% ci [-0.016; 0.147], p = 0.1). There was no association between hamd-17 scores or ymrs scores and mrna expression levels of ptgds and akr1c3 in depressive and manic / hypomanic bipolar disorder patients, respectively (model b-4). Post hoc subgroup analysis of patients not taking acetylsalicylic acid (n = 35) or statins (n = 33) during the study period revealed no difference in either ptgds or akr1c3 mrna expression between affective states (data not shown). Ptgds and akr1c3 mrna expression levels were weakly correlated [r(239) = 0.299, p <0.001]. This study investigated alterations of mrna expression of ptgds and akr1c3 between bipolar disorder and healthy control subjects and is the first study in a larger cohort to explore possible state - related alterations in expression of ptgds and akr1c3 . We did this by employing a longitudinal design that allowed for assessment of gene expression in various affective states and incorporated both intra - individual and inter - individual alterations in mixed model analyses . In accordance with our hypothesis, ptgds mrna expression was altered in rapid - cycling bipolar disorder patients in euthymic, depressed, and manic / hypomanic states compared with healthy control subjects, with ptgds expression down - regulated in patients compared with healthy control subjects . No difference in akr1c3 mrna expression between patients and healthy control subjects contrary to our hypothesis, mrna expression of both ptgds and akr1c3 did not differ between affective states in bipolar disorder patients . The finding of down - regulated levels of ptgds mrna expression in peripheral blood in the current study is supported by findings in post - mortem brain tissue where both genes have been found down - regulated in frontal brain regions in bipolar disorder patients compared with healthy control subjects (stanley medical research institute online genomics database). It would be of interest to elucidate possible single nuclear polymorphisms related to the ptgds locus, which could further lead to mapping of potential expression quantitative trait loci . This analysis could prove especially useful in investigating differentially - regulated genes of interest between affective states in bipolar disorder . Our results of comparable expression levels of ptgds and akr1c3 across affective states in bipolar disorder patients are in contrast to the findings in two studies by the same group involving a total of five rapid - cycling disorder patients (begemann et al ., 2008; gurvich et al ., 2014), where ptgds and akr1c3 expression were found to be down - regulated in depressive episodes compared with manic episodes . Our study consisted of a larger cohort of rapid - cycling disorder patients (n = 37), likely constituting a more representative sample of rapid - cycling bipolar disorder patients, involved a more rigorous methodological approach, such as transparent statistical analyses adjusted for relevant covariates, and used several reference genes that were tested for stability . Our cohort also represents a heterogeneous population of patients, and it is possible that subgroups of patients could exhibit a different pattern of gene expression . In the study finding ptgds down - regulated in adhd patients compared with bipolar disorder patients (marn - mndez et al ., 2012), patients were both adolescents and adults and there was no information about the affective state or medication of participants . Our finding of down - regulation of ptgds could possibly represent a compensatory mechanism in reaction to an activated aa cascade in rapid - cycling bipolar disorder . Specifically, the down - regulation of ptgds activity could represent a counter reaction to up - regulated pla2 or cox activity, as increases in pla2 activity in in vivo (noponen et al ., 1993) and of pla2 and cox-2 mrna and protein levels in post - mortem bipolar brain tissue (rao, bazinet, et al ., 2007) this is in line with findings from a recent study of bipolar disorder patient post - mortem brain tissue, where decreased expression of cox-1 and cytosolic prostaglandin e synthase were speculated to be compensatory to the increased expression of cox-2 and membrane prostaglandin e synthase also demonstrated in the study (kim et al ., 2011). The relatively weak correlation observed between ptgds and akr1c3 mrna expression levels may indicate that the role of the ptgds and akr1c3 enzymes in the aa cascade are not closely interrelated or that they are possibly differentially influenced by medication on a transcriptional level . The finding of down - regulated ptgds expression in rapid - cycling bipolar disorder patients across all affective states is also consistent with preclinical evidence suggesting down - regulation of the aa cascade as a mechanism of action for these medications . The majority of patients in our study were treated with lithium, anticonvulsants, or antipsychotics that, in preclinical studies, have been demonstrated to down - regulate the aa cascade (bazinet, 2009; rapoport et al ., 2009), and it is possible that prolonged treatment with these medications not only results in normalizing an up - regulated aa cascade in these patients but even leads to down - regulation of the cascade below normal activity . While there are no studies of the effect of mood - stabilizing medications on the mrna expression of ptgds and akr1c3 specifically, medication may also down - regulate mrna expression of these genes . It is possible that under such circumstances, state - related alterations in ptgds and akr1c3 mrna expression do not occur . In the present study we did not find an effect of individual medication groups on either ptgds or akr1c3 mrna expression levels . However, the majority of the patients (78.4%) were treated with two or more medication groups and it is therefore difficult to assess the impact of individual medications . Along those lines, it cannot be excluded that treatment with multiple medication groups adds to the possible down - regulating effect of these medications on mrna expression of ptgds and akr1c3 . Investigation of larger cohorts may be necessary to elucidate the effect of individual medications on ptgds and akr1c3 mrna expression and of the aa cascade regulation in general . Such an effect is not only suggested by post - mortem brain findings but also by in vivo preclinical studies demonstrating that antipsychotics down - regulate aa metabolism (cheon et al ., 2011; modi et al ., the present finding of aberrations in the aa cascade is in line with the current hypothesis on the pathophysiological background of bipolar disorder involving disturbances within several inter - related pathways, such as inflammatory system dysregulation (goldstein et al ., 2009), oxidative and nitrosative stress pathways (maes et al ., 2011), impairments in neuroplasticity (duman and monteggia, 2006), and mitochondrial dysfunction (clay et al ., 2011). Specifically, up - regulation of pla2 and cox-2 activity is observed upon activation of the inflammatory response system (bauer et al ., 1997; adibhatla and hatcher, 2007), which has been demonstrated to be activated in bipolar disorder (munkholm et al ., 2013), and elevated expression of pla2 and increased aa turnover have been found in animal models of neuroinflammation (rao, ertley, et al ., 2007). Activation of the aa cascade can, conversely, also induce inflammatory responses and the production of pro - inflammatory cytokines (munoz and costa, 2013). Accumulation of aa generates intracellular reactive oxygen species (magder, 2006), which can generate a state of oxidative stress that has been suggested to contribute to systemic toxicity (kapczinski et al ., 2010) and neuroprogression in bipolar disorder . Further, possible neuroprotective effects of mood - stabilizing treatment may be mediated through inhibition of the aa cascade (rapoport et al ., 2009) and aa cascade dysregulation may be involved in the pathophysiology underlying the neuroprogressive changes suggested in bipolar disorder (berk, 2009) with excess aa possibly inducing apoptosis by damaging mitochondria (saitoh et al ., the mechanisms through which these pathways interact with disturbances in the aa cascade and relate to the clinically distinct affective states that constitute bipolar disorder, however, is unclear . Genes expressed in the brain are to a large extent also expressed in peripheral blood, indicating that peripheral blood potentially could serve as a surrogate tissue (liew et al ., 2006; le - niculescu et al ., 2009) the peripheral blood transcriptome may thus reflect system - wide biology, and it has further been demonstrated that a significant amount of single nuclear polymorphism expression relationships are conserved between the brain and peripheral blood lymphocytes (iwamoto et al ., 2011). It is unclear, however, to what extent gene expression in peripheral blood reflects gene expression changes in the brain, and ultimately whether peripheral blood can function as a neural probe (chana et al ., 2013). First, we specified relatively low cut - offs and waived duration criteria in defining affective states . It is possible that in these patients where ptgds and akr1c3 mrna expression was down - regulated in both a manic / hypomanic and a depressive state as well as a euthymic state, alterations between depressive and manic / hypomanic states would only be present in more severe episodes . Second, it is possible that state - related alterations in the mrna expression of ptgds and akr1c3 could be observed in drug - nave patients . It is, however, likely not feasible to include a large cohort of unmedicated rapid - cycling bipolar disorder patients, due to the severity of illness of these patients . Third, our sample size was relatively modest, and given the naturalistic design, not all patients experienced episodes of all polarities and the contribution of between - subject variation was therefore relatively large . Since the number of hypotheses tested was relatively small and hypotheses, outcomes, and covariates were specified a priori and other tests were treated as hypothesis - generating, we did not correct statistical analyses for multiple testing, which may be considered appropriate (streiner and norman, 2011; panda et al ., 2013) but may be regarded as a limitation . Finally, the included rapid - cycling bipolar disorder patients had relatively long durations of illness and it is possible that the prolonged illness courses could have induced sustained down - regulation of these enzymes to a degree where more subtle expression changes between affective episodes do not occur . While the study could possibly suggest that ptgds mrna down - regulation in bipolar disorder patients is trait related, it is thus possible that the expression pattern could differ in patients with a more benign course or in patients in early stages of the disorder . Therefore it would be of interest to study mrna alterations in a larger cohort including bipolar disorder patients in early, intermediary, and late stages of the disorder . In conclusion, we demonstrated down - regulation of mrna expression of ptgds in depressed, manic / hypomanic, and euthymic states in rapid - cycling bipolar disorder patients compared with healthy control subjects, with no alterations between affective states . The results suggest a role for aberrant regulation of ptgds and of aa cascade and prostaglandin pathway dysregulation in rapid - cycling bipolar disorder . Lars vedel kessing has within the preceding three years been a consultant for lundbeck and astrazeneca . Maj vinberg has been a consultant for lundbeck, astrazeneca, eli lilly and servier.
To date, technetium-99 m ethyl cysteinate dimer (tc-99m - ecd or bicisate) is one of the most essential single - photon emission - computed tomography (spect) imaging agents in hospitals . According to the practice guidelines of the american college of radiology (acr) and the european association of nuclear medicine neuroimaging committee (enc), clinical indications of tc-99m - ecd include evaluating the regional cerebral blood flow (rcbf) in patients with (i) cerebrovascular diseases, (ii) transient ischemic attack, (iii) various forms of dementia, (iv) symptomatic traumatic brain injury, (v) encephalitis, (vi) vascular spasm following subarachnoid hemorrhage, (vii) inflammation, (viii) epileptic foci, and (ix) lacunar infarctions [1, 2]. The indications of tc-99m - ecd in spect brain perfusion imaging of neuropsychiatric disorders and chronic fatigue syndrome have not been fully characterized [1, 2]. However, investigations of the conversion in patients of mild cognitive impairment (mci) to alzheimer's disease (ad), the functional compensation mechanism in incipient ad, the mechanism for suppression of parkinsonian tremor by thalamic stimulation, the mechanism by which thyroid hormone availability affects cerebral activity, brain glucose metabolism in hypothyroidism, reduction in the bifrontal regions and diffusion - weighted imaging of creutzfeldt - jakob disease [8, 9], quantitation and differentiation in patients with tourette's syndrome [1012], and abnormal rcbf in patients with sjgren's syndrome were reported . For clinical implements, tc-99m - ecd is obtained by radiolabeling of active pharmaceutical ingredient (api), that is, l - cysteine, n, n -1,2-ethanediylbis-, diethyl ester, dihydrochloride (ecd) with tc-99 m . Radiochemical purity (rcp) of tc-99m - ecd is used for the quality control (qc) purpose [1416]. Although the characteristics of tc-99m - ecd, such as in vivo kinetics and biodistribution studies in healthy human [15, 17], pharmacological studies in primates [14, 18], uptake, clearance, and brain retention [1922], biotransformation, metabolites, and stability [14, 21, 23], have been well - investigated, the chemical properties (such as purity and content) of ecd in ecd kit (vial a), that is, api in drug product, which might significantly disqualify the efficacy of tc-99m - ecd have not been much discussed . Moreover, no analytical method for the determination of content and uniformity of ecd in ecd kit has been published . Analysis of the content and uniformity of ecd in ecd kit is a relevant requirement for the pharmaceutical qc in processes of mass fabrication . In the stability study of mikiciuk - olasik and bilichowski, they demonstrated that ecd decomposed as soon as it was dissolved in phosphate buffer solutions ., showing that the composition of ecd kit is the major obstacle to determine stability of ecd in (non)aqueous solutions . Methods for the determination of sulfur, including eschka method, gas chromatography - mass spectrometry (gc - ms), inductively coupled plasma atomic emission spectrometry (icp - aes), instrumental neutron activation analysis (inaa), x - ray fluorescence [27, 28], and elemental analyzer coupled with a thermal conductivity detector (ea - tcd) or an isotope ratio mass spectrometer (ea - irms), have been developed . We here presented a direct solid sample determination method of ecd in ecd kit without sample dissolution to avoid the rapid degradation of ecd in aqueous solution using elemental analyzer (ea) coupled with a nondispersive infrared detector (ndir). Ecd (purity: 97.53%) was obtained from abx (radeberg, germany). Coal standard (eltra coal standard no . 92510 - 50; c: 76.6%, s: 3.07%) was purchased from eltra (neuss, germany). All chemicals and reagents were of analytical grade and used as received without further purification . An elemental analyzer (ea) (vario el cube, elementar analysensysteme gmbh, hanau, germany), equipped with a microbalance (mettler - toledo xp6, mettler - toledo gmbh, giessen, germany), a nondispersive infrared detector (ndir), and a thermal conductivity detector (tcd) was employed for the measurement of sulfur . The microbalance was connected to control a personal computer (pc) of the ea for automatic transmission of the sample weight to the pc . The measurement of sulfur was switched to ndir photometer in operation mode of chns . Since the ndir detector is sensitive to water vapor, the measured gas was dried with a u - tube filled with sicapent (phosphorus pentoxide drying agent) before entering the ndir . For ea analysis, the samples were sealed in a tin container and were dropped automatically into a combustion tube filled with catalytic material (wo3 granulate) and maintained at a temperature of 1150c . As the sample entered the combustion tube, a fixed amount of oxygen was injected into the helium carrier . After passing through a reduction tube (silver wool, corundum balls, and copper) at a temperature of 900c, elements of nitrogen, carbon, sulfur, and hydrogen in the samples were converted into gases of nitrogen, carbon dioxide, sulfur dioxide, and water, respectively . The mixture of gases was separated by gas chromatographic column, and the tcd or ndir signals of co2, h2o, and so2 were recorded . Data were acquired and processed with software from elementar (vario el version of 1.3.1 ., the preparation of ecd kit (vial a) was done according to the procedure of walovitch et al ., which was freeze - dried under an n2 headspace and contained 0.90 mg ecd, 72 g sncl22h2o, 360 g na2edta2h2o, and 24 mg mannitol . Compositions of ecd calibration standards (stdecd), blanks (bkkit), and qc samples (qcecd: qc - l, qc - m, qc - h) for method validation were prepared by isotope application division, institute of nuclear energy research (iner, taoyuan, taiwan) and summarized in table 2 . Ecd kit and kit blank samples were grounded by using an agate mortar for 40 seconds before determination . Coal calibration standards (stdcoal) were freshly prepared daily by weighing 1.00 to 3.50 mg of coal standard . Coal qc samples (qccoal) were prepared in the same way as the coal calibration standards by weighing 2.00 0.20 mg of coal standard . The method was modified and validated according to the international conference on harmonization (ich) guidelines for the validation parameters of analytical method, including specificity, linearity, precision, accuracy, stability, robustness, and system suitability . Three tin blanks (tin container without sample) and three 7.60 mg kit blanks (table 2) were analyzed . Peak areas appeared on the retention time of sulfur were determined to evaluate the specificity (selectivity) of the method in resolution between sulfur and other elements . The calibration curves of five coal standards (1.08 to 3.39 mg) were plotted against the peak areas . The linearity was evaluated by the linear least squares regression method with three coal qc samples determined at concentration of 2.10 mg . The precision of the method was assessed by the same batch of ecd kit at five concentrations (1.08 to 3.39 mg) and three qc samples determined at concentration of 2.10 mg . Intraday precision (repeatability) and inter - day precision (reproducibility) were evaluated by one analyst within one day and on two different days, respectively . Ecd quality control (qc) samples of low (qc - l), medium (qc - m), and high (qc - h) concentration at 0.23, 0.27, and 0.31 mg / vial (nominal weight of ecd per vial of ecd kit, table 2) and one coal qc sample at concentration of 2.15 mg were analyzed by the proposed method . Experimental values (sulfur(mg)exp or sulfur(%)exp) were obtained by interpolation to the linear least squares regression equation of a fresh prepared calibration curve (1.08 to 3.45 mg) and compared to the theoretical values (sulfur(mg)nominal or sulfur(%)nominal): (1)recovery yield (%) = sulfur(mg)exp sulfur(mg)nominal100%, or (2)recovery yield (%) = sulfur(%)exp sulfur(%)nominal100% . The bench - top stabilities were examined by analyzing 2.05 0.05 mg of coal standards and 7.52 0.03 mg of ecd kit samples for three consecutive days . The samples were kept in an autosampler at ambient temperature for ea analysis over this period . Experimental data were obtained by comparing the linear least squares regression equations of calibration curves . The robustness of an analytical method is a basic measurement of its capacity to remain unaffected by small variations in method parameters . In this case, method robustness was evaluated through the effects of dosing time of oxygen, temperatures of combustion tube and reduction tube . The system suitability was assessed by the triplicate analyses of tin blanks and kit blanks with acceptance criterion of 5,000 counts . Various sulfur forms are presented in coal, that is, pyrite, ferrous sulfate, gypsum, organic sulfur, and elemental sulfur [26, 28, 31]. For direct solid sample analysis of sulfur, effects of matrix, chemical form, and homogeneity of the analyte in sample are relevant to the reliability of analytical results [3234]. The matrix effect on the determination of sulfur was examined as shown in table s1 in supplementary material available online at doi:10.1155/2011/196238 . The average peak area of kit blanks was ten times higher than that of tin blanks . The linear least squares regression equations of coal standard without and with the existence of kit blanks were y = 1.565 10x + 3.174 10 and y = 1.547 10x + 8.932 10, respectively . Determination of different concentration ecd standards (0.78 to 1.07 mg, table 2) in kit blank using coal for calibration curve were shown in supplemental table s2 . Again, no significant difference of inter - day study coal standard curves was found . In supplemental table s1, it is shown that the peak areas on the retention time of sulfur were 248 11 and 2438 642 for tin blanks and kit blanks, respectively . Data are expressed as average sd . Although the peak areas of kit blanks were higher than those of tin blanks, the areas were approximately half of the acceptance criterion of system suitability (5000 counts). Standard curves were constructed by plotting peak areas (counts) against the amounts of coal standard and were linear over the range of 1.08 to 3.39 mg (x in weight of sulfur = 0.0330.104 mg). The linear least squares regression equation of the standard curve in this range was y = 1.615 10x + 4.747 10, with a correlation coefficient (r) of 0.9993 . Table 3 provides the results of repeatability, reproducibility, and accuracy of the proposed method . The intraday precisions of sulfur weight (%) in coal qc samples were 0.60% to 2.25% . The inter - day precisions of sulfur weight (%) and slope of the calibration curve in coal qc samples were 1.69% and 1.56%, respectively . Average recovery yield of ecd in ecd qc samples was 101.62% 1.45% (r.s.d . The samples for bench - top stability study were kept in the ea autosampler under ambient environment for a three - consecutive - day experiment (table 4). Average recovery yields for the determination of sulfur in coal qc samples and ecd in ecd qc samples were 100.88% 1.46% (r.s.d . However, recovery yields of qcecd increased gradually from 96.02% 2.33% (day 1) to 102.31% 1.63% (day 3). The method robustness was evaluated through the effects of dosing time of oxygen, temperatures of combustion tube and reduction tube as shown in table 5 . Optimal dosing time of oxygen, temperatures of combustion tube and reduction tube were 120 sec, 1150c and 900c, respectively . The acceptance criterion of system suitability was assessed by triplicate analyses of the tin blanks and kit blanks for peak area and was set at 5000 counts . The determined (experimental) value of ecd by the proposed method gradually increased from 0.934 0.021 mg (batch 1) to 0.984 0.007 mg (batch 3). No significant matrix effect of kit blank on the peak area, linearity of calibration curve, and selectivity of sulfur was found (table s1). The findings suggest that coal standard (without being spiked into kit blank) is more convenient and stable (table s2) than ecd standard to construct the calibration curve . In this investigation, background peak area of sulfur is attributed to the sample moisture and usage of ea tubes such as sicapent tube, combustion tube, and reduction tube . Although the background peak area of sulfur is variable, the proposed method has sufficient selectivity (resolution) to the sulfur determination . The system suitability can be simply assessed by background peak areas of tin blanks and kit blanks . Background of coal standard and ecd kit can be deducted by tin and kit blanks, respectively . Although samples of multiple batches can be assayed within one single day, background peak area of each batch should be determined separately . Each analytical batch should consist of tin blanks, kit blanks, coal qc samples, calibration coal standards, and unknown samples . Coal standards are grounded and dried under 110~120c for at least 2 hours before determination and prepared for the standards curve freshly . The number of qc samples (in multiples of three) depends on the total number of samples in a batch . Table s3 demonstrates that triplicate qc samples analyses are necessary to ensure quality of the assay for a batch within 1020 samples . Acceptance criterion is suggested to set at least 67% (2 out of 3) of qc samples, which should be within 5% of their respective nominal value, and 33% of the qc samples may be outside 5% of nominal value . Nominal content of ecd in each ecd kit vial is 0.900 0.135 mg / vial, which is equal to the weight of sulfur in the range of 0.0330.104 mg / vial . Therefore, one - third to half of content of ecd kit was suggested to sample for ea analysis . The observation of three - day stability study of ecd kit in table 4 (recovery yields of qcecd increased gradually) is difficult to explain, but it might be related to the degradation of ecd in ecd kit due to the moisture . For example, an intermolecular sulfur - sulfur bonding compound was found in our preliminary forced degradation study . In table 5, the results of method robustness evaluation further support the optimal conditions of table 1 . Additionally, the results of method validation in tables 3, 4, and 5 indicate the potential of this method in pharmaceutical qc . Ecd kit, where purity of ecd should be determined prior to mass fabrication processes . Based on the test specification in practice guidelines of the american college of radiology (acr) and the european association of nuclear medicine neuroimaging committee (enc), the radiochemical purity (rcp) determinations of tc-99m - ecd should be performed on each vial prior to injection and can also be used to verify the quality of ecd kit [1, 2]. Since the composition of ecd kit may cause degradation of ecd as soon as it is dissolved in (non)aqueous solutions, the best way to adopt for the quantitation is highly restricted to a method of direct solid samples analysis . This investigation provides a method for the intended purpose, for example, routine qc of chemical manufacturing . Ecd is one of the diamino dithiol (dadt) derivatives to form stable complexes with radiorhenium or radiotechnetium . Therefore, this method can be also a useful tool to investigate the qc quantitation and properties of thiol - contained derivatives . Finally, this research not only enhances our understanding of ecd kit about its stability but also raises some questions that require further investigation, especially the degradation pathways, degradation compounds of ecd in ecd kit and a more stable ecd kit, formulation design.
Human investigations focusing on concentrated ambient particles have shown acute lung inflammation and changes in both blood indices and heart rate . It is an urgent priority to establish in vivo bioassays for the detection of hazards related to fine particles, which can be inhaled into deep lung tissue by humans . Histopathologic observation of lung tissue from f344 rats treated with 4 mg / rat quartz i.t . On day 28 . Arrows in the figures indicate typical findings . A, neutrophil infiltration in the walls; b, neutrophil infiltration in the spaces of alveoli; c, pulmonary edema; d, pulmonary fibrosis; e, histiocytic macrophage infiltration; f, restructuring of walls; g, granuloma . A, immunostaining of brdu on day 1; b, immunostaining of inos on day 28 after i.t . Of 4 mg / rat quartz . In quartz dust - exposed construction workers, obstructive and restrictive loss of lung function has been detected, as has chronic obstructive pulmonary disease (copd). These are associated with inflammatory cell responses characterized by alveolitis with recruitment of inflammatory cells, particularly neutrophils, and may result in pulmonary fibrosis and impaired lung function . Intratracheal instillation (i.t .) Of quartz into rats produces an inflammatory reaction followed by histological changes characteristic of lung fibrosis, similar to the above noted human conditions . Reported a no observed adverse effect level (noael) for quartz of between 0.03 and 0.13 mg / m (40 year exposure) based on an autopsy study for humans showing that lung burdens between 0.7 and 1.7 g of quartz are associated with macules only and not simple coal worker s pneumoconiosis . In order to establish an appropriate bioassay for detection of lung damage after particle inhalation, several experiments were performed in rats using quartz as a typical particle lung toxicant . Histopathologically, neutrophil infiltration in the walls and in the spaces of alveoli, pulmonary edema, pulmonary fibrosis, macrophage infiltration in the alveoli, restructuring of walls of the alveoli and granuloma production were all assessed (fig . 1). These parameters of inflammatory change were scored as follows: 0, no change; 1, weak; 2, moderate; and 3, severe . For objective assessment, 5-bromo-2-deoxyuridine (brdu) incorporation and expression of inducible no synthase (inos) were also immunohistochemically examined (fig . Antibodies specific for brdu provide a sensitive method for detecting dna replication for dna repair and cell proliferation in situ, while inos is associated with development of lung damage, inflammation, granulomas and fibrosis induced by inhalation of silica . These markers are generally associated with inflammation and may increase at different times after particle inhalation . In this review, we summarize some pilot data from a sequential analysis study, a dose response study and a vehicle assessment study conducted to establish a bioassay model featuring instilled quartz as the positive control . Finally, the toxicity of a series of different particle types, including nanoparticles and diesel exhaust particles, was also tested with our new bioassay . In order to establish an appropriate bioassay for detection of lung damage after fine particle inhalation, sequential histopathological changes were examined after i.t . Of quartz with a particle diameter of not more than 7 m (dq-12, deutzche montan technologie, gmbh, germany, 4 mg / rat), as a typical lung toxic agent, in f344 male rats . Experimental design for the sequential analysis study . A total of 50, 10-week - old animals were separated into two groups ., i.t . Of 4 mg / rat quartz suspended in 0.2 ml saline ., a total of 50, 10-week - old animals were separated into two groups (fig . Twenty - five rats were exposed to the material suspended in saline (0.2 ml) using a specially designed aerolizer (penn century, pa, usa), and subgroups were sacrificed 1, 3, 7, 14 and 28 days thereafter . (0.2 ml) as a control group and were sacrificed on the same days . Both groups received intraperitoneal injections (i.p .) Of 100 mg / kg b.w . Brdu before sacrifice and underwent assessment of lung histopathology with immunohistochemical demonstration of brdu, inos and matrix metalloproteinase-3 (mmp-3, employed as a marker of fibrotic change). In this experiment, after quartz treatment, lungs on day 1 demonstrated acute inflammatory changes with neutrophil infiltration, while granulation - like changes with giant cells and macrophages in the alveoli were evident on day 28 . Furthermore, the numbers of brdu positive cells were found to gradually decrease after the initial peak on day 1 . Monoclonal antibodies specific for brdu provide a sensitive method for detecting dna replication for dna repair and cell proliferation in situ . The numbers and areas of inos positive cells, in contrast, increased with time up to day 28, i. e., throughout the experimental period . Previous studies show that inos is expressed by many cells within the lung parenchyma after exposure to various inflammatory stimuli and is also associated with neovascularization and proliferation . Furthermore, inos activity in primary tumor tissues in cases of fresh human gynecological and breast cancers correlates positively with the tumor grade . Generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with development of lung damage, inflammation, granulomas and fibrosis induced by inhalation of silica . Experimental design for validation of the sequential analysis study . A total of 108, 10-week - old f344/ducrj male rats were randomly divided into 8 groups ., i.t . Of 4 mg / rat test substances, quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black, suspended in 0.2 ml saline or pg - cmc for carbon black ., the results suggest that days 1 and 28 after intratracheal instillation of fine test particles are the most appropriate day for detection of acute and subacute inflammatory changes, respectively . Furthermore, brdu on day 1 and inos on day 28 proved to be suitable end - point markers for this purpose . The quartz - treated group demonstrated severe toxicity, while the other hydrotalcite, potassium octatitanate, palladium oxide and carbon black - treated groups all exhibited relatively mild toxicity . The toxicities of fine particles from various materials (quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black) were examined using our in vivo bioassay with a special focus on the correlations between immunohistochemical and histopathological findings . A total of 108, 10-week - old f344/ducrj male rats were randomly divided into 8 groups (fig . Of the 5 test particles (4 mg / rat), quartz, hydrotalcite, potassium octatitanate, palladium oxide and carbon black, respectively, suspended in 0.2 ml vehicle (saline or 10% propylene glycol and 1% sodium carboxymethyl cellulose in saline: pg - cmc) with an aerolizer, and subgroups of 7 rats were sacrificed on days 1 and 28 thereafter . Groups 6 and 7 were similarly exposed to saline and pg - cmc, respectively, as vehicle controls, while group 8 was maintained untreated . Histopathological changes and immunohistochemically assessed brdu labeling indices and inos levels were applied as end points . From the scoring indices generated by the comparative histopathological and immunohistochemical assessment (fig . 5), the quartz treated group demonstrated severe toxicity, while the other particle treated groups all exhibited relatively mild toxic effects . Biochemical analyses at different time points following instillation of different materials earlier demonstrated that all of the exposed groups developed granulomas . Alveolar lipoproteinosis and pulmonary fibrosis were most severe in the quartz treated lungs and progressed with time . The present results for brdu immunohistochemistry demonstrated that, in all groups, proliferation was enhanced on day 1 due to particle exposure but had returned to almost normal values by day 28 . In contrast, inos values were higher at the latter time point in the quartz and hydrotalcite groups . This time course of change in inos expression may be important in terms of toxicity assessment, and it may be necessary to examine a later time point (estimated to be about 8 months) after i.t . To clarify the influence of change with time . However, in this experiment, expression of mmp-3 was not associated with any of the subchronic changes, such as granulation, collagenization or fibrosis, after particle instillation . Generally, bronchoalveolar lavage (balf) with markers of inflammation is often used to assess the lung toxicity of instilled test particles in rats . In our experiment, histopathological findings and 3 different immunohistochemical markers (brdu, inos and mmp-3) were selected and were scored in order to provide an objective assessment . Compared with balf, this approach has the advantage of allowing detailed investigation of lung damage; it also has disadvantages, for example, in that rats must be sacrificed at each time point and sequential changes cannot be followed at the individual animal level . The present study featured comparison of 5 different particles in one experiment using an in vivo bioassay model . There has hitherto been no data available concerning instillation or inhalation of hydrotalcite and palladium oxide . Tismo has been evaluated after inhalation, and similarly, quartz and carbon black have been evaluated after inhalation and instillation . In this experiment, quartz exerted much stronger toxicity than the other test particles, which did not greatly differ in their effects . The dose of 4 mg / rat of quartz instilled directly into the trachea in this experiment was selected based on data reported previously, and dose dependence at higher levels is currently being investigated in our laboratory . There are biologically different responses to inhalation and instillation, but given the rapidity with which different particles can enter the lung, acute effects may be most important . The advantages of assessment of particle toxicity using in vivo bioassays such as i.t . Have been stressed . Method is clearly useful for detection of acute and subacute pulmonary particle effects using a histopathological scoring system and markers like brdu and inos . A total of 40, 10-week - old male f344 rats were randomly separated into 4 groups of 10 rats ., i.t . Of 4 mg, 2 mg or 1 mg / rat quartz suspended in 0.2 ml saline ., i.t . Of 0.2 ml saline (control). S(5), sacrifice of 5 rats . The dose of 4 mg quartz used by mercer et al . (2003) is too large to allow for assessment of slight changes . In fact, the toxicities of the 5 materials in our validation experiment were found to be only high (quartz only) or low (other particles), with no intermediate toxicity . In many reports of toxicity assessment of fine particles using quartz as a positive control, the doses employed have been less than 4 mg . In our study, a total of 40, 10-week - old male f344 rats were randomly separated into 4 groups of 10 rats each (fig . 6) and exposed by intratracheal instillation to 4 mg, 2 mg, 1 mg and 0 mg (control) quartz suspended in saline (0.2 ml) using a specially designed aerosolizer . Subgroups of 5 rats each were sacrificed on days 1 and 28 thereafter, and histopathological examination was performed on the lungs . In the quartz treated groups, granulomas were observed at 2 mg and 4 mg, with only mild inflammation was observed at 1 mg . A dose of 4 mg does not induce any novel extra alterations, and 1 mg is too weak to cause changes in histopathological parameters on day 28 . A dose of 2 mg quartz was thus suggested to be the most appropriate dose for sensitive detection of acute and subchronic inflammatory changes . Experimental design for the vehicle assessment study (dry powder assessment). A total of 20, 10-week - old male f344 rats were randomly separated into 2 groups of 10 rats ., i.t . Of 4 mg / rat quartz powder with 0.2 ml air using a dp-4 insufflator (dry powder insufflator)., s(5), sacrifice of 5 rats . In our experiments, test particles were suspended in saline, but this is associated with considerable agglutination . To prevent this as much as possible, we also tested the efficacy of a dry powder instillation method using a dry powder insufflator without any vehicle . A total of 20, 10-week - old male f344 rats were randomly separated into 2 groups of 10 rats each (fig . Groups 1 and 2 were exposed to 4 mg and 0 mg (air only control) quartz powder, without suspension in any vehicle, and 2 ml air using another type of aerolizer, the dp-4 insufflator (dry powder insufflator, penn century, pa, usa), and sacrificed on days 1 and 28 thereafter . Histopathological examination revealed that 4 mg quartz dry powder also caused inflammation, but it was mild compared with the previous experiment result with the same dose suspended in 0.2 ml saline . While the dry powder instillation method may be suitable for experiments requiring dry powder formulations, for example, to increase stability in vaccine studies, instillation by this method may weaken the effects . Because it is difficult to maintain precise dose and it is impossible to control loss due to expiration air after intratracheal instillation . There are also problems in regard to exposure of researchers since there is possibility of aspiration . The pilot experiments suggested that a dose of 2 mg quartz suspended in 0.2 ml saline was suggested to be most appropriate for sensitive detection of acute and subchronic inflammatory changes . Using this as a control, examination of the toxicity of a series of particles these materials were selected to perform this validation study and for their variety of characteristics, including shape, composition and particle diameters with the nanometer order or not . Histopathological and immunohistochemical analysis of brdu incorporation and inos were performed after exposure of the lungs . Experimental design for the toxicity assessment of a series of different particle types (experiments 13). [experiment 1], i.t . Of 2 mg / rat quartz, titanium dioxide, hydrotalcite or -cyclodextrin suspended in 0.2 ml saline or k2pdcl4 and na2pdcl4, suspended in 0.2 ml distilled water . [experiment 2], i.t . Of 2 mg / rat test substances, quartz, titanium dioxide, cuo, cuo nanoparticles, mno2, nio or nio nanoparticles suspended in 0.2 ml saline ., i.t . Of 0.2 ml saline (control). [experiment 3], i.t . Of 2 mg / rat quartz, diesel standard powder, diesel powder, c6h10o4pd or caco3 suspended in 0.2 ml saline or neutralized na2pdcl4 (final ph was 6.5) in 20 mm phosphate buffer and 5 n nacl . Experiment 1: a total of 108 rats were randomly separated into 9 groups of 12 animals in each (fig . Mg / rat quartz, titanium dioxide, hydrotalcite and -cyclodextrin suspended in 0.2 ml saline, and groups 5 and 6 were exposed by i.t . To 2 mg / rat distilled water was employed as a vehicle because only the bases, na and k, of k2pdcl4 and na2pdcl4 differed . Groups 79 were maintained as controls (saline, distilled water or untreated). Subgroups of 6 rats were sacrificed on days 1 and 28 . Experiment 2: a total of 106 rats were randomly separated into 9 groups (12 rats each in group 18, 10 rats in group 9). Mg / rat quartz, titanium dioxide, cuo, cuo nano, mno2, nio and nio nano suspended in 0.2 ml saline, and groups 8 and 9 were maintained as controls (saline or untreated). Some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment . The ranking order is cuo> quartz> neutralized na2pdcl4> nio> hydrotalcite> mno2> diesel> titanium dioxide (in experiment 2)> -cyclodextrin> diesel standard> titanium dioxide (in experiment 1)> caco3 . Experiment 3: a total of 96 rats were randomly separated into 8 groups of 12 rats each . Mg / rat quartz, diesel standard powder, diesel powder, c6h10o4pd and caco3 suspended in 0.2 ml saline, and group 5 was exposed to neutralized na2pdcl4 (final ph was 6.5) in 20 mm phosphate buffer and 5 n nacl . Groups 7 and 8 were maintained as vehicle controls (saline or vehicle of group 5). Subgroups of 6 rats were sacrificed on days 1 and 28 . In order to assess toxicity to lungs for each fine particle material and to screen for hazard in a relatively simple way, toxicity points for each particle or chemical were calculated from histopathological and the immunohistochemical brdu and inos findings (table 1) and placed in order . More detailed information on total toxicity points has previously been reported by yokohira et al .. the results of the evaluation of particle toxicity by total toxicity points are outlined below, and summarized results for fine particle toxicity are detailed in fig . Only cuo caused greater toxicity than quartz; the other particles had relatively minor effects . Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, the ranking order could be clarified as follows: cuo> quartz> neutralized na2pdcl4> nio> hydrotalcite> mno2> diesel> titanium dioxide (in experiment 2)> -cyclodextrin> diesel standard> titanium dioxide (in experiment 1)> caco3 . The various particles employed in the present study, quartz, titanium dioxide, hydrotalcite and -cyclodextrin, k2pdcl4, na2pdcl4, cuo, cuo nano, mno2, nio, nio nano, diesel standard powder, diesel powder, c6h10o4pd and caco3, were chosen based on likelihood of human exposure . Hydrotalcite is employed as an antacid, and -cyclodextrin is employed as a food additive and in the pharmaceutical field to improve dissolution, chemical stability and bioavailability . Caco3 is calcium carbonate, is commonly referred to as chalk, and in industrial plants, workers may breathe in k2pdcl4, na2pdcl4, c6h10o4pd (the preceding 3 particles are used as catalysers), cuo, cuo nanoparticles, mno2, nio or nio nanoparticles . All rats instilled with k2pdcl4 or na2pdcl4 solution died from severe hemorrhage and edema of the lungs following intratracheal instillation in experiment a. the ph values of the k2pdcl4 and na2pdcl4 solutions were strongly acidic at 3.2, and this presumably was a major contributor to lung damage . The lungs instilled with neutralized na2pdcl4 also demonstrated severe inflammatory changes on day 1 . The findings for lungs treated with c6h10o4pd were almost the same, severe hemorrhage and edema, as those for k2pdcl4 and na2pdcl4 . However, the ph value of the c6h10o4pd solution was 4.9, which was not sufficiently acidic to lead to death . Reported that pdso4 exerts significant effects on human epithelial lung cells, and we have previously confirmed pdo toxicity in rats . Based on the available results, it is possible that not only strong acids but also palladium itself has toxic potential in the lung . In the case of cuo and nio, the diameters of the particles were in the micrometer and nanometer ranges . With the same volume of material, in general, it is likely that nanoparticles exert a greater impact because of the area and characteristics of the surfaces . For example, an oral administration study in mice indicated that titanium dioxide is retained in the liver, spleen, kidney and lung tissues after uptake through the gastrointestinal tract . However, no remarkable histopathological changes were seen in the liver and kidneys after treatment with nanoparticles of cuo or nio in the present study . Also, nio nanoparticles induced only mild inflammatory change in the lungs on day 1 . Similarly found limited histopathological effects on the lungs with 13.0 mg nio powder / kg b.w .. further examination is necessary to clarify the causes of death observed here . To establish a bioassay model for detection of lung toxicity due to fine particles for screening purposes, quartz is known as a typical lung toxic agent and provides a reliable positive control . Titanium dioxide was classified as belonging to group 2b (possibly carcinogenic to humans) in a recent international agency for research on cancer (iarc) publication, but hext et al . Concluded from an overview of epidemiology studies and toxicology studies in mice, rats and hamsters that titanium dioxide dust should not exert carcinogenic effects in the human lung . On the other hand, titanium dioxide has carcinogenic potential, and exposure may result in formation of dna adducts . With earlier assessment of acute or subacute findings, the severity of toxicity differed according to the instilled particles: quartz> 80:20 anatase: rutile and ultrafine titanium dioxide> fine - sized rutile titanium dioxide = ultrafine rutile titanium dioxide . Use of titanium dioxide as a negative control should be considered, where possible, for experimental purposes . In both experiments 1 and 2, however, based on the total points in these experiments, at least 3 rough grades of toxicity level, severe, moderate and mild, could be determined . It is expected that further investigations to obtain more particle toxicity data will allow more sensitive assessment of the toxicity of particles in the future . The results of several pilot experiments, performed in rats using quartz as a typical lung toxic particle, suggest that days 1 and 28 after i.t . Of 2 mg of fine test particles suspended in 0.2 ml vehicle are the most appropriate for detection of acute and subacute inflammatory changes, respectively . Furthermore, brdu on day 1 and inos on day 28 proved to be suitable end - point markers for this purpose . Examination of the toxicity of a series of particles could be performed with the developed bioassay . Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, a ranking order could be clarified . Our bioassay does suffer from a weakness in that even low doses of some particles may lead to death . Method has been proposed as the most reliable route for assessing the pulmonary toxicity of particles in rodents, although there are biologically different responses to inhalation and instillation . Another limitation of this bioassay is that the toxicity of particles can only be detected at an early stage because the experimental period is limited to 28 days . In this experiment, a dose response study was performed for quartz, not for risk assessment, but for improvement of the bioassay, and this study was not performed with other test particles . For risk assessment, more experiments, including dose response studies for each test particle with hazard characterization, should be conducted . With in that said, the bioassay was originally designed to be used for hazard identification at an early stage and to rank the toxicities of various particles given at single representative concentrations . The approach adopted clearly is also useful for detection of acute and subacute pulmonary particle characteristics using a histopathological scoring system and markers like brdu and inos for screening purposes.
The insulin - like growth factor binding proteins (igfbps) are a superfamily comprised of six proteins (igfbp-1 to 6) that bind to igfs with high affinity . The existence of igfbps was discovered in the 1960s, and most studies were performed after the cloning of six igfbps in the early 1990s . Igfbps are not only involved in igf action, but also appear to regulate igf - independent functions including inhibition or activation of cell growth and induction of apoptosis . Igfbps interact with the acid - labile subunits and glycoproteins as well as insulin - like growth factors (igfs) in serum . Recently, another group of proteins which have a low affinity with igfs was discovered and named igfbp- related proteins (igfbp - rps) even though their structures are similar to the classical igfbps . Igfbps are regulated by proteases released from several tissues, and their igf binding affinity is negatively affected by proteolytic cleavage as well as phosphorylation of igfbps . The characteristics of the six igfbps are summarized in table 1 . When igfs are secreted and circulate freely, they are unstable and easily degraded . To achieve functional stability, they need to bind igfbps for effective circulation in the blood . Lately, igfbps have demonstrated multiple functions as potential players in pathophysiological diseases including cancer, metabolic disease and neuronal disease . In this review, furthermore, we overview the transcriptional regulation of igfbp-1 and discuss current evidence that supports the effects of igfbp-1 gene expression on metabolic signaling . Understanding the transcriptional regulation of igfbps will expand our knowledge and research of the pathogenesis of metabolic disease and identify potential therapeutic targets against metabolic diseases . Igfbp-1, among the igfbp family, is produced dominantly in the liver and kidney . The n- and c - terminals of igbfp-1 are highly conserved among the igfbp isoforms and contribute to binding with igf . Igfbp-1 has central linker domains affected by post - translational modifications including proteolysis and phosphorylation . Igfbp-1 can affect igf-1 activity on cellular responses and independently activates igf-1 action on cell signaling . Igfbp-1 not only is an important determinant of igf activity, but also enhances glucose uptake in peripheral tissues and reduces glucose output in liver . The phosphorylated form of igfbp-1 shows an enhanced affinity to igf-1 and ii, thereby reducing the utility of igfs . Regulation of igf activity by igfbps, however, is very complicated in both stimulatory and inhibitory mechanisms . Because the affinity of igf for igf receptor is limited by that of igfbp-1 to igfs, igfbp-1 inhibits free igf-1 levels and reduces igf signaling through the igf receptor . This result indicates that high concentrations of igfbp-1 limit igf-1 bioavailability and activity of igf-1 with regard to metabolism . Igfbp-1 is also stimulated to very high concentrations during intensive exercise and in catabolic conditions, which is not completely explained by known regulators such as insulin and glucocorticoids . The role of amp activated protein kinase (ampk), an important signaling system for lipid and carbohydrate metabolism, in the regulation of igfbp-1 was studied in h4-ii - e rat hepatoma cells . Arsenic (iii) oxide and 5-aminoimidazole-4-carboxamide - riboside (aicar) were used as activators . Ampk was identified as a novel regulatory pathway for igfbp-1 by stimulating secretion and blocking the inhibitory effect of insulin . The mammalian igfbp genes have been duplicated by chromosome copying since 1993 when locations of igfbp-1 and 2 genes were identified in the same regions as housekeeping gene clusters on human chromosomes 7 and 2 . The evolutionary relationship between the different igfbp isoforms is well explained by genome duplication in vertebrates . Along with the historical aspect for the analysis of igfbp evolution, igfbps form part of the growth - activating hormonal axis with igfs, which are released into the blood by the action of growth hormone . Igfbp-1 is located on the short arm of chromosome 7 and encoded by a 5.31 kb gene . Igfbp-1 has a molecular weight of about 25 kda and circulates in plasma as a heterodimer complex with igf-1 or ii . Igfbp-1 is present in the amniotic fluid at concentrations 100500 times higher than in plasma . Although igfbps are secreted into the blood and the physiological function of igfbps has been studied and is achieved via igf-1 and ii binding, this review focuses on igfbp gene expression regulated by transcription factors to help achieve metabolic homeostasis . From the point of view of igfbp gene expression regulation of igfbp gene expression has been shown to be affected by hormones and growth factor - dependent molecular mechanisms, and it has been speculated that the dynamic regulations of igfbp gene expression may suggest the modulation of igf actions by systemic local effectors in various tissues . In this review, first, tamura et al . Suggested that igfbp-1 gene expression was regulated by camp in human endometrial stromal cells (escs). Camp is a commonly used decidualization stimulus, which is involved in the process of differentiating fibroblastoid stromal cells of the estrogen - primed endometrium by progesterone . Decidualization of stromal cells is induced by biochemical stimuli in vitro and in vivo camp is known to be an activator of igfbp-1 and increases igfbp-1 gene expression in escs . Two camp response elements (cres) have been found on the igfbp-1 promoter (fig . In addition to camp, the effect of metformin on decidualization was revealed in escs . However, tamura et al . Found a new function of metformin during decidualization processing through regulation of igfbp-1 . The tamura group showed that igfbp-1 gene expression was reduced by metformin treatment, and il-8 and il-1 were stimulated after long - term exposure to metformin . Even though the effect of metformin on igbfp-1 mrna expression was revealed, two progesterone response elements (pres) were identified; in a long - term primary culture system, progesterone receptor (pr) directly activates igfbp-1 gene transcription by binding to pres (fig . 2). Mutation of either the 5 or 3 half - site of the putative pre1 site (from 193 to 179 bp) reduced the promoter activity . Endogenous pr alone is insufficient to activate pre1, indicating that the pre1 site of the igfbp-1 promoter mediates direct activation of pr upon transcription, specifically in decidualized stromal cells . The primary physiological functions of igfbp-1 appear to be the suppression of igf action and a role in hypoglycemia . Moreover, igfbp-1 in liver may act as an igf - independent survival factor, resulting in pro - apoptotic signal reduction . Recently, degenhardt et al . Found that igfbp-1 was produced by peroxisome proliferator - activated receptor (ppar)- agonists independently of insulin . The direct activator of gene expression by ppar ligands requires at least one binding site of ppar protein as a ppar response element (ppre) on the promoter of the ppar target gene . A previous study has suggested that insulin inhibits hepatic igfbp-1 gene expression, and that igfbp-1 levels rapidly declined after feeding . Because igfbp-1 mrna expression increases during fasting, glucagon seems to stimulate plasma insulin level . These results suggest that igfbp-1 gene transcription might play a key role in a complicated metabolic regulation . Moreover, igfbp-1 gene expression is also activated by glucocorticoids as well as hepatocyte nuclear factor (hnf)-1 . Suh and his colleague found a glucocorticoid response element (gre) located between 91 and 77 from the transcription start site of the rat igfbp-1 promoter . Also, three accessory regulatory sites for a hepatocyte nuclear factor-1 at 62/50, an insulin response element at 108/99, and an upstream site at 252/236 might be involved in dexamethasone - dependent activation . Specifically, suh et al . Showed that hnf-1 and -1 transcription factors extracted from h4-ii - e cells could bind to the palindromic hnf-1 response element . In addition, hnf-1 synergistically stimulates igfbp-1 gene expression with the glucocorticoid receptor bound to the gre, suggesting a possible mechanism for glucocorticoid - specific regulation . In addition to the effect of glucocorticoid as a transcription factor of igfbp transcription through gre, igfbp-1 gene expression is regulated by caat / enhancer - binding proteins (c / ebps) (fig . The effects of insulin for gene expression are mediated through an insulin response element (ire) on the promoter of the target gene . C / ebps are mainly expressed in the liver and adipose tissue where they play key roles in metabolism and cell differentiation . They found that c / ebp protein forms a complex with other factors and inhibits insulin from binding to ire without interacting directly with the ire . Owing to inhibition of igfbp-1 expression by insulin, as mentioned above, this complex formation correlates with the capacity of insulin to control igfbp-1 promoter activity . Among the six igfbps, igfbp-1 represents the most important member with regard to insulin and glucose metabolism . It is mainly produced in the liver and binds insulin - like growth factor-1 and ii . Hence, there is considerable evidence that dysregulation of igfbp-1 may contribute to metabolic disease . In this section, dysregulation of igfbp-1 as well as igfs in serum and cervical secretions induces metabolic disease and cardiovascular disease . Therefore, measurement of igfbp-1 level in various body fluids might be used in clinical diagnosis for such afflictions as insulin resistance, cardiovascular risk, hypoglycemia, and growth failure including fetal growth, pre - eclampsia, laron dwarfism, and ruptured fetal membranes . On the other hand, serum igf-1 is reduced while igfbp-1 level is increased in type 1 diabetic patients . . Serum level of igfbp-1 is associated with metabolic nutritional factors and increases igf-1 concentration, resulting in reciprocal regulation of plasma insulin level . Various evidences suggest that malfunction of the igfbp-1 system is involved in the long - term complications of diabetes mellitus . Low levels of igfbp in serum identify insulin resistance as a marker of insulin sensitivity . Therefore, igfbp-1 level can correlate well with common indicators of insulin resistance in obese children and adults . Recent studies demonstrated that secretion of igfbp-1 is activated by proinflammatory cytokines like il-1 and tnf in hepg2 liver hepatocarcinoma cell lines . In addition, il-6 upregulates mrna expression and secretion of igfbp-1 in a dose - dependent manner in hepatocytes which are co - cultured with liver macrophages . Therefore, the concentration of igfbp-1 regulated by cytokines may be involved in the initiation and progression of cardiovascular disease . This article aimed to discuss the regulation of the igfbp-1 in metabolic diseases like diabetes and cardiovascular disease . In addition, a complex diagram for cis - elements of transcription factors on the igfbp-1 promoter have emerged suggesting that the molecular mechanism of their expression is dependent on transcription factors in cells and tissues . Although roles of igfbp-1 in the pathogenesis and progression of disease are complicated, the literature clearly indicates functional significance of igfbp-1 gene expression in vivo and in vitro . Therefore, the knowledge of igfbp-1 system regulation including its gene expression may offer a wide variety of interesting developments that are involved in the future strategy of igfbp - mediated therapy.
Gunshot wounds to the upper cervical spine are rare in south korea due to gun control . Due to the location and nature of these injuries, patients can present with concomitant airway damage, esophageal perforation, vascular injury, or spinal cord injury6). Therefore, gunshot wounds to the upper cervical spine without neurological deficits are rarely observed . To our knowledge, there is no published report that describes surgical removal of a bullet from the anterior arch of the c1 vertebra in south korea . In this report, we describe our experience with the surgical removal of a bullet located in the c1 anterior arch using a transoral approach . A 48-year - old man was admitted to the emergency room because of a self - inflicted single gunshot wound to the mouth . The patient was awake and alert without dyspnea or broken teeth . A review of the patient's past medical history showed that he suffered from depressive disorder for 6 years and had received treatment for this condition . During the physical examination, aerodigestive involvement was not detected, and no neurologic abnormalities were found in the cranial nerves or cerebellar system . The bullet had passed along the right side of the mouth and exit wound was on the dorsal portion of the hard palate . A single bullet lodged in the anterior arch of c1 was found on the simple lateral radiograph and computed tomographic (ct) scans (fig . 1). Fortunately, the dural sac, trachea, esophagus, and vertebral artery were undamaged . The patient was placed on the operating table with the head in extension while under general anesthesia and with orotracheal intubation . A self - retaining oral retractor was placed over the teeth and expanded to keep the mouth open . To obtain an operational view, the soft palate was divided with an incision along the midline extending from the junction with the hard palate to the base of the uvula . An incision was made in the posterior pharyngeal mucosa from the base of the clivus to the upper border of the third cervical vertebra . Pharyngeal mucosa, pharyngeal constrictor musculature, and longus colli and longus capitis musculature were sequentially incised . A lead bullet about 1 cm in length was removed from the c1 anterior arch . The patient had no neurologic sequelae and dynamic (flexion and extension) radiographs and ct scans did not reveal any instability . Various clinical findings can accompany gunshot penetration into the cervical spine . In our patient, he had been injured by gunshot from an air pistol that used low velocity bullets . In general, low velocity bullets cause relatively little soft tissue trauma due to their low residual kinetic energy after penetrating the skin . If a bullet strikes a bone, most of its energy may be spent in fracturing that bone6). The consequences of gunshot entry into the upper cervical spine are frequently devastating, including spinal cord injury, vertebral artery injury, and even death resulting from airway dysfunction4,5,7). Moreover, a projectile can cause tissue trauma without coming into direct contact with the tissue through the concussive effect of the bullet2). The clinical presentation of patients with major vascular injury, multiple penetrating wounds, or other life - threatening injuries typically obscures subtle signs of aerodigestive penetration1). Patients with cervical spine gunshot injuries rarely sustain a fracture alone without neurologic deficits or retain vertebral missile fragments . Some authors have recommended a non - surgical approach for treating gunshot wounds in the cervical spine with the belief that further manipulation increases the chance of additional tissue trauma subsequent infection11). However, missiles retained in the anterior portion of c1-c2 vertebrae can cause both short - term and long - term complications even in stabilized patients who do not show signs of neurological deficits4). These complications include migration of the bullet fragments, central nervous system infections, osteomyelitis, or abscess formation . Migration anteriorly would lead to possible aspiration of the foreign body, and dorsal migration would lead to possible spinal cord compression or inflammation of the epidural space6). Atlanto - occipital subluxation is another possible complication if an extensive inflammatory reactions result in ligamentous laxity12). Romanick et al.10) reported frequent infection in civilians sustaining colonic injury resulting from low velocity, low thoracic, and lumbar spinal gunshot penetration . They recommended early bullet removal and debridement of the spine and missile tract because it was felt that the bullets could become coated with bacteria and serve as a nidus of infection4,10). Moreover, lead poisoning can result from a retained bullet or missile although this is relatively rare3,8,9). Therefore, early surgical removal of the bullet and debridement was performed in our patient without incurring any neurological complications . We report a rare case of gunshot injury to the c1 anterior arch . Early removal of bullets could prevent early or late complications despite the intact neurological status of the patient.
It is performed for functional or cosmetic reasons, which is why the ophthalmologist has to be trained to satisfy the patients needs, as well as manage any possible complications, because even when the surgeon may have plenty of experience, complications may arise . Some of the most frequent complications are lagophthalmos and cicatricial ectropion, a product of the shortening of the anterior lamella . Another complication, infrequent but severe, is retrobulbar hemorrhage, which requires immediate treatment . Besides, the wound may become infected and there may be asymmetry, palpebral ptosis and, among other less frequent complications, diplopia [1, 2]. For this last one, we revised the literature and made a new presentation . A 61-year - old female, type a personality, consulted us for eyelid surgery . After 24 h, the patient was consulted for horizontal diplopia . In her ophthalmic exam, she presented a visual acuity of 10/10 in both eyes and an intraocular pressure of 10 mm hg . A cover test presented et6 in the primary position, et6 in the supraversion, et8 in the infraversion, et14 vd+2 in the levoversion and orthotropia in the dextroversion, linked to paresis of the right vi nerve (fig . Mg / l . We performed brain and orbit mris, which showed no variations . The frequency of diplopia after blepharoplasty is unknown . However, taking the large amount of blepharoplasties performed and the small amount of diplopia cases reported into account, we estimate that the frequency is very low . Even though there are cases of horizontal diplopia, most of the literature describes vertical and torsional diplopia . Within the last one, there are 2 patterns: superior oblique muscle dysfunction, which is associated to upper blepharoplasty, and inferior rectus / oblique rectus dysfunction associated to lower blepharoplasty . The first one may be due to a lesion in the trochlea, caused by brown syndrome or a traumatic lesion of the superior oblique muscle, caused by hematoma or anesthesia toxicity [4, 5, 6, 7, 8, 9]. In the second pattern, a lesion can occur in the inferior rectus muscle or in the inferior oblique muscle . Part of the route of the inferior oblique muscle goes between the internal and medial fat pad and it is necessary to identify it during surgical dissection to avoid causing a lesion . However, the muscle is robust, and some lesions are compensated and subclinical [4, 5, 6, 7, 8, 9]. This is a case of diplopia of unknown origin . Although microvascular mononeuropathy of the vi cranial nerve is already known, we cannot confirm the diagnosis because the patient's only risk factors were dyslipidemia and type a personality . In our experience, it is the second case of diplopia following blepharoplasty in more than 5,000 surgeries . The other case was due to a lesion in the inferior oblique muscle . In this article, we want to highlight the importance of very well knowing the anatomy in order to perform a careful dissection, using as little cauterization as possible . We believe that when we assist the patient with empathy and devotion, we get better results.
They are similar to amphetamine, 3,4-methylenedioxymethamphetamine (ecstasy, mdma) and cathinone (the amphetamine found in khat) structurally and pharmacologically . All drugs in this category share certain common structural similarities, namely a beta - keto substituent to phenethylamine . Methedrone, methylone, and mdvp (the 3ms) seem to be particularly widespread and problematic . The three molecules are likely to be the active agents found in illicit products referred to as bath salts structural analogues of cathinone did not appear on the united states' illicit drug market until 2010, but they have been popular drugs of abuse in europe since 2003 . While there are countless other analogues that need attention, these three agents account for most police seizures, and the drugs themselves seem to be associated with the most toxicity . The term products sold as bliss, blizzard, blue silk, charge+, hurricane charlie, ivory snow, ivory wave, ocean burst, pure ivory, purple wave, red dove, snow leopard, star dust, vanilla sky, white dove, white knight, white rush, and white lightening . Synthetic cathinone products are also marketed as plant food / fertilizer, insect repellant, pond cleaner, and vacuum fresheners . Synthetic cathinones are commonly distributed in powder, crystal, and liquid forms, but they are also available and abusable in the tablet and capsule forms . Distributers have marketed some synthetic cathinone tablets and capsules as ecstasy, a practice that may account for many - reported case of toxicity . According to the u.s . Drug enforcement administration (dea) databases, and reports from state forensic laboratories that analyze seized ecstasy tablets, these tablets contain synthetic cathinones, alone or in combination with other drugs . Generally these tablets and capsules are sold in retail outlets and on the internet in conjunction with the more widely recognized bath salts . Of course, legitimate drugs such as bupropion, diethylpropion and pyrovalerone would not only qualify, as bath salt like substances they are considered as legal medications . Each of the 3 m drugs has been linked to varying degrees of agitation, hyper arousal, confusional states, and various types of psychotic behavior, up to and including a syndrome indistinguishable from excited delirium syndrome . Components share common molecular mechanisms of action with cocaine (see below on excited delirium syndrome). Among the hundreds of cathinones that have been synthesized, or can be synthesized, three molecules in particular appear to account for the content of most products sold as bath salts . These mephedrone was initially synthesized in 1928 but did not become a recreational drug until in 2003 . It had first gained notoriety in europe, especially in the u.k ., because of the remarkably high incidence of hospital admissions, even deaths, associated with its use . The european monitoring centre for drugs and drug addiction (emcdda) indicates that over the first quarter of 2010, mephedrone was detected in some 20 e.u . Mephedrone is synthesized by the bromination of substituted propiophenone and subsequent reaction with the appropriate, amine or via reduction of the hydroxyl of the substituted ephedrine to form mephedrone . Like all cathinones, mephedrone can be administered by almost any route, though it is most often used orally at doses between 100 to 200 mg . Pharmacokinetic studies are lacking, but peak effects are said to be observed roughly two hours after the drug has been taken . If the stomach is full, absorption is delayed and so is the onset of action . Nasal insufflation is said to require smaller doses of drug and produces quicker onset of action, usually in less than 30 minutes . Rectal use has been reported and is said to produce the quickest onset of action . To a greater or lesser degree all 3ms produce the effects generally associated with sympathetic excitation: anxiety, tachycardia, hypertension, sweating, and flushing, all as a consequence of the cathinone s ability to block norepinephrine reuptake . Unlike simple amphetamines, intensified sensory experiences may also occur, and even moderate sexual arousal has been reported, but explanations for these responses remain lacking . At higher doses, perceptual changes have been reported . Hospital admissions are relatively common, but when they occur, they are treated no differently than other hyperadrenergic states, namely with benzodiazepines and supportive care . Individual case reports describing more severe toxicity (confusion, psychosis, chest pain) have been published [9 - 11], but these complications almost always occur in the setting of poly - drug abuse, making it impossible to attribute causation to any one drug . In a case where mephedrone was the only drug detected, a 22-year - old man who took 0.2 g orally, followed by 3.8 g intramuscularly, required hospitalization . He developed all the signs and symptoms of a hyper sympathetic state, and finally delusional psychosis, but the psychosis persisted for only a few hours . Six hours later his vital signs returned to normal and he was discharged . Since mephedrone was the only drug that he had used, this suggests a relative lack of toxicity, at least compared to the other 3ms . Higher blood levels of mephedrone are associated with increased mortality . In the u.k . Over 45 cases of suspected deaths from mephedrone have been reported, most confirmed by toxicology testing; however the reports contain very few details, and most of those who died were poly - drug abusers . One report describes a 36-year - old man arrested after having injured himself severely by smashing windows in a rage of fury . He died despite resuscitation attempts . At autopsy mild cerebral edema was evident, but otherwise no other obvious cause of death was apparent . Toxicologic analysis showed a high concentration of mephedrone in femoral blood (5.1 mg / l) and traces of cocaine, mdma, and oxazepam . Many of the symptoms displayed by this patient (particularly the glass - breaking behavior) resemble symptoms of excited delirium syndrome that, until recently, was a disease more or less confined to cocaine and methamphetamine abusers (see below). In a second autopsied case l respectively; cocaine and its metabolites were also present, and hair testing disclosed prior exposure to methadrone, ketamine, and mdma . It is difficult to know what to make of such reports since a microscopic examination of the heart was not, so far as can be determined, performed . The possible presence of underlying heart disease, even at the molecular level (i.e. A channelopathy), makes it impossible to classify the cause of death with any certainty . However, the paper is not without value as it was the first to report mephedrone concentrations in the bile, lung, and brain (1.29, 0.79, and 0.89 mg / l (kg) respectively . Mephedrone was initially synthesized in 1928 but did not become a recreational drug until in 2003 . It had first gained notoriety in europe, especially in the u.k ., because of the remarkably high incidence of hospital admissions, even deaths, associated with its use . The european monitoring centre for drugs and drug addiction (emcdda) indicates that over the first quarter of 2010, mephedrone was detected in some 20 e.u . Mephedrone is synthesized by the bromination of substituted propiophenone and subsequent reaction with the appropriate, amine or via reduction of the hydroxyl of the substituted ephedrine to form mephedrone . Like all cathinones, mephedrone can be administered by almost any route, though it is most often used orally at doses between 100 to 200 mg . Pharmacokinetic studies are lacking, but peak effects are said to be observed roughly two hours after the drug has been taken . If the stomach is full, absorption is delayed and so is the onset of action . Nasal insufflation is said to require smaller doses of drug and produces quicker onset of action, usually in less than 30 minutes . Rectal use has been reported and is said to produce the quickest onset of action . To a greater or lesser degree all 3ms produce the effects generally associated with sympathetic excitation: anxiety, tachycardia, hypertension, sweating, and flushing, all as a consequence of the cathinone s ability to block norepinephrine reuptake . Unlike simple amphetamines, intensified sensory experiences may also occur, and even moderate sexual arousal has been reported, but explanations for these responses remain lacking . At higher doses, perceptual changes have been reported . Hospital admissions are relatively common, but when they occur, they are treated no differently than other hyperadrenergic states, namely with benzodiazepines and supportive care . Individual case reports describing more severe toxicity (confusion, psychosis, chest pain) have been published [9 - 11], but these complications almost always occur in the setting of poly - drug abuse, making it impossible to attribute causation to any one drug . In a case where mephedrone was the only drug detected, a 22-year - old man who took 0.2 g orally, followed by 3.8 g intramuscularly, required hospitalization . He developed all the signs and symptoms of a hyper sympathetic state, and finally delusional psychosis, but the psychosis persisted for only a few hours . Six hours later his vital signs returned to normal and he was discharged . Since mephedrone was the only drug that he had used, this suggests a relative lack of toxicity, at least compared to the other 3ms . Higher blood levels of mephedrone are associated with increased mortality . In the u.k . Over 45 cases of suspected deaths from mephedrone have been reported, most confirmed by toxicology testing; however the reports contain very few details, and most of those who died were poly - drug abusers . One report describes a 36-year - old man arrested after having injured himself severely by smashing windows in a rage of fury . He died despite resuscitation attempts . At autopsy mild cerebral edema was evident, but otherwise no other obvious cause of death was apparent . Toxicologic analysis showed a high concentration of mephedrone in femoral blood (5.1 mg / l) and traces of cocaine, mdma, and oxazepam . Many of the symptoms displayed by this patient (particularly the glass - breaking behavior) resemble symptoms of excited delirium syndrome that, until recently, was a disease more or less confined to cocaine and methamphetamine abusers (see below). In a second autopsied case l respectively; cocaine and its metabolites were also present, and hair testing disclosed prior exposure to methadrone, ketamine, and mdma . It is difficult to know what to make of such reports since a microscopic examination of the heart was not, so far as can be determined, performed . The possible presence of underlying heart disease, even at the molecular level (i.e. A channelopathy), makes it impossible to classify the cause of death with any certainty . However, the paper is not without value as it was the first to report mephedrone concentrations in the bile, lung, and brain (1.29, 0.79, and 0.89 mg / l (kg) respectively . It first appeared in the netherlands, mixed with mccp (meta - chlorophenylpiperazine) as the main component of a designer drug called explosion . According to un drug monitors, methylenedioxypyrovalerone (mdpv) and methylone are among the most popular synthetic cathinones (united nations office of drugs and crime). Mdma and mccp are both semi - synthetic derivatives methcathinone, like the 3ms . Methylone acts on the plasma membrane catecholamine transporter and has a weak effect on the vesicular monoamine transporter . It is metabolized by n - demethylation, reduction of the keto group, and oxidation of the tolyl moiety . If 4-hydroxy-3-(hmmc) is also present, that would confirm the use of methylone, as hmmc is by far the most abundant of methylone s major metabolites . Suggest metabolism is mainly via cyp2d6 with minor contributions from cyp1a2, cyp2b6, and cyp2c19 . Very little is known about methylone pharmacokinetics but there are unsettling reports that when methylone is co - ingested with mdvp, bizarre behavior, including a number of suicides, deaths, highly violent crimes and delirium have occurred . In 2012, cawrse described the tissue distribution in four fatalities . All four cases had detectable levels of methylone; heart blood concentrations were mostly at or below 1 mg / l (0.118, 0.060, 0.740, and 1.12 analysis of several other tissue samples showed that methylone does not sequester in a particular tissue type after death . Two cases also had mdpv present, but insufficient data was collected to reach any conclusions . In 2014, mcintyre reported the toxicology findings in a 19-year - old woman who had drowned . Concentrations of methylone found in the peripheral blood, central blood, vitreous, liver and gastric contents were measured at 3.4 mg / l 3.4 mg / l, 4.3mg / l, 11 mg / kg, and 1.7 mg, respectively . No other amphetamine - like compounds (including ecstasy) were detected . While this information may prove to be useful to death investigators, it is important not to forget that dna resequencing was not performed, and the young woman might well have died from a previously undiagnosed channelopathy (type l1 is prominently associated with drowning deaths). The same could be said of carbone s report of sudden death in another otherwise healthy 19 year - old . A thorough examination of the heart, indeed the entire autopsy, showed no significant abnormalities . Mg / l an order of magnitude lower compared to earlier reported cases . In this case, attributing sudden death to methylone, without first seeking a channelopathy, may be premature . Pearson described three deaths where the deceased exhibited seizure - like activity and elevated body temperatures (103.9, 105.9 and 107f). One of the three individuals was hospitalized only to die of multisystem failure, metabolic acidosis, rhabdomyolysis, acute renal failure and disseminated intravascular coagulation . The laboratory results for this patient over the 24 hour period of hospitalization were significant for increased lactate, liver transaminases, creatinine, myoglobin, creatine kinase and clotting times, with decreased ph, glucose and calcium . Peripheral blood methylone concentrations in the three fatal cases were 0.84, 3.3 and 0.56 mg / l . In conclusion, it appears that peripheral blood methylone concentrations in excess of 0.5 mg / l may result in lethal toxicity, including hyperthermia and other sympathomimetic - like symptoms . The pathophysiology of methylone - related deaths is also poorly understood, but some in vitro evidence is emerging, the results of which seem to explain the myriad of symptoms observed . Symptoms seem to fall on a scale somewhere between serotonin - syndrome and excited delirium . Further more, the greater the methylone concentration, the greater the agitation produced . Both the psychological and physiological abnormalities appear to be dose related [26, 27]. In vivo animals studies show methylone has a high affinity for 5h(2a) receptors, comparable to that of mdma . If methylone inhibits dopamine uptake in the same fashion as mdma, which it almost certainly does, that action may account for many of the observed psychological symptoms . Mdpv is a derivative of pyrovalerone, which is a psychoactive drug that was used to treat chronic lethargy and fatigue . Mdpv differs from other synthetic cathinones because it contains a pyrrolidine ring, which makes the drug a potent uptake blocker at dopamine and norepinephrine transporters, in much the same fashion as methylone . Although mdpv, mephedone and methylone are now controlled drugs, a group of mdpv derivatives remains legal . The most frequently encountered are referred to as pyrrolidinophenones and alpha - pyrrolidinovalerophenone (alpha - pvp) is the one most frequently encountered . In studies using rat brain synaptosomes, alpha - pvp acts as a potent uptake blocker of dopamine and norepinephrine transporters, comparable in activity to mdpv, it is also a catecholamine transporter blocker, though not as potent as mpvd . It may also explain why mdpv, and all of its analogs, induce typical stimulant effects at lower doses, but bizarre behaviors at higher doses . Among the 3ms a hungarian study published in 2013 described heterogeneous symptoms in five mdpv abusers; delusional behavior was frequent . Some of these patients had psychiatric history but others did not; the majority were chronic intravenous drug abusers . It appears that at least some of the behavioral abnormalities induced by mdpv may require a pre - existing substrate, such as chronic poly - drug abuse . Though nothing can be concluded from a solitary case report, it appears that mdpv may, in some respects, behave more like an amphetamine than the other two cathinones discussed here . A 2013, case report described a 27 year - old male, with no past medical history . He was brought to an emergency room because of increasing agitation and admitted he had been injecting and inhaling bath salts . The salts were found to contain a combination of mephedrone and methylenedioxypyrovalerone (mdpv). On presentation, he was tachycardic, hypotensive and febrile . His initial lab tests showed an elevated white count, and increased levels of creatinine and creatinine phosphokinase . The study disclosed a dilated cardiomyopathy with an ejection fraction (ef) of 15 - 20% and global hypokinesia . At a 20-week follow up, the patient stated that he had stopped abusing bath salts and was asymptomatic . Additional cases have not been report with the 3ms, but reversible cardiomyopathy is commonly seen in methamphetamine abusers . The concentration range for blood methylenedioxypyrovalerone was 10640 ng / ml, with an average value of 109 ng / ml . When peripheral and heart blood values were available, the average heart - to - peripheral blood ratio was 1.48, with a range of 1.3 to 1.7 . The highest mdpv concentration occurred in a suicide by hanging and the highest methylone concentration was in a driver killed in a collision . After reviewing the results, it appeared to the authors that blood concentration does not predict either fatalities or impairment . Exds seems to occur more frequently in mdpv users than with others 3 m drugs . Cocaine, methamphetamine and synthetic cathinones all bind to the dopamine transporter (dat), a membrane spanning protein that pumps dopamine out of the synapse back into presynaptic terminal where it is stored and later released . Mdpv is a very high potency dat reuptake inhibitor, even more powerful than cocaine . Dopaminergic pathways originate in the midbrain and provide input into the cortical and subcortical regions of the brain . Abnormally high dopaminergic transmission is known to cause psychosis and schizophrenia . The finding that stimulant drugs increase dopamine levels by more than tenfold is probably the mechanism by which these cathinone derivatives cause temporary psychosis, though it is not the mechanism that causes full blown excited delirium (exds). In order for excited delirium to occur, this notion is supported by experimental data showing that mephedrone does not, itself, damage striatal nerve endings, but enhances the neurotoxicity of methamphetamine and amphetamine . Given that 3 m users are often poly - drug abusers, neurotoxicity would seem likely . Another substrate could be extreme environmental stressors that also act to prevent dopamine reuptake [39, 40]. When reuptake of dopamine is blocked, dopamine remains trapped in the synapse, leading to a hyperdopaminergic state, which can cause violent behavior, delirium, agitation and sudden death if, and when, the neurocardiac axis is activated . This sequence has been repeatedly demonstrated in the murine models using various cathinone derivatives [41, 42]. This particular mechanism might explain the unexpected deaths of drug users when they are being taken into custody, but the hypothesis has yet to be tested . Epidemiologic studies by many different agencies have detected the increasing use, and proliferating variety of new synthetic psychoactive drugs . Today, the three most commonly encountered are mephedrone, methylone, and mdpv (bath salts). Toxicity, most often manifested as psychosis or even fatal excited delirium, seems to be dose related, and more likely to occur in chronic drug abusers . No unique pathologic lesions have been identified, and the clinical toxicology of these drugs is poorly characterized . The underlying molecular disorder, however, closely resembles chronic cocaine toxicity and, in cases of exds may be well identical.
Fxs patients exhibit neurological symptoms that include learning disabilities, social anxiety, attention deficits, hyperarousal, hypersensitivity, autism, and epilepsy . Notwithstanding the complexity of neurophysiological and behavioral alterations, fxs is caused by the silencing, deletion, or loss - of - function mutation of a single gene, fmr1 . As a result, fmrp (fragile x mental retardation protein), its protein product, is not expressed in the majority of cases or is non - functional in the rare cases with a point mutation [2, 3, 4]. Fmrp is an mrna - binding protein that regulates several aspects of mrna metabolism such as nuclear export, transport to synaptic terminals, activity - dependent ribosome stalling and gene expression [6, 7, 8]. Although much of fmrp activity is thought to be related to regulation of synaptic function [9, 10, 11], little is known about the potential defects in neuronal function caused by the absence of fmrp, in particular how these neurophysiological alterations lead to impairment in neuronal computations and behavior in patients with fxs . Initial studies revealed that dendritic spine number is increased in the cortex of fxs patients [12, 13]. In fact, dendritic abnormalities are the most consistent anatomical correlates of intellectual disability . Studies on animal models of fxs showed that fmrp regulates neuronal branching [15, 16, 17] as well as dendritic spine morphology and density [11, 18]. In addition to defects in synaptic structure and axonal branching, impairments in animal behavior have been observed [11, 16]. However, further studies showed that neuroanatomical and behavioral defects can be genetically uncoupled, suggesting that unknown impairments in neuronal circuit function may underlie behavioral deficits . Fmrp regulates translation of mrnas at synapses, some of which encode proteins involved in synaptic plasticity [19, 20]. Importantly, the absence of fmrp leads to abnormally enhanced group 1 mglur (metabotropic glutamate receptor) signaling, which results in exaggerated long - term depression, with a net loss of ampa and nmda receptors [22, 23]. Additionally, enhanced group 1 mglur signaling contributes to the elongation of dendritic spines in rodent models of fxs [18, 24] and leads to increased intrinsic neuronal excitability through the downregulation of potassium channels controlling resting membrane potential and action potential afterhyperpolarization [25, 26]. Moreover, fmrp directly influences neuronal excitability by regulating expression of potassium channels [27, 28] and by interacting with potassium channels in a translation - independent manner . Nevertheless, the recent failure of fxs clinical trials targeting group 1 mglur signaling has led the field to re - examine the group 1 mglur hypothesis . Loss of fmrp was shown to increase network - level hyperexcitability in the rodent cortex [31, 32], which has been associated with the symptoms observed in fxs patients, such as hypersensitivity, hyperarousal, hyperactivity, anxiety, and epilepsy . Interestingly, absence of fmrp downregulates gabaa receptor subunits in both mice and flies [34, 35]. Furthermore, the enzymes for gaba synthesis and degradation, gaba membrane transporters, a gaba receptor scaffolding protein, and a protein that regulates gabab receptor signaling are downregulated in the absence of fmrp [36, 37]. These observations suggest a tantalizing, yet poorly understood, link between gabaergic signaling, network hyperexcitability, and behavioral deficits in fxs models and patients . In contrast to the group 1 mglur component of fxs, the potential effects of altered synaptic inhibition on neuronal circuit excitability and how these changes might impact sensory computations and animal behavior remain unexplored . In this study, we explore the changes in neuronal circuit function and connectivity underlying fxs by using a combination of behavioral assays, functional brain imaging, optogenetics, and electrophysiology in a fly fxs model . We focused on the drosophila melanogaster olfactory system, which is a well - understood and genetically tractable neuronal circuit . Specifically, we evaluated olfactory computations in the antennal lobe, a circuit constituted by excitatory projection neurons, which receive synaptic input from their cognate olfactory receptor neurons, as well as inhibitory local interneurons involved in mediating lateral inhibition . We find that the absence of dfmrp, the fly homolog of the human fmrp, results in reduced olfactory attraction and aversion . Calcium imaging data show that antennal lobe projection neurons have broader odor tuning in dfmr1 flies, leading to reduced specificity in odor coding and alterations in olfactory representations . Consistent with these results, we observe that lateral inhibition across olfactory glomeruli, as well as the inhibitory connections between local interneurons and projection neurons, are impaired in dfmr1 flies . Finally, downregulation of gaba receptors in projection neurons is sufficient to produce olfactory behavioral defects . We propose that absence of dfmrp leads to defective lateral inhibition across olfactory glomeruli, which, in turn, results in impaired odor coding and olfactory behaviors . Dfmr1 flies were previously shown to have learning deficits in olfactory behavioral assays . The authors suggested that this was not due to a sensory deficit; however, no detailed analysis of olfactory processing was performed . To evaluate whether the olfactory system of drosophila melanogaster is affected by the absence of fmrp, we conducted olfactory attraction and aversion assays (figures 1a and s1a s1c). Ethyl acetate is known to induce attraction in flies, whereas benzaldehyde induces aversion [40, 41]. We presented these odors to starved flies and quantified attraction and aversion by counting the number of flies in odorized and non - odorized sections of the behavioral arena, before and during odor delivery . We found that dfmr1 flies exhibit significantly weaker olfactory attraction and aversion compared to wild - type (wt) flies (figures 1b, 1c, 1h, 1i, 1l, and 1 m). We observed that dfmr1 flies spend less time exploring the quadrant odorized with the attractive odor ethyl acetate (figures 1b, 1h, and 1l). Similarly, dfmr1 flies were not repelled as much as wt flies by the aversive odor benzaldehyde (figures 1c, 1i, and 1 m). Furthermore, impaired olfactory performance in dfmr1 flies can be restored by the genomic construct of dfmrp (figures 1d, 1e, 1h, 1i, 1l, and 1 m). To test whether reduced olfactory performance was due to the absence of dfmrp in the antennal lobe circuit, we knocked down dfmrp expression specifically in excitatory antennal lobe projection neurons . Downregulation of dfmrp in the antennal lobe projection neurons led to a significant impairment of olfactory behaviors (figures 1f, 1 g, and 1j1 m), confirming the role of dfmrp for antennal lobe circuit function and olfactory behaviors . Similar results were obtained by knocking down dfmrp expression in inhibitory antennal lobe local interneurons (figure s1e). Reduced performance of dfmr1 flies in olfactory behaviors suggests that odor coding is compromised in these animals . To evaluate whether olfactory computations are affected by the absence of dfmrp, we measured the odor responses of antennal lobe projection neurons using calcium imaging (figures 2a, 2b, and s2) in wt and dfmr1 flies (figure 2d). We extracted the location of individual glomeruli using independent component analysis, which is effective in identifying even the sister glomeruli across antennal lobes with very similar locations and response profiles (figures 2c and s3). Next, we investigated the glomerular activation patterns of projection neurons and compared the representations of 24 odors in wt and dfmr1 flies . We observed that overall responsiveness of olfactory glomeruli is significantly altered, with more excitatory and fewer inhibitory odor responses in dfmr1 flies (figures s4a s4d). Specifically, wt flies exhibited more inhibitory responses, more silent glomeruli, and more strong excitatory responses, whereas dfmr1 flies presented an increased number of weak excitatory responses (figures s4a s4d), reflecting that not only are dfmr1 projection neurons hyperexcitable, but excitation of strongly responding neurons is also impaired . This indicates a deficit in contrast enhancement of olfactory representations, which might be a consequence of reduced lateral inhibition . To further evaluate olfactory coding, we carried out a pairwise comparison of odor - evoked glomerular activation patterns using two commonly used and complementary measures of similarity, cosine distance and euclidean distance . Cosine distance compares odor responses regardless of amplitude, while euclidean distance takes the strength of odor responses into account . High cosine and euclidean distances indicate increased difference among odor representations and, hence, greater specificity in odor encoding . Our results show significantly lower cosine and euclidean distances between pairs of odors in dfmr1 flies (figures 3a3d). This indicates that loss of dfmrp causes odor - evoked glomerular activation patterns to become less distinct from each other and, therefore, harder to discriminate . Reduced odor specificity of glomerular activation patterns could explain why dfmr1 flies are impaired in both attractive and aversive olfactory behavioral tasks . What underlies the increased similarity among odor representations in dfmr1 flies? To answer this question, we visualized odor selectivity by plotting the responses of each glomerulus normalized to its maximum odor response . We observed that dfmr1 glomeruli have broader response profiles and, thus, reduced odor selectivity, represented by warmer colors (figure 3e). To quantify this, we calculated the lifetime sparseness, a measure of response selectivity, of all glomeruli . We found that dfmr1 glomeruli have significantly lower lifetime sparseness values (figure 3f), suggesting they are less odor selective . Complementary to this, we computed the population sparseness, which is a measure of the number of glomeruli activated by a single odor . A high population sparseness value signifies that few glomeruli were activated by a given odor, whereas a low population sparseness value signifies that many glomeruli were similarly activated . We found that the antennal lobe of dfmr1 flies has significantly lower population sparseness (figure 3 g), which is consistent with our complementary analysis showing reduced response to background (signal - to - noise) ratios (figure s4d) and increased correlations across antennal lobe glomeruli in dfmr1 flies (figure s4e). Our results reveal an impairment in olfactory coding and odor selectivity in dfmr1 flies due to broader tuning and reduced odor selectivity of antennal lobe projection neurons . This reduced odor selectivity can, in principle, arise from less selective glomerular innervation patterns of individual projection neurons in dfmr1 flies . However, we did not observe changes in glomerular morphology or size in any of the genetically identified projection neurons of dfmr1 flies (figure s5). The lack of any obvious morphological alterations in projection neurons, combined with our observations of increased excitatory and reduced inhibitory odor responses, suggests that defective lateral interactions among antennal lobe neurons might be responsible for the reduced specificity of olfactory representations in dfmr1 flies . In the fly antennal lobe, lateral interactions across olfactory glomeruli were shown to mediate the spread of both excitation, through gap junctions, and inhibition, through local interneurons . It has been shown that, when odors are mixed, lateral interactions across antennal lobe glomeruli can alter odor representations, both through lateral excitation and lateral inhibition . To compare the level of lateral interactions in wt and dfmr1 flies, we applied mixtures of odorants, in which the concentration of one of the components is kept constant while the concentration of the other mixture component is gradually increased (figures 4a and s6a). This, in turn, will change the odor - evoked activity patterns, creating new odor representations depending on the degree of lateral interactions among all recruited neurons . We observed that the odor representation of the component with fixed concentration became progressively different with increasing concentrations of the second mixture component . These mixing - related changes in odor representations were more pronounced in wt flies than in dfmr1 flies (figures 4a4c and s6a s6c). Our results showed that, on average, wt flies exhibited significantly more mixture - related suppression, whereas dfmr1 flies exhibited significantly more mixture - related excitation (figures 4d, 4e, s6d, and s6e). This suggests that, while lateral inhibition is impaired in dfmr1 flies, lateral excitatory interactions might be spared (figure s7). In line with this, our results suggest that populations of individual glomeruli in wt flies have a significantly larger variety of both inhibitory and excitatory effects at all mixture concentrations, when compared to dfmr1 flies (figures 4f and s6f). Altogether, these results support the idea that lateral inhibitory interactions are impaired in the antennal lobe of dfmr1 flies, which eventually results in reduced contrast across odor representations and, therefore, poorer performance in olfactory behaviors . Our findings using odor mixtures point to reduced lateral inhibition among olfactory glomeruli in dfmr1 flies . In line with this, several components of the gabaergic transmission machinery are reported to be downregulated in mouse and fruit fly models of fxs [34, 35]. Moreover, gabaergic signaling appears to be disrupted in the brains of autistic patients, a recurrent phenotype in fxs . All this evidence led to the hypothesis that reduced inhibition may be a major mechanism underlying neuronal deficits in fxs . However, direct in vivo physiological evidence that inhibitory connections between neurons are impaired in any in vivo model of fxs is lacking . In the fruit fly antennal lobe, lateral inhibition across olfactory glomeruli is mediated by gabaergic local interneurons that can act on both olfactory receptor neuron terminals and on projection neurons [44, 51, 52]. To directly test the action of local interneurons on the activity of projection neurons, we performed intracellular recordings of projection neurons while optogenetically stimulating gabaergic local interneurons expressing channelrhodopsin-2 (figure 5a) [53, 54]. Optogenetic activation of local interneurons consistently hyperpolarized the membrane potential of wt projection neurons (figures 5b5d). In contrast, dfmr1 projection neurons exhibited significantly smaller or no hyperpolarization in their membrane potential (figures 5b5d). Importantly, we observed that dfmr1 projection neurons exhibit a prominent excitation upon optogenetic local interneuron stimulation (figure 5c), which is mediated by the gap junctions between local interneurons and projection neurons . During these recordings, we kept the antennae dry and the olfactory nerve intact, which ensures that the olfactory receptor neurons are undamaged and sustain a healthy level of background activity . As previously shown, this remaining olfactory receptor neuron background firing results in prominent subthreshold synaptic activity and spontaneous action potential firing in our recorded projection neurons (figures 5b, 5e, and 5 g). The optogenetic activation of local interneurons reduced the firing rate of wt projection neurons significantly more than that of dfmr1 projection neurons (figures 5e5h). In line with the remaining gap - junction - mediated lateral excitation, we observed a slight increase in projection neuron firing rates of dfmr1 flies (figures 5e and 5f). The observed impaired inhibition in the projection neurons of dfmr1 flies could, in principle, be the consequence of a less effective optogenetic activation of local interneurons . To rule out this possibility, we recorded the responses to optogenetic activation in local interneurons expressing channelrhodopsin-2, both in wt and dfmr1 flies (figures 6a and 6b). Our results showed that optogenetic stimulation elicited significantly larger depolarization and higher firing rates in dfmr1 local interneurons, when compared to wt local interneurons (figures 6c6h). Furthermore, optogenetic stimulation of local interneurons consistently inhibit the local interneurons that do not express channelrhodopsin-2 in wt flies (figures 6i6k). By contrast, little or no inhibition was observed in dfmr1 local interneurons not expressing channelrhodopsin-2 (figures 6i6k). These results indicate that the reduced inhibition observed in dfmr1 projection neurons (figure 5) cannot be due to less effective optogenetic activation of dfmr1 local interneurons . In fact, our results suggest that optogenetic stimulation is more effective in activating dfmr1 local interneurons, especially at the later phase of the stimulation, presumably, due to less effective gabaergic inhibition across dfmr1 local interneurons . In summary, these experiments revealed that deficient inhibition of dfmr1 projection neurons (figure 5) is due to less effective gabaergic inhibition from local interneurons onto the whole antennal lobe circuit, at the level of both projection neurons and local interneurons . We observed that dfmr1 flies present less strongly activated glomeruli (figures s4a s4d), which could be caused by reduced lateral excitation . We, therefore, recorded lateral excitatory responses in projection neurons of flies, in which the antennae were removed and, hence, did not present spontaneous activity (figure s7a). Optogenetic activation of local interneurons produced an excitatory response in both wt and dfmr1 projection neurons (figures s7b and s7c). However, lateral excitatory responses decay faster in dfmr1 projection neurons (figures s7b and s7c) and were smaller in amplitude (figure s7d). This observation could, in part, explain the lower incidence of strongly activated glomeruli upon odor stimulation in dfmr1 flies (figures s4a s4d). Our observations, indicating that inhibition is reduced in both the projection neurons (figure 5) and the local interneurons (figure 6) of the antennal lobe, suggest that lack of inhibition is the neurophysiological cause of the behavioral abnormalities observed in the absence of dfmrp (figure 1). We directly tested this idea by knocking down the expression of the gabaergic rdl receptor . Downregulation of rdl receptors selectively in projection neurons (figure 7a) or in local interneurons (figure 7b) resulted in lower olfactory behavioral performance in fruit flies . Taken together with the previously reported decreased expression of gabaa receptors in the absence of fmrp, the electrophysiological and behavioral evidence presented in this study strongly suggests that reduced inhibition of neuronal circuits contributes to the pathophysiology of fxs . Since the discovery of reduced gabaa receptor subunit expression in the absence of fmrp, accumulated evidence has pointed toward alterations in gabaergic transmission as a key component in the neurophysiology of fxs [50, 56]. In fact, intracellular recordings on acute brain slices suggested that reduced inhibitory input from interneurons onto pyramidal neurons could result in an excitation / inhibition imbalance [35, 57]. Whether this is true in vivo and we tested this using the fruit fly antennal lobe circuit and demonstrate that gabaergic connections established by local interneurons, which mediate lateral inhibition [44, 51, 58], are impaired in a drosophila melanogaster model of fxs . Moreover, we show that deficits in gabaergic lateral inhibition leads to increased circuit excitability, which results in reduced stimulus selectivity in projection neurons we postulate that similar deficits in lateral inhibition impair neuronal computations in other sensory modalities . In consonance with this, it has been reported that circuit hyperexcitability leads to behavioral alterations in tactile, auditory, and olfactory tasks in mouse models of fxs [32, 59, 60]. Our results indicate that, in the absence of dfmrp, neuronal computations are impaired in the antennal lobe of drosophila melanogaster . This is in apparent contradiction with a previous study showing long - term memory defects in dfmr1 flies and no sensory deficits . As we report, responses to many odors are still elicited in projection neurons of dfmr1 flies . We suggest that this difference may be due to a more extensive and quantitative analysis of behavior and physiology in our study that revealed defects that may have not been previously detected . Alternatively, the penetrance and severity of phenotypes in fmr1 mutant animals, both mice and flies, can be sensitive to genetic background . It is possible that the previous study did not account for this . At any rate, both null alleles and rnai flies analyzed using behavioral, imaging, and electrophysiological approaches revealed that dfmr1 mutants exhibit reduced odor specificity and, thus, deficient olfactory processing . Lateral inhibition across drosophila olfactory glomeruli has been proposed to be important for increasing contrast among odor representations and, therefore, for discriminating odors [44, 48]. Interestingly, such a mechanism has been suggested to be relevant for other sensory modalities . In this winner - take - all model, glomeruli with most prominent odor responses would strongly activate surrounding interneurons, spreading inhibition to nearby weakly activated glomeruli . The spread of lateral inhibition, in turn, would inhibit the odor responses of weakly activated glomeruli, while strongly activated glomeruli remain as the unique encoder of the particular odor . This model also suggests that the lack of many weakly activated glomeruli, in addition to few strongly responding but very odor - specific glomeruli, enhances the separation of odor response patterns from one another . In line with this model, we observed that lack of lateral inhibition in the antennal lobe of dfmr1 flies, indeed, leads to an increase in the number of weakly activated and less odor - specific glomeruli . By contrast, wt flies present more inhibitory and less weak excitatory responses, sparing strongly responding olfactory glomeruli that are more odor specific . This is probably a consequence of reduced lateral inhibition, which is important for contrast enhancement of odor representations . Additionally, the slight decrease in lateral excitation observed in dfmr1 flies could result in less strongly represented glomeruli . Importantly, defects in olfactory processing have been observed in other animal models of fxs, as well as in human patients, which display hypersensitivity to smells and to other sensory modalities involving lateral inhibitory mechanisms such as tactility and audition . Beyond the olfactory system, several studies have shown that cns neurons are hyperexcitable in the absence of fmrp [31, 32, 57]. Since activation of the group 1 mglur signaling pathway results in increased neuronal excitability [25, 26], circuit hyperexcitability has been attributed to the constitutively enhanced group 1 mglur signaling observed in mouse fxs models . Here, we provide the first direct in vivo evidence showing that defects in lateral gabaergic inhibition significantly contribute to circuit hyperexcitability . This is consistent with downregulation of proteins involved in gabaergic transmission both in fruit flies and in rodents [34, 36]. Thus, reduced inhibition could be a consequence of decreased gaba release from local interneurons, reduced expression of postsynaptic gaba receptors, or both . Further studies of protein expression profiles for the specific neuron types are needed to elucidate this . It is possible that this mechanism might explain phenotypes observed in fxs patients such as hypersensitivity, hyperarousal, hyperactivity, and epilepsy, all of which reflect hyperexcitable brain states . In summary, we demonstrate that lateral inhibition within the antennal lobe is strongly affected in dfmr1 flies due to impaired inhibitory connections from local interneurons onto projection neurons and other local interneurons . The lack of this lateral inhibition on projection neurons is probably the major cause for their increased excitability and reduced odor specificity . We propose that this compromised olfactory coding consequently leads to impaired olfactory behaviors in dfmr1 flies . More generally, we provide the missing in vivo evidence that the lack of dfmrp has a direct impact on sensory processing and animal behavior through a weakening of lateral inhibitory connections, which broadens response tuning of principal neurons . This mechanism might be ubiquitously present in the brain of fxs patients . For instance, reduced gabaergic inhibition could produce hyperexcitable neuronal circuits in fxs patients, which not only explains symptoms such as hypersensitivity, hyperarousal, or epilepsy but also potentially contributes to the misprocessing of information across the brain, which would have severe effects on human behavior . Finally, given the overlap between the phenotypes of fxs and those of other neurological diseases, such as autism, rett syndrome, or dravet syndrome, and their corresponding perturbations in gabaergic transmission [33, 49, 63], it is possible that similar mechanisms involving reduced lateral inhibition are also present in these neurological syndromes, which are yet to be discovered . L.m.f ., b.a.h ., and e.y . Conceived the study, designed the experiments, and wrote the manuscript.
A 77-year - old male patient was referred by his primary ophthalmologist, who suspected an inadvertent globe perforation . The patient was a myope and had been previously diagnosed to have nuclear sclerosis grade iii in both eyes . The patient was scheduled for a left eye cataract surgery for which peribulbar anesthesia was administered, following which the intraocular pressure was found to be hard and on indirect ophthalmoscopy, vitreous hemorrhage was noted . The surgery was canceled and an ultrasound b - scan of the eye confirmed vitreous hemorrhage and a rhegmatogenous retinal detachment; however, the ultrasound showed intact ocular coats and no obvious vitreous incarceration . With a working diagnosis of an occult - globe perforation causing a retinal detachment and vitreous hemorrhage, a small incision cataract surgery was performed to extract the cataractous lens; following which an encircling silicone band was placed; vitrectomy, fluid air exchange, endolaser and silicone oil injection were completed uneventfully . The globe was thoroughly examined, and no obvious scleral dehiscence was found . At the end of the surgery, the intraocular pressure was maintained with no obvious signs of leakage or hypotony . However, on taking off the surgical drapes, both the periorbital regions were swollen; with the right side significantly more affected than the left side [fig . Crepitus could be palpated all over the swollen area, which, on closer examination was found to be extending up to the patient's chest [fig . There was no pain, and the patient's vitals were found to be stable . The patient was closely monitored and at the end of 1-week, the subcutaneous emphysema had resolved completely . The operated eye recovered well, and subsequently underwent silicone oil removal and had an uneventful recovery . External photograph taken immediately after the surgery demonstrating the bilateral subcutaneous emphysema (a), extending up to the level of the chest on both sides (b and c; black arrowheads) cervicofacial surgical emphysema develops where air enters into the fascial planes of the head and neck . These planes consist of loose connective tissue, which harbor potential spaces between layers of muscles and other structures . Once air enters the deep soft tissue under pressure, such as passage through syringes or high - pressure tubings, it follows the path of least resistance through the connective tissue, along the fascial planes, reaching distant spaces . In our case however, as the primary ophthalmologist had suspected, the 24 g needle used to administer the peribulbar anesthesia had caused an occult globe perforation; perhaps a double perforation [fig . 2]. Given the beveled tip of the needle and the oblique angle of entry, we postulate that the scleral entry wound could have possibly been a flap - like shelved entry, which acted as a one - way valve during the fluid gas exchange . Fluid air exchange is usually performed while maintaining an intraocular pressure of 30 mm hg, which would have allowed the air to escape through the sclera and into the subconjunctival space and into the orbit . The needle tract which passed through the skin, the subcutaneous tissue, orbital septum and through the orbital fat would have provided the path of least resistance for the air to escape and enter the subcutaneous plane . The laxity of the subcutaneous tissue, especially in a 77-year - old patient could have further contributed it . On retrospective questioning, the patient denied any episode of sneezing, trauma, coughing or nose blowing in the interim period between the initial injection and the subsequent surgery . (a) line drawing showing the needle tract, which served as the path of least resistance for the escaping air . Points a and b indicate the possible points of scleral perforation and point c indicates the site of the retinal break . (b) line drawing showing the possible route taken by the air to escape, through the scleral opening and conjunctiva (d), into the orbital tissue . And through the orbital septum (e) and muscular plane and into the subcutaneous plane (f). The passage of the air being facilitated by the previously created needle tract (a) ophthalmic procedures that have reported to have caused emphysema include balloon dacryoplasty and orbital decompression surgery . Damasceno et al . Have reported a case of subcutaneous emphysema involving the orbit, mediastinum, and face after pars plana vitrectomy with the fluid gas exchange . However, the patient had an old orbital fracture and the authors hypothesize that in the presence of an old orbital fracture, the high gas pressure during fluid gas exchange allowed air to travel from the eye into the soft tissue, orbit, and chest . Venous - air embolism has also been reported to be one of the rare complications of intra - ocular surgery involving a fluid gas exchange . The incidence of globe perforation has been reported in as many as 0.75% of retrobulbar and peribulbar injections . Surgical emphysema could be a rare, but possible complication while performing vitreoretinal surgery in cases of inadvertent globe perforation while administering peribulbar anesthesia . Serious complications of cervicofacial emphysema include the involvement of retropharyngeal, mediastinal and peritoneal spaces, which may in turn lead to cardiopulmonary distress.
The success rate in restoring a class ii cavity lesion depends upon the type of dental material used for restoration as well as the skill with which an operator performs the procedure . The clinician's main concern when placing direct, posterior, resin - based composite restorations would be to counter polymerization shrinkage stress and its consequent outcomes . Polymerization shrinkage causes stress at the tooth - restoration interface as the elastic modulus of the restorative resin increases during light activation . The detrimental effects of polymerization shrinkage stress include bond failure, cuspal flexure, interfacial gap formation and subsequent microleakage . The resulting marginal discoloration is often misdiagnosed as recurrent caries at the margins leading to unnecessary restoration replacement and further tooth tissue loss . Furthermore, the occurrence of recurrent cervical caries in posterior teeth has been reported to be eight times higher than the recurrent decay at occlusal margin, of class ii composite restorations . The incremental layering technique and use of low - modulus intermediate liner material such as flowable composites however, every dentist desires a posterior composite resin with handling properties similar to dental amalgam . In this context bulk fill composites. These materials are recommended for insertion in a 4-mm bulk due to their high reactivity to light curing . However, the potential for development of internal and marginal discrepancies exists with bulk placement and the proportion of gaps relative to use of conventional 2-mm increments needs to be ascertained . If the bulk fill restorative materials are to provide a true clinical advantage, then they require high depths of cure while simultaneously demonstrating a decrease in internal stress, and subsequently enhanced adaptation to the tooth substrate . The aim of this in vitro study was to investigate the cervical marginal and internal dentinal adaptation among different posterior bulk fill restorative material systems in class ii cavities . The research hypotheses tested were: there would be no significant differences in marginal and internal adaptation at the cervical tooth restoration interface in cavities restored using either the bulk fill or the incremental fill technique.bulk fill composites of different viscosities provide the same marginal adaptation quality before and after thermo - cycling (tmc) in restored class ii cavities . There would be no significant differences in marginal and internal adaptation at the cervical tooth restoration interface in cavities restored using either the bulk fill or the incremental fill technique . Bulk fill composites of different viscosities provide the same marginal adaptation quality before and after thermo - cycling (tmc) in restored class ii cavities . Forty intact, non - carious, unrestored human maxillary first premolars, extracted for therapeutic reasons were collected for the study . The teeth were hand scaled and kept in 0.05% thymol solution at 37c for no longer than 1 month before using . The teeth were examined to ensure that they were free of defects under an operating microscope at 20 magnification . Eighty box - only class ii cavities with parallel walls were prepared, with the distal cervical margin established one mm below the cej . The overall dimensions of the cavities were standardized as follows: 5 mm wide bucco - lingually and 2 mm deep axially . The cavities were cut using coarse diamond points under profuse water cooling, and finished with finishing diamond points (one pair of diamond points per four cavities). The teeth were randomly assigned to the four experimental groups (n = 10). A metal matrix band was adopted and a dental probe was used to place marks on the outer surface of the matrix, allowing height control of the subsequently placed increments . Adhesive procedures were performed with tetric - n bond (ivoclar vivadent) adhesive system following the manufacturer's instructions . Total - etch dentine bonding system was used in all the groups to reduce variability in results . Three commercial bulk fill composite systems were tested and one conventional composite that required 2-mm increments was used as control . I sonic fill (kerr / sybron orange, ca); increment thickness-4 mm gr . Ii sdr (dentsply, konstanz, germany) + ceram x mono; increment- 4 mm + 2 mm gr . Iii tetric n ceram bulk fill (ivoclar vivadent); increment thickness- 4 mm gr . Iv tetric n flo + tetric n ceram (ivoclar vivadent); increment- 1 mm + 2 mm sonic fill composite was inserted by sonic activation using the propriety hand - piece . Each 4-mm increment was light - cured for 40 s while the 2-mm increments were cured for 20s with a led light curing unit (bluephase c8, ivoclar vivadent) with output irradiance of approximately 800 mw / cm held in contact with the coronal edge of the matrix band . After removal of the matrix band, the restorations were light - cured from their buccal and lingual aspects for an additional 20 s on each side . The polishing procedure was conducted under 20 magnification with flexible disks (soflex pop - on, 3 m espe, st . Impressions of the tooth - restoration interface were made using a polyvinyl - siloxane material (virtual light body, ivoclar vivadent ag, fl schaan, liechtenstein) and epoxy resin (diemet - e, erkodent, germany) replicas were obtained . All specimens were submitted to 2,500 thermal cycles by alternating immersion in water at + 5 8c and + 55 8c with a dwell time of 2 min and transfer time of 5 s in each path . Scanning electron microscopy (sem) evaluation (jeol, jsm 6510 sem) was carried out at a 500 magnification . After evaluating the marginal adaptation, the teeth were embedded in a slow self - curing acrylic resin material and sectioned mesio - distally up to the cej . Only one of the two coronal segments was utilized for investigating the internal dentinal adaptation . The sections were polished and etched for 2 min using a 37% phosphoric acid etching gel (total etch, ivoclar vivadent), rinsed and dried . Impressions were taken and epoxy resin replicas were obtained . Drying and impression processes were carried out carefully to limit dentine dehydration . Results for the marginal adaptation (gap - free margin), before and after tmc were expressed as a percentage of the entire margin length in enamel and dentine . The composite / tooth interface was divided into three regions and measurements of marginal gap widths in each region were made at four points at 500 magnification . The largest marginal gap width in each region was recorded in micrometers (m), and the mean gap widths for each of the enamel and dentin margins were calculated . Material statistical evaluation was performed using the minitab version 5.0 software with one - way anova and tukey's post hoc test at a 5% level of significance . The differences between marginal quality, before and after tmc were assessed with a paired t - test . Forty intact, non - carious, unrestored human maxillary first premolars, extracted for therapeutic reasons were collected for the study . The teeth were hand scaled and kept in 0.05% thymol solution at 37c for no longer than 1 month before using . The teeth were examined to ensure that they were free of defects under an operating microscope at 20 magnification . Eighty box - only class ii cavities with parallel walls were prepared, with the distal cervical margin established one mm below the cej . The overall dimensions of the cavities were standardized as follows: 5 mm wide bucco - lingually and 2 mm deep axially . The cavities were cut using coarse diamond points under profuse water cooling, and finished with finishing diamond points (one pair of diamond points per four cavities). The teeth were randomly assigned to the four experimental groups (n = 10). A metal matrix band was adopted and a dental probe was used to place marks on the outer surface of the matrix, allowing height control of the subsequently placed increments . Adhesive procedures were performed with tetric - n bond (ivoclar vivadent) adhesive system following the manufacturer's instructions . Total - etch dentine bonding system was used in all the groups to reduce variability in results . Three commercial bulk fill composite systems were tested and one conventional composite that required 2-mm increments was used as control . I sonic fill (kerr / sybron orange, ca); increment thickness-4 mm gr . Ii sdr (dentsply, konstanz, germany) + ceram x mono; increment- 4 mm + 2 mm gr . Iii tetric n ceram bulk fill (ivoclar vivadent); increment thickness- 4 mm gr . Iv tetric n flo + tetric n ceram (ivoclar vivadent); increment- 1 mm + 2 mm sonic fill composite was inserted by sonic activation using the propriety hand - piece . Each 4-mm increment was light - cured for 40 s while the 2-mm increments were cured for 20s with a led light curing unit (bluephase c8, ivoclar vivadent) with output irradiance of approximately 800 mw / cm held in contact with the coronal edge of the matrix band . After removal of the matrix band, the restorations were light - cured from their buccal and lingual aspects for an additional 20 s on each side . The polishing procedure was conducted under 20 magnification with flexible disks (soflex pop - on, 3 m espe, st . After finishing procedures, impressions of the tooth - restoration interface were made using a polyvinyl - siloxane material (virtual light body, ivoclar vivadent ag, fl schaan, liechtenstein) and epoxy resin (diemet - e, erkodent, germany) replicas were obtained . All specimens were submitted to 2,500 thermal cycles by alternating immersion in water at + 5 8c and + 55 8c with a dwell time of 2 min and transfer time of 5 s in each path . Scanning electron microscopy (sem) evaluation (jeol, jsm 6510 sem) was carried out at a 500 magnification . After evaluating the marginal adaptation, the teeth were embedded in a slow self - curing acrylic resin material and sectioned mesio - distally up to the cej . Only one of the two coronal segments was utilized for investigating the internal dentinal adaptation . The sections were polished and etched for 2 min using a 37% phosphoric acid etching gel (total etch, ivoclar vivadent), rinsed and dried . Impressions were taken and epoxy resin replicas were obtained . Drying and impression processes were carried out carefully to limit dentine dehydration . Results for the marginal adaptation (gap - free margin), before and after tmc were expressed as a percentage of the entire margin length in enamel and dentine . The composite / tooth interface was divided into three regions and measurements of marginal gap widths in each region were made at four points at 500 magnification . The largest marginal gap width in each region was recorded in micrometers (m), and the mean gap widths for each of the enamel and dentin margins were calculated . Material statistical evaluation was performed using the minitab version 5.0 software with one - way anova and tukey's post hoc test at a 5% level of significance . The differences between marginal quality, before and after tmc were assessed with a paired t - test . Table 1 demonstrates the values of marginal and internal adaptation expressed as percentage of gap - free continuous margin (% cm). Analysis of variance (anova) test was performed and showed that there were no significant differences when considering the cervical enamel margin between the groups (p = 0.900 before and p = 0.739 after tmc) [table 3]. For dentine interface the differences in gap - free margin extent were found between groups depending on the bulk fill type (p = 0.000 before and after tmc). The tukey's post hoc test was performed to confirm the results of anova test between each group for different interfaces . With respect to the cervical dentin interface, there was no significant difference between group i, group ii and group iv (control group) (p 0.05). Both before and after tmc; group iii (tetric n bulk fill) showed a very highly significant difference (p 0.001) with group i (sonic fill) and group iv (control). A highly significant difference (p = 0.008) was noted between group ii and group iii before tmc which changed to a very highly significant difference (p = 0.000) after tmc [table 4]. Also for internal adaptation to dentine, group iii exhibited significantly less areas of gap - free transition compared to other restorative systems (p <0.05) [tables 1 and 3]. Mean values and standard deviation for marginal gap values for all the experimental groups are presented in table 2 . Statistically significant difference in gap width at cervical dentin margin was evident only after tmc (p = 0.042) [tables 3 and 4]. Intra - group comparison showed that gap free margin length deteriorated for all the restorative materials after thermo - cycling; the difference being statistically significant (p <0.05) [table 5]. Group i (sonic fill) was the only material where the marginal gap width did not show significant variation before and after tmc (p = 0.115 and p = 0.069 for cervical enamel and dentin interfaces, respectively . Results of marginal and internal adaptation at the cervical tooth - restoration interface expressed as percentages of continuous margins% cm (mean) sd before and after thermo - cycling (tmc) results of marginal gap values at the cervical tooth - restoration interface expressed in micro - meter (m) before and after thermo - cycling (tmc) anova test results for the experimental groups before and after thermo - cycling (tmc) tukey's post hoc test for comparison between the experimental groups paired t test results for comparison within each group before and after thermo - cycling (tmc) representative sem (500) micrograph of margins in cervical enamel . (a) continuous margin, (b) noncontinuous margin representative sem (500) micrograph of noncontinuous margins in cervical dentine . (a) continuous margin, (b) non - continuous margin the recent introduction of bulk - filled restorative materials has reignited the debate of bulk vs. incrementally placed composites as the effect of shrinkage stress may be more pronounced with bulk fill since the entire mass polymerizes at one time rather than in small increments . An ideal bulk fill composite would be one that could be placed into a preparation having a high c - factor design and still exhibit very little polymerization shrinkage stress, while maintaining a high degree of cure throughout . Currently, bulk fill materials are available in different viscosities, which is low, variable or medium . The present study investigated whether bulk fill composites of different viscosities provide the same marginal and internal adaptation quality when used to restore class ii cavities . In all the experimental groups, mesial class ii cavity was limited above cej and the distal proximal box was maintained one mm below the cej, where in the challenge to dentin adaptation could be analyzed . The results of this study demonstrated that all materials under investigation exhibited satisfactory marginal adaptation before tmc, particularly in enamel . This could be attributed to the fact that the enamel margins were not bevelled and it has been reported that cervical marginal adaptation is inferior to that observed in proximal and occlusal enamel margins . However, the results reinforce the fact that phosphoric acid etching remains the most reliable mode of pre - treatment to achieve fatigue resistant enamel bonds . The lower percentage of gap - free margins in dentine gives an indication of an increased potential for gap formation and microleakage . The inferior adaptation to dentine observed in group iii tetric n ceram bulk fill when compared with the other experimental groups could be attributed to the restricted flow of the material in the cavity . The curing depth of 4 mm is achieved mainly due to the patented photo - initiator, ivocerin, which is far more reactive than conventional initiators . The stiffer composites help in restoring good contacts in posterior teeth; however, they may not adequately adapt to internal areas and cavosurface margins at the cervical joint . Sdr (smart dentin replacement) (dentsply, konstanz, germany) was introduced to the market as flowable bulk fill composite which incorporates a new stress decreasing resin technology . However, it requires a conventional posterior composite to be cured on top of the 4-mm thick flowable base . A novel resin composite system, sonic fill system (kerr / kavo), was recently introduced . The bulk placement is facilitated by a specialized hand - piece, which delivers sonic energy at varying intensities . As sonic energy is applied through the hand - piece, the incorporated modifier causes the viscosity to drop (up to 87%), during the composite insertion . When the sonic energy is stopped, the composite returns to a more viscous, non - slumping state that is more suitable for carving and contouring . The low viscosity of sdr and sonic fill system, which facilitates plastic flow during the early phases of polymerization could be responsible for the better adaptation exhibited by these restorative materials . The use of flowable resin liners with a low modulus of elasticity under composites as the first increment has become increasingly accepted over the past few years . They serve as a stress - absorbing layer during polymerization shrinkage of the subsequent increment and act by reducing the effect of the c - factor . However, the benefit of using flowable composites as gingival increment for reducing polymerization contraction stress is still a matter of controversy . Considering bulk fill placement technique, it has been demonstrated that surefil sdr showed better internal adaptation than conventional composites in high c - factor cavities . Another study showed similar levels of microleakage of bulk fill (surefil sdr and x - tra base) and standard (grandioso, voco) composites . This study infers that the critical issue regarding internal and marginal gap formation is still a matter of concern . Though composite may be cured to enhanced depths, the potential for developing any post - insertion sensitivity that is related to gap formation at the pulpal floor, and the resulting hydraulic movement of fluids that will occupy this space upon occlusal loading is still present . Within the limitations of this in vitro study, it can be concluded that: both bulk fill restorative materials and incrementally layered composite resulted in a similar proportion of gap - free, marginal interface in enamel.all the experimental groups except for tetric n ceram bulk fill demonstrated similar dentin adaptability when compared with the control group . Thus, the viscosity of the bulk fill restorative material influenced the proportion of gap - free marginal interface and the internal adaptation in dentine . Both bulk fill restorative materials and incrementally layered composite resulted in a similar proportion of gap - free, marginal interface in enamel . All the experimental groups except for tetric n ceram bulk fill demonstrated similar dentin adaptability when compared with the control group . Thus, the viscosity of the bulk fill restorative material influenced the proportion of gap - free marginal interface and the internal adaptation in dentine.
Leishmaniasis refers to a variety of diseases caused by more than 20 species of intracellular protozoan parasites belonging to the genus leishmania . The clinical spectrum of the disease ranges from simple self - limiting cutaneous ulcers to severe disfiguring mucocutaneous, and even to a fatal visceral disease known as kala azar . About 98 tropical and subtropical countries are known to be endemic for this disease, with 350 million people at risk and overall prevalence of 12 million worldwide (1). Cutaneous leishmaniasis (cl) is commonly caused by l. major and l. tropica (2). Visceral leishmaniasis (vl), the most life threatening form, is caused by l. infantum and in very rare occasions by l. tropica (2, 3). In vl fever and hepato - splenomegaly are the main clinical signs in which leishmania parasite is dispersed to the internal viscera like spleen, liver and bone marrow (4). Based on the leishmaniasis clinical symptoms, it is evident that the host immunity factors, leishmania species, and in some cases the leishmania strain, determines the measure of pathogenecity (5). . Has about 8000 genes among only 78 genes are restricted to individual species (6). In spite of a few species parasite genes implicated in pathogenesis and clinical presentation, the parasite gene expression rates differ greatly among species (6). In leishmaniasis, parasites are challenged by the host immune conditions throughout their life cycle such as temperature increase of visceral tissues (liver, spleen or bone marrow). Such challenges causes leishmania experience biochemical changes in which post transcriptional modification are activated and may eventuate into the emergence of the leishmaniasis pathogencity (714). Analysis of proteome is most commonly performed by a combination of 2-de and mass spectrometry (ms). 2-de method could separate proteins in first and second dimensions according to their isoelectric and molecular weight points . With the help of 2-de and the ms, a variable mixture of proteins is separated, visualized and then identified (1516). In this preliminary study, we compared the proteome mapping, in three iranian isolates of leishmania species including l. tropica, l. major and l. infantum, with immobilized ph gradient stripes with linear ph 47 . Moreover, liquid chromatography (lc) - mass spectrometry was used for identification of a number of differentially expressed proteins among the three species . Ef653267, l. major (jn860745) and l. infantum (jx289853) compared and were analyzed . Promastigote forms recovered from the iranian leishmania parasite bank located in leishmaniasis lab, school of public heath, tehran university of medical sciences (tums). The identity of these strains was already obtained by other molecular dna based methods (2, 17). Cell culture promastigotes recovered from liquid nitrogen (196 c), were mass cultured in rpmi1640 medium (gibco, life technologies gmbh, frankfurt, germany) supplemented with 15% heat inactivated fetal bovine serum (gibco, germany) and 100u / ml penicillin and 100ug / ml streptomycin (gibco, germany) and incubated at 24c . Parasites were harvested washed in sterile phosphate buffered saline (pbs, ph: 7.27.4) and were used for protein extraction . Proteomics analysis was performed on l. tropica, l. major and l. infantum, three species at the time of study . Promastigotes were harvested by centrifugation at 3000rpm, 4 c and 20 minutes and washed three times in sterile pbs (ph: 7.27.4) in the same condition for 10 minutes . The cells were resuspended in 5 mm tris hcl, ph 7.8, containing 1 mm phenylmethylsulfonyl fluoride (pmsf (merck, germany)). Proteins were precipitated by 10% (w / v) trichloroacetic acid (merck, germany) in acetone (merck) with 0.07% (w / v) dithiothreitol (dtt) (merck) for 1 hour at 20c . The samples were then centrifuged at 17500 g (hettich, germany) for 15 minutes at 4 c and the pellets were washed with ice - cold acetone containing 0.07% dtt, incubated at 20c for 1 h and centrifuged at 4 c . The samples were then solubilized in lysis buffer (9.5 m urea (merck), 2% (w / v) chaps (merck), 0.8% (w / v) ampholyte (bio - rad, usa) ph 310, 1% (w / v) dtt) (18, 19). The concentration of protein was measured by the bradford assay with bovine serum albumin bsa (merck) as the standard (20). For analytical and preparative gels, 120 g and ief was carried out on the 18 cm immobilized ph gradient (ipg) strips (ph 47) (bio - rad, usa). Ipg strips were rehydrated overnight by loading the samples diluted with rehydration buffer containing 8 m urea, 4% chaps, 2% ampholyte, 50 mm dtt, and traces of bromophenol blue (merck). Isoelectric focusing was conducted at 20 c with mutiphor ii and a drystrip kit (ge healthcare, germany). The running condition was as follows: 300 v for 90 minute, followed by 500 v for 90 min, 1000 v for 3 h and finally 3500 v for 16 h. the focused strips were equilibrated twice in equilibration solution . The first equilibration was performed in a solution containing 6 m urea, 20% (w / v) glycerol, 2% (w / v) sds (merck), 1% (w / v) dtt, and 50 mm tris - hcl (merck) buffer, ph 8.8 . The second equilibration was performed in a solution with 2.5% (w / v) iodoacetamide (merck). Separation in the second dimension was performed by sds - page in a vertical slab of acrylamide (merck) (12% total monomer, with 2.6% cross - linker) using a protean ii multi cell (biorad). The protein spots in analytical and preparative gels were visualized by silver nitrate (merck, germany) and coomassie brilliant blue cbb/ g-250 (sigma, germany) respectively (19 - 21 - 22). Gs-800 densitometer (bio - rad) was used for scanning of silver stain gels . Gels were analyzed using the melanie 6 software (genebio, geneva, switzerland). The molecular masses of protein on gels were determined by co electrophoresis of standard protein markers (ge heathcare) and pi of the proteins were determined by migration of the protein spots on 18 cm ipg (ph 47, linear) strips . 2-de per sample (each species) was run for three biologically independent replicates, percent volume of each spot was estimated and analyzed by one - way analysis of variance (anova) sas software, and means were compared by the lsd test at p 0.01 . Spots were only considered to be significantly different in abundance at least between two leishmania species when / at p 0.01 . The protein spots of interest were excised from coomassie brilliant blue (cbb) stained gels and analyzed using an amazon ion trab ms / ms (bruker daltonics) mass spectrometer . Briefly, peptides were solubilized in 0.5% formic acid and fractionated on a nano flow uhplc system (thermo rslcnano) before online analysis by electrospray ionisation (esi) mass spectrometry on an amazon ion trap ms / ms (bruker daltonics). Peptide separation was performed on a pepmap c18 reversed phase column (lc packings), using a 5 85% v / v acetonitrile gradient (in 0.5% v / v formic acid) run over 45 min . At a flow rate of 0.2 mass spectrometric (ms) analysis was performed using a continuous duty cycle of survey ms scan followed by up to ten ms / ms analyses of the most abundant peptides, choosing the most intense multiply charged ions with dynamic exclusion for 120s . Ms data was processed using data analysis software (bruker) and the automated matrix science mascot daemon server (v2.1.06) (23). Protein identifications were assigned using the mascot search engine to interrogate in house databases of protein sequences for l. major . Ef653267, l. major (jn860745) and l. infantum (jx289853) compared and were analyzed . Promastigote forms recovered from the iranian leishmania parasite bank located in leishmaniasis lab, school of public heath, tehran university of medical sciences (tums). The identity of these strains was already obtained by other molecular dna based methods (2, 17). Cell culture promastigotes recovered from liquid nitrogen (196 c), were mass cultured in rpmi1640 medium (gibco, life technologies gmbh, frankfurt, germany) supplemented with 15% heat inactivated fetal bovine serum (gibco, germany) and 100u / ml penicillin and 100ug / ml streptomycin (gibco, germany) and incubated at 24c . Parasites were harvested washed in sterile phosphate buffered saline (pbs, ph: 7.27.4) and were used for protein extraction . Proteomics analysis was performed on l. tropica, l. major and l. infantum, three species at the time of study . Promastigotes were harvested by centrifugation at 3000rpm, 4 c and 20 minutes and washed three times in sterile pbs (ph: 7.27.4) in the same condition for 10 minutes . The cells were resuspended in 5 mm tris hcl, ph 7.8, containing 1 mm phenylmethylsulfonyl fluoride (pmsf (merck, germany)). Proteins were precipitated by 10% (w / v) trichloroacetic acid (merck, germany) in acetone (merck) with 0.07% (w / v) dithiothreitol (dtt) (merck) for 1 hour at 20c . The samples were then centrifuged at 17500 g (hettich, germany) for 15 minutes at 4 c and the pellets were washed with ice - cold acetone containing 0.07% dtt, incubated at 20c for 1 h and centrifuged at 4 c . The samples were then solubilized in lysis buffer (9.5 m urea (merck), 2% (w / v) chaps (merck), 0.8% (w / v) ampholyte (bio - rad, usa) ph 310, 1% (w / v) dtt) (18, 19). The concentration of protein was measured by the bradford assay with bovine serum albumin bsa (merck) as the standard (20). For analytical and preparative gels, 120 g and 1.2 mg of extracted promastigotes proteins were loaded respectively . Ief was carried out on the 18 cm immobilized ph gradient (ipg) strips (ph 47) (bio - rad, usa). Ipg strips were rehydrated overnight by loading the samples diluted with rehydration buffer containing 8 m urea, 4% chaps, 2% ampholyte, 50 mm dtt, and traces of bromophenol blue (merck). Isoelectric focusing was conducted at 20 c with mutiphor ii and a drystrip kit (ge healthcare, germany). The running condition was as follows: 300 v for 90 minute, followed by 500 v for 90 min, 1000 v for 3 h and finally 3500 v for 16 h. the focused strips were equilibrated twice in equilibration solution . The first equilibration was performed in a solution containing 6 m urea, 20% (w / v) glycerol, 2% (w / v) sds (merck), 1% (w / v) dtt, and 50 mm tris - hcl (merck) buffer, ph 8.8 . The second equilibration was performed in a solution with 2.5% (w / v) iodoacetamide (merck). Separation in the second dimension was performed by sds - page in a vertical slab of acrylamide (merck) (12% total monomer, with 2.6% cross - linker) using a protean ii multi cell (biorad). The protein spots in analytical and preparative gels were visualized by silver nitrate (merck, germany) and coomassie brilliant blue cbb/ g-250 (sigma, germany) respectively (19 - 21 - 22). Gs-800 densitometer (bio - rad) was used for scanning of silver stain gels . Gels were analyzed using the melanie 6 software (genebio, geneva, switzerland). The molecular masses of protein on gels were determined by co electrophoresis of standard protein markers (ge heathcare) and pi of the proteins were determined by migration of the protein spots on 18 cm ipg (ph 47, linear) strips . 2-de per sample (each species) was run for three biologically independent replicates, percent volume of each spot was estimated and analyzed by one - way analysis of variance (anova) sas software, and means were compared by the lsd test at p 0.01 . Spots were only considered to be significantly different in abundance at least between two leishmania species when / at p 0.01 . The protein spots of interest were excised from coomassie brilliant blue (cbb) stained gels and analyzed using an amazon ion trab ms / ms (bruker daltonics) mass spectrometer . Briefly, peptides were solubilized in 0.5% formic acid and fractionated on a nano flow uhplc system (thermo rslcnano) before online analysis by electrospray ionisation (esi) mass spectrometry on an amazon ion trap ms / ms (bruker daltonics). Peptide separation was performed on a pepmap c18 reversed phase column (lc packings), using a 5 85% v / v acetonitrile gradient (in 0.5% v / v formic acid) run over 45 min . At a flow rate of 0.2 mass spectrometric (ms) analysis was performed using a continuous duty cycle of survey ms scan followed by up to ten ms / ms analyses of the most abundant peptides, choosing the most intense multiply charged ions with dynamic exclusion for 120s . Ms data was processed using data analysis software (bruker) and the automated matrix science mascot daemon server (v2.1.06) (23). Protein identifications were assigned using the mascot search engine to interrogate in house databases of protein sequences for l. major . Protein extracts from the three leishmania species including l. tropica, l. major and l. infantum were separated by 2-de gel electrophoresis . Multiple gels from the three independent replications were run to ensure reproducibility of the protein homogenates on the 2-de gels . Show a representative example of the proteins separated / detected on a 2-de gel in l. tropica, l. major and l. infantum, where a total weight of 120 g of proteins had been applied . The gel images were analyzed by melanie software, and the percent volume of the spots was estimated and compared across the gels . We succeeded in detecting 600 100 spots on the 2-de gels, from which 638, 590, and 546 spots were statistically analyzed across the replicates in l. tropica, l. major, and l. infantum respectively . A number of 478 spots could be paired in all of the three species (supplementary table 1). The numbering corresponds to the 2-de gel in figure 1./ accession number in swiss - prot./ experimental pi and molecular weight./ theoretical pi and molecular weight./ mascot score./ nd: not detected (spot) however 34, 33, and 4 protein spots of l. tropica, l. major, and l. infantum were identified as leishmania species - specific spots (fig . 2). Altogether, 265 protein spots exhibited reproducible quantitative (p 0.01) changes across the three samples in leishmania species (supplementary table 2). Among them, 35, 22 and 11 protein spots were different between the l. tropica and l. major (presented by a), l. tropica and l. infantum (presented by b), l. major and l. infantum (presented by c) respectively . Seven protein spots were different between all of the three species (presented by abc). Within differentially expressed proteins, which were detected on the analytical gels, we could reliably detect and excite a total number of 28 protein spots on cbb - stained preparative gels . Due to the lack of the protein amount, the remaining proteins could not be detected . The excised protein spots were then analyzed by lc / ms leading to the identification of 24 proteins (table 1). These proteins were classified in multiple categories according to their species, functions, and biological processes: cell motility and cytoskeleton, cell signaling and vesicular trafficking, intracellular survival / nucleotid metabolism, protein synthesis, oxidative stress defense, microtubule motor movement proteolysis, lipid metabolism, amino - acid biosynthesis, protein ubiquitination / proteolysis, transport, stress related proteins / protein folding and hypothetical proteins (unknown) (table 1). It is worth noting that some of these proteins are hypothetical and their functions in leishmania still remain to be elucidated . The clustering of protein expression pattern of differentially expressed proteins in l. tropica, l. major and l. infantum is presented in fig . All quantitative information is showed using a color scale in which the color ranges from green for the highest down - regulation to red for the highest up - regulation . A comparison among the 3 species revealed that the changes in expression pattern were more pronounced in l. infantum compared with l. tropica and l. major . In addition, the number of up regulated proteins was higher than that of down - regulated proteins . The functional annotation of the 3 species identified proteins in l. tropica, l. major and l. infantum classified by biological function and processes described in table 1 . The biological function pie charts of the cutaneous species (l. tropica and l. major) are highly similar whereas, the pie chart for the biological function of the visceral species (l. infantum) is different from those of cutaneous species (fig . Most of the proteins functionalities are observed in the following categories: intracellular survival / nucleotid metabolism and cell motility/ cytoskeleton (2022%). Moreover, the least functionalities are observed among cell signaling/ vesicular trafficking and lipid metabolism (5%). Moreover, in the l. infantum species, most functionality are observed among protein synthesis and cell motility/ cytoskeleton (15%) and least of them are observed among amino - acid biosynthesis and oxidative stress defense (7%). Protein extracts from the three leishmania species including l. tropica, l. major and l. infantum were separated by 2-de gel electrophoresis . Multiple gels from the three independent replications were run to ensure reproducibility of the protein homogenates on the 2-de gels . Show a representative example of the proteins separated / detected on a 2-de gel in l. tropica, l. major and l. infantum, where a total weight of 120 g of proteins had been applied . The gel images were analyzed by melanie software, and the percent volume of the spots was estimated and compared across the gels . We succeeded in detecting 600 100 spots on the 2-de gels, from which 638, 590, and 546 spots were statistically analyzed across the replicates in l. tropica, l. major, and l. infantum respectively . A number of 478 spots could be paired in all of the three species (supplementary table 1). The numbering corresponds to the 2-de gel in figure 1./ accession number in swiss - prot./ experimental pi and molecular weight./ theoretical pi and molecular weight./ mascot score./ nd: not detected (spot) however 34, 33, and 4 protein spots of l. tropica, l. major, and l. infantum were identified as leishmania species - specific spots (fig . 2). Altogether, 265 protein spots exhibited reproducible quantitative (p 0.01) changes across the three samples in leishmania species (supplementary table 2). Among them, 35, 22 and 11 protein spots were different between the l. tropica and l. major (presented by a), l. tropica and l. infantum (presented by b), l. major and l. infantum (presented by c) respectively . Seven protein spots were different between all of the three species (presented by abc). Within differentially expressed proteins, which were detected on the analytical gels, we could reliably detect and excite a total number of 28 protein spots on cbb - stained preparative gels . Due to the lack of the protein amount, the remaining proteins could not be detected . The excised protein spots were then analyzed by lc / ms leading to the identification of 24 proteins (table 1). These proteins were classified in multiple categories according to their species, functions, and biological processes: cell motility and cytoskeleton, cell signaling and vesicular trafficking, intracellular survival / nucleotid metabolism, protein synthesis, oxidative stress defense, microtubule motor movement proteolysis, lipid metabolism, amino - acid biosynthesis, protein ubiquitination / proteolysis, transport, stress related proteins / protein folding and hypothetical proteins (unknown) (table 1). It is worth noting that some of these proteins are hypothetical and their functions in leishmania still remain to be elucidated . The clustering of protein expression pattern of differentially expressed proteins in l. tropica, l. major and l. infantum is presented in fig . All quantitative information is showed using a color scale in which the color ranges from green for the highest down - regulation to red for the highest up - regulation . Black color indicates no changes in expression pattern of the 3 leishmania species . A comparison among the 3 species revealed that the changes in expression pattern were more pronounced in l. infantum compared with l. tropica and l. major . In addition, the number of up regulated proteins was higher than that of down - regulated proteins . The functional annotation of the 3 species identified proteins in l. tropica, l. major and l. infantum classified by biological function and processes described in table 1 . The biological function pie charts of the cutaneous species (l. tropica and l. major) are highly similar whereas, the pie chart for the biological function of the visceral species (l. infantum) is different from those of cutaneous species (fig . Most of the proteins functionalities are observed in the following categories: intracellular survival / nucleotid metabolism and cell motility/ cytoskeleton (2022%). Moreover, the least functionalities are observed among cell signaling/ vesicular trafficking and lipid metabolism (5%). Moreover, in the l. infantum species, most functionality are observed among protein synthesis and cell motility/ cytoskeleton (15%) and least of them are observed among amino - acid biosynthesis and oxidative stress defense (7%). The aim of this preliminary study was to apply 2-de, which is a valuable method in the proteomics arena to analyze the protein profile patterns of the three iranian cutaneous and visceral leishmania species including l. tropica, l. major and l. infantum to search for species - specific leishmania proteins . In recent years by completion of genome sequencing leishmania parasites coupled with protein separation techniques analysis has given us an insight in better understanding the mechanisms of pathogenesis of leishmania species . Proteomics approaches at the protein expression level provide additional information for further analysis with a biological function . Moreover, comparative proteomics has been successful in determining the virulence biomarkers (24). Overall, the proteome 2-de maps of l. tropica, l. major and l. infantum were strikingly similar in terms of protein distribution and positioning . By using 18-cm ipg strips at the pi47 range in 2-de comparative analysis, we successfully identified more than 700 protein spots for all the three species . These numbers represent about 9% of total proteins in leishmania genome projects (25). The analysis and discussion about the detected proteins in this study could be categorized into three parts . First, we discuss comprehensively about the biological function of proteins whose expression abundances were common among each of the three species . In the second and third part, we represent the biological function of the proteins which are different and absent / present in the three different species . As it is described in table 1 and venn diagram (fig . 2), in the first part we have studied a total number of 478 proteins among which we will discuss the common ones . Among the common proteins of three leishmania species, which were surveyed, we would like to mention significant proteins such as beta tubulin and adf / coflin . Adf / cofilin is existent in all eukaryotic organisms and has been involved in cell motility and cytokinesis . Leishmania parasites express only one isoform of adf / cofilin, which is essential for flagellar assembly and motility (26). Beta tubulin is known as one of the members of distinct microtubule networks in leishmania and is implicated in locomotion, cell shape and division (27). Within other common proteins we could name tryparedoxin, calpain - like cysteine peptidase, and calmodulin putative from the groups of oxidative stress defense, intercellular survival / proteolysis, cell signalling, and vesicular trafficking, respectively . Tryparedoxins are special thiol disulfide oxidoreductases related to thioredoxins, which play a crucial role in hydroperoxide detoxification cascades of kinetoplastida (28). It participates in calcium signaling pathways that regulate multiple critical processes such as growth and proliferation (29, 30). In addition, it is an actin / microtubule - binding protein which interacts with the cytoskeleton (3133). Another common protein could be calcium channel protein that is a member of the transporter group . A huge number of proteins exist under the group of hypothetical proteins whose biological mechanisms are still not well discovered . The second group of proteins, which were studied, was the ones, which were different in all three leishmania species . The differences in proteins mainly occur between l. tropica and l. major and the least differences occur between l. major and l. infantum (fig . It is an interesting point that the proteins, which are different between l. tropica and l. infantum are less than those of l. major and l. tropica . L. tropica is the main cause of dry cutaneous leishmaniasis lesions; whereas, l. major is the main cause of wet cl lesion and l. infantum causes visceral leishmaniasis . Recently there have been reports of viscerotropic forms caused by l. tropica in either humans or dogs infected to visceral leishmaniasis in iran and different parts of the world (34, 2). Among significant proteins which are different in l. tropica and l. major we could mention calcium channel proteins, tryparedoxin and elongation factor from the groups of oxidative stress defense and protein synthesis respectively whose mechanism have been described previously . Recently, it was demonstrated that leishmania ef-1alpha acts as a virulence factor (35). This protein could diffuse into the cytosol of infected macrophages, where it is able to activate tyrosine phosphatase-1 leading to macrophage deactivation (31). Among the proteins different in l. tropica and l. infantum we could mention proteins such as calmadolin and trypardoxin in which the former role is to transfer the material into the cells and the latter role is defending the host cell against oxidative factors and preventing its death . Nevertheless, some of these proteins have a specific domain and have a different mechanism such as hypothetical protein, conserved contains nucleoside 2-deoxyribosyltransferase domain, that have a role in nucleotide metabolism . Among the most significant proteins between l. major and l. infantum another group of proteins, found in this study, was too rare and introduced as absent / present . However, further studies are needed to define precise biological function for the mentioned proteins in the process of pathogenesis . Moreover, the examination of these species must be repeated with other strains in order to sanction the results . In addition, such differences must be evaluated and approved by other methods such as real time pcr and western blotting with monoclonal antibodies . The analysis of proteome mapping of 3 leishmania species including, l. tropica, l. major and l. infantum by 2-de and mass spectrometry demonstrated that the vast majority of leishmania proteins are commonly expressed among 3 species . Therefore, differentiation, virulence and pathogenesis may be related not only to the immunity situation of the host but also to the differentiation expression of a number of proteins like stress related proteins / protein folding and protein ubiquitination / proteolysis . It must be pointed out that further studies must be undertaken using western blotting or real time pcr in order to support the results of the current study.
The online version of this article (doi:10.1007/s00249 - 009 - 0412 - 6) contains supplementary material, which is available to authorized users . Analytical ultracentrifugation is an analytical absolute technique applying centrifugal force to fractionate the sample and optical detection systems to detect the concentration distribution of the sample inside the ultracentrifuge cell . These experiments yield a number of important physico - chemical quantities of the sample like size, shape, density, molar mass, sedimentation and diffusion coefficient, interaction constants and stoichiometry etc . And many of them in form of distributions . However, often the available information depends on the applied optical detection system . Optical systems that have been constructed are uv / vis absorption, rayleigh interference, schlieren and the lavrenko optics (lavrenko et al . Development of new optical detection systems can expand the possible use of auc for different types of samples . Size distributions can be determined with turbidity optics (mchtle 1992; mller 1989; scholtan and lange 1972). Fluorescence optics has expanded the use for extremely diluted labeled samples even in presence of other solutes at a much higher concentration (macgregor et al . The only commercially available machine at the moment is the beckman xl - i which is equipped with a uv / vis absorption optics and rayleigh interference optics (giebeler 1992). Recently, the uv / vis absorption detection was significantly improved by the development of a multiwavelength uv / vis absorption detector (mwl - auc). By essentially replacing the monochromator with a spectrograph, the mwl - auc delivers an entire uv / vis spectrum for each radial point instead of a single wavelength reading (bhattacharyya et al . This technology has a number of advantages over the commercial absorption optics detecting at a single wavelength with time - consuming wavelength scanning, thus excluding the study of all fast processes, obtaining full uv / vis spectra at each point in the ultracentrifuge cell rather than a radial concentration profile at a single wavelength can give much more structural information about the sample, allows for averaging and can even decrease the experimental time when modern fast ccd based spectrometers are used (bhattacharyya et al . Successful optical, mechanical, radial scan, linearity and noise tests of the mwl detector have been published recently (strauss et al . 2008). Combined with the fractionating power of the auc, application of the mwl detector with its additional structural and/or compositional information on light absorbing samples can yield distributions of the individual components in complex mixtures with respect to composition and size / density related to different chromophores . This can start with relatively straightforward issues like sample homogeneity and purity but can then get increasingly complex in case of composite and/or interacting samples . Especially for such complex samples mwl - auc has a huge potential, as spectral discrimination can synergistically enhance the hydrodynamic resolution (balbo et al . This is also an important issue for any colored industrial product composed of at least two components which at least slightly differ in their uv / vis spectra . In this work, we will show the capabilities of mwl - auc for the analysis of an industrial composite sample of -carotene and gelatin . This system was investigated before with x - ray scattering, uv / vis absorption spectroscopy, foqels (fiber - optic quasi - elastic light scattering), microelectrophoresis and on basis of these results, a core - shell structure was presented (auweter et al . The core structure with 120 nm diameter consists of partially crystallized, partially amorphous -carotene as active ingredient . This hybrid structure self - assembles in a carefully tuned co - precipitation of gelatin (from an aqueous solution) and the active ingredient (from a lipophilic solvent). Such particulate formulations can transport an active ingredient that is not water soluble across an aqueous phase with high bioavailability, in this case provitamin a. these particles are not persistent, but disassemble and get digested quickly in biological media . -carotenes can precipitate as h - aggregate or j - aggregate; the two morphologies do not interconvert and are regarded to be kinetically stable over years . The h - aggregate is observed in precipitation of dilute solutions (0.3 weight%) whereas the j - aggregate is observed at higher concentration (1.0 weight%). (1999) calculated a 40 nm hypsochromic shift observed for an h - aggregate and a bathochromic shift in j - aggregates . This results in a significant color change from yellow to red of the product depending on the precipitation conditions and hybrid particle size (figs . 1, 2). This color change is the basis for the industrial application of the -carotenes as pigments for food applications.fig . 1dashed line uv / vis spectrum of the core - shell -carotene / gelatin sample with 0.05 g / l concentration; solid line uv / vis spectrum of gelatin at 1 g / lfig . 1999), right side color change of -carotene / gelatin microparticles due to particle size and structure dashed line uv / vis spectrum of the core - shell -carotene / gelatin sample with 0.05 g / l concentration; solid line uv / vis spectrum of gelatin at 1 g / l left side assumed structure of the -carotene microparticle system (auweter et al . 1999), right side color change of -carotene / gelatin microparticles due to particle size and structure not only the purity of the sample concerning the color characteristics (brilliance of color due to steep absorbance profiles) or sample homogeneity (different species or unbound gelatin) is of interest for the industrial application but furthermore any possible transitions between different structures . This is a problem which can be advantageously solved in a single mwl - auc experiment, which we will describe in this work . The -carotene product was obtained in powder form as a laboratory sample from basf se, ludwigshafen . An aqueous dispersion in water the uv / vis spectrum of the dispersion and of the free gelatin is shown in fig . 1 . In contrast to conventional sedimentation velocity experiments, where the sample between boundary and bottom of the cell is only diluted by radial dilution, the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . The reservoir is filled with a small amount of concentrated sample . Column and sample sectors are filled with d2o with a density which is higher than that of the sample solution and lower than the density of the dispersed solute . We have prepared a 20 g / l solution and deposited 15 l of the solution into the cell reservoir . After preliminary experiments, this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with od s <1.4 in the experiment without too much dilution which causes noisy data . After cell assembly, the auc was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the d2o column . Forty scans were taken with a time interval of 90 s and a radial step size of 50 m to observe the full sedimentation of the sample . The selected wavelength range was 250750 nm . In the prototype setup, we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 od (see fig . 3i) after the experiment, while cleaning the cell, we saw some precipitate in the cell reservoir . Thus, not all particles were transferred to the sample column, but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.fig . 3three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . I scan 1 (1.5 min); ii scan 10 (15 min); iii scan 18 (27 min); iv scan 40 (60 min) three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . I scan 1 (1.5 min); ii scan 10 (15 min); iii scan 18 (27 min); iv scan 40 (60 min) each of the 40 scans produces a 3-dimensional graph . We have radial position as x - dimension, wavelength as y - dimension and absorbance as z - dimension . In the present contribution, we will perform a semi - quantitative evaluation based on simple model - free transformations of the data without any prior knowledge . For evaluation, we have converted the radial position (r) to the sedimentation coefficient s by using eq, rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{s}} = \frac{{{\text{ln}}\,(r / r_{\text{m}})}} {{\omega^{2} t}} $$\end{document} the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . A multiwavelength auc as described in bhattacharyya et al . (2006) and strauss et al . In contrast to conventional sedimentation velocity experiments, where the sample between boundary and bottom of the cell is only diluted by radial dilution, the sample in band centrifugation is diluted additionally by fractionation in the pure solvent . The reservoir is filled with a small amount of concentrated sample . Column and sample sectors are filled with d2o with a density which is higher than that of the sample solution and lower than the density of the dispersed solute . We have prepared a 20 g / l solution and deposited 15 l of the solution into the cell reservoir . After preliminary experiments, this concentration was chosen to ensure that the individual components of the mixture are detected in as many as possible scans with od s <1.4 in the experiment without too much dilution which causes noisy data . After cell assembly, the auc was accelerated to 5,000 rpm for 3 min to transfer the sample in the reservoir via capillaries to overlay the d2o column . Forty scans were taken with a time interval of 90 s and a radial step size of 50 m to observe the full sedimentation of the sample . The selected wavelength range was 250750 nm . In the prototype setup, we apply the spectrum acquired for an empty cell as a reference for the calculation of the absorption leading to a baseline offset of 0.05 od (see fig . 3i) after the experiment, while cleaning the cell, we saw some precipitate in the cell reservoir . Thus, not all particles were transferred to the sample column, but some big particles remained in the reservoir as they already must have completely sedimented upon speeding up the rotor to 5,000 rpm.fig . 3three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . I scan 1 (1.5 min); ii scan 10 (15 min); iii scan 18 (27 min); iv scan 40 (60 min) three - dimensional plots of the raw data from a band sedimentation experiment with -carotene detected with the mwl detector . I scan 1 (1.5 min); ii scan 10 (15 min); iii scan 18 (27 min); iv scan 40 (60 min) each of the 40 scans produces a 3-dimensional graph . We have radial position as x - dimension, wavelength as y - dimension and absorbance as z - dimension . In the present contribution, we will perform a semi - quantitative evaluation based on simple model - free transformations of the data without any prior knowledge . For evaluation, we have converted the radial position (r) to the sedimentation coefficient s by using eq . 1 . In eq . 1, rm is the radial position of the meniscus and t is the run time integral.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{s}} = \frac{{{\text{ln}}\,(r / r_{\text{m}})}} {{\omega^{2} t}} $$\end{document} the 3d absorption dataset can now be projected either onto the wavelength or sedimentation coefficient axis to better visualize the spectral changes with different sedimentation coefficients or sedimentation coefficient distributions at different wavelengths . In principle, the entire dataset can be evaluated globally, and efforts are underway to incorporate such routine into the ultrascan evaluation software package (demeler 2005). However, even then we are confronted with a confounded polydispersity of both optical and colloidal / hydrodynamic properties . In the left side of fig . 2, the assumed core - shell structure of a -carotene microparticle is shown (auweter et al . 1999). Such a complex hybrid particle exhibits several levels of polydispersity, which impact the distribution of sedimentation coefficients observed in an auc . Oil content, of the inner core;concentration of the adsorbed protection colloid (gelatin);degree of swelling of the gelatin . Oil content, of the inner core; concentration of the adsorbed protection colloid (gelatin); degree of swelling of the gelatin . Parameters 1 and 2 determine the optical properties and bioavailability that are decisive for the commercial application profile . For smallest particle sizes, -carotene is an h - aggregate while for the biggest particle sizes -carotene forms j - aggregates . Intermediate particle sizes are assumed to integrate h- and j - aggregates in differing ratio (auweter et al . Parameters 3 and 4 determine the thickness of the protection colloid layer, which is typically 40 nm in pure water . The buoyant density of gelatin is rather high (above 1.3 g / cm), and cannot be matched with a non - interfering solvent such as heavy water . All parameters 14 enter into the calculation of the effective density and the hydrodynamic diameter of the hybrid particle . The frictional force under sedimentation depends on the ion concentration and ph because the gelatin may collapse or swell thus changing the effective frictional forces (and thus changing the observable sedimentation constant) although the chemical composition and buoyant density, which in principle could be measured in a krattky gauge or density gradient, stay the same . The swelling of the gelatin corona alone impedes an exact conversion from measured sedimentation constants to hydrodynamic diameters . Considering that also parameters 1 and 2 contribute to the polydispersity in the observable distribution of sedimentation coefficients, we decided to limit ourselves to a conservative evaluation on the level of sedimentation fractions, not sizes . We now discuss the optical properties that result from the specific colloidal microstructures as discussed above . Due to different preparation conditions, the morphology of the -carotene core changes . H- and j - aggregates have different uv / vis spectra, shown as visual impression on the right side of fig . 2 . Figure 1 dot line curve shows the uv / vis spectrum of 0.05 g / l product without any ultracentrifugation . Four peaks at 288, 449, 478 and 518 nm can be seen . The three peaks in the visible can be attributed to the 1ag (s0)1bu+ (s2) transition with the vibrational progression 20, 10, 00 of the c c stretch vibration along the alternatingly double bonded electronically conjugated backbone of the carotenoid (polivka and sundstrom 2004).the uv peak partially can be attributed also to the carotenoid transition 1ag1ag+, which is forbidden by symmetry, but becomes allowed in the crystalline assembly . The spectrum of the composite particle indicates the -carotene j - aggregate (auweter et al . 1999). It can be seen that gelatin only contributes to the uv region of the spectra below 280 nm . However, the contribution of gelatin is vanishing compared to the three times stronger absorption of the composite sample at 20 times lower overall concentration . Another component that presumably contributes to the uv absorption is the ascorbylpalmitat dispersant that is added during the co - precipitation . If we put all these 40 scans in sequence, we can form a 3d movie of the sedimentation process . Figure 3.1 shows scan 1 where particles just have been transferred from the reservoir to the sample column . The baseline offset is 0.05 (purple) due to the absorption calculation with an empty cell as reference ., there is an overlay of two peaks, one is the uv peak of -carotene (see fig . 2) and the other is the uv signal of gelatin . After 15 min of sedimentation, fractionation of the sample is obvious and the first sedimentation fraction proceeds to the bottom of the cell . Scan 10 (15 min) is the last scan where the entire particle range can be seen before the first particles reach the bottom . If we compare the height of the peak in the uv and visible region at different radial positions, the ratio changes . This is the first important result, demonstrating that the sample is not homogenous . Instead the observed effect can be explained by a higher content of stabilizing agent that induces smaller particle sizes . 1) does not exactly match the gelatin absorption and that the expected contribution of gelatin is weak at the applied concentrations, hinting at a combined action of both gelatin and the ascorbylpalmitat added during the co - precipitation in particle synthesis . The third part of fig ., the fastest particles have sedimented already . In the fourth part of fig . 3, we see the last fraction that remained after 60 min of sedimentation, which is mainly composed of gelatin . However, some -carotene absorption is still visible, which seems to be solubilized in small amount by the excess gelatin or excess ascorbylpalmitat . We do not detect free gelatin in the analysis . In an independent experiment we measured the characteristic sedimentation behavior of gelatin with the interference optics of the beckman xli auc at 44,000 rpm . This confirms our assignment that the last fraction cannot be pure gelatin . To summarize the global evaluation, fig . We can differentiate particles, observe the full uv / vis spectra of the particles and can already draw conclusions about the different components in the complex sample mixture without any further evaluation, as the y - axis shows the full uv / vis wavelength range . We can also use projections of the data onto individual axes and proceed thus to a more quantitative evaluation . In order to calculate the full s - distribution of all particles, we have selected scan 10 for all further evaluation as this scan shows fractionation of the mixture while no particles are yet lost due to complete sedimentation . More information is potentially available with a global evaluation of the entire dataset . In fig . 4, the s - distribution of the particles is shown for 5 different representative wavelengths out of 330 (250750 nm with a wavelength resolution of 1.5 nm). We have selected the wavelengths according to the peaks of the -carotene microparticles: 260, 280, 450, 480 and 520 nm in fig . 4sedimentation coefficient distributions at different wavelengths sedimentation coefficient distributions at different wavelengths the s - distribution is obviously very broad . Due to the chemical heterogeneity of the particles and the resulting density distribution, however, all important sample characteristics can be discussed for the s - distributions . From fig . 4, we conclude that there are at least three fractions in the sample: small hybrid particles with s <25 s, a main fraction around 100 s and larger particles around 200 s. their absorption spectra (and chemical composition) are clearly different as can be seen in fig . 5.fig . 5top normalized uv / vis spectra of particles with different sedimentation coefficients, bottom zoom the range around 450 nm and peak positions of 10.6 s (448 nm) up to 232 s (439 nm) top normalized uv / vis spectra of particles with different sedimentation coefficients, bottom zoom the range around 450 nm and peak positions of 10.6 s (448 nm) up to 232 s (439 nm) in fig . 5, seven representative uv / vis spectra are shown . The spectra agree well with those of pure h - aggregates (auweter et al . However, the original sample contained j - aggregates too (fig . 1, dashed line). We believe that the j - aggregates were already precipitated before the first scan in the auc cell was taken . Indeed precipitation of particulate material inside the reservoir of the vinograd cell was observed visually after cell disassembly following the experiment . However, irrespective of the actual nature of the particulate material that remained in the vinograd cell reservoir, the result stands for itself that the coloristic polydispersity (fig . 1) is not due to an intra - particle but inter - particle distribution of morphologies (fig . This result is contrary to the previous assumption that is sketched in fig . 2, where h / j - aggregates would coexist in the particles.fig . 6ad hoc structural model of the -carotene microparticle system on basis of the presented auc results . The different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before (fig . 2) (auweter et al . 1999) but instead separate particles contain pure h- or j - aggregates and the concentration ratio between these particles determines the colour of the final sample . Note the difference to fig . 2 ad hoc structural model of the -carotene microparticle system on basis of the presented auc results . The different color of the samples does not originate from the intraparticular coexistence of h- and j - aggregates as assumed before (fig . 2) (auweter et al . 1999) but instead separate particles contain pure h- or j - aggregates and the concentration ratio between these particles determines the colour of the final sample . 2 the peak around 520 nm slightly shifts to lower wavelength with increasing sedimentation coefficient and the peak height also decreases . Therefore, this excitation of -carotene microparticles decreases with increasing sedimentation coefficient . For the peak at 450 nm, only the spectral shift to lower wavelength is observed with increasing sedimentation coefficient . We suspect that the displacement of the electronic potential energy surfaces changes, such that the frank factors change for the vibrational progression, due to the changing incorporation of the chromophore into the partially crystalline assembly . As discussed above, an overlay of the signal from -carotene, gelatin and the ascorbylpalmitat a shift of the peak maximum to higher wavelength with increasing sedimentation coefficient is detected . In addition, a drastic decrease of the peak height relative to the 450 nm peak is observed with increasing sedimentation coefficient . The particles that sediment slower show stronger uv absorption, which we attribute to a higher content of ascorbylpalmitat, and hence smaller particle diameters due to the formation process in co - precipitation auweter et al . 1999). To our knowledge, it is the first time that relatively small steps of spectral shift among an h - aggregate are shown for composite particles . Although the spectral changes appear to be continuous, that does not exclude defined and different spectra for different particle populations as the detected raw signals are those of a sample band, which is broadened by polydispersity in size, composition and diffusional broadening . Industrial -carotene gelatin composite particles are a highly heterogeneous system both in particle size and chemical composition . Fractionation of this mixture in mwl - auc allows to resolve individual components in the mixture and detect compositional changes in an experiment, which takes only 1 h. this proves the power of mwl - auc as a direct technique that can differentiate particles with respect to size and uv / vis spectra . Although our current analysis does not allow to unambiguously assign the spectra to defined particles as the particle density, swelling, composition and size maybe varying simultaneously, the presented multiwavelength analysis allows insights into this complex system, which were not possible before by other techniques . We do not further evaluate the directly experimentally determined sedimentation coefficients as for our sample, in addition to the above mentioned polydispersity, ph effects as well as charge interactions between the colloids also have to be taken into account . Despite these restrictions, we have shown the existence of h - aggregates inside a sample that was previously known as j - aggregate and have detected spectral changes of different h - aggregate populations as well as changes in the electronic potential energy surfaces of different hybrid particles . We restricted ourselves to a semi - quantitative evaluation based on simple model - free transformations of the data of 1 out of 40 scans without any prior knowledge . Clearly, even richer phenomena can potentially be discovered with a global evaluation of the entire dataset . Below is the link to the electronic supplementary material . Supplementary information (avi 10.4 mb).
A 37-year - old woman presented with a 3-month history of exertional dyspnea, cough and one recent episode of hemoptysis . At the time of admission the axial and coronal ct scans were obtained with using 16-channel multidetector ct after iv administration of contrast media . A mildly enhancing left hilar mass with an endobronchial protrusion and an extension along the left lingular segmental bronchus was noted (figs . 1b, c). Bronchoscopy revealed a mutilobular, hypervasacular mass obstructing the lumen of the left lingular segmental bronchus (fig . A careful biopsy was taken from the peripheral portion because the tumor tended to bleed . This tumor was believed to be either an endobronchial bronchogenic carcinoma or another type of hypervascular tumor . The pathologic diagnosis was chronic inflammation with granulation tissue, which was not in accordance with the radiologic findings . We subsequently performed a left upper sleeve lobectomy with dissection of the mediastinal lymph nodes . The gross examination showed an endobronchially growing solid mass along the bronchial lumen, the so - called toothpaste figure, and this mass measured 73 cm in dimension . The mass showed a yellowish brown granular appearance with infiltration into the bronchial wall . The microscopic examination revealed that the tumor consisted of nests of epitheloid cells in an organoid or alveolar pattern, and this was surrounded by a delicate, richly vascular reticulin network, producing the classic' zellballen' or basket pattern (fig . The tumor cells showed marked nuclear pleomorphism and up to one or two mitotic figures/10 high power fields . Immunohistochemical staining for chromogranin, which is a marker for neuroendocrine tumors, was strongly positive . After surgery, a ct evaluation of the neck and abdomen demonstrated no abnormal findings . The biochemical study revealed the following: the urine epinephrine level was 2.3 g / day (0 - 20 for the normal range), and the norepinephrine level was 37.3 g / day (15 - 80 for the normal range). Paragangliomas are rare neuroendocrine tumors arising from neuroectodermally derived paraganglionic cells that are scattered throughout the body . Paragangliomas have been described in virtually all organs, including the orbits, nasal cavity, thyroid, heart, urinary bladder, gallbladder, liver, biliary system, kidneys, prostate, urethra, spermatic cord, uterus, ovaries, vagina, vulva, cauda equina and lungs . The most common site is the superior paraaortic region between the diaphragm and the lower renal poles (approximately 46% of all cases), and particularly in and around the renal hilus (6). In contrast, primary pulmonary tumors are extremely rare (<1%) (6). Most paragangliomas are benign, but a small percentage of these tumors produce distant metastases or they invade the nearby structures in the manner of malignant tumors (6). Although some reports have stated that a diagnosis of malignant paraganglioma can only be made after metastasis has occurred, some findings such as an extraadrenal location, macroscopic nodularity and tumor necrosis indicate a paraganglioma's malignant potential (7). Metastatic paragangliomas are more frequent in the lung than a primary pulmonary paraganglioma . Since primary pulmonary paragangliomas are rare and they present no pathognomic clinical features, they are usually observed as asymptomatic solitary nodules and they can be suspected of being a primary lung malignancy . The reported incidence of metastasis of a pulmonary paragangliomas has varied and the incidence appears to be related to the length of the follow - up period (8). The thoracic manifestations of paragangliomas include well - enhancing mediastinal masses, metastatic parenchymal nodules, lymphadenopathy from malignant paragangliomas, and pulmonary edema as a complication of epinephrine - producing paragangliomas . The much less common manifestations include a primary mass in the lung, heart, esophagus and/or trachea (5). Primary pulmonary paraganglioma was first reported by heppleston in 1958 (9), and only 19 cases have been reported since then in the english literature (2). Three of these cases were malignancies with lymph nodes metastases (8). In the remaining cases, the lesions behaved in a benign manner . Most patients are free of symptoms and hypertension, and the tumors are often discovered incidentally on routine chest radiographs (8). Two distinct forms of primary pulmonary paragangliomas have been reported (4, 8). The first and more common form consists of multiple minute tumors, in proximity to the pulmonary veins . The reported incidence of malignancy for a pulmonary paraganglioma is approximately 18%, which is lower than that for paraganglioma in other locations (20 - 50%). . Therefore, a lifetime of follow - up with careful, long - term observation by checking the urinary catecholamines and performing imaging studies is essential . While pulmonary involvement by an adrenal or extraadrenal pheochromocytoma is uncommon, endobronchial involvement like that seen in our case is very rare . Only one case of hilar and subcarinal lymphadenopathies with endobronchial metastases from recurrent adrenal pheochromocytoma and another case of a 0.9 cm primary endobronchial paraganglioma of the lung functioning extra - adrenal paragangliomas represent more than 10% of all pheochromocytomas (4). Among all the reported cases of pulmonary paraganglioma, two cases developed hypertension and the patients died from a cardiac disorder that was believed to be associated with the functional tumors . There was a report of a case of functioning metastases of a nonfunctioning paraganglioma (10). They assumed that a hypothetical phenotypic heterogeneity of the primary tumor could explain the difference in the biological behavior of the primary tumor and its metastases (10). The endobronchial paraganglioma in our patient manifested on ct as a hypervascular endobronchial mass, and this manifestation was similar to that of an endobronchial carcinoid or bronchogenic carcinoma . Therefore, we think paraganglioma should be considered when making the differential diagnosis of an enhancing endobronchial mass.
A 65-year - old man with no medical history was transferred from another hospital because of pain in the right side of chin and ear . He complained of frequent paroxysms of severe stabbing pain in the right side of his mandible radiating to the right ear . His pain either occurred spontaneously or was triggered by chewing and tooth brushing and lasted few seconds in each pain attack . The patient was diagnosed as tn at another hospital and was prescribed carbamazepine (200 mg a day) with only partial relief of the pain . He had a pain - free period for about 1 month before visiting our clinic and stopped medication on his own decision . In the last few days prior to the admission, his condition had got worse in terms of increase of frequency and severity of pain . Neurological and nasopharyngeal assessments were normal findings . Computed tomography of the neck and magnetic resonance imaging (mri) of the brain revealed also normal . His pain paroxysms had occurred four or five times daily with 100 of visual analogue scale (vas, 0 is no pain and 100 is imaginary the worst pain) at the first visit . He underwent mandibular nerve block with alcohol after the test block with 1% of mepivacaine . He still complained of right ear pain even though right v3 innervated area was anesthetized and pain free after the v3 alcohol block . Ten minutes after the procedure, paroxysmal pain in right ear developed and he experienced faintness and got loss of consciousness with convulsive movement for about 1 minute . He was consulted with a neurologist to exclude seizure disorders . Brain mri was not able to be performed due to convulsive movement during the pain attack . His echocardiogram was normal, however, bradycardia followed by asystole during pain attacks was revealed in the continuous electrocardiography . According to a careful asking about the pain characters following the v3 block with alcohol, he presented that his pain occurred in the soft palate, uvula and throat and was triggered by swallowing or mechanical stimulation of the right side of the pharynx . Bradycardia and asystole accompanied with sometimes seizure - like activity occurred always with pain attack . Based on his altered pain characters and cardiac symptoms with no specific etiology originated from heart, carbamazepine (100 mg twice a day) was started for control of pain as well as asystole . Regardless of increasing dose of carbamazepine up to 400 mg a day, severe bradycarida, asystole and syncope preceded by paroxysmal pain continued four to five times in one hour . A temporary cardiac pacemaker was implanted via left subclavian vein by a cardiologist to prevent cerebral ischemia during asystole . Although syncope and seizure - like activity disappeared after the insertion of a temporary pacemaker, intensity or frequency of pain in the throat and ear was unchanged . Carbamazepine increased up to 600 mg a day to reach pain control for 4 days after the implantation of temporary cardiac pacemaker . On the 5 day of hospitalization, he underwent implantation of a permanent pacemaker to prevent bradycardia and asystole by a cardiologist . He was achieved pain free and no cardiac symptoms after the implantation of permanent pacemaker with daily 600 mg of carbamazepine and has maintained symptom free condition for 4 months of follow - up . Gpn is an uncommon form of facial pain (0.2 to 1.3% of the facial pain) that occurs approximately one hundred times less frequently than tn . It was first described by weisenberg in 1910 in a patient with tumor affecting cerebellopontine angle . The other secondary causes of gpn are following; cerebellopontine angle tumor, carcinoma of the laryngeal and nasopharyngeal tumors, parapharyngeal abscess, multiple sclerosis, trauma, direct carotid puncture, paget's disease, calcified sylohyoid ligament, and chiari i malformation . Pain characters are almost similar to tn, however, pain originates in the ear, posterior pharynx, tonsillar area and base of the tongue innervated by glossopharyngeal nerve . Even though majority of gpn patients report that swallowing is a prominent trigger factor, some of tn patients also have it as a pain - provocating factor . Because of the similar pain characters between gpn and tn, especially in the v3 division, it could be misdiagnosis gpn as tn . Concurrent ipsilateral tn and gpn is rare, representing 10.0% to 46.7% of gpn cases but only 0.3% to 0.5% of tn cases . The pain is triggered by pharyngeal movement such as coughing, gargling and swallowing, especially cold liquids, as well as by touching in the above mentioned zones . While in tn, the pain is confined to the face, and is triggered by facial movement, such as chewing and speaking . In our patient he visited our clinic with known tn and presented tn symptom at the first visit, however, he could express the gpn component of pain when tn component of pain disappeared after the v3 alcohol block . The association between gpn and syncope is very rare phenomenon and could be life - threatening . It was reported that 217 patients diagnosed for gpn and only 4 were found having associated syncope postulating mechanism is originated from the idea of a close connection between the glossopharyngeal and vagus nerves with circulatory system . It has been suggested that intense afferent impulses from the sensory fibers of the glossopharyngeal nerve may stimulate the dorsal motor nucleus of the vagus nerve either by way of central collateral pathways or through an " artificial synapse " along the peripheral course of the glossopharyngeal nerve as it travels with the nerve of hering . In neuralgia pain condition like gpn, extremely severe pain could activate the vagoglossopharyngeal reflex resulting in bradycardia, hypotension and syncope . The basic goal of treatment of gpn with cardiac manifestations should be focused on control of the pain, which could be a main cause of bradycardia and syncope . First choice of medical treatment is carbamazepine and other medical trial is gabapentin although theoretically any membrane stabilizer could be used . The problem with using carbamazepine is the possibility of tachyphyaxis due to need of long - term use . In our case carbamazepine was effective at a moderate dosage (600 mg / day) in abolishing paroxysmal pain . Although percutaneous radiofrequency thermocoagulation through oval foramen has been tried, it carries the risk of injury to the whole vagus nerve . Kondo reported that immediate complete relief was achieved in 67 to 79% of patients, and partial relief in 10 to 25% of patients obtained, while long - term complete relief was obtained in 58 to 76% and partial relief in 15 to 18% of patients . However, manipulation of lower cranial nerves can be associated with morbidity; dysphagia, hoarseness, facial paresis, hearing disturbance associated 7th, 8th, 9th and 10th caranial nerve disturbances, and cerebrospinal fluid leakage . A temporary pacemaker should be used for emergency management of syncopal attacks caused by bradycardia and asystole originated from gpn like our patient until plasma concentration of carbamazepine reaches the therapeutic range . A temporary transvenous cardiac pacemaker implantation was described first by khero and mullins in 1971 to treat the reflex cardiac syncope while waiting for surgical section of the nerve . Regarding permanent pacemaker implantation, the available literatures are quite controversial, because spontaneous recovery in gpn is not uncommon and relapses may occur . However, once severe bradycardia and asystole relapse again in unexpected time, the patient might be at risk of a fatal outcome . It should be individualized to determine to implant a permanent pacemaker in patients with gpn associated with neurocardiogenic syncope . In our case, a permanent pacemaker underwent to prevent heart and brain damage with the reasons of that every pain attack leaded to asystole and relatively old patient's age . In conclusion, a temporary pacemaker combined with medical treatment should consider as an initial treatment modality in patients with gpn associated with asystole and syncope . Furthermore, if the disorder is longstanding and severe, a permanent pacemaker combined with medical treatment could be a safe treatment option.
Opportunistic sampling of mammalian roadkill took place over a two - year period near the university of oklahoma campus (norman, ok, usa). Carcasses deemed fresh (generally determined to have been struck by motor vehicles no more than 10 h prior to sampling) were selected, and those with one or more intact orifices (i.e., mouth, nose, ear, eye, and rectum) or gastrointestinal tracts were sampled roadside with sterile swabs . The mammalian roadkill carcasses that were sampled included (in order of increasing frequency) the following: skunk (mephitis mephitis), armadillo (dasypus novemcinctus), deer (odocoileus virginianus), raccoon (procyon lotor), squirrel (sciurus carolinensis), and opossum (dideiphis virginiana). Sampling of these carcasses led to the generation of 3659 bacterial isolates (figure 2). Not only were opossum carcasses the most frequently encountered, they were also a particularly rich source of morphologically unique bacteria, accounting for 39% (1425) of the roadkill - associated isolates collected (figure 2). The phylogenetic diversity of the opossum microbiome was further evaluated by sequencing 16s rrna gene libraries . Swabs taken from five opossum body sites were used to generate 13 994 sequences, which clustered into 73 operational taxonomic units (otus) at 97% sequence similarity (figure 3). Members of the gammaproteobacteria dominated the communities from the mouth, nose, and ear, constituting greater than 90% of each community . The upper gastrointestinal tract also contained a sizable population of gammaproteobacteria (72.4%) and bacilli (21.7%). The rectum was notably more diverse containing large populations of fusobacteria (47.2%), clostridia (19.1%), and bacteroidia (10.8%). The gammaproteobacteria were largely represented by two abundant operational taxonomic units, one of which was unclassified and another assigned most closely to the genus pseudomonas . (a) breakdown of the 3659 roadkill microbiome isolates based on the source organisms from which they were derived . (b) distribution of the isolates based on the locations / orifices on the carcasses from which they were derived . (c) categorization of the isolate data illustrating the percent contribution of each body site to the overall bacterial collection prepared from the different animal species (note that the colors and categories used to construct the slices within each of the pie charts in panel c are the same as those used for the pie chart in panel b). All of the bacterial isolates were individually cultured in two or more broth media, and a library of their ethyl - acetate - soluble natural products was prepared . One of the opossum ear samples showed appreciable activity in an assay designed to identify compounds that inhibited c. albicans biofilm formation . However, upon closer scrutiny, this sample was determined to be derived from a culture containing two bacteria that each produced slightly different - sized white, opaque, mucoidal colonies . When the two microorganisms were obtained in pure culture, the 16s rrna genes of both isolates were sequenced, and the bacteria were identified as members of the genera pseudomonas and serratia . Representative colonies of both isolates were analyzed in situ by laser ablation electrospray ionization mass spectrometry (laesims). (figure 4), which was determined to be viscosin (1) based on its ms / ms fragmentation pattern relative to an authentic standard . Its assignment was later confirmed by a single - crystal x - ray diffraction experiment of the metabolite following its scale - up production and purification (figure 5). Laesims analysis of the serratia sp . Isolate led to the purification of serrawettin w2 (2), as well as three co - metabolites that could not be dereplicated (i.e., m / z values and fragmentation patterns did not produce any reasonable matches to reported bacterial natural products). Although the information derived from the lc - ms investigation of the compounds (ms data, lc retention times, and uv vis pda profiles) enabled us to postulate that the serratia sp . Metabolites were analogues of 2, we decided to pursue their scale - up purification for structure confirmation and bioactivity testing . Phylogenetic diversity of mammalian microbiome bacteria from different orifices / body sites of a roadkill opossum carcass . The inset shows bacterial colonies growing on the surface of an agar plate . The plate was placed inside of the laesims chamber for mass spectrometry profiling . A subset of representative colonies was selected (indicated by red arrows), and a virtual grid was laid over these colonies using the instrument s software to target where laser ablation would occur . The light blue polygons show where mass data were collected from the colonies within the range of m / z 2002000 . The presented mass data were derived from the circled colony (average of several locations taken from the colony and subtracted from mass data obtained from a blank [uncolonized] portion of the plate), which reveals prominent single and doubly charged sodium adduct ions for viscosin (1). Ortep rendering of viscosin (1) illustrating the metabolite s absolute configuration (determined by refinement of the flack parameter; the water molecule identified near atom n2 has been removed from the figure for clarity; however, the position of the water can be found in the supporting information). The numbering system used for this structure reflects the atom assignments used in the supporting information and in the cambridge structural database (ccdc 1511786). Upon partitioning of the liquid cultures of the serratia sp . Isolate, the combined ethyl acetate layer was determined to retain the putative serrawettin analogues, as well as the biofilm inhibition activity . Laesims - guided fractionation of the organic layer by hp20ss vlc, as well as preparative and semipreparative c18 hplc resulted in the purification of compounds 35, along with 2, whose structure was subsequently confirmed by ms experiment, h and c nmr data (tables 1 and 2, respectively), and marfey s analysis . Analysis of hresims data for compound 3 provided a prominent ion with m / z 740.4235 [m + na] that supported a molecular formula of c37h59n5o9 . This indicated that compound 3 likely differed from 2 by the loss of a ch2 unit . H tocsy data for 3 versus 2 revealed that the spin system associated with the isoleucine residue was altered . Inspection of the h (table 1) and h h dqfcosy nmr data for 3 showed that the amide proton (8.28, d, j = 7.22, 1 h) coupled with the -proton (3.73, t, j = 7.76, 7.76, 1 h), which coupled to a methine (1.95, m, 2 h) that in turn coupled with methyl protons (0.84, m, 6 h). These results could be accounted for if the isoleucine residue in 2 changed to a valine residue in 3 . Subsequent investigation of the metabolite by hydrolysis followed by marfey s analysis confirmed that the isoleucine residue was no longer present and instead an l - valine had been incorporated . Thus, metabolite 3 was assigned the trivial name serrawettin w4 in recognition of its structural relationship to metabolite 2 . A similar structure determination strategy was applied to compound 4 after observing that the hresims data contained a prominent ion with m / z 726.4073 [m + na], which corresponded to a molecular formula of c36h57n5o9 . This indicated that the metabolite was deficient for two ch2 units relative to compound 2 . H tocsy data for 4 provided strong evidence that metabolites 2 and 4 shared identical macrocycles . This was supported by hydrolysis followed by marfey s analysis, which confirmed the presence of the same amino acid residues in 2 and 4 . Turning our attention to the ms result, as well as h nmr data attributable to the hydrocarbon chain, it was determined that the lipid - derived portion of 4 was missing two ch2 units relative to 2 . With the structure of metabolite 4 confirmed, the new compound was given the trivial name serrawettin w5 . An evaluation of metabolite 5 by hresims provided an ion with m / z 770.4346 [m + na] that enabled us to determine its molecular formula was c38h61n5o10 . This represented an increase of one oxygen atom in 5 relative to compound 2 . Focusing on the h nmr data (table 1) for the amino acid portion of the macrocycle in 5, it was readily apparent that the aromatic protons associated with the phenylalanine residue in 2 were altered and that one hydrogen bonded to a carbon was missing . The new aromatic spin - coupled network appeared as an aabb system, which led us to propose that 5 incorporated a tyrosine residue in place of the phenylalanine in 2 . The assignments of the carbon and hydrogen spins for the tyrosine were subsequently confirmed by hmbc experiment, as well as hydrolysis followed by marfey s analysis, which together established the presence of an l - tyrosine in 5 . Metabolite 5 was assigned the trivial name serrawettin w6 . With the stereochemical assignments of the amino acid residues completed, we focused on securing the configurations of the c-3 positions in each compound . Applying biosynthetic - based logic to the problem, it appeared reasonable to presume that the stereochemistries of the metabolites c-3 positions were set upon incorporation of either 3-hydroxydecanoic acid (2, 3, and 5) or 3-hydroxyoctanoate (4). A survey of the chemical literature concerning the spectroscopic properties of -hydroxy fatty acids revealed a decisive trend in their chiral - optical data (table s1). Namely, in chloroform, r - configured -hydroxy fatty acids displayed consistent levorotatory activities, whereas in ethanol, these molecules exhibited dextrorotatory properties . Returning to the acid hydrolysates prepared from 25, we were able to purify milligram quantities of enantiopure -hydroxy fatty acids from each sample . Specific rotation values were obtained for the products in both ethanol and chloroform, which enabled us to assign r configurations to the c-3 positions in 25 . The genome of the serratia sp . Isolate was sequenced to link the identified natural products to their prospective biosynthetic gene cluster . Over 5.4 million reads were generated and assembled into 35 contiguous sequences after quality control . The annotated 16s rrna gene was extracted and aligned to otus classified as gammaproteobacteria, which we had observed from the opossum ears . These showed close (98%) homology to an otu classified as a member of the enterobacteriaceae . A total of eight potential secondary - metabolite - producing biosynthetic gene clusters were identified by antismash analysis, including four nrps clusters . One of the candidate clusters presented a near - perfect match to what was required to construct metabolites 25 (si figure s69). The purified compounds were evaluated for their abilities to inhibit growth and biofilm production of c. albicans . None of the metabolites were able to kill fungal cells at concentrations up to 100 m . Isolate proved to be the most potent inhibitor of c. albicans biofilm formation, with an ic50 value of 4.6 1.0 m . The serratia sp . Metabolites exhibited a range of biofilm inhibition capabilities, with 2 being the most potent (ic50 = 7.7 0.7 m), followed by 4 (ic50 = 13.4 0.2 m), 5 (ic50 = 29.2 0.4 m), and 3 (ic50 = 59.8 5.7 m). The abilities of these bacterial cyclic lipodepsipeptides to limit biofilm formation, while not impacting yeast cell survival, hint at the possibility of alternative chemical options for controlling c. albicans infections by means of biofilm modulation . The use of opportunistic sampling to humanely investigate the microbiomes of a broad range of animals offers an intriguing method to accelerate the exploration of this resource for new natural products . Notably, the discovery pipeline presented here is an enabling tool for identifying bacterial natural products from nonhominid mammals . One potential concern centers on how well our collection strategy accurately reflects the native microbiomes of living mammals since the microbiomes of humans are known to change as the time since death increases . While our investigation did not test this point specifically, we did make efforts to sample only recently deceased mammals and took care to avoid orifices that were markedly compromised by collisions with vehicles . Therefore, we remain confident that the isolates obtained in this study are representative members of mammalian microbiomes and not taxa simply derived from the immediate environment or strictly enhanced via decomposition . Further studies are anticipated to provide additional microbiome - associated bacteria, their natural products, and associated biosynthetic gene clusters, which might serve as candidates for future microbiome engineering endeavors . Indeed, colony profiling by laesims suggests the presence of many bacteria - derived peptide - like products that we anticipate will possess potentially useful therapeutic properties . Nmr data were obtained on varian vnmr spectrometers (agilent technologies, inc ., santa clara, ca, usa) with broadband and triple resonance probes at 25 0.5 c unless otherwise noted . Optical rotation measurements were made on a rudolph research autopol iii automatic polarimeter (rudolph research analytical corp . Uv data were measured with a hewlett - packard 8452a diode array spectrophotometer (agilent technologies, inc . ). Ir spectra were obtained on a bruker vector 22 ft - ir spectrometer (bruker corp ., billerica, ma, usa). Accurate mass electrospray - ionization mass spectrometry data were collected on an agilent 6538 high - mass - resolution qtof mass spectrometer (agilent technologies, inc . ). Hplc separations were carried out on a shimadzu system using a scl-10a vp controller (shimadzu scientific instruments inc ., columbia, md, usa) and gemini 5 m c18 and hexyl - phenyl columns (110, 250 21.2 mm and 250 10.0 mm, respectively, phenomenex inc ., torrance, ca, usa) with flow rates of 10 or 4 ml / min . All chemicals and solvents used in the study were of research grade quality or better . A 48 km section of oklahoma state highway 9 from the university of oklahoma campus, norman, ok, to the city of tecumseh, ok, was used for this study . This route was chosen because it traverses a state park (lake thunderbird state park), a significant quantity of acreage devoted to agriculture, and forested land, which are all prime habitats for many of oklahoma s major mammalian species . Additionally, the highway is heavily traveled (a major east west thoroughfare from arkansas to texas) with posted speed limits of 65 mph (105 kph). The juxtaposition of having an active highway that intersects such a variety of mammalian habitats made this a prime location for near daily animal vehicle collisions . A sampling permit was obtained from the state of oklahoma (scientific collector permit #5250), and the university of oklahoma iacuc was informed of the experiments, resulting in the assignment of an internal case - tracking number (r11 - 021). When fresh roadkill was encountered (generally less than 10 h old), the intact orifices, which included the mouth, ear, nose, and rectum, were sampled with sterile cotton - tipped swabs and then immediately plunged into sterile phosphate - buffered saline (pbs) buffer . When opportunities presented themselves, samples were also taken by swabbing the inner portions of the lower gastrointestinal tract . The cotton tips of the swabs were placed into 1.0 ml of pbs and vortexed . These suspensions were diluted 10 with pbs, and 50 l aliquots were spread onto three types of solid agar media (10% tryptic soy agar, 10% brain heart infusion agar, and dm7 agar) and incubated at 30 c for 3 days . Colony picking was performed using sterile toothpicks and the inoculum used to prepare streak plates on dm7 medium . Single colonies were picked with toothpicks, which were then dipped into tryptic soy broth containing 15% glycerol and incubated at room temperature for 1 week . Cultures of isolates were used to generate samples for cryogenic preservation at 80 c, as well as inoculate various media for further chemical and bioassay studies . For these experiments, 5 l aliquots were inoculated into the wells of three 24-well plates containing 50% tryptic soy broth, 50% brain heart infusion broth, and dm7 broth and incubated for 7 days at 30 c . These cultures were subjected to partitioning three times against equal volumes of ethyl acetate . The organic layers from each sample were combined, and the solvent was removed in vacuo . Extracts were evaluated for bioactivities, and those determined to be active were examined by lcms and laesims . Isolates were obtained from swabbing the ear canal of a roadkill opossum encountered on oklahoma state highway 9 near norman, ok . While the original colony selected for the study was thought to be a single isolate, subsequent cultivation of the bacteria led us to determine that the culture contained two taxa, which were separated through isolation on a solid agar medium and analyzed in pure culture . A single colony of the serratia sp . Was used to inoculate 1/10th strength tryptic soy broth medium, which, after 16 h, was used to obtain genomic dna by means of an xpedition soil / fecal dna miniprep kit following the manufacturer s directions (zymo research, irvine, ca, usa). The genomic dna was quantified using the qubit br assay (life technologies, carlsbad, ca, usa), and approximately 1 g was sheared to a mean insert size of 500 bp and sequenced using pe250 v2 chemistry on the illumina miseq platform (illumina, san diego, ca, usa). The reference strain c. albicans sc5314 was cultured in brain - heart infusion medium (bhi, becton dickinson) or rpmi-1640 plus mops medium [rpmi-1640 medium (sigma) buffered to ph 7.0 with 0.17 m mops (3-(n - morpholino)propanesulfonic acid, sigma)] as required . Sequence data used for 16s rrna gene analysis are available at the ncbi sra under accession number srx1601902 . Genome is available under the accession number gca_001643155.1, and the raw reads used to assemble the genome are available for download at the ncbi sra under the accession number srx1585056 . The opossum carcass was sampled with sterile cotton swabs, and the swabs were frozen at 20 c until dna extraction was performed . The samples were thawed and then homogenized (biospec products, bartlesville, ok, usa) for 30 s. community genomic dna was extracted using the mobio power biofilm dna extraction kit (mobio laboratories, inc ., libraries of bacterial and archaeal 16s rrna gene fragments were amplified from each dna extraction by pcr with primers that spanned the v4 region between positions 519 and 802 (e. coli numbering), producing a 300 bp fragment . These primers evenly amplify a broad range of both the bacteria and archaea . The forward primer (m13l-519f: 5-gta aaa cga cgg cca gcacmg ccg cgg taa-3) contained the m13 forward primer (in bold), followed by the 16s rrna gene - specific sequence (underlined). The reverse primer (785r: 5-tac nvg ggt atc taa tcc-3) was taken directly from s - d - bact07850b - a-18 in klindworth et al . Each 50 l pcr consisted of 3 u of recombinant taq polymerase (thermo fisher scientific inc ., grand island, ny, usa), 1.5 mm mgcl2, 0.1 m dntps (thermo fisher scientific inc . ), 0.2 m of each primer, and 4 l of a dna extraction . Initial amplification was conducted using the following protocol: initial denaturation at 95 c for 2 min, followed by 30 cycles of denaturation at 95 c for 45 s, annealing at 52 c for 45 s, and extension at 72 c for 45 s, followed by a final extension at 72 c for 5 min . The amplified 16s rrna gene fragments in each library were purified using ampurexp paramagnetic beads according to the manufacturer s protocols (beckman coulter, inc ., indianapolis, in, usa). A second, six - cycle pcr was used to add a unique 12 bp barcode to each amplicon library using a unique forward primer containing the barcode + m13 forward sequence (53) and the 785r primer using reaction conditions identical to those listed above . The resulting barcoded pcr products were quantified using the qubit hs assay (life technologies), pooled in equimolar amounts, and concentrated to a final volume of 80 l using amicon ultra-0.5 ml 30k centrifugal filters (merck millipore corp ., the pooled library was then submitted for sequencing on the miseq platform using pe250 v2 chemistry (illumina). After sequencing, reads were merged using pear, demultiplexed in qiime, filtered by quality, and clustered into otus using uparse . The mapping file used to demultiplex the samples is present as a supplementary table . The taxonomy of each otu was assigned using uclust and the silva database (release 123). For the genomic sequence, reads were first quality trimmed, and sequence adapters were removed using peat before assembly within spades using kmer values of 21, 33, 55, 77, 99, and 127 . After assembly, contiguous sequences less than 300 bp in length were discarded, and contaminating phix sequences were removed after visualization of the genome assembly graph in bandage . The assembled genome was annotated using the ncbi pgap annotation pipeline, and putative metabolite clusters were identified using antismash . Single colonies of the serratia sp . Isolate were transferred to dm7 and vortexed . Aliquots of this suspension were inoculated into 40 high - aeration shake flasks (2 l ultra yield flask) containing 1 l of dm7 . The flasks were shaken at 135 rpm for 7 days at room temperature on an innova 5000 shaker . The dm7 broth consisted of (per liter) monopotassium phosphate, 2.0 g; ammonium chloride, 1.5 g; magnesium sulfate heptahydrate, 0.5 g; glycerol, 12.6 g; myo - inositol, 0.4 g; monosodium glutamate, 5.0 g; sodium fluoride, 0.084 g; iron(ii) sulfate heptahydrate, 0.025 g; zinc(ii) sulfate heptahydrate, 0.01 g; cobalt(ii) chloride, 0.01 g; calcium carbonate, 0.25 g; and p - aminobenzoate, 0.001 g; with the ph of the final solution adjusted to 7 . The broth from the liquid culture of the pseudomonas sp . Was partitioned against ethyl acetate . During the partitioning process, crystals of 1 formed on the inner surface of the separatory funnel . The crystals were rinsed from the funnel under a stream of ethyl acetate, and the solvent was removed by leaving the crystals overnight in an open vial at room temperature . A colorless needle - shaped crystal of 1 of dimensions 0.26 0.04 0.04 mm was selected for structure analysis . Intensity data for this compound were collected using a diffractometer with a bruker apex ccd area detector and graphite - monochromated mo k radiation (= 1.541 78). The sample was cooled to 100(2) k. cell parameters were determined from a nonlinear least - squares fit of 2010 peaks in the range 4.2 <<68.2. A total of 39 420 data were measured in the range of 3.626 <<71.353 using and oscillation frames . The data were corrected for absorption by the empirical method giving minimum and maximum transmission factors of 0.835 and 0.972 . The data were merged to form a set of 11 566 independent data with r(int) = 0.145 and a coverage of 98.5% . The orthorhombic space group p212121 was determined by systematic absences and statistical tests and verified by subsequent refinement . The structure was solved by direct methods and refined by full - matrix least - squares methods on f. the positions of hydrogens bonded to carbons were initially determined by geometry and were refined using a riding model . Hydrogens bonded to nitrogens and oxygens were located on a difference map, and their positions were refined independently with x - h restraints . A total of 767 parameters were refined against 51 restraints and 11 566 data to give wr(f) = 0.3772 and s = 1.238 for weights of w = 1/[(f) + (0.1800p) + 30.0000p], where p = [fo + 2fc]/3 . The final r(f) was 0.1281 for the 11 016 observed, [f> 4(f)] data . The largest shift / s.u . The final difference map had maxima and minima of 0.783 and 0.891 e /, respectively . The absolute configuration of the structure was determined by refinement of the flack parameter . The broth from each culture flask was pooled and partitioned three times against equal volumes of ethyl acetate . The solvent was removed in vacuo to generate a crude organic residue (10.3 g). The organic material was processed by vlc over hp20ss resin using a step gradient going from water to methanol to generate six fractions . Fraction 4 was further separated by preparative c18 hplc (gradient elution with meoh h2o, 75:25, to 100% organic in 30 min) to yield three subfractions (13). Subfraction 2 was further separated by semipreparative c18 hplc (mecn h2o, 55:65) to provide four subfractions (a d). Subfraction 2c was further purified by semipreparative c18 hplc (mecn h2o, 60:40) to yield 2 (46.7 mg, 4.53% yield). Subfraction 2b was further purified by semipreparative c18 hplc (mecn h2o, 65:35) to yield 3 (47.1 mg, 4.57% yield). Subfraction 2a was further purified by semipreparative hexyl - phenyl hplc (mecn0.1% formic acid in h2o, 65:35) to yield 4 (2.8 mg, 0.271% yield) and 5 (3.7 mg, 0.359% yield). Samples of the metabolites (300 g) were incubated overnight in 6 m hcl (500 l) at 110 c . The hydrosylates were dried under nitrogen gas, and 20 l of 1 m nahco3 was added to each sample to facilitate neutralization . Next, 100 l of 1% 1-fluoro-2,4-dinitrophenyl 5-l - alanine amide was added to the samples, and the resulting solutions were heated at 45 c for 1 h. the reaction mixtures were neutralized with 20 l of 1 m hcl and diluted with 500 l of mecn . The samples were centrifuged and analyzed by lc - ms (100, c18 kinetex 2.6 m, 75 3.0 mm column with gradient elution using mecn with 0.1% formic acid in h2o, 10:90, to 100% organic over 15 min . ). Standards eluted as l - isoleucine at 7.96 min, d - isoleucine at 8.19 min, l - valine at 7.68 min, d - valine at 7.68 min, l - leucine at 7.78 min, d - leucine at 8.26 min, l - phenylalanine at 7.80 min, d - phenylalanine at 8.13 min, l - serine at 7.57 min, d - serine at 8.13 min, l - tyrosine at 8.57 min, d - tyrosine at 8.88 min, l - threonine at 8.53 min, l - allo - threonine at 5.73 min, and d - allo - threonine at 5.90 min . The hydrosylates were partitioned with equal portions of hexanes (3). The hexanes were removed in vacuo, the resultant compound was suspended in 2 ml of chloroform or ethanol, and their specific rotation values were determined . The effects of compounds on the growth of c. albicans were tested using the method described in the nccls 2008 clsi m27-a3 guidelines with the following modifications . Cells of c. albicans sc5314 were cultured in bhi medium (becton dickinson co., franklin lakes, nj, usa) at 37 c overnight . The cells were pelleted by centrifugation, washed with sterile pbs (ph 7.4), and resuspended in rpmi-1640 plus mops medium . Test compounds were prepared in dmso at stock concentrations of 2 mm before being diluted in rpmi-1640 plus mops for testing . Aliquots of yeast suspension (100 l containing 2.5 10 cells ml) were added to the medium containing the diluted compounds or dmso [final concentrations did not exceed 2% (v / v)] before being transferred to 96-well plates (corning). After 48 h of incubation at 37 c, the viability of the yeast was measured using the xtt assay . In brief, yeast cells were treated with 0.1 mg ml xtt at 37 c for 1 h. absorbance measurements were taken at 492 nm using a microplate reader (infinite m200). The minimum inhibitor concentrations (mic) for growth were defined as the lowest antifungal concentrations that caused 85% reduction in metabolic activity . For measuring biofilm formation, the medium was aspirated and the wells were washed twice with sterile pbs to remove nonadherent cells . Fresh medium (100 l of rpmi-1640 plus mops) was then added back to each well . The 50% inhibitory concentration (ic50) values for biofilm inhibition were calculated using graphpad prism 5 . White, powdery solid; []d20 = 7.3 (c 0.164, chcl3); uv (meoh) max (log) 206 (3.52); ir (film) max 3600, 3516, 3412, 3003, 2965, 2926, 1715, 1425, 1362, 1221, 1092, 903, 783, 527; h nmr (dmso - d6, 600 mhz) refer to table 1; c nmr (dmso - d6, 151 mhz) refer to table 2; hresims m / z 730.4402 [m h] (calcd for c38h60n5o9, 730.4391). White, powdery solid; []d20 = 7.4 (c 0.270, chcl3); uv (meoh) max (log) 206 (3.17); ir (film) max 3607, 3524, 3412, 3003, 2967, 2925, 1713, 1707, 1427, 1361, 1221, 1094, 903, 783, 527; h nmr (dmso - d6, 600 mhz) refer to table 1 . C nmr (dmso - d6, 151 mhz) refer to table 2; hresims m / z 740.4235 [m + na] (calcd for c37h59n5o9na, 740.4210). White, powdery solid; []d20 = 7.6 (c 0.245, chcl3); uv (meoh) max (log) 204 (3.05); ir (film) max 3603, 3520, 3412, 3003, 2968, 2926, 1714, 1427, 1362, 1221, 1094, 905, 783, 523; h nmr (dmso - d6, 600 mhz) refer to table 1 . C nmr (dmso - d6, 151 mhz) refer to table 2; hresims m / z 726.4073 [m + na] (calcd for c36h57n5o9na, 726.4054). White, powdery solid; []d20 = 7.4 (c 0.140, chcl3); uv (meoh) max (log) 206 (3.11); ir (film) max 3609, 3528, 3412, 3003, 2695, 2924, 1746, 1712, 1422, 1362, 1222, 1092, 903, 785, 530; h nmr (dmso - d6, 600 mhz) refer to table 1 . C nmr (dmso - d6, 151 mhz) refer to table 2; hresims m / z 770.4346 [m + na] (calcd for c38h61n5o10na, 770.4316).
A 35-year - old male patient was presented at our outpatient clinic with dyspnea and hemoptysis . He had been lost to follow - up until recently, and no imaging study was performed for the patient until he presented with complaints . His physical examination revealed central cyanosis and clubbing, and there was loudness in p2 on auscultation . Chest radiography revealed cardiomegaly, severe dilation of the main pulmonary artery, and dilation of the pulmonary branch arteries (fig . Hemoglobin, hematocrit and bnp values were 19 gr / dl, 58% and 986 pg / ml, respectively . Transthoracic echocardiography showed an aneurysm of the main pulmonary artery of approximately 7.4 cm, dilation in left and right pulmonary arteries, and a turbulent flow between the pulmonary artery and descending aorta with color doppler (fig . Pulmonary valve, in parasternal long axis, was seen as it were tricuspid aortic valve that was classically observed in parasternal short axis view (fig . Contrast - enhanced, multislice computed tomography (ct) with 3 dimensional reconstruction of the patient s chest revealed a giant aneurysm of the pulmonary trunk with a maximum diameter of 79 mm and a patent ductus arteriosus (pda) (fig . Systolic and mean pulmonary arterial pressures measured during catheterization were 140 and 95 mmhg, respectively . The underlying causes include congenital heart diseases, pulmonary embolism, trauma, connective tissue disorders (marfan s syndrome, ehler - danlos syndrome), systemic vasculitis, and pulmonary hypertension [1 - 3]. Pda is the most frequently associated congenital anomaly, followed by ventricular and atrial septal defects [4, 5]. Paas have high morbidity and mortality due to the thinning of the arterial wall related to increased wall stress and eventual rupture . The formation of an aneurysm in the presence of marked pulmonary artery hypertension suggests that the mechanical stress on the vascular wall which elevates intraluminal pressures outweighs the protective deposition of connective tissue proteins, and the medial smooth muscle cell hypertrophy observed in pulmonary hypertension . The low pressure condition is much more benign, with low risk of arterial dissection and rupture even for large dilations, which may have diameters of up to 16 cm [7, 8]. Although the relationship between aneurysm size and risk of rupture in low - pressure pulmonary artery aneurysms is not well defined, decades without rupture have been observed in some cases [9, 10]. In contrast, paas secondary to pulmonary arterial hypertension pose a significant risk for the patient; and according to laplace s law, the size of these aneurysms is an important determinant of progression and rupture [11, 12]. Treatment of the low - pressure condition is based more on right ventricular function and associated pulmonary valve stenosis or regurgitation rather than the size of the aneurysm . It is treated primarily by aneurysmorrhaphy or by allograft implantation with or without pulmonary valve replacement [7, 13]. However, if significant pulmonary arterial hypertension or connective tissue disease is present, which increases the risk of rupture, surgical treatment must be considered, particularly in patients with symptoms or progressive changes . Although a heart - lung transplant is rarely the treatment of choice, it appears to be necessary in the present case with eisenmenger s syndrome . Conservative treatment is advocated for asymptomatic patients without a causative cardiac lesion and pulmonary hypertension [14, 15]. The disadvantage of conservative treatment, however, is the potential for dissection . There are many other factors to consider during the treatment decision, including the patient s age, life expectancy, symptoms and functional status . Minimizing the risks for rupture (i.e. Pulmonary hypertension) and annual imaging studies, including ct scans, in addition, serial echocardiograms may help to monitor cardiac function and changes in pulmonary hypertension pressures . However, one must be vigilant about symptoms that portend a possible dissection and emergent surgical intervention . Our patient had a known prior diagnosis of a cardiac defect, but duration of the pulmonary artery dilation was unknown . In many patients a chest radiograph is obtained for unrelated symptoms, which reveals vascular dilation or a hilar / mediastinal mass; and a definitive diagnosis usually requires further radiological evaluation . Echocardiogram is a reasonable tool for initial investigation due to its ease of use, low cost and convenient accessibility . Further non - invasive imaging studies such as ct angiography can be used if echocardiography fails to detect an intimal flap, or to provide information related to dissection [1, 18]. In conclusion, we present an adult patient with a giant pulmonary artery aneurysm secondary to untreated pda leading to eisenmenger s syndrome . The high risk of acute pulmonary artery dissection and rupture resulting from the progressive enlargement of the aneurysm with concomitant pulmonary artery thrombosis in the setting of eisenmenger s syndrome dictates treatment by heart - lung transplant, which, our patient currently awaits . While waiting for the heart - lung transplantation, we initiated treatment with bosentan according to the current guidelines to improve symptoms.
Cdd, also known as heller's syndrome or disintegrative psychosis, is a rare condition (1.7 cases per 100,000) characterised by late onset (> 3 years of age) of developmental delays in language, social function and motor skills . It is grouped with the pervasive developmental disorders (pdds) and is related to the better known and more common disorder of autism . Although recognised for many years, research on this condition is less advanced than that in autism . The present case report describes a child who developed cdd at the age of 6 years after an episode of chicken pox . Master d, 8 years and 1 month old child, was admitted with chief complaints of hyperactivity, increased talkativeness and no social reciprocity since last 2 years . History of present illness revealed an episode of fever with rash, diagnosed to be chicken pox for a period of 15 days about 2 years ago . After 15 days of asymptomatic period, the patient developed fearfulness, decreased social interaction and decreased speech production for next 2 months . After this, gradually patient developed new symptoms continuous irrelevant speech, verbal stereotypy, markedly increased motor activity, no social and emotional reciprocity, no eye to eye contact, decreased performance in activities of self help . The above mentioned symptoms developed within a period of 1 month and were stable after that . Electroencephalogram (eeg) showed occasional bursts of spike and sharp wave discharges, though clinically there was no evidence of seizure disorder . In the ward, child would remain hyperactive most of the time and would run here and there without any purpose . When someone would try to talk to him, he would not make eye to eye contact and would continuously utter irrelevant things . He would repeat some phrase multiple times without any meaning . To confirm the diagnosis, autism diagnostic interview revised (adi - r) was applied and a diagnosis of cdd was made on the basis of following items onset of symptoms after the age of 3 years, normal development of language before illness, loss of language and loss of purposive hand movement, loss of motor skills and loss of self help skills . Vineland adaptive behaviour scales (vabs), 2 edition (vineland ii) was applied to actuate the age of the skills before and after the illness and it also showed significant loss of skills in each sub - domains of communication, daily living and social skills . Various viral infections have been known as the first event leading to the development of cdd . To the author's knowledge cdd this case represents a rare and interesting complication of chicken pox in a boy of age 6 years with normal development, developing cdd within short span of time . The typical age of onset of cdd is 3 - 4 years . Though case reports have mentioned age of onset between 1 and 9 years . In our patient, age of onset . Such late age of onset has been reported in few cases . In childhood disintegrative disorder, there significant loss of previously acquired skills in at least two of the following areas expressive or receptive language, social skills, bowel or bladder control, play behaviour or motor skills . In our patient also, there was marked loss of most of the above mentioned skills . The core features of cdd are loss of communication skills, marked regression of reciprocal interactions and the onset of stereotyped movements and compulsive behavior . In our patient, these features were present . In most cases the intellectual deficit becomes static or shows some amount of recovery . In our patient the patient showed significant regression of milestones within short span of time and non - progressive course after that . The present case report aims to alert the clinicians about the possibility of developing cdd, when seeing a case of chicken pox, especially when there is presence of regression of developmental milestones.
The genus georgenia was established within the family bogoriellaceae subdivided into genera bogorilla and georgenia . Members of genus georgenia are reported to be gram - positive, motile or non - motile, non - endospore forming, aerobic or facultative anaerobic, oxidase and catalase positive actinobacteria . They are also identified on the basis of chemotaxonomic properties such as fatty acid profiling, polar lipids, amino acids, peptidoglycan as well as isoprenoid quinones with mk-8(h(4)) as the predominant menaquinone and less frequently by polyamines . They are having unique characteristics containing anteiso - c15:0 as a predominant fatty acid and dna g + c contents range from 69.7 to 72.9 mol% . At the time of writing the genus comprises ten different recognized species namely georgenia muralis, georgenia ruanii, georgenia thermotolerans, georgenia soli, georgenia halophila, georgenia daeguensis, georgenia satyanarayani, georgenia sediminis, georgenia deserti and georgenia ferrireducens; however, no one amongst them is reported as plant pathogen . Georgenia sp . Strain sub25 was isolated from infected leaves of solanum lycopersicum l. pure culture was maintained on nutrient agar containing 0.5% salt concentration with beef extract . The isolate was confirmed as phytopathogen by pathogenicity test on healthy leaves of s. lycopersicum l. and fulfilled koch's postulates . Genome sequencing of this strain was performed with high throughput ion torrent personal genome machine with ion torrent server (torrent suite v3.2). The annotation of the genome was performed using the ncbi prokaryotic genomes automatic annotation pipeline (pgaap) (http://www.ncbi.nim.nih.gov/genome/annotation_prok/) utilizing genemark, glimmer, and trnascan - se tools and using the rapid annotations using subsystems technology (rast) server with the seed database . Strain sub25 was gram positive actinobacteria, rod in shape and about 0.81.2 m in diameter; colonies were observed in yellow color, non - spore forming, oxidase and catalase positive . Optimum growth and ph was observed at 37 c temperature and at 7.0 ph . On the basis of 16s rrna gene sequencing analysis the total length of the genome was found to be 48, 50, 495 base pairs, allocated into 796 contigs having> 500 bp and 1852 contigs 500 bp with 88.5x coverage . Pathogenicity is not reported till date in any member of genus georgenia . According to rast annotated, georgenia sp . Type ii secretion system is mediated by conserved, multi - component secretion system, which span both inner and outer membranes and proteins are transported . This secretion system encodes a novel genomic island, which encompasses the tad (tight adherence) gene cluster, shown to be essential for colonization of surfaces by a human pathogen actinobacillus actinomycetemcomitans, . The majority of tad genes were shown to be essential for tenacious biofilm formation and synthesis of bundled flp pili (fibrils) that mediated adherence . The pilin subunit flp remains inside the cell in various tad - mutants, indicating that they encode a secretion system for export and assembly of fibrils . Homologous gene clusters have been detected in a wide range of bacterial and archaeal species, and their sequence characteristics indicate possible horizontal transfer . Strain sub25 having genes related to wide spread colonization and type ii / iv secretion system protein tadc, atpase tadz / cpae, is associated with flp pilus assembly and atp hydrolase tada / virb11/cpaf, tada subfamily genes . In addition to these, five genes for potassium metabolism, sixteen genes for nitrogen metabolism, ten genes for iron metabolism, 59 genes for phosphorous metabolism along with 342 genes for carbohydrate metabolism, 127 genes for fatty acid metabolism and 202 genes for protein metabolism are also present . It also contains seven genes that are resistance towards cobalt zinc cadmium (fig . The whole genome shotgun project has been deposited at ddbj / embl / genbank under the accession no.
Nurse practitioners (nps) and clinical nurse specialists (cnss) have practiced for over 50 years in the united states, followed closely by canada and the united kingdom, and the roles are increasingly being implemented in other countries . The quest for improved quality of care and control of healthcare costs are important drivers in the decision to implement these roles . We conducted a systematic review to assess the evidence of cost - effectiveness of np and cns roles . Nps are defined as rns who have additional education in recognized programs, preferably at the graduate level . They demonstrate advanced competencies to practice autonomously and collaboratively to perform assessments, order laboratory and diagnostic tests, diagnose, prescribe medications and treatments, and perform procedures, as authorized by legislation and their regulatory scope of practice, as well as performing an advanced nursing role that includes consultation, collaboration, education, research, and leadership . Cnss are registered nurses (rns) with a graduate degree in nursing who have expertise in a clinical specialty and perform an advanced nursing role that includes practice, consultation, collaboration, education, research, and leadership . Those working in alternative roles provide similar services to those for whom they are substituting, usually physicians . Those working in complementary roles provide additional services that are intended to complement or extend existing services . The intention of the alternative role is typically to reduce cost or workload or to address workforce shortages while maintaining or improving the quality of care; in contrast, the intention of the complementary role is to improve the quality of care . During the 1970s, the first randomized controlled trials (rcts) of nps demonstrated their safety and effectiveness, as well as patient satisfaction with the np role [617]. Nps improved resource utilization and access to care [14, 1820], increased primary care services in the community, and reduced costs . Over the past 30 years, a number of literature reviews and systematic reviews have summarized the findings of studies evaluating nps [2125]. The reviews have consistently shown no difference in the health outcomes of patients receiving np care when compared to patients receiving physician care, but often both quality of care and patient satisfaction are higher with np care . Most rcts of cns roles have been published since 1980 except one . In 1977, pozen and colleagues found that the cns increased the knowledge of heart disease in patients with myocardial infarction resulting in an increased rate of return to work and a reduction in smoking . Literature reviews and systematic reviews of cnss [25, 27] reveal that cnss are associated with reductions in hospital length of stay, readmissions, emergency room visits, and costs, as well as improvements in staff nurse knowledge, functional performance, mood state, quality of life, and patient satisfaction . Study findings are consistent that nps and cnss, either in alternative or complementary provider roles, deliver high quality patient care that results in high patient satisfaction . To address a question that often surfaces, are nps and cnss cost - effective?, we conducted a systematic review of rcts of np and cns cost - effectiveness (defined broadly to also include studies measuring health resource utilization) entitled a systematic review of the cost - effectiveness of nurse practitioners and clinical nurse specialists: 1980 to july 2012 . The purpose of this paper is to report on the methodological strengths and threats to internal and external validity of these rcts . We sought rcts of np and cns cost - effectiveness between january 1980 and july 2012 . Due to inconsistencies in the use of titles and lack of role clarity for these two roles, we developed specific criteria to decide if the role was an np, a cns, or an rn in an expanded role . To be deemed an np, the nurse had to have completed a formal postbaccalaureate or graduate np education program or be licensed as an np . To be deemed a cns, the nurse had to have completed a graduate degree and the role had to be reflective of the cns role definition . If necessary, we contacted the lead author and/or experts in advanced practice nursing from the country where the study was conducted to determine eligibility . The principal outcomes of interest in this review were objective measures of health system utilization . These included length of stay, rehospitalization, costs of healthcare (e.g., hospital, professional, and family costs), and health resource use (e.g., diagnostic tests and prescriptions). Because it is important to examine health system utilization in the context of patient and provider outcomes, we also extracted data on all patient (e.g., mortality, morbidity, quality of life, and satisfaction with care) and provider (e.g., quality of care and job satisfaction) outcomes . Participants were patients of any age receiving care in all types (e.g., teaching and nonteaching, public and private), sizes (e.g., small, medium, and large), and locations (e.g., rural and urban) of hospitals or community settings (e.g., long - term care, primary care, and home care). Substantive developments since 1980 (e.g., training, payment models, and scope of practice of nps) have reduced the relevance of pre-1980 studies to modern - day policy . In consultation with a policy advisor, we chose to exclude pre-1980 studies from this review . Studies were also excluded if (1) the np or cns education failed to meet our criteria or if we could not contact the author for clarification despite repeated attempts; (2) the np or cns was part of a multicomponent or multidisciplinary intervention in which the impact of their contribution could not be isolated from other healthcare providers on the team; (3) the study evaluated a very specific intervention (e.g., cognitive behavioural therapy) that was delivered by an np or cns but could be delivered by other clinicians, such as an rn; (4) the control group was also exposed to an np or cns during the study; (5) a measure of health system utilization was not included; (6) true randomization was not used (randomization was predictable, for example, assignment by day of hospital admission and alternating assignment). A search was conducted to identify all relevant published and unpublished rcts reported from january 1980 to july 2012 . Medical librarians conducted a comprehensive search of the literature using cinahl, embase, global health, healthstar, medline, allied and complementary medicine database (amed), cochrane library database of systematic reviews and controlled trials register, database of abstracts of reviews of effects (dare), health economics evaluation database (heed), and web of science . Relevant medical subject headings (mesh) keywords, inclusive suffixes, and search strings formed the search strategy (appendix). In addition, the following methods were used to identify primary studies: handsearching of 16 high - yield journals, checking reference lists of all relevant papers and reviews, contacting authors of an early list of relevant studies, searching personal files, reviewing bibliographies, and searching websites of nursing research and professional organizations and national, provincial / state, and territorial governments . We uploaded all identified citations to a web - based reference management program (refworks) and removed duplicate entries . Two - member teams independently screened titles and abstracts of these citations for relevance using prespecified criteria . Translators assisted with the review of all citations in languages other than french or english . The full - text of a published paper and/or study report was obtained if it appeared to meet the inclusion criteria, if an abstract was unavailable, or if it was not possible to determine relevance from the title and abstract review . In instances where a study was reported in more than one paper, we grouped the study's papers in a constellation and collectively reviewed them . Two - member teams independently screened these full - text papers for eligibility based on the inclusion criteria . Two team members (ad and kr) independently assessed the methodological quality of the studies for internal validity and disagreements were resolved through discussion and consensus . The internal validity of each study was assessed using a slightly modified version of the cochrane risk of bias criteria; modifications to the criteria were three - fold . First, we did not assess for blinding of participants and personnel because the nature of np and cns interventions precludes this possibility . Second, outcome assessment and completeness of outcome data were evaluated separately for objective and subjective outcomes within a study . We looked for evidence of key outcomes that would typically be measured for each study's research question . Third, if outcomes had more than 20% missing data, we judged the study to be at high risk of bias for incomplete outcome data . We assessed studies, assigning a high, low, or unclear risk of bias for each of the following eight questions: (1) to avoid selection bias, was the strategy used for random sequence generation likely to produce comparable groups (e.g., random number table, computer random number generator)? (2) to avoid selection bias, was a method used to conceal the allocation sequence so that group allocation could not be foreseen in advance (e.g., sequentially numbered, opaque, sealed envelopes; central allocation office)? (3) to avoid detection bias, was an appropriate method / source used to collect objective (e.g., mortality) measures (e.g., death records, blinding of outcome assessor, trained chart abstracter)? (4) to avoid detection bias, was an appropriate method used to collect subjective (e.g., quality of life) measures (e.g., blinding of outcome assessor; use of reliable, valid, established self - administered questionnaires)? (5) to avoid attrition bias, was outcome data complete for the objective measures (i.e., complete for 80% of sample; missing data balanced between groups; missing data imputed using appropriate methods)? (6) to avoid attrition bias, was outcome data complete for the subjective measures (i.e., complete for 80% of sample; missing data balanced between groups; missing data imputed using appropriate methods)? (7) to avoid reporting bias, were all outcomes described in the methods section of the study reported in the results and were all key outcomes reported? (8) were other biases detected in the study (e.g., contamination bias in which the control group had exposure to the intervention)? We sought clarification from 40 of the 43 study authors when there were insufficient details in the paper to determine the risk of bias and we received 28 (70%) responses . An overall risk of bias was assigned to each study as follows: low risk of bias (at risk in 0 - 1 category), moderate risk of bias (at risk in 2 - 3 categories), high risk of bias (at risk in 46 categories), and very high risk of bias (at risk in 7 - 8 categories). External validity refers to the generalization or applicability of the study to other circumstances . To assess external validity, two team members independently assessed the generalizability of the study population, intervention, control, and outcomes (pico). Historically, rcts of nps and cnss have been criticized because the number evaluated in any study has been small (e.g., one or two nps) causing concern that those willing to be evaluated may be atypical in training, experience, knowledge, skills, or practice characteristics . We consulted with our policy advisor and together decided that 10 nps or cnss either within a single study or across studies combined in meta - analyses would be a reasonable minimum sample necessary to generalize results to similar np or cns roles . As reported in a separate paper, we applied the quality of health economic studies (qhes) instrument [3133] to evaluate the economic analyses in each study . The quality of the body of evidence for individual outcomes was evaluated using the grading of recommendations assessment, development and evaluation (grade) system [34, 35] and gradepro software . A trained research assistant (kr) extracted data from each study into a summary table regarding general information (i.e., author, country, setting, language of publication, and publication status), characteristics of the study (design and group allocation), characteristics of the participants (number per group, sex, ages, and health conditions), characteristics of the intervention (number and type of nps or cnss, education and training, specific role, and comparison intervention), outcomes (health system, patient, and provider), length of follow - up, proportion followed to study completion, and study findings . If the findings of a single study were reported in two or more papers, they were extracted as one study . Team members checked the accuracy of extractions and discrepancies were resolved through discussion and consensus . Studies were categorized into the following six groupings: np - outpatient, np - transition, np - inpatient, cns - outpatient, cns - transition, and cns - inpatient . In a transition role, the np or cns could provide time - limited services designed to ensure healthcare continuity, avoid preventable poor outcomes among at - risk populations, and promote the safe and timely transfer of patients from one level of care to another or from one type of setting to another [37, page 747]. Within these groupings, studies were further categorized into alternative or complementary np or cns role function . The strengths and threats to internal and external validity of the included rcts the searches yielded 4,397 unique records of which 3,981 were excluded during title and abstract review . Based on full - text review of the remaining 416 papers, 351 were excluded based on reasons listed in figure 1 . The remaining 65 papers described 43 relevant rcts (28 studies reported in single papers and 15 studies reported in 37 papers). All studies were published in english . In general, the control intervention was usual care . The distribution of the 43 rcts across groupings was np - outpatient (n = 11), np - transition (n = 5), np - inpatient (n = 2), cns - outpatient (n = 11), cns - transition (n = 13), and cns - inpatient (n = 1). We summarize the results by grouping beginning first with a brief overview of the study characteristics (tables 1 and 2) followed by a description of threats to internal validity (figures 2 and 3). Eleven rcts of nps in outpatient care [3848] met our inclusion criteria (table 1). They were conducted in the united states (n = 7), united kingdom (n = 2), or the netherlands (n = 2). Six studies evaluated nps in alternative provider roles and five in complementary provider roles . The number of nps ranged from one to 20 in np alternative provider studies and from one to four in np complementary provider studies . Some of the trials were quite large with over 1000 patients, while most of the trials examining specific patient populations tended to be much smaller . Overall, six of the 11 rcts were judged to be at low risk of bias (in other words, the methods were of high quality), four at moderate, and one at high risk of bias (figure 2). With regard to selection bias, nine studies used a random sequence generation process that was likely to produce comparable groups; for two studies, we had insufficient information about the sequence generation process to permit judgement, despite contact with one of the authors . Seven trials used an adequate process to conceal allocation so that participants and those enrolling participants could not foresee the group to which the next patient would be assigned . We judged one study as unclear because there was insufficient information and three at high risk of selection bias . All the rcts were judged to be at low risk of detection bias with respect to objective outcome measures (e.g., blood levels and medical records abstraction) and all but two trials were assessed at low risk of detection bias for subjective measures because most used established validated self - report instruments (e.g., sf-12 and sf-36). Two studies were judged as unclear, one because they used self - reported dietary intake and physical activity which can be subject to recall and social desirability bias and the other because clinicians self - recorded the length of time they spent with each patient . Seven rcts were judged to be at low risk of attrition bias for the objective measures; one study reported a follow - up rate less than 80% for a blood cholesterol measure, and two did not report all follow - up rates . The risk of attrition bias for subjective measures was high or unclear for six studies due to failure to report follow - up rates or poor response rates for at least one self- or interviewer - administered questionnaire by last follow - up . One study was judged at high risk of reporting bias because they did not report any patient outcomes such as child's health status, quality of life, or parent satisfaction in a study of the appropriateness of follow - up care after attendance at an emergency department . We rated one study at high risk of other bias because there was substantial baseline imbalance which was not adjusted for in the analyses . Five rcts evaluated nps in a transition role [4953] (table 1). Three studies were conducted in the us, one in canada, and one in the uk . One study evaluated nps in an alternative provider role and four in complementary provider roles . One or two nps were evaluated in each study . The number of patients included in the trials ranged from 54 to 750 and they were conducted at between one and 10 sites . Overall, two studies were judged to be at low risk of bias and three at high risk of bias (figure 2). The trials assessed to be at low risk of selection bias used a random number generator that revealed the intervention assignment when a patient was ready for allocation and a computer generated sequence concealed in sequentially numbered, opaque, sealed envelopes . The other three studies provided insufficient information to fully judge random sequence generation and allocation concealment . All five rcts were judged to be at low risk of detection bias for objective measures as they used abstraction of hospital administrative records or blinded outcome assessment . With respect to subjective measures, two trials were at high risk of detection bias because patients self - reported their smoking cessation success and the np, who delivered the intervention, also collected baseline and outcome data from the comparison groups during a guided interview . All but one trial were at low risk of attrition bias for objective measures as they followed over 80% of participants and this was balanced across comparison groups within each study . With respect to subjective data, three trials scored high for risk of attrition bias due to poor response rates to self- or interviewer - administered questionnaires . Two studies identified outcomes that they planned to measure but did not report, placing them at risk of reporting bias . Two rcts of nps in inpatient settings met our inclusion criteria, both of which evaluated the np in an alternative provider role [54, 55] (table 1). The number of nps in the trials ranged from 2.5 to 4.5 full - time equivalent nps . The number of patients included in the trials ranged from 381 to 821 and each study was conducted at one site . Overall, the two studies were judged to be at low risk of bias (figure 2). Both studies were at low risk of selection bias having used acceptable random sequence generation processes (table of random numbers; computer random number generator) and having concealed allocation through the use of sequentially numbered, sealed, opaque envelopes . Both were at low risk of detection bias as they relied on medical record and hospital database extraction of objective data such as mortality, medical complications, and length of hospital stay . In cases where study participants completed questionnaires, there were reliable, valid measures such as the sf-36 and the minnesota infant development inventory (midi). Both studies were judged to be at low risk of attrition bias for the objective measures but at high risk of attrition bias for the subjective measures . While many of the primary objective outcome data were available for all study participants (e.g., mortality, complications, and length of stay), subjective self - report measures often had response rates less than 80% . We judged both studies to be at low risk of reporting bias and other biases . Eleven rcts [5666] addressed the cns role in delivering outpatient care (table 2). Six studies were conducted in the us, two in the uk, two in the netherlands, and one in china . Four trials evaluated one to six cnss in the alternative provider role, while seven trials evaluated one to nine cnss in the complementary provider role . The number of patients included in the trials ranged from 20 to 643 and the studies were conducted at between one and six sites . Overall, five of the eleven studies were assessed at low risk, four at moderate risk, and two at high risk of bias (figure 3). While seven studies used valid methods to generate the random sequence and were at low risk of selection bias, we judged the remaining four to be at unclear risk of bias because the authors did not include this information in their papers and we did not receive responses to our request for further details . With respect to allocation concealment, five trials were assessed at low risk of selection bias (e.g., central allocation and sealed envelopes) and three at unclear risk of bias because methods were not described . We judged one at high risk of bias because the patients were randomly assigned by the cns to one of the study groups by drawing the next allocation from an envelope; using this method, it is possible that the drawn assignment could be returned to the envelope and redrawn if allocation was deemed unsuitable . Two studies used cluster randomization and allocation concealment was not applicable as the clusters were all randomized at one time . All the studies were rated as low in risk of detection bias for objective outcome measures (e.g., mortality and rehospitalization). Of the nine studies that included subjective outcomes, eight were judged at low risk of bias as they used established, validated instruments, or blinded outcome assessment and one was at high risk of bias because the cns who delivered the intervention also collected data from both groups before and after the intervention via telephone interviews . With respect to attrition bias, two of the studies judged at unclear risk of bias provided insufficient information to assess the completeness of all objective outcome measures and one, judged at high risk of bias, did not have cost data for at least 80% of the study participants . While seven trials were judged to be at low risk of reporting bias, four were judged at high risk because they did not fully report all the outcomes they collected or did not collect all patient - important outcomes that would have been expected (e.g., patient / parent satisfaction with care and quality of life). Finally, one trial was judged at high risk of other bias because they did not adjust for cluster randomization . Thirteen rcts [6779] evaluated the cns in the delivery of transition care in the us (n = 12) and in the uk (n = 1) (table 2). The number of patients included in the trials ranged from 40 to 375 and the studies were conducted at between one and six sites . Overall, three of the thirteen trials were at low risk, eight at moderate risk, and two at a high risk of bias (figure 3). All but one trial used valid methods to generate the random sequence and all but one trial concealed allocation . All trials were rated at low risk of detection bias for objective measures, except one . In this study, the risk of bias was unclear because healthcare utilization outcomes were based on self - report rather than medical record review data . For subjective measures, two trials were judged to be at unclear risk of detection bias because the validity of their scales was not described, and, in one trial, treatment adherence was based on self - report rather than objective measures, such as pill counts and was assessed at high risk of bias . For objective measures, six trials had a low risk of attrition bias but, for five trials, the risk was unclear and, for two, it was high . For subjective measures, four trials had a low risk of attrition bias but, for six trials, the risk was unclear and, for three, it was high . For those studies in which it was unclear, the response rates were not specified or data were imputed; for those at high risk of bias, the follow - up rate was less than 80% . Of the 13 trials, four were at high risk of reporting bias, three of which did not report on all outcomes measured and one of which did not include a measure of health status . One study was at unclear risk of bias because it was unclear if measures reported at baseline should have been reported as outcomes . Finally, seven trials were assessed at high risk of other bias because there were baseline differences between the groups for which adjustments were not made . Only one study, conducted in the us in 1990, evaluated the cns delivering inpatient care (table 2). Two cnss participated in the study, which included 107 patients and was conducted at one site . Overall, the risk of bias for this study was judged as moderate (figure 3). The study, however, was judged to be at high risk of attrition bias because over 20% of patients were dropped from the study after randomization as the intervention they received was changed (e.g., sitters discontinued and control group receiving cns consultation) resulting in unequal distribution of patients in the two groups . The study was also at high risk of reporting bias because they did not report whether the cns and staff nurse intervention influenced patient risk behaviours as intended . Contamination bias was possible because the same staff nurses who received coaching from the cns for intervention group patient management and for charting nursing observations cared for the control group and might have provided the same patient management and charting strategies for them . Because the associated risk of bias was unknown, we judged this as unclear other bias . Overall, we assessed that 18 of the 43 trials (42%) were at low risk, 17 (39%) at moderate risk, and 8 (19%) at high risk of overall bias (figures 2 and 3). Figures 4 and 5 summarize the studies by type of bias . With respect to the np trials, many studies were at high risk of detection bias with incomplete (<80%) follow - up for subjective outcomes (e.g., self - administered scales). In cns trials, a number of studies were at high risk of reporting bias because they either did not report on all outcomes measured or did not include a key outcome that we would have expected . A number of studies (especially smaller studies) had baseline differences with no mention of adjusting the analyses to account for these differences . Some of the potential threats to validity may not in reality be threats, but rather it may be an issue of lack of reporting . There were many instances that we rated categories as unclear risk of bias because there was insufficient information in the paper or from the author to permit judgment of low or high risk of bias . Of the 43 rcts, 70% of the studies were conducted in the united states (n = 30) and the remainder in four other countries: the united kingdom (n = 6; 14%), the netherlands (n = 4; 9%), canada (n = 2; 5%), and china (n = 1; 2%). Given that healthcare systems and np and cns education, role implementation, and scope of practice vary internationally, applicability of study findings from one country to another may be compromised . Some rcts evaluating np and cns roles were conducted across many sites which may enhance generalizability . However, many trials were conducted in single sites, which likely limits the generalizability of study findings . Of the 43 rcts, 13 (30%) studies were published prior to the year 2000 . Given the substantive progress that has occurred in the development of np and cns roles and dynamic changes in healthcare systems internationally, the results of these studies may be less relevant to current - day policy . Although we found a substantial number of eligible rcts, when broken down by grouping, we identified only one dated rct of cnss in the nontransitional care role for inpatient settings . This rct evaluated two cnss providing consultation for a small very particular population of medical - surgical patients requiring sitters due to the risk of self - harm or unpredictable behaviour . Similarly, we identified only two rcts of nps in the nontransitional care role in inpatient settings, both of which were published over 10 years ago . One study evaluated nps caring for a homogeneous population of critically ill infants in a canadian hospital and the other evaluated nps caring for a heterogeneous population of adults admitted to general medical wards in a us - based hospital . Given the existence of only three fairly dated rcts of nps or cnss in inpatient settings and somewhat specific populations, caution is needed in generalizing these results to nps and cnss in other inpatient settings . Nine (21%) trials were conducted with small numbers of patients (n <100) with specific health conditions . The larger studies with patients experiencing common conditions are more readily generalizable to the general population than smaller trials with patients experiencing a specific condition . However, one of the larger trials limited study entry to poor, non - english speaking hispanic people which may limit the generalizability of the findings to other patients seeking primary healthcare . Twenty - seven (63%) of the rcts evaluated one or two nps or cnss, 9 (21%) evaluated three to five, four (9%) evaluated six to nine, and three (7%) evaluated 10 or more all of which were np - outpatient studies . The small number of nps and cnss evaluated in any study raises concern that the results may not be generalizable to colleagues in similar roles . In some cases when study outcomes were similar we were able to combine study findings which increased the number of nps or cnss evaluated for that outcome . About two - thirds of the studies (n = 29; 67%) specified that they evaluated experienced nps or cnss (i.e., nps or cnss who had completed their training at least one year before the evaluation and/or had graduate degrees). One study posed concern, as it compared novice nps who had completed a two - year advanced nursing practice graduate degree in the previous two months with general practitioners who had an average of 16 years work experience . Most studies used reliable and valid outcome measures to evaluate patient - important outcomes such as health status, quality of life, and satisfaction with care which strengthens the generalizability of the findings; however, some studies had very short - term follow - up periods (e.g., two weeks after the patient appointment) which may compromise generalizability of study findings over the long term [39, 48, 60]. The purpose of this paper was to report on the methodological strengths and threats to internal and external validity of rcts of np and cns cost - effectiveness . Based on a comprehensive search of the international literature, we identified 43 rcts, evaluating nps (n = 18) and cnss (n = 25). While 43 rcts sound like a large number of evaluations of nps and cnss, categorizing the studies by np or cns role (i.e., alternative or complementary) and by setting (i.e., outpatient, transition, or inpatient) reveals the areas where further research is still required . For example, we found only one rct of the cns in a nontransitional role in the inpatient setting and only two rcts of the np in a nontransitional role in the inpatient settings, both of which were alternative provider roles . Of the 43 rcts, 70% (n = 30) were conducted in the united states with far fewer conducted in four other countries (canada, china, the netherlands, and united kingdom). In 2011, newhouse et al . They chose to restrict the review to studies conducted in the united states to enhance the applicability of study findings to the united states healthcare system . A recent systematic review that also includes studies conducted outside the united states therefore, we chose to broaden our search to include international studies in order to learn more about where np and cns role evaluations have been conducted and how the roles are being enacted globally . Our assessment of the risk of bias revealed that about two - fifths (n = 18; 42%) of the 43 studies were at low risk of bias, close to the same number (n = 17; 39%) were at moderate risk of bias, and about one - fifth (n = 8; 19%) at high risk of bias . When examined by date, 31% of the 13 rcts published before the year 2000 were at high risk of bias compared to 13% of the 30 rcts published in or after the year 2000 that were at high risk of bias which may mean that study validity is improving over time . In many cases it was unclear if the authors met the risk of bias criteria because the required information was not reported in the paper consequently, we rated a large number of categories as unclear risk of bias . To permit complete and accurate assessment of risk of bias, researchers are encouraged to use a guide such as the cochrane risk of bias criteria when planning and reporting future studies . A clear brief description of the sequence generation (e.g., random number table; computer random number generator) is needed to allow the reader to determine if the process should provide comparable groups . A description of allocation concealment (e.g., sequentially numbered, opaque, and sealed envelope) is important for the reader to determine if allocation to groups could be manipulated . While blinding of participants is not possible in a study incorporating nps or cnss, a description of procedures used to blind outcome assessors and/or the description of valid outcome measures is needed to assess the quality of the study . Completeness of outcome data for each outcome measure and group, including the description of missing data and details of all participants excluded, lost to follow - up (e.g., dropped out of study or died), or reincluded at each stage, also needs to be reported . If researchers do not report outcomes that were measured or key outcomes that would be expected, a clear description is needed of the reasons for failing to report the outcome . A description of how any other biases were managed that threaten the quality of the study should also be reported . More detailed recommendations for reporting rcts can be found in the consolidated standards of reporting trials (consort) 2010 statement [81, 82]. When authors are faced with cutting back on the number of words in a publication, a suggestion is to reduce the introductory sections to provide sufficient space to describe in detail the strategies used to prevent or minimize threats to internal validity . As others have found [5, 24], a challenge in conducting this systematic review was determining the fidelity of the intervention . The definition of the role and the education, training, and experience of the nps or cnss were often inadequately described or missing . When we contacted authors for this information, we found that some studies were conducted with rns who had received as little as a few weeks of training or one course and were then called nps . Over half (n = 27; 63%) of the 43 studies evaluated only one or two nps or cnss and only three trials, all of nps in outpatient settings, evaluated 10 or more . Approximately two - thirds of the studies (n = 29; 67%) evaluated experienced nps or cnss . Researchers are encouraged to include a detailed description of the nps or cnss being evaluated in their study (role in the context of an internationally accepted definition, education, experience in the role, and training for the specific intervention if applicable). Furthermore, evaluations of these roles should not be initiated while the nps or cnss are still novices but rather when they have had sufficient experience in their role (i.e., at least 12 months). Challenging as it is, researchers are encouraged to plan multisite studies, to increase the number of nps or cnss evaluated, to increase the number of patients enrolled in the study, and to account for variations in practice to enhance the generalizability of study findings . Restricting this review to rcts may be viewed as a strength or limitation, depending on the perspective of the reader . Health service settings are complex and research is confounded by multiple variables that challenge the ability to evaluate the effectiveness of an intervention, such as np and cns roles . When feasible, randomization of participants to intervention and control groups is considered the optimal design to control known and unknown complexities and confounding variables [8386]. The quality of evidence in this review demonstrates that it is feasible to conduct well - designed rcts to evaluate the effectiveness of np and cns roles in a variety of settings, remuneration mechanisms, and patient populations . Strengths of our review include use of numerous strategies to identify all rcts in any language (published or unpublished) that met our inclusion criteria, contact with authors and international expert advisors when it was unclear whether a study met our inclusion criteria, use of current education and credentialing criteria to verify that the trial was indeed evaluating an np or cns, use of duplicate assessment by independent reviewers and a consensus process for every stage of the review, use of an internationally recognized and established tool to assess the overall risk of bias of each trial and contact with authors when additional information was required to make our assessment, use of an established tool (quality of health economic studies) to evaluate the health economic analysis in each study, use of grade to evaluate outcome - specific quality of evidence, consideration of external as well as internal validity, grouping of trials by type (np or cns), setting (inpatient, transition, or outpatient), and role (alternative or complementary), and conducting meta - analyses whenever possible . In future publications, we will summarize our assessment of the quality of the economic analyses of each rct and outcome - specific quality of evidence using grade for each of the six groupings . With respect to limitations, despite our attempts to identify all relevant rcts, we may have missed some relevant studies or included some that do not meet our criteria based on author responses, advisor advice, or our interpretation of the description of the education or role . With respect to generalizability, we did not use a specific tool to assess threats to external validity but did consider the country and year of publication, number of nps or cnss in the study, the number of settings, and characteristics of the population, setting, intervention, and outcomes . We do not know how the exclusion of observational studies that investigate the effectiveness of np and cns roles may have influenced our findings . This paper builds on the body of knowledge regarding quality of rcts of np and cns cost - effectiveness (defined broadly to also include studies measuring health resource utilization). We have used an international lens and inclusion criteria that meet today's definitions of the np and cns roles . While almost half the rcts were found to be at low risk of bias, incomplete reporting of study methods and lack of details about np and cns education, experience, and roles make it difficult to fully evaluate the internal and external validity of studies of these roles . Future studies that adhere to current standards for internal validity, such as cochrane risk of bias, consort [81, 82], and grade [34, 35], will contribute to a stronger body of evidence to address policy makers' questions regarding the cost - effectiveness of np and cns roles.
Wnt proteins comprise a large family of secreted glycoproteins that regulate key developmental processes including cell - fate determination, proliferation, motility and the establishment of the primary axis of the body during vertebrate embryogenesis [1 - 3]. Defects in wnt signaling are also implicated in a host of pathologies including cancer and neural tube defects . Wnt ligands can transform cells, and mutations in components of the wnt signaling pathway, such as -catenin, have causative roles in colon cancers in humans, while mutations in dishevelled (dsh) are implicated in neural - fold closure disorders . To date, 18 wnt ligands have been identified in humans . This large number of ligands is paralleled by an equally impressive number of receptors and co - receptors, which are encoded in the frizzled and low - density - related lipoprotein receptor 5/6 (lrp5/6) gene families, which have ten and two members, respectively, in the human genome . Through intensive studies spanning over two decades, a molecular pathway for wnt signaling has emerged (figure 1). Upon binding of wnt to its receptor, either frizzled or a complex comprising frizzled and lrp5/6, a signal is transduced to the cytoplasmic phosphoprotein dsh . There are three dsh proteins in mammals (dsh-1, dsh-2, and dsh-3), and dsh family members in all organisms are comprised of three highly conserved domains: an amino - terminal dix domain (named for dsh and axin), a central pdz domain (named for postsynaptic density-95, discs - large and zonula occludens-1), and a carboxy - terminal dep domain (for dsh, egl-10 and pleckstrin). At the level of dsh, the wnt signal branches into three separate pathways, the so - called canonical, non - canonical or planar cell polarity (pcp), and wnt - capathways (figure 1). In all three pathways dsh is a key transducer of the wnt signal that operates at the plasma membrane or in the cytoplasm . But now, a new study suggests that dsh also functions within the nucleus . To put this study in context for canonical signaling, which mediates gene induction events (figure 1a), wnt signaling utilizes the dix and pdz domains of dsh to induce the stabilization of cytosolic -catenin; this allows for cytoplasmic accumulation and subsequent translocation of -catenin into the nucleus . Regulation of -catenin stability is mediated via a complex of proteins including axin, glycogen synthase kinase 3 (gsk3), gsk3-binding protein (gbp) and casein kinase 1 (ck1). In the absence of wnt stimulation, -catenin is targeted for degradation through the proteosomal pathway via the -transducin repeat containing protein (-trcp), but -catenin is stabilized when a wnt signal is received . In the nucleus, -catenin forms complexes with members of the lef / tcf family of transcription factors and other factors, and mediates transcription of wnt target genes . The non - canonical or pcp pathway mediates cell polarity, cell movements during gastrulation, and other processes, by signal transduction through the pdz and dep domains of dsh, leading to a modification of the actin cytoskeleton (figure 1b). At the level of dsh, two independent and parallel pathways activation of rho requires the formin - homology protein daam1 that binds to the pdz domain of dsh, leads to the activation of the rho - associated kinase rock, and mediates cytoskeletal re - organization . Rac activation is independent of daam1, requires the dep domain of dsh, and stimulates jun kinase (jnk) activity . Other dsh - binding molecules that influence the pcp pathway include strabismus and prickle, but their mechanisms of action remain incompletely understood . The wnt - capathway (figure 1c) wnt signaling through frizzled receptors leads to the release of intra - cellular cain a process mediated through heterotrimeric g - proteins and involving numerous other molecules, including phospholipase c (plc), calcium - calmodulin - dependent kinase 2 (camk2) and protein kinase c (pkc). With such a daunting number of wnt ligands and frizzled receptors, two challenging questions that remain unanswered are whether (and if so which) wnt ligands are specific to particular pathways, and how signals are channeled to each pathway . Notably, some wnt ligands are known to activate both canonical and non - canonical pathways such as wnt3a, whereas others such as wnt5a appear to be specific to non - canonical signaling . Equally elusive is the understanding of how the signal is transmitted from the receptor / coreceptor complex to dsh, although two recent studies have revealed a direct interaction between dsh and frizzled . Most importantly, the way in which dsh couples and distributes wnt signaling into the three signaling branches remains at best poorly understood . Dsh occupies a key position at the crossroads of all branches of the wnt signaling cascade . It has been proposed that both the subcellular localization of dsh and the choice of effector molecules downstream of dsh govern the selectivity of specific pathway activation . Dsh - localization studies in drosophila and recently caenorhabditis elegans have shown a correlation between localization of dsh at the membrane and activation of the pcp pathway . Indeed, mutations in the dep domain, which is required for pcp signaling, show impaired membrane localization that is correlated with impaired pcp signaling . These studies have furthered the hypothesis that the membrane localization of dsh is required for at least one output of wnt signaling . In unstimulated cells, dsh localizes to punctate vesicular structures in the cytoplasm by a process that requires the dix domain; in response to certain wnt ligands, dsh translocates to the plasma membrane or to the perinuclear / nuclear area, and the membrane localization in all cases studied requires the dep domain . The significance of nuclear / perinuclear localization remained unclear, but it is noteworthy that a number of components of the canonical signaling pathway, such as apc, axin, and gsk3, appear to traffic between the cytoplasm and the nucleus along with -catenin [26 - 28]. This multitude of studies forms the background for the current paper by sokol and colleagues in journal of biology, which identifies two additional domains in dsh that modulate both its subcellular distribution and its ability to activate canonical wnt signaling . The first newly identified domain, located carboxy - terminal to the dep domain, modulates localization of dsh through its action as a nuclear export signal . Dsh protein lacking this domain or harboring a mutation in a critical lysine residue strongly accumulates in the nucleus of both xenopus embryos and cultured mammalian cells . Surprisingly, however, these mutant proteins retain their ability to mediate canonical signaling as effectively as wild - type dsh . Pharmacological agents impeding nuclear export, and cellular fractionation studies, further provide evidence that endogenous dsh enters the nucleus, supporting the view that dsh shuttles between the cytoplasmic and nuclear compartments . The authors then identified a second domain, located just carboxy - terminal to the pdz domain, that is required for nuclear localization; the sequence of this domain is atypical for a nuclear localization sequence (nls). Mutation of this second domain abolished nuclear accumulation of dsh in the presence of nuclear export inhibitors and, remarkably, impaired the ability of dsh to induce -catenin stabilization and to transduce the canonical wnt signal . Interestingly, replacement of this atypical nls with the prototypical nls of the t antigen from the simian virus sv40 redirected dsh to the nucleus and largely restored wnt signaling . The authors further bolster their findings by demonstrating that stimulation of cultured mammalian cells with wnt3a results in the accumulation of a portion of endogenous dsh (dvl2 in this case) in / around the nucleus . So what is the role for dsh in the nucleus and is the wnt field ready to accommodate such a role for dsh? This new finding comes as a surprise, because dsh has been studied extensively over the past two decades and its nuclear localization remained unappreciated . To support their conclusions the authors showed that dsh is found in nuclear fractions, but this approach is not fully conclusive for one may argue that dsh exhibits perinuclear localization and co - fractionates with the outer nuclear envelope . The strongest evidence for a nuclear role for dsh comes from experiments in which nuclear import and export are manipulated, showing that import is critical for function . Yet, when the basic conclusion of a nuclear localization and function of dsh is accepted, several questions remain . If dsh function is required in the nucleus for canonical wnt signaling, why is no hyperactivation of the pathway observed by targeting dsh to the nucleus? The authors note this point and postulate that a' steady state' rather than just localization is required for function . However, one would at least be compelled to posit that -catenin, which should be stabilized by such dsh - targeted approaches, should increase signaling, and this was not observed . Perhaps a more salient question is why many studies have observed dsh translocation to the plasma membrane in response to wnt stimulation or frizzled expression, but have not detected dsh in the nucleus . It is possible that a small but selective pool of dsh translocates to the nucleus to mediate canonical signaling while most dsh goes to the membrane . Yet, if this is the case, is the membrane relocalization of the majority of dsh just a gratuitous cellular behavior without meaning? It is possible that nuclear dsh acts in transcriptional regulation independent of -catenin to mediate wnt signaling, as sokol and colleagues have previously suggested for the dsh - binding protein frodo . Perhaps the nuclear localization of dsh will indeed provide clues to elucidate the final frontier of understanding the diverse mechanisms of wnt regulation of gene transcription in the nucleus . A schematic representation of the wnt signal transduction cascade . (a) for the canonical pathway, signaling through the frizzled (fz) and lrp5/6 receptor complex induces the stabilization of -catenin via the dix and pdz domains of dishevelled (dsh) and a number of factors including axin, glycogen synthase kinase 3 (gsk3) and casein kinase 1 (ck1). -catenin translocates into the nucleus where it complexes with members of the lef / tcf family of transcription factors to mediate transcriptional induction of target genes . (b) for non - canonical or planar cell polarity (pcp) signaling, wnt signaling is transduced through frizzled independent of lpr5/6 . Utilizing the pdz and dep domains of dsh, this pathway mediates cytoskeletal changes through activation of the small gtpases rho and rac . (c) for the wnt - capathway, wnt signaling via frizzled mediates activation of heterotrimeric g - proteins, which engage dsh, phospholipase c (plc; not shown), calcium - calmodulin kinase 2 (camk2) and protein kinase c (pkc). This pathway also uses the pdz and dep domains of dsh to modulate cell adhesion and motility . Note that for the pcp and capathways dsh is proposed to function at the membrane, whereas for canonical signaling dsh has been proposed to function in the cytoplasm; a recent study implicates nuclear localization of dsh in this pathway

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