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a 55-year - old man, presented to our hospital, complained of a large pelvic mass, which was found incidentally during a routine ultrasonography. the patient had no history of abdominal or pelvic discomfort, nor any abdominal or pelvic surgery. in addition, the patient had no relevant medical or family history except for diabetes mellitus. upon a digital rectal examination, a large mass with a stone - like hardness was palpated at 5 - 6 cm above the anal verge. a single - phase helical ct (lightspeed ultra ; ge healthcare, milwaukee, wi), spanning the whole abdominopelvic cavity, was performed. the ct scanning parameters included a beam collimation of 8 1.25 mm, a reconstruction interval of 1.25 mm, a pitch of 1.35, a rotation time of 0.8 seconds, a table speed of 13.5 mm / rotation, 120 kv and 160 mas. transverse ct images were obtained 70 seconds after the intravenous injection of non - ionic contrast material (120 ml) at a rate of 3 ml / sec (ultravist 300 ; bayer schering pharma, berlin, germany). the ct images revealed a large, well - defined mass in the presacral space of the pelvic cavity, which seemed to have a long stalk arising from the left gluteus medius muscle (fig. the stalk traversed along the surface of the left iliac bone, and proceeded through the left sciatic foramen, and ended up connecting with the mass. the ct attenuation generated an easily recognizable mass, which was mainly composed of fatty tissue and calcification, with most of the stalk becoming calcified. some focal areas of intermediate attenuation were identified as a mixture between fatty tissue and calcification, and occupied only a limited portion of the mass. mr imaging (horizon 1.5 t ; ge healthcare, milwaukee, wi) was also performed with the use of a torso coil. on both the t1-weighted (366/8, echo train length of zero, 5-mm slice thickness, 2-mm gap, 256192 matrix, 24-cm field of view, 2 signal acquired, and sequence duration of 3 - 4 minutes) and t2-weighted (4300/84, echo train length of ten, 5-mm slice thickness, 2-mm gap, 512256 matrix, 24-cm field of view, 2 signal acquired, and sequence duration of 3 - 4 minutes) mr images, the mass was revealed to mainly consist of the hyperintense and signal void areas that represent fatty tissue and calcification, respectively (fig. 1c - e). also seen are the focal areas of intermediate signal intensity, as shown on the ct images. the gadolinium - enhanced t1-weighted mr images obtained 30 seconds, 60 seconds, and 3 minutes after the intravenous injection of contrast material (gadovist ; bayer schering pharma, berlin, germany), and the focal areas of the intermediate signal intensity around the calcifications observed at 30 and 60 seconds post - injection, became isointense as the surrounding fat tissue at 3 minutes post - injection. the main mass was resected using a transabdominal approach, whereas the stalk was removed through the sciatic foramen. the surgical specimen consisted of a 13 cm, well - circumscribed, hard mass, with a severely calcified external surface. the cut surface of the resected specimen revealed a mass mixed with fatty tissue and calcification (fig. a histopathological examination revealed that the mass was composed of lipoma with chondroid metaplasia (fig. the term mesenchymoma was originally defined by stout (7) in 1948 to describe tumors containing at least two mesenchymal tissues not normally found together. the tumors can be classified as benign or malignant, and most of the benign varieties have been called angiomyolipomas, angiolipomas, chondrolipomas, osteolipomas, and other names depending on the predominant tissue form in the tumor. however, fibrous tissue is found in all mesenchymal tumors, and is not counted as one of the elements (6). conversely, if a mesenchymal tumor is well - defined or encapsulated and composed predominantly of one type of mesenchymal tissue, along with one or more minor element, the diagnosis should reflect the predominant mesenchymal tissue (6). in our case, the diagnosis of a chondrolipoma was appropriate, since the mass was composed of a sufficiently high proportion of both fatty tissue and extensively calcified chondroid tissue, though it was well - defined and encapsulated. two possible explanations for the pathogenesis of cartilage and bone formation in benign mesenchymomas have been proposed (5, 6, 8, 9). the first is that cartilage arises from chondro - osseous metaplasias of adipose tissue, presumably as a result of mechanical stress or trophic disturbance. the close proximity or contact with bone and a large joint, myxoid background, or a lipodystrophy - like change, which are often associated with these tumors, suggest a metaplastic process caused by a trophic disturbance or mechanical stress (8 - 10). a previous immunohistochemical study supported the explanation that the pattern of expression for growth factor-, latent transforming growth factor- binding protein-1 transforming, and bone morphogenetic protein might play an important role in the transformation of multipotential cells into the chondrolipoma (11). considering the proximity of the identified chondrolipoma to the iliac bone of our case study, we agree with the first explanation of chondroid - osseous metaplasia, which states that the mass may be a result of a micro - trauma. our mass occupied both the inside and outside of the pelvic cavity ; however, we could not clarify whether its primary site was the pelvic cavity or the left buttock. according to previously reported cases the only reported case of an abdominal chondrolipoma in a human occurred in the small bowel (8), whereas a chondrolipoma was once documented in the pelvic cavity of a dog (12). considering these reports, we thought that in our case, a chondrolipoma in the buttock might be extended to the pelvic cavity through the sciatic foramen, though the proportion of the mass in the pelvis was greater than in the buttock. there are few reports pertaining to the imaging features of chondrolipomas of various organs, with most cases describing their mr features (5, 9, 13, 14). for these reports, chondroid and fatty tissues were described as intermediate and high signal intensity areas on t1-weighted images, respectively. both chondroid and fatty tissues were identified as having a higher signal intensity area than the surrounding muscle, whereas chondroid tissue was depicted as having a higher signal intensity area than fatty tissue on t2-weighted images. signal void areas corresponding to calcifications were only found in a small portion of these reported cases. for the ct images, fatty tissue was depicted as a low attenuation area (less than -30 hounsfield units), whereas chondroid tissue was depicted as an intermediate attenuation area. in our case, the mass was depicted to be primarily composed of fatty tissue and calcification, which matched the ct attenuation and mr signal intensity. also, we found that additional focal areas of intermediate attenuation or signal intensity corresponded to chondroid tissue. these findings were different from those described in previously reported cases whereby, in our case, the calcification was much more extensive, and the signal intensity of chondroid tissue on the t2-weighted images was intermediate. moreover, we found extensive calcification located peripherally in a linear and rosary pattern. a previous report also cited the observation of chondroid tissue in a rosary pattern (9). despite the very limited number of reported cases, we propose that the rosary pattern of either the chondroid tissue or calcification could be a characteristic finding of chondrolipomas, and in our case, the calcification in a rosary pattern was likely a consequence of the dystrophic change involving the chondroid tissue. moreover, the signal intensity of chondroid tissue was intermediate on the t2-weighted images, and could be explained by the presence of fibrous tissue within the chondroid tissue (fig. 1i), which reduced the signal intensity of chondroid tissue below the fatty tissue. it is interesting that the focal areas of intermediate signal intensity around the calcifications showed delayed enhancement on the gadolinium - enhanced mr images. (9) described the presence of enhancing foci between the fatty tissue and chondroid tissue in a chondrolipoma, without clarifying the histopathological findings. also, there have been no reports of chondrolipomas with delayed enhancement. in our case, however, keeping in mind that the location of these focal areas corresponded to chondroid tissue, and that chondroid tissue is usually not enhanced, even on the delayed phase, the delayed enhancement of these focal areas may be attributed due to sufficient fibrous tissue within the chondroid tissue. despite the characteristic imaging findings of chondrolipomas, fatty tumors such as lipomas or liposarcomas, which contain chondro - osseous differentiation, the treatment of choice for chondrolipomas is surgical excision (5, 8, 14). conclusively, we experienced the only reported human case of a chondrolipoma in the pelvic cavity, and found that it had characteristic imaging features consistent with fatty tissue and calcification, as well as the areas corresponding to cartilage. | a chondrolipoma is an extremely rare form of a benign mesenchymal tumor containing mature cartilage and fatty tissue. chondrolipomas may be found in almost any part of the body, particularly in the connective tissue of the breast, head and neck area, as well as in the skeletal muscle. however, to the best of our knowledge, chondrolipomas located in the pelvic cavity have not been reported. in this case report, we describe a case of a chondrolipoma in the pelvis, and show that it has its own characteristic imaging findings, which included the composition of fatty tissue and calcification in most parts, as well as some focal areas of chondroid tissue based on the ct and mr findings. |
esthesioneuroblastoma, or olfactory neuroblastoma, was first described by berger, in 1924 as l'esthsioneuropithliome olfactif.1 since then several hundred patients have been described in case series, single - institution reviews, and meta - analyses.2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 patient presentation is often nonspecific, including congestion and sinusitis - like symptoms, which makes diagnosis challenging, such that most patients have advanced disease at the time of diagnosis.15 18 27 the gold standard for the treatment of esthesioneuroblastomas is craniofacial resection with histologically proven disease - free margins, with the use of radiotherapy.2 7 8 10 12 20 25 the use of chemotherapy, neoadjuvant therapy, neck dissection, and irradiation, however, remains controversial.5 6 7 11 14 22 esthesioneuroblastoma has an established propensity for being locally aggressive with the possibility of distal metastases and leading to decades - delayed recurrences.5 11 12 20 in an effort to prolong disease - free survival, most advocate for radical resection with clear margins, which has been shown to double disease - free survival, especially in the setting of advanced kadish tumors.2 5 7 28 additionally, greater tumor extent often necessitates substantial resections, which have both been associated with complication rates as high as 33%.28 29 30 more extensive disease, meticulous surgical resection, and aggressive adjuvant therapies together increase the likelihood of adverse events, including cerebrospinal fluid (csf) leak, neurologic deficits, and infectious complications. we performed a retrospective chart review to describe the history of a single patient with kadish d esthesioneuroblastoma, who underwent endoscopic - assisted craniofacial resection followed by adjuvant radiation and chemotherapy. his course was complicated by multiple infections and csf leaks necessitating several skull base reconstructions. we describe his case to elucidate the multiple and successive escalations for reconstruction available to skull base surgeons, even in the face of malignancy, radiation, chemotherapy, and infection. a 61-year - old man, with a several decade history of chronic sinusitis, presented with a 5-month history of nasal congestion, decreased sense of taste and smell, and intermittent yellow nasal drainage, which failed to resolve with antibiotics, scant epistaxis, left ptosis, and medial periorbital edema. there was a homogenously enhancing, erosive left skull base lesion with extension into the right nasal cavity, bilateral ethmoid, left maxillary, frontal, and sphenoid sinuses with anterior cranial fossa extension and leptomeningeal enhancement (fig. 1). two mildly enlarged fludeoxyglucose (fdg)avid left parapharyngeal lymph nodes were seen on positron emission tomography computed tomography (pet - ct). coronal and axial t1-weighted contrasted mri showing large, avidly enhancing skull base mass centered in the left nasal cavity. the lesion abuts and laterally displaces the left orbit and has extensive intracranial invasion with leptomeningeal enhancement but without evidence of cavernous sinus invasion. he subsequently underwent a combined level 1 transbasal and endoscopic endonasal approach for complete, margin - free resection. at the start of the procedure, the bilateral middle and superior turbinates were removed, and tumor was resected from the nasal cavity, ethmoid and sphenoid sinuses, and frontal recess. this was followed by a transbasal level 1 craniotomy with removal of the medial orbital bar and both intra- and extradural tumor resection and resection of the skull base dura. all margins were negative and reconstruction was performed with a large vascularized pericranial flap on - lay, four standard and two firm nasopores (polyganics, the netherlands), and nasal trumpets. the patient was discharged home on postoperative day (pod) 5 without any perioperative complications. the patient then received intensity - modulated radiation therapy (imrt) with 54 gy to the tumor bed and 70 gy for his cervical lymph node disease, with two cycles of cisplatin and etoposide concurrently. after completing his therapy, 3.5 months after surgery pet - ct 6 months after surgery revealed decreased size and activity of the parapharyngeal nodes as well. his postoperative course was complicated by radiation - induced dysphagia, treated with temporary percutaneous endoscopic gastrostomy (peg) tube placement, and nasal crusting, managed with periodic debridements and antibiotics when indicated. coronal and axial t1-weighted contrasted mri 3.5 months after surgery and concurrent radiation and chemotherapy showing extensive resection without evidence of residual or recurrent disease. he was regularly followed by the neurosurgical, otolaryngology, radiation, and medical oncology teams. nearly 1 year after surgery, he presented with progressive left eye ptosis and painful forehead and left canthal lesions. magnetic resonance imaging (mri) showed avid enhancement and a neofrontal sinus in the epidural space (fig. he was taken to the operating room 15 months after his initial resection for endoscopic debridement, biopsies to rule out intranasal recurrence, and fine - needle aspiration (fna) of the forehead masses. he was found to have significant mucopurulence, osteoradionecrosis of portions of his frontal bone, which were then removed, and shrinking of his pericranial flap with an airspace between the pericranium and frontal bone (fig. he then underwent fna of the forehead mass and left endoscopic dacryocystorhinostomy (dcr). no malignancy was found, but forehead cultures grew proteus mirabilis and klebsiella pneumoniae for which he was treated with intravenous (iv) metronidazole and ceftriaxone for 8 weeks. after extensive multidisciplinary discussions, the decision was made to proceed with removal of the necrotic bone flap. coronal, sagittal, and axial, respectively, t1-weighted contrasted mri demonstrating frontal bone osteoradionecrosis with creation of a neofrontal sinus within the epidural space and enhancement concerning for infection. intraoperative endoscopic images showing frontal sinus mucopurulence and osteonecrotic bone, as well as defect in pericranial flap coverage over the right - sided anterior skull base. seventeen months after initial resection, the patient underwent craniectomy, irrigation and debridement, titanium mesh cranioplasty, and skull base reconstruction with an autologous fascia lata graft. there was no gross extracranial infection, but an obvious epidural collection with fibrinous exudate, and osteomyelitis and necrosis of the orbital bar. we also encountered a focal dehiscence in the pericranium, allowing communication between the endonasal and intracranial spaces. after extensive irrigation and debridement, cranial reconstruction was performed with titanium mesh, which was contoured to the nasal bridge and provided medial orbital bar reconstruction. he was discharged home on pod 5 with 4 weeks of iv metronidazole and ceftriaxone. cultures revealed mycobacterium chelonae for which he was treated with additional iv amikacin, iv tigecycline, and po clarithromycin for an additional 2 weeks, and then continued on po doxycycline and clarithromycin for 12 weeks. four months later, ct revealed a persistent intracranial air pocket with an area of presumed continued nasal - intracranial communication. after completing his antibiotic regimen, the patient underwent cranial reconstruction with a custom polyetheretherketone (peek) bone flap with an orbital bar extension and vascularized left radial forearm free flap, to cover the anterior skull base defect, 5 months after the craniectomy (22 months after initial surgery). reconstruction required partial takedown of the pericranial flap laterally to access the skull base posteriorly to the level of the planum sphenoidale. we encountered a small area of csf leak around the left orbit that was reconstructed using primary closure and tisseel fibrin sealant (baxter international inc., deerfield, illinois, united states). the vascularized radial forearm myofascial flap was anastomosed to the superficial temporal artery and vein and was found to be well perfused and laying freely. it was secured to the dura and skull base, and nasal endoscopy confirmed complete skull base closure. the patient experienced acute altered mental status and disinhibition, ct revealed a large bifrontal epidural hematoma, and the patient was taken emergently to the operating room for evacuation (fig. another small csf leak was noted and also repaired with primary closure, tisseel fibrin sealant, and placement of a lumbar drain. his mental status gradually returned to baseline and he was discharged home on pod 7. coronal, sagittal, and axial, respectively, t1-weighted contrasted mri performed immediately after skull base reconstruction, vascularized myofascial flap, and peek cranioplasty. he returned to the emergency department 2 days after discharge after reports of strange disinhibited behavior. ct revealed intracranial air with a new endonasal - intracranial focus of communication, suspicious for tension pneumocephalus (fig. 7). he was taken to the operating room for yet another skull base reconstruction where the new defect was clearly noted. an alloderm (lifecell corp., woodlands, texas, united states) graft was cut to size and sutured both to the planum sphenoidale and the periorbita bilaterally. additional alloderm was used as an on - lay over the friable frontal lobe dura and secured with evicel fibrin sealant (johnson & johnson wound management, somerville, new jersey, united states). the myofascial flap was reapproximated and reconstruction was again confirmed with nasal endoscopy (fig. he had a gradual return to his intact neurologic baseline and was discharged home on pod 9. coronal ct scan showing two foci (arrows) of breakdown in the skull base reconstruction with extensive bifrontal pneumocephalus. the patient returned 1 month later to the emergency department (ed) with complaints of 2 days of severe progressive headache and subgaleal fluid collection. he was treated with 5 days of csf drainage via a lumbar drain and discharged on hospital day 8, after observation for two days after drain removal. unfortunately, he returned with recurrence of the subgaleal fluid collection, and moderate amount of fluid expressed from a forehead pustule. imaging revealed increase in the subgaleal and epidural fluid, and he was once again admitted for an epidural - peritoneal shunt, without immediate perioperative complications. however, the epidural fluid culture later returned as enterobacter cloacae and was treated with 8 total weeks of iv meropenem. he also complained of abdominal pain concerning for infectious or chemical peritonitis, while abdominal ct revealed fat infiltration and a right lower quadrant fluid loculation. he was reluctant to have his shunt removed due to concern for fluid reaccumulation and possible subsequent infection. seven weeks after shunt placement, the patient presented to the ed with rhinorrhea and pneumocephalus and was taken for shunt removal and epidural drain placement. four days later, he underwent yet another skull base reconstruction with repositioning of the still viable myofascial flap, additional buttressing with an abdominal fat graft, and insertion of lumbar drain. his postoperative course was complicated with abdominal hematoma that was evacuated at bedside, and he was discharged home on pod 8. after the aforementioned 8 weeks of iv meropenem, the patient was transitioned to 4 weeks of po levofloxacin and continued to be monitored with imaging and clinical nasal endoscopy. mri performed 26 months after initial resection then revealed right temporoparietal and falcine nodular dural enhancement concerning for metastatic disease (fig. coronal and axial t1-weighted contrasted mri showing the right - sided temporoparietal convexity and parfalcine nodular enhancing lesions (arrows) suspicious for metastatic disease. the first, and most commonly used, staging system for esthesioneuroblastoma was described in 1976 by kadish and was later modified by foote to include involvement of cervical lymph node and distant metastases.19 high kadish classification, especially with cervical lymph node involvement, is known to be a profound negative prognosticator, cutting survival in half and increasing the rate of distal metastasis by 35%.4 17 19 23 on the other hand, aggressive surgical resection, with clear margins, is a strong predictor of disease freedom.2 7 18 28 anterior skull base tumors with positive margins were found to have double the incidence of local recurrence and half the survival of complete resections.7 28 the widespread use of craniofacial approaches has improved our ability to obtain complete resections with histologically disease - free margins, even in the setting of extensive, high kadish stage tumors.2 7 10 12 23 25 craniofacial resection has been shown to increase progression - free survival from 37.5 to 82%.7 14 23 28 despite the favorable outcomes with extensive resection, single - modality treatment, including surgery in isolation, led to poor results and higher rates of both local recurrence and distal metastatic disease.15 18 besides radical resection, the highest control rates were found with the addition of radiation therapy. dulguerov performed a meta - analysis of 390 patients which ultimately showed the best outcomes in esthesioneuroblastoma occurred when margin - free resection was followed by radiotherapy ; this has also been corroborated by several large studies.7 12 20 although controversial, many groups argue for the addition of chemotherapy as well, to achieve the longest duration of tumor freedom.18 24 multimodality treatment is especially recommended in those with high kadish stage esthesioneuroblastoma.15 although aggressive management of these persistent malignant tumors is advocated, there are treatment effects and complications that should be considered. postoperative complication rates for craniofacial approaches can affect one in three patients undergoing anterior craniofacial resection based on an international collaborative study, with wound complications in 18% of patients.28 although there have been significant advances in skull base reconstruction, especially from endoscopic approaches, csf leak still remains a significant concern.12 31 some studies advocate the use of synthetics, whereas others use vascularized nasoseptal flaps, and still other groups encourage the use of gasket seals.5 12 31 ultimately there are numerous options available to the surgeon, which can be used simultaneously or even sequentially, should the need arise. unfortunately, prior radiation treatment and wide intracranial tumor extension increase the risks of complications, including infection and leak.28 29 30 patients requiring postoperative radiation therapy will likely also incur decreased vascularization and increased fibrosis.32 33 over time, most free flaps, without vascularization, will reabsorb, and these flaps can be salvaged by buttressing them with vascularized flaps from the outset.32 additionally, in the setting of radiation, allografts have been shown to have a high rate of extrusion.32 34 pedicled pericranial flaps are the most frequently used and easily accessible flaps and have been proven successful.34 nasoseptal flaps have also been shown to have tremendous success, even in the setting of wide extensive tumors.31 32 however, in the setting of malignancy, they can only be safely used if histologically confirmed to be tumor - free. unfortunately, in our case, this was not enough to prevent breakdown, with subsequent nasal - intracranial communication and an epidural, neofrontal sinus. pedicled myocutaneous and myofascial flaps have also been used with success, including the pectoralis major, latissimus dorsi, and trapezius muscles. they have both the ability to fill dead space, dampen the effect of csf and brain pulsations, and have a rich vascular supply.32 although this provided a good salvage reconstruction for our patient, it was not without its own complication, a symptomatic epidural hematoma requiring emergent evacuation. free tissue transfer and synthetic allografts are also available options, particularly when supplementing more robust vascularized flaps, in the setting of chemotherapy and radiation. studies have shown that greater tumor extent, especially in the setting of intracranial extension, cervical lymph node involvement, and distal metastases are associated with greater rates of recurrence and poorer prognoses.4 17 19 23 in light of this, initial treatment, both medical and surgical, should be aimed at curing the primary disease. measures should be taken to ensure adequate skull base reconstruction from the outset, as well as any risk mitigating actions that can be performed. nevertheless, sequential escalation of varied repair techniques is possible and often necessary in these challenging cases. early use of vascularized flaps should be considered after aggressive skull base resections, radiation, and chemotherapy. ultimately, there are several options available to surgeons, and although precautions should be taken whenever possible, risk of wound breakdown, leak, or infection should not preclude radical surgical resection and aggressive adjuvant therapies in the setting of malignancy. | introduction advanced kadish stage esthesioneuroblastoma requires more extensive resections and aggressive adjuvant therapy to obtain adequate disease - free control, which can lead to higher complication rates. we describe the case of a patient with kadish d esthesioneuroblastoma who underwent multiple surgeries for infectious, neurologic, and wound complications, highlighting potential preventative and salvage techniques. case presentation a 61-year - old man who presented with a large left - sided esthesioneuroblastoma, extending into the orbit, frontal lobe, and parapharyngeal nodes. he underwent margin - free endoscopic - assisted craniofacial resection with adjuvant craniofacial and cervical radiotherapy and concomitant chemotherapy. he then returned with breakdown of his skull base reconstruction and subsequent frontal infections and ultimately received 10 surgical procedures with surgeries for infection - related issues including craniectomy and abscess evacuation. he also had surgeries for skull base reconstruction and csf leak, repaired with vascularized and free autologous grafts and flaps, synthetic tissues, and csf diversion. discussion extensive, high kadish stage tumors necessitate radical surgical resection, radiation, and chemotherapy, which can lead to complications. ultimately, there are several options available to surgeons, and although precautions should be taken whenever possible, risk of wound breakdown, leak, or infection should not preclude radical surgical resection and aggressive adjuvant therapies in the treatment of esthesioneuroblastoma. |
studies from the united states, europe and hong kong indicate that young children are at risk for serious illness and hospitalization from influenza virus infection.,,,, in the united states, 80% of influenzaassociated hospitalization in children occurs in those 38c and cough or sore throat in the absence of other known causes (e.g., urinary tract infections, dengue fever etc). patients reporting an onset of symptoms more than 5 days before admission were excluded. the study was explained to caregivers and written informed consent was obtained. we then obtained a medical history, conducted a physical examination and collected respiratory swab specimens. all patients were tested using one of two rapid influenza diagnostic tests and also influenza viral culture upon admission. other medical investigations and treatments were provided by attending physicians according to the routine standard of care. these tests detect influenza type a and yield results in 1530 min, but are not able to detect influenza type b. nasal swabs were collected with plain cotton swab for both rapid tests. nasopharyngeal swabs for influenza virus isolation were collected with dacron swab and placed in viral transport media composed of nacl 1 gm, k2hpo4 1 gm, beef extract 1 gm, peptone 3 gm, add dw2 up to 200 ml, add antibiotics to make final concentration as follow ; penicillin 1000 unit / ml, streptomycin 1000 g / ml, fungizone 5 g / ml and ph was adjusted to 7.0 0.2 with 1 n naoh, vtm then transported chill to the thai national influenza center, national institute of health, ministry of public health for influenza virus isolation. the specimens were cultured using madin darby canine kidney (mdck) cells and observed for cytopathic effect. isolated viruses were confirmed as influenza and typed by reference antisera for influenza a or b with immunofluorescent staining technique. patients with a positive rapid test were treated with oseltamivir (tamiflu) according to the recommended doses per kilogram. because tissue cell culture is considered the gold standard diagnostic method, only children with a positive viral culture were included in this analysis. clinical findings of influenza and noninfluenza patients were compared using chisquare test and considered significant at p 38.6c, conjunctival injection and stridor were significantly higher in influenza patients (p 38.5c) (100%), cough (100%), constitutional symptoms (anorexia, irritability, and lethargy) (100%), rhinorrhea (86%), vomiting (71%), respiratory distress (43%), and seizure (14%). the features were similar to cultureconfirmed patients except the fever ; all seven patients had high fever (t > 38.5c) while in cultureconfirmed influenza the majority (59%) had high fever and the rest had lowgrade or no fever (table 2). the clinical diagnosis on admission for these children with cultureconfirmed influenza included lower respiratory tract infection (pneumonia, bronchitis, bronchiolitis, and viral croup), upper respiratory tract infection (rhinopharyngitis and pharyngitis), influenza, viral myositis, and gastroenteritis (table 3). clinical diagnosis on admission among 39 influenza patients, 20 (51%) required oxygen therapy. two patients needed mechanical ventilation ; one case was previously healthy and the other had underlying sotos syndrome with recurrent pneumonia. the length of the hospital stay was 124 days, the highest percentage was between 610 days (23.1%) and 48.8% required hospital stays of 5 days or longer (table 4). length of hospital stay rapid tests for influenza a was evaluated compared to viral tissue cell culture. directigen flu a was tested in 394 patients and now flu a was tested in 62 patients (according to changes in the test supplied by the department of communicable diseases control). the sensitivity of directigen flu a was 50% and specificity was 98% while now flu a had sensitivity 33% and specificity 98% (5, 6). sensitivity and specificity of directigen flu a sensitivity and specificity of now flu a positive viral cultures for influenza a but not influenza b (34 from 39 totals) were used to evaluate sensitivity and specificity of the rapid tests, since the tests specified for influenza a diagnosis. directigen flu a was tested in 394 patients and now flu a was tested in 62 patients (according to changes in the test supplied by the department of communicable diseases control). the sensitivity of directigen flu a was 50% and specificity was 98% while now flu a had sensitivity 33% and specificity 98% (5, 6). sensitivity and specificity of directigen flu a sensitivity and specificity of now flu a positive viral cultures for influenza a but not influenza b (34 from 39 totals) were used to evaluate sensitivity and specificity of the rapid tests, since the tests specified for influenza a diagnosis. during 20042005, 9% of children 5years old that were hospitalized with lower respiratory tract infection or ili were culturepositive for influenza virus infection. because our study did not use the more sensitive rtpcr or serological laboratory diagnostic methods, influenza was isolated throughout the year round and peak activities occurred during june to october and january to march, a finding that was consistent with published reports.,, of interest, peak influenza activity in thailand usually follows the beginning of school year in the middle of may each year. school aged children are believed to play a major role in the propagation of seasonal influenza epidemics.,, the clinical manifestations of influenza infection in our study are similar to those previously described. fever, cough, rhinorrhea, and constitutional symptoms are the most common presenting symptoms. influenza in young children was significantly associated with highgrade fever (temperature > 38.6c). and we found unusual diagnoses included viral myositis and gastroenteritis which shows that in young children influenza virus infection may not present with respiratory tract infection. in our study, seizures were presented in four (10.3%) of influenza patients probably due to highgrade fevers. neurological manifestations of influenza virus infection, including febrile seizures, encephalitis, and meningitis have been reported and warrant further study in thai children., while the rapid test is proved very useful to identify influenza patients to conduct research and guide treatment decisions, the sensitivity of the tests was less than that reported elsewhere., the sensitivity of now flu a test and directigen flu a were only 33% and 50%, respectively. these markedly low figures were probably due to two reasons ; first, most of the patients in our study were enrolled at the 4th or 5th day of illness, a time when influenza viral antigen may no longer be present in the nasopharynx in sufficient quantities to yield a positive rapid test. second, we used of a nasal swab specimen rather than a nasal wash, a nasopharyngeal aspirate or a nasopharyngeal swab specimen that is recommended by the manufacturers. this finding underscores the importance of proper training and supervision of clinical staff who use rapid influenza diagnostic tests. in conclusion, our study demonstrates that influenza virus infection is an important cause of hospitalization in healthy thai children < 5 years of age. clinical characteristics of influenza in young children are highgrade fever, cough, constitutional symptoms, rhinorhea, and abnormal breath sounds. especially during the months of july to october and january to march when influenza activity appears to be highest, influenza infection should be considered in the differential diagnosis when caring for patients with acute upper or lower febrile respiratory infection. rapid tests for influenza a had a moderate sensitivity and high specificity that can help guide clinical treatment and the wider application of these tests in thailand should be considered. further research on influenza infection in thai children using rtpcr, serology, and tissue cell culture laboratory diagnostic method is needed to improve clinical care, inform vaccine introduction decisions, and to develop improved practices for nonpharmaceutical interventions to control seasonal epidemics. | background studies in north america and europe have shown that young children are at increased risk of serious complications and hospitalization from influenza infection. in thailand, however, influenza is commonly considered a mild infection that rarely requires hospitalization. an improved understanding of the burden of serious complications from influenza infection in young children is needed to inform clinical treatment and vaccination guidelines. methods we conducted a prospective study of children 05 years of age with lower respiratory tract infection or influenzalike illness admitted to a pediatric tertiarycare hospital in bangkok, thailand during july 2004 to july 2005. all respiratory specimens were tested for influenza using a rapid antigen test and tissue cell culture. results thirtynine of 456 (8.6%) hospitalized children had culturepositive influenza. eighty percent of hospitalized influenza patients had no underlying chronic illnesses. nineteen (49%) influenza patients required hospital stays of 5 days or more and two patients required mechanical ventilation. influenza activity demonstrated bimodal seasonal variation with peak activity from august to october and january to april. cough was present in 38 (97%) cases and fever > 38.5c was significantly associated with influenza. conclusion influenza is an important cause of hospitalization in children < 5 years of age in thailand. children < 5 years should be considered as a target group when establishing clinical guidelines for antiviral treatment and influenza vaccination. |
sleep loss and sleep disorders are among the most common health problems, but are frequently overlooked. it is estimated that about one - third of people in the general population suffer from a chronic disorder of sleep and wakefulness.1 insomnia is a subjective complaint that sleep is difficult to initiate or maintain, or that it is nonrefreshing or of poor quality and quantity.2 insomnia affects approximately 20%40% of older adults at least a few nights per month.3,4 it is an important concern in elderly patients, not only because it is common, but also because it can cause multiple daytime impairments, such as difficulty sustaining attention or alertness, a slowed response time, impaired memory and concentration, and decreased performance. insomnia is also associated with a shortened lifespan because of an increased risk of heart disease, stroke, cancer, and suicide.5,6 insomnia is mostly treated with benzodiazepine and nonbenzodiazepine hypnotic drugs, which potentiate the central nervous system suppressant activity of gamma aminobutyric acid a receptors in the brain. however, residual daytime effects, development of dependence, and withdrawal symptoms associated with most hypnotic drugs are of considerable concern in public health.7 lactuca sativa (garden lettuce), a member of the compositae family, is a very popular salad herb in egypt.8,9 its seed oil has been in use in folk medicine since ancient times as a sleeping aid, as well as to relieve pain and inflammation.10,11 a crude methanol / petroleum ether extract from l. sativa seeds has demonstrated a time - dependent and dose - dependent analgesic effect in a formalin test and also dose - dependent anti - inflammatory activity in a carrageenan model of inflammation.12 l. sativa seed oil (sedan, pharco pharmaceutical company, alexandria, egypt) is marketed as a soft gelatin capsule containing 1000 mg of the purified expressed oil. gas liquid chromatography shows that the l. sativa seed oil capsule is comprised mainly of oleic acid (61.5%), stearic acid (20.4%), palmitic acid (9.7%), myristic acid (2.8%), cispalmitoleic (1.2%), behenic acid (0.5%), and lignoseric acid (0.3%), with b - sitosterol and b - amryn also present. despite its well - known safety over thousands of years of use, l. sativa seed oil has only recently been subjected to extensive pharmacological and toxicological investigation. it was found to have significant sedative, analgesic, and anti - inflammatory activity in a murine model,13 and in the same study was shown to have significant anticonvulsant activity against convulsions induced by pentylenetetrazole and to potentiate the hypnotic effect of barbiturates. the oral ld50 was found to be 19.75 ml / kg.13 the scientific data from this animal research in addition to the extensive anecdotal experience of successive generations of traditional egyptian herbalists, prompted our research group, which has a special interest in traditional herbal medicines, to undertake a pilot clinical study to evaluate the sedative and hypnotic effects of l. sativa seed oil in patients complaining of sleeping difficulty with or without anxiety. this was a prospective single - blind, randomized, placebo - controlled trial performed in two outpatient clinics in alexandria, one at the mamorah psychiatric hospital and the other at the green clinical research center. sixty consecutive patients presenting with insomnia or sleeping difficulty were recruited for the study after careful medical history - taking and a physical examination. patients were included if they were aged over 18 years and had a presenting complaint of insomnia, defined as frequently upset sleep quantity / quality or difficulty in initiating or sustaining a normal deep sleep pattern. dementia of the alzheimer s type was not an exclusion criterion if the patient had a supportive partner / caregiver. patients with concomitant organic or neuropsychiatric disease necessitating use of tranquilizers, sedatives, anticonvulsants, or psychotropic agents were excluded. patients who were concomitantly receiving any other conventional or alternative medical intervention which might influence the study outcome, including all other herbal products, hypnotherapy, acupuncture, homeopathy, and yoga, were also excluded. patients were asked not to use any of these concomitant interventions during the course of the study, and were excluded from the analysis if they did so. critically ill patients with severe heart, renal, or liver dysfunction were excluded, as were patients whose condition precluded verbal communication, unless they were accompanied by a supportive partner / caregiver. the protocol was approved by the local research ethical committee (iorg0006902 green clinic and research center irb # 1), and each subject signed a consent form. group 1 (n = 30) received one sedan capsule every night for 1 week and group 2 (n = 30) received a matching placebo. randomization was done using a software - generated block randomization technique and the patients were blinded to their allocated drug. double - blinding was not possible in this pilot study because the baseline examination, supply of medication, and final assessment were all carried out by the same investigator at each clinic. the effect of sedan vs placebo on both the modified state - trait anxiety inventory (stai) score14 and a sleeping difficulty score was assessed at the end of 1 week of treatment. the modified stai scale comprises 20 questions, each of which is answered on a five - point scale, from 1 (nil) to 4 (very much). the sleeping difficulty scale was a 10-item questionnaire based on the items of the leeds sleep evaluation questionnaire15 and assessed subjective patient estimates of and satisfaction with sleep quality using a three - point rating scale (0 = rare / nil, 1 = sometimes, and 2 = often). the higher the total score on both scales, the worse the patient s insomnia was deemed to be. both questionnaires were administered verbally at baseline and at the end of 1 week of treatment. both instruments were translated into the arabic language by the investigators and had been validated previously in patients with anxiety and insomnia treated at mamorah psychiatric hospital (unpublished data). clinical global impression of change (cgi - c) scores were also recorded by the investigator at baseline after 1 week of treatment. the cgi - c is a seven - item symptom rating scale (0 = not assessed, 1 = very much improved, 2 = much improved, 3 = minimally improved, 4 = no change, 5 = minimally worse, 6 = much worse, 7 = very much worse). data were analyzed using the computer software package spss (v 14 ; spss, inc, chicago, il). sample size was calculated using sigmastat (v 3.5 for windows ; systat software, inc, chicago, il). the minimum sample size needed for each treatment group was calculated to be 23 subjects to detect a difference of 3.0 points in mean sleep scale scores with a standard deviation (sd) of 3.5, 80% power, and a 0.05 two - tailed level of significance. a paired - samples t - test was done to compare the mean scores for each group before and after treatment. an independent - samples t - test was done to test for a significant difference between any improvements in mean score in each treatment group. multivariate analysis of covariance with treatment as the main independent factor and the study center as a controlling factor was used in full factorial model testing for main effects plus any possible interaction effects. baseline pretreatment scores were used as covariates to control for initial group differences potentially affecting the dependent outcome variables, ie, any differences between baseline and posttreatment stai and sleep scores, and their interaction. parametric statistical methods were utilized after ascertaining the normality of distribution of the dependent variables by kolmogorov the effect of sedan vs placebo on both the modified state - trait anxiety inventory (stai) score14 and a sleeping difficulty score was assessed at the end of 1 week of treatment. the modified stai scale comprises 20 questions, each of which is answered on a five - point scale, from 1 (nil) to 4 (very much). the sleeping difficulty scale was a 10-item questionnaire based on the items of the leeds sleep evaluation questionnaire15 and assessed subjective patient estimates of and satisfaction with sleep quality using a three - point rating scale (0 = rare / nil, 1 = sometimes, and 2 = often). the higher the total score on both scales, the worse the patient s insomnia was deemed to be. both questionnaires were administered verbally at baseline and at the end of 1 week of treatment. both instruments were translated into the arabic language by the investigators and had been validated previously in patients with anxiety and insomnia treated at mamorah psychiatric hospital (unpublished data). clinical global impression of change (cgi - c) scores were also recorded by the investigator at baseline after 1 week of treatment. the cgi - c is a seven - item symptom rating scale (0 = not assessed, 1 = very much improved, 2 = much improved, 3 = minimally improved, 4 = no change, 5 = minimally worse, 6 = much worse, 7 = very much worse). data were analyzed using the computer software package spss (v 14 ; spss, inc, chicago, il). sample size was calculated using sigmastat (v 3.5 for windows ; systat software, inc, chicago, il). the minimum sample size needed for each treatment group was calculated to be 23 subjects to detect a difference of 3.0 points in mean sleep scale scores with a standard deviation (sd) of 3.5, 80% power, and a 0.05 two - tailed level of significance. a paired - samples t - test was done to compare the mean scores for each group before and after treatment. an independent - samples t - test was done to test for a significant difference between any improvements in mean score in each treatment group. multivariate analysis of covariance with treatment as the main independent factor and the study center as a controlling factor was used in full factorial model testing for main effects plus any possible interaction effects. baseline pretreatment scores were used as covariates to control for initial group differences potentially affecting the dependent outcome variables, ie, any differences between baseline and posttreatment stai and sleep scores, and their interaction. parametric statistical methods were utilized after ascertaining the normality of distribution of the dependent variables by kolmogorov smirnov goodness of fit test. two patients from group 1 and one from group 2 withdrew from the study after randomization, and three patients in each group were lost to follow - up. one patient from group 2 was not included in the statistical analysis as a result of recording errors made on the sleep scoring sheet. twenty - five patients from each group were finally assessed, and their results were subjected to statistical analysis. the two groups were well matched for age, gender, and baseline sleep scores (see table 1). cronbach s alpha coefficients for the 20 items on the modified stai scale and for the ten items of the sleeping difficulty scale were 0.835 and 0.764, respectively, indicating good reliability. there was a statistically significant improvement in mean scores for both the modified stai and the sleeping difficulty questionnaires in both groups after treatment (tables 2 and 3). however, improvements in both the mean modified stai score and mean sleep score were significantly greater in group 1 than in group 2 by independent - samples t - test (p < 0.05). multivariate analysis was undertaken using treatment allocation as the main independent factor and the study center as a controlling factor in a full factorial model, with baseline scores as covariates (tables 4 and 5). treatment allocation and baseline scores (covariates) had statistically significant effects on the outcome variables, while both the study center and its two - way interaction with treatment (group, center) did not. univariate effects of the corrected model and the treatment groups adjusted for baseline severity on both the stai and sleep score showed statistically significant differences, while the study center and its two - way interaction with the groups did not. eighteen out of the 25 patients who completed the protocol (72%) in group 1 rated their insomnia as very much or much improved on the cgic scale, versus 5 (20%) in the control group (ie, 60% vs 16.7% respectively of the 30 intention - to - treat patients) (risk ratio : 3.6 ; 95% confidence interval : 1.588.18). to our knowledge, this is the first published study of the effect of l. sativa seed oil on a sleeping disorder. we found that l. sativa seed oil significantly improved both stai and sleep scores, with no apparent side effects at the dose strength used. being clinicians with a special interest in complementary alternative medicine, we undertook this pilot research on a pragmatic hypothesis - generating basis, rather than as a confirmatory or explanatory study, and hope that our findings will help pave the way for other investigators to join in the attempt to bridge the gap between alternative medicine and evidence - based medicine. this study has a number of limitations, including a small size, a short follow - up duration, and a single - blind design. the obvious reason for this is that, unlike patented new chemical entities with research funding from the pharmaceutical industry, quality research involving alternative medicines tends to suffer from financial and logistic constraints. in our opinion, alternative medicines, in particular products already available as over - the - counter dietary supplements, should be subjected to more translational research and clinical trials in human subjects, and the results made available in the scientific literature. the criteria for publishing such studies would need to be different to those used with the registration trials for new chemical entities. in conclusion, l. sativa seed oil was found to be a useful sleeping aid and may be a hazard - free line of treatment, especially in geriatric patients suffering mild - to - moderate forms of anxiety and sleeping difficulties. did you feel in need of frequent naps or a lie - down during the day ? | background : lactuca sativa (garden lettuce) is a popular salad herb. it has been in use in folk medicine since ancient times as both an appetite stimulant and as an aid to sleep. l. sativa seed oil (sedan) has demonstrated a pronounced sedative effect and potentiated the hypnotic effect of barbiturates in animal models. it also exhibited significant analgesic and anti - inflammatory activities. in this study, we evaluated the sedative and hypnotic effects of l. sativa in patients suffering from insomnia.methods:sixty patients suffering from insomnia with or without anxiety were randomized to receive capsules containing l. sativa seed oil 1000 mg (n = 30) or placebo (n = 30). all patients were asked to complete a verbal questionnaire before the start of the trial and 1 week after starting treatment.results:improvements in the modified state - trait anxiety inventory and the sleep rating scale scores were significantly greater in patients receiving l. sativa seed oil compared with those on placebo (p < 0.05). no side effects were found to be attributable to l. sativa seed oil at the given dosage.conclusion:l. sativa seed oil was found to be a useful sleeping aid and may be a hazard - free line of treatment, especially in geriatric patients suffering from mild - to - moderate forms of anxiety and sleeping difficulties. |
dural arteriovenous fistula (davf), also called dural arteriovenous shunt or malformation, consists of acquired lesions within the dura mater.11)19) the etiology and natural history of these lesions remain unknown.18)19)23) however, the clinical presentations and assessed risk of the lesions have been correlated with the associated patterns of venous drainage. cortical venous reflux is considered to be one important predictor of clinical course.7)8)18) some authors have reported that some patients with davf show conversion of angiographic venous drainage pattern.4)11)12)18)19) of these studies, most cases have exhibited chronological progression with spontaneous occlusion of the arteriovenous shunt. only a small percentage of cases (about 1.7 - 4%) showed conversion to an aggressive lesion with symptomatic aggravation.1118)19) therefore, in cases with worsening symptoms and neurological status, close clinical and radiological follow - up is particularly necessary. we examined a case of spontaneous venous drainage pattern conversion from borden type ii to type iii in a patient with davf. the patient presented with worsening neurological deficit and newly developed seizure within a four month period. the lesion was suspected to be caused by sinus thrombosis and rerouting of cortical venous reflux following venous infarction with mild hemorrhagic transformation. a 64-year - old female patient was admitted to our institute with mild right hemiparesis, numbness on the right upper extremity, nausea, vomiting, and dizziness. a magnetic resonance image (mri) scan of the brain showed multiple small, round and tubular low - signal intensity regions on the left frontal lobe, a finding suggestive of vascular malformation. initial digital subtraction angiography (dsa) identified a hypervascular lesion and arteriovenous shunting lesion on both external carotid artery (eca) angiograms (fig. both lesions suggestive of davf showed abnormal retrograde flow from the middle meningeal artery (mma) and the superficial temporal artery into the superior sagittal sinus, including cortical venous reflux via the arteriovenous shunt (borden classification ii). we planned to treat the lesions with an endovascular approach because they were correlated with the patient 's symptoms and showed cortical venous reflux. however, the patient and her family refused further treatment and requested hospital discharge. at 4 months after hospital discharge, the patient was readmitted with repeated seizure that had been occurring for 3 weeks. the most recent seizure was a generalized tonic - clonic seizure with head turning and eyeball deviation to the right side ; postictal confusion was also shown for 10 minutes. brain mri revealed subacute focal cerebral infarction with minimal hemorrhagic transformation on the left frontal lobe (fig. antiepileptic drugs were initially loaded, and follow - up dsa was performed after 5 days when the patient 's seizures were controlled by medication. abnormal venous reflux into the superior sagittal sinus was not present in the right eca angiogram. unexpectedly, only venous drainage directly into the subarachnoid cortical vein was observed, without contrast filling in the venous sinus in the left eca angiogram (borden classification iii). we hypothesized that the hemiparesis and seizure arose from venous infarction with congestion of the left frontal lobe due to progression and rerouting of the cortical venous reflux after venous sinus thrombosis. we planned to treat the lesions with trans - arterial endovascular embolization with onyx (a nonadhesive liquid embolic agent ; ev3 neurovascular, irvine, ca, usa). onyx embolization was performed with successful obliteration of the arteriovenous shunt through the superselected left mma in a single session (fig. the glasgow outcome scale score was 2 when the patient was discharged, an improvement over the hemiparesis from her status at admission. over a clinical follow - up period of 3 years, the patient did not experience any major problems. intracranial davf constitutes 10 - 15% of all intracranial vascular malformations, yet the natural course and pathogenesis of the lesions remain controversial.14)18)19)23) it is generally accepted that the presentation of davf is dictated by the location and pattern of venous drainage. moreover, the cortical venous reflux pattern is an important predictor of clinical course.7)8)17) davfs with an aggressive course have characteristic angiographic features such as cortical or leptomeningeal venous drainage and an associated varix.17) in a meta - analysis, awad.2) reported a hemorrhage rate of 88% and a severe neurologic deficit in 12% of 100 aggressive cases among 377 patients with davf. a previous study of borden types ii and iii davfs reported an annual hemorrhage rate of 8.1% and an annual mortality rate of 10%.22) another recent study reported an annual incidence of hemorrhage of 7.4% for patients presenting with an intracranial hemorrhage and 1.5% for those not presenting with hemorrhage.19) these discrepancies between the studies are likely due to demographic differences, different patient inclusion criteria, and publication bias. knowledge about the natural disease course and classification based on the venous drainage pattern can inform decisions regarding the most appropriate treatment modality for these patients. however, davf is a dynamic disease that can undergo spontaneous angiographic venous drainage pattern conversion. kim.11) reported pattern conversion as assessed by angiography in 18 of 112 cases (16.1%). among these cases, the conversion rate to an aggressive lesion was 4% ; all of these cases were associated with occlusion of the ipsilateral draining vein. moreover, several reports have described spontaneous closure of davf without any aggressive treatment.1)5)12) on the other hand, a few cases of conversion to aggressive lesions have been reported, and one study reported that benign avfs have a 2% potential for angiographically verified conversion.18) several theories have been suggested to explain the conversion of venous drainage pattern, including sinus thrombosis, change of sinus wall structure, and flow dynamics.2)12)16) cerebral sinus thrombosis can cause spontaneous regression with symptom resolution and can also force retrograde venous reflux through the cortical veins, predisposing the patient to an aggressive clinical course.11)18)21) thus, cerebral sinus thrombosis can promote the occlusion of venous outflow and result in dangerous rerouting. a histological study of davf suggested that microscopic thrombosis is always present, and that localizing thrombosis at the compartment or accessory mural channel of the drainage sinus may cause shunt occlusions.15) stenosis and thrombosis of the venous outlets have been reported to forecast later worsening of davfs. our case also showed stenosis of the venous outlets and ultimately showed spontaneous closure of the shunt into the superior saggital sinus and aggravated cortical venous reflux from borden type ii to type iii. endovascular embolization has become the first line of treatment in patients with high - risk davfs, and surgery is considered only if endovascular treatments fail or are unfeasible. trans - arterial approaches with embolic materials such as glue (nbca : n - butyl cyanoacrylate), particles (pva : polyvinyl alcohol), or onyx are performed to enable the embolic material to be pushed through the shunt into the proximal venous outlet. in recent studies, onyx has demonstrated multiple advantages over other materials, with reported occlusion rates of 62.5 - 80%.6)13)20) however, in cases of potentially dangerous occult eca - internal carotid artery or vertebral artery anastomosis, development of ischemic cranial nerve palsies, or in fistulas with multiple feeders, a trans - venous approach is recommended.17) trans - venous approaches can be performed with detachable coils and particles in the most proximal venous outlet and have been the treatment of choice, especially for carotid cavernous davfs. the shunt locations and cortical venous reflux patterns may predict the clinical manifestations and drainage patterns of davfs. superior saggital sinus davfs, as in our case, represent about 8 - 13% of all intracranial fistulae.10) these davfs are often accompanied by hemorrhage or progressive neurological deficits.24) these fistulae are characterized by bilateral supply from the mma, ophthalmic artery, or posterior meningeal artery with critical venous drainage and require aggressive treatment.10) despite the variety of treatment modalities available, trans - arterial embolization is recommended in superior saggital sinus davfs with adjuvant surgery or radiosurgery following failed embolization.3)9) in our case, we performed onyx embolization through the mma, and successful obliteration was achieved in a single session. due to the properties and increased predictability of onyx, more prolonged injections through single feeders like the mma can be performed compared with other embolic materials.14) moreover, the excellent flow control enables greater injection control, even when the vessel diameter is very small. with the newer and easier - to - navigate micro - catheters and the advent of onyx, trans - arterial embolization has become the accepted initial treatment for davf although most cases of venous drainage pattern conversion in davf show benign clinical courses and do not develop symptoms, aggressive conversion sometimes occurs. therefore, close clinical and radiological follow - up is needed to detect these lesions. our results indicate that endovascular embolization with onyx is a reliable treatment with acceptable safety and efficacy. | we report a case of dural arteriovenous fistula (davf) that showed spontaneous conversion of venous drainage pattern from borden type ii to type iii within a four month period of follow - up. upon admission, the patient presented with aggravated neurologic status and newly developed seizure. after admission, endovascular embolization was performed through the middle meningeal artery with onyx. complete obliteration of dural arteriovenous shunt was confirmed by angiography, and the patient 's clinical symptoms improved. although most cases of davf show benign clinical course and conversion pattern, close follow - up is required to detect potential aggravation. |
study participants for the gwas were drawn from two studies, the shanxi upper gastrointestinal cancer genetics project (shanxi) and the linxian nutrition intervention trial (nit), a prospective cohort. for the second phase, we genotyped additional subjects from shanxi and nit as well as subjects from the shanghai men 's health study (smhs), the shanghai women 's health study (smhs), and the singapore chinese health study (schs) (supplementary table 1). the shanxi study controls were matched on age and sex for the case - control portion, while for the nit controls were selected as a case - cohort and frequency matched on age and sex. for the smhs, swhs, and schs cohorts, controls were alive, free of upper gastrointestinal tract cancer, and matched to cases as described in supplementary table 1. for the shanxi and nit study, tumor anatomic location was known for all cases and > 85% of cases had pathologic confirmation. for the three cohorts added in the second phase, the proportion with anatomic location in the stomach is given in supplementary table 1 and pathologic confirmation was available for > 95% of cases. all examined esophageal cancers were squamous cell carcinomas (escc) and all examined gastric cancers (gc) were adenocarcinomas. cardia cancers were located in the proximal 3 cm of the stomach, while noncardia cancers were those in the remainder of the stomach. gastric cancers without location information were included in total gc analyses but excluded from gc anatomic subsite analyses. each of the five participating studies obtained informed consent from subjects and from their studies institutional review board(s). genome - wide scanning was attempted on 6,384 samples using the illumina 660w quad chip. after excluding 8 samples with no observed intensity data, the 6,376 remaining samples were analyzed, including 4,987 from the shanxi study and 1,389 from the nit. clustering was performed with 1,270 previously scanned caucasian samples in order to improve calling for low maf snps in the east asian samples. participants were excluded because of : 1) completion rate lower than 94% (n=485 samples) ; 2) abnormal heterozygosity values of less than 25% or greater than 30% (n=53, among which 36 were also excluded for low completion rates ; 3) discordant expected duplicates (n=3 pairs) ; 4) concordant unexpected duplicates (n=5 pairs, all from shanxi) ; 5) gender discordance (n=55, all from shanxi) ; 6) phenotype exclusions (due to ineligibility or incomplete information) (n=46). we checked for relatedness among study subjects using genotypes for all subject pairs with identity - by - state greater than 45%. these were input into glu qc.ibds module (http://code.google.com/p/glu-genetics/) to estimate the identity - by - degree ratio and infer the degree of relatedness (12nd degree). we found 20 full sibling pairs, 2 parent - child pairs, and 22 half - sibling pairs. this level of relatedness was not surprising because of the geographic proximity of subjects accrued in the two studies. we selected and retained one from each of the 1st degree relative pair and excluded the other (n=22) for the pca but included all for the association analyses. for the 132 known duplicate pairs the concordance was 99.98%. using 12,000 snps in low linkage disequilibrium (pair - wise r 85% of cases had pathologic confirmation. for the three cohorts added in the second phase, the proportion with anatomic location in the stomach is given in supplementary table 1 and pathologic confirmation was available for > 95% of cases. all examined esophageal cancers were squamous cell carcinomas (escc) and all examined gastric cancers (gc) were adenocarcinomas. cardia cancers were located in the proximal 3 cm of the stomach, while noncardia cancers were those in the remainder of the stomach. gastric cancers without location information were included in total gc analyses but excluded from gc anatomic subsite analyses. each of the five participating studies obtained informed consent from subjects and from their studies institutional review board(s). genome - wide scanning was attempted on 6,384 samples using the illumina 660w quad chip. after excluding 8 samples with no observed intensity data, the 6,376 remaining samples were analyzed, including 4,987 from the shanxi study and 1,389 from the nit. clustering was performed with 1,270 previously scanned caucasian samples in order to improve calling for low maf snps in the east asian samples. participants were excluded because of : 1) completion rate lower than 94% (n=485 samples) ; 2) abnormal heterozygosity values of less than 25% or greater than 30% (n=53, among which 36 were also excluded for low completion rates ; 3) discordant expected duplicates (n=3 pairs) ; 4) concordant unexpected duplicates (n=5 pairs, all from shanxi) ; 5) gender discordance (n=55, all from shanxi) ; 6) phenotype exclusions (due to ineligibility or incomplete information) (n=46). we checked for relatedness among study subjects using genotypes for all subject pairs with identity - by - state greater than 45%. these were input into glu qc.ibds module (http://code.google.com/p/glu-genetics/) to estimate the identity - by - degree ratio and infer the degree of relatedness (12nd degree). we found 20 full sibling pairs, 2 parent - child pairs, and 22 half - sibling pairs. this level of relatedness was not surprising because of the geographic proximity of subjects accrued in the two studies. we selected and retained one from each of the 1st degree relative pair and excluded the other (n=22) for the pca but included all for the association analyses. for the 132 known duplicate pairs the concordance was 99.98%. using 12,000 snps in low linkage disequilibrium (pair - wise r<0.004)11, we identified and excluded two subjects with less than 90% asian ancestry based on structure analysis (http://pritch.bsd.uchicago.edu/structure.html)26 (supplementary fig. for all subjects in the genome - wide scan phase, we attempted 657,364 genotype assays. for analysis, we removed snps with a call rate < 90%. 4) for case - control analyses were separately examined for gc and escc and there was no evidence for significant problems with population substructure or case - control matching : the unscaled for gc and escc were 0.990 and 0.989 respectively, while 1000 for gc and escc were 0.995 and 0.994, respectively28. the illumina infinium genotype probe cluster plots for select snps (rs2274223 and rs3781264) are shown in supplementary figure 5. after completion of the genome - wide phase, we selected six snps at10q23, two at 1q22, and two at 22q12 for taqman genotyping in our second phase. all ten snps were at or near genome - wide significance for total gc, escc, or both. for the selected snps, we successfully optimized eight taqman assays (abi), while two failed manufacturing or validation. for the second phase using taqman, we included samples from the shanxi and nit study that had not been scanned or failed qc metrics in the genome - wide phase as well as samples from three prospective cohort studies of subjects of chinese ethnicity (smhs, swhs, and schs) (supplementary table 1). in total after standard quality control metrics were applied, the sample completion rate overall was 98.8.%. we report trend models (tables 1, 2), but also fit genotype models for comparison (supplementary table 3). all reported p - values are based on two - sided tests. in the second and combined phases, logistic regression models were adjusted for age, sex, and study. because previous studies reported an interaction between risk of escc, alcohol or tobacco consumption, and variants marking the adh1b or aldh2 gene loci, we fit models both adjusted for and stratified on these factors (supplementary table 4). data analysis and management was performed with glu (genotyping library and utilities version 1.0), a suite of tools available as an open - source application for management, storage and analysis of gwas data. | we conducted a genome - wide association study of gastric cancer (gc) and esophageal squamous cell carcinoma (escc) in ethnic chinese subjects in which we genotyped 551,152 single nucleotide polymorphisms (snps). we report a combined analysis of 2,240 gc cases, 2,115 escc cases, and 3,302 controls drawn from five studies. in logistic regression models adjusted for age, sex, and study, multiple variants at 10q23 had genome - wide significance for gc and escc independently. a notable signal was rs2274223, a nonsynonymous snp located in plce1, for gc (p=8.40109 ; per allele odds ratio (or) = 1.31) and escc (p=3.85109 ; or = 1.34). the association with gc differed by anatomic subsite. for tumors located in the cardia the association was stronger (p=4.19 1015 ; or= 1.57) and for those located in the noncardia stomach it was absent (p=0.44 ; or=1.05). our findings at 10q23 could provide insight into the high incidence rates of both cancers in china. |
many use trimeric fusion proteins that share structural characteristics with cellular proteins of similar function 1. since refolding of most viral fusion proteins is irreversible, triggering must be strictly regulated. while many enveloped viruses, including influenza-, rhabdo-, alpha- and flaviviruses, take advantage of low ph in the endosomal compartment to trigger fusion 2, the filovirus ebola uses proteases 3. other enveloped viruses, including measles virus (mv) and hiv fuse directly with the plasma membrane at neutral ph 1,4. mv, while being targeted for eradication, still causes more than 150,000 deaths yearly 5,6. mv is a member of the paramyxoviridae 4,7, a family including deadly emerging viruses like hendra and nipah, and prevalent human viruses like mumps, parainfluenza, and respiratory syncytial viruses. moreover, the mv entry system has become a paradigm for targeting oncolysis and therapeutic gene delivery 810 because it can be triggered by cell surface proteins differing in size, trans - membrane organization, and quaternary structure 11. since the mechanism triggering mv cell entry is not understood, we set out to characterize it the attachment protein mediates receptor binding and triggers a refolding event of the metastable fusion (f) protein that results in membrane fusion 4,7. the attachment proteins are named hemagglutinin (h) for mv and the other members of the genus morbilliviruses, glycoprotein (g) for the henipaviruses, and hemagglutinin - neuraminidase (hn) for most other genera. while the h and g proteins bind proteinaceous receptors, hn proteins bind sialic acid 12. mv h is a type ii transmembrane glycoprotein comprised of an amino - terminal cytoplasmic tail, a membrane - spanning segment, and an extracellular membrane - proximal stalk region connected to a large cuboidal head with six - blade beta - propeller structure contacting the receptors (figure 1a - c). mv h proteins form dimers stabilized by two inter - subunit disulfide bonds 13 (cys139 and cys154, figure 1a, d) located at the top of the long helical stalk 14, below the base of the cuboidal heads. it was recently shown that h - tetramers or higher oligomeric forms sustain fusion - support function 15. specific interactions of the attachment and f proteins of paramyxoviridae are required for membrane fusion because heterotypic glycoprotein pairs can not mediate this process 16,17. in particular, the stalk regions of different hn proteins determine the specificity for the cognate f proteins 4,7. similarly, the mv h - stalk region interacts directly with the f protein head 18, an observation suggesting that the h - oligomer is much taller than the f - trimer (figure 1d). these f - h interactions are relevant to late phases of the fusion triggering process. novel information revealing structural diversity of the attachment proteins of paramyxoviridae prompted us to analyze early triggering phases. in particular, the interface of the h - dimer 19 was found to be much smaller than that of hn - dimers 2022 (1070 versus 1800 in a direct comparison 23), and the h protein head crystallized as a monomer when cys154 was omitted 24. moreover, while the h - heads lean about 40 away from the symmetry axis, and tilt sideways slightly (schematics in figure 1c ; see also figure 1d, left) 19, the hn - heads are more upright 2022. the biology of these attachment proteins also differs : while h hetero - oligomerizes with f in the endoplasmic reticulum 25, hn and f travel separately to the cell surface 26. as to fusion triggering, the small interface area of the h - dimer suggested to us that the heads might move relative to each other : figure 1c illustrates a re - alignment movement, but adjustment or rotation movements are also possible. movement hypothesis by adding covalent cross - links that stabilize the dimer in a single conformation. we also noted that the footprints of the three mv receptors 2735 are located distal to the dimer interface, such that two bound receptors per dimer would have enough leverage to force the heads to move. twist hypothesis by directing a membrane - anchored receptor to bind tags inserted in strategic locations on the h - head. indeed, only binding close to the locations used by the natural receptors, and distal to the h - dimer interface, efficiently triggers membrane fusion. to test the movement hypothesis we sought to stabilize the h - dimer interface by adding inter - subunit disulfide bonds. we note that the heads have a solid beta - propeller structure, and may thus move as rigid bodies. moreover, cd46 binding does not induce substantial conformational changes of the h - head 36, consistent with a rigid structure suited to conduct torsion. we identified amino acid pairs across the interface whose c - alpha atoms are less than 7.5 apart (table 1) and mutated these to cysteine. we sought to introduce two classes of disulfide bonds : single bonds located on the two - fold symmetry axis of the dimer and involving the same residue on both molecules (table 1, top four lines), and twin bonds located on both sides of the axis and involving two different residues (table 1, bottom four lines). twin cysteine bonds, forming a triangle with cys154, would provide more resistance to a force, increasing the energy barrier for fusion triggering ; larger triangles would provide more resistance than smaller triangles. figure 2a shows the location of the two key single bonds (t273c t273c, blue and w327c w327c, green) and the two key twin bonds (k236c g264c, brown / red and p330c l161c, orange / yellow) in a side view of the dimer interface. the dimerization capacity of the mutated h - proteins was then assessed (table 1 and figure 2c). we note that the cysteine substitutions are distal from known receptor binding epitopes, but to control for the ability of each position to tolerate mutations we also made alanine substitutions, and assayed fusion triggering by the alanine mutants. where twin bonds were involved, we ensured that neither one of the cysteine substitutions in the pair had an effect on fusion function. as listed in table 1 (fifth column), only one of the alanine mutants, l203a was fusion deficient, and this residue was eliminated from further consideration. the fact that all the other alanine mutations were fusion - competent implies that the corresponding mutant proteins have intact receptor binding function. since total expression levels of certain mutants were reduced (figure 2c), we sought to measure their cell surface expression. we measured the mean fluorescence intensity (mfi) by facs analysis for each mutant (supplementary table 1), which was 4768% as that of the standard h - nse protein. even with this reduction in cell surface protein levels, we note the mv fusion system is very efficient, and that decreased h surface expression may not necessarily translate in decreased fusion 13. we also probed the mutants with a conformation - specific monoclonal antibody that recognizes the h - noose epitope 37. the mfi measured with this antibody was similar to that recorded with the anti - h cocktail (supplementary table 1), indicating that the mutants had similar conformations to the unmodified h - protein. we then assessed whether covalent bonds are formed, taking advantage of an h protein backbone with mutations in both cysteine residues (cys139 and cys154) that normally mediate covalent inter - subunit cross - linking. while this mutant forms minimal amounts of stable dimers, given time it does support low levels of membrane fusion 13. c154a mutant loses the ability to form detectable amounts of stable h - dimers, as assayed by non - reducing sds - page. c154a background the only inter - subunit bridges are those formed at positions with added cysteines. figure 2d (lanes 14) show that cysteine substitutions at 236 + 264, 273, 327 or 330 + 161 restore the dimerization capacity of the h - protein, demonstrating that these substitutions allow inter - subunit disulfide bonds under physiological conditions. c154a (lanes 14) or the normal h - backbone (lanes 58), indicated that the cysteine substitution mutants alone are less efficient at forming dimers. this is not surprising as the introduced cysteines may not promote covalent linkage as efficiently as the two stalk cysteines. this analysis also revealed that k328c and k236c+l265c did not yield cross - linked dimers, and these residues were eliminated from further consideration (table 1, seventh column). moreover, p330c+e162c dimerized but was not pursued further because it is similar to p330c+l161c. thus, analysis focused on two single - bond and two twin - bond cysteine mutants. after confirming disulfide bond formation, h - protein mutants were co - expressed with f - protein to test the hypothesis that movement of the subunits is required to allow membrane fusion. figure 3a shows that the most distal twin bonds (k236c+g264c, 32 from fusion pivot, 533 stabilized area) abrogated fusion. on the other hand, the two single bonds (22 and 16 from the pivot) and the pivot proximal twin bond (10 from pivot, 68 stabilized area) had little effect on fusion, in this assay performed 24 hours after transfection. this analysis was repeated using a luciferase - based quantitative fusion assay that provided additional 12- and 18-hour time points. at 12-hours the positive control showed high fusion levels, while all the mutants had fusion levels at or near background (figure 3b). at 18-hours the fusion functions of the t273c, w327c and p330c l161c mutants the fusion function of the k236c+g264c mutant was reduced by a factor of 13 compared to the wild type protein, while fusion efficiency of the other three mutants was comparable to wild type h, which syncytia had already begun to lyse. these results indicate that the single bonds at 273 and 327, and the twin bond at 330 + 161 interfere with fusion temporarily. however, given time fusion is observed, possibly because occasionally disulfide bonds are reduced. we then assessed whether eliminating the disulfide bonds with a reducing agent restores fusion function. cells expressing the k236c+g264c mutations were treated with 1,4-dithio - dl - threitol (dtt). in the presence of dtt, fusion function was restored in the k236c+g264c h mutant that previously exhibited no fusion (figure 3c, compare lower left and right panels). similar results were observed for the t273c and w327c mutants when treated with dtt 12-hours post - transfection (data not shown), indicating that this protein is available in sufficient quantities and retains the capacity to interact with receptors. we next assessed whether the efficiency of fusion triggering depends on interface - distal receptor binding on the h - dimer. we took a systematic approach and inserted a short hexahistidine tag in different positions at the top half of the h - dimer. the hexahistidine tag directs binding to vero - his cells which express a membrane - anchored single - chain antibody that recognizes the six - histidine peptide38. the insertion points were chosen taking into consideration structural constraints, including n - linked oligosaccharide chains at n168, n187, n200 and n215, and availability of surface accessible loops (figure 4a, b). the tags were inserted in eight exposed loops (figure 4a, b, yellow residues). we used mutant 9 with a carboxy - terminal tag as the positive control 38. mutants 5 and 6 are located on the top of the h - dimer, between the h - residues in close contact with cd46, as identified based on the h - cd46 co - crystal 36 (highlighted in light blue) and those identified by functional assays to be relevant for slam - dependent fusion 2729 (highlighted in red). mutants 1, 3, 7 and 8 are all located near the top of the h - dimer. while mutant 1 is close to the cd46 footprint, and mutant 7 close to the slam footprint, mutants 3 and 8 are located away from both receptor binding areas. mutant 2 is also located away from these binding areas and close to the interface. mutant 4 is the only one in the bottom half of the dimer. to identify functional mutants, dimerization capacity, expression levels, and accessibility of the his - tag protein extracts from transfected vero cells were separated on sds - page under non - reducing conditions, and immunoblotted. since mutants 2 and 4 dimerized poorly (figure 4c), they were excluded from further analysis. surface expression levels of the remaining mutants, measured by facs, were at 3999% of standard h - nse (supplementary table 2, third column), which was considered acceptable for all mutants. to determine the accessibility of the his - tag we compared the mfi generated by an anti - his antibody to that generated by an anti - h cocktail (supplementary table 2, last column). all but one mutant had mfi ratios close to 1, indicating that their his - tags were equivalently accessible. since mutant 3 had a lower (0.65) mfi ratio, it was not considered when drawing conclusions. thus, hexahistidine tagging of exposed protein loops is compatible with efficient protein folding and dimerization, and allows display of accessible tags in a majority of the constructs. we then measured the fusion efficiency of the dimerization - competent mutants and the control mutant, 9, in cell lines expressing either the anti - hexahistidine antibody as the designated receptor, or the natural receptor slam, or the vaccine strain receptor, cd46. since none of the hexahistidine tags were predicted to interfere with cd46-binding, these fusion assays were considered positive controls. on the other hand, slam - dependent fusion assays were expected to further define the slam footprint. finally, hexahistidine receptor - dependent fusion assays were expected to reveal the h - dimer surface area poised for fusion triggering. for consistency in host cell type, we used vero cells, or vero cells expressing anti - hexahistidine (vero - his), or slam (vero - slam). to focus exclusively on entry through one receptor, we generated a second set of tag mutants in an h - protein backbone exclusively recognizing slam (h - wt, figure 5a, first and second column). the h - nse vaccine lineage backbone was used only to measure cd46-dependent fusion (h - nse, figure 5a, third column). this analysis indicated that, with the exception of mutants 7 and 8, which were reduced in cd46-specific fusion (fusion scores of 2 and 1, respectively), all the mutants fully retained cd46-dependent fusion function. analysis of slam - dependent fusion revealed that the h - slam contact area may extend to the region defined by mutants 5 and 6, because these two mutants are specifically impaired in this fusion function (figure 5a, first column and figure 5b, red cylinders ; fusion score of 1 and 0, respectively). the slam contact area on the top of the h - dimer may extend also to the position defined by mutant 1 (fusion score of 1). we then assessed the efficiency of membrane fusion triggering through the membrane - anchored anti - hexahistidine antibody. figure 5a, center column and figure 5b, yellow cylinders, show that both mutants (5 and 6) in which the tags are located between the slam and cd46 footprints, had fusion scores of 2 each. mutants 1 and 7, with tags located close to only one of the receptor footprints, and closer to the interface than mutants 5 and 6, had fusion scores of 1 each. thus, both hexahistidine tags inserted within the slam - footprint trigger fusion through an artificial receptor, almost as efficiently as the control mutant. to better compare the fusion efficiency of mutants 5 and 6, the luciferase - based quantitative fusion assay was used. figure 5c documents fusion efficiency in vero cells expressing either the membrane - anchored anti - hexahistidine antibody (yellow bars) or slam (red bars). mutant 5 was about 65% as effective as positive control mutant 9 in inducing fusion of vero - his cells, and mutant 6 was about 50% as effective. thus, an artificial receptor must bind proximal to the locations used by the natural receptors to effectively trigger fusion. while preferential accessibility of this area of the h - head may influence this striking co - localization of functional binding sites, our findings are also fully consistent with the twist hypothesis proposing that receptors have more leverage to force the heads to move while engaging an interface - distal location. to test the movement hypothesis we sought to stabilize the h - dimer interface by adding inter - subunit disulfide bonds. we note that the heads have a solid beta - propeller structure, and may thus move as rigid bodies. moreover, cd46 binding does not induce substantial conformational changes of the h - head 36, consistent with a rigid structure suited to conduct torsion. we identified amino acid pairs across the interface whose c - alpha atoms are less than 7.5 apart (table 1) and mutated these to cysteine. we sought to introduce two classes of disulfide bonds : single bonds located on the two - fold symmetry axis of the dimer and involving the same residue on both molecules (table 1, top four lines), and twin bonds located on both sides of the axis and involving two different residues (table 1, bottom four lines). twin cysteine bonds, forming a triangle with cys154, would provide more resistance to a force, increasing the energy barrier for fusion triggering ; larger triangles would provide more resistance than smaller triangles. figure 2a shows the location of the two key single bonds (t273c t273c, blue and w327c w327c, green) and the two key twin bonds (k236c g264c, brown / red and p330c l161c, orange / yellow) in a side view of the dimer interface. the dimerization capacity of the mutated h - proteins was then assessed (table 1 and figure 2c). we note that the cysteine substitutions are distal from known receptor binding epitopes, but to control for the ability of each position to tolerate mutations we also made alanine substitutions, and assayed fusion triggering by the alanine mutants. where twin bonds were involved, we ensured that neither one of the cysteine substitutions in the pair had an effect on fusion function. as listed in table 1 (fifth column), only one of the alanine mutants, l203a was fusion deficient, and this residue was eliminated from further consideration. the fact that all the other alanine mutations were fusion - competent implies that the corresponding mutant proteins have intact receptor binding function. since total expression levels of certain mutants were reduced (figure 2c), we sought to measure their cell surface expression. we measured the mean fluorescence intensity (mfi) by facs analysis for each mutant (supplementary table 1), which was 4768% as that of the standard h - nse protein. even with this reduction in cell surface protein levels, we note the mv fusion system is very efficient, and that decreased h surface expression may not necessarily translate in decreased fusion 13. we also probed the mutants with a conformation - specific monoclonal antibody that recognizes the h - noose epitope 37. the mfi measured with this antibody was similar to that recorded with the anti - h cocktail (supplementary table 1), indicating that the mutants had similar conformations to the unmodified h - protein. we then assessed whether covalent bonds are formed, taking advantage of an h protein backbone with mutations in both cysteine residues (cys139 and cys154) that normally mediate covalent inter - subunit cross - linking. while this mutant forms minimal amounts of stable dimers, given time it does support low levels of membrane fusion 13. c154a mutant loses the ability to form detectable amounts of stable h - dimers, as assayed by non - reducing sds - page. c154a background the only inter - subunit bridges are those formed at positions with added cysteines. figure 2d (lanes 14) show that cysteine substitutions at 236 + 264, 273, 327 or 330 + 161 restore the dimerization capacity of the h - protein, demonstrating that these substitutions allow inter - subunit disulfide bonds under physiological conditions. c154a (lanes 14) or the normal h - backbone (lanes 58), indicated that the cysteine substitution mutants alone are less efficient at forming dimers. this is not surprising as the introduced cysteines may not promote covalent linkage as efficiently as the two stalk cysteines. this analysis also revealed that k328c and k236c+l265c did not yield cross - linked dimers, and these residues were eliminated from further consideration (table 1, seventh column). moreover, p330c+e162c dimerized but was not pursued further because it is similar to p330c+l161c. thus, analysis focused on two single - bond and two twin - bond cysteine mutants. after confirming disulfide bond formation, h - protein mutants were co - expressed with f - protein to test the hypothesis that movement of the subunits is required to allow membrane fusion. figure 3a shows that the most distal twin bonds (k236c+g264c, 32 from fusion pivot, 533 stabilized area) abrogated fusion. on the other hand, the two single bonds (22 and 16 from the pivot) and the pivot proximal twin bond (10 from pivot, 68 stabilized area) had little effect on fusion, in this assay performed 24 hours after transfection. this analysis was repeated using a luciferase - based quantitative fusion assay that provided additional 12- and 18-hour time points. at 12-hours the positive control showed high fusion levels, while all the mutants had fusion levels at or near background (figure 3b). at 18-hours the fusion functions of the t273c, w327c and p330c l161c mutants were reduced by a factor of 6 compared to the positive control. at 24-hours the fusion function of the k236c+g264c mutant was reduced by a factor of 13 compared to the wild type protein, while fusion efficiency of the other three mutants was comparable to wild type h, which syncytia had already begun to lyse. these results indicate that the single bonds at 273 and 327, and the twin bond at 330 + 161 interfere with fusion temporarily. however, given time fusion is observed, possibly because occasionally disulfide bonds are reduced. we then assessed whether eliminating the disulfide bonds with a reducing agent restores fusion function. cells expressing the k236c+g264c mutations were treated with 1,4-dithio - dl - threitol (dtt). in the presence of dtt, fusion function was restored in the k236c+g264c h mutant that previously exhibited no fusion (figure 3c, compare lower left and right panels). similar results were observed for the t273c and w327c mutants when treated with dtt 12-hours post - transfection (data not shown), indicating that this protein is available in sufficient quantities and retains the capacity to interact with receptors. we next assessed whether the efficiency of fusion triggering depends on interface - distal receptor binding on the h - dimer. we took a systematic approach and inserted a short hexahistidine tag in different positions at the top half of the h - dimer. the hexahistidine tag directs binding to vero - his cells which express a membrane - anchored single - chain antibody that recognizes the six - histidine peptide38. the insertion points were chosen taking into consideration structural constraints, including n - linked oligosaccharide chains at n168, n187, n200 and n215, and availability of surface accessible loops (figure 4a, b). the tags were inserted in eight exposed loops (figure 4a, b, yellow residues). we used mutant 9 with a carboxy - terminal tag as the positive control 38. mutants 5 and 6 are located on the top of the h - dimer, between the h - residues in close contact with cd46, as identified based on the h - cd46 co - crystal 36 (highlighted in light blue) and those identified by functional assays to be relevant for slam - dependent fusion 2729 (highlighted in red). mutants 1, 3, 7 and 8 are all located near the top of the h - dimer. while mutant 1 is close to the cd46 footprint, and mutant 7 close to the slam footprint, mutants 3 and 8 are located away from both receptor binding areas. mutant 2 is also located away from these binding areas and close to the interface. mutant 4 is the only one in the bottom half of the dimer. to identify functional mutants, dimerization capacity, expression levels, and accessibility of the his - tag protein extracts from transfected vero cells were separated on sds - page under non - reducing conditions, and immunoblotted. since mutants 2 and 4 dimerized poorly (figure 4c), they were excluded from further analysis. surface expression levels of the remaining mutants, measured by facs, were at 3999% of standard h - nse (supplementary table 2, third column), which was considered acceptable for all mutants. to determine the accessibility of the his - tag we compared the mfi generated by an anti - his antibody to that generated by an anti - h cocktail (supplementary table 2, last column). all but one mutant had mfi ratios close to 1, indicating that their his - tags were equivalently accessible. since mutant 3 had a lower (0.65) mfi ratio, it was not considered when drawing conclusions. thus, hexahistidine tagging of exposed protein loops is compatible with efficient protein folding and dimerization, and allows display of accessible tags in a majority of the constructs. we then measured the fusion efficiency of the dimerization - competent mutants and the control mutant, 9, in cell lines expressing either the anti - hexahistidine antibody as the designated receptor, or the natural receptor slam, or the vaccine strain receptor, cd46. since none of the hexahistidine tags were predicted to interfere with cd46-binding, these fusion assays were considered positive controls. on the other hand, finally, hexahistidine receptor - dependent fusion assays were expected to reveal the h - dimer surface area poised for fusion triggering. for consistency in host cell type, we used vero cells, or vero cells expressing anti - hexahistidine (vero - his), or slam (vero - slam). to focus exclusively on entry through one receptor, we generated a second set of tag mutants in an h - protein backbone exclusively recognizing slam (h - wt, figure 5a, first and second column). the h - nse vaccine lineage backbone was used only to measure cd46-dependent fusion (h - nse, figure 5a, third column). this analysis indicated that, with the exception of mutants 7 and 8, which were reduced in cd46-specific fusion (fusion scores of 2 and 1, respectively), all the mutants fully retained cd46-dependent fusion function. analysis of slam - dependent fusion revealed that the h - slam contact area may extend to the region defined by mutants 5 and 6, because these two mutants are specifically impaired in this fusion function (figure 5a, first column and figure 5b, red cylinders ; fusion score of 1 and 0, respectively). the slam contact area on the top of the h - dimer may extend also to the position defined by mutant 1 (fusion score of 1). we then assessed the efficiency of membrane fusion triggering through the membrane - anchored anti - hexahistidine antibody. figure 5a, center column and figure 5b, yellow cylinders, show that both mutants (5 and 6) in which the tags are located between the slam and cd46 footprints, had fusion scores of 2 each. mutants 1 and 7, with tags located close to only one of the receptor footprints, and closer to the interface than mutants 5 and 6, had fusion scores of 1 each. thus, both hexahistidine tags inserted within the slam - footprint trigger fusion through an artificial receptor, almost as efficiently as the control mutant. to better compare the fusion efficiency of mutants 5 and 6, the luciferase - based quantitative fusion assay was used. figure 5c documents fusion efficiency in vero cells expressing either the membrane - anchored anti - hexahistidine antibody (yellow bars) or slam (red bars). mutant 5 was about 65% as effective as positive control mutant 9 in inducing fusion of vero - his cells, and mutant 6 was about 50% as effective. thus, an artificial receptor must bind proximal to the locations used by the natural receptors to effectively trigger fusion. while preferential accessibility of this area of the h - head may influence this striking co - localization of functional binding sites, our findings are also fully consistent with the twist hypothesis proposing that receptors have more leverage to force the heads to move while engaging an interface - distal location. we have used information from the static crystal structures of the attachment proteins of four paramyxoviruses to formulate and test two hypotheses aiming at inferring the mechanism of a necessarily dynamic process, such as the triggering of membrane fusion. our findings are consistent with the following model of h - based fusion triggering : first, two membrane - anchored receptor molecules bind the h - dimer heads opposite to the interface. this movement transmits a signal to the f protein, triggering irreversible f - trimer refolding and membrane fusion. our model provides a framework for interpreting recently published structural information documenting one type of interaction between two external cd46 short consensus repeats (scr) and the h - heads 36 (figure 1d ; scr12 are shown in pale green). surprisingly, cd46 binds h in an upside - down orientation : scr2 points towards the viral membrane. very recently, almost the entire cd46 ectodomain (scr 14) was co - crystallized with the adenovirus knob. in this complex cd46 when this structure is modelled into the h - dimer, scr 4 is parallel to the membranes (figure 1d). remarkably, the slam footprint suggests that its h - binding domain is positioned tangentially to the h - dimer interface (figure 5b) and thus also parallel to the membrane. we propose that tangential engagement sustains efficient transfer of forces arising from the receptor s lateral movement in the plasma membrane to h - heads. these observations also suggest that cd46 and the h - oligomer initially interact at another angle, or in a different conformation, than those shown in figure 1d. indeed, there are indications that these molecules have structural plasticity and assume different conformations during the binding and fusion processes. in particular, it was observed that cd46 domains 3 and 4 enhance binding of domains 1 and 2 to mv particles, but impair binding to soluble h 40. since the domain 12 interface possesses some flexibility 41, and flexibility may also be a feature of the similarly - sized domain 23 interface 39, it is conceivable that before h - binding cd46 is shaped as a hook (figure 1d ; scr 12 shown in pale green, linked to yellow ovals representing scr 34). the hockey stick could be an alternative conformation occurring after binding and facilitating membrane fusion. a precedent for this is the piv5 hn - tetrameric ectodomain : electron microscopy revealed a staggered arrangement of the heads, and it was proposed that flexibility of the stalk is important in membrane fusion 42. h - tetramers may bind cd46 molecules in the hook conformation, and subsequently these complexes may adjust to form a scaffold of optimal height to support fusion. such a scaffold was previously proposed based on the analysis of the fusion - support efficiency of receptor molecules of variable length 43. based on the analysis of the h - oligomer s intracellular transport, it was proposed that paramyxoviruses binding either proteinaceous receptors (h - class), or sialic acid (hn - class), regulate fusion by different mechanisms 25. this suggestion was strengthened by the documentation of additional structural and functional differences between h- and hn - class proteins 12. we now propose that h - heads switch partners to transmit the fusion - triggering signal, while hn - heads must use another mechanism. we know 1d) 19, and we propose that they make them rotate 90 about their stalk. h - heads will then switch partners while the stalk remains covalently linked with the stalk of the original partner, cross - linking two h - dimers into an irregular h - tetramer. since the stalks of these h - tetramers would have anomalous contacts with f - trimers 18, their accumulation would progressively de - stabilize the viral envelope, eventually triggering membrane fusion. our model considers that fusion is supported by tetrameric or higher order h - oligomers 15, and implies that alteration of residues critical for formation of the interface between dimers would interfere with transmission of the fusion - triggering signal. however, we do not know whether this interface is formed between only one subunit in each dimer, analogous to the staggered arrangements of hn pairs - of - dimers revealed by electron microscopy 42, or between both subunits in each dimer, analogous to the tightly packed, quadratic arrangements of hn - head crystals 2022. towards identifying residues in this interface and testing this aspect of the model we generated 10,000 docked poses of h - head pairs - of - dimers using the program zdock 44 (n.o. and w.b., we expressed mutated h - proteins, focusing initially on mutants representing tightly packed poses, but all mutated proteins maintained fusion function, and formed tetramers as documented by blue native gel electrophoresis (c.k.n. since we have identified many potential interface residues, we plan iterative cycles of mutagenesis and functional tests. hn - based triggering can not be accounted by head partner switch because intermolecular disulfide bonds in the hn - dimer interface do not block fusion and thus both heads of a hn - dimer must move as a unit 45. since receptors engage these units from the top 2022, hn - head dimeric units may slide relative to another rather than rotate. sliding would not cross - link subunits within hn - tetramers, but may catenate the tetramers, which would liberate individual f - trimer spikes and trigger fusion 46. | the measles virus entry system, constituted of attachment (hemagglutinin, h) and fusion proteins, operates based on a variety of natural and targeted receptors. however, the mechanism triggering fusion of the viral envelope with the plasma membrane is not understood. here we tested a model considering that the two heads of an h - dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion - triggering signal. indeed, stabilizing the h - dimer interface by additional inter - molecular disulfide bonds prevented membrane fusion, an effect reversed by a reducing agent. moreover, a membrane - anchored designated receptor efficiently triggered fusion, provided it engaged the h - dimer at locations proximal to where the natural receptors bind, and distal to the h - dimer interface. we discuss how receptors may force h - heads to switch partners and transmit the fusion - triggering signal. |
success of endodontic treatment relies on the accurate determination of the working length and adequate enlargement of the root canal. showed that often canals are improperly cleaned and attributed this to inadequate instrumentation due to the fact that the root canal diameter is larger than the instrument caliber used in each particular case. in addition with shaping, determination of the anatomic diameter is also related to cleaning of the root canal system. determination of the anatomical diameter is based solely on clinician 's ability to detect the apical narrowing by tactile sense which is an empirical and inaccurate method. tan and messer. stated that traditional methods for determination of the anatomic diameter at the apical third have under estimated the real diameter of this region. without adequate scientific evidence decision ca thus the concept of cleaning the apical canal to three sizes larger than the first file to bind is not based on the scientific evidence. there are many factors that affect the determination of minimal initial working width at working length, these are ; canal shape, canal taper, canal curvature, canal content, canal wall irregularities and instruments for determining initial working width. preflaring of the cervical and middle thirds of the root canal improves anatomical diameter determination ; the instrument used for preflaring plays a major role in determining the anatomical diameter at the wl. thus, the objective of this study was to investigate the influence of cervical preflaring using different and currently used instruments on the accuracy of apical file size determination. tooth selection and preparation 80 extracted human permanent maxillary first molars displaying normal pulp chambers, patent root canals, fully formed apices without any sign of resorption were collected. maxillary molars with degree of curvature of the mesiobuccal root between 10 and 15 were utilized. all teeth were placed in 0.1% thymol solution and taken out 24 hours before use. the cusps of the teeth were cut horizontally to get a plane occlusal zone to determine the working length precisely. standard access cavities were performed and the pulp tissue was removed with a barbed broach, avoiding contact with the root canal walls. canals were then irrigated with copious 2.5% sodium hypochlorite solution (qualigens fine chemicals, navi mumbai, india). sizing of canals : after final irrigation with normal saline, the canal was dried. to maintain the patency of canal, an iso 06/.02 k - file (dentsply) was inserted until it came out of the apex. after maintaining patency an iso 08/0.02 k - file was inserted with gentle pressure in watch winding motion until the tip of file was visible at the apex. the overall canal length was thus determined by placing tip of the file at the apex and working length was established 1 mm short of this length. cervical and middle third flaring- after working length determination, the teeth were divided in to eight groups (n = 10). one group was taken as control group that is without pre - flaring, other seven groups received pre - flaring of root canals. all rotary instruments were used in continuous rotary motion with the help of x - smart endomotor (dentsply / maillefer, ballaigues, switzerland). the cervical and middle third of canals were pre - flared 3 - 4 mm short of the working length. gates - glidden drills (dentsply maillefer, size iso 90 - 110) were used until the binding sensation was felt in the middle third. protaper files (dentsply maillefer) sx (iso 20, taper 3.5 - 19%), s1 (iso 17, taper 2 - 11%), s2 (iso 20, taper 4 - 11.5%) were used in respective order. the flexmaster intro file (vdw) (iso 22/0.11) was initially used for flaring followed by iso 25/0.06 and 25/0.04 files respectively. an iso size 25/0.08 hyflex cm file (coltene) was used to flare the canal followed by an iso 20/0.04 file. determination of initial apical file- after pre - flaring of all teeth was done, hand files were inserted in to the mesio - buccal root canal starting with k - file iso 08/0.02 at the working length. an iso 10/0.02 file was then inserted till the working length. at iso 10, the file size was increased in increment of 5 iso units until slight friction was felt at the working length. the first file that had binding sensation at the working length was noted and fixed with methacrylate in the root canal. one millimeter of the root apex was cut horizontally with a microcutter so that the remaining tooth was at the working length. the apical sections were visualized using stereo microscope and images were recorded digitally for each specimen. the analysis of the images obtained was performed on a computer using the image pro plus software (media cybernetics, usa). it was used to determine the diameter of the root canal and diameter of the initial apical file. the largest and smallest diameter of the root canal and the largest diameter of the instrument were recorded. the data were submitted to anova test and post hoc tests bonferroni multiple comparisons, to assess the effect of pre - flaring techniques on the discrepancies found between the diameter of the binding instrument and the anatomic diameter of the root canal. cervical preflaring and the type of instrument had a significant effect on initial apical file size determination. preflaring with la group burs leads to the most accurate determination of the initial apical file size. in this group, the maximal apical root canal diameter and the diameter of the initial apical file had the lowest discrepancy (mean 0.015 mm 0.015) followed by hyflex group (mean 0.017 mm 0.017) and there was no statistical difference between these groups. following hyflex group were race instruments (mean 0.020 mm 0.020). protaper group showed greater discrepancy than the former groups (mean 0.028 mm 0.028). after protaper came flexmaster group (mean 0.035 mm 0.035) followed by k3 (mean 0.048 mm 0.048) and gatesglidden (mean 0.051 0.051) which showed comparable results [figure 1 ]. stereomicroscopic pictures of transverse sections of root canals at the wl with the iaf fixed in the root canal to show the discrepancies of root canal diameter and diameter of the iaf of (a) control group (b) la group (c) hf group (d) rc group (e) pt group (f) fm group (g) k3 group (h) gg group in order of minimal discrepancies between the initial apical file diameter and apical root canal diameter. discrepancies (in mm) between the diameter of initial apical file and canals at the working length, for different groups the instrument binding technique for determining anatomical diameter at working length is not precise ; preflaring of the cervical and middle thirds improve the determination of the anatomical diameters at the working length and the type of instrument play an important role. the increase in file size after preflaring can be explained by realizing that, within a canal, irregularities and curvature produced contacts with the file and interfere with its progression toward the apex. early flaring, regardless of the method used, removes these contacts, opens the space and reduces file contact ; thus, a file progresses more easily towards the apex after flaring. comes to a stop only when the diameter of the canal begins to apply pressure against the instrument. the point in the apical region of the canal at which preparation and obturation should be terminated is determined by several variables. and the canal is supposed to be prepared has been a controversial issue in the endodontic field. grossman also stated that the canal should be enlarged at least three sizes greater than its original diameter. however this concept needs to be reviewed, as it is ineffective and may leave canal walls untouched when no preflaring is performed. mechanical (that is rotary flaring) reduces treatment time, but is accompanied by a risk of complications. over enthusiastic use, inappropriate size and excessive depth can result in lateral perforations, ledges and instrument breakage. the roots of the teeth in the present study were embedded in methacrylate to preserve the apical region and to avoid any destruction during root canal preparation and cutting the root. the apical foramen was not covered by methacrylate to be able to precisely measure tooth length and exactly cut 1 mm of the apex with the iaf fixed in the root canal. in the present study, cervical preflaring of the root canal significantly increased the accuracy of determining the initial apical diameter by the initial apical file compared with non - flared root canals. the size of initial apical file in the control group was three times smaller than that in la and hf groups. each system has its own unique design feature and hence has a characteristic preparation technique. taper of instruments, used for preflaring, is a determining factor in the accuracy for determination of initial apical file. from all specimens evaluated, the root canal preflared with la axxess instruments presented the least discrepancies. this could be attributed to the configuration, metal alloy properties and mode of operation. moreover the 0.06 taper, safe end and flute design of la axxess burs have been associated with complete removal of cervical dentin projections without causing canal perforations and transportations. the 0.08 taper of the hyflex cm files could be associated with the good results. they are manufactured by a unique process that controls the material memory, making the files extremely flexible but without the shape memory of other niti files. this gives the file the ability to follow the anatomy of the canal very closely. these advanced features of hyflex cm files increase its efficiency to remove the dentin projections from cervical and middle thirds of root canals more effectively and quickly. this is the first study to compare the effect of preflaring with this new and unique file system. similarly the greater taper of race instruments that is, 0.10 and 0.08, effectively created the path for the initial apical file to progress without touching canal walls (curvature) and dentin projections, this could be attributed to its good performance in reducing the discrepancy between the apical canal diameter and initial apical file diameter. these results are in agreement with previous study, showing better results of race files over protaper and flexmaster. flexmaster intro file has a 11% taper but its simple k - file type design and less flute space for effective debris removal can be associated with its lack of performance as compared to previous instruments. these results are in agreement with previous study. showing better results of protaper over flexmaster. k3 files having 0.10 and 0.08 taper has a positive cutting and rake angle but its low flexibility as compared to other files could reflect its results in reducing the discrepancies between the initial apical file and apical canal diameter. although the results of k3 file are comparable with gates glidden drills. apical shaping is easier when early flaring is used, from the data presented, one can speculate that early flaring reduces the discrepancy between the initial apical file diameter and apical canal diameter. an appropriate apical sizing method can help operator to avoid unnecessary enlargement of the apex. there is further need of research to develop methods for optimal determination of root canal size in all dimensions while respecting the complexity of apical anatomy. although early preflaring of the root canal could not guarantee that the instruments bound only at the working length. clinically, it is not always possible to straighten the coronal two - thirds of root canal because sometimes the radius of root curvatures is long. however, it must not be forgotten that the capability of the endodontist in applying all available information is also a determinant for success. cervical preflaring plays a vital role in reducing the discrepancy between initial apical file diameter and apical canal diameter. taper, cross sectional design and flexibility of the instrument used for preflaring plays a vital role in determining its effect. la axxess burs and hyflex cm files showed best results in comparison to all groups compared in this study. | aim : to investigate the influence of cervical preflaring using different rotary instruments on apical file size determination.materials and methods : extracted human molar teeth were randomly divided in to eight groups (n = 10) : control group (cg) ; la axxess group (la) ; hyflex group (hf) ; gatesglidden group (gg) ; protaper group (pt) ; race group(rc) ; flexmaster group (fm) ; and k3 group (k3). patency was maintained and working length was established under magnification. all instruments were used according to manufacturer 's instructions. steriomicroscopic images were taken to determine the discrepancies in diameters. proplus software (usa) was used to determine the diameter of the root canal. anova test and post hoc tests bonferroni multiple comparisons were used for statistical analysis.results:canals preflared with la axxess burs showed the best results. control group that is, the canals with no cervical preflaring showed the maximum discrepancy between the initial apical file diameter and apical canal diameter.conclusion:cervical preflaring plays an important role in reducing the discrepancy between initial apical file diameter and apical canal diameter. |
a primiparous 26 year old mother, 41 weeks of gestational age wanted a vaginal delivery. to provide analgesia during the delivery, an epidural catheter was inserted in the l4-l5 interspace using the loss - of - resistance technique. no cerebrospinal fluid or blood leakage was observed and the patient did not experience paresthesia of the lower extremity, numbness, or pain. at the time 0.15% levobupivacaine 9 mg and fentanyl 50 ug was administered, the cervical os was dilated to about 2 cm, effaced 100%, and labor pains were 1 to 2 minutes apart with a visual analogue scale of 4 to 5 points. nothing unusual was observed during the administration of the drugs, so a mixture of 97 ml of 0.125% levobupivacaine and 150 ug of fentanyl was administered at a 10 ml / h flow rate via the epidural space using the pca infusion pump (automed 3300, ace - medical, seoul, korea). the mother gave birth to a 3.03 kg male newborn baby without issue other than the use of the vacuum. the neonate did not cry, had pale skin, and had no response to stimulation. as the apgar score did not improve, the child was immediately moved to the neonatal intensive care unit. the total amount of drugs administered to that specified point, totaled 82 ml of the 0.125% 97 ml of levobupivacaine and 150 ug of fentanyl mixture during 82 min. the patient experienced no ambulatory or urinary discomfort immediately post delivery but 11 hours afterward the patient reported hypoesthesia, numbness, tingling, and muscle weakness in both lower extremities enough to have trouble standing. cauda equina syndrome caused by an epidural hematoma was suspected so a lumbar spine x - ray and lumbosacral mri were taken, the mri results only showed a l3 through l5 intervertebral disc protrusion (fig. 1). the patient reported the same symptoms the next day as well as trouble detecting the desire to urinate and defecate. on postpartum day 2, after consulting a neurologist, myelopathy was suspected so a whole spine mri was taken but no abnormalities were found (fig. strength in the lower extremities recovered enough to walk with support and limited hip, back of the thigh, calves and feet sensations returned. however, the patient still had trouble detecting the need to urinate / defecate and urinary retention, incontinence and constipation persisted. 15 days following delivery, an emg showed that the motor nerve conduction by both tibial nerves were normal but right - sided compound muscle action potential was reduced. while hoffmann 's reflex on the left - side was not undulated in terms of tibial nerve damage, peripheral neuropathy was suspected. after improvement in sensory nerve function, the patient left by her own volition to follow - up at another facility. 21 days after delivery, neurological observation revealed : hip flexion / extension was 4+/4 on the right, 4-/4 on the left ; 4+/4 for right hip abduction / adduction, 4-/4 for the left ; 4+/4 + for both left and right knee flexion / extension ; ankle dorsiflexion / plantarflexion 5/5 for left and right ;, and big toe dorsiflexion / plantarflexion 5/5 for left and right, stable enough to walk without discomfort. but perianal area sensations and the lateral side of calf was both 60/60, and the urination / defecation problem did not improve. the neurologist had an impression that the lumbosacral plexus was injured so steroid pulse therapy with 500 mg of methyl prednisolone was started and further tests were scheduled. the urodynamic study results showed bladder filling, a pressure of 6 mmh2o (normal value < 10 mmh2o) and normal bladder compliance and capacity. but the first desire to void bladder volume was 373 ml (normal value 175 - 250 ml) and the normal desire to void bladder volume was 591ml (normal value 350 - 450 ml), while the patient could not detect a strong desire to void. during urination, residual urine was 600 ml, allowing for a final diagnosis of flaccid neurogenic bladder with a severely reduced contractility of bladder sensation and detrusor muscle. impaired rectal sensation was concluded with anorectal manometry results at rectal sensory threshold volume for first desire to defecate measured as 30 ml (normal value 17 - 23 ml), anal pressure reduced to 32 mmhg (normal value 59 - 74 mmhg) at resting, and 12 mmhg (normal value 80 - 160 mmhg) while squeezing. in terms of defecography, the anorectal angle, which is deeply related to the pudendal nerve, also, the angle was 123 degrees at straining and 126 degrees at defecating which was higher than normal values, and a tendency for proctocolic intussusceptions was observed when defecating, showing a problem in contraction and relaxation. pudendal nerve terminal motor latency (pntml) was also planned but was not initiated due to the request of the patient. the patient continued to experience dyschezia, but voiding difficulties improved and self voiding urine was 300 - 400 ml with residual urine of 50 - 70 ml. the patient decided to follow up on an outpatient basis while continuing treatment options like biofeedback physiotherapy, but she has not fully recovered currently at 7 month. an epidural block using local anesthetics is commonly used for analgesia for pain relief of the vaginal delivery process. and when complications arise, epidural blocks are rarely the cause. according to holdcroft., neurological complications for those that receive an epidural block or anesthesia occur at a low rate of 1 out of 13,007 patients, while those caused by the obstetric procedure itself or other causes can occur at rates of 4 - 6 times that. if neurological complications from an epidural block occur, it can present as paresthesia and a reduction of muscular strength. nerve damage can be caused by the tuohy needle, the catheter, spinal cord ischemia, accidental injection of neurotoxic agents, infection or injection of local anesthetics into the epidural space. also neurological complications from an epidural hematoma can occur, but such cases are rare at a rate of 1 : 150,000. most hematoma cases occur when a patient has problems with coagulation, therefore preeclamptic patients with a risk of coagulopathy have a higher risk. in these patients the time of injection and removal of the catheter is very important. also, 24 hours following a catheter removal, a thorough neurological examination should be taken. if complications arise, sharp leg and back pains can occur followed by a loss of sensation and strength in both extremities, incontinence, lack of tension of the sphincter ani, and a loss of motor reflexes. if hematoma is suspected, computer tomography or mri should be taken and after a hematoma diagnosis, surgical decompression is needed to commence in 6 hours. the epidural needle can enter a blood vessel, pierce the intervertebral foramen or get tangled in the spinal nerve root and cause complications. if the spinal nerve root is injured during the procedure, in most cases, unconscious leg movement and radiculopathy occurs, in which the procedure should be immediately stopped and the placement of the needle or catheter should be properly adjusted. complications resulting from improper epidural catheter placement can be more severe than that of direct damage from a needle or drugs. recovery from injury of the nerve root of the spine takes a long time, usually months after damage, so extra care should be taken. during the case in question, because there was no pain or paresthesia until the catheter was removed, the likelihood of nerve damage by the tuohy needle or epidural catheter seems unlikely. also, no neurological symptoms were observed during the injection of the drugs, so nerve damage from the toxicity of the drugs also seems unlikely. if neurological complications are suspected, taking a thorough history of the symptoms is vital to the differentiation of the cause of the damage. the onset, area of the symptom, presence of radiating pain or pain during the procedure should be inquired. also, through physical examination, it should be recognized whether the pain or the neurologic deficit follows the dermatome or the pathways of the peripheral nerves. because changes in the emg shows after 2 weeks of the injury, if changes occur in the first week of delivery, it means the mother had neurologic problems before the delivery. during vaginal delivery, various factors can lead to nerve damage such as improper vacuum or forceps operation, inappropriate position of the mother, and pressure from the fetal head. examples include : damage to the lumbar plexus, sacral plexus, femoral nerve, obturator nerve, common peroneal nerve, and tibial nerve are examples. the pudendal nerve can also receive damage during vaginal delivery, and denervation of the sphinter ani can cause trouble with defecation. it is not known if such neurological damage is caused by extension of the pudendal nerve or direct pressure from the fetal head at the small branch of the nerve or neuromuscular junction. the sacral plexus is derived from the anterior rami of spinal nerves l4, l5, s1, s2, s3, and s4. each of these anterior rami gives rise to anterior and posterior branches which provide motor and sensory nerves for the posterior thigh, most of the lower leg, the entire foot, and part of the pelvis. if damages to the sacral plexus were to occur, urination and defecation disorders similar to cauda equina syndrome can occur, which is due to damage to the automatic nervous system of the rectum and bladder. the pelvic splanchnic nerve from the ventral roots, through the parasympathetic fiber and pudendal plexus, sends arousal signals to the bladder 's destrusor muscle, while it sends repression signals to the internal sphincter muscle of the urethra and the smooth muscle of the rectum. the sensation of pain and expansion from the bladder and lower rectum is sent to the central nervous system, it passes through the pudendal nerve and posterior rami to be terminated at the anterolateral column of the s2 - 4 spine. the pudendal nerve also includes motor fibers and relays repression or arousal signals to the external sphincter of the urethra and anus. in this case, urological testing showed a flaccid neurogenic bladder, which was consistent with the t11, t12, l1 and l2 sympathetic nerve signaling the bladder, while s2 through s4 parasympathetic nerve signals were being blocked, causing detrusor muscle relaxation, and persistent arousal of the internal sphincter muscle of the urethra. also severe degradation of the desire to void and defecate showed there may have been problems with parasympathetic signaling between the bladder and rectum. upon evaluation of the patient 's anorectal function, an anorectal angle greater than the normal value and proctocolic intussusception during defecation was observed by defecography. there is no specific way of evaluating pelvic splanchnic nerve damage, while the pntml test exists for pudendal nerve damage. the pntml test has its limitations in that even while nerve damage has progressed severely, motion conduction ability can be maintained since motion conduction can occur through the small diameter axon of the pudendal nerve. other testing methods excluding pntml only examines end - organ functions, and it can be said is a practical and accurate method of measuring pudendal nerve function does not exist. through a combination of results from physical examinations and consequent emg, mri, urodynamic study, anorectal manometry, defecography as well as patient urination / defecation disorder symptoms, we were able to conclude, the sacral plexus, in which the splanchnic nerve and pudendal nerve originates, was injured. when a mother experiences neurological symptoms after epidural analgesia for a cesarean section or vaginal delivery, the anesthesiologist is the first to be called. since neurologic regional anesthesia related complications have a lower incidence than obstetric origins, an anesthesiologist may initially have difficulties handling such cases. but it is important for the anesthesiologist to keep in mind that neurological damage during delivery is most often caused by peripheral neuropathy from the obstetric process. though rare, neurological consequences can occur from anesthesia, so if neurological complications do occur, even while there was nothing unusual during the procedure, it is important to decide on the proper course of management after a thorough history, physical examination, and diagnostic tests. | a 26 year old, healthy, 41 week primiparous woman received a patient - controlled epidural analgesia (pcea) and experienced paraplegia 11 hours later after a vaginal delivery. this was thought to be the result of complications from pcea but there was no specific abnormality on magnetic resonance imaging (mri) of the lumbosacral spine. on an electromyography (emg) study performed 15 days following delivery, signs of tibial neuropathy were present and peripheral nerve injury during vaginal delivery was suspected. motor weakness and hypoesthesia of both lower extremities improved rapidly, but a decrease in the desire to urinate or defecate, followed by urinary incontinence and constipation persisted, we suspected the sacral plexus had been severely damaged during vaginal delivery. seven months later, the patient 's conditions improved but had not fully recovered. |
the patient was a 28-year - old woman who was admitted to hospital because of a hepatic mass found incidentally on screening sonography. she had no previous surgical and medical problems. a physical examination and the laboratory findings on admission revealed no abnormalities. the serum alpha - fetoprotein level was 1.2 ng / ml and the carcinoembryonic antigen level was 1.3 ng / ml. test for the hepatitis - b antigen was negative and the test for hepatitis - b antibodies was positive. ct of the abdomen showed a mass in the segment 6 measuring approximately 4.03.8-cm with a capsular retraction. the hepatic helical ct scan during the hepatic arterial phase showed a heterogeneously enhancing mass with a lobulated margin. the hepatic helical ct scan during the portal phase showed an isodense mass with a hypodense central scar (fig. these findings were compatible with the focal nodular hyperplasia except for a central scar extension to the hepatic capsule causing a retraction. the t1-weighted axial mr image of the liver demonstrated a slightly hypointense mass demarcated by a thin hypointense rim with a central hypointense scar. the t2-weighted axial mr image showed a slightly hyperintense mass with a hypointense central scar. the contrast - enhanced t1-weighted axial mr image revealed a marked enhancement of the tumor with a nonenhancing central scar (fig. the gross specimen showed a nodular configuration with a dense fibrotic central scar. on the cut surface, a central dense fibrotic scar with radiating fibrous septa dividing the lesion into smaller nodules the central scar extended to the surface, and the surface of the liver parenchyma adjacent to the mass was retracted. the resected specimen showed a depressed thickened stellate scar, slightly eccentrically positioned, with tapering fibrous septa, which radiated through the mass, dividing it into multiple lobules. focal nodular hyperplasia (fnh) is found predominantly in women during the third to fifth decade of life. however, it has been reported to occur in both sexes and in all age groups. the cause of fnh is not well understood, but a congenital vascular malformation or vascular injury has been suggested to be an underlying mechanism for the hepatocelluar hyperplasia (2). fnh is classically seen as a solitary, homogeneous, and slightly hypoattenuating or isoattenuating area compared to the normal liver on unenhanced ct (1). in approximately 20% of patients, a central low - attenuating scar may be observed (6). on enhanced ct, fnh shows an immediate and intense enhancement, with the exception of a central scar with a delayed enhancement, possibly due to abundant fibrous stroma (6). the atypical imaging findings of fnh include hemorrhage, necrosis, fat accumulation on an unenhanced ct, rapid contrast washout, delayed contrast accumulation, or the absence of a central scar (1, 2). the mr imaging of typical fnh is iso- or slightly hypointense mass with hypointense scar on the t1-weighted mr imaging and a slightly hyperintense mass with a hyperintense scar on the t2-weighted image. intensely and immediately enhancing mass with a delayed enhancing central scar is a classic finding on the gadolinum enhanced mr (1, 2). according to vilgrain (7), when the firosis, edema, vessels, and inflammation of the central scars were carefully studied, obliterative vascular changes were observed in both hyperintense and hypointense scars on the t2-weighted images. they reported that the distribution of fibrosis, inflammation, and vessels were not different in the hyper- and hypointense scars, but most of the hyperintense scars had predominant edema, whereas the hypointense scars showed an absence of or a lower degree of edema (7). a similar correlation between the pathology findings and mr imaging was done to evaluate the central scars of the primary liver tumor (8). rummeny (8) described three pathologic types of the central scars : (a) vascular scars composed mainly of vascular channels penetrating the collagenous tissue ; (b) inflammatory scars with edema, necrosis, hepatocellularity and loose connective tissue ; and (c) collagenous scars composed predominantly of dense, sclerotic collagen. vascular and inflammatory scars appeared hypointense relative to the liver parenchyma on the t1-weighted images, and hyperintense on the t2-weighted images, while collagenous scars were hypointense on both the t1- and t2-weighted images. in our case, the microscopic findings showed a collagenous scar composed mainly of dense, sclerotic collagen, which explains the cause of the hypointensity on the t2-weighted image and a minimal enhancing central scar on the enhanced t1-weighted image. the cut surface of a typical fnh revealed a central stellate scar with radiating fibrous septa dividing the lesion into smaller nodules. the central scar contained thick - walled vessels that provided arterial blood supply to the lesion (2). grossly, the resected specimen showed a depressed thickened stellate scar, slightly eccentrically positioned, with tapering fibrous septa that radiated through the mass, dividing it into multiple lobules. microscopically, numerous fibrous bands of varying thickness divided the lesion, surrounding the nodules of the hyperplastic hepatocytes and converging at the central scar. several large blood vessels, which exhibited varying degrees of myointimal hyperplasia, mural thickening and intimal narrowing, were observed within the dense collagenous scar. these radiating septa contained numerous thin - walled vessels, lymphocyte infiltrates and numerous proliferated bile ducts, which were closely apposed to the periseptal hepatocytes. the hepatocytes nodules were composed of normal parenchymal elements, which were irregularly arranged with plates of one or two cells ' thickness but the normal lobular architecture was not seen. a capsular retraction of the liver adjacent to the hepatic mass is an uncommon finding. soyer. (3) reported that the prevalence of the capsular retraction adjacent to the hepatic tumor was 2% and all tumors were malignant. seo (4) suggested that the prevalence was approximately 12% in malignant hepatic masses. these findings have been observed in cases of hepatic epitheloid hemangioendothelioma (heh), hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and metastases from colon and breast cancers (3 - 5, 10). the capsular retraction of heh was most likely due to the tumor fibrous reaction, which distorted the tumor margin and the adjacent liver capsule (5). the main factors causing the capsular retraction in malignant hepatic tumors are a portal venous obstruction in the hepatocelluar carcinoma, and hepatic atrophy followed by a bile duct obstruction in a cholangiocarcinoma (4). soyer. (3) reported that this finding was never associated with a benign tumor, supporting the hypothesis that a retraction of the liver capsule adjacent to a hepatic tumor is a finding specific to malignant tumors. a capsular retraction with hemangiomas in cirrhotic livers has also been described (10). although blachar (10) reported that the hemangioma was the only benign neoplasm associated with a capsular retraction, this study found another benign hepatic mass with a capsular retraction. this is the first report showing a capsular retraction in focal nodular hyperplasia. in this case, the central fibrotic scar within the tumor was extended to the surface, and a retraction of the liver capsule adjacent to the tumor was made. | focal nodular hyperplasia (fnh) is characterized by the presence a central scar with radiating fibrous septa. our case had a capsular retraction, which was the result of an extension of the central scar to the surface. in addition, a hypointense scar on the t2-weighted image and a minimal enhancing central scar on the enhanced t1-weighted image, which was due to dense, sclerotic collagenous tissue, were observed. we report the first case of fnh with a capsular retraction. |
immunoglobulin g4 -related disease (igg4rd) is a novel disease entity associated with high serum igg4 levels. the kidney is a distinct target organ affected by this disease entity and we report one such patient with an unusual presentation. a 57-year - old gentleman was referred with high grade intermittent fever with chills since 3 months. he was a longstanding type 2 diabetic, on oral hypoglycemic agents with a fairly good sugar control. the complete blood count of the patient showed an hemoglobin 11 g / dl, total leukocyte count 10,100 (polymorphs 85, lymphocytes 13% and eosinophils 2%) and a normal platelet count of 2.73 lacs. liver function tests were essentially normal with a bilirubin of 0.7 mg / dl, alanine aminotransferase / alt of 36 and 31 iu / l, serum albumin 3.84 g / dl, and serum globulin 2.46 g / dl. renal function tests showed bun 55 mg / dl, serum creatinine 1.5 mg / dl, and normal serum electrolytes. the urine analysis was essentially bland with no albumin, 1 - 2 pus cells, and no red cells. test results during these 3 months revealed an erythrocyte sedimentation rate of 90 mm at the end of 1 h and two high c - reactive protein levels of 35.8, 24, and 18.5 mg / l (normal up to 6 mg complete blood count, urine examination, chest radiology, and 2d echo performed to investigate the cause of the fever were all normal. a whole body computed tomography (ct) scan revealed multiple lymph nodes measuring 1.5 - 4 cm in the mediastinum and retrocarinal region reported to be of inflammatory / infective etiology. the contrast ct abdomen revealed heterogeneous enhancement of both kidneys with hypodensities at the lower pole [figure 1 ]. contrast computed tomography abdomen showing heterogeous enhancement of the kidneys with hypodensities at the lower pole given the clinical presentation and the clinical possibilities of vasculitis and lymphoma, and the kidney findings on ct, a renal biopsy was performed. the conspicuous feature was presence of expansile interstitial fibrosis with a plasma cell rich interstitial infiltrate that were accompanied by a few lymphocytes and occasional eosinophil [figure 2a ]. the fibro inflammatory process was seen to extend into the peri - nephric tissue and had resulted in significant tubular loss. the glomeruli revealed diffuse capillary wall thickening but there were no changes of diffuse or nodular diabetic glomerulopathy. igg4 immunohistochemistry was performed which showed numerous igg4 expressing plasma cells amounting to at least 15 per high power field (hpf) [figure 2b ]. the diagnosis of igg4 mediated nephropathy was made on the basis of the clinical history, ct scan findings, histopathology, immunohistochemistry, and serology. he was initiated on a dose of 40 mg prednisolone (0.5 mg / kg / day) and his fever defervesced within 24 h. the steroids were continued and tapered over the next 5 months. his serum creatinine touched a baseline of 1.2 mg / dl at the end of 2 months therapy with steroids. although an attempt was being made to taper down the steroids further, his fever recurred at a steroid dose of 5 mg / day. he was therefore initiated on mycophenolate mofetil (mmf) with a dose of 1 g b.i.d to which his fever responded again. g / l to 2.04 followed by 1.55 g / l by the end of the 4 month. the patient continued to be on a dose of 2 g of mmf per day. the ct scan repeated after 6 months showed a significant regression in the mediastinal and retrocarinal lymphadenopathy and resolution of the original hypodense foci in the kidneys. (a) expansile interstitial fibrosis (h and e, 100), (b) plasma cells with expression for immunoglobulin g4 (400s) igg4rd is an increasingly recognized syndrome of unknown etiology comprised a collection of disorders that share specific clinical, serologic, and pathologic features. the commonly shared features include tumor like swelling of the involved organs, a lymphoplasmacytic infiltrate rich in igg4 positive plasma cells, and variable degrees of fibrosis. renal involvement in igg4rd is relatively uncommon when compared with autoimmune pancreatitis which is the most studied group with the latter being linked to elevated serum igg4 concentrations as early as 2001. the myriad of renal presentations include hydronephrosis secondary to retroperitoneal fibrosis that may represent extra - renal extension of the fibro inflammatory pathology and tubulointerstitial nephritis. rarely, patients show multiple tumor like low density areas on imaging like our patient. the renal biopsy in such cases reveals aggregates of lymphocytes and plasma cells in the renal interstitium. asymptomatic lymphadenopathy is common, occurring in 80% of patients and may be the only initial manifestation. mediastinal, hilar, intra - abdominal, and axillary lymphadenopathy are the common sites and are readily detectable on gallium 67 scanning. in fact, the combination of renal involvement and lymphadenopathy should arouse clinical suspicion of igg4-related disease. it is more often described as occurring in middle aged and older men. the majority of patients with igg4-rd have elevated serum igg4 concentrations, but the range varies widely. approximately 30% of patients have normal serum igg4 concentrations, despite classic histopathologic and immunohistochemical findings. however, as a corollary it has been found that up to 40% of patients with igg4rd have peripheral eosinophilia. immune complex deposits in the pancreas, kidneys, and certain other affected tissues have been reported. the number of igg4-positive plasma cells per hpf regarded as sufficient for the diagnosis of igg4rd varies somewhat from tissue to tissue ; generally, the minimum for making the diagnosis for most tissues is from 30 to 50 igg4-positive cells / hpf. however, only 10 igg4 positive plasma cells / hpf may be sufficient in some organs or tissues, including the kidney. there are increased numbers of t regulatory cells (tregs) in peripheral blood and increased levels of cytokines produced by tregs, including interleukin (il)-10 and transforming growth factor- in affected tissues. although igg4 concentrations become lower with glucocorticoid treatment in the great majority of patients in whom they are elevated at baseline, they remain above normal values in most patients. a multicenter study from japan showed that igg4 levels failed to normalize in 115 of 182 patients (63%) treated with glucocorticoids. azathioprine and mmf have been used for patients who require chronic therapy to avoid the adverse effects of steroids. b cell depletion therapy with rituximab is an effective treatment for patients refractory to glucocorticoids and standard immunosuppression. the natural history of igg4rd has not been well defined. several types of lymphomas especially non - hodgkin 's variety have been reported in these patients 3 - 5 years after the diagnosis of igg4rd. pyrexia of unknown origin (puo) is a diagnostic challenge. in the general population infections however, unlike in the young, a precise diagnosis can be made 87 - 95% of the times. in a study carried out in elderly patients, infections constituted 25 - 35%, connective tissue disorders constituted 25 - 35%, and malignancies accounted for 12 - 23% of the cases. as many of these diseases are treatable it is recommended that etiology of puo in elderly should be investigated further. in this patient, puo was the presenting manifestation and the ct finding of wedge shaped hypodensities within the kidney, steered the investigative algorithm towards the kidney as the organ of interest. as renal infections, vasculitis, and septic emboli could present in this fashion with fever and renal hypodensities, the kidney biopsy showed a clinically unsuspected diagnosis for the presenting symptoms. igg4rd is a newly recognized fibro inflammatory condition characterized by tumefactive lesions, a dense lymphoplasmacytic infiltrate, and storiform fibrosis of the tissue. high index of suspicion is required especially with rare extra pancreatic manifestations of this disease. timely diagnosis, treatment, and disease monitoring will play a significant role in influencing the long - term outcome of the patient. | pyrexia of unknown origin is a challenging clinical problem. infections, malignancies, and connective tissue diseases form the major etiologies for this condition. we report a case of a 57-year - old diabetic male who presented with fever of unknown origin for several months. the course of investigations led to a kidney biopsy which clinched the cause of his fever as well as the underlying diagnosis. the light microscopy findings of expansile storiform fibrosis with a dense inflammatory infiltrate suggested the diagnosis which was confirmed by positive staining of immunoglobulin g4, the dense lympho - plasmacytic infiltrate and elevated serum igg4 concentrations. a course of steroids followed by mycophenolate mofetil as maintenance immunosuppression rendered the patient afebrile with improvement of renal function. |
our laboratory has developed a method of particle exposure whereby anesthetized rats intratracheally inhale, at a regulated breathing rate and pressure, an aerosolized test material. this method is capable of delivering considerable doses in a short time period and, unlike the commonly used method of intratracheal instillation, does so with an even particle distribution throughout the lung. early studies comparing the response of male fischer 344 rats exposed to tio2 particles of two differing primary particle sizes showed that at similar particle doses animals exposed by the two methods showed differences in response, as measured by bronchoalveolar lavage (bal) parameters. building on this, we sought to study the roles that macrophage inflammatory protein-2 (mip-2) and tumor necrosis factor alpha (tnf - alpha), two cytokines thought to have proinflammatory roles in the lung, may play in the differences observed. increases in mip-2 protein levels in the lavaged cells, but not the supernatant, were observed in those groups where increased polymorphonuclear cells (pmn) in the lung lavage were found, but not in those where no increase in pmn levels was observed. bal tnf - alpha levels, measured by enzyme - linked immunosorbent assay, showed no apparent correlation with cellular or biochemical bal parameters for either particle size or dosing method. increases in immunocytochemical staining for tnf - alpha, compared to unexposed controls, were observed in several particle - exposed groups. thus, it appears that increased bal mip-2 protein levels, but not tnf - alpha, correlate well with the inflammatory response, as measured by pmn numbers in lavaged cells, for both exposure systems.imagesfigure 5. bfigure 5. afigure 5. dfigure 5. c |
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the hav is a non - enveloped, positive - stranded rna virus that was first identified using electron microscopy in 1973, and is classified within the genus hepatovirus of the picornavirus family.1 hepatitis a occurs predominantly in children, where it is a self - limiting and usually asymptomatic infection. while almost all acute hepatitis a infections subside spontaneously without severe complications, some infections can be relapsing, prolonged, and involve cholestasis and acute kidney injury. hav - induced fulminant hepatitis is rare, with a reported incidence rate of below 0.5%.2 recently, the incidence of hepatitis a in korean adults has increased rapidly due to the decreased seroprevalence of anti - hav. thus, there is an increasing number of symptomatic adult hepatitis a patients with an unusual clinical course.3 drug rash with eosinophilia and systemic symptoms syndrome (dress), also known as drug - induced hypersensitivity syndrome (dihs), is a severe idiosyncratic reaction to drugs, mainly antiepileptics and antibiotics, and can occasionally result in acute liver failure.4 the characteristics of dress syndrome include skin rash, fever, lymph node enlargement, and internal organ involvement. although the pathophysiology of dress syndrome remains unclear, it may be due to a defect in detoxification of the causative drug, immunological imbalance, or infections such as human herpes virus type 6 (hhv6).5 the present report describes the case of a 22-year - old male with hepatitis a involving prolonged cholestasis and renal injury who developed dress syndrome due to antibiotic and/or antiviral treatment. to our knowledge, this is the first report of histopathologically confirmed dress syndrome following severe relapsing hepatitis a in an adult. in january 2010, a 22-year - old male presented with acute renal failure at another hospital, and was diagnosed with acute hepatitis a. he was treated conservatively at that hospital for one month, during which time the symptoms and laboratory findings improved. at that time three days prior to the end of treatment, he developed a sudden fever and liver enzyme levels began rising again. one day after the administration of oseltamivir, he developed a whole body skin rash with intense itching. records from the previous hospital admission indicated a persistent fever accompanied by a skin rash, and an abdomino - pelvic ct showed gall bladder wall thickening. consequently, cefotaxime and metronidazole antibiotics were prescribed due to the suspicion of acute cholecystitis just one day before his arrival at our hospital. the patient was referred to our hospital on february 9th due to the cholestatic hepatitis a, fever and skin rash symptoms. the initial vital signs were as follows : blood pressure 112/72 mmhg, heart rate 113/min, respiratory rate 20/min, and body temperature 38.0. laboratory findings showed a white blood cell count of 15,900/mm, a hemoglobin level of 11.9 g / dl, a platelet count of 321,000/mm, an eosinophil count of 2,150/mm (8% [upper limit of normal (uln) 7% ]), a serum protein level of 5.0 g / dl, a serum albumin level of 1.9 g / dl, a serum aspartate aminotransferase (ast) level of 152 iu / l (uln 40 iu / l), a serum alanine aminotransferase (alt) level of 187 iu / l (uln 40 iu / l), a serum creatinine level of 1.3 mg / dl (uln 1.4 mg / dl), a serum total bilirubin level of 25.8 mg / dl (uln 1.2 mg / dl), a serum direct bilirubin level of 15 mg / dl (uln 0.5 mg / dl), and a prothrombin time (pt) (%) level of 22.4% (normal, 70 - 140%). the patient tested negative for herpes virus, cytomegalovirus (cmv), hepatitis b and c, and positive for igg antibodies against the epstein barr virus (ebv) and hav. the patient was treated using conservative management for the hepatitis a with prolonged cholestasis, including the administration of cefotaxime for 4 - 5 days for the persistent fever and jaundice. by the 4th day after admission to our hospital, the fever and skin rash symptoms worsened, prompting a skin biopsy (fig. the biopsy revealed perivascular lymphocytic and eosinophilic infiltration, consistent with drug eruption (fig. the peripheral blood eosinophil count was 32.4% (6,059/mm), and laboratory findings showed a total bilirubin level of 23 mg / dl, an ast level of 110 iu / l, an alt level of 142 iu / l and a serum creatinine level of 4.4 mg / dl. upon consideration of the overall findings, we made a diagnosis of dress syndrome and prolonged cholestatic hepatitis a with renal azotemia. therefore, cefotaxime administration was immediately ceased, and high - dose steroid (hydrocortisone 80 mg tid daily) and immunoglobulin (1 g / kg / day for 2 days) were administered. after a week of high - dose steroid administration, the fever and skin rash improved, and prednisolone was then continued at a maintenance dose. a cmv antigenemia test returned positive findings (41/200,000 wbc), and a cmv pcr showed 1,097,500 copies / ml. the steroid dose was rapidly reduced and ganciclovir was administered (fig. the steroid was discontinued on march 28th, and ganciclovir was ceased on april 7th. approximately 10 days after steroid discontinuation, the serum alt level rapidly rose to 1,200 iu / l. we performed further blood cultures and pcr assays for the detection of hav, cmv and ebv, yet these tests all returned negative findings. therefore, we commenced steroid pulse therapy (methylprednisolone 1 mg / kg / day) on april 14th. the patient improved and the steroid dose was tapered gradually such that it was 20 mg daily in the form of oral prednisone by the time of discharge on april 28th. the incidence of acute hepatitis a in adults has increased rapidly during the past 10 years in korea. less than 30% of infected children are symptomatic, while approximately 80% of infected adults manifest severe hepatitis with markedly elevated liver enzymes. however, recently in korea, the incidence of unusual patterns of severe hepatitis and renal insufficiency has increased as the age of patients with acute hepatitis a has increased.3 the unusual clinical manifestations of acute hepatitis a include protracted cholestasis, relapse and complications involving renal failure.6 relapsing hepatitis a occurs in 3 - 20% of acute hepatitis a patients,7 and is characterized by a biphasic or polyphasic peak of liver enzyme elevation. relapses occur some weeks or months after the apparent recovery, and are marked by the return of symptoms and liver enzyme abnormalities. it generally takes less than 3 weeks for a patient to recover from a recurrent episode. in most cases, transaminases and bilirubin levels usually show a marked decrease but not a complete normalization during this period. the symptoms are usually milder in a relapse episode, although cholestasis can occur.8,9 the current patient first presented at another hospital with renal azotemia, and was diagnosed with acute hepatitis a. over a treatment period of 4 weeks, the transaminase levels showed marked improvement, although they never completely returned to normal levels. this patient 's recurrent episode developed with more severe symptoms than the initial episode, possibly due to the development of dress syndrome. extrahepatic manifestations are unusual in hepatitis a, and renal manifestations are even less common. acute kidney injury complicating non - fulminant hepatitis a is reported to occur in only 1.5 - 4.7% of hepatitis a patients.2 kidney injury may be the result of direct renal toxicity due to increased bilirubin levels, cryoglobulinemia, alterations in renal blood flow due to endotoxemia, and peripheral immune complex - mediated damage. in the cholestatic variant of hepatitis a, while transaminase levels reduce to normal levels, bilirubin levels increase to above 15 mg / dl, and may persist in that range for more than 8 weeks. this variant can be characterized by pruritus, persistent anorexia, diarrhea and weight loss.10 although corticosteroids hasten the resolution of prolonged cholestatic hepatitis a, they may predispose to hepatitis relapse.11 in the current patient, the bilirubin level decreased and normalized after the administration of corticosteroid. however, symptoms returned following the cessation of steroid treatment, which is not generally observed when corticosteroids are used to treat recurrent hepatitis a.8 hence, we considered that the patient was experiencing dress syndrome recurrence rather than hepatitis a recurrence. the detection of hav rna in the plasma after 20 days of illness is a predictor of prolonged cholestasis, but it is not a predictor of relapsing hepatitis.12 acalculous cholecystitis may often be a hepatitis a complication, as in the present case, and often manifests transiently with spontaneous recovery.2 the current patient presented with various forms of atypical manifestations associated with acute hepatitis a, including cholestasis and renal injury. dress syndrome is a severe delayed hypersensitivity drug reaction that manifests 2 - 8 weeks after exposure to the triggering drug. our patient was diagnosed with dress syndrome according to the definition of bocquet.13 the diagnostic criteria include the simultaneous presence of 3 conditions : 1. at least one of the following systemic abnormalities : enlarged lymph nodes, hepatitis (transaminases > 2 uln), interstitial nephropathy, interstitial lung disease or myocardial involvement. the hypersensitivity reaction is associated with high levels of il-5 and eosinophil accumulation, and may justify the use of steroids. a recent report suggests that certain drugs may stimulate a massive expansion of drug - specific cytotoxic t cells that destroy hepatocytes via a cell - contact - dependent mechanism.14 liver involvement in dress syndrome is common and may range from a transitory increase in liver enzymes to liver necrosis with fulminant hepatic failure.4 the overall mortality in dress syndrome is approximately 10%.15 dress syndrome occurs frequently with concomitant viral infection, especially hhv6.16 it may be that viruses interfere with the clearance of the triggering drug and thus induce accumulation of a metabolite. in the present case, it is still unclear whether dress syndrome followed acute hepatitis a or developed simply after receiving the antibiotics and antiviral agent. however, hepatitis a virus infection appeared to have triggered dress syndrome development by reducing the hepatic clearance of drugs, leading to an accumulation of drugs and their metabolites. in a type of feedback loop, it then appeared that the dress syndrome caused the recurrent hepatitis symptoms to be worse than the initial symptoms. at this stage our future plan is to perform patch tests using oseltamivir, cefotaxime and metronidazole to identify the causative drug once prednisone treatment is ceased. a recent report showed that the most common causes of dress syndrome in korea were antibiotics, followed by anticonvulsants.17 this may reflect that antibiotics may be being over - prescribed in korea. to date, the only undisputed treatment for dress syndrome is prompt withdrawal of the offending drug. systemic corticosteroid administration is particularly recommended in patients with life - threatening visceral manifestations that do not respond to drug withdrawal.18 as in the present patient, others have reported cases where dress syndrome recurred after a short period of steroid treatment, which has led some investigators to suggest that high - dose systemic steroid may be helpful, and that tapering should then be performed slowly.4 the final diagnosis in the present case was dress syndrome due to antibiotic and/or antiviral treatment following hepatitis a with cholestatic features and renal injury. there has been only one previous report of a histopathologically confirmed case of dress syndrome following hepatitis a, and that was in a child.19 to our knowledge, the present report is the first to describe a histopathologically confirmed case of dress syndrome following severe cholestatic hepatitis a in an adult. | hepatitis a virus (hav) infections occur predominantly in children, and are usually self - limiting. however, 75 - 95% of the infections in adults are symptomatic (mostly with jaundice), with the illness symptoms usually persisting for a few weeks. atypical manifestations include relapsing hepatitis, prolonged cholestasis, and complications involving renal injury. drug rash with eosinophilia and systemic symptoms (dress) syndrome is a severe, drug - induced hypersensitivity reaction characterized by skin rash, fever, lymph - node enlargement, and internal organ involvement. we describe a 22-year - old male who presented with acute kidney injury and was diagnosed with prolonged cholestatic hepatitis a. the patient also developed dress syndrome due to antibiotic and/or antiviral treatment. to our knowledge, this is the first report of histopathologically confirmed dress syndrome due to antibiotic and/or antiviral treatment following hav infection with cholestatic features and renal injury. |
hypercholesterolaemia is a major risk factor for the development of atherosclerosis and coronary heart disease (chd). reducing low - density lipoprotein cholesterol (ldl - c) with statins lowers the risk of chd events and all - cause mortality and there is a clear relation between the degree of absolute ldl - c lowering and the degree of cardiovascular event reduction. comparative data of intensive vs. standard - dose statin treatment suggest that the lower the ldl - c concentration, the greater the benefit in high cardiovascular risk patients. mmol / l (1 year) ] or on a lower dose provided the reason for doing so was documented. statin dose had to be stable for 4 weeks before the screening visit and use of other llt was not permitted. at screening, patients with documented cardiovascular disease (cvd) and ldl - c 1.8 mmol / l (70 mg / dl) or no documented history of cvd but who were at high cardiovascular risk and had ldl - c 2.6 mmol / l (100 mg / dl) were eligible to participate. the study was performed in accordance with the principles of the declaration of helsinki and all applicable amendments by the world medical assemblies, and the international conference on harmonization guidelines for good clinical practice. eligible patients entered a screening period of up to 3 weeks before randomization during which they were trained to self - inject using a prefilled pen (autoinjector), vital signs were taken, a 12-lead electrocardiogram was performed, and fasting blood and urine samples were obtained. whose triglycerides exceeded 4.5 mmol / l, the central laboratory automatically measured ldl - c using the beta - quantification method (medpace reference laboratories ; cincinnati, oh, usa ; leuven, belgium ; singapore). other lipid parameters [total cholesterol, high - density lipoprotein cholesterol (hdl - c), triglycerides, apolipoprotein b, and lipoprotein a ] were measured directly by the central laboratory (medpace reference laboratories). eligible patients were randomized to alirocumab or ezetimibe through an interactive voice response system (almac company), using a permuted - block design with a 2:1 allocation ratio. to attain balance between arms for factors that may have influenced treatment response, patients were stratified according to history of myocardial infarction or ischaemic stroke, intensity of statin treatment, and geographic region. after randomization, patients entered a double - blind, double - dummy treatment period lasting 104 weeks. patients were randomized to either subcutaneous (sc) alirocumab 75 mg (in 1 ml volume) every 2 weeks (q2w) (plus oral placebo for ezetimibe daily) or 10 mg oral ezetimibe daily (plus placebo sc q2w for alirocumab) and continued to receive their background statin therapy. the dose in the alirocumab arm (only) was automatically increased, per protocol, at week 12 to 150 mg q2w (1 ml volume) if the week-8 ldl - c value was 1.8 the study is ongoing at the time of writing, and randomized treatment will continue until week 104, followed by an 8-week post - treatment observational period. patients were instructed to remain on a stable diet [national cholesterol education program adult treatment panel iii (ncep atp iii) therapeutic lifestyle changes diet or equivalent ] and to maintain the same daily statin dose throughout the study. the primary endpoint was percent change in calculated ldl - c from baseline to week 24, using all ldl - c values from week 24 regardless of adherence to treatment [intent - to - treat (itt) approach ]. principal secondary efficacy endpoints included : percent change in calculated ldl - c from baseline to week 24 (on - treatment analysis), and from baseline to weeks 12 (itt / on - treatment analysis) or 52 (itt analysis) ; percent change in apolipoprotein b, non - hdl - c, total cholesterol, lipoprotein a, hdl - c, fasting triglycerides, and apolipoprotein a-1 from baseline to week 24 (itt analysis), and proportion of patients reaching calculated ldl - c 3 uln8/470 (1.7)1/240 (0.4) creatine kinase > 3 uln13/467 (2.8)6/236 (2.5)data are n (%) or n / n (%). chd, coronary heart disease ; q2w, every 2 weeks ; sae, serious adverse event ; sc, subcutaneous ; teae, treatment - emergent adverse event ; uln, upper limit of normal.teaes are adverse events that developed or worsened or became serious during the teae period [defined as the time from the first dose of double - blind study treatment to the last injection plus 70 days (10 weeks), as the residual effect of alirocumab was expected until 10 weeks after the last injection].one patient was not on maximally tolerated statin therapy.alirocumab 75 mg sc q2w with a dose increase to 150 mg q2w at week 12 if week 8 ldl - c was 1.8 mmol / l (70 mg / dl).10 mg / day oral ezetimibe.both deaths in the alirocumab arm were due to cardiovascular events (cardiac arrest and sudden cardiac death). of the four deaths in the ezetimibe arm (malignant lung neoplasm, suicide, defect conduction intraventricular plus sudden cardiac death, and sudden death one patient was counted in two categories), two were due to cardiovascular events.accidental overdose was an event suspected by the investigator or spontaneously notified by the patient (not based on systematic injection / capsule counts) and defined as at least twice the intended dose within the intended therapeutic interval (i.e. 2 injections from the double - blind treatment kit administered in 3 uln8/470 (1.7)1/240 (0.4) creatine kinase > 3 uln13/467 (2.8)6/236 (2.5)data are n (%) or n / n (%). chd, coronary heart disease ; q2w, every 2 weeks ; sae, serious adverse event ; sc, subcutaneous ; teae, treatment - emergent adverse event ; uln, upper limit of normal.teaes are adverse events that developed or worsened or became serious during the teae period [defined as the time from the first dose of double - blind study treatment to the last injection plus 70 days (10 weeks), as the residual effect of alirocumab was expected until 10 weeks after the last injection].one patient was not on maximally tolerated statin therapy.alirocumab 75 mg sc q2w with a dose increase to 150 mg q2w at week 12 if week 8 ldl - c was 1.8 mmol / l (70 mg / dl).10 mg / day oral ezetimibe.both deaths in the alirocumab arm were due to cardiovascular events (cardiac arrest and sudden cardiac death). of the four deaths in the ezetimibe arm (malignant lung neoplasm, suicide, defect conduction intraventricular plus sudden cardiac death, and sudden death one patient was counted in two categories), two were due to cardiovascular events.accidental overdose was an event suspected by the investigator or spontaneously notified by the patient (not based on systematic injection / capsule counts) and defined as at least twice the intended dose within the intended therapeutic interval (i.e. 2 injections from the double - blind treatment kit administered in 40% of patients fail to achieve the target, leaving them at substantially increased risk of a major cardiovascular event. initial data from the improve it trial suggest that further lowering of ldl - c with the non - statin agent ezetimibe reduces cardiovascular events, but this is being studied in several large outcomes trials with other agents, including with alirocumab. the data presented here suggest that addition of alirocumab to a treatment regimen with maximally tolerated statins will provide substantial lowering of ldl - c so that many more patients can achieve ldl - c goals than by adding ezetimibe. furthermore, the maximum ldl - c response to a pcsk9 inhibitor is greater with combination therapy, as in combo ii, vs. monotherapy (i.e. with no background lipid - lowering therapies), indicating a possible additive effect, or synergy, with these two classes of drugs, as also suggested in studies involving evolocumab. the combo ii study included a strategy of individualized goal attainment, with a pre - planned dose - increase in patients who failed to reach the ldl - c target by week 8. we hypothesized that most patients would gain substantial lipid lowering (50%) even with the starting dose, and this proved correct. approximately 80% of patients treated with alirocumab did not require a dose increase. of note, the 18% alirocumab - treated patients who required a dose increase had much higher mean baseline ldl - c values vs. patients who did not require an increase. the dose increase at 12 weeks led to an additional mean reduction of 10.5% in ldl - c. furthermore, the absolute reduction in ldl - c by week 24 was slightly greater in the dose - increase group (1.6 vs. 1.5 alirocumab was generally well tolerated, with no evidence of an excess of teaes, serious adverse events, or deaths in this ongoing study. injection site reactions occurred more frequently in the alirocumab arm ; these were mild in intensity in all but one case with moderate intensity. the rate of adjudicated cardiovascular events was slightly higher with alirocumab (4.8%) vs. ezetimibe (3.7%). cardiovascular outcomes will be assessed in an ongoing study (http://clinicaltrials.gov/show/nct01663402) and in a pooled analysis from overall odyssey program. this study was limited to high cardiovascular risk patients with inadequately controlled hypercholesterolaemia, but will complement the range of data emerging from the odyssey program. further research is needed to evaluate the efficacy of alirocumab in different racial groups. while the primary endpoint in this study was ldl - c reduction at 24 weeks, the study will continue up to 104 weeks to maximize available safety data and generate information on the durability of alirocumab lipid - lowering effects. in this population of high cardiovascular risk patients with inadequately controlled ldl - c on maximally tolerated doses of potent statins, alirocumab produced significantly greater reductions in ldl - c vs. ezetimibe using a dose - increase approach, with a comparable safety profile. conflict of interest : c.p.c. reports personal fees from sanofi, personal fees from regeneron pharmaceuticals, inc., during the conduct of the study ; grants from accumetrics, grants from arisaph, grants from astra zeneca, grants from boehringer - ingelheim, personal fees from csl behring, personal fees from essentialis, grants and personal fees from glaxosmithkline, grants from janssen, grants and personal fees from merck, grants and personal fees from takeda, personal fees from lipimedix, personal fees from bms, personal fees from pfizer, outside the submitted work. b.c. reports personal fees from sanofi / regeneron pharmaceuticals, inc., personal fees from amgen, outside the submitted work., during the conduct of the study ; personal fees from amgen, personal fees from sanofi / regeneron pharmaceuticals, inc., personal fees from aegerion, personal fees from astra zeneca, personal fees from msd, personal fees from pfizer, personal fees from servier, personal fees from unilever, grants from amgen, grants from sanofi / regeneron pharmaceuticals, inc., grants from eli lilly, grants from novartis, grants from aegerion, outside the submitted work. reports grants, personal fees and non - financial support from sanofi / regeneron pharmaceuticals, inc., during the conduct of the study ; grants, personal fees, non - financial support, and other from pfizer inc., grants, personal fees, and non - financial support from sanofi aventis, grants and non - financial support from novartis pharmaceuticals, grants and other from eli lilly & company, grants and other from roche pharmaceuticals, grants from boehringer ingelheim, grants from astrazeneca lp, grants and other from regeneron pharmaceuticals, inc., outside the submitted work | aimsto compare the efficacy [low - density lipoprotein cholesterol (ldl - c) lowering ] and safety of alirocumab, a fully human monoclonal antibody to proprotein convertase subtilisin / kexin 9, compared with ezetimibe, as add - on therapy to maximally tolerated statin therapy in high cardiovascular risk patients with inadequately controlled hypercholesterolaemia.methods and resultscombo ii is a double - blind, double - dummy, active - controlled, parallel - group, 104-week study of alirocumab vs. ezetimibe. patients (n = 720) with high cardiovascular risk and elevated ldl - c despite maximal doses of statins were enrolled (august 2012may 2013). this pre - specified analysis was conducted after the last patient completed 52 weeks. patients were randomized to subcutaneous alirocumab 75 mg every 2 weeks (plus oral placebo) or oral ezetimibe 10 mg daily (plus subcutaneous placebo) on a background of statin therapy. at week 24, mean se reductions in ldl - c from baseline were 50.6 1.4% for alirocumab vs. 20.7 1.9% for ezetimibe (difference 29.8 2.3% ; p < 0.0001) ; 77.0% of alirocumab and 45.6% of ezetimibe patients achieved ldl - c < 1.8 mmol / l (p < 0.0001). mean achieved ldl - c at week 24 was 1.3 0.04 mmol / l with alirocumab and 2.1 0.05 mmol / l with ezetimibe, and were maintained to week 52. alirocumab was generally well tolerated, with no evidence of an excess of treatment - emergent adverse events.conclusionin patients at high cardiovascular risk with inadequately controlled ldl - c, alirocumab achieved significantly greater reductions in ldl - c compared with ezetimibe, with a similar safety profile.trial registrationclinicaltrials.gov identifier : nct01644188. |
information about irs was obtained from the replicate 6-month rcts (studies co405 and co406 or gout 1 and 2 ; identifier : nct00325195) and the subsequent open - label extension (ole) study data sets (c0407 ; identifier nct01356498). briefly, these studies enrolled patients with refractory gout and sua of 8.0 mg / dl or greater, who either had failed or had contraindications to allopurinol treatment, and who had at least 1 or more of the following : 3 or more self - reported gout flares over the previous 18-month period, 1 or more tophus, or gouty arthropathy. eligible patients were randomized in a 2:2:1 ratio to receive 12 biweekly, 2-hour intravenous (iv) infusions containing either pegloticase 8 mg every 2 weeks (biweekly treatment group), pegloticase 8 mg alternating with placebo at successive infusions (monthly treatment group), or placebo. patients were required to discontinue any pretrial urate - lowering therapies 1 week prior to the rct treatment period. prophylaxis against gout flares with colchicine or a nonsteroidal anti - inflammatory drug was started 1 week before the first infusion and continued throughout the rct. all patients received ir prophylaxis prior to each infusion in the rct and ole studies as follows : the nonsedating h1-antihistamine fexofenadine, 60 mg on the night preceding and the morning of infusion ; acetaminophen, 1000 mg the morning of infusion ; and hydrocortisone, 200 mg iv given immediately prior to infusion. patients completing treatment in either rct were eligible to enroll in the ole study during which all patients received either biweekly or monthly pegloticase infusions. while still blinded to rct treatment, the investigator and patient decided whether pegloticase would be administered biweekly or monthly at ole study entry. changes in dosing frequency from monthly to biweekly or vice versa were allowed once after week 25 and a second time after the data from the rcts were unblinded. flare prophylaxis was required for the first 3 months of the ole study and continued at investigator discretion thereafter. for both the rcts and ole study, approval of the protocol was obtained from central (integreview, austin, tx) or local institutional review boards, and informed consent was obtained from all patients before any study - related procedures were performed. the primary efficacy end point in the rcts was the proportion of responders in each treatment group achieving pua of less than 6.0 mg / dl for 80% of the total time or greater during months 3 and 6 combined. plasma uric acid concentration determinations were completed prior to infusions and at 6 additional times during months 3 and 6. patients who did not meet the pua criteria, or who discontinued early, were classified as nonresponders. safety assessments included physical examination and adverse event updates every 2 weeks and laboratory testing every month. safety was the primary end point in the ole ; evaluation for adverse events included physical examination at each biweekly or monthly visit and laboratory testing performed every 12 weeks. infusion - related reactions were defined in the study protocols as any infusion - related adverse event or cluster of temporally related events that occurred during or within 2 hours after drug infusion that could not be reasonably attributed to another cause. these events prompted a standardized assessment, which included a thorough dermatologic and cardiopulmonary examination and a 12-lead electrocardiogram. vital signs were evaluated every 30 minutes until resolution or stabilization of the event. besides the interventions implemented by the investigator to manage the patient s medical condition, the infusion rate could be slowed by half, interrupted and restarted at the same or slower rate, or stopped. if the event resolved, the infusion could proceed to completion at the original rate, at a reduced rate, or with increased volume of diluent. however, the infusion was to be stopped if the ir failed to resolve within 1 hour, or if the patient, in the investigator s opinion, was at risk for anaphylaxis. in the ole, prednisone, 20 mg on the night before the next infusion, could be added to the prophylaxis regimen if a patient had experienced an ir, and further pegloticase treatment was to be given. information about irs was obtained from the replicate 6-month rcts (studies co405 and co406 or gout 1 and 2 ; identifier : nct00325195) and the subsequent open - label extension (ole) study data sets (c0407 ; identifier nct01356498). briefly, these studies enrolled patients with refractory gout and sua of 8.0 mg / dl or greater, who either had failed or had contraindications to allopurinol treatment, and who had at least 1 or more of the following : 3 or more self - reported gout flares over the previous 18-month period, 1 or more tophus, or gouty arthropathy. eligible patients were randomized in a 2:2:1 ratio to receive 12 biweekly, 2-hour intravenous (iv) infusions containing either pegloticase 8 mg every 2 weeks (biweekly treatment group), pegloticase 8 mg alternating with placebo at successive infusions (monthly treatment group), or placebo. patients were required to discontinue any pretrial urate - lowering therapies 1 week prior to the rct treatment period. prophylaxis against gout flares with colchicine or a nonsteroidal anti - inflammatory drug was started 1 week before the first infusion and continued throughout the rct. all patients received ir prophylaxis prior to each infusion in the rct and ole studies as follows : the nonsedating h1-antihistamine fexofenadine, 60 mg on the night preceding and the morning of infusion ; acetaminophen, 1000 mg the morning of infusion ; and hydrocortisone, 200 mg iv given immediately prior to infusion. patients completing treatment in either rct were eligible to enroll in the ole study during which all patients received either biweekly or monthly pegloticase infusions. while still blinded to rct treatment, the investigator and patient decided whether pegloticase would be administered biweekly or monthly at ole study entry. changes in dosing frequency from monthly to biweekly or vice versa were allowed once after week 25 and a second time after the data from the rcts were unblinded. flare prophylaxis was required for the first 3 months of the ole study and continued at investigator discretion thereafter. for both the rcts and ole study, approval of the protocol was obtained from central (integreview, austin, tx) or local institutional review boards, and informed consent was obtained from all patients before any study - related procedures were performed. the primary efficacy end point in the rcts was the proportion of responders in each treatment group achieving pua of less than 6.0 mg / dl for 80% of the total time or greater during months 3 and 6 combined. plasma uric acid concentration determinations were completed prior to infusions and at 6 additional times during months 3 and 6. patients who did not meet the pua criteria, or who discontinued early, were classified as nonresponders. safety assessments included physical examination and adverse event updates every 2 weeks and laboratory testing every month. safety was the primary end point in the ole ; evaluation for adverse events included physical examination at each biweekly or monthly visit and laboratory testing performed every 12 weeks. infusion - related reactions were defined in the study protocols as any infusion - related adverse event or cluster of temporally related events that occurred during or within 2 hours after drug infusion that could not be reasonably attributed to another cause. these events prompted a standardized assessment, which included a thorough dermatologic and cardiopulmonary examination and a 12-lead electrocardiogram. vital signs were evaluated every 30 minutes until resolution or stabilization of the event. besides the interventions implemented by the investigator to manage the patient s medical condition, the infusion rate could be slowed by half, interrupted and restarted at the same or slower rate, or stopped. if the event resolved, the infusion could proceed to completion at the original rate, at a reduced rate, or with increased volume of diluent. however, the infusion was to be stopped if the ir failed to resolve within 1 hour, or if the patient, in the investigator s opinion, was at risk for anaphylaxis. in the ole, prednisone, 20 mg on the night before the next infusion, could be added to the prophylaxis regimen if a patient had experienced an ir, and further pegloticase treatment was to be given. a total of 225 patients were randomized, and 212 received at least 1 infusion of study treatment in the rcts. one hundred fifty - one (96%) of the 157 patients who completed either of the rcts elected to enter the ole study (149 received pegloticase, and 2 chose to be followed up by observation only). a total of 6389 infusions were administered during the rcts and the ole study (rct biweekly, 852 ; monthly, 846 ; placebo, 502 ; ole, 4189). eighty - five patients were allocated to biweekly pegloticase in the rcts and received a median of 36 infusions (range, 163) during combined rct and ole participation ; 84 patients were initially allocated to monthly pegloticase and received a median of 23 pegloticase infusions (range, 157). all 39 patients randomized to placebo and completing 1 of the rcts started pegloticase in the ole study ; 23 received biweekly treatment with a median of 20 pegloticase infusions (range, 251), and 16 received monthly pegloticase with a median of 7 infusions (range, 126). overall, 91 patients (44%) received more than 30 pegloticase infusions, and 40 patients (19%) received more than 50 infusions. infusion - related reactions were reported for 94 (45%) of 208 patients treated with pegloticase during the phase 3 trials. two (5%) of 43 patients experienced at least 1 ir while receiving placebo (both during the rcts). an ir occurred during the first pegloticase infusion in 10 (5%) of the 208 pegloticase - treated patients and in 1 (2%) of 43 patients receiving their first placebo infusion. among the other 84 patients with at least 1 ir, these events were distributed over time as shown in figure 1. among patients with at least 1 ir, 93 (97%) had symptoms that arose during the infusion, and 3 (3%) had symptoms with onset in the 2-hour observation period after the infusion. infusion - related reaction was the basis for discontinuation from study drug for 20 patients in the rcts and for 11 patients in the ole study. among these 11, no patient who was a responder in the rcts reported an ir as the basis for discontinuation during the ole study. number of first irs with pegloticase by treatment week and sua. in the pooled study population from the rcts and ole, the most common symptoms defining ir (incidence > 5% and excluding irs associated with placebo infusions) were chest discomfort (n = 32 ; 15%), flushing (n = 24 ; 12%), dyspnea (n = 23 ; 11%), back pain (n = 19 ; 9%), hyperhidrosis (n = 18 ; 9%), nausea (n = 18 ; 9%), erythema (n = 18 ; 9%), urticaria (n = 17 ; 8%), chest pain (n = 17 ; 8%), pruritus (n = 16 ; 8%), rash (n = 13 ; 6%), muscle spasms (n = 13 ; 6%), headache (n = 12 ; 6%), and abdominal pain (n = 11 ; 5%). other symptoms, occurring at rates of 2% to 5%, included dizziness (5%), vomiting (5%), pain (4%), chills (3%), hypertension (3%), hypotension (3%), tachycardia (3%), feeling hot (2%), musculoskeletal discomfort (2%), and wheezing (2%). most irs were rated mild or moderate in severity, although 12 (7%) of 169 patients who started pegloticase during the rcts and 11 (7%) of 149 patients who received pegloticase in the ole had irs judged to be severe by the investigator (table 1). of note, in patients experiencing more than 1 pegloticase - related ir, no increase in ir severity was seen over time with treatment. incidence of infusion reactions by severity in the rct and ole populations all irs (including those classified retrospectively as anaphylaxis) in the rcts and ole study resolved with supportive measures, which included slowing or stopping the infusion and/or providing fluids, diphenhydramine, corticosteroids, and/or analgesics. table 2 summarizes the adjustments to the infusions made by investigators in response to irs during phase 3 testing. as not all reports of potential irs were accompanied by a description of the investigator actions, the information is presented both as a percentage of all irs (n = 381) and as a percentage of those irs with infusion adjustments recorded (n = 264). epinephrine was administered to 3 patients : 1 for wheezing, 1 for lip swelling, and 1 for an ir without blood pressure change. none of the patients with irs required intubation, mechanical ventilation, vasopressors, or hospitalization within 2 hours after infusion, and there were no infusion - related deaths. adjustments made to infusions by investigators during the rcts and ole a cluster or constellation of symptoms likely to be of particular concern to clinicians was defined post hoc as the occurrence of stridor, wheezing, perioral or lingual edema, or hemodynamic instability with or without rash or urticaria. twelve (7%) of 169 patients treated with pegloticase in the rcts experienced such defined symptom clusters. two patients had irs with a constellation of defined symptoms during the first infusion, 3 during the second or third infusion, 5 during the fifth infusion, and the remaining 2 during their seventh and ninth infusions. most irs with a symptom constellation were moderate in severity, except for 1 rated mild and 3 rated severe by investigators. for 9 of these 12 patients (including the 2 affected during the first infusion), the ir occurred when sua was greater than 6 mg / dl, whereas sua data were not available at the time of these symptoms in 2 patients, and sua was less than 6 mg / dl in the 1 remaining patient. eighteen patients had irs with a constellation of defined symptoms during the ole, including 11 patients who were initially allocated to placebo in the rct and started pegloticase in the ole. in all instances pegloticase is immunogenic, and antipegloticase antibodies were detectable in 89% of patients treated in the rcts. in a post hoc analysis, the formation of high - titer antibodies (titer exceeding 1:2430) was significantly associated with loss of the urate - lowering efficacy of pegloticase (p 6 mg / dl by definition) shows an even higher rate of patients with documented loss of response prior to their first ir (95% and 80% with biweekly and monthly dosing, respectively). the relationship between loss of urate - lowering efficacy and increased risk of irs was not apparent during the conduct of the phase 3 clinical trials because investigators and patients were blinded to uric acid levels. entry into the ole study required completion of the rct, so that investigator s blinding was maintained for many patients for several months into the ole study. as noted previously, 94 patients in the pooled study population had at least 1 ir related to an infusion of pegloticase. of these, 81 patients (86%) had sua of greater than 6 mg / dl (including the 10 patients who experienced an ir at their first infusion), and 13 patients (14%) had sua of less than 6 mg / dl at the time of their first ir (fig. 1). when considered on a per - infusion rather than a per - patient basis, the rate of irs for patients with sua of less than 6 mg / dl was 0.5 per 100 infusions in the rcts and 0.8 per 100 infusions in the ole (fig. 2). considering all irs during the studies (including multiple irs for individual patients), 91% of those reported in the rcts and 88% in the ole study occurred when sua was greater than 6 mg / dl. discontinuation of pegloticase therapy in patients who lose urate - lowering response offers a valuable approach for mitigating the risk of ir. using data from the rcts as an example, 36 (42%) of 85 patients treated with biweekly pegloticase achieved the primary urate - lowering end point, and 22 (26%) of 85 patients in this dosing group had at least 1 ir. these results occurred in the absence of specific rules for stopping pegloticase treatment. a post hoc analysis of the biweekly pegloticase dosing group in the rcts illustrated the impact of several possible stopping rules based on sua. as shown in table 3, a stopping rule based on 2 consecutive uric acid measurements of greater than 6 mg / dl reduced the risk of ir by nearly half (from 26% to 14%) while having little effect on the treatment response rate (from 42% to 41%). use of a stopping rule based on a single sua measurement of greater than 6 mg / dl would have resulted in a much greater reduction in ir risk (to 8%), but fewer patients would have remained in the study to achieve the primary end point (36%). comparable changes in ir risk and response rates were seen for stopping rules based on 1 or 2 consecutive sua measurements of greater than 7 mg / dl. stopping rules to evaluate benefit (treatment response) versus risk (irs) with biweekly pegloticase based on data pooled from the rcts serious adverse events are defined by the us food and drug administration (fda) as those that are life threatening, require initial or prolonged hospitalization, lead to disability or permanent damage, cause death, or jeopardize the patient such that medical or surgical intervention is needed to prevent 1 of these outcomes. in the pegloticase - treated pooled population, 22 (11%) of the 208 patients had a serious ir, including 6 (7%) of 85 patients who received biweekly pegloticase in the rct, 9 (11%) of 84 patients who received monthly pegloticase, and 7 (18%) of 39 patients who were initially allocated to placebo and then started pegloticase in the ole study. serious irs occurred more frequently at early time points, with 2 episodes at the first infusion of pegloticase, 14 episodes through 6 months, and the remaining 6 episodes occurring after 6 months. as noted above, no serious ir occurred in the placebo groups of the rcts. there was no prespecified definition of anaphylaxis in the pegloticase study protocols, and there were no reports of anaphylaxis in the database from investigators who participated in the rcts. however, a post hoc analysis was conducted during the fda review process, in which the following diagnostic criteria for anaphylaxis were used : skin or tissue mucosal involvement and either airway compromise and/or reduced blood pressure with or without associated symptoms, which showed a temporal relationship to the pegloticase or placebo infusion with no other identifiable cause. using this definition, the fda identified 14 cases (5%) of anaphylaxis or potential anaphylaxis among the 273 patients tested in the complete phases 2 and 3 clinical development program. among patients treated with biweekly pegloticase in all clinical trials irrespective of dose, 8 (7%) of 123 met the fda s clinical criteria for anaphylaxis. four of these patients were identified by the fda as receiving biweekly pegloticase in the rcts and are presented in table 4. summary of 4 irs listed in pegloticase label an additional and independent post hoc analysis was conducted using the national institute of allergy and infectious disease / food allergy and anaphylaxis network criteria for anaphylaxis with an unknown allergen. these criteria mandate acute onset of a reaction over minutes to several hours with involvement of the skin, mucosal tissues, or both (eg, hives, pruritus, flushing, or swollen lips, tongue, or uvula) and either respiratory compromise (eg, dyspnea, wheezing, stridor, or hypoxemia) and/or reduced blood pressure or associated symptoms of end - organ dysfunction (eg, hypotonia, syncope, or incontinence). applying these criteria, 3 (4%) of 85 patients treated with biweekly pegloticase met the national institute of allergy and infectious disease / food allergy and anaphylaxis network clinical criteria for anaphylaxis. a total of 225 patients were randomized, and 212 received at least 1 infusion of study treatment in the rcts. one hundred fifty - one (96%) of the 157 patients who completed either of the rcts elected to enter the ole study (149 received pegloticase, and 2 chose to be followed up by observation only). a total of 6389 infusions were administered during the rcts and the ole study (rct biweekly, 852 ; monthly, 846 ; placebo, 502 ; ole, 4189). eighty - five patients were allocated to biweekly pegloticase in the rcts and received a median of 36 infusions (range, 163) during combined rct and ole participation ; 84 patients were initially allocated to monthly pegloticase and received a median of 23 pegloticase infusions (range, 157). all 39 patients randomized to placebo and completing 1 of the rcts started pegloticase in the ole study ; 23 received biweekly treatment with a median of 20 pegloticase infusions (range, 251), and 16 received monthly pegloticase with a median of 7 infusions (range, 126). overall, 91 patients (44%) received more than 30 pegloticase infusions, and 40 patients (19%) received more than 50 infusions. infusion - related reactions were reported for 94 (45%) of 208 patients treated with pegloticase during the phase 3 trials. two (5%) of 43 patients experienced at least 1 ir while receiving placebo (both during the rcts). an ir occurred during the first pegloticase infusion in 10 (5%) of the 208 pegloticase - treated patients and in 1 (2%) of 43 patients receiving their first placebo infusion. among the other 84 patients with at least 1 ir, these events were distributed over time as shown in figure 1. among patients with at least 1 ir, 93 (97%) had symptoms that arose during the infusion, and 3 (3%) had symptoms with onset in the 2-hour observation period after the infusion. infusion - related reaction was the basis for discontinuation from study drug for 20 patients in the rcts and for 11 patients in the ole study. among these 11, no patient who was a responder in the rcts reported an ir as the basis for discontinuation during the ole study. number of first irs with pegloticase by treatment week and sua. in the pooled study population from the rcts and ole, the most common symptoms defining ir (incidence > 5% and excluding irs associated with placebo infusions) were chest discomfort (n = 32 ; 15%), flushing (n = 24 ; 12%), dyspnea (n = 23 ; 11%), back pain (n = 19 ; 9%), hyperhidrosis (n = 18 ; 9%), nausea (n = 18 ; 9%), erythema (n = 18 ; 9%), urticaria (n = 17 ; 8%), chest pain (n = 17 ; 8%), pruritus (n = 16 ; 8%), rash (n = 13 ; 6%), muscle spasms (n = 13 ; 6%), headache (n = 12 ; 6%), and abdominal pain (n = 11 ; 5%). other symptoms, occurring at rates of 2% to 5%, included dizziness (5%), vomiting (5%), pain (4%), chills (3%), hypertension (3%), hypotension (3%), tachycardia (3%), feeling hot (2%), musculoskeletal discomfort (2%), and wheezing (2%). most irs were rated mild or moderate in severity, although 12 (7%) of 169 patients who started pegloticase during the rcts and 11 (7%) of 149 patients who received pegloticase in the ole had irs judged to be severe by the investigator (table 1). of note, in patients experiencing more than 1 pegloticase - related ir, no increase in ir severity was seen over time with treatment. incidence of infusion reactions by severity in the rct and ole populations all irs (including those classified retrospectively as anaphylaxis) in the rcts and ole study resolved with supportive measures, which included slowing or stopping the infusion and/or providing fluids, diphenhydramine, corticosteroids, and/or analgesics. table 2 summarizes the adjustments to the infusions made by investigators in response to irs during phase 3 testing. as not all reports of potential irs were accompanied by a description of the investigator actions, the information is presented both as a percentage of all irs (n = 381) and as a percentage of those irs with infusion adjustments recorded (n = 264). epinephrine was administered to 3 patients : 1 for wheezing, 1 for lip swelling, and 1 for an ir without blood pressure change. none of the patients with irs required intubation, mechanical ventilation, vasopressors, or hospitalization within 2 hours after infusion, and there were no infusion - related deaths. a cluster or constellation of symptoms likely to be of particular concern to clinicians was defined post hoc as the occurrence of stridor, wheezing, perioral or lingual edema, or hemodynamic instability with or without rash or urticaria. twelve (7%) of 169 patients treated with pegloticase in the rcts experienced such defined symptom clusters. two patients had irs with a constellation of defined symptoms during the first infusion, 3 during the second or third infusion, 5 during the fifth infusion, and the remaining 2 during their seventh and ninth infusions. most irs with a symptom constellation were moderate in severity, except for 1 rated mild and 3 rated severe by investigators. for 9 of these 12 patients (including the 2 affected during the first infusion), the ir occurred when sua was greater than 6 mg / dl, whereas sua data were not available at the time of these symptoms in 2 patients, and sua was less than 6 mg / dl in the 1 remaining patient. eighteen patients had irs with a constellation of defined symptoms during the ole, including 11 patients who were initially allocated to placebo in the rct and started pegloticase in the ole. in all instances pegloticase is immunogenic, and antipegloticase antibodies were detectable in 89% of patients treated in the rcts. in a post hoc analysis, the formation of high - titer antibodies (titer exceeding 1:2430) was significantly associated with loss of the urate - lowering efficacy of pegloticase (p 6 mg / dl by definition) shows an even higher rate of patients with documented loss of response prior to their first ir (95% and 80% with biweekly and monthly dosing, respectively). the relationship between loss of urate - lowering efficacy and increased risk of irs was not apparent during the conduct of the phase 3 clinical trials because investigators and patients were blinded to uric acid levels. entry into the ole study required completion of the rct, so that investigator s blinding was maintained for many patients for several months into the ole study. as noted previously, 94 patients in the pooled study population had at least 1 ir related to an infusion of pegloticase. of these, 81 patients (86%) had sua of greater than 6 mg / dl (including the 10 patients who experienced an ir at their first infusion), and 13 patients (14%) had sua of less than 6 mg / dl at the time of their first ir (fig. 1). when considered on a per - infusion rather than a per - patient basis, the rate of irs for patients with sua of less than 6 mg / dl was 0.5 per 100 infusions in the rcts and 0.8 per 100 infusions in the ole (fig. 2). considering all irs during the studies (including multiple irs for individual patients), 91% of those reported in the rcts and 88% in the ole study occurred when sua was greater than 6 mg / dl. discontinuation of pegloticase therapy in patients who lose urate - lowering response offers a valuable approach for mitigating the risk of ir. using data from the rcts as an example, 36 (42%) of 85 patients treated with biweekly pegloticase achieved the primary urate - lowering end point, and 22 (26%) of 85 patients in this dosing group had at least 1 ir. these results occurred in the absence of specific rules for stopping pegloticase treatment. a post hoc analysis of the biweekly pegloticase dosing group in the rcts illustrated the impact of several possible stopping rules based on sua. as shown in table 3, a stopping rule based on 2 consecutive uric acid measurements of greater than 6 mg / dl reduced the risk of ir by nearly half (from 26% to 14%) while having little effect on the treatment response rate (from 42% to 41%). use of a stopping rule based on a single sua measurement of greater than 6 mg / dl would have resulted in a much greater reduction in ir risk (to 8%), but fewer patients would have remained in the study to achieve the primary end point (36%). comparable changes in ir risk and response rates were seen for stopping rules based on 1 or 2 consecutive sua measurements of greater than 7 mg / dl. stopping rules to evaluate benefit (treatment response) versus risk (irs) with biweekly pegloticase based on data pooled from the rcts serious adverse events are defined by the us food and drug administration (fda) as those that are life threatening, require initial or prolonged hospitalization, lead to disability or permanent damage, cause death, or jeopardize the patient such that medical or surgical intervention is needed to prevent 1 of these outcomes. in the pegloticase - treated pooled population, 22 (11%) of the 208 patients had a serious ir, including 6 (7%) of 85 patients who received biweekly pegloticase in the rct, 9 (11%) of 84 patients who received monthly pegloticase, and 7 (18%) of 39 patients who were initially allocated to placebo and then started pegloticase in the ole study. serious irs occurred more frequently at early time points, with 2 episodes at the first infusion of pegloticase, 14 episodes through 6 months, and the remaining 6 episodes occurring after 6 months. as noted above, no serious ir occurred in the placebo groups of the rcts. there was no prespecified definition of anaphylaxis in the pegloticase study protocols, and there were no reports of anaphylaxis in the database from investigators who participated in the rcts. however, a post hoc analysis was conducted during the fda review process, in which the following diagnostic criteria for anaphylaxis were used : skin or tissue mucosal involvement and either airway compromise and/or reduced blood pressure with or without associated symptoms, which showed a temporal relationship to the pegloticase or placebo infusion with no other identifiable cause. using this definition, the fda identified 14 cases (5%) of anaphylaxis or potential anaphylaxis among the 273 patients tested in the complete phases 2 and 3 clinical development program. among patients treated with biweekly pegloticase in all clinical trials irrespective of dose, 8 (7%) of 123 met the fda s clinical criteria for anaphylaxis. four of these patients were identified by the fda as receiving biweekly pegloticase in the rcts and are presented in table 4. summary of 4 irs listed in pegloticase label an additional and independent post hoc analysis was conducted using the national institute of allergy and infectious disease / food allergy and anaphylaxis network criteria for anaphylaxis with an unknown allergen. these criteria mandate acute onset of a reaction over minutes to several hours with involvement of the skin, mucosal tissues, or both (eg, hives, pruritus, flushing, or swollen lips, tongue, or uvula) and either respiratory compromise (eg, dyspnea, wheezing, stridor, or hypoxemia) and/or reduced blood pressure or associated symptoms of end - organ dysfunction (eg, hypotonia, syncope, or incontinence). applying these criteria, 3 (4%) of 85 patients treated with biweekly pegloticase met the national institute of allergy and infectious disease / food allergy and anaphylaxis network clinical criteria for anaphylaxis. these data from the rcts and ole study show that the risk of ir with pegloticase is associated with loss of urate - lowering efficacy as reflected by sua of greater than 6 mg / dl. for patients who maintain therapeutic response to pegloticase with sua of less than 6 mg / dl, the risk of irs is relatively low (1:2430) antipegloticase antibodies. in a post hoc analysis, only 2% of patients with antibody titers exceeding 1:2430 maintained a urate - lowering response to pegloticase compared with 63% of patients who were treated for at least 2 months without developing high - titer antibodies (p < 0.001). these findings imply that the risk of irs may be associated with antibody titer. indeed, the incidence of irs was higher among patients who developed high - titer antibodies compared with those who had titers that did not exceed 1:2430 (60% vs 19% ; p < 0.001). it is, however, important to note that antibody titers at the time of a first ir (versus the final highest titer) were not a reliable predictor of urate response and ir risk. the mechanism(s) pegloticase induces production of antibodies of the immunoglobulin m and immunoglobulin g isotypes, which appear to target the polyethylene glycol moiety rather than the uricase. in the rcts, these antibodies did not neutralize pegloticase activity in vitro except in 1 patient. from a practical perspective, the risk of ir and anaphylaxis with pegloticase therapy is likely to be reducible through routine monitoring of sua levels prior to each pegloticase infusion, and then discontinuing pegloticase therapy if sua exceeds 6 mg / dl, particularly when 2 consecutive measurements show sua greater than 6 mg / dl or an ir occurs. in fact, early postapproval safety surveillance in the united states has demonstrated a 69% reduction in the rate of ir compared with the ir rate recorded during the rcts. the stopping rules exercise shown in table 3 provides a framework for appreciating the shift in risk - benefit that is possible with preinfusion uric acid guided treatment within the rct population and should be augmented in real - world practice with clinical experience and individualized care. all patients treated in the phase 3 studies were premedicated with h1-antihistamines and corticosteroids and then monitored closely for signs and symptoms of irs during the 2-hour infusion of pegloticase. monitoring is recommended even if no adverse events were observed during or after previous infusions, and sua testing should be done as near as is practical to each successive infusion. in the event of an ir, besides any interventions implemented to manage the patient s medical condition, the infusion rate could be slowed by half or stopped. if the reaction resolved, then the infusion can proceed to completion, either at the original rate, at a reduced rate, or using an increased volume of diluent. in no instance during the clinical trials did a second ir occur upon restarting the infusion on the same day. however, the infusion should be stopped and not restarted if the ir fails to resolve within 1 hour, or if the patient is believed to be at risk for an anaphylactic reaction. if a severe or serious ir occurs, the infusion should be stopped, and supportive treatment with antihistamines, fluids, corticosteroids, analgesics, and/or epinephrine provided. physician discretion should guide further treatment if a patient experiences an ir and their preinfusion sua is less than 6 mg / dl. in summary, monitoring sua levels, stopping pegloticase if urate - lowering treatment response is lost, and monitoring patients during infusions are effective steps that should be taken to minimize the risk of irs in patients treated with pegloticase. monitoring preinfusion uric acid levels in patients treated with pegloticase provides critical information on the response to therapy and the appropriateness of continuing treatment. understanding the relationship between uric acid response and irs seen in the pegloticase clinical trials combined with ongoing postapproval pharmacovigilance data can continue to inform patient management and minimize ir risk. | backgroundin clinical trials of pegloticase, a pegylated uricase developed for treatment of gout refractory to conventional therapy, infusion - related reactions (irs) were the second most frequent adverse event reported.objectivethe objective of this study was to provide a detailed account of irs with pegloticase therapy.methodsdata from 2 replicate, 6-month randomized trials and an open - label extension study were pooled. infusions of pegloticase (8 mg) were administered biweekly or monthly ; all patients received prophylaxis (antihistamine, acetaminophen, and corticosteroid) and were tested for urate levels prior to each infusion. an ir was defined by protocol as any otherwise unexplained adverse event or cluster of temporally related events occurring during or within 2 hours of infusion.resultsinfusion-related reactions occurred in 94 (45%) of 208 patients receiving pegloticase ; 10 patients reported irs at first infusion and 84 during subsequent infusions. chest discomfort (15%), flushing (12%), and dyspnea (11%) were the most common symptoms. most irs were rated mild or moderate ; 7% were rated severe. all irs resolved with slowing, interrupting, or stopping the infusion. no patient required blood pressure or ventilatory support. infusion - related reactions were associated with loss of pegloticase urate - lowering efficacy : 91% of all irs occurred in patients with preinfusion serum uric acid concentrations (sua) greater than 6 mg / dl. for patients sustaining preinfusion sua of less than 6 mg / dl, irs occurred in fewer than 1 per 100 infusions.conclusionsphase 3 trial data combined with post hoc analyses demonstrated that knowledge of sua preceding each pegloticase infusion and cessation of therapy when urate - lowering efficacy is lost provide a means to optimize the safety of pegloticase in clinical practice. |
pyrimidinones have been paid increasing attention, due to their various therapeutic and pharmacological properties, such as antiviral, antibacterial, antihypertensive, and antitumor effects. more recently, they emerged as integral backbones of several calcium blockers, antihypertensive agents, -1a - antagonists, and neuropeptide y (npy) antagonists. pyrimidinone derivatives are found as core units in many marine alkaloids (batzelladine and carambine), which have been found to be potent to hiv - gp-120 cd4 inhibitors. due to the remarkable biological utilization, the pyrimidinones attract many researchers as well as academicians. recently, several methods improved the procedure using phosphorus pentoxide - methanesulfonic acid, potassium ter - butoxide (t - buok), ammonium dihydrogen phosphate, silica - gel, mesoporous molecular sieve mcm-41, cyanuric chloride, nano - bf3sio2, silica gel - supported polyphosphoric acid, zirconium(iv) chloride, and indium(iii) bromide as catalysts. however, some of these one - pot procedures generally require strong protic or lewis acids, prolonged reaction times, and high temperature. consequently, there is a scope for further modification towards mild reaction condition, increased variation of the substituents, and improved yields. microwave promoted solvent - free reactions are well known as environmentally benign methods that also usually provide improved selectivity, enhanced reaction rates, cleaner products, and manipulative simplicity. however, these procedures are practically limited as the solvents in microwave oven at elevated temperatures create high pressures, which may cause explosion. to circumvent these problems, there is a need for the development of newer methods which proceed under mild and solvent free condition. nowadays solvent - free reactions gained much importance in organic synthesis because of the high yields and shorter reaction times. earlier reported procedures for the synthesis of pyrimidine derivatives typically involved longer reaction time and fewer yields. in the present communication, we would like to describe the advantages of dry reaction techniques coupled with microwave activation and their applications to organic synthesis. reactions were routinely monitored by thin layer chromatography (tlc) on silica gel (precoated f 254 merck plates) and visualized the products under uv light (254 nm). h nmr spectra were determined in cdcl3 or dmso - d6 solutions with a bruker advance ii 400 mhz spectrometer and signals recorded in parts per million () downfield from tetramethylsilane as internal standard. ir spectra were recorded on perkin elmer ft - ir spectrometer (spectrum rx i) using kbr pellet technique. melting points were recorded in open capillaries on labindia melting point apparatus and were uncorrected. anti - inflammatory activity has been carried out in institute of pharmacy, vikram university, ujjain. a mixture of aldehyde 1 (1 mmol), ethyl cyanoacetate 2 (1.2 mmol), guanidine nitrate 3 (1.5 mmol), and 2 - 3 drops of piperidine was subjected to microwave irradiation at 60% power in 600 w microwave oven for 5 min. (successive irradiation of 3040 sec with cooling intervals of time, the temperature being 90100c). on completion of reaction, indicated by tlc, the mixture was cooled and quenched with water (3 10 ml). white crystals ; mp : 218220c ; ir (kbr vmax / cm) : 3478 (nh), 3090 (c h), 2260 (cn), 1690 (c = o), 1617 (c = h nmr (300 mhz, cdcl3) 8.56, 2.0 (s, 2h nh), 7.277.40 (m, 5h, ar), 4.1 (d, ch, j = 8.4 hz), 3.97 (d, ch, j = 11.5 hz). c nmr (cdcl3) : 168.4, 153.3, 143.5, 128.5, 126.9, 126.7, 116.8, 43.2, 42.4. molecular weight : 214.22 ; mass (m / z) : 214 (m) ; c11h10n4o (214.09) ; cacld. c, 61.67 ; h, 4.71 ; n, 26.15 ; o, 7.47 ; found. c, 60.07 ; h, 4.13 ; n, 25.87 ; o, 7.08. 2-amino-6-(4-methoxyphenyl)-4-oxo-1,4,5,6-tetrahydropyrimidine-5-carbonitrile (4b). light yellow crystals ; mp : 110112c ; ir (kbr vmax / cm) : 3318 (nh), 3055 (c h), 2190 (cn), 1710 (c = o), 1620 h nmr (300 mhz, cdcl3) : 8.32 (s, 2h, nh2), 6.947.18 (m, 4h, ar), 3.9 (d, ch, j = 14.4 hz), 3.72 (d, ch, j = 11.5 hz), 3.50 (s, 3h, och3), 2.0 (s, 2h nh). c nmr (cdcl3) : 166.7, 159.1, 153.5, 136.2, 127.4, 116.3, 115.0, 56.1, 43.6, 44.8. molecular weight : 244 ; mass (m / z) : 243 (m) ; c12h12n4o2 (244.10) ; cacld. c, 59.01 ; h, 4.95 ; n, 22.94 ; o, 13.10 ; found. c, 58.87 ; h, 4.63 ; n, 22.65, o, 12.72. yellow crystals ; mp : 127 - 128c ; ir (kbr vmax / cm) : 3460 (nh), 3080 (arom. c h), 2943 (methyl c h), 1590 (c = c), 1290 (aryl och3), 1670 (c = o), 1567 (c = n), 2310 (cn).h nmr (300 mhz, cdcl3) : 6.746.96 (m, 3h, ar), 2.0, 8.56 (s, 2h, nh), 3.97 (d, ch, j = 8.4 hz), 4.1 (d, ch, j = 11.5 hz), 3.83 (s, 2h, methylene proton). c nmr (cdcl3) : 168.4, 153.3, 149.6, 147.8, 136.8, 121.9, 118.9, 116.8, 109.8, 43.2, 42.7, 56.1. molecular weight : 274.28 ; mass (m / z) : 248 (m) ; c13h14n4o3 (274.11) ; cacld. c, 56.93 ; h, 5.14 ; n, 20.43 ; o, 17.50 ; found. c, 56.14 ; h, 5.04 ; n, 21.98 ; o, 17.19. 2-amino-6-(4-nitrophenyl)-4-oxo-1,4,5,6-tetrahydropyrimidine-5-carbonitrile (4d). yellow crystals ; mp : 162164c ; ir (kbr vmax / cm) : 3440 (nh), 3080 (c h), 2327 (cn), 1640 (c = o), 1560 (c = n), 1567 (c = c), 1523 (n = o). h nmr (300 mhz, cdcl3) : 7.558.21 (dd, 4h, ar), 2.0, 8.56 (s, 2h, nh), 3.97 (d, ch, j = 11.5 hz), 4.1 (d, ch, j = 8.4 hz). c nmr (cdcl3) : 168.4, 153.3, 149.6, 145.9, 123.7, 123.4, 116.8, 43.2, 42.4. molecular weight : 259.22 ; mass (m / z) : 259 (m) ; c11h9n5o3 (259.07) ; cacld. c, 50.97 ; h, 3.50 ; n, 27.02 ; o, 18.52 ; found. c, 50.73 ; h, 3.24 ; n, 26.92 ; o, 18.32. yellow crystals ; mp : 9395c ; ir (kbr vmax / cm) : 3420 (nh), 3111 (ar c h), 2360 (cn), 1720 (c = o), 1593 (c = n). h nmr (300 mhz, cdcl3) : 5.726.69 (m, 3h, ar), 2.0, 8.56 (s, 2h, nh), 3.02 (m, 1h, ch), 3.9 (m, 1h, ch). c nmr (cdcl3) : 168.4, 153.3, 130.5, 118.0, 116.8, 108.5, 107.7, 43.9, 42.8. molecular weight : 203.20 ; mass (m / z) : 203 (m) ; c9h9n5o (203.08) ; cacld. c, 53.20 ; h, 4.46 ; n, 34.47 ; o, 7.87 ; found. n, 33.94 ; o, 7.59. 2-amino-6-(furan-2-yl)-4-oxo-1,4,5,6-tetrahydropyrimidine-5-carbonitrile (4f). light yellow crystals ; mp : 7678c ir (kbr vmax / cm) : 3427 (nh), 3121 (c h), 2230 (cn), 1690 (c = o), 1615 (c = h nmr (300 mhz, cdcl3) : 7.92 (s, 2h, nh2), 6.547.67 (m, 3h, ar), 4.1 (d, ch, j = 11.5 hz), 3.82 (d, ch, j = 8.4 hz), 2.0 (s, 2h, nh). c nmr (cdcl3) : 164.5, 153.6, 149.9, 142.1, 116.5, 111.1, 110.4, 44.3, 40.7. molecular weight : 204 ; mass (m / z) : 203 (m) ; c9h8n4o2 (204.06) ; cacld. c, 52.94 ; h, 3.95 ; n, 27.44 ; o, 15.67 ; found. c, 52.81 ; h, 3.78 ; n, 26.97 ; o, 15.41. yellow crystals ; mp : 190192c ; ir (kbr vmax / cm) : 3450 (nh), 3120 (c h), 2310 (cn), 1690 (c = o), 1590 (c = h nmr (300 mhz, cdcl3) : 10.8 (s, 1h, nh), 8.22 (s, 2h, nh2), 7.117.69 (m, 5h, ar), 3.7 (d, ch, j = 14.0 hz), 3.4 (d, ch, j = 8.0 hz), 2.0 (s, 2h, nh). c nmr (cdcl3) : 164.3, 154.1, 133.2, 126.4, 124.3, 122.5, 120.6, 114.7, 117.1, 110.9, 43.1, 45.4. molecular weight : 253 ; mass (m / z) : 252 (m) ; c13h11n5o (253.10) ; cacld. c, 61.65 ; h, 4.38 ; n, 27.65 ; o, 6.32 ; found. c, 61.16 ; h, 3.99 ; n, 27.34 ; o, 5.89. yellow crystals ; mp : 148150c ; ir (kbr vmax / cm) : 3470 (nh), 3111 (arom. c h), 2360 (cn), 1720 (c = o), 1542 (c = c), 1593 (c = h nmr (300 mhz, cdcl3) : 5.726.69 (m, 3h, ar), 2.0, 8.56 (s, 2h, nh), 3.90 (s, 1h, ch3), 3.97 (d, ch, j = 8.4 hz), 4.1 (d, ch, j = 11.5 hz). c nmr (cdcl3) : 168.4, 153.3, 132.1, 122.5, 116.8, 108.6, 108.4, 44.2, 40.3, 35.2. molecular weight : 217.23 ; mass (m / z) : 217 (m) ; c10h11n5o (217.10) ; cacld. c, 55.29 ; h, 5.10 ; n, 32.24 ; o, 7.37 ; found. c, 54.94 ; h, 4.92 ; n, 31.91 ; o, 7.03. colony bred healthy rats of wistar strain and albino mice procured from local market from ujjain were used for the study. they were housed in standard polypropylene cages under room temperature (24 2c), relative humidity (60%70%), and exposed to 12 : 12 hours light : dark cycle. the rats were fed nutrilab rodent feed and drinking water filtered through an aqua guard water filter system adlibitum. the anti - inflammatory activity was determined in vivo using the carrageenan - induced rat paw edema test [5, 11 ]. a solution of 0.1 ml of 1% carrageenan (sigma - aldrich, dorset, uk) in saline was injected subplantarly in the right hind paw of the rats 1 h after ip administration of compounds. the paw thickness was measured from the ventral to the dorsal surfaces using a dial caliper immediately prior to carrageenan injection and then at each hour, up to 4 h after the subplanar injection. the edema was calculated as the thickness variation between the carrageenan and saline treated paw. anti - inflammatory activity was expressed as the percent of inhibition of the edema when compared with the control group. the data was statistically analyzed by one way analysis of variance (anova) followed by tukey multicomparison test. antibacterial activity of the prepared compounds 4d, 4e, 4f, 4 g, and 4h were tested by the disk diffusion method. the sterile disks were impregnated with the test compounds (250 mg / ml). agar plates were uniformly surface inoculated with fresh broth culture of staphylococcus aureus, basillus subtilis, escherichia coli, and pseudomonas aeruginosa. the impregnated disks were placed on the medium suitably spaced apart, and the plates were incubated at 30c for 1 h to permit good diffusion and were then transferred to an incubator at 37 2c for 24 h. the zones of inhibition were measured on mm scale. the results of antimicrobial activity tests are listed in table 3. antifungal susceptibility test was done by disk diffusion method using sabouraud 's dextrose agar medium. after sterilization, the medium was inoculated with candida albicans, aspergillus flavus, and aspergillus niger. the standard antifungal agent clotrimazole (100 g / ml), solvent control (0.5% v / v tween 80), and the newly synthesized compounds 4d, 4e, 4f, 4 g, and 4h in a concentration of 100 g / ml were then added by sterile micropipette. the plates were then incubated at 37c for 24 h and the diameter of zone of inhibition was measured and recorded in table 3. in the view of the above mentioned limitations of the reported method, pharmacological importance of dihydropyrimidinones and our ongoing endeavors to conduct organic synthesis under solvent free conditions, we describe an expeditious solvent less microwave accelerated approach for the rapid assembly of 2-amino dihydropyrimidinones. aromatic aldehydes (1a h, 1 mmol) on reaction with ethyl cyanoacetate (2, 1.2 mmol) and guanidine nitrate (3, 1.5 mmol) using dry conditions yielded corresponding 2-amino dihydropyrimidinones (scheme 1). as far as our interest in investigating the facile, rapid, and expeditious solvent - less methodology for 2-amino dihydropyrimidinone, we tried the reaction of benzaldehyde (1a, 1 mmol), with ethyl cyanoacetate (2, 1.2 mmol) and guanidine nitrate (3, 1.5 mmol) by varying microwave power from 150 watts to 750 watts. it was observed that by increase in power up to 600 watts, there was increase in yield and shortened reaction time. beyond the 600 watts there was no significant change in reaction time and yield. in order to evaluate the generality of this model reaction, we then prepared a range of 2-amino dihydropyrimidinone derivatives under the optimized reaction conditions. in all cases, aryl aldehydes and heteroaryl aldehydes with substituents carrying either electron - donating or electron - withdrawing groups reacted successfully and gave the expected products in good to excellent yields in relatively short reaction times. the formation of the product takes place when aryl aldehydes were reacted with ethyl cyanoacetate to form arylmethylene ethylcyanoacetate, which subsequently added with guanidine followed by cyclization and tautomerization to form the desired product. the results of the anti - inflammatory activity of compounds 4d, 4e, 4f, 4 g, and 4h were summarized in table 2. from the results, it is evident that compounds 4c, 4f, and 4h as well as indomethacin as the reference drug induced significant anti - inflammatory activity after 3 and 4 h in comparison to control and almost all of the tested compounds were shown moderate to good anti - inflammatory activity. the mic values of the test solutions are recorded in table 3 which is recorded in zones of inhibition in mm for the bacteria and fungi. the antimicrobial screening has shown that compounds 4d, 4f, 4 g, and 4f have displayed moderate activity against gram + ve bacteria tested, that is, s. aureus and b. subtilis. compounds 4d and 4h have shown broad spectrum activity against gram ve bacteria tested, that is, e. coli and p. aeruginosa while compound 4e has shown poor activity against both gram + ve and gram ve bacteria tested. compound 4d has shown good antifungal activity against all the tested fungi, that is, c. albicans, a. flavus, and a. niger while the remaining compounds 4e and 4f have shown good activity, and compounds 4 g and 4h have shown moderate activity against all fungi tested. in summary, we have described one - pot synthesis of 2-amino dihydropyrimidinone derivatives via a three component cycloaddition reaction under microwave irradiation. another advantage of this method is excellent yields in shorter reaction time with high purity of the products. the synthesized compounds have shown good anti - inflammatory, antibacterial, and antifungal activities. | a simple protocol for the efficient preparation of aryl and heteroaryl substituted dihydropyrimidinone has been achieved via initial knoevenagel, subsequent addition, and final cyclization of aldehyde, ethylcyanoacetate, and guanidine nitrate in the presence of piperidine as a catalyst in solvent - free under microwave irradiation. the synthesized compounds showed a good anti - inflammatory, antibacterial, and antifungal activity. |
neoplastic cell lines used in this study were te-1, te-5, and te-8, which were established from well-, poorly-, and moderately - differentiated escc, respectively. these cell lines were purchased from riken cell bank (tsukuba, japan) and cultured in rpmi-1640 medium supplemented with 10% fetal bovine serum. het-1a, an sv40 large t antigen - harboring normal human esophageal squamous cell line, was purchased from american type culture collection (manassas, va, usa) and cultured using the begm kit (lonza, basel, switzerland) but without addition of the ga-1000 (gentamycin - amphotericin b mix) provided with the kit. total rna was isolated from these cells using isogen reagent (wako, osaka, japan) according to the manufacturer 's protocol. rna purity was evaluated by the rna integrity number (rin), a representative index to assess rna quality determined using the agilent 2100 bioanalyzer (agilent, santa clara, ca, usa). the rin value of all rna samples used in this study was shown to be 10. the rna samples were then subjected to ribosomal and mitochondrial rna depletion using ribo - zero rrna removal kit (illumina). maltiplex rna - seq libraries were generated for samples using truseq stranded total library preparation kit (illumina). sequencing was carried out on hiseq2500 (illumina) according to the protocol of 2 125 bp run. as an output of this sequencing, 53 million to 66 million raw reads trimming and filtering of the reads to remove adaptor sequences and low - quality nucleotides were performed using trimmomatic (ver. 0.32) (http://www.usadellab.org/cms/?page=trimmomatic).table 1overview of sequencing results and statistics.table 1sampleraw readscalled bases (mbp)%q30aaverage qualityhet-1a53,656,462670793.5535.25te-153,331,576666693.2735.16te-558,934,174736793.2435.16te-865,921,940824093.2835.16aquality score 30, an index to predict the probability of an error in base calling. overview of sequencing results and statistics. quality score 30, an index to predict the probability of an error in base calling. for the individual cell lines, 90.392.3% of the filtered reads were mapped to the genome. the statistics of sequencing and mapping results are summarized in table 1 and table 2, respectively.table 2overview of mapping.table 2sampleraw reads (m)cleaned reads (mbp)reads mapped (m)mapping rates (%) het-1a53.744.340.691.5te-153.342.938.890.5te-558.947.042.891.0te-865.954.650.392.1 assembly of transcriptomes from rna - seq data and quantification of their expression levels were performed using cufflinks (ver. fragments per kilobase of exon per million fragments mapped (fpkm) values are used as an index of expression level. 1). using fpkm values of all annotated genes, the values of correlation coefficients between het-1a and te-1/5/8 are calculated to be 0.234 (het-1a vs. te-1), 0.323 (het-1a vs. te-5), and 0.401 (het-1a vs. te-8). correlation coefficients among the three escc - derived cell lines range from 0.419 to 0.697. these values possibly indicate that the individual escc - derived cell lines show a more similar gene expression pattern to each other than to het-1a. when analysis objects are filtered with the criteria of fpkm > 1 or > 10 for both of paired samples, the values of correlation coefficients increase to 0.8040.956.fig. neoplastic cell lines used in this study were te-1, te-5, and te-8, which were established from well-, poorly-, and moderately - differentiated escc, respectively. these cell lines were purchased from riken cell bank (tsukuba, japan) and cultured in rpmi-1640 medium supplemented with 10% fetal bovine serum. het-1a, an sv40 large t antigen - harboring normal human esophageal squamous cell line, was purchased from american type culture collection (manassas, va, usa) and cultured using the begm kit (lonza, basel, switzerland) but without addition of the ga-1000 (gentamycin - amphotericin b mix) provided with the kit. total rna was isolated from these cells using isogen reagent (wako, osaka, japan) according to the manufacturer 's protocol. rna purity was evaluated by the rna integrity number (rin), a representative index to assess rna quality determined using the agilent 2100 bioanalyzer (agilent, santa clara, ca, usa). the rin value of all rna samples used in this study was shown to be 10. the rna samples were then subjected to ribosomal and mitochondrial rna depletion using ribo - zero rrna removal kit (illumina). maltiplex rna - seq libraries were generated for samples using truseq stranded total library preparation kit (illumina). sequencing was carried out on hiseq2500 (illumina) according to the protocol of 2 125 bp run. as an output of this sequencing, 53 million to 66 million raw reads trimming and filtering of the reads to remove adaptor sequences and low - quality nucleotides were performed using trimmomatic (ver. 0.32) (http://www.usadellab.org/cms/?page=trimmomatic).table 1overview of sequencing results and statistics.table 1sampleraw readscalled bases (mbp)%q30aaverage qualityhet-1a53,656,462670793.5535.25te-153,331,576666693.2735.16te-558,934,174736793.2435.16te-865,921,940824093.2835.16aquality score 30, an index to predict the probability of an error in base calling. overview of sequencing results and statistics. quality score 30, an index to predict the probability of an error in base calling. the filtered reads were aligned to the ensembl human genome grch38. p7 as a reference (ftp://ftp.ensembl.org/pub/release-85/fasta/homo_sapiens/dna/) using bowtie (ver. the statistics of sequencing and mapping results are summarized in table 1 and table 2, respectively.table 2overview of mapping.table 2sampleraw reads (m)cleaned reads (mbp)reads mapped (m)mapping rates (%) het-1a53.744.340.691.5te-153.342.938.890.5te-558.947.042.891.0te-865.954.650.392.1 assembly of transcriptomes from rna - seq data and quantification of their expression levels were performed using cufflinks (ver. 2.2.1). fragments per kilobase of exon per million fragments mapped (fpkm) values are used as an index of expression level. 1). using fpkm values of all annotated genes, the values of correlation coefficients between het-1a and te-1/5/8 are calculated to be 0.234 (het-1a vs. te-1), 0.323 (het-1a vs. te-5), and 0.401 (het-1a vs. te-8). correlation coefficients among the three escc - derived cell lines range from 0.419 to 0.697. these values possibly indicate that the individual escc - derived cell lines show a more similar gene expression pattern to each other than to het-1a. when analysis objects are filtered with the criteria of fpkm > 1 or > 10 for both of paired samples, the values of correlation coefficients increase to 0.8040.956.fig. | esophageal cancer (ec) is the eighth most common cancer globally in 2012 and predominantly occurs in the man (enzinger and mayer, 2003 ; conteduca., 2012). ec is classified mainly into two types, esophageal squamous cell carcinoma (escc) and esophageal adenocarcinoma, accounting for 6070% and 2030% of all ec cases, respectively. in a previous statistical study it was reported that the numbers of new ec cases and ec - related deaths worldwide in 2008 were estimated to be 482,300 and 406,800, respectively (jemal., 2011). this high mortality rate is largely due to the characteristics of ec such as frequent distant / local metastasis and poor subjective symptoms leading to difficulty with early diagnosis. patients affected with ec diagnosed at late stages mostly have unsatisfactory prognosis, even though various therapeutic options are available. therefore, there is an urgent need to develop effective methods that enable the early detection of ec (orringer, 1993), prompting us to search for novel biomarkers for ec. here, we provide datasets from rna - seq analysis of het-1a, a normal human esophageal squamous cell line (stoner., 1991), and te-1, te-5, and te-8, which are well-, poorly-, and moderately - differentiated escc - derived cell lines, respectively (nishihira., 1993). the raw data of these experiments have been deposited at dna data bank of japan (ddbj) under the accession ids drr084199, drr084200, drr084201, and drr084202. |
human parainfluenza virus type 1 (hpiv-1), which is a member of the family paramyxoviridae, is an enveloped virus with a single - stranded rna genome [1, 2 ]. hpiv-1 infects the upper and lower respiratory tract and causes acute respiratory infections (aris), ranging from mild infections, such as the common cold and laryngitis, to severe infections, such as croup, pneumonia, and bronchiolitis. hpiv-1 is responsible for almost half of all hospitalizations due to aris both in patients younger than 5 years old and in the elderly ; additionally, hpiv-1 is the most common cause of infectious laryngotracheitis (croup) in children [36 ]. the therapy used to treat symptoms of inflammation is based on glucocorticoid and ephedrine, also humidifying the airway ; however, this is not always effective [79 ]. there is no evidence indicating that mitogenic signal activation is required in the early stages of infection [10, 11 ]. il-8 is a mediator responsible for the recruitment of neutrophils that participate in the local inflammatory infiltrate, contributing to airway closure [12, 13 ]. it has been reported that epithelial cells, alveolar macrophages, and neutrophils secrete il-8 [1417 ]. other authors have reported that infection with respiratory syncytial virus (rsv), varicella - zoster virus, and smallpox virus activates il-8 secretion without viral replication [18, 19 ]. these observations indicate that the interaction between the virus and its receptor is sufficient to promote the signalling pathways that activate the il-8 gene ; however, replication is necessary in other viruses, such as vaccinia virus and rhinovirus [2123 ]. it has been shown that viruses have different effects on the regulation of il-8 expression and secretion. the most prominent examples include the filoviruses marburg and ebola and arenaviruses, such as lassa and junin. other viruses such as rsv, adenovirus, vaccine virus, and herpes virus secrete il-8 immediately [24, 25 ]. the primary signalling pathways that elicit a response by chemokines are the mapks and transcription factor nf kappa b pathways. mapks are a family of proteins that activate kinases through a cascade of intracellular phosphorylation events and signal transduction from the cell surface to the nucleus. they are composed of three well - characterized subfamilies : extracellular signal - regulated kinases (erk1/2), c - jun n - terminal kinases (jnks), and p38 mitogen - activated proteinkinases (p38). each subfamily includes a kinase that sequentially acts on three proteins : mekk, mek, and mapk. the mapk family substrates in the cytoplasm and nucleus include additional kinases, transcription factors, phospholipases, and cytoskeletal proteins. erk1/2 is associated with anabolic processes, such as cell division, growth, and differentiation, while jnk and p38 are associated with cellular responses to stress conditions, death, and inflammation [2628 ]. the molecular mechanisms in which larynx epithelial cells play a role and their active involvement in the inflammatory infiltration response to infection by hpiv-1 through production of small chemoattractants that recruit neutrophils have not been investigated sufficiently to generate a strategy that counteracts pathogenesis and to determine whether viral replication is necessary. in this study, an in vitro model of hpiv-1 infection of hep-2 and a549 cells was used to simulate the upper and lower airways. the aim of this study was to determine whether viral replication induces the il-8 secretion and mapk kinase signalization. the hep-2 human laryngeal epithelioma type 2 cells (atcc ccl-23, usa, which reported a contamination with hela cells) and a549 human alveolar type ii - like epithelial cells (atcc, ccl-185, usa) were acquired from american type culture collection (manassas, va, usa). cell lines were propagated in 25 cm flasks (nunc thermo scientific inc., turnberry, usa) and petri dishes with eagle 's minimal essential medium (mem), supplemented with 10% foetal bovine serum and antibiotics (penicillin 100 u / ml, streptomycin 100 g / ml, amphotericin - b 1 g / ml) from invitrogen life technologies (gaithersburg, md). cells were maintained at 37c in 5% co2 and were stored and grown in accordance with the manufacturer 's instructions. virus strain was obtained from american type culture collection, manassas, va, usa. confluent monolayers of hep-2 and a549 cells in 25 cm flasks were inoculated with hpiv-1 and incubated at 37c in 5% co2 until a cytopathic effect was observed 48 hours after inoculation (formation of syncytia). one aliquot of the virus was transferred to 1.8 ml cryotubes after titration and stored at 70c, and another aliquot was used to infect cell monolayers at a 0.01 multiplicity of infection (moi). uninfected hep-2 and a549 cell monolayers were used as controls. a portion of hpiv-1 (5 ml of virus at a titre of 6 10 pfu) in serum - free medium (rpmi) was placed in a sealed box and irradiated at a distance of 10 cm for 10 minutes with optimal radiation levels (1,200 units 100 j / cm) using an fb - uvxl 1000 uv cross - linker fisher biotech (thermo fisher scientific inc., ky, usa) as previously described. haemagglutination assays were performed in 96-well plates to verify the haemagglutinin activity of hpiv-1 using serial dilutions of o - positive human blood (1 : 2, 1 : 4, etc.). hep-2 and a549 cells were infected with live and inactive hpiv-1 at an moi of 0.01, supernatant was collected at different times (30, 45, 60, 90, and 120 minutes and 12 and 24 hours), and uninfected hep-2 and a549 cells were used as controls. il-8 concentrations were quantitated by elisa according to the procedure described by kittigul. (1998) using anti - human cxcl8/il-8 monoclonal antibodies from r&d systems, inc.. optical density at 450 nm was measured on an elisa reader spectrophotometer from thermo labsystems (santa rosa, ca, usa, and helsinki, finland). the expression of mrna for il-8 was determined by reverse transcription - polymerase chain reaction (rt - pcr). total rna was isolated by using sv total rna isolation system (promega, madison, usa) following the manufacturer 's protocol. a 0.5 g portion of total rna was reverse - transcribed for subsequent pcr amplification for each pair of primers in a volume of 20 l, including 200 u m - mlv rt reverse transcriptase (invitrogen, ca, usa), 50 u of rnase inhibitor (sigma), 0.2 g of random primer, 10 mm of dntp mix (invitrogen, ca, usa), and 5x first - strand buffer (250 mm tris - hcl, ph 8.3 ; 375 mm kcl ; 15 mm mgcl2) provided by life technologies.. a 20 l portion of the rt products was then brought to a volume of 20 l containing 10 mm of each dntp, 1 u of taq polymerase (promega, madison, usa), 10 pmol of both the upstream and downstream pcr primers, and 1 x pcr buffer (proprietary formulation supplied at ph 8.5 containing blue dye and yellow dye) provided by promega. the primers for il-8 are as follows : forward, 5-atg act tcc aag ctg gcc gtg gct-3 and reverse, 5-tct cag ccc tct tca aaa act tct-3 with a product of 289 bps. for glyceraldehyde-3-phosphate dehydrogenase (gapdh) ; the forward primer was 5-ccagccgagccacatcgctc-3 ; and the reverse primer was 5-atgagccccagccttctccat-3, giving a 390 bps pcr product. the gapdh gene was amplified in the same reaction to serve as the reference gene. amplification was carried out in a biometra personal thermal cycler after an initial denaturation at 94c for 3 min. this was followed by 35 cycles of pcr using the following temperature and time profile : denaturation at 94c for 40 s, primer annealing at 58c for 40 s, primer extension at 72c for 1 min, and a final extension of 72c for 6 min. the pcr products were visualised by electrophoresis on a 2% agarose gel and visualized by uv transillumination. hep-2 and a549 cells were infected with live or inactive hpiv-1 at an moi of 0.01 with the same kinetics as that described above in the previous assays. cells were harvested, and total protein was extracted and quantified by the bradford method. cells were boiled at 100c for 10 minutes in a buffer solution supplemented with -mercaptoethanol. the obtained proteins were separated on 15% sds - page gels and transferred to a 0.45 m nitrocellulose membrane from bio - rad laboratories (hercules, ca) for 3.5 hours, and western blot assays were performed. transfer efficiency was determined by staining with 0.1% ponceau red in 5% glacial acetic acid from sigma - aldrich (st. louis, mo), and measurements were performed using laemmli 's sds - page system. membranes were washed, blocked, and probed overnight with mapk phospho - erk1/2 (sc-81492), mapk erk1/2 (sc-292838), phospho - p38 mapk (sc-7973), p38 mapk (sc-535), phospho - jnk (sc-135642), and jnk (sc-571) antibodies (1 : 3 000 dilution) purchased from santa cruz biotechnology (santa cruz, ca). the bound antibody was coated with a peroxidase - conjugated goat anti - mouse igg secondary antibody (sigma - aldrich) (1 : 3 000) for 2 hours. membranes were washed and incubated with a luminal chemiluminescent substrate from santa cruz biotechnology, before exposure to x - ray film. the band intensity was quantified using the quantity one (bio - rad) image analysis software. for the best reproduction of larynx conditions, assays were conducted using hep-2 cells that were pretreated with the kinase inhibitors pd980559 for erk1/2, sb203580 for p38 mapk, and sp600125 for jnk from calbiochem millipore (san diego, ca). the concentrations used were from 5 to 20 m (1.3 to 5.2 l) in dmso ; the same dmso concentration was added to control cells and was incubated at 37c. one ml of hpiv-1 at an moi of 0.01 was added, and infected cells were incubated under the same conditions. supernatants from hep-2 cells without inhibitors collected at the same infection time were used as controls. when the mapk inhibitor was used, cells were pretreated with the inhibitor 1 hour before infection. because mapk inhibitors were diluted in dmso, equal concentrations of dmso were added to the untreated infected cells as control. after treatment with the inhibitor, the analysis done in the western blot bands was performed in the quantity one software from bio - rad. individual quantification of the bands was made from the intensity of light generated in them, due to the fluorescence emitted in the radiographic plate. data were tested for normality (shapiro - wilk) and since data did not show normal distribution, kruskal - wallis test was used to compare the different treatments. the assays were performed in triplicate, and the results were statistically analysed using statistica software 5.0 (stat soft, tulsa, oklahoma). a p value < 0.05 was considered to indicate a statistically significant difference. the inactivated viruses were created to demonstrate whether viral replication was involved in the activation of the secretion of il-8 or viral structure alone was sufficient to generate the response to this chemokine. the virus was inactive with uv radiation ; plaque assays were performed to verify that the virus was found inactive and will not be replicated. exponential virus dilutions are presented in figure 1 to determine the number of infectious particles. (a) the first two rows correspond to the viral activity with the inactive virus, where there was no replication due to the absence of pfu. in the remaining rows of live virus activity in both cell types was evaluated. panel (b) corresponds to viral dilutions and panel (c) refers to the number of infective particles in a dilution type cellular. because the inactive virus did not replicate, haemagglutination tests were made showing that inactive virus particles had the same ability to agglutinate erythrocytes and the active virus (l). in both lytic and haemagglutination assays exponential dilutions these tests are often used to demonstrate the viral replication, for the presence of hemagglutinin glycoprotein or viable structures. il-8 secretion was quantified during hpiv-1 infection suggesting that infection of cell lines with live hpiv-1 induced a gradual increase in il-8 secretion. the highest il-8 secretion in hep-2 and a549 was observed at 12 hours after infection, whereas the cells with inactive virus showed a marginal response at 24 hours (2.05 and 2.0). the higher secretion of il-8 was found using infected hep-2 cells followed by infected a549 cells (table 1), compared with the cells with inactive virus where a minimal response was achieved. in the case of hep-2 cells and a549 cells treated with inactive virus, no il-8 secretion was observed neither in the uninfected cells used as controls. the mrna expression of il-8 in a549 and hep-2 cells was determined by rt - pcr analysis. in the analysis, uninfected cells, cells infected with hpiv - infected cells, and hpiv-1 inactive were used. the increased expression of il-8 originated from infected cells, followed by uninfected cells and the inactivated virus, and the expression in the latter two was almost equal. infection was performed at different times and behavior for each was similar in both cell lines (figure 3). gapdh was constitutively expressed in both cell lines and expression did not vary by infection. to establish whether the inactive virus stimulated the phosphorylation of mapks (erk, jnk, and p38) western blot tests were made (figure 4). the two different cell lines hep-2 cell line for the upper portion of the tract and the a549 for the bottom line of the same tract were used to simulate the respiratory tract. western blot assays were performed to determine the phosphorylation status of the mapks erk1/2, jnk, and p38. in hep-2 cells, infection with live hpiv-1 promoted early erk1/2 phosphorylation, beginning at 60 minutes of interaction (figures 5(a) and 5(b) ; band intensities were quantified through densitometric analysis). however, erk phosphorylation was higher at 12 hours than at any other selected time and decreased at 24 hours. in the case of inactivated virus, the 12-hour band was only in lane 3 because no evidence of phosphorylation was observed in the kinetics selected times. the same criteria were applied to the western blot figures ; only a band corresponding to a 12-hour interaction was observed with no increase in phosphorylation before and after this time point. controls consisted of uninfected cells, which showed no significant changes. in a549 cells infected with live hpiv-1, the detection of erk1/2 phosphorylation began at 60 minutes of interaction ; a small phosphorylation signal was observed at 90 minutes of interaction (figures 5(c) and 5(d)), increasing slightly at 120 minutes and remaining constant until the 24-hour time point. phosphorylation was slightly detected for the inactivated virus and control samples. in controls (uninfected cells), inactive virus samples, and samples that were incubated for 30 and 45 minutes phosphorylation was only slightly detected. with inactive viruses, the result was similar to the previous western blot (figures 5(c), lane 4, and 5(d), lane 3). jnk phosphorylation in hep-2 cells also began at 60 minutes and gradually increased until the 90-minute time point but decreased at 120 minutes. by 12 and 24 hours, phosphorylation ceased (figures 6(a) and 6(b)). as in hep-2 cells, jnk phosphorylation in a549 cells began at 60 minutes, increased at 120 minutes, and decreased at 12 and 24 hours, with no further increase (figures 6(c) and 6(d), lanes 9 and 10). likewise, p38 phosphorylation in the hep-2 cell line began at 30 minutes (figures 7(a) and 7(b)), increased again at 120 minutes, and subsequently remained stable until the 24-hour time point. in a549 cells, p38 phosphorylation levels were similar, beginning at 30 minutes (figures 7(c) and 7(d)) but with a gradual increase from the 90-minute time point and achieving a maximum at 12 and 24 hours. the kinase inhibition assay was performed on the cell line hep-2, derived from human laryngeal epithelioma, given that is a suitable target for the in vitro virus assay. our results suggest that p38, erk1/2, and jnk are activated following hpiv-1 infection. therefore, to determine whether these kinases are required for il-8 gene expression, elisas were performed using the supernatant from hep-2 cells pretreated with erk1/2, jnk, and p38 kinase inhibitors and infected with hpiv-1 at the following time points : 45, 60, and 90 minutes and 12 and 24 hours (figure 9). results demonstrate that the p38 inhibitor repressed il-8 production upon viral infection, whereas erk1/2 and jnk mapk inhibitors did not inhibit il-8 production (figures 9(a) and 9(b)), suggesting that p38 mapk signalling is involved in il-8 production. figure 9(c) depicts the average of three experiments with hep-2 cells infected with hpiv-1 and inhibitors. controls without inhibitors for each kinase were included for all time points. treating the cells with jnk and erk1/2 inhibitors did not impair il-8 production, but treating cells with an inhibitor of p38 mapk significantly impaired il-8 secretion in most of the incubation times (before 24 h). erk1/2 and jnk inhibitors were not different from the control group (table 2). total inhibitory dose mapks (erk1/2, jnk, and p38) were evaluated, showing that the optimal concentration to suppress phosphorylation was 20 m (figure 8). in the present study, an in vitro model with two cell lines that were infected with hpiv-1 at different time points was used to determine whether an increase in il-8 secretion occurred. these results are consistent with reports that other respiratory viruses, including influenza a virus (h3n2) and rsv, induce il-8 secretion from bronchial epithelial cells [36, 37 ]. it is known that il-8 is responsible for the recruitment of neutrophils and macrophages and that it performs other functions, such as adhesion and degranulation. likewise, the presence of il-8 in inflammatory infiltrates in lung tissues has been reported. studies performed with different viruses have revealed that epithelial cells infected with specific viruses release il-8 without requiring viral replication, as what occurs in rsv infections [18, 19 ]. contact with the cellular receptor is sufficient to trigger il-8 secretion, whereas other viruses only promote il-8 synthesis during replication [21, 23 ]. in this study, assays using both live and inactivated hpiv-1 were performed to determine whether replication - independent il-8 release occurs with hpiv-1, a virus from the same family as rsv. the results indicate that contact between the virus and target cell is insufficient, even when haemagglutination activity is intact in the inactivated virus, and viral replication is required to stimulate il-8 secretion. previous reports have shown that il-8 secretion is involved in mapk pathway activation [12, 16 ]. these kinases respond to different ligands and stimuli and are involved in signalling pathways and cellular processes, such as proliferation, differentiation, inflammation, immune response, and apoptosis. hpiv-1 does not use the jnk pathway for il-8 activation, whereas p38 and erk are phosphorylated when il-8 is present in the supernatant after infection. the results of this study showed that hpvi-1 does not use the jnk pathway for activation of il-8, while the mapks p38 and erk are phosphorylated by the production of il-8 after the infection, similar results have been described after rsv infection (virus belonging to the paramyxoviridae family) in human lung endothelial cells where erk phosphorylation enabled il-8se creation. therefore, to define which kinases are required for il-8 secretion, the same assays were performed using specific inhibitors for each kinase. the results showed that, even in the presence of specific inhibitors of erk and jnk, il-8 was still secreted, but there was no secretion in the presence of p38 inhibitor. in this case, a complete suppression of il-8 secretion was observed, which suggests that p38 mapk is the pathway through which hpiv-1 induces il-8 production. it is known that the inhibitor sb203580 is a potent, cell - permeable, selective, and reversible inhibitor of p38 and isoforms. inhibition is competitive with atp and requires substrates to accommodate the fluorophenyl ring structure of the pyridinyl imidazole in their atp - binding pocket. structures of other p38 isoforms, jnks, and erk1/2 do not allow inhibitor binding at equivalent positions [38, 39 ]. many members of the paramyxovirus family have been shown to be potent inducers of chemokines of the cxc family such as interleukin-8 (il-8), including respiratory syncytial virus (rsv), sendai virus, human parainfluenza virus type 2 (hpiv-2), hpiv-3, and newcastle disease virus. the receptor - binding glycoprotein is hn in parainfluenza virus and mumps virus, h in measles virus, and g in pneumoviruses (rsv and metapneumovirus). the second glycoprotein is the fusion protein (f), which catalyses membrane fusion (viral and cellular). it has been suggested that hn glycoproteins form a complex that is initiated by the binding hn, activating the fusion protein that acts immediately in such a way that the virus can enter the cell. however, it must be noted that glycoprotein hn acts as both a haemagglutinin (ha) and a neuraminidase (na), binding to sialic acid molecules through its ha activity, mediating the enzymatic cleavage of sialic acid and promoting fusion through its neuraminidase activity. glycoprotein h of morbillivirus only has haemagglutinin activity, and glycoprotein g has neither of these two activities [4144 ]. such differences are likely related to signalling pathways that are activated when viruses interact with the cell. although hpiv-1 and rsv belong to the same family, the two viruses have different glycoproteins, which may explain why rsv uses erk kinase and hpiv-1 uses p38 kinase. following virus binding to its cellular receptor, the extracellular stimulus is transmitted to the nucleus to activate the expression of various genes involved in a given biological activity. the virus activates these pathways according to the coevolution of the parasite - host complex, and it is likely that the virus also can take advantage of the activation of the pathway to provide a better environment and thus freely replicate. viruses, such as herpesviruses, hepatitis b virus, hiv, influenza virus, coxsackie virus, and vaccinia virus, activate the mapk pathway, particularly erk, in the early stages of infection. in this study, we found that mapks are activated during the early stages of infection (60 minutes). it is possible that the activation of the p38 kinase pathway following hpiv-1 infection plays a similarly essential role during other stages of infection. importantly, the stimulus by hpiv-1 increased and was maintained up to 24 hours ; therefore, it was detectable at the first stages of infection, even when cytopathic effect was clearly visible in at least 90% of the culture. several authors have asserted that kinase activation could be a requirement for signalling regulation associated with different biological functions during infection with other viruses. for example, the first events during hiv-1 infection depend on the mapk / erk pathway, which improves viral infectivity. the mechanism by which hpiv-1 stimulates p38 activation needs to be further investigated. however, a product of any of the virus genes may be involved in the process, considering that no kinase activation was observed when viruses were uv - irradiated. additionally, it is possible that mapks also influence virus replication, promoting the synthesis of some proteins necessary for the virus. children with croup are regularly admitted to the emergency department with their lives at risk due to airway closure. currently, the treatment for patients with moderate to severe croup is oral dexamethasone at 0.6 mg / kg (maximum 1012 mg) doses, which acts on these and additional cell types involved in the immune response. however, data show that p38 mapk inhibitors may reverse glucocorticoid insensitivity and have beneficial effects on glucocorticoid concentrations in patients with asthma as well as airway inflammation, in addition to its role as a treatment substitute. a better understanding of hpiv-1 pathogenesis will guide the design of strategies and therapies allowing the inhibition of excessive cell recruitment and il-8 synthesis to reduce the uncontrolled inflammatory process. for now, the exact mechanism in inflammatory and immune processes must be defined, and further research is required to understand whether these pathways are significant for hpiv-1 pathogenesis. an in vitro model with two cell lines infected with hpiv-1 at different time points was used to determine whether an increase in il-8 production occurred. the results showed that il-8 production gradually increased during the time points assessed and that viral replication is required for il-8 secretion. no kinase activation was observed when viruses were inactivated, suggesting that a product of any of the virus genes may be involved in the process. when a p38 inhibitor was used, il-8 concentrations decreased, suggesting that hpiv-1 induces il-8 secretion through p38. additionally, it is possible that mapks also affect virus replication, promoting the synthesis of some proteins necessary for the virus. however further studies are needed to understand whether these pathways are significant for hpiv-1 pathogenesis, in order to develop strategies and therapies allowing the inhibition of excessive cell recruitment and il-8 synthesis to reduce the uncontrolled inflammatory process. | human parainfluenza virus type 1 (hpiv-1) is the most common cause of croup in infants. the aim of this study was to describe molecular mechanisms associated with il-8 production during hpiv-1 infection and the role of viral replication in mapk synthesis and activation. an in vitro model of hpiv-1 infection in the hep-2 and a549 cell lines was used ; a kinetic - based elisa for il-8 detection was also used, phosphorylation of the mitogen - activated protein kinases (mapks) was identified by western blot analysis, and specific inhibitors for each kinase were used to identify which mapk was involved. inactivated viruses were used to assess whether viral replication is required for il-8 production. results revealed a gradual increase in il-8 production at different selected times, when phosphorylation of mapk was detected. the secretion of il-8 in the two cell lines infected with the hpiv-1 is related to the phosphorylation of the mapk as well as viral replication. inhibition of p38 suppressed the secretion of il-8 in the hep-2 cells. no kinase activation was observed when viruses were inactivated. |
1. this bioequivalence study demonstrates that the pharmacokinetic properties of gabapentin 500 mg and zolpidem tartrate 10 mg are unaffected when both treatments are taken simultaneously, compared with each drug taken alone, in healthy participants.2. both treatments were well tolerated when taken alone and in combination, in a population of healthy participants. gabapentin was first developed to treat seizure disorders and neuropathic pain, and is now being investigated as a potential sleep aid for occasional disturbed sleep. studies suggest that low - dose gabapentin (250 mg [1, 2 ] and 500 mg) can improve objective and subjective measures of sleep in adults using a model of occasional disturbed sleep [1, 2 ]. the exact mechanism by which gabapentin improves sleep is unknown, but is likely related to its well - characterized binding affinity to the 2 subunit of voltage - activated calcium channels, and subsequent modulatory effects on neurotransmitter release [3, 4 ]. the pharmacokinetic profile of gabapentin as an immediate - release formulation has been well defined across the therapeutic dose range (3001,800 mg / day, with a recommended dose of 9001,800 mg / day for approved indications). for example, after single - dose oral administration (300 or 600 mg) [57 ], gabapentin reaches peak plasma concentration (cmax) in 23 h [time to reach cmax (tmax) ] [5, 8 ], and with multiple dosing, achieves steady state within 2448 h. plasma concentrations of gabapentin do not increase proportionally with increasing doses owing to absorption in only a limited segment of the small intestine. gabapentin is eliminated unchanged through the kidney, with a terminal half - life (t) of 57 h. gabapentin is largely unaffected by alteration in hepatic function or cytochrome p450 (cyp450) activity [6, 9 ], and there are few known drug drug interactions, including no interaction observed with other antiepileptic drugs (lamotrigine, vigabatrin or oxcarbazepine) or with a contraceptive regimen of norethindrone acetate and ethinyl estradiol. the most common adverse events (aes) observed following treatment with gabapentin (9001,800 mg / day) (not seen at an equivalent frequency among placebo - treated patients) were dizziness, somnolence and peripheral edema in adults with post - herpetic neuralgia, and somnolence, dizziness, ataxia, fatigue and nystagmus in adults with partial seizures with and without secondary generalization. gabapentin s overall pharmacokinetic and pharmacodynamic profile makes for a predictable and well - tolerated drug. there is potential for individuals with sleep disturbances to self - medicate with multiple sleep products. zolpidem, an imidazopyridine agent, is widely prescribed for the short - term management of insomnia. it is absorbed rapidly, with bioavailability of 70 %, and reaches cmax in < 1 h. in addition, zolpidem is rapidly metabolized before being eliminated primarily in bile, urine and feces [15, 16 ]. because metabolism occurs via successive cyp450 enzymes, zolpidem has a high potential for drug drug interactions with agents that inhibit or induce cyp450 enzymes. although gabapentin is not metabolized via the cyp450 pathway, there is potential for co - administration with gabapentin, and therefore, any potential effects of zolpidem on the pharmacokinetic properties of gabapentin, and vice versa, warrant investigation. drug interaction of gabapentin with zolpidem in healthy participants, when administered individually and in combination. participants were randomized in an open - label, three - period crossover design to single - dose gabapentin (500 mg), zolpidem tartrate (10 mg), and the combination (gabapentin 500 mg + zolpidem tartrate 10 mg), between november 5 and 21, 2001. the study was conducted in compliance with the declaration of helsinki and the international conference on harmonisation good clinical practice guidelines. protocol, consent documents and protocol amendments were all approved by the relevant institutional review board at participating centers. healthy men and women (of non - child - bearing potential or those using an acceptable contraceptive) aged 18 years and weighing 50100 kg (110220 lbs) were recruited. standard exclusion criteria were imposed, including, but not limited to, participants with a known sensitivity to either drug ; a positive pregnancy test on screening ; a history of drug or alcohol abuse ; or on physical examination, any clinically significant disease or abnormality that was deemed likely to affect the properties of either drug under investigation. venous blood (210 ml per participant) was collected in heparinized collection tubes pre - dose (0 h) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 18, 24 and 36 h post - dose. samples were centrifuged and the plasma stored in labeled, screw - capped, polypropylene tubes at 20 c within 1 h of collection. gabapentin plasma concentrations were determined by validated liquid chromatography tandem mass spectrometry methods using 1-(amino - methyl)-cycloheptaneacetic acid (pd0403609 - 0000) as an internal standard (bioassay laboratories, houston, tx, usa). the analytical range for detection of gabapentin was 0.02510.0 g / ml, with precision [% coefficient of variation (cv) ] 15.5 % and accuracy [% relative error (re) ] of 0.26.7 %. zolpidem plasma concentrations were determined by a validated high - throughput liquid chromatography (hplc) with fluorescence detection, using trazodone as an internal standard (covance laboratories, inc., the analytical range of detection for zolpidem was 1.00400 ng / ml, with precision (% cv) 14 % and accuracy (% re) of 2.05.0 %. the cmax and tmax were observed directly from the plasma concentration time profile of each participant. time curve from time zero to time of last quantifiable concentration (tlqc) (auc0tlqc) was evaluated using the linear trapezoidal method. the t was calculated using the terminal rate constant determined from the terminal log - linear phase of the concentration data were reviewed and accepted by quality - control specialists before being used for pharmacokinetic analyses. safety evaluations were conducted throughout the study and included monitoring of aes, vital signs, 12-lead electrocardiograms, and physical examination including blood pressure, respiratory, and pulse rate. laboratory tests were conducted at screening only. a sample size of 36 participants completing the study was estimated on the basis of estimates of intra - subject cv for zolpidem 10 mg, which is larger than that for gabapentin [18, 19 ]. the sample size provided at least 80 % power to demonstrate bioequivalence with respect to auc0 and cmax, assuming a cv of 33 % and no underlying difference between the combination and each drug administered alone. log - transformed auc0 and cmax were analyzed using analysis of variance (anova) with treatment sequence, period and treatment as fixed effects and subject - within - sequence as a random effect. results from the anova were used to calculate 90 % confidence intervals (cis) for the ratios (test / reference) of least - squares mean treatment values, where administration of gabapentin and zolpidem together was the test treatment and administration of gabapentin alone and zolpidem alone were the reference treatments. lack of an interaction was concluded if the estimated 90 % cis for the ratios (test / reference) of adjusted means for both auc0 and cmax fell within the 80125 % accepted range for bioequivalence. participants were randomized in an open - label, three - period crossover design to single - dose gabapentin (500 mg), zolpidem tartrate (10 mg), and the combination (gabapentin 500 mg + zolpidem tartrate 10 mg), between november 5 and 21, 2001. the study was conducted in compliance with the declaration of helsinki and the international conference on harmonisation good clinical practice guidelines. protocol, consent documents and protocol amendments were all approved by the relevant institutional review board at participating centers. all participants provided written, informed consent prior to randomization. healthy men and women (of non - child - bearing potential or those using an acceptable contraceptive) aged 18 years and weighing 50100 kg (110220 lbs) were recruited. standard exclusion criteria were imposed, including, but not limited to, participants with a known sensitivity to either drug ; a positive pregnancy test on screening ; a history of drug or alcohol abuse ; or on physical examination, any clinically significant disease or abnormality that was deemed likely to affect the properties of either drug under investigation. venous blood (210 ml per participant) was collected in heparinized collection tubes pre - dose (0 h) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 18, 24 and 36 h post - dose. samples were centrifuged and the plasma stored in labeled, screw - capped, polypropylene tubes at 20 c within 1 h of collection. gabapentin plasma concentrations were determined by validated liquid chromatography tandem mass spectrometry methods using 1-(amino - methyl)-cycloheptaneacetic acid (pd0403609 - 0000) as an internal standard (bioassay laboratories, houston, tx, usa). the analytical range for detection of gabapentin was 0.02510.0 g / ml, with precision [% coefficient of variation (cv) ] 15.5 % and accuracy [% relative error (re) ] of 0.26.7 %. zolpidem plasma concentrations were determined by a validated high - throughput liquid chromatography (hplc) with fluorescence detection, using trazodone as an internal standard (covance laboratories, inc., madison, wi, usa). the analytical range of detection for zolpidem was 1.00400 ng / ml, with precision (% cv) 14 % and accuracy (% re) of 2.05.0 %. non - compartmental methods were employed to estimate pharmacokinetic parameters. the cmax and tmax were observed directly from the plasma concentration time profile of each participant. time curve from time zero to time of last quantifiable concentration (tlqc) (auc0tlqc) was evaluated using the linear trapezoidal method. the t was calculated using the terminal rate constant determined from the terminal log - linear phase of the concentration data were reviewed and accepted by quality - control specialists before being used for pharmacokinetic analyses. safety evaluations were conducted throughout the study and included monitoring of aes, vital signs, 12-lead electrocardiograms, and physical examination including blood pressure, respiratory, and pulse rate. a sample size of 36 participants completing the study was estimated on the basis of estimates of intra - subject cv for zolpidem 10 mg, which is larger than that for gabapentin [18, 19 ]. the sample size provided at least 80 % power to demonstrate bioequivalence with respect to auc0 and cmax, assuming a cv of 33 % and no underlying difference between the combination and each drug administered alone. log - transformed auc0 and cmax were analyzed using analysis of variance (anova) with treatment sequence, period and treatment as fixed effects and subject - within - sequence as a random effect. results from the anova were used to calculate 90 % confidence intervals (cis) for the ratios (test / reference) of least - squares mean treatment values, where administration of gabapentin and zolpidem together was the test treatment and administration of gabapentin alone and zolpidem alone were the reference treatments. lack of an interaction was concluded if the estimated 90 % cis for the ratios (test / reference) of adjusted means for both auc0 and cmax fell within the 80125 % accepted range for bioequivalence. three participants were withdrawn because of positive urine drug screens (one prior to the second treatment period and two prior to the third treatment period), and one participant did not return for the third treatment period. because of these withdrawals, 39 participants received gabapentin (500 mg), 38 received zolpidem tartrate (10 mg), and 38 received the combination and were included in the pharmacokinetic analyses (table 1). during the study, no participant took or received concomitant therapy that was judged to have the potential to affect pharmacokinetic parameters.table 1demographic characteristics of randomized participants at baseline (n = 40)characteristicnumber (%) or mean standard deviation male : female19:21age, years34.1 8.1 range1845race white24 (60) black, non - hispanic7 (18) other9 (23)weight (kg)68.3 9.7 range51.492.7body mass index (kg / m)25.4 2.7 range20.732.1 unless otherwise specified demographic characteristics of randomized participants at baseline (n = 40) unless otherwise specified the mean plasma concentration time profiles for gabapentin and zolpidem following administration alone or in combination are shown in figs. 1 and 2, respectively. for gabapentin + zolpidem combination versus gabapentin alone, the mean pharmacokinetic parameters were cmax 4.61 versus 4.72 g / ml, tmax 4.63 versus 3.64 h and auc0 53.4 versus 51.0 g h / ml (table 2). the 90 % cis for the treatment ratio of cmax (92.0102.0) and auc0 (99.0109.0) for the combination versus gabapentin alone were within the 80125 % range accepted for bioequivalence (table 2). for the combination versus zolpidem alone, the mean pharmacokinetic parameters were cmax 154.0 versus 138.0 ng / ml, tmax 1.45 versus 1.84 h and auc0 912.0 versus 854.0 ng h / ml (table 3). the 90 % cis for the treatment ratio of cmax (104.0118.0) and auc0 (98.1112.0) for the combination versus zolpidem alone were within the 80125 % range accepted for bioequivalence (table 3). the t of gabapentin or zolpidem was also similar when administered alone versus in combination, and averaged ~5.5 h for gabapentin and 3.4 h for zolpidem.fig. time profiles following administration of zolpidem tartrate alone and with gabapentintable 2summary of pharmacokinetic parameters for gabapentin co - administered with zolpidem tartrate (test) and for gabapentin alone (reference)parameter (units)gabapentin + zolpidem (test) [mean (sd)]gabapentin (reference) [mean (sd)]ratio (%) 90 % confidence interval lowerupper n 3838 c max (g / ml)4.61 (1.11)4.72 (1.18)97.092.0102.0auc0tlqc (g h / ml)52.5 (12.6)50.2 (12.1)104.098.8109.0auc0 (g h / ml)53.4 (12.9)51.0 (12.4)104.099.0109.0 t max (h)4.63 (1.95)3.64 (1.17) t (h)5.47 (1.06)5.49 (0.86) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed datatable 3summary of pharmacokinetic parameters for zolpidem co - administered with gabapentin (test) and for zolpidem tartrate alone (reference)parameter (units)zolpidem + gabapentin (test) [mean (sd)]zolpidem (reference) [mean (sd)]ratio (%) 90 % confidence interval lowerupper n 3837 c max (ng / ml)154 (49.9)138 (43.9)111.0104.0118.0auc0tlqc (ng h / ml)898 (381)842 (338)105.098.0112.0auc0 (ng h / ml)912 (386)854 (341)105.098.1112.0 t max (h)1.45 (0.94)1.84 (1.23) t (h)3.47 (1.17)3.44 (1.21) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed data mean gabapentin plasma concentration time profiles following administration of gabapentin alone and with zolpidem tartrate mean zolpidem plasma concentration time profiles following administration of zolpidem tartrate alone and with gabapentin summary of pharmacokinetic parameters for gabapentin co - administered with zolpidem tartrate (test) and for gabapentin alone (reference) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed data summary of pharmacokinetic parameters for zolpidem co - administered with gabapentin (test) and for zolpidem tartrate alone (reference) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed data co - administration of gabapentin and zolpidem was well tolerated. five participants reported seven treatment - emergent aes ; all were mild in severity (table 4). no deaths, serious or severe aes, or discontinuations due to aes were reported. in addition, no clinically significant changes in vital signs were recorded pre- versus post-study.table 4summary and frequency of aes for participants treated with gabapentin alone, zolpidem alone, or gabapentin + zolpidemgabapentinzolpidemgabapentin + zolpidemevaluable participants393838 participants [n (%) ] reporting aes1 (2.6)3 (7.9)1 (2.6) participants [n (%) ] withdrawing due to aes0 (0)0 (0)0 (0) participants [n (%) ] with treatment - related aes 1 (2.6)3 (7.9)1 (2.6)number of aes142number of serious aes000number of aes by intensity mild142 moderate / severe000participants (n) reporting ae (by type) dizziness011 headache110 vomiting010 ae adverse event considered by the investigator to be related to treatment summary and frequency of aes for participants treated with gabapentin alone, zolpidem alone, or gabapentin + zolpidem considered by the investigator to be related to treatment three participants were withdrawn because of positive urine drug screens (one prior to the second treatment period and two prior to the third treatment period), and one participant did not return for the third treatment period. because of these withdrawals, 39 participants received gabapentin (500 mg), 38 received zolpidem tartrate (10 mg), and 38 received the combination and were included in the pharmacokinetic analyses (table 1). during the study, no participant took or received concomitant therapy that was judged to have the potential to affect pharmacokinetic parameters.table 1demographic characteristics of randomized participants at baseline (n = 40)characteristicnumber (%) or mean standard deviation male : female19:21age, years34.1 8.1 range1845race white24 (60) black, non - hispanic7 (18) other9 (23)weight (kg)68.3 9.7 range51.492.7body mass index (kg / m)25.4 2.7 range20.732.1 unless otherwise specified demographic characteristics of randomized participants at baseline (n = 40) unless otherwise specified the mean plasma concentration time profiles for gabapentin and zolpidem following administration alone or in combination are shown in figs. 1 and 2, respectively. for gabapentin + zolpidem combination versus gabapentin alone, the mean pharmacokinetic parameters were cmax 4.61 versus 4.72 g / ml, tmax 4.63 versus 3.64 h and auc0 53.4 versus 51.0 g h / ml (table 2). the 90 % cis for the treatment ratio of cmax (92.0102.0) and auc0 (99.0109.0) for the combination versus gabapentin alone were within the 80125 % range accepted for bioequivalence (table 2). for the combination versus zolpidem alone, the mean pharmacokinetic parameters were cmax 154.0 versus 138.0 ng / ml, tmax 1.45 versus 1.84 h and auc0 912.0 versus 854.0 ng h / ml (table 3). the 90 % cis for the treatment ratio of cmax (104.0118.0) and auc0 (98.1112.0) for the combination versus zolpidem alone were within the 80125 % range accepted for bioequivalence (table 3). the t of gabapentin or zolpidem was also similar when administered alone versus in combination, and averaged ~5.5 h for gabapentin and 3.4 h for zolpidem.fig. time profiles following administration of zolpidem tartrate alone and with gabapentintable 2summary of pharmacokinetic parameters for gabapentin co - administered with zolpidem tartrate (test) and for gabapentin alone (reference)parameter (units)gabapentin + zolpidem (test) [mean (sd)]gabapentin (reference) [mean (sd)]ratio (%) 90 % confidence interval lowerupper n 3838 c max (g / ml)4.61 (1.11)4.72 (1.18)97.092.0102.0auc0tlqc (g h / ml)52.5 (12.6)50.2 (12.1)104.098.8109.0auc0 (g h / ml)53.4 (12.9)51.0 (12.4)104.099.0109.0 t max (h)4.63 (1.95)3.64 (1.17) t (h)5.47 (1.06)5.49 (0.86) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed datatable 3summary of pharmacokinetic parameters for zolpidem co - administered with gabapentin (test) and for zolpidem tartrate alone (reference)parameter (units)zolpidem + gabapentin (test) [mean (sd)]zolpidem (reference) [mean (sd)]ratio (%) 90 % confidence interval lowerupper n 3837 c max (ng / ml)154 (49.9)138 (43.9)111.0104.0118.0auc0tlqc (ng h / ml)898 (381)842 (338)105.098.0112.0auc0 (ng h / ml)912 (386)854 (341)105.098.1112.0 t max (h)1.45 (0.94)1.84 (1.23) t (h)3.47 (1.17)3.44 (1.21) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed data mean gabapentin plasma concentration time profiles following administration of gabapentin alone and with zolpidem tartrate mean zolpidem plasma concentration time profiles following administration of zolpidem tartrate alone and with gabapentin summary of pharmacokinetic parameters for gabapentin co - administered with zolpidem tartrate (test) and for gabapentin alone (reference) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed data summary of pharmacokinetic parameters for zolpidem co - administered with gabapentin (test) and for zolpidem tartrate alone (reference) auc area under the concentration time curve, c max peak plasma concentration, sd standard deviation, t terminal half - life, tlqc time of last quantifiable concentration, t max time to peak plasma concentration ratio of mean values, and the confidence interval for the ratio expressed as percentage, based on the model fitted to natural log - transformed data five participants reported seven treatment - emergent aes ; all were mild in severity (table 4). no deaths, serious or severe aes, or discontinuations due to aes were reported. in addition, no clinically significant changes in vital signs were recorded pre- versus post-study.table 4summary and frequency of aes for participants treated with gabapentin alone, zolpidem alone, or gabapentin + zolpidemgabapentinzolpidemgabapentin + zolpidemevaluable participants393838 participants [n (%) ] reporting aes1 (2.6)3 (7.9)1 (2.6) participants [n (%) ] withdrawing due to aes0 (0)0 (0)0 (0) participants [n (%) ] with treatment - related aes 1 (2.6)3 (7.9)1 (2.6)number of aes142number of serious aes000number of aes by intensity mild142 moderate / severe000participants (n) reporting ae (by type) dizziness011 headache110 vomiting010 ae adverse event considered by the investigator to be related to treatment summary and frequency of aes for participants treated with gabapentin alone, zolpidem alone, or gabapentin + zolpidem considered by the investigator to be related to treatment gabapentin is being investigated as a potential sleep aid for occasional disturbed sleep on the basis of clinical evidence from healthy volunteers and patients with insomnia [1, 2, 2023 ]. the present study demonstrated that the pharmacokinetics of gabapentin (500 mg) and a commonly prescribed prescription sleep aid, zolpidem tartrate (10 mg), were unaffected when both drugs were administered simultaneously, compared with each drug taken alone. in addition, both drugs at the doses stated were well tolerated when taken alone or in combination in this group of healthy individuals. zolpidem is widely prescribed in clinical practice for short - term management of insomnia, and is generally safe and well tolerated [24, 25 ]. although not recommended, the nature of occasional sleep disturbance may result in individuals self - medicating with multiple sleep aids to improve sleep [26, 27 ]. of concern, many prescription sleep aids are associated with significant residual next - day effects due to long half - lives, which could be further prolonged with a change to the pharmacokinetic elimination parameters. our data demonstrate that administration of gabapentin with zolpidem was bioequivalent to administration of gabapentin alone, as the 90 % cis for cmax (rate of absorption) and auc0 (extent of absorption) comparing single drug administration versus each drug administered in combination were within the 80125 % range accepted for bioequivalence. in addition, administration of gabapentin or zolpidem alone or in combination did not change the t for either drug. therefore, no prolonging of next - day effects of zolpidem would be expected, although pharmacodynamic studies would be needed to confirm this assumption. overall, our study found no new safety signals for single - dose use of these two drugs when co - administered. our observations also support what is known about the pharmacokinetic properties of both drugs, as drug drug interactions would not be expected between gabapentin and zolpidem. for example, zolpidem is metabolized via cyp3a4, and acts as a mild mechanism - based inactivator of human cyp3a in vitro [29, 30 ]. in contrast, gabapentin is non - metabolized and is eliminated renally primarily as unchanged drug, showing plasma clearance that is proportional to creatinine clearance. it should be noted that the present study was conducted in a small study population of healthy individuals, using only a single dose, and should be interpreted in light of this. in addition, our observations might not be extrapolated to elderly participants, or those with reduced renal function, as data have shown that age - related changes in renal function can alter the pharmacokinetics of gabapentin [31, 32 ]. the pharmacokinetic properties of gabapentin 500 mg and zolpidem tartrate 10 mg are unaffected when both treatments are taken simultaneously, compared with each treatment taken alone. all treatments were well tolerated when taken alone or in combination in this study population. furey are full - time employees of, and hold stock options in, pfizer inc. | objectivegabapentin is being investigated as a potential treatment for occasional disturbed sleep. this study assessed the pharmacokinetics and tolerability of gabapentin 500 mg and the commonly prescribed sedative / hypnotic zolpidem tartrate 10 mg, administered separately and in combination.methodsforty healthy participants (19 male, 21 female) were randomized into this three - period crossover study [mean (range) age 34.1 (1845) years, weight 68.3 (51.492.7) kg ; 60 % white ]. participants were dosed with gabapentin alone (n = 39), zolpidem tartrate alone (n = 38), and the combination (gabapentin + zolpidem) (n = 38) over three treatment periods, which were separated by 7 days. blood samples were collected pre - dose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 18, 24 and 36 h post - dose. plasma concentrations of each drug were assayed using validated methods. pharmacokinetic parameters were estimated from plasma concentration time data using standard non - compartmental methods.resultsfor gabapentin + zolpidem combination versus gabapentin alone, mean pharmacokinetic parameters were peak plasma concentration (cmax) 4.61 versus 4.72 g / ml, time to cmax (tmax) 4.63 versus 3.64 h and the area under plasma concentration time curve extrapolated to infinity (auc0) 53.4 versus 51.0 g h / ml. for the combination versus zolpidem alone, mean pharmacokinetic parameters were cmax 154 versus 138 ng / ml, tmax 1.45 versus 1.84 h and auc0 912 versus 854 ng h / ml. the 90 % confidence intervals for cmax (rate of absorption) and auc0 (extent of absorption) comparing the combination versus single drug administration fell within the 80125 % range accepted for bioequivalence. all treatments were well tolerated.conclusionthe pharmacokinetics of gabapentin 500 mg and zolpidem tartrate 10 mg are unaffected when both drugs are taken simultaneously, compared with each drug taken alone. |
latissimus dorsi exercises are advocated to provide muscle balance to chest and shoulder press exercises. like many strength training exercises, beliefs persist regarding the influence of exercise variations on muscle recruitment patterns. anecdotally, two beliefs assert that using a supinated grip during the performance of a pulldown will preferentially activate the biceps brachii over the latissimus dorsi when compared to the traditionally forward grip anterior pulldown. a second belief suggests that performance of the seated row increases activation of the middle trapezius / rhomboids when compared with the lat pulldown. it has also been suggested that the seated row performed with scapulae retraction may alter middle trapezius and rhomboid activity when compared with no scapular retraction. signoreli investigated the influence of grip width and line of pull during the lat pulldown on latissimus dorsi and other muscle group 's electromyographic (emg) activity. the authors found that using a pronated wide grip while pulling anterior to the head resulted in the greatest myoelectric activity of the latissimus dorsi when compared to widegrip pulldowns pulled posterior to the head, pulldowns using a supinated grip and pulldowns using a close grip. scapula protraction is thought to tilt the glenoid fossa forward, influencing stability by tilting the glenoid fossa and changing the orientation of the inferior glenohumeral ligament. protraction of the scapula with the addition of the anterior load increases the strain on the inferior glenohumeral ligament. the authors found that the one arm row activated the middle trapezius to 79% of its maximum however, the authors did not state whether the subject was encouraged to retract the scapula or allow protraction. currently no studies have compared the influence of different shoulder extension / adduction exercises on lattissimus dorsi, biceps brachii and middle trapezius / rhomboid myoelectric activity. this study aims to determine the influence of forearm supination, angle of pull and scapula retraction during common latissimus dorsi exercises on the myoelectric activity of latissimus dorsi, middle trapezius and biceps brachii. twelve healthy males (average age (standard deviation) 27.09 years(1.23), average height (sd) 179.08 cm(3.75), average weight (sd)78.25 kg (5.23)), with greater than 6 months of weight training experience, with out back pain or upper limb injuries were recruited from a convenience sample of college students. subjects signed an informed consent form approved by the internal review board of the canadian memorial chiropractic college (cmcc). the muscle activation level, expressed as a percentage of a maximum voluntary contraction (mvc), of the latissimus dorsi (ld), biceps brachii (bb) and middle trapezius / rhomboid muscle (mtr) groups during a series of different exercise tasks was quantified. disposable bipolar ag - agcl disc surface electrodes with a diameter of one cm were adhered bilaterally over the muscle groups with a centre to centre spacing of 2.5 cm. for the right biceps brachii electrodes were placed on the middle of the muscle belly when the elbow was flexed at 90 degrees. for the latissimus dorsi, electrodes were placed one cm lateral to the inferior border of the right scapula. a pair of electrodes was adhered superiorly to the skin above the middle trapezius and rhomboid minor between the spine of the scapula and the 2thoracic spinous process. the amplifier had a cmrr of 10,000:1 (bortec emg, calgary ab, canada). raw emg was band pass filtered (10 and 1000 hz) and a / d converted at 2000 hz using a national instruments data acquisition system and collected using emg acquisition software (delsys, boston ma). three different maximal voluntary contractions for the three muscle groups studied were collected for each subject. for the latissimus dorsi, subjects were required to perform a 3 second maximal isometric lat pull down against an immovable resistance. for the biceps brachii subjects were required to perform a maximum isometric bicep curl (i.e. attempted elbow flexion) against an immoveable object with the arm at 90 degrees of flexion. the maximum voluntary contraction to recruit the middletrapezius / rhomboid muscle required the participants to perform a maximum isometric scapular retraction against experimenter provided manual resistance. the muscle activity during the exercise tasks was then subsequently expressed as a percentage of the peak activity found during the previously described normalization tasks. during all exercises subjects used the same weight on a standard lat pulldown and seated row pulley machine. this weight was chosen by the subject based on their perceived ability to perform between 10 and 12 reps until failure for the pronated grip lat pulldown. for each exercise, two repetitions of a ten second isometric contraction were performed. following each repetition, wide grip pull down (wgp) : from a seated position with the thighs restricted, subjects used an overhand grip on a straight pull down bar at 150% of the bi acromial distance (bad). the weight was pulled into an isometric position with the arms at 90 degrees of shoulder flexion and elbow flexion. (essentially a bar position which finds the bar 12 inches above eye level). 2. reverse grip pull down (rgp) : from a seated position with thighs fixed subjects used an underhand grip on a straight bar at 100% bad. the isometric contraction was held at a position with 90 degrees of shoulder forward flexion and 90 degrees of elbow flexion (essentially a bar position which finds the bar 12 inches above eye level) 3. seated row, shoulders retracted (srr) : subjects started from a seated position, arms extended with forearms at a mid pronated position 6 inches apart. the participants pulled the weight to a position where the shoulder was at 0 degrees of flexion and 90 degrees of elbow flexion with maximal scapular retraction. during the isometric portion of the exercise, seated row, shoulders slack (srr) : subjects performed the same movement as exercise # 3 however the subject was instructed to allow the scapula to roll forward during the isometric hold portion of the exercise. during all of these exercises the isometric portion (the portion that was analysed) was preceded by a concentric contraction that positioned the subjects arm and then followed by an eccentric contraction where the participant lowered the weight to the stack. the root mean square (sliding window of 128 ms with an overlap of 64 ms) of the raw emg during each exercise task and the normalization tasks was calculated using an emg analysis software package (delsys, boston usa). the average activity was then calculated for the middle two seconds of the isometric portion of each exercise and repetition. the average of the two repetitions for each exercise was then calculated for each subject and presented as a percentage of the maximum activity found during the normalization tasks. separate repeated - measures anova with post - hoc tukey tests were then used to determine the influence of exercise type on muscle activity within the latissimus dorsi, biceps brachii and middle trapezius / rhomboids. twelve healthy males (average age (standard deviation) 27.09 years(1.23), average height (sd) 179.08 cm(3.75), average weight (sd)78.25 kg (5.23)), with greater than 6 months of weight training experience, with out back pain or upper limb injuries were recruited from a convenience sample of college students. subjects signed an informed consent form approved by the internal review board of the canadian memorial chiropractic college (cmcc). the muscle activation level, expressed as a percentage of a maximum voluntary contraction (mvc), of the latissimus dorsi (ld), biceps brachii (bb) and middle trapezius / rhomboid muscle (mtr) groups during a series of different exercise tasks was quantified. disposable bipolar ag - agcl disc surface electrodes with a diameter of one cm were adhered bilaterally over the muscle groups with a centre to centre spacing of 2.5 cm. for the right biceps brachii electrodes were placed on the middle of the muscle belly when the elbow was flexed at 90 degrees. for the latissimus dorsi, electrodes were placed one cm lateral to the inferior border of the right scapula. a pair of electrodes was adhered superiorly to the skin above the middle trapezius and rhomboid minor between the spine of the scapula and the 2thoracic spinous process. the amplifier had a cmrr of 10,000:1 (bortec emg, calgary ab, canada). raw emg was band pass filtered (10 and 1000 hz) and a / d converted at 2000 hz using a national instruments data acquisition system and collected using emg acquisition software (delsys, boston ma). three different maximal voluntary contractions for the three muscle groups studied were collected for each subject., subjects were required to perform a 3 second maximal isometric lat pull down against an immovable resistance. for the biceps brachii subjects were required to perform a maximum isometric bicep curl (i.e. attempted elbow flexion) against an immoveable object with the arm at 90 degrees of flexion. the maximum voluntary contraction to recruit the middletrapezius / rhomboid muscle required the participants to perform a maximum isometric scapular retraction against experimenter provided manual resistance. the muscle activity during the exercise tasks was then subsequently expressed as a percentage of the peak activity found during the previously described normalization tasks. during all exercises subjects used the same weight on a standard lat pulldown and seated row pulley machine. this weight was chosen by the subject based on their perceived ability to perform between 10 and 12 reps until failure for the pronated grip lat pulldown. for each exercise, two repetitions of a ten second isometric contraction were performed. following each repetition, wide grip pull down (wgp) : from a seated position with the thighs restricted, subjects used an overhand grip on a straight pull down bar at 150% of the bi acromial distance (bad). the weight was pulled into an isometric position with the arms at 90 degrees of shoulder flexion and elbow flexion. (essentially a bar position which finds the bar 12 inches above eye level). 2. reverse grip pull down (rgp) : from a seated position with thighs fixed subjects used an underhand grip on a straight bar at 100% bad. the isometric contraction was held at a position with 90 degrees of shoulder forward flexion and 90 degrees of elbow flexion (essentially a bar position which finds the bar 12 inches above eye level) 3. seated row, shoulders retracted (srr) : subjects started from a seated position, arms extended with forearms at a mid pronated position 6 inches apart. the participants pulled the weight to a position where the shoulder was at 0 degrees of flexion and 90 degrees of elbow flexion with maximal scapular retraction. during the isometric portion of the exercise, 4. seated row, shoulders slack (srr) : subjects performed the same movement as exercise # 3 however the subject was instructed to allow the scapula to roll forward during the isometric hold portion of the exercise. during all of these exercises the isometric portion (the portion that was analysed) was preceded by a concentric contraction that positioned the subjects arm and then followed by an eccentric contraction where the participant lowered the weight to the stack. the root mean square (sliding window of 128 ms with an overlap of 64 ms) of the raw emg during each exercise task and the normalization tasks was calculated using an emg analysis software package (delsys, boston usa). the average activity was then calculated for the middle two seconds of the isometric portion of each exercise and repetition. the average of the two repetitions for each exercise was then calculated for each subject and presented as a percentage of the maximum activity found during the normalization tasks. separate repeated - measures anova with post - hoc tukey tests were then used to determine the influence of exercise type on muscle activity within the latissimus dorsi, biceps brachii and middle trapezius / rhomboids. table 1 shows the average activity found for each muscle for the four different exercise tasks. the latissimus dorsi musle activity was higher during the seated row with a protracted scapulae than the activity found during a wide grip pulldown and a reverse grip pulldown. the level of protraction / retraction did not influence latissimus dorsi activity during the seated row exercise. the level of forearm supination had no influence on latissimus dorsi activity during the pulldown exercise. average myoelectric activity (expressed as % mvc) for each muscle studied across 4 different shoulder extension exercises. this row indicates which exercise [denoted by a superscripted column number [i.e 1 = pulldown exercise ] results in statistically significant [p <.05 ] muscle activity. lats = latissimus dorsi, biceps = biceps brachii, ratio lats : biceps is the average muscle activation ratio between the latissimus dorsi and biceps brachii for each exercise tested. slack = seated row allowing scapulae protraction, retract = seated row with encouragement to retract the scapulae during the seated row the act of retracting the scapula did not influence middle trapezius / rhomboid muscle activity. however, performing the seated row with the scapula not retracted resulted in an increased myoelectric activity when compared with the reverse grip pulldown. a trend also existed for increased activity in the middle trapezius / rhomboids during the seated row with retraction although this was not statistically significant. significant differences were found when comparing the ratio between latissimus dorsi activity and biceps brachii activity across exercises. this ratio is the average ratio for each subject not the ratio of the group average. the wide grip lat pulldown had a siginificantly higher ratio than the reverse grip lat pulldown, as did the seated row with protracted scapula. the belief that a wide grip during the lat pulldown preferentially recruits the latissimus dorsi over the biceps brachii does not appear to be supported. however, there was a statistically significant change in the latissimus dorsi : biceps brachii ratio between the two pulldown exercises. the statistically significant change in the latissimus dorsi to biceps ratio occurred because of the slight non - statistically significant decrease in latissimus dorsi activity when changing from the wide grip to the reverse grip position of the lat pulldown being coupled with the slight non - statistically significant increase in biceps activity when changing from the wide grip to the reverse grip lat pulldown exercise. these results suggest that slight changes occur when changing grip position but these changes are small and may have no weight training significance. to state, as many clinicians and personal trainers do, that the wide grip pulldown preferentially trains the back and the close grip supinated pulldown preferentially trains the biceps is unsupported. while not investigated in this study, differences in strength between the exercises may be due to the different mechanical advantages / disadvantages of one exercise or possibly to differences in the recruitment levels of the forearm flexors which may be most affected by grip position. additionally, it appeared that the seated row slightly increased latissimus dorsi activity without decreasing biceps brachii activity as seen by no difference in the latissimus dorsi : biceps brachii ratio when compared with the wide grip lat pulldown. a related limitation to this study is that only one portion of the latissimus dorsi was studied.. however, this study investigated different exercises, it is possible that the muscle activation of the latissimus dorsi in our current study merely shifted to another portion of the muscle group when moving from the wide grip lat pulldown to the reverse grip or seated row. this deserves further study. a second aim of the study was to determine the influence of scapula retraction during the seated row on middle trapezius / rhomboids activity. while the seated row exercise did recruit the middle trapezius / rhomboid to a greater extent than either lat pulldown exercises, actively retracting the scapula did not result in an increase activity of the middle trapezius / rhomboids. this suggests that the middle trapezius / rhomboids is active regardless of the position of the scapulae or another muscle functions to cause scapula retraction. it is possible that deep fibres of the rhomboid may become more active during the scapula retraction and this increased activity was not picked up by the electrodes or the lower trapezius may function to cause scapula retraction. the lower trapezius has been shown to be recruited to 74% mvc during shoulder horizontal extension. a limitation of the study is related to the different amount of weight lifted by each subject. participants were asked to choose a weight where they would experience muscle failure between 1012 repetitions. ten to twelve repetitions was chosen because this represents what is commonly done during many strength training programs. however, at this repetition level, the participant 's muscle activity was often less than 3040 % of the mvc. the moderately low level of muscle activity (3040% mvc) during this repetition level suggests that large fast twitch fibres (type ii) may not be recruited during this exertion level, especially if fatigue and the associated type ii fibre recruitment is not achieved. having participants choose their weight for repetitions suggest they may be cautious and underestimate their capabilities. this may carry over to strength training individuals who may underestimate their strength and lift loads at a less than optimum level for recruiting large muscle fibres. additionally, it is unknown whether the changes in muscle activation ratios between the latissimus dorsi and biceps brachii occurs at higher levels of muscle activation. it is possible that one muscle may achieve fatigue and maximum activation while the other does not achieve fatigue and is not maximally stressed. it is unknown whether it is the biceps or latissimus dorsi that is the limiting factor during the performance of the lat pulldown or seated row. the wide grip lat pulldown demonstrated a small but non - significant increase in the activity of the latissimus dorsi compared with the supinated grip pulldown. this same small increase is seen in biceps muscle when using a supinated grip versus the wide grip during the lat pulldown. due to the small changes in muscle activity there appears to be very little difference in muscle activity between the wide grip lat pulldown and the supinated grip lat pulldown for the biceps and latissimus dorsi muscles. additionally, the seated row while recruiting the latissimus dorsi and biceps brachii more or equally effectively as the lat pulldown also recruits the middle trapezius / rhomboid muscle group to a greater extent. actively retracting the scapula does not appear to increase activation levels of the middle trapezius / rhomboid muscle group. | backgroundexercise beliefs abound regarding variations in strength training techniques on muscle activation levels yet little research has validated these ideas. the purpose of the study is to determine muscle activation level, expressed as a percent of a normalization contraction, of the latissimus dorsi, biceps brachii and middle trapezius / rhomboids muscle groups during a series of different exercise tasks.methodsthe average muscle activity during four tasks ; wide grip pulldown, reverse grip pull down [rgp ], seated row with retracted scapula, and seated rows with non - retracted scapulae was quantified during two 10 second isometric portions of the four exercises. a repeated measures anova with post - hoc tukey test was used to determine the influence of exercise type on muscle activity for each muscle.results & discussionno exercise type influenced biceps brachii activity. the highest latissimus dorsi to biceps ratio of activation occurred during the wide grip pulldown and the seated row. highest levels of myoelectric activity in the middle trapezius / rhomboid muscle group occurred during the seated row. actively retracting the scapula did not influence middle trapezius / rhomboid activity.conclusionvariations in latissimus dorsi exercises are capable of producing small changes in the myoelectric activity of the primary movers. |
spinal dural ateriovenous fistula (sdavf) is the lesion within dural leaflets due to direct arterial - to - venous shunt. dural ateriovenous fistula (davf) at the craniocervical junction is rare, as an uncommon type of sdavf, which has been documented in sevral case reports2,3,4,8) we report a patient who presented brainstem dysfunction due to davf at the craniocervical junction that was cured by transarterial onyx embolization. it is a very rare case when symptoms are caused by abnomal venous drainage. on the other hand, onyx embolization for davf in this area a 46-year - old female patient presented with a history of one month 's duration of vertigo, gait, nausea, vomiting and dysphagia, which developed progressively. she was referred for a brain mri study with a preliminary diagnosis of brainstem infarction in a local hospital. t2-weight mri and flair image showed high signal intensity swelling from pons to medulla oblongata (fig. low signal intensity and partial enhancement in the same territory were detected on t1-weight and contrast enhanced imagings (fig. neurologic examination revealed attenuation of left - side gag reflex and right - side nasolabial groove. tongue extension moved to the right and muscular strength of left limbs was iv in this case. spinal mri disclosed abnormal flow void at ventral surface from craniocervical junction to cervical cord, suggesting engorged vessels (fig. cerebral angiography performed five days after admission revealed davf with a meningeal branch originated from the radicular artery of the right c2 segment of va as a feeding vessel, draining via abnormally hypertrophic pontomesencephalic veins ascending into basal vein retrogradely and descending into the anterior spinal vein, anterior internal vertebral venous plexus and vertebral artery venous plexus (fig. arterial feeder originated from external carotid artery (eca) was not disclosed from bilateral eca angiography. a diagnosis of davf at the craniocervical junction with venous congestion of brainstem was made. the patient underwent a transarterial endovascular embolization with onyx-18 (ev3, irvine, ca, usa). intravenous heparin was given before the guiding catheter was advanced to keep activated clotting time from 200 to 300 seconds. a six - french envoy guiding catheter (cordis endovascular, miami lakes, fl, usa) and a marathon microcatheter (ev3, irvine, ca, usa) were used. onyx-18 was injected using real - time digital subtraction fluoroscopic mapping after the microcatheter lumen was flushed with 0.3 ml dimethyl sulfoxide (dmso, ev3, irvine, ca, usa). the injection was terminated when the angiogram confirmed complete obliteration of the fistula (fig. low - molecular - weight heparin (lmwh) injection, aspirin and clopidogrel were administrated after endovascular treatment. meanwhile, aspirin (100 mg) and clopidogrel (75 mg), once a day, were administrated for one month. this patient has been followed - up for six months and recovered completely without any neurological deficits. cerebral angiography performed at six months after embolization showed complete obliteration of the fistula and disappearance of abnormal venous drainage (fig. sdavf is a common type of spinal vascular malformation in adult that usually affects the lumbar or lower thoracic spine. review of the literature revealed that davfs occuring at the craniocervical junction had been called davfs at the foramen magnum and often present with progressive myelopathy or sah2,3,4,8). only one case in this anatomical area has been reported to involve brainstem dysfunction10). brainstem dysfunction as an initial symptom has been documented in several cases of ccfs or intracranial davfs1,6,9,12,15,17,18). congestion caused by venous hypertension was also considered as a probable pathological mechanism. in here we describe a case presenting with brainstem dysfunction at onset. a cerebral angiogram confirmed a davf at the craniocervical junction that was supplied by the meningeal branch originated from the radicular artery of the right va with ascending and descending venous drainage. davf ought to be considered for these dysfunctions to avoid catastrophic outcomes such as brainstem hemorrhage or necrosis which might result from erroneous diagnosis followed by incorrect therapy8,11,15). brainstem venous congetion may be caused by elevated venous pressure secondary to arterial pressure via fistula. in previously reported cases of sdavfs, three major anomalous venous drainage patterns were recognized : ascending, descending and undetected8,13). ascending venous drainage into intracranial sinus was considered to account for sah caused by venous hypertension. moreover, a descending venous drainage was considered for myelopathy. ascending along with descending venous drainage the previous two cases of cervical davfs and one of lumbar spinal davf that presented brainstem dysfunctions demonstrated a similar ascending venous drainage (table 1)10,19,20). venous flow drains into the transverse sinus, the petrosal sinus and the straight sinus under normal physiological conditions14). in a retrograde ascending venous drainage system of the cervical davf if the venous flow increases precipitously, an elevated hemodynamic stress, varix formation or even hemorrhage may occur5,8). however, this patient and the other three cases reported with ascending drainage presented braistem dysfunction rather than hemorrhage (table 1). we found that retrograde flow in each case was slow and the draining volume was not very high. under this condition, normal brainstem venous drainage would be stagnant, resulting in congestion and edema, similarly to myelopathy caused by venous hypertension as it is seen in some cases of sdavfs7). therefore, the aim of treatment is to obliterate abnormal shunt and venous drainage as well as relieving venous congestion. onyx, as a novel liquid embolic agent, has being an alternative option in the treatment of cerebral and spinal vascular malformations16). surgical approach would be difficult and at high risk when the fistula and draining veins are located at the ventral side of the foramen magnum. this patient was treated with endovascular treatment using transarterial onyx embolization, which is safer and more controllable than former endovascular theraputic approaches. onyx-18 was injected with a " hold - reinjection " technique to avoid glue migration to the proximal trunk of the feeding artery under real - time digital subtraction fluoroscopic mapping. anticoagulant and antiplatelet therapy were performed to prevent thrombosis caused by blood flow stasis in the arterialized drainage vein due to closure of a fistula. this patient recovered completely without any neurological deficits as determined during the follow - up period. brainstem anomalous signals of t1-weight, t2-weight and contrast - enhanced t1-weight imaging had disapeared by the latest mri study, which verified the lesion congestion rather than infarction. retrograde ascending venous drainage of the davf at the craniocervical junction, when venous flow is not high, may induce brainstem venous congestion. congestion could be reversible if davf is considered as a differential diagnosis for brainstem dysfunction of undetermined origin and appropriate treatment was performed promptly to avoid poor outcomes. moreover, onyx embolization is an alternative option for the treatment of davf at the craniocervical junction. | dural ateriovenous fistula (davf) at the craniocervical junction is rare. we report a patient presenting with brainstem dysfunction as an uncommon onset. brainstem lesion was suggested by magnetic resonance image study. angiogram revealed a davf at a high cervical segment supplied by the meningeal branch of the right vertebral artery, with ascending and descending venous drainage. complete obliteration of the fistula was achieved via transarterial onyx embolization. clinical cure was achieved in the follow - up period ; meanwhile, imaging abnormalities of this case disappeared. accordingly, we hypothesize that a brainstem lesion of this case was caused by craniocervical davf, which induced venous hypertension. thus, venous drainage patterns should be paid attention to because they are important for diagnosis and theraputic strategy. |
acetylcysteine is well established as a safe and effective antidote for paracetamol poisoning, although there is uncertainty regarding the indications for treatment and most effective administration protocol [13 ]. a treatment nomogram was originally devised by prescott to allow identification of patients at significant risk of acute liver injury, the so - called 200-line plotted between 200 mg / l (1320 mol / l) at 4 hours and 30 mg / l (200 mol / l) at 15 hours. as a modification, 100-line was plotted 50% lower so that treatment was indicated by lower paracetamol concentrations in patients with individual risk factors for paracetamol toxicity, such as malnourishment, chronic excess ethanol intake, or prior use of enzyme - inducing drugs [57 ]. these have long been established in clinical practice in the united kingdom as standard and high - risk nomograms to indicate the need for acetylcysteine after acute paracetamol overdose. other protocols have been adopted elsewhere, for example, the rumack nomogram or 150-line is used in the united states for all patients irrespective of individual risk factors and is plotted 25% lower than the standard prescott nomogram and extrapolated to 24 hours. the rationale for treating only patients at increased risk of toxicity is based, at least in part, upon the high rate of occurrence of adverse effects of acetylcysteine particularly amongst patients with comparatively low paracetamol concentrations [9, 10 ]. a notable exception to this practice is the routine treatment of all paracetamol overdose patients in denmark, irrespective of paracetamol concentration or risk factors [9, 11 ]. despite widespread acceptance of the nomogram method (1) it is valid only for estimating paracetamol exposure up to 15 hours after single time - point ingestion and can not be applied where there has been repeated or staggered ingestion over > 1 hour. 200-line or 100-line in an individual patient may be difficult due to a lack of objective assessment of risk factors such as malnutrition or chronic alcohol excess [57, 12, 13 ]. (3) the nomogram fails to identify all patients that may develop toxicity and, for example, around 1 in 4400 patients develops acute liver failure despite paracetamol concentrations below the the medicines and healthcare products regulatory agency (mhra) undertook a major review of acetylcysteine between 2011 and 2012 and substantially amended its marketing authorisation with effect from 3rd september 2012 onward. amendments included (1) abandoning assessment of individual patient risk factors, (2) application of the 100-line to all cases so that the 200-line is obsolete, and (3) treating all patients after staggered overdose or where the time of ingestion is unclear, irrespective of the paracetamol concentrations. the regulatory amendment was based solely on a risk - benefit basis, and resource implications were beyond the remit of the mhra review. few clinical data were available to inform the likely number of additional patients that might require treatment. therefore, the present study sought to examine the impact of the effect of the marketing authorisation amendment on patterns of acetylcysteine administration and hospitalisation after single time - point and staggered paracetamol overdose. york hospital serves a catchment population of around 350 thousand people, and receives approximately 70 thousand emergency department attendances per year. adults that present after paracetamol overdose are assessed in a dedicated observation area and may be discharged home after completion of medical and psychiatric assessment or may be admitted to the acute medical unit if ongoing medical treatment is required or the patient is too drowsy or intoxicated to allow detailed psychiatric assessment. the study population consisted of consecutive patients aged 16 years that presented to the emergency department due to paracetamol overdose between september 2011 and april 2013 inclusive. data collected were age, gender, weight, date and time of ingestion, paracetamol dose, serum paracetamol concentration, acetylcysteine administration, and duration of hospital episode. paracetamol concentrations are determined using an enzymatic hydrolysis method (olympus diagnostics, southall, united kingdom) and performed using an au2700 automated analyser (beckman coulter, high wycombe, united kingdom). the test principle is based on paracetamol hydrolysis by aryl acylamidase to yield p - aminophenol and acetic acid ; p - aminophenol further reacts with o - cresol and ammoniacal copper sulphate to form indophenol. the quantity of indophenol is determined using spectrophotometry at a wavelength of 600 nm and is directly proportional to the amount of paracetamol in the sample. the assay is specific for paracetamol and is not subject to interference by its major metabolites. a before - after analysis was used to compare patterns of paracetamol overdose and acetylcysteine administration around the marketing authorisation amendment on 3rd september 2012. if patients presented 1 hour (54% before versus 80% after, p = 0.0498), whereas treatment of other overdose patterns was broadly similar before and after the licence change (table 2). patients were considered in one of three treatment groups : (1) those that ingested a nontoxic dose (< 8 grams) and did not require paracetamol concentration to be checked, (2) those that did not require acetylcysteine treatment, and (3) those that received acetylcysteine treatment. the duration of hospital stay in each treatment group did not change as a result of the marketing authorisation update (table 3). however, across the study population the median duration of hospital stay increased after the license amendment from 15 to 24 hours (p = 0.0159) due to an increased number of patients that required acetylcysteine treatment (table 3). the overall duration of hospital stay was 15696 hours before september 2012 and 11358 hours after, representing an additional 5.1 hours (95% confidence interval 4.36.0 hours) for every patient that presented to hospital (p < 0.0001). these data show that the mhra amendment to the acetylcysteine marketing authorisation led to a significant change in clinical practice after september 2012. as expected, there was a substantial increase in the number of patients receiving treatment if paracetamol concentrations were between the 200-line and 100-line, indicating a high level of awareness and implementation of the new recommendations in respect of acute single time - point overdose. the legislative changes in relation to late or staggered overdose or where the time of ingestion was uncertain also led to an increase in the proportion of patients treated (53% to 74%). this indicates a poorer level of awareness and implementation of the updated guidance in relation to staggered overdose and delayed presentations, which are that patterns of paracetamol overdose with the worst outcome. this might be explained by poor communication between the regulatory authorities and prescribers, as previously been suggested. clinicians would be expected to make reference to the treatment nomogram in management of acute, single time - point overdose, and consultation with an up - to - date resource such as toxbase or the british national formulary might have alerted to the mhra amendments. in contrast, clinicians may be less likely to access additional resources when faced with a late or staggered overdose. fewer patients presented late or after a staggered ingestion than after acute overdose, so that experiential learning might take longer to be implemented into clinical practice. the magnitude of that change equated to an additional 5.1 hours for every patient presenting to hospital after paracetamol overdose, which correspond to an additional 102 bed days per year and costs of 3550,000 at york hospital alone. if the same findings were extrapolated to the united kingdom, then this would represent an additional cost of around 7.08.5 million annually. indeed, the eventual cost is likely to be even greater once the marketing authorisation amendments concerning late and staggered overdoses are fully implemented. the amendment was based primarily upon a risk - benefit analysis that primarily sought to lessen the occurrence of paracetamol - induced liver failure. clinical experience indicates that acute liver injury is rare in patients that do not meet the criteria for acetylcysteine based upon the 150-line after acute paracetamol overdose, and there have been calls for the united kingdom to adopt this approach as used in the united states, australia, and new zealand [19, 20 ]. in order to examine the impact of the mhra update and its cost effectiveness, multicentre studies will need to recruit very large patient numbers to examine the occurrence of paracetamol - induced liver failure. there are challenges to applying conventional research methods to the study of paracetamol poisoning, but recent studies demonstrate the feasibility of clinical research in this patient group and are encouraging [3, 21 ]. a limitation is that the findings are based on data from one hospital and might not be generalised to other institutions. notwithstanding, paracetamol accounts for around 40% of all overdose presentations at york hospital, which is broadly similar to that at other hospitals in the united kingdom. a further potential limitation is that the study period extended to only 8 months after the mhra update, whereas the legislative changes might perhaps take longer than this to become fully implemented into clinical practice [23, 24 ]. the findings indicate that the update to the marketing authorisation for acetylcysteine has led to a significant change in clinical practice. there has been a significant increase in antidote administration after paracetamol overdose, resulting in a significantly prolonged hospital this is a financially costly intervention, and further studies are needed to examine clinical outcomes so that the cost - effectiveness of the recent mhra update can be addressed. | in september 2012, the medicines and healthcare products regulatory agency (mhra) substantially amended the marketing authorisation for acetylcysteine following an extensive review. the present study examined the impact of this license change on patterns of acetylcysteine use in patients presenting to hospital after paracetamol (acetaminophen) overdose. between september 2011 and april 2013, 785 consecutive patients presented to york hospital due to paracetamol overdose, and a before - after analysis was used to compare outcomes. there were 483 patients before and 302 patients after the license amendment, and age, gender, acute or staggered overdose pattern, and dose were similar in both groups. in the patients with paracetamol concentrations between the 100-line and 200-line, a significantly higher proportion received acetylcysteine treatment (51% before versus 98% after, p = 0.0029), as expected. a modest increase was also observed in relation to late or staggered overdose or cases where the time of ingestion was uncertain (53% versus 74%, p = 0.0430). the median duration of hospital stay increased across the entire study population, from 15 to 24 hours (p = 0.0159) due to the increased proportion of patients requiring acetylcysteine treatment. the findings indicate that the mhra amendment is a financially costly intervention, and further studies are needed to examine clinical outcomes so that its cost effectiveness might be addressed. |
the disease of dental caries dates back to ancient times and is the most common disease besetting human race. in spite of various preventive methods, dental caries it has been shown to be a very useful adjunct to restorative dentistry because of its unique ability to release fluoride, which is mainly responsible for its cariostatic action. moreover, glass ionomer cement bonds chemically to enamel and dentin, thereby reducing the need for a retentive cavity preparation ; thus, also preserving the sound tooth structure following the principle of because of these properties, glass ionomer cement is the material of choice in atraumatic restorative treatment (art). because atraumatic restorative treatment (art) is practiced using hand instruments only, there is a possibility of insufficient caries removal ; therefore, such kind of cavities require a restorative material with good antibacterial efficacy. thus, therapeutic benefits may be gained by reinforcing glass ionomer cements with additional antibacterial agents. such agents should not affect the physicomechanical properties of the glass ionomer cement. among the antiseptics, a new antibacterial agent of interest is triclosan, a broad spectrum antimicrobial, which has been extensively used in mouthwashes and dentifrices. compressive stress results when the body is subjected to two sets of forces in the same straight line but directed toward each other. shear bond strength is the maximum amount of force required to break the interface between a bonded restoration and the tooth surface with the failure occurring in or near the adhesive interface. tensile stress results in a body when it is subjected to two sets of forces that are directed away from each other in the same straight line. considering the importance of compressive strength, diametral tensile strength and shear bond strength of restorative materials, the purpose of the study undertaken was to evaluate and compare the compressive strength, diametral tensile strength, and shear bond strength of gic type ix, chlorhexidine - incorporated gic, and triclosan - incorporated gic. this in vitro study was carried out in the department of pedodontics and preventive dentistry, d.j. college of dental sciences and research, modinagar, ghaziabad, in collaboration with apex laboratories, mohanagar. for the evaluation of compressive strength and diametral tensile strength restorative pellets of gic type ix, 0.5, 1.25, and 2.5% concentrations of chlorhexidine - incorporated gic and triclosan - incorporated gic were prepared. for the evaluation of shear bond strength the desired concentrations of gic powder, chlorhexidine salt, and triclosan powder were obtained using an analytical digital scale. chlorhexidine - gic (chx - gic) : three different proportions of chx - gic were prepared based on the concentration of chlorhexidine. 0.5% chx - gic : prepared by adding 0.015 g of chlorhexidine to 2.985 g of glass ionomer powder;1.25% chx - gic : prepared by adding 0.037 g of chlorhexidine to 2.96 g of glass ionomer powder;2.5% chx - gic : prepared by adding 0.075 g of chlorhexidine to 2.925 g of glass ionomer powder. 0.5% chx - gic : prepared by adding 0.015 g of chlorhexidine to 2.985 g of glass ionomer powder ; 1.25% chx - gic : prepared by adding 0.037 g of chlorhexidine to 2.96 g of glass ionomer powder ; 2.5% chx - gic : prepared by adding 0.075 g of chlorhexidine to 2.925 g of glass ionomer powder. triclosan - gic (t - gic) : similarly, 0.5, 1.25, and 2.5% concentrations of triclosan - incorporated gic were prepared. autoclavable plastic molds of standardized dimensions (4 mm diameter, 6 mm thickness) were used for the preparation of all restorative pellets. immediately after mixing, the material was placed into plastic molds by a plastic instrument and covered with acetate strips on both sides. this assembly was then placed into an incubator at 37 1c and 95 5% relative humidity for 1 h to simulate oral conditions. the pellets were then removed from the molds and ground on 500-grit silicon carbide paper for finishing. occlusal dentin samples were obtained from 35 primary human molars (five molars for each group). the buccal enamel was reduced to a flat surface using a diamond disk, and was polished thereafter. the dentin surface was conditioned using polyacrylic acid for 10 s followed by air - water spray for 10 s. then, the restorative materials from various groups were mixed and placed on the buccal surface of the tooth. a block of glass ionomer cement of dimension 4 3 2 mm was prepared. after that the specimens were stored in an incubator at 37 1c and 95 5% relative humidity for 24 h. thereafter, the division of samples was done accordingly [table 1 ]. for the compressive strength testing, restorative pellets were placed with the flat ends up between the plates of the instron universal testing machine (instron 1500hdx). a compressive load was applied at a crosshead speed of 1 mm / min until the restorative pellet fractured. for the diametral tensile strength testing, the restorative pellets were placed on the instron universal testing machine such that the diameter of the pellet coincided with the direction of the force. a crosshead speed of 1 mm / min was used and the force was applied until the pellets fractured. the specimens were placed in the lower assembly of the instron universal testing machine one by one. a sharp knife - like mandrel was attached to the upper assembly and was suspended downwards toward the glass ionomer cement block. the force with which the restoration block was dislodged was recorded and shear bond strength was calculated. the data was statistically analyzed using independent t - test and intercomparison among various groups was done using dunnett test and post hoc test. for the evaluation of compressive strength and diametral tensile strength restorative pellets of gic type ix, 0.5, 1.25, and 2.5% concentrations of chlorhexidine - incorporated gic and triclosan - incorporated gic were prepared. for the evaluation of shear bond strength the desired concentrations of gic powder, chlorhexidine salt, and triclosan powder were obtained using an analytical digital scale. chlorhexidine - gic (chx - gic) : three different proportions of chx - gic were prepared based on the concentration of chlorhexidine. 0.5% chx - gic : prepared by adding 0.015 g of chlorhexidine to 2.985 g of glass ionomer powder;1.25% chx - gic : prepared by adding 0.037 g of chlorhexidine to 2.96 g of glass ionomer powder;2.5% chx - gic : prepared by adding 0.075 g of chlorhexidine to 2.925 g of glass ionomer powder. 0.5% chx - gic : prepared by adding 0.015 g of chlorhexidine to 2.985 g of glass ionomer powder ; 1.25% chx - gic : prepared by adding 0.037 g of chlorhexidine to 2.96 g of glass ionomer powder ; 2.5% chx - gic : prepared by adding 0.075 g of chlorhexidine to 2.925 g of glass ionomer powder. triclosan - gic (t - gic) : similarly, 0.5, 1.25, and 2.5% concentrations of triclosan - incorporated gic were prepared. autoclavable plastic molds of standardized dimensions (4 mm diameter, 6 mm thickness) were used for the preparation of all restorative pellets. immediately after mixing, the material was placed into plastic molds by a plastic instrument and covered with acetate strips on both sides. this assembly was then placed into an incubator at 37 1c and 95 5% relative humidity for 1 h to simulate oral conditions. the pellets were then removed from the molds and ground on 500-grit silicon carbide paper for finishing. occlusal dentin samples were obtained from 35 primary human molars (five molars for each group). the buccal enamel was reduced to a flat surface using a diamond disk, and was polished thereafter. the dentin surface was conditioned using polyacrylic acid for 10 s followed by air - water spray for 10 s. then, the restorative materials from various groups were mixed and placed on the buccal surface of the tooth. a block of glass ionomer cement of dimension 4 3 2 mm was prepared. after that the specimens were stored in an incubator at 37 1c and 95 5% relative humidity for 24 h. thereafter, the division of samples was done accordingly [table 1 ]. for the compressive strength testing, restorative pellets were placed with the flat ends up between the plates of the instron universal testing machine (instron 1500hdx). a compressive load was applied at a crosshead speed of 1 mm / min until the restorative pellet fractured. for the diametral tensile strength testing, the restorative pellets were placed on the instron universal testing machine such that the diameter of the pellet coincided with the direction of the force. a crosshead speed of 1 mm / min was used and the force was applied until the pellets fractured. the specimens were placed in the lower assembly of the instron universal testing machine one by one. a sharp knife - like mandrel was attached to the upper assembly and was suspended downwards toward the glass ionomer cement block. the force with which the restoration block was dislodged was recorded and shear bond strength was calculated. the data was statistically analyzed using independent t - test and intercomparison among various groups was done using dunnett test and post hoc test. the mean values of compressive strength, diametral tensile strength, and shear bond strength of various groups are depicted in table 2. independent t - test revealed that gic type ix (group 1) showed the highest values followed by 0.5% t - gic (group 3a) and the least values were found in 0.5% chx - gic (group 2c). mean values of compressive strength, diametral tensile strength, and shear bond strength (mpa) of various groups table 3 depicts the intragroup comparison of compressive strength, diametral tensile strength, and shear bond strength of various concentrations of chx - gic. dunnett test showed that 0.5% chx - gic was significantly better (p 0.05) in compressive strength, diametral tensile strength, and shear bond strength existed between gic type ix (group 1), 0.5% chx - gic (group 2a) and 0.5% t - gic (group 3a). art has been practiced using hand instruments only, therefore, there is a possibility of insufficient caries removal. literature has shown that microorganisms have been found to be viable for at least a period of two years under the glass ionomer cement restoration. thus, therapeutic benefits may be gained by reinforcing glass ionomer cements with additional antibacterial agents. chlorhexidine diacetate is a more stable antibacterial material, not prone to decomposition, and can be easily blended with gic. triclosan at low concentrations acts as a bacteriostatic and at high concentrations as bactericidal. to evaluate which concentration of chlorhexidine and triclosan is best suited for our purpose various concentrations were used, i.e. 0.5%, 1.25%, and 2.5%. all the concentrations have been shown to provide adequate antibacterial property to gic type ix. in an in vitro study in 2008, trkn. used 0.5%, 1.25%, and 2.5% concentrations of chlorhexidine - containing gic to study the long - term antibacterial effects and physical properties. in another study by deepalakshmi., glass ionomer cements containing chlorhexidine and cetrimide at concentrations of 1% and 2% were used to evaluate their antibacterial and physical properties. the results of the current study demonstrated that the mean value of compressive strength, diametral tensile strength, and shear bond strength was found to be the highest in gic type ix. the above result may be attributed to the fact that gic type ix is characterized by having smaller glass particles and higher powder to liquid ratio. this is said to give the gic type ix higher strength, greater wear resistance, and increased flexural strength. this result was in accordance to a study done by ahluwalia. in 2012, in which the authors found that the diametral tensile strength of gic type ix (12.61 mpa) was higher than 1% chx - gic (12 mpa). when the various concentrations of t - gic and chx - gic were compared with each other, it was found that 0.5% t - gic and 0.5% chx - gic have the highest values of compressive strength, diametral tensile strength, and shear bond strength in their respective groups. the common finding in both the intragroup comparisons points out that the physical properties abruptly decrease in a concentration - dependent manner. in addition, the results manifest that 1.5% and 2.5% concentration of antibacterials incorporated into gic type ix might not be suitable for clinical use because increase in the concentration adversely affects the physical properties of the parent material. because of the vitrification of gic with chlorhexidine and triclosan at higher concentrations, many of these carboxylic (cooh) groups are prevented from participating in these coordination complexes. a possible reason for the decrease in physical properties at a concentration of more than 2% chlorhexidine can be attributed to the fact that cationic salts hamper the setting reaction of the polyacrylic acid glasses, thereby extending the setting time, because of interfered proton attack and leaching of ions from the glasses. the intercomparison between gic type ix (control), 0.5% chx - gic (group 2a), and 0.5% t - gic (group 3a) discernibly signified that 0.5% t - gic and 0.5% chx - gic exhibit physical properties comparable to gic type ix, which indicates that these maybe considered as viable options for use in pediatric dentistry along with physical properties within the higher acceptable range. it can be concluded that the compressive strength, diametral tensile strength, and shear bond strength values of 0.5% t - gic and 0.5% chx - gic were comparable to gic type ix. we recommend that further studies should be conducted to test various antibacterial glass ionomer cements in a randomized clinical trial. because a smaller sample size was used in this study, further studies with a larger sample size are required to authenticate the results. clinical impact of various other factors such as occlusal forces needs to be investigated in further studies. | aim : to comparatively evaluate the compressive strength, diametral tensile strength, and shear bond strength of glass ionomer cement type ix, chlorhexidine - incorporated glass ionomer cement, and triclosan - incorporated glass ionomer cement.materials and methods : in this study, glass ionomer cement type ix was used as a control. chlorhexidine diacetate, and triclosan were added to glass ionomer cement type ix powder, respectively, in order to obtain 0.5, 1.25, and 2.5% concentrations of the respective experimental groups. compressive strength, diametral tensile strength, and shear bond strength were evaluated after 24 h using instron universal testing machine. the results obtained were statistically analyzed using the independent t - test, dunnett test, and tukey test.results:there was no statistical difference in the compressive strength, diametral tensile strength, and shear bond strength of glass ionomer cement type ix (control), 0.5% triclosan - glass ionomer cement, and 0.5% chlorhexidine - glass ionomer cement.conclusion:the present study suggests that the compressive strength, diametral tensile strength, and shear bond strength of 0.5% triclosan - glass ionomer cement and 0.5% chlorhexidine - glass ionomer cement were similar to those of the glass ionomer cement type ix, discernibly signifying that these can be considered as viable options for use in pediatric dentistry with the additional value of antimicrobial property along with physical properties within the higher acceptable range. |
the mucosal folds in the body of the stomach are usually less than 1 cm thick. occasionally these folds, also referred to as rugae, may thicken and enlarge, a condition known as hypertrophic gastropathy. while mntrier 's disease is most commonly associated with hypertrophic gastric folds, a variety of other diseases have also been associated. these include zollinger - ellison syndrome, gastric lymphoma, diffuse gastric cancer, polyposis and eosinophilic gastroenteritis. rarely, infections with human cytomegalovirus (cmv) or helicobacter pylori (hp) may cause enlargement of the gastric folds [3, 4 ]. although awareness of these mimics of mntrier 's disease is crucial, it may have an important bearing on patient management and outcome. we report a patient with hypertrophic gastropathy caused by hp who showed dramatic response to therapy. a 52-year - old man undergoing screening for gastric cancer was found to have grossly enlarged gastric mucosal folds (fig. there was no history of upper abdominal pain, vomiting, anorexia or significant weight loss. laboratory testing revealed hemoglobin 15.1 g / dl, total protein 6 g / dl, serum albumin 3.9 g / dl, total leucocyte count 9,320/l, eosinophils 5.8%, serum gastrin 87 iu / ml and lactate dehydrogenase 155 u / l. upper gastrointestinal endoscopy showed marked thickening of the gastric folds in the gastric proximal body (fig. contrast - enhanced computed tomography (cect) was performed mainly to investigate if other intra - abdominal lesions such as neuroendocrine tumor, lymphadenopathy or metastatic disease might be responsible for the thickened gastric folds. except for the thickened upper body of the stomach, endoscopic ultrasonography (eus) was performed to determine the origin of the thickened gastric mucosal folds and whether the layers of the gastric wall were preserved. eus showed that the thickening in the proximal body was confined to the mucosal layer and that the muscularis propria and serosal layers were normal. whole - body gallium scan for the presence of neoplastic lesions was normal. since gastric lymphoma, especially mucosa - associated lymphoid tissue lymphoma, was considered in the differential diagnosis, the interleukin 2 receptor serum level was measured and found to be mildly elevated at 783 u / ml (normal < 520 u / ml). significant neutrophilic infiltration was seen in the mucosa, and there was no evidence of malignancy or cmv infection (fig. the ratio of gastric crypts to glands was not determined because that assessment required a deeper biopsy. the absence of symptoms and the finding of normal serum albumin indicated that mntrier 's disease was unlikely, and the diagnosis of hypertrophic gastropathy associated with hp infection was considered. the patient received hp eradication therapy consisting of 1 week of treatment using lansoprazole, amoxicillin and clarithromycin. he underwent a barium examination 5 months later, which revealed normal gastric mucosal folds (fig. gastroscopy showed that the thickened gastric mucosal folds previously seen in the proximal body were normal (fig. a repeat mucosal biopsy was negative for hp organisms, and the neutrophilic infiltration seen on the initial biopsy was minimal (fig. infection with hp is common, with about half of the world 's population harboring this bacterium in their stomach. since hp infection is associated with important clinical diseases such as peptic ulcer disease, gastric lymphoma and gastric carcinoma, therapy is warranted for its eradication. other conditions sometimes linked with hp infection include idiopathic thrombocytopenic purpura, iron deficiency anemia, scleroderma, idiopathic urticaria and migraine. eradication therapy is generally recommended only for idiopathic thrombocytopenic purpura and unexplained iron deficiency anemia because evidence for association with hp is weak for the other conditions. recently, hypertrophic gastropathy associated with hp has been proposed as an indication for treatment. the dramatic improvement in gastric fold morphology and histology that was seen after eradication therapy in our patient provides additional support that patients with hp - associated hypertrophic gastropathy should be treated. these patients may be at increased risk of gastric cancer, which underlines the importance of prompt treatment. since hypertrophic gastric mucosal folds can be associated with many conditions, a thorough clinical and diagnostic evaluation is required to exclude other causes. patients with mntrier 's disease are usually symptomatic and present with epigastric pain, vomiting, weight loss and low serum albumin level as a result of loss of proteins from the stomach. patients with lymphoma and carcinoma commonly have anorexia and weight loss. in patients with zollinger - ellison syndrome, the serum gastrin levels may be very high, multiple ulcers may be seen on gastroduodenoscopy, and abdominal imaging may show a tumor. hence, serum albumin level, total and differential leukocyte count (eosinophilia in hypereosinophilic disorders), serum gastrin level, cmv serology and abdominal imaging (cect, eus) play an important role in the evaluation of patients with hypertrophic gastropathy. our patient was asymptomatic, had a normal serum albumin and was tumor - free on imaging, which indicated that the conditions considered in the differential diagnosis were less likely. the definitive diagnosis is derived from histological examination of the gastric mucosa, and therefore biopsy, preferably using a sufficiently large forceps to obtain a deep sample, is crucial. mntrier 's disease is characterized by foveolar hyperplasia and scanty parietal cells, lymphoma and carcinomas manifest infiltration by neoplastic cells, inclusion bodies may be seen with cmv infection, and eosinophilic gastroenteritis shows infiltration with eosinophils. evidence of hp infection by itself may not be sufficient to establish a cause - effect relationship with hypertrophic gastric folds. the significant neutrophilic infiltration of the mucosa, exclusion of other causes and the resolution of thickened gastric mucosal folds and inflammation after bacterial eradication therapy in our patient strongly suggested that hp was the cause of his hypertrophic gastropathy. there are also a few case reports describing the association of hp with lymphocytic and granulomatous gastritis, indicating that hp can cause non - neoplastic enlarged gastric folds [11, 12 ]. bacterial components may induce a distinct cytokine profile that may promote cellular proliferation and enlargement of the gastric folds. one report suggested that increased production of interleukin-1 beta and hepatocyte growth factor during hp infection may result in gastric fold thickening. the mutagenicity of gastric juice is increased in patients with hp infection and enlarged gastric folds, which may explain the increased risk of cancer in these patients. in conclusion, hp infection should be considered in the differential diagnosis of patients with hypertrophic gastropathy because the infection is easy to treat and the outcome is gratifying. | infection with helicobacter pylori (hp) is common in many parts of the world. while most patients are asymptomatic, it causes peptic ulcer disease and malignancy in some of them. other rare conditions have occasionally been reported in association with this infection. we report a case of hypertrophic gastropathy caused by hp in a 52-year - old asymptomatic patient. he was found to have marked enlargement of the gastric mucosal folds on radiological imaging and endoscopy. a gastric mucosal biopsy showed hp colonization associated with neutrophilic inflammation. after exclusion of neoplasia, other infections and infiltrative disorders, hp was thought to be the cause of the gastric fold hypertrophy. the patient responded well to hp eradication therapy, with normalization of the gastric mucosal folds. hp infection should be considered in the differential diagnosis of hypertrophic gastropathy and treated accordingly. |
oligoribonucleotides were synthesized on an mm12 synthesizer (bioautomation corp., irving, tx) on a 50 nmol scale with 2otbdmsphosphoramidites (thermo fisher), using a 1000 unylinker support (chemgenes, wilmington, ma). coupling time was 290 s for regular 2otbdms phosphoramidites, 2180 s for the dnb phosphoramidite 5, and 3240 s for 2opropargyl rc phosphoramidite. isolated 5dnbprotected oligonucleotides were dried in a speedvac and redissolved in 200 l rnasefree water prior to irradiation for 10 min at 365 nm in a vilber lourmat biolink blx crosslinker (58 w). final rphplc purification yielded 5phosphate oligoribonucleotides ready for the introduction of functional groups by click chemistry (e.g., psoralen, cy5), and for enzymatic ligation experiments with t4 rna ligase 2 (m0239, new england biolabs) (see the supporting information for details). as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | abstractlong structured rnas are useful biochemical and biological tools. they are usually prepared enzymatically, but this precludes their sitespecific modification with functional groups for chemical biology studies. one solution is to perform solidphase synthesis of multiple rnas loaded with 5terminal phosphate groups, so that rnas can be concatenated using template ligation reactions. however, there are currently no readily available reagents suitable for the incorporation of the phosphate group into long rnas by solidphase synthesis. here we describe an easytoprepare phosphoramidite reagent suitable for the chemical introduction of 5terminal phosphate groups into long rnas. the phosphate is protected by a dinitrobenzhydryl group that serves as an essential lipophilic group for the separation of oligonucleotide byproducts. the phosphate is unmasked quantitatively by brief uv irradiation. we demonstrate the value of this reagent in the preparation of a library of long structured rnas that are sitespecifically modified with functional groups. |
dyke - davidoff - masson syndrome (ddms) was originally characterized by its radiologic features, which include cerebral hemiatrophy and ipsilateral skull hypertrophy with hyperpneumatization of the paranasal sinuses and mastoid cells.1,2 although seizure is one of the core symptoms along with hemiparesis and mental retardation, few papers described long - term course. we present one typical and one suspicious case within one family and summarize published cases concerning seizures. a 26-year - old man was referred for consultation about his recurrent seizures and hemiparesis. he could express meaningful words at the age of 3, and showed a hemiplegic gait since he first started walking. the patient had experienced seizures since the age of 4. from the age of 4 to the time of referral, his seizures were yearly events regarding generalized tonic - clonic (gtc) seizure and weekly regarding aura - only. neither febrile convulsion, nor meningoencephalitis occurred. he experienced focal motor seizures in the left arm and leg, independently or prior to a gtc seizure. in some instances, he noted an auditory aura with or without being followed by a brief alteration of consciousness or secondary generalization. his brain magnetic resonance image (mri) 1a, b, and c. repetitive electroencephalographies (eegs) demonstrated continuous irregular theta slow activities and lower amplitude over the right hemisphere (fig. also, he had not experienced febrile seizure or meningoencephalitis. from the age of 2 to the last year before a first visit, he experienced seizures with a frequency of approximately one per year. its semiologies varied as follows : generalized seizures, often preceded by visual or cephalic auras, or an auditory aura such as tinnitus. the generalized seizures and auditory auras had recently frequently recurred at one or two per month. the eeg showed continuous theta or delta activity in the right hemisphere, which was accompanied by occipital sharp waves (fig. a 26-year - old man was referred for consultation about his recurrent seizures and hemiparesis. he could express meaningful words at the age of 3, and showed a hemiplegic gait since he first started walking. the patient had experienced seizures since the age of 4. from the age of 4 to the time of referral, his seizures were yearly events regarding generalized tonic - clonic (gtc) seizure and weekly regarding aura - only. neither febrile convulsion, nor meningoencephalitis occurred. he experienced focal motor seizures in the left arm and leg, independently or prior to a gtc seizure. in some instances, he noted an auditory aura with or without being followed by a brief alteration of consciousness or secondary generalization. his brain magnetic resonance image (mri) 1a, b, and c. repetitive electroencephalographies (eegs) demonstrated continuous irregular theta slow activities and lower amplitude over the right hemisphere (fig. also, he had not experienced febrile seizure or meningoencephalitis. from the age of 2 to the last year before a first visit, he experienced seizures with a frequency of approximately one per year. its semiologies varied as follows : generalized seizures, often preceded by visual or cephalic auras, or an auditory aura such as tinnitus. the generalized seizures and auditory auras had recently frequently recurred at one or two per month. the eeg showed continuous theta or delta activity in the right hemisphere, which was accompanied by occipital sharp waves (fig. the case 1 displayed the characteristic radiological and clinical findings that are consistent with a diagnosis of ddms. based on the long - term history, the stationary pattern in this patient regarding the seizure occurrence, his cognition and the hemiparesis, made rasmussen encephalitis unlikely, which has a progressive nature of seizure and neurologic deficit, despite of the similarity of the imaging findings. the patients with sturge - weber syndrome usually have typical skin lesions which is also incompatible with this patient. however, diagnosing the second case as ddms may be debatable, because case 2 did not show the definite hemispheric atrophy, skull thickening and mental retardation consistent with ddms, like his brother s. furthermore, asymmetries of the ventricles have been known to be a normal variant in some population, particularly the occipital horns.3 nonetheless, the result of eeg indicating continuous irregular slow activity over one hemisphere, hippocampal atrophy and small caudate body in the same side with that of small ventricle might support a possibility of abnormal or dysfunctional hemisphere rather than normal variant. notably, the first case had just yearly seizures even though the imaging is more typical and severe than the second case, who had drug - resistant seizures and was considered as a potential candidate for surgery. taken previous cases (table 1) and ours together, the severity of seizure and radiological findings seem to be unrelated with each other. of interest, the both patients were brothers and they had another relative suffering from epilepsy (fig. conventionally, the congenital causes of ddms include congenital malformation, intrauterine vascular occlusion and infection,1,413 but genetic cause, neither the case of ddms in a epilepsy family, has not been previously reported. for the semiological aspect, case 1 showed several different types of seizure, which were focal motor or auditory with / without secondary generalization. this common semiological feature was consistent with that of a previous report that documented familial temporal lobe epilepsy (tle) with an auditory aura.14 contrary to their similar semiology, discordant data showing occipital epileptiform discharges on eeg and abnormal medial temporal area on mri, do not support this assumption. another possibility is that hs, as a common image finding between the two brothers, might be the sole familial proportion of genetic inheritance. in addition, the different degree of cerebral asymmetry could be a reflection of different seizure - induced neo - cortical change between two brothers. however, this seems unlikely because the seizure frequencies were similar in the two brothers and the onset of hemiparesis in case 1 preceded the seizure onset. a previous study of patients with cerebral hemiatrophy showed that the hs in the study subjects was strongly related with a history of febrile convulsion.15 since there was no history of febrile convulsion in the brothers of our study, it does not appear that febrile convulsion intrudes to the explanation of our familial epilepsy case. there also might have been the coexistence of ddms and common non - genetic tle, and the unrevealed environmental factors occurring in this family might have affected the development of epilepsy, as is often the case of families with various diseases. in order to confirm the possible genetic component in ddms, a detail family history should be sought in a patient with the features of ddms and further reports are mandatory. | dyke - davidoff - masson syndrome (ddms) has cerebral hemiatrophy and compensatory ipsilateral skull thickening, and is manifested by recurrent seizures and hemiparesis. we present one case with typical ddms, who had a brother suffering from epilepsy with mild imaging abnormality relevant to ddms and similar seizure semiology. a 26-year - old man had a history of developmental delay, mental retardation, hemiparesis and recurrent seizures. his brother, 23-year - old man had also experienced recurrent seizures, but he had no neurological deficits. older brother experienced focal motor seizures with / without secondary generalization. sometimes, he noted an auditory aura. mri demonstrated the hemispheric atrophy with the adjacent bony hypertrophy. the seizures of younger brother were mainly of the auditory type and the mri showed mild hemispheric atrophy with hippocampal sclerosis without any bony change. our sibling cases might have a familial predisposition and support the idea that clinical courses and radiological findings of ddms are varied even within one family. |
all materials for cell culture including fetal bovine serum (fbs) and opti - mem were obtained from gibco brl (grand island, ny, usa). other media supplements, such as epidermal growth factor (egf), ascorbic acid, rpmi 1640 vitamin mixture, insect lipid, and chondroitin sulfate, were purchased from sigma chemical (st. louis, mo, usa). nerve growth factor (ngf) was obtained from r&d systems (minneapolis, mn, usa). human eyes were obtained in accordance with the tenets of the declaration of helsinki and proper informed consent. human corneal endothelial cells were isolated by trypsin digestion of the posterior portions of excised cornea. after enzymatic digestion at 37 for 10 to 15 min, the endothelium was removed by gentle scraping, and seeded onto a tissue culture dish in opti - mem supplemented with 5 ng / ml of egf, 20 ng / ml of ngf, 20 g / ml of ascorbic acid, 0.005% insect lipid, 200 mg / l of cacl2, 0.02% chondroitin sulfate, 1% rpmi 1640 vitamin mixture, and 8% fbs. pa317 amphotropic packaging cell lines stably transfected with plxsn 16 e6-e7 (hpv 16 e6/e7) were purchased from american type culture collection (atcc, manassas, va, usa). cells were grown to 70 - 80% confluence, and supernatants were collected for 24 hr and stored in aliquots at -80. the primary endothelial cells were infected with 1 ml of virus stock in 3 ml of medium containing polybrene (sigma, mo, usa) at 4 l / ml for 24 hr. the virus was then removed and the medium replaced by opti - mem supplemented with 8% fbs and other supplements as described above. selection media with 200 g / ml g418 (duchefa, amsterdam, netherlands) was added after 72 hr. one of the established cell lines was seeded onto 100 mm - diameter culture dishes at a density of 510 cells / dish. every two days for 14 days, cells were detached by 0.05% trypsin/0.5 mm ethylenediaminetetraacetic acid (edta), and counted in a hemocytometer in triplicate. for doubling time determination, the formula tc=0.3t / log (a / a0) was used where tc = doubling time, t = initial time, a = the number of cells at time t of proliferation, and a0=the number of cells at an initial time point. human amniotic membrane from donated human placenta tested by hepatitis or hiv infection was pretreated with 0.025% trypsin - edta to remove amnion cells, followed by lyophilization and sterilization using eo gas. lyophilized amniotic membranes (lam) were installed on a teflon ring, and 110 cells / support ihcen were seeded onto the amnion side of a denuded lam. cells were maintained using opti - mem for six days by changing the growth media every two days. corneal endothelial origin of the primary and transformed cells was confirmed by reverse transcription - polymerase chain reaction (rt - pcr). primary cells and established cell lines were cultured as above, and total rna was extracted with trizol reagent (gibco brl, ny, usa). the first strand of cdna was synthesized from two g of the total rna in a 20 l reaction mixture containing superscript ii rnase, h reverse transcriptase, and oligo (dt)18 primer. target genes (table 2) were then amplified, pcr products were run on a preparative 2% agarose gel, and the bands were photographed. an avidin biotin complex technique was selected for staining sectioned specimens (2 - 4 m). paraffin sections were deparaffinized in xylene, rehydrated through decreasing ethanol concentrations and quenched for endogenous peroxidase. cryostat sections were placed on gelatinized slides, fixed in cold acetone and then rinsed in tris - buffered saline. nonspecific background was eliminated by incubating tissue sections with non - immuno serum (histostatin - plus kits, reagent a ; zymed laboratories, ca, usa). sections were then incubated with monoclonal mouse anti - human collagen iv (santa cruze biotechnology inc. ca, usa) and anti - human na / k atpase antibody (santa cruze biotechnology inc. ca, usa) overnight at 4, followed by extensive washing in 0.05 m tris - buffered saline (ph 7.6) before the addition of a biotinylated secondary antibody (reagent b). sections were washed again and incubated for one hour with peroxidase - conjugated streptavidin (reagent c). the presence of peroxidase was revealed by adding a substrate - chromogen (3-amino-9-ethycarbazole) solution (reagent d) for immunohistochemistry, and an fitc conjugated rabbit anti - mouse igg for immunofluorescence. all materials for cell culture including fetal bovine serum (fbs) and opti - mem were obtained from gibco brl (grand island, ny, usa). other media supplements, such as epidermal growth factor (egf), ascorbic acid, rpmi 1640 vitamin mixture, insect lipid, and chondroitin sulfate, were purchased from sigma chemical (st. louis, mo, usa). nerve growth factor (ngf) was obtained from r&d systems (minneapolis, mn, usa). human eyes were obtained in accordance with the tenets of the declaration of helsinki and proper informed consent. human corneal endothelial cells were isolated by trypsin digestion of the posterior portions of excised cornea. after enzymatic digestion at 37 for 10 to 15 min, the endothelium was removed by gentle scraping, and seeded onto a tissue culture dish in opti - mem supplemented with 5 ng / ml of egf, 20 ng / ml of ngf, 20 g / ml of ascorbic acid, 0.005% insect lipid, 200 mg / l of cacl2, 0.02% chondroitin sulfate, 1% rpmi 1640 vitamin mixture, and 8% fbs. pa317 amphotropic packaging cell lines stably transfected with plxsn 16 e6-e7 (hpv 16 e6/e7) were purchased from american type culture collection (atcc, manassas, va, usa). cells were grown to 70 - 80% confluence, and supernatants were collected for 24 hr and stored in aliquots at -80. the primary endothelial cells were infected with 1 ml of virus stock in 3 ml of medium containing polybrene (sigma, mo, usa) at 4 l / ml for 24 hr. the virus was then removed and the medium replaced by opti - mem supplemented with 8% fbs and other supplements as described above. selection media with 200 g / ml g418 (duchefa, amsterdam, netherlands) was added after 72 hr. one of the established cell lines was seeded onto 100 mm - diameter culture dishes at a density of 510 cells / dish. every two days for 14 days, cells were detached by 0.05% trypsin/0.5 mm ethylenediaminetetraacetic acid (edta), and counted in a hemocytometer in triplicate. for doubling time determination, the formula tc=0.3t / log (a / a0) was used where tc = doubling time, t = initial time, a = the number of cells at time t of proliferation, and a0=the number of cells at an initial time point. human amniotic membrane from donated human placenta tested by hepatitis or hiv infection was pretreated with 0.025% trypsin - edta to remove amnion cells, followed by lyophilization and sterilization using eo gas. lyophilized amniotic membranes (lam) were installed on a teflon ring, and 110 cells / support ihcen were seeded onto the amnion side of a denuded lam. cells were maintained using opti - mem for six days by changing the growth media every two days. corneal endothelial origin of the primary and transformed cells was confirmed by reverse transcription - polymerase chain reaction (rt - pcr). primary cells and established cell lines were cultured as above, and total rna was extracted with trizol reagent (gibco brl, ny, usa). the first strand of cdna was synthesized from two g of the total rna in a 20 l reaction mixture containing superscript ii rnase, h reverse transcriptase, and oligo (dt)18 primer. target genes (table 2) were then amplified, pcr products were run on a preparative 2% agarose gel, and the bands were photographed. an avidin biotin complex technique was selected for staining sectioned specimens (2 - 4 m). paraffin sections were deparaffinized in xylene, rehydrated through decreasing ethanol concentrations and quenched for endogenous peroxidase. cryostat sections were placed on gelatinized slides, fixed in cold acetone and then rinsed in tris - buffered saline. nonspecific background was eliminated by incubating tissue sections with non - immuno serum (histostatin - plus kits, reagent a ; zymed laboratories, ca, usa). sections were then incubated with monoclonal mouse anti - human collagen iv (santa cruze biotechnology inc. ca, usa) and anti - human na / k atpase antibody (santa cruze biotechnology inc. ca, usa) overnight at 4, followed by extensive washing in 0.05 m tris - buffered saline (ph 7.6) before the addition of a biotinylated secondary antibody (reagent b). sections were washed again and incubated for one hour with peroxidase - conjugated streptavidin (reagent c). the presence of peroxidase was revealed by adding a substrate - chromogen (3-amino-9-ethycarbazole) solution (reagent d) for immunohistochemistry, and an fitc conjugated rabbit anti - mouse igg for immunofluorescence. the morphological characteristics of isolated primary human corneal endothelial cells (phcen) and ihcen were observed using an inverted microspcope. ihcen were polygonal to slightly elongated in shape, similar to phcen, but were siginificantly different from dendritic corneal fibroblasts and small globular - shaped epithelial cells (fig. stable expression of e6/e7 mrna was observed in ihcen, but not in phcen by rt - pcr analysis. messenger rna of the corneal epithelial cell marker, keratin 12, was not expressed in phcen and ihcen (fig. 2). proliferative characteristics of ihcen were elucidated by cell counting every two days for 14 days. the cell growth curve of ihcen showed typical s - curves : minimal growth for the initial seven days, geometrical growth until day 14, and decreased cell numbers after 14 days. 3). ihcen showed a more extended life span (over passage 30), and more rapid proliferation than phcen (data not shown). transformed traits, except for the transducing of hpv 16 e6/e7 oncogenes, were examined by rt - pcr of several channel proteins including voltage - dependent annion channel 3 (cdac3), sodium bicarbonate cotransporter member 4 (slc4a4), chloride channel protein 3 (clcn3), fibroblast growth factor (fgf)-1, type iv collagen (col iv), and na / k atpase in phcen and ihcen. mrnas for all of the mentioned proteins were expressed in ihcen, with similar expression patterns observed in phcen. in order to elucidate the efficiency of lam for the human corneal endothelial niche, messenger rna expressions of several channel proteins and na / k atpase were observed in established ihcen cultivated on lam. moreover, their expressions were stronger than in cells grown on a plastic culture dish (fig. 5). immunohitochemical staining of col iv and immunofluorescence of na / k atpase in ihcen cultivated on lam also showed positive expressions results similar to rt - pcr (fig. the morphological characteristics of isolated primary human corneal endothelial cells (phcen) and ihcen were observed using an inverted microspcope. ihcen were polygonal to slightly elongated in shape, similar to phcen, but were siginificantly different from dendritic corneal fibroblasts and small globular - shaped epithelial cells (fig. stable expression of e6/e7 mrna was observed in ihcen, but not in phcen by rt - pcr analysis. messenger rna of the corneal epithelial cell marker, keratin 12, was not expressed in phcen and ihcen (fig. 2). proliferative characteristics of ihcen were elucidated by cell counting every two days for 14 days. the cell growth curve of ihcen showed typical s - curves : minimal growth for the initial seven days, geometrical growth until day 14, and decreased cell numbers after 14 days. 3). ihcen showed a more extended life span (over passage 30), and more rapid proliferation than phcen (data not shown). transformed traits, except for the transducing of hpv 16 e6/e7 oncogenes, were examined by rt - pcr of several channel proteins including voltage - dependent annion channel 3 (cdac3), sodium bicarbonate cotransporter member 4 (slc4a4), chloride channel protein 3 (clcn3), fibroblast growth factor (fgf)-1, type iv collagen (col iv), and na / k atpase in phcen and ihcen. mrnas for all of the mentioned proteins were expressed in ihcen, with similar expression patterns observed in phcen. in order to elucidate the efficiency of lam for the human corneal endothelial niche, ihcen were cultured on lam and harvested for characterization. messenger rna expressions of several channel proteins and na / k atpase were observed in established ihcen cultivated on lam. moreover, their expressions were stronger than in cells grown on a plastic culture dish (fig. 5). immunohitochemical staining of col iv and immunofluorescence of na / k atpase in ihcen cultivated on lam also showed positive expressions results similar to rt - pcr (fig. although the condition of the corneal endothelium is essential for corneal transparency, it is difficult to treat endothelial - damging corneal diseases, since the corneal cells have a very restricted regenerative capacity. in order to overcome these limitations, corneal endothelial cell transplantation had been attempted by several groups, but insurance of a sufficient number of endothelial cells and the development of appropriate endothelial substrates are still not guaranteed. therefore, this experiment was conducted to establish immortalized human corneal endothelial cells (ihcen) by stable introduction of hpv 16 e6/e7, and to investigate the biological characteristics of an established corneal endothelial cell line cultivated on lyophilized human amniotic membrane, an ideal coreneal endothelial substrate. ihcen stably transfected by pa317 amphotropic packaging cell lines with plxsn 16 e6/e7 showed stable expression of e6/e7 mrna. there was no corneal epithelial cell contamination observed by rt - pcr analysis of keratin 12, a corneal epithelial cell marker (fig. 2). ihcen showed morphological similarity with phcen, a longer life span (over passage 30), and more rapid proliferation than phcen (data not shown). 3), was faster than that of a previously established rabbit corneal endothelial cell line in our laboratory (51.307.3 hrs).26 several immortalization techniques on human corneal endothelial cells by transfection of various oncogenes including sv40 large t - antigen or hpv 16 e6/e7 had been previously reported,27,28 but there were no reports elucidating the typical cell growth properties such as growth curve or doubling time. introduction of e6/e7 oncogenes facilitates the degradation of p53 and progression into the s phase synergistically.29 - 32 moreover, their stability was also confirmed by observing which gene was non - carcinogenic when injected into nude mice.33 therefore, we were able to successfully obtain abundant populations of highly proliferative and stably immortalized human corneal endothelial cells. furthermore, it was also identified that established ihcen maintained normal corneal endothelial functions similar to phcen by confirming vdac3, clcn3, clc4a4, and na / k atpase mrna expressions (fig. corneal transparency is maintained by uniform structure, avascularity, and hydration of the stroma. the mechanism that maintains corneal hydration has an active component, hco3-, which is transported from the stroma to the aqueous humor. a number of different hypotheses have been proposed to describe the molecular mechanisms that drive the net hco3 flux. common to these models is the recognition that the net hco3 flux is coupled to and energized by a basolateral na / k atpase in the corneal endothelium.34 the tendency to swell is due to the presence in the stromal matrix of non - diffusible, negatively charged molecules such as glycosaminoglycans. ion transport across the endothelium is thought to involve the active transport of anions from the stroma towards the aqueous humor, followed by the mainly passive diffusion of cations. the osmotic effect of this transendothelial ion transport counters the excess osmotic potential of the stroma.34 at hydration levels outside the normal range, the cornea loses its transparency. thus the positive expressions of several channel proteins including vdac3 and na / k atpase mrna in ihcen means that stably established ihcen did not lose their normal endothelial functions, and can maintain them during ex vivo expansion. essential factors for normal endothelial functions such as channel proteins or na / k atpase were positively expressed in cultivation not only on plastic culture dishes, but also on lam. furthermore, the expression patterns on lam were stronger than that on plastic culture dishes. these results suggest that human amniotic membrane can act as the ideal endothelial niche for ihcen and phcen. the amniotic membrane has a specific advantage that makes it a good substrate for endothelial cell growth. it is a biomaterial different from biochemical substrates such as gelatin membrane or coated hydrolens, it increases cell adherence by abundant extracellular matrix proteins including integrin and lamin, and is easily transplanted since its physical properties are similar to descement 's membrane. furthermore, our results demonstrate that amniotic membrane acts as a good substrate for the maintenance of corneal endothelial functions. this study indicates that a sufficient immortalized cell line having characteristics similar to phcen is useful for in vitro studies of human corneal endothelial functions. furthermore, it is expected that the culture of corneal endothelial cells on an amniotic membrane, an ideal corneal endothelial niche, is useful for the basic investigation of not only drug response or toxicological evaluation, but also for corneal endothelial cell transplantation or the development of reconstructed bioartificial cornea. | purposeto establish the immortalized human corneal endothelial cell line (ihcen) by transducing human papilloma virus (hpv) 16 e6/e7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (lam).methodsprimary human corneal endothelial cells (phcen) were infected using a retroviral vector with hpv 16 e6/e7, and transformed cells were clonally selected by g418. growth properties and characteristics of ihcen were compared with phcen by cell counting and rt - pcr of vdac3, slc4a4, clcn3, fgf-1, col iv, and na+/k+ atpase. ihcen were cultured on lam. messenger rna expressions of vdac3, clcn3, and na+/k+ atpase, and protein expressions of na+/k+ atpase and col iv in ihcen cultivated on lam were investigated by rt - pcr, immunofluorescence, and immunohistochemical staining, respectively.resultssuccessful immortalization was confirmed by stable expression of hpv 16 e6/e7 mrna by rt - pcr, and ihcen exhibited typical corneal endothelial morphology. doubling time of ihcen was 30.1510.96 hrs. both ihcen and phcen expressed vdac3, clcn3, slc4a4, fgf-1, col iv, and na+/k+ atpase. ihcen cultivated on lam showed stronger expression of vdac3, clcn4, and na+/k+ atpase mrna than on plastic culture dish. immunohistochemical staining and immunofluorescence revealed the positive expression of na+/k+ atpase and col iv.conclusionsihcen were successfully established, and lam is a good substrate for the culture of human corneal endothelial cells. |
the diagnosis of uti is very often missed in young children due to minimal and nonspecific symptoms. the developing renal cortex in young children is vulnerable to renal scarring resulting in hypertension and chronic renal failure. these morbidities in adults often have their origin in childhood. a clinically suspected case of uti this helps in guiding a clinician about the appropriate radio / nuclear imaging evaluation, choice of antimicrobial agent, duration of treatment and need of chemoprophylaxis. etiological agents of uti are variable and usually depend on time, geographical location and age of patients. however, escherichia coli, proteus mirabilis, enterobacter agglomerans, citrobacter frreundii and klebsiella pneumonia account for over 70% of cases. the exact information on etiology and resistance pattern of community - acquired pediatric utis in a region is usually not available, and if available it is outdated as antimicrobial sensitivity patterns are bound to change over a period of time. moreover, the data would also help authorities to formulate antibiotic prescription policies, at least for a region. a prospective study was performed on 800 children with suspected uti attending pediatric outdoor patient clinic, admitted to pediatric ward, paediatric intensive care unit (picu) and neonatal intensive care unit (nicu) of a tertiary care centre (1 day admission) from january 2012 to july 2014. the study was conducted in accordance with the guidelines of the international conference on harmonization / world health organization good clinical practice standards and the principles of the declaration of helsinki. an ethical review board approved the study protocol for this site, and the patients granted written informed consent before any study procedures. all children up to 18 years of age with urinary symptoms alone (frequency, dysuria, suprapubic pain), fever with urinary symptoms, fever without urinary symptoms, pain in abdomen with no previous history of uti were included. neonates with features of sepsis (i.e., poor feeding, jaundice or altered sensorium) were also included. the following cases were excluded : previous history of urinary infection (documented previous urinary culture sensitivity report), known urinary malformations (according to prenatal ultrasound and previous medical records), chronic illness, or current prophylactic treatment / pretreated with antibiotics (within 4 weeks of presentation) [figure 1 ]. flowchart showing patients enrolment in study for all suspected cases of uti urine microscopy, gram staining and culture were done for the patients admitted to the pediatric ward, picu or nicu on 1 day and on the same day on opd basis. urine sample (10 ml) was collected by suprapubic aspiration in neonates, mid - stream clean - catch and at the time of first insertion / in and out sampling in the rest of children. the processing of urinary samples consisted of urine microscopy, gram staining, and culture. wet mount microscopy was done on a well - mixed uncentrifuged urine sample to detect white blood cells (wbcs), red blood cells, yeast and epithelial cells. the presence of at least 1 organism per oil immersion field in uncentrifuged urine corresponds to a colony of 10 cfu / ml. urine culture was done on cysteine lactose electrolyte deficient agar (cled media [hi - media, mumbai, india ] [semi - quantitative method ]) and colony count done after overnight incubation at 37c. numbers of colonies obtained were multiplied by 1000 to obtain the colony forming units (cfu)/ml. antibiotic sensitivity was performed using kirby - baurer disk diffusion method following the clinical laboratory standards institute guidelines. e. coli atcc 25922, staphylococcus aureus atcc 29213, pseudomonas aeruginosa atcc 27853, e. faecalis atcc 29212 strains were tested for cefotaxime (30 mcg), cefpirome (30 mcg), amikacin (30 mcg), cotrimoxazole (25 mcg), norfloxacin (10 mcg), nitrofurantoin (300 mcg), sparfloxacin (5 mcg), nalidixic acid (30 mcg) and novobiocin (30 mcg). diagnostic threshold for significant bacteriuria was considered as 10 cfu / ml for clean - catch voiding in girls (repeat testing if 10,000100,000 cfu / ml), 10 cfu for clean - catch voiding in boys and catheter sampling (repeat testing if 100010,000 cfu / ml), any number of colonies for gram - negative bacilli and > 10 cfu / ml for gram - positive cocci in suprapubic aspiration. pyuria was defined as 5 wbcs / hpf of unspun urine, which corresponds to a colony count of > 10 organisms / ml in fresh uncentrifuged urine. data were analyzed separately for four age groups : infants (01 year), toddlers (> 15 year), preteens (> 512 year) and teens (> 1218 year). data management and statistical analysis were performed using spss software version 17.0 (spss inc., a total of 800 children with suspected uti were evaluated, out of which 720 were enrolled in this study. the most common age group presented with suspected uti was preteens 45.5% (328/720), followed by teens 32.2% (232/720), infants 15% (108/720) and toddlers 7.2% (52/720), respectively. the presenting symptoms were urinary symptoms alone 29.2% (210/720), fever without urinary symptoms 23.1% (166/720), fever with urinary symptoms 18.7% (134/720), pain in abdomen 23.3% (168/720), and features of sepsis 5.8% (42/720) [figure 2a ]. (a) distribution of various presenting symptoms in cases recruited, (b) distribution of various presenting symptoms in culture positive cases, (c) sex wise distribution of culture proven cases presented with fever and urinary symptoms, (d) sex wise distribution of culture proven cases presented with urinary symptoms alone, (e) sex wise distribution of culture proven cases presented with fever without urinary symptoms, (f) sex wise distribution of culture proven cases presented with pain in abdomen a total of 192 cases (26.7%) were culture positive. the most common age group was preteens 52.1% (100/192), followed by teens 27.1% (52/192), infants 8.33% (16/192) and toddlers 12.5% (24/192), respectively. fever with urinary symptoms was equally seen in males and females (34 in each) [figure 2c ]. other symptom categories were urinary symptoms alone (n = 64 ; m:24, f:40), fever without urinary symptoms (n = 36 ; m:18, f:18) and pain abdomen (n = 20 ; m:8, f:12) [figure 2d f ]. age and gender wise distribution and frequency of uropathogens isolated from community - acquired infection a total of 312 cases showed pyuria and culture negativity, 180 cases showed pyuria (on direct microscopy) and culture positivity, 12 cases had positive cultures with no pyuria and 23 cases of candiduria. a significant difference of direct microscopy and culture positivity is gained on chi - square test with p 0.001. out of 192 culture positive cases, 16 (8.3%) had an infection with 2 types of bacteria whereas 176 (91.7%) had an infection with single organism [figure 3a ]. of the 192 cases, uti was more frequently found in females 104 (54.2%) as compared to males 88 (45.8%) [figure 3b ]. out of total cases, we isolated a sum of 208 organisms. (a) percentage distribution of number of organisms in culture proven urinary tract infection, (b) sex - wise distribution of culture positive cases the predominant isolates were e. coli (42.3%), which showed significance (= 46.46, p < 0.01) [table 1 ]. the predominant isolates were e. coli 42.3% (88/208) followed by enterococci 13.5% (28/208), s. aureus 11.5% (24/208) and klebsiella spp. a panel of selected drugs on the most commonly found organisms confirmed antimicrobial sensitivity patterns [table 2 ]. among all the antimicrobials cotrimoxazole showed 100% coverage against cons and good coverage against other organisms except psedomonas auroginosa and acenitobacter spp. this study demonstrates the distribution and antibiotic susceptibility pattern of microbial species isolated from pediatric patients with suspected uti from a tertiary care center. among the children attending our center with symptoms suggestive of uti, a significant percentage (26.7%) mashouf. in a cross - sectional study performed on 912 children with uti admitted to the pediatric department of a hospital in iran had 34.2% cases with culture - proven uti. a majority of pathogens were isolated from female subjects (54.2%) in our study. t akram. in their study analyzed age and gender - wise data of the prevalence of uropathogens in community - acquired urinary infections. data from other international studies on pediatric patients also report that utis are more common in females, which is similar to our finding. however unlike to our study, kalantar. in his prospective study of 1696 children aged up to 5 years reported male to female ratio of 1.07:1. included children up to 12 years of age and found that uti is more common in males (77.8%). in our study, population majority of cases presented with sterile pyuria was found in significant numbers (43.3%) in our study. in the past international studies there are three possible explanations for this : (i) specimen collection postantibacterial therapy, (ii) any inflammatory condition of bladder, (iii) infection by a fastidious organism not detected by overnight incubation on a primary isolation medium. tuberculosis (tb) which is common in our country for adults but seldom described in children could be a possible etiology. sterile pyuria due to genitourinary tb is reported from singapore, in a population where tb is uncommon and genitourinary tb is a rarity. hence, a detailed evaluation and a proper follow - up are required to address sterile pyuria. some of them could be having other diseases with vasculitis like kawasaki 's disease, which are known to have sterile pyuria. e. coli (42.3%) was the leading etiology of pediatric uti at our center. data from the above studies showed that e. coli are consistently found predominant uropathogen irrespective of country, community or hospital setting. being detected in 22.0% cases and in various parts of the world as 14.0%, 14.5% and 21.0% cases. their low incidence could be justified by the studies like akram. also from north india, where no such microorganism was found in children from 0 to 19 years and abdulhadi. however, in contrast to our findings, taneja. demonstrated p. aeruginosa (10.9%) and acinetobacter spp. inclusions of surgical cases with more chances of indwelling catheters may be responsible for the higher incidence. unlike to prospective studies where enterobacter aerogenes 3.8% and streptococci spp. 1.7% from northern india and retrospective analysis where 3.8% and 2.3% respectively from a multi - speciality hospital in south - africa, these microbes were not found in samples from our centre. gram - positive organisms have received more attention recently as a cause for bacteriuria and uti. coagulase negative staphylococcus, s. aureus, streptococci, and enterococci have been reported in small numbers by various authors, but they are recognized as important causes of uti. we found similar occurrence rate as 13.5%, 11.5% and 5.8% for enterococci, s. aureus, and coagulase - negative staphylococcus, respectively. we found a valuable laboratory data on antibiotic susceptibilities of uropathogens and allows comparison of the situation in our area with that in other countries and other regions of our country. we found that the most active antibiotics against all the gram - negative isolates were nitrofurantoin, amikacin, and cefotaxime. these findings are contrary to studies from africa, south - asia, and some middle east countries which showed that these drugs are less potent against gram - negative organisms. in a prospective study conducted on a total of 524 subjects have shown lower resistance rates which corresponds to results of our study. similar susceptibility patterns of nitrofurantoin and cefotaxime have been found in a study by sharmin. our study, similar to kalantar. and mashouf. demonstrated the extremely low susceptibility of gram - negative organisms to the first - line agents like fluoroquinolones and cotrimoxazole, which are frequently used antibiotics in the settings of uti in our population. nalidixic acid showed high resistance to all isolates especially gram - negative organisms analogous to studies from different parts of the world. we observed a significant degree of antibiotic resistance among the uropathogens isolated. among the gram - negative organisms, there was a tendency towards multidrug - resistance defined as resistance to one or more of the extended - spectrum cephalosporins and fluoroquinolones. however, the numbers of this isolate are very less in our study (only 4). the possible reason of high level of antibiotic resistance among the uropathogens isolated could be due to high level of antibiotics used in our region. coagulase - negative staphylococcus was the only organism susceptible (66.7%) to novobiocin whereas other gram - positive organisms were highly resistant at our center. it may be worthwhile using this drug in our country as other centers are not using it. this study is not placebo controlled as only patients with symptoms included and that only from a single center. the worldwide trend of empirically treating community - acquired uti may not apply for specific geographical regions, where decreased susceptibility rates are documented for common urinary pathogens. international guidelines are no longer applicable for treating community - acquired uti in a region, which have always the tendency of changing antimicrobial sensitivity over a period of time. the development of specific guidelines based on local susceptibility patterns is necessary to serve as a guide for empirical treatment before or in the absence of urine culture report when proper diagnostic modalities are limited in rural and sub - rural areas. however, it 's very important to mention here that clinical use of any antibiotic for uti can not be recommended based on this study as further studies are warranted showing its effectiveness clinically on pediatric uti patients. development of regional surveillance programs is necessary periodically to enable the development of community - acquired uti guidelines. other organisms grown in significant numbers were e. fecalis, klebsiella spp. and s. aureus. prospective, regional studies should be ensured periodically to identify bacteriological profile and antibiotic sensitivity patterns to be applicable for children with uti for that geographic area at that particular period of time. | introduction : development of regional surveillance programs is necessary for the development of community - acquired urinary tract infection (uti) guidelines, especially for sub - urban and rural areas where empirical treatment is the mainstay in the absence of proper diagnostic modalities. our aim was to evaluate the bacteriological profile and antibiotic sensitivity patterns in children with uti prospectively from a tertiary care center.methods:a total of 800 children up to 18 years of age with suspected uti attending our center were included. for all suspected cases urine microscopy, gram staining, and culture were done. antibiotic sensitivity was performed on selected antimicrobials using disk diffusion method following clinical laboratory standards institute guidelines.results:majority of pathogens were isolated from female (54.2%) patients. pre - teens (52.1%) and teens (27.1%) were most commonly affected age group. the most common presentation in culture - proven uti was fever with urinary symptoms (33.3%). in a group of 192 patients 26.7% had proven uti. escherichia coli (42.3%) was the most common aetiological agent, followed by enterococcus fecalis (13.5%), klebsiella spp. (11.5%) and staphylococcus aureus (11.5%). most active antibiotics against gram - negative isolates were nitrofurantoin, cefotaxime, and amikacin. gram - positive isolates were sensitive to nitrofurantoin, cotrimoxazole, and novobiocin.conclusion:e. coli was the commonest isolate. the organisms grown in significant numbers were e. fecalis, klebsiella spp. and s. aureus, causing uti in 018 years of age group. gram - negative isolates were sensitive to nitrofurantoin, amikacin, and cefotaxime. gram - positive isolates were sensitive to nitrofurantoin, cotrimoxazole, and novobiocin. prospective, regional studies are ensured periodically to explain bacteriological profile and antibiotic sensitivity patterns to be applicable for children with uti over that geographic area. |
the general view of the cellular plasma membrane has evolved over the last twenty years from that of a homogeneous arrangement of lipids with embedded proteins towards that of a mosaic of microdomains, each having a specific protein and lipid composition. over the last couple of decades, evidence has accumulated for organisation of the plasma membrane into lipid - based microdomains or lipid rafts. a new model of membrane architecture has been suggested in which the membrane is patchy with segregated cholesterol - rich portions, called lipid rafts. lipid rafts are envisaged as islands of highly ordered saturated lipids and cholesterol that are laterally mobile in the plane of a more fluid disordered bilayer of largely unsaturated lipids [3, 4 ]. the hallmark of the lipid raft hypothesis are the spontaneous partitioning of lipids and proteins in discrete membrane domains, a behaviour based on their physicochemical characteristics and the possibility to recover these microdomains and their associated protein machinery as detergent - resistant entities using biochemical flotation experiments. microdomains appear as small dynamic structures that can aggregate into larger platforms in response to various stimuli. currently, lipid rafts are thought to allow different protein - lipid and protein - protein interactions that temporarily compartmentalise the plasma membrane. lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface. they seem to provide the necessary microenvironment in order for certain specialised signalling events to take place. recent studies have shown the importance of lipid raft formation in the acquired immune response. major histocompatibility complex- (mhc-) restricted t - cell activation seems to be facilitated by lipid raft formation. furthermore, we have recently found that mediators of the innate immune response also concentrate in lipid rafts in order to facilitate signal transduction [7, 8 ], thus suggesting that both the acquired and innate immune systems utilise membrane partitioning as means of activation against invading pathogens. crucial receptors for both innate and acquired immunity seem to oligomerize in nonrandom membrane structures, bringing together their signalling machinery. thus accumulation of receptors within these floating islands on the cell membrane seems to bring together intracellularly all the adaptor molecules that are necessary for signalling. in this paper, we will investigate further the mechanisms of innate immune recognition and review past and current literature that leads us to believe that membrane partitioning and lipid rafts play a central role in innate immune activation. the innate immune system constitutes the most archaic part of our immune defences and has survived through years of evolution. its function is thought to be the recognition of invading pathogens, the activation of inflammation to control the pathogen, and the subsequent activation of the acquired immune response. as part of its mechanism of activation, the innate immune system employs germ - lined encoded receptors, called pattern recognition receptors (prrs) in order to sense pathogens. these prrs recognise a restricted collection of microbial signatures, able to sense different types of microbial pathogens ranging from bacteria and viruses to fungi and spirochetes. lipid rafts seem to be a key feature of the innate immune response, playing a crucial role in phagocytosis, receptor - receptor as well as receptor - pathogen associations as well as signal transduction. families of prrs, such as the toll - like receptor family (tlr) as well as the c - type lectin family seem to localise in lipid rafts for their function thus demonstrating the importance of this membrane partitioning for the function of the innate immune response. the tlr receptor family were the first pattern recognition receptors to be identified [9, 10 ]. this family of at least ten germ - line encoded receptors is able to sense microbial signatures and trigger activation leading to proinflammatory cytokine secretion. tlrs are expressed on immune cells and are able to distinguish a great variety of microbial ligands, such as cell wall components like lipopolysaccharide (lps) from gram - negative bacteria and lipoteichoic acid from gram - positive bacteria, bacterial flagellin, cpg dna, and viral dna or single stranded rna. all identified tlrs are type i transmembrane proteins, whose intracellular domains contain regions homologous to the intracellular domains of il-1r and are referred to as tir domains. these intracellular domains are able to trigger signalling pathways known to activate the nuclear factor kappa b (nf-b) [12, 13 ], which in turn leads to the secretion of proinflammatory cytokines such as tnf-, il-6, and il-8. tlr4 was found to recognise bacterial lipopolysaccharide (lps) or endotoxin [14, 15 ] ; tlr2 was found to recognise lipoteichoic acid (lta) and peptidoglycan ; tlr3 was able to sense double - stranded viral rna ; tlr5 was found to recognise bacterial flagellin, tlr7 and tlr8 to sense single stranded viral rna, whereas tlr9 to recognise bacterial cpg dna. in addition, tlr2 was found to recognise different motifs including several components of gram - positive bacteria such as peptidoglycan, lipoteichoic acid (lta), lipoarabinomannan, lipoproteins, and different lps from certain gram - negative bacteria, yeast, spirochete, and fungi [28, 29 ] through its unique ability to heterodimerize with tlrs 1 and 6. studies using diacylated and triacylated lipoproteins have revealed that diacylated lipoproteins require tlr2/6 heterodimers for activation, whereas triacylated lipoproteins induce activation of the innate immune system independently of tlr6 and mainly through tlr2/tlr1 heterodimers [25, 3135 ]. the membrane distribution of tlrs as well as their intracellular trafficking has only now begun to be investigated. most tlrs (tlr1, tlr2, tlr4, tlr5, and tlr6) seem to activate cells by engaging their ligands on the cell surface, whereas tlr3, tlr7, tlr8, and tlr9 seem to trigger signalling intracellularly. these tlrs have been shown to reside in the er and to recognise their ligands once they have been endocytosed [36, 37 ]. investigations into the innate immune recognition of bacterial endotoxin led to the discovery of the tlr family. tlr4 is the most studied tlr, mainly because of its involvement with sepsis and septic shock. sepsis is a paradoxical and complex disorder that results from an overreaction of our innate immune system to bacterial infections. the mechanisms that are designed to protect the host against infection by bacterial pathogens, either gram - negative or gram - positive, can lead to oversecretion of cytokines and fatal sepsis syndrome. it is now widely accepted that the overreaction of the host occurs at the level of the innate immune system and is directly linked to the recognition of bacterial cell wall components, such as lipopolysaccharide (lps) from gram - negative bacteria or lipoteichoic acid (lta) from gram - positive bacteria. thus the recognition of bacterial products by the innate immune system under certain conditions seems to be detrimental for the host. in the last twenty - five years, great leaps forward in our understanding of one of the seminal discoveries has been the identification of a serum protein, lipopolysaccharide - binding protein (lbp), which binds lps or lta and delivers it to its cellular targets. probably the most important discovery has been that the main family of receptors employed by the innate immune system are the toll - like receptors (tlrs). as far as sepsis and bacterial recognition is concerned, tlr4 seems to be the central sensor of gram - negative bacterial products [15, 39 ], whereas tlr2 seems to be the key receptor in activating the immune system against gram - positive bacteria. in addition to the involvement of tlrs, other accessory molecules seem to be involved. cd14 is believed to act as a transfer molecule for both gram - negative and gram - positive bacteria [40, 41 ]. in the case of lps recognition, it has been further shown that a soluble molecule, md-2, as well as activation clusters involving several other receptors [4345 ]. in the case of lta recognition, tlr2 seems to form receptor clusters as well, comprising of at least cd14, tlr2, tlr6, and cd36. thus we are moving away from the single - receptor model of activation, and a more complex picture is emerging. the mechanism that leads to activation seems to involve the careful interplay of several receptor molecules as well as serum proteins. therefore such a complex orchestration of events requires a nonrandom membrane architecture specifically geared to bring receptor molecules together and trigger activation within the lipid bilayer and lipid rafts or membrane microdomains seem to provide this platform. prrs employed by the innate immune system have been shown to have the ability to bind and recognise conserved products of pathogens that are unique to the invading microorganisms but not to the host, it is becoming increasingly apparent that the model of a single prr recognising foreign antigen is an oversimplified one. with the discovery of the toll - like receptors as the main signal transducing molecules of the innate immune system, an onslaught of research has shown that prrs are part of multicomponent sensor apparatuses. tlrs have been shown to function as homo- or heterodimers and to even form functional interactions with non - tlr molecules. many of these interactions are highly stable, whereas others are transient, forming dynamic associations in response to specific stimuli. whether homotypic, heterotypic, stable, or transient, these different protein combinations generate considerable functional diversity for the innate immune system by triggering distinct signalling cascades leading to cellular activation. there are a number of examples that suggest that tlr associations are required for cellular activation. tlr4 seems to form a complex with at least two other molecules, cd14 and md2, in order to recognise bacterial lps. in addition, it seems to associate with a toll - like receptor homologue rp105, which acts as a negative regulator of tlr4 responses. tlr2 has been found to heterodimerize with tlr1 or tlr6 for recognition of yeast components and to associate with tlr1 for the recognition of bacterial lipoproteins. in addition, tlr2 has been shown to also interact with scavenger receptors in order to recognise lipoproteins and most recently it was shown that tlr2 associates with cxcr4, which acts as a negative regulator of tlr2 responses (figure 1). figure 1 depicts the possible model of tlr activation, and how tlrs and other receptors are organized in lipid rafts on the cell surface before and after stimulation. these heterodimers preexist and are not induced by the ligand (figure 1(a)). tlr2/6 heterodimers are recruited within lipid rafts and associate with lipid raft - resident proteins cd14 and cd36 upon ligand engagement. binding of appropriate microbial substances leads to energy - dependent clustering of heterotypic receptors and activation of intracellular signaling cascades that lead, for example, via nf-b to production and secretion of proinflammatory cytokines (figure 1(b)). functional associations of tlrs with non - tlr molecules have also been demonstrated, for example, tlr2 association with dectin-1 is required for macrophage and dendritic cell activation by -glucan - containing particles. more recently, functional interactions of tlr2 and cd36 have been shown to be involved in the recognition of diacylglycerides. tlr4 seems to be the best example of tlrs associating with non - tlr molecules. as it has already been mentioned, tlr4 has been shown to form at least a trimolecular complex with cd14 and md2 in order to recognise bacterial lps. the possibility that additional receptor components such as heat shock proteins [43, 50 ], cxcr4, or cd55 have been suggested to be part of this activation cluster, possibly acting as additional lps transfer molecules. shapes of lps induce the formation of different activation clusters, involving the association of tlr4 with a variety of molecules mentioned above, which seems to determine lps responses. recent structural studies have shed some light onto tlr associations, supporting the hypothesis of cluster formation, since all tlrs that have been crystallised have been found to be in a dimer formation, thus the hypothesis has been put forward that dimerisation or clustering might be a common feature of the tlrs and might be essential for signalling. structural studies of tlrs have been an attractive area of research since structural information is crucial in understanding receptor function. in 2005, it was surprising, that although the structure did not have a ligand, tlr3 was crystallised as a dimer. in 2007 and 2008, three structures of tlr - ligand complexes were revealed, tlr1-tlr2-lipoprotein, tlr4-md-2-eritoran, and tlr3- dsrna [5355 ]. prior to the publication of the crystal structures, gayand gangloff suggested a possible model of activation, where dimerization was ligand induced. these observations have suggested the hypothesis that dimerization of the ectodomains forces the intracellular tir domains to dimerize, and this initiates signalling by recruiting the intracellular adaptor molecules, such as myd88, mal, trif, and tram in order to initiate signalling. the structures of the tir domains of tlr1, tlr2, and tlr10 have been revealed. interestingly, the tir domain of tlr10 was shown to be involved in a homodimeric interaction. however, it is not certain whether the structure seen in the crystal corresponds to a physiologically relevant dimer of tlr10 tir domains because they have been found to exist as monomers in solution. interestingly it has been recently suggested that myd88 interacts with irak4 in an 8 : 4 ratio in solution, suggesting that maybe there is higher oligomer formation. in order for such higher oligomers to be formed and in order to have such a well - orchestrated accumulation of receptors and signalling machinery membrane partitioning seems to be crucial for the formation of these tlr multicomponent sensor apparatuses. tlr4 was the first one to be shown to be recruited to lipid rafts upon stimulation by bacterial lps. within these membrane microdomains it was shown that tlr4 formed clusters with non - tlr molecules that tailored the immune response against the particular pathogen [43, 5962 ]. it was subsequently shown that this accumulation in lipid rafts also influenced its internalization and targeting. tlr4 was found to accumulate in lipid rafts, to internalize in a lipid - raft - dependent manner and to be targeted to the golgi apparatus. this intracellular targeting was shown to be independent of signalling, thus suggesting that accumulation in lipid rafts only facilitated ligand recognition and signalling that was initiated at the cell surface and not in the intracellular compartments where tlr4 was targeted to. more recently it had been proposed that the molecular mechanism for signalling by the tlrs must involve a series of protein conformational changes initiated by dimerization of their extracellular domains. it was suggested that this receptor - receptor association of the extracellular domains forced the association of the cytoplasmic domains as well. recently proved this experimentally, demonstrating that the death domains of human myd88, one of the adaptor proteins used by all but one of the tlrs, and irak4 assemble into closed complexes with stoichiometries of 7 : 4 and 8 : 4, which they called the myddosome. the ability to form 7 : 4 or even 8 : 4 stoichiometries suggests a mechanism by which clusters of activated receptors concentrate in lipid rafts and their intracellular machinery clusters as well, forming a signalling platform that seems to be crucial for tlr activation. cell membranes display a tremendous complexity of lipid and proteins designed to perform the functions cells require. lipid rafts were originally proposed as an explanation for a nonrandom membrane architecture and their function was originally thought to be linked with membrane trafficking. however, rafts proved to be able to influence organization of membrane receptors and bioactivity as well as membrane trafficking. it is now emerging that this membrane partitioning might play a major role in protein uptake and intracellular routing. it is becoming more apparent that this differential sorting on the cell surface might pre - dispose the intracellular fate of a given molecule. since the discovery of clathrin - coated pits by roth and porter in 1964, as specialised sites for the selective recruitment of specialised molecules that are internalised into eukaryotic cells, clathrin - independent endocytic pathways have now emerged. endocytic pathways that do not rely on the formation of clathrin coated pits include the earliest identified pathways such as phagocytosis, macropinosis, and caveolae., it has been shown that lipid rafts play a crucial role in the phagocytic uptake of latex microspheres, suggesting that these specialised microdomains on the plasma membrane are necessary for endocytosis and phagocytosis. furthermore, caveolae which is defined as small, uncoated invaginations in the plasma membrane containing the plasma protein caveolin 1 has been shown to be able to bind cholesterol and to be resistant to detergent extraction, and this has led to the suggestion that caveolae might constitute a type of lipid raft. lipid rafts are increasingly becoming linked with clathrin - independent endocytosis, since nearly all molecules that are known to be internalised independently of clathrin are found in biochemically defined rafts. it has been suggested that raft components might be taken up preferentially by clathrin - independent endocytosis. the extent to which these different pathways require lipid rafts to operate or are somehow selective for lipid rafts is currently the subject of intensive investigation. recently, nichols. have described a rapid lipid - raft - dependent targeting from the cell surface to the golgi apparatus. in addition, a new clathrin - independent mechanism has been described that can lead to delivery of receptor molecules from the plasma membrane to caveolin-1-containing endosomes, termed the idea that different types of endocytosis have markedly different functions is beginning to become apparent. ultimately we have to speculate that sorting at the plasma membrane might predispose the intracellular route that a molecule might take. if that is the case, then where are the raft - associated molecules, such as tlr4, targeted to ? and most importantly why ? tlr2 has also been found to reside in lipid rafts after stimulation by gram - positive bacterial products and to be similarly targeted to the golgi apparatus (figure 1). the question that remains is whether lipid raft association is common for all tlrs expressed at the cell surface ? if this is the case, do they all follow the same intracellular route ? do different signalling cascades require differential targeting of tlrs and their ligands ? in the case of the er - resident tlrs, very little evidence of their trafficking upon stimulation exists. to date only tlr9 has been found to translocate from the er to lysosomes in response to its ligand, cpg dna. based on the findings for tlr9, a hypothesis has been put forward that er - resident tlrs might become accessible to endosomal and lysosomal compartments after the er fuses with sites of microbial entry. if this is the case, then it would seem that er membrane fusion might be critical for microbial recognition by er - resident tlrs. the regulation of endosome dynamics is crucial for fundamental cellular functions, such as nutrient intake / digestion, membrane receptor recycling / degradation, antigen presentation, cell migration, and intracellular signaling [7476 ]. the system is also utilised by various pathogens to bud in and from the cell. in addition to the function as a distribution centre, it has been proposed that the endosome system serves as an intracellular signalling station. in the case of innate immunity this is certainly the case, since tlr3, tlr7, tlr8, and tlr9 all reside within the endosome waiting to capture incoming pamps and trigger signalling. endosomes are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance. the question that remains is whether in addition to these morphologically distinguishable regions, endosomal membranes are further subcompartmentalized into membrane lipid rafts or microdomains. lipid rafts have mostly been studied at the plasma membrane, mainly due to accessibility for microscopy and biophysical methods. characterisation of lipid rafts has also been extensively based on their resistance to detergent solubilisation, although this has inherent limitations, as well as fluorescent microscopy. although most studies have focused on the existence of lipid rafts on plasma membranes, many intracellular organelles appear to contain raft - like domains [8184 ]. due to its low cholesterol content, the endoplasmic reticulum was originally thought not to contain cholesterol - dependent microdomains. however, recently several studies have reported their existence [85, 86 ]. raft - like domains have been described in the golgi and trans - golgi network [87, 88 ], along the endocytic pathway as well as in the endosomes [8991 ]. although potential roles of lipid rafts for the outer membrane have been demonstrated, including endocytosis, exocytosis, vesicle formation, and signalling, the functions of lipid rafts in the processes of endosomal membrane dynamics are currently unknown. it has been suggested that protein and lipid sorting into and out of the endosomes may be controlled by endosomal membrane partitioning, but whether these microdomains control signalling and in particular tlr signalling has not been investigated. since most extracellular tlrs have been shown to be recruited to lipid rafts upon ligand activation, it is safe to assume that endosomal tlrs act in a similar manner, especially since the existence of cholesterol - dependent microdomains on the endosomal membranes has been proven (figure 2). thus it is safe to assume that membrane partitioning control both extracellular and intracellular tlr - dependent signalling. we are proposing a model for endosomal tlr activation, where ligation of endosomal tlrs by their respective ligands can lead to clustering within lipid rafts at the endosomal membrane and activation of intracellular signaling cascades that lead via nf-b to production and secretion of proinflammatory cytokines (figure 2(b)). c - type lectin receptors (clrs), such as dectin 1, are a family of pattern recognition receptors which bind -glucan found in the cell walls of pathogenic fungi such as candida albicans. in particular dectin 1 has been shown to mediate the phagocytosis of yeast and yeast - derived particles, such as zymosan, activating the production of inflammatory cytokines [9294 ]. interestingly, dectin-1 possesses an immunotyrosine - activated motif (itam) in its cytoplasmic tail, suggesting that it is capable of mediating signalling analogous to the bcr and tcr. since both the bcr and tcr have been found to reside in lipid rafts it was suggested that dectin-1 might also be recruited there upon activation. a recent study has revealed that dectin-1 and possibly other clrs are recruited to lipid rafts upon activation and raft integrity is important for signalling. thus suggesting that recruitment to lipid rafts is a common feature for most prrs, including tlrs and clrs. cell membranes are complicated in composition but precise in purpose : to selectively compartmentalize its constituents in order to coordinate cellular functions. in this way, the membrane is able to compartmentalize, segregate receptors as well as their signalling machinery and create oligomeric signalling platforms in order to transduce signals. once the required function has subsided, these segregated islands are involved in internalization and membrane trafficking, thus bringing the whole function to a close. the innate and acquired immune systems seem to utilise this membrane partitioning for their functions. in this paper, we have extensively looked at the use of this membrane partitioning by the innate immune system and most particular by the tlrs. the molecular mechanism involved in lps recognition and tlr signalling in general, the plasma membrane seems to be heterogeneous and to coalesce to more stable membrane - ordered assemblies upon activation by ligands. this partitioning of the membrane and the assembly of more stable raft platforms in the functionalized state must be initiated by raft - resident proteins, which form protein - lipid as well as protein - protein interactions. the tlrs and other prrs associate with the raft - resident proteins and are recruited to these floating islands forming higher oligomers, both extracellularly as well as inside the cell, concentrating their signalling machinery which finally leads to a functional, focused, and coordinated activation of the innate immune system. the lifetime of these domains and the length of the response will depend on their size and factors that may stabilise or destabilise them. these factors will include not only lipid - lipid, lipid - protein and protein - protein interactions both in the plane of the membrane but also elements of the cytoskeleton, pericellular matrix adjacent to the membrane as well as transmembrane and cytoplasmic domains of the receptors involved. in the case of tlrs, the association of the tir domains intracellularly would stabilise the ectodomains extracellularly and provide the molecular scaffold that will recruit the adaptor molecules that contribute to signalling. the challenge for the future will be to visualise the assembly and stoichiometry of these large and transient oligomeric complexes in vivo. thus refining existing biophysical methods, such as single particle tracking (spt), fluorescence correlation spectroscopy (fcs), and fluorescence resonance energy transfer (fret) will help us reveal these dynamic nanoassemblies of sterol, sphingolipid, and proteins in living cell and provide us with the first dynamic picture of the innate immune response. | in the last twenty years, the general view of the plasma membrane has changed from a homogeneous arrangement of lipids to a mosaic of microdomains. it is currently thought that islands of highly ordered saturated lipids and cholesterol, which are laterally mobile, exist in the plane of the plasma membrane. lipid rafts are thought to provide a means to explain the spatial segregation of certain signalling pathways emanating from the cell surface. they seem to provide the necessary microenvironment in order for certain specialised signalling events to take place, such as the innate immune recognition. the innate immune system seems to employ germ - lined encoded receptors, called pattern recognition receptors (prrs), in order to detect pathogens. one family of such receptors are the toll - like receptors (tlrs), which are the central sensing apparatus of the innate immune system. in recent years, it has become apparent that tlrs are recruited into membrane microdomains in response to ligands. these nanoscale assemblies of sphingolipid, cholesterol, and tlrs stabilize and coalesce, forming signalling platforms, which transduce signals that lead to innate immune activation. in the current paper, we will investigate all past and current literature concerning recruitment of extracellular and intracellular tlrs into lipid rafts and how this membrane organization modulates innate immune responses. |
one of the great challenges for wave propagation is the efficient and stable computation of waves in unbounded domains. the crucial point for these computations is that the numerical scheme avoids any reflections at the boundaries, even in case the diameter of the computational domain is just a fraction of a wavelength. since the eighties of the last century, several numerical techniques have been developed to deal with this topic : infinite elements, dirichlet - to - neumann operators based on truncated fourier expansions, absorbing boundary conditions, etc. the advantages and drawbacks of these different approaches have been widely discussed in literature, see e.g.. especially higher order absorbing boundary conditions (abcs) have gained increasing interest, since new methods do not involve high order derivatives [6,2124,26,27,25,28,8 ]. an alternative approach to approximate free radiation is to surround the computational domain by an additional damping layer and guarantee within the formulation, that no reflections occur at its interface with the computational domain. this so - called perfectly matched layer (pml) technique was first introduced by berenger using a splitting of the physical variables and considering a system of first order partial differential equations (pdes) for electromagnetics. since then, there has been much research work on this technique which subsequently was applied to different pdes. in the framework of time - harmonic wave propagation, the pml can be interpreted as a complex - valued coordinate stretching. therewith, a pml formulation for a linear pde in frequency domain can be considered as a straightforward approach. however, in time domain most pml formulations require a first order hyperbolic system, e.g.,. the difficulty arising for the second order wave equation in time domain is, that an inverse fourier transform of its frequency representation will lead to convolution integrals, see e.g.. a method to avoid convolution integrals e.g., in a pml method for the second - order elastodynamic equations has been formulated. the basic idea of the formulation is to decompose the gradient operator in terms of components perpendicular and parallel to the interface, and then split the mechanical displacement in four variables. however, as noticed in the resulting equations need special treatment for the time stepping and additional memory is needed for the split - field variables. furthermore, such split - field pml methods suffer from numerical instability, see e.g.. once a pml formulation has been obtained, the question of stability arises, which is a topic of strong ongoing research. a stability analysis is not trivial and in general it has to be performed for each new formulation. several works have analyzed the properties of the pml technique, such as among others. e.g., in a time - domain analysis of pml methods for wave equations in 2d by using the cagniard - de hoop method has been presented. the main result is to validate the modified fundamental solution extended to the absorbing layers. this method is easily applicable to the wave equation with any time - dependent point source. however, the evaluation is not easy for general initial value problems of the wave equation, because those in general include not only propagating but also evanescent waves. our stability analysis investigates the evolution of the energy over time and we are able to show decay of an upper bound on the energy for our formulation, thus achieving long term stability. it should be noted that the pml formulation under consideration has been first published in, where a finite difference time domain (fdtd) method on space time staggered grids with explicit time stepping has been used. in we have discussed this formulation in a finite element (fe) setting and have shown numerically that omitting terms in the time domain formulation (calling it an almost pml) provides long term stability when applying it to a computational setup with a thin damping layer. in this contribution, we further investigate in our fe formulation, use unstructured grids with tetrahedra, apply an implicit newmark time stepping method and provide a detailed discussion of our long time stability proof based on energy analysis. furthermore, we perform rigorous numerical tests with different damping layer thicknesses and damping functions. the results show that for a thick enough damping layer long time stability is achieved. a key novel feature of our approach is that the analysis not only works for a special space discretization but applies to arbitrary space discretizations such as geometrically flexible unstructured finite element grids as demonstrated in the numerical case study and the application example of flow induced sound. numerical case studies and an application to the computation of sound generated by the flow around a side view car mirror are presented in section 4, and finally we summarize our main achievements in section 5. we consider the time dependent wave equation in an unbounded three dimensional rectangular domain dr3(1)1c2p2t2-p=0with the speed of sound c>0 and zero initial conditions. in order to model free radiation, we surround the propagation region of interest prop by a pml of thickness li in each coordinate direction i and denote this region by pml. to obtain the correct pml formulation of the wave equation within pml first we transform (1) to the frequency domain(2)jc2p-p=0with p the fourier transform of p and the angular frequency. according to we introduce the complex change of variables inside pml(3)xi(xi)=xi+1j0xii(x)dx;xi{x, y, z},where the damping function i is positive inside pml and vanishes in prop. hence, we obtain the following relationsxixi=1+ij=iandxi=1ixi.therewith, the modified helmholtz equation, which has to be solved in pml, reads as(4)xyzjc2p-yzx1xpx-xzy1ypy-xyz1zpz=0with(5)x=1+xj;y=1+yj;z=1+zj.before we proceed, we use the fact that l/xk=0 for kl to rewrite (4) by(6)xyzjc2p-xyzxpx-yxzypy-zxyzpz=0.now, we investigate in the first term of (6) and expand the terms i by (5) to obtain(7)xyzjc2p=jc21+xj1+yj1+zjp=1c2(j)2+j(x+y+z)+xy+xz+yz+xyzjp.for an inverse fourier transformation of (7) we recognize, that the last term will result in a time integral of p. therefore, we introduce the first auxiliary variable according to(8)v = pj.in a next step, we analyze the second term in (6) and start with(9)ii=yzxpx=1+yj1+zj1+xjpxnow, we perform the following rearrangements and use also (8)(10)ii=(y+j)(z+j)j(x+j)px=yzj+(y+z)+j(x+j)pxxx+jpx=yzj+(y+z-x)(x+j)px+px=1x+jyzvx+(y+z-x)px+px.the same procedure is performed on the third and fourth term in (6). having an inverse fourier transform in mind, we introduce a vectorial auxiliary variable u with the following relations(11)ux=1x+jyzvx+(y+z-x)pxuy=1y+jxzvy+(x+z-y)pyuz=1z+jxyvz+(x+y-z)pz.now, we are ready to apply the inverse fourier transform to (6), (8), (11) and achieve at the following coupled system of partial differential equations(12)1c22pt2+pt+p+v-p-u=0(13)ut+au+bp - cv=0(14)vt = pwith(15)=x+y+zc2;=xy+xz+yzc2;=xyzc2,(16)a=x000y000z;c=yz000xz000xy,(17)b=x-y-z000y-x-z000z-x-y. the proper choice of the damping functions is of great importance, especially in order to obtain a very robust and efficient pml - technique. assuming a layer thickness of l, e.g., in y - direction, the damped wave will be totally reflected at the outer boundary of the pml - region and this reflected wave at the interface between propagation and pml region takes the value(18)pr=p0e-(2/c)cos0ly(y)dy = p0r.a reasonable choice of the reflection factor r has to be chosen by a trade - off between the necessity of sufficient reduction of reflected waves according to (18) and possible disturbances of the numerical solution by a too rapid damping in a narrow pml region. in our computations in addition, in order to get rid of the dependence of the overall damping on the speed of sound c, we will choose y directly proportional to c. using (18), we obtain the following relations:constant dampingyconst =- clnr2lcos.inverse distance dampingyinverse = cl - y. as shown in, the inverse distance damping function leads to an optimal damping behavior for the acoustic wave equation in the frequency domain, and compared to other damping functions does not need specification of the reflection coefficient r. this is also confirmed by many of our computations for the wave equation in time. however, we have also obtained instable results, especially for thin damping layers. in section 4 we provide rigorous numerical tests for different damping layer thicknesses and the two above defined damping functions. for deriving boundedness of solutions (p, v, u) of (12)(14) in an appropriate norm (related to the acoustic energy) we proceed as follows:1.test the system with appropriate multipliers to derive energy estimates;2.combine these estimates to assess the time evolution of a scalar valued function (t), more precisely, to show that (t) is nonincreasing over time ; can be interpreted as a lyapunov function for the system (12)(14);3.prove that by a proper choice of the parameters defining, the energy can be bounded by a fixed multiple of. test the system with appropriate multipliers to derive energy estimates ; combine these estimates to assess the time evolution of a scalar valued function (t), more precisely, to show that (t) is nonincreasing over time ; can be interpreted as a lyapunov function for the system (12)(14) ; prove that by a proper choice of the parameters defining, the energy can be bounded by a fixed multiple of. in the course of this derivation, it will turn out that instabilities may emerge in regions where c is strictly positive. thus we will end up with considering a reduced pml (rpml) where we just set c0. we consider the bounded computational domain, which includes the propagation region prop and the attached pml layer pml. for the weak formulation, we introduce appropriate test functions,q and, and describe space and time dependence by considering p(t),v(t) and u(t) as elements of the function space v and h, respectively, for each time t0. we assume homogeneous dirichlet boundary conditions on p(t),v(t) and the test functions,q, but not on u(t),.p(t)==0on,pt(t)=v(t)=q=0on,i.e.,p(t),,v(t),qv = h01(),u(t),h = l2()3t0.in addition, we introduce the matrix d(19)d=y+z000x+z000x+y, so that we can express b by a - d. therewith, the weak formulation of our coupled system of pdes (12)(14) becomes(20)2pt2(t)dx+pt(t)dx+p(t)dx+v(t)dx+p(t)dx+u(t)dx=0v,(21)ut(t)dx+(au(t))dx+(bp(t))dx-(cv(t))dx=0h,(22)vt(t)qdx-p(t)qdx=0qv, for all t0. the relation according to (22) will be used explicitly as v(t)/t = p(t). in here,,0 are scalars, a, b, cd are diagonal matrices b = a - d with a, c, d0 (meaning positive semidefinite here) and supp{}pml, {,,,a, b, c, d } (see (15)(17)). now we insert different test functions into (20) and (21), and integrate with respect to time, using the fact that, e.g.0tp(s)pt(s)dxds=120tddt|p(s)|2dxds=12|p(t)|2dx-12|p(0)|2dx.setting(23)=p(s)/tin(20),=p(s)in(20)(with some possibly space dependent factor0c2),=p(t)in(21),=fu(t)in(21)(with some possibly space dependent diagonal matrix0f),and using the notation2=||2dx;2=||2dx,1,2=12dx;1,2=12dx, we therewith obtain(24)121cpt(t)2 - 121cpt(0)2+0tpt(s)2ds+12p(t)2 - 12p(0)2+0tv(s),pt(s)ds+12p(t)2 - 12p(0)2+0tu(s),pt(s)ds=0,(25)0t1c22pt2(s),p(s)ds+12p(t)|2 - 12p(0)|2+0tp(s)2ds+12v(t)2 - 12v(0)2+0tp(s)2ds+0tu(s),p(s)ds=0,(26)0tut(s),p(s)ds+0t(au(s)),p(s)ds+0ta1/2p(s))2ds-0td1/2p(s))2ds-12c1/2v(t))2 + 12c1/2v(0))2=0,(27)12f1/2u(t)2 - 12f1/2u(0)2+0t(fa)1/2u(s)2ds+0tfbp(t),u(s)ds-0tfcv(t),u(s)ds=0,for all t0. 2nd step : conclude monotonicity of some lyapunov function. in a next step, we will compute the sum of the four above equations (24), so that the term 0tu(s)/t,p(s)ds cancels out. furthermore, we define the following function of time(29)(t)121cpt(t)2+p(t)2+f1/2u(t)2++p(t)2+v(t)2-c1/2v(t))2 + 2v(t),p(t)+2u(t),p(t)+2c2pt(t),p(t).remark 1here we wish to point out that the sixth term on the right hand side in (29) suggests that it is favorable to set c=0 from a stability point of view. indeed, even if we would add the time integral of (20) with =v(s), which would give us a term 12v(t)2 on the left hand side for possible control of -c1/2v(t))2, we would end up with a term 0tu(s),v(s)ds, which we can not control since we have no means for obtaining an estimate of 0tv(s)2ds, especially for long times when this expression gets larger than sups[0,t]v(s)2. here we wish to point out that the sixth term on the right hand side in (29) suggests that it is favorable to set c=0 from a stability point of view. indeed, even if we would add the time integral of (20) with =v(s), which would give us a term 12v(t)2 on the left hand side for possible control of -c1/2v(t))2, we would end up with a term 0tu(s),v(s)ds, which we can not control since we have no means for obtaining an estimate of 0tv(s)2ds, especially for long times when this expression gets larger than sups[0,t]v(s)2. using (28) and the above definition according to (29), we obtain the following relation for the sum of (24)(27)(30)(t)+0t(fa)1/2u(s)2ds+0t-c2pt(s)2ds+0tsign(-)||-|p(s)|2dxds+0tsign(i+b)||i+b|p(s)|2dxds=(0)-0t(i+a+fb)p(s),u(s)ds+0tfcv(t),u(s)ds(0)+120t(fa)1/2u(s)2ds+120t((fa)1/2)(i+a+fb)p(s)2ds+120t((fa)1/2)fcv(s)2dsor if c=0(31)(t)+0t(fa)1/2u(s)2ds+0t-c2pt(s)2ds+0tsign(-)||-|p(s)|2dxds+0tsign(i+b)||i+b|p(s)|2dxds=(0)-0t(i+a+fb)p(s),u(s)ds(0)+0t(fa)1/2u(s)2ds+0t12((fa)1/2)(i+a+fb)p(s)2ds, provided(32)u(s)n(fa),s(0,t)orr(i+a+fb)n(fa)(and for (30) additionally u(s)n(fc),s(0,t)). in here, for some matrix m, we denote by r(m), n(m), and m the range, the orthogonal complement of the nullspace, and the generalized inverse, respectively, and for some positive semidefinite matrix m we denote by m1/2 the square root, being defined by the relation m1/2m1/2=m. for deriving (30) and (31), we have exploited the identity0t(i+a+fb)p(s),u(s)ds=0t((fa)1/2)(i+a+fb)p(s),((fa)1/2)u(s)ds, that holds under condition (32), and whose right hand side can then be estimated via the cauchy schwarz inequality. studying (30) as well as (31), we see that for(33)c=0,0c2,-0,i+b0,and(34)4fa(i+b)-(i+a+fb)20 in all of, we get(t)(0).note that since here time = 0 can be replaced by time = st, this shows monotone decrease of the function. 3rd step : estimate energy by. reconsidering the definition of (t) (29), we see that in case c=0 it is an upper bound for a multiple of the energy(35)(t)121cpt(t)2+e1/2p(t)2+e1/2u(t)2with 0,e0,e0, provided that additionally to (33) and (34)(36)i - f-10and1-cp2+2c2-(+)i - f-10,where cp is the constant in the poincar friedrichs inequalityv = h01():cp.namely, we can estimate(t)=121cpt(t)2+p(t)2+f1/2u(t)2++p(t)2+v(t)2 + 2v(t),p(t)+2u(t),p(t)+2c2pt(t),p(t)121cpt(t)2+p(t)2+f1/2u(t)2++p(t)2+v(t)2-v(t)2-p(t)2-(f - e)1/2u(t)2-(f - e)-1/2p(t)2 - 1-cpt(t)2-c1-p(t)2121cpt(t)2+e1/2p(t)2+e1/2u(t)2,where the last two inequalities hold if(37)f - e>0andi - e-(f - e)-10and+2c2(1-)-(+)p(t)2i - e-(f - e)-1p(t)i.e., if(38)f - e>0andi - e-(f - e)-1-cp2+2c2(1-)-(+)i0. it remains to interpret conditions (32)(34), (36), (38) i.e., to find,f,,e,e such that they can be satisfied : according to (15)(17) we havec2=x+y+z, c2=yz+zx+xy, c2=xyz, aii=i, bii=i-j-k, dii=j+k;i,j,k{x,y,z}and setting c=0, conditions (33) are satisfied for=x+y+z, sincec2(-)=(yz+zx+xy)(x+y+z)-xyz0.since f will be chosen as a diagonal matrix, condition (34) can be reformulated as04fa(i+a - d)-((i+a)+f(a - d))2=4(i+a)af-4adf-((i+a)+af - df)2=4(i+a)af-4adf-(i+a)2-a2f2-d2f2 - 2(i+a)af+2(i+a)df+2adf2=-(i+a)2+(2(i+a)(a+d)-4ad)f-(a - d)2f2,where the diagonal entries of the right hand side are (with the abbreviation a = aii=i, d = dii=j+k, f = fii,=a+d)-(2a+d)2+(2(2a+d)(a+d)-4ad)f-(a - d)2f2=-(2a+d)2 + 2(2a2+ad+d2)f-(a - d)2f20f-ff+where(39)f=2a2+ad+d2(2a2+ad+d2)2-(2a+d)2(a - d)2(a - d)2.note that in here, the square root gives a real number since2a2+ad+d2|(2a+d)(a - d)|=|2a2-ad - d2|and we have 0f-f+. therefore, f = fii0 can indeed be chosen so that (34) is satisfied. condition (36) additionally requires f = fii1 which is enabled by the fact that f+1 due to(2a2+ad+d2)2-(2a+d)2(a - d)2(a - d)2-(2a2+ad+d2)=-a2 - 3ad, with strict inequality unless a vanishes, hence(40)2a2+ad+d2+(2a2+ad+d2)2-(2a+d)2(a - d)2(a - d)2,with strict inequality if a>0. (in case a = d=0, condition (34) is trivially satisfied for any f and we can choose an arbitrary f > i.) to see the right hand part of (36) we use the fact that we have chosen =c2 and (33) in(41)(+)--2c2=-=-0,so that the right hand part of (36) is implied by the left hand part. concerning (38), we get from estimate (41) that it follows from(42)f - e0andi - e-(f - e)-1-cp22c21-i0. in order to check the nullspace condition (32) and to identify regions in which the matrices e,e or at least some of their entries can be chosen strictly positive, we distinguish between the following subdomains:(a)0={:x()=y()=z()=0}prop : on this subdomain the quantities,,,a, b, c vanish. since (13) together with u(0)=0 implies u(t)0=0 for all t>0, we directly obtain that the respective part of (t), namely (29) with 2 replaced by 0l2(0)2, just equals 0(t)121cpt(t)02+p(t)02, so we can choose1,ei, ei, on0.(b)xyz={:x()>0andy()>0andz()>0}:since on this subdomain a is strictly positive definite, choosing a positive f we will automatically satisfy the nullspace condition (32). we can select f = diag(f11,f22,f33) with fii = f+=fi+ according to (39) with a=i>0 which implies fii>1 (cf. the observation following (40)). with this choice, =x+y+z>0 ande=12(f - i)>0,e=14i+12(f - i)-1(f - i)>0,=1+,onxyz, with 14c2cp22mini{1,2,3}(1 + 12(fii-1))-1(fii-1)>0, we also get (42), which by (41) is sufficient for (38).(c)x={:x()>0andy()=z()=0 } (analogously for y,z):here it can be seen that the nullspace condition (32) indeed restricts the first component of f : since n(fa)n(a)t = r{0}{0 }, which in general will not contain u(s), we have to make sure that (i+a+fb)r{0}{0 }, which by fii1 (from (36)), x (from (33)) is equivalent to f22=f33=1,=x>0, so that we have to pute22=e33=e22=e33=0onx.still, application of the argument from (b) and direct consideration of (38) shows that we can choosee11=12(f11 - 1)>0,e11=141 + 12(f11 - 1)-1(f11 - 1)>0,=1+>0,onx, with 14c2cp22(1 + 12(f11 - 1))-1(f11 - 1)>0.(d)yz={:x()=0andy()>0andz()>0 } (analogously for zx,xy):this case can be treated similarly to (c), which yields a choice f11=1,fjj>1,=y+z>0,e11=e11=0onx.ejj=12(fjj-1)>0,ejj=141 + 12(fjj-1)-1(fjj-1)>0,=1+>0,onyz, for j=2,3, with 14c2cp22mini{2,3}(1 + 12(fii-1))-1(fii-1)>0. since (13) together with u(0)=0 implies u(t)0=0 for all t>0, we directly obtain that the respective part of (t), namely (29) with 2 replaced by 0l2(0)2, just equals 0(t)121cpt(t)02+p(t)02, so we can choose1,ei, ei, on0. xyz={:x()>0andy()>0andz()>0 } : since on this subdomain a is strictly positive definite, choosing a positive f we will automatically satisfy the nullspace condition (32). we can select f = diag(f11,f22,f33) with fii = f+=fi+ according to (39) with a=i>0 which implies fii>1 (cf. the observation following (40)). with this choice, =x+y+z>0 ande=12(f - i)>0,e=14i+12(f - i)-1(f - i)>0,=1+,onxyz, with 14c2cp22mini{1,2,3}(1 + 12(fii-1))-1(fii-1)>0, we also get (42), which by (41) is sufficient for (38). x={:x()>0andy()=z()=0 } (analogously for y,z) : here it can be seen that the nullspace condition (32) indeed restricts the first component of f : since n(fa)n(a)t = r{0}{0 }, which in general will not contain u(s), we have to make sure that (i+a+fb)r{0}{0 }, which by fii1 (from (36)), x (from (33)) is equivalent to f22=f33=1,=x>0, so that we have to pute22=e33=e22=e33=0onx.still, application of the argument from (b) and direct consideration of (38) shows that we can choosee11=12(f11 - 1)>0,e11=141 + 12(f11 - 1)-1(f11 - 1)>0,=1+>0,onx, with 14c2cp22(1 + 12(f11 - 1))-1(f11 - 1)>0. yz={:x()=0andy()>0andz()>0 } (analogously for zx,xy) : this case can be treated similarly to (c), which yields a choice f11=1,fjj>1,=y+z>0,e11=e11=0onx.ejj=12(fjj-1)>0,ejj=141 + 12(fjj-1)-1(fjj-1)>0,=1+>0,onyz, for j=2,3, with 14c2cp22mini{2,3}(1 + 12(fii-1))-1(fii-1)>0. thus, we have proven the following stability result for a modified version of the pml system (12)(14):theorem 1consider the system (12)(14) with homogeneous dirichlet boundary conditions on p, homogeneous initial conditions on u, and c,,,,a, b, according to (15)(17), in l(),l()33, respectively, but with c0.there exist a space dependent strictly positive function >0 and positive semidefinite diagonal matrix functions e,e as well as a nonnegative monotonically non - increasing time dependent function such that(t)121cpt(t)2+e1/2p(t)2+e1/2u(t)2holds for any solution (p, v, u)w(r+;v, h) of (20)(22), wherew(i, v, h)=(c2(i;v)c1(i;l2())c(i;v))(c2(i;l2())c1(i;v))c(i;h),v = h01(),h = l2()3,v=h-1() (the dual of v).more precisely we have e()=diag(e11(),e22(),e33() and e()=diag(e11(),e22(),e33()) with (upon identification 1x,2y,3z)(43)eii>0,eii>0oni, where(44)i={:i()>0}{:x()=y()=z()=0 } consider the system (12)(14) with homogeneous dirichlet boundary conditions on p, homogeneous initial conditions on u, and c,,,,a, b, according to (15)(17), in l(),l()33, respectively, but with c0. there exist a space dependent strictly positive function >0 and positive semidefinite diagonal matrix functions e,e as well as a nonnegative monotonically non - increasing time dependent function such that(t)121cpt(t)2+e1/2p(t)2+e1/2u(t)2holds for any solution (p, v, u)w(r+;v, h) of (20)(22), wherew(i, v, h)=(c2(i;v)c1(i;l2())c(i;v))(c2(i;l2())c1(i;v))c(i;h),v = h01(),h = l2()3,v=h-1() (the dual of v). more precisely we have e()=diag(e11(),e22(),e33() and e()=diag(e11(),e22(),e33()) with (upon identification 1x,2y,3z)(43)eii>0,eii>0oni, where(44)i={:i()>0}{:x()=y()=z()=0 } note that it suffices to impose homogeneous initial conditions on u in 0={:x()=y()=z()=0 } only.remark 2here we have assumed that a weak solution (p, v, u)w(r+,v, h) of (12)(14) exists. indeed, existence and uniqueness at a first glance should be establishable for c0 along the usual lines (cf., e.g.,) : 1.galerkin approximation, i.e., replacing vvh by a sequence of finite dimensional nested subspaces vnvnhn whose union is dense in vvh, and which satisfy hn for all vn (cf. (23)) ; local in time existence of the galerkin solutions (pn, vn, un)w([0,t],v, h) follows from linearity of the system;2.uniform energy estimates of the galerkin solutions in w([0,t];v, h), which can be done as above, replacing the test and ansatz spaces by their finite dimensional counterparts vn, hn, using the topology of the infinite dimensional spaces v, h on them ; the additional c(0,t;v) bound on 2pnt2 follows directly from (20).3.these uniform estimates imply existence of weakly convergent subsequence whose weak limits by linearity solve the system (20)(22) in a weak sense provided the coefficients are in l() ; global in time existence follows from the uniform energy estimates.however, there is a gap in this proof : since, e.g., e22=e33=e22=e33=0 in x={:x()>0andy()=z()=0 }, we only get boundedness of the l2(x) norm of the first component of pn and of un, so only this component converges weakly, while the other two components might diverge. still we get existence of a very weak solution, i.e., (p, v, u)(c2(0,t;(h2()))c1(0,t;l2()))c2(0,t;l2())c1(0,t;(h1()3)) such that(45)2pt2(t)dx+pt(t)dx+p(t)dx+v(t)dx-p(t)dx+u(t)dx=0h2(),(46)ut(t)dx+(au(t))dx-p(t)(b)dx=0h1()3,(47)vt(t)=p(t)in this way.corollary 1let the assumptions of theorem 1 be satisfied with additionally bw1,()33 and mini{1,2,3}infii()>0, where i={:i()>0}.then for any t>0 there exists a solution (p, v, u)(c2(0,t;(h2()))c1(0,t;l2()))c2(0,t;l2())c1(0,t;(h1()3)) of (45)(47), t(0,t), for which a nonnegative monotonically non - increasing time dependent function exists such that(t)1cpt(t)2for all t(0,t).proofwe proceed as described in remark 2 with the additional assumption vnh2(),hnh1() on the test spaces. along the lines of the proof of theorem (1) we obtain uniform boundedness of pntc(0,t;l2())=2vnt2c(0,t;l2()) for the galerkin solutions (pn, vn, un) of (20)(22). from this and the time differentiated version of (21) with =unt (note that c=0) we obtain uniform boundedness of untc(0,t;(h1()3)). altogether we have existence of a uniform constant k>0 such that for arbitrarily fixed finite time t:pntc(0,t;l2())=2vnt2c(0,t;l2())k, pnc(0,t;l2())=vntc(0,t;l2())k(1+t),vnc(0,t;l2())k(1+t)2,untc(0,t;(h1()3))kunc(0,t;(h1()3))k(1+t),2pnt2c(0,t;(h2()))k(1+t)2,where we have used (45) to obtain the latter estimate. the function here is set to 1min with from theorem 1, where min = min{1,infii }, i=14c2cp22(1 + 12(fii-1))-1(fii-1), fii = min{2,2a2+ad+d2+(2a2+ad+d2)2-(2a+d)2(a - d)2(a - d)2}>1 a=i, d=j+k, j, ki,=a+d. here we have assumed that a weak solution (p, v, u)w(r+,v, h) of (12)(14) exists. indeed, existence and uniqueness at a first glance should be establishable for c0 along the usual lines (cf., e.g.,) : 1.galerkin approximation, i.e., replacing vvh by a sequence of finite dimensional nested subspaces vnvnhn whose union is dense in vvh, and which satisfy hn for all vn (cf. (23)) ; local in time existence of the galerkin solutions (pn, vn, un)w([0,t],v, h) follows from linearity of the system;2.uniform energy estimates of the galerkin solutions in w([0,t];v, h), which can be done as above, replacing the test and ansatz spaces by their finite dimensional counterparts vn, hn, using the topology of the infinite dimensional spaces v, h on them ; the additional c(0,t;v) bound on 2pnt2 follows directly from (20).3.these uniform estimates imply existence of weakly convergent subsequence whose weak limits by linearity solve the system (20)(22) in a weak sense provided the coefficients are in l() ; global in time existence follows from the uniform energy estimates. galerkin approximation, i.e., replacing vvh by a sequence of finite dimensional nested subspaces vnvnhn whose union is dense in vvh, and which satisfy hn for all vn (cf. (23)) ; local in time existence of the galerkin solutions (pn, vn, un)w([0,t],v, h) follows from linearity of the system ; uniform energy estimates of the galerkin solutions in w([0,t];v, h), which can be done as above, replacing the test and ansatz spaces by their finite dimensional counterparts vn, hn, using the topology of the infinite dimensional spaces v, h on them ; the additional c(0,t;v) bound on 2pnt2 follows directly from (20). these uniform estimates imply existence of weakly convergent subsequence whose weak limits by linearity solve the system (20)(22) in a weak sense provided the coefficients are in l() ; global in time existence follows from the uniform energy estimates. however, there is a gap in this proof : since, e.g., e22=e33=e22=e33=0 in x={:x()>0andy()=z()=0 }, we only get boundedness of the l2(x) norm of the first component of pn and of un, so only this component converges weakly, while the other two components might diverge. still we get existence of a very weak solution, i.e., (p, v, u)(c2(0,t;(h2()))c1(0,t;l2()))c2(0,t;l2())c1(0,t;(h1()3)) such that(45)2pt2(t)dx+pt(t)dx+p(t)dx+v(t)dx-p(t)dx+u(t)dx=0h2(),(46)ut(t)dx+(au(t))dx-p(t)(b)dx=0h1()3,(47)vt(t)=p(t)in this way. let the assumptions of theorem 1 be satisfied with additionally bw1,()33 and mini{1,2,3}infii()>0, where i={:i()>0}. then for any t>0 there exists a solution (p, v, u)(c2(0,t;(h2()))c1(0,t;l2()))c2(0,t;l2())c1(0,t;(h1()3)) of (45)(47), t(0,t), for which a nonnegative monotonically non - increasing time dependent function exists such that(t)1cpt(t)2for all t(0,t). we proceed as described in remark 2 with the additional assumption vnh2(),hnh1() on the test spaces. along the lines of the proof of theorem (1) we obtain uniform boundedness of pntc(0,t;l2())=2vnt2c(0,t;l2()) for the galerkin solutions (pn, vn, un) of (20)(22). from this and the time differentiated version of (21) with =unt (note that c=0) we obtain uniform boundedness of untc(0,t;(h1()3)). altogether we have existence of a uniform constant k>0 such that for arbitrarily fixed finite time t:pntc(0,t;l2())=2vnt2c(0,t;l2())k, pnc(0,t;l2())=vntc(0,t;l2())k(1+t),vnc(0,t;l2())k(1+t)2,untc(0,t;(h1()3))kunc(0,t;(h1()3))k(1+t),2pnt2c(0,t;(h2()))k(1+t)2,where we have used (45) to obtain the latter estimate. the function here is set to 1min with from theorem 1, where min = min{1,infii }, i=14c2cp22(1 + 12(fii-1))-1(fii-1), fii = min{2,2a2+ad+d2+(2a2+ad+d2)2-(2a+d)2(a - d)2(a - d)2}>1 a=i, d=j+k, j, ki,=a+d. according to our stability analysis, we have no estimate on the l2-wrt time norm of ||v|| (cf. therefore, we will also numerically investigate the case, where c is set to zero (i.e. we omit cv in (13)) and will denote this reduced formulation by rpml. we are aware of the fact that this rpml formulation will not achieve perfect matching. however, as numerical results will demonstrate, the additional error compared to the full pml formulation is small, and it strongly increases the stability in case of thin damping layers. furthermore, we want to state that the rpml is a true pml in case of 1d as well as 2d computations. in these cases, we do not need the additional scalar auxiliary variable v and so c is not present. e.g., in 2d we just need the auxiliary vector variable u=(ux, uy)t leading to a total number of just three unknowns in the pml region. this can be also seen by analyzing (12)(14), e.g., assuming waves in the xy - plane. then and c get zero (z=0) resulting in just three scalar equations for p, ux, uy. so an error just occurs in 3d, when waves propagate towards corners, where all three damping coefficients are active. the setup of our numerical example for investigating the pml formulation is displayed in fig. 1. on the surface of the sphere we set a dirichlet boundary condition for all nodes according to the following functionf(t)=ddte-2(f0t-1)2.we choose the propagation region to be /2 (being the wavelength given by =c / f0 with c the speed of sound) and use pml regions with /8,/4 and /2. the propagation as well as pml region are discretized with tetrahedra finite elements of first order having an average edge length of about /16. thus, the pml is discretized in thickness direction with two finite elements for the /8 layer, with four finite elements for the /4 layer, and with eight finite elements for the /2 layer, respectively. furthermore, we use an implicit newmark time stepping scheme with =0.25 and =0.5 and we choose a time step size of t=1/(80f0). for the first investigations concerning the accuracy, we use the inverse distance damping functions as given in section 2. for this simple setup however, to just measure the error introduced by the pml, we compute a reference solution using the same computational mesh in the propagation region and use instead of the pml region an additional large propagation region. we consider the evolution of the l2-error at each time step tn(48)errorpml(tn)=1npropi=1nproppi(tn)-pnref(tn)2.in (48) pi denotes the solution at each fe node i using the pml formulation, piref the reference solution at fe node i and nprop the number of nodes within the propagation region. in a first investigation, we computed the acoustic pressure over time applying the pml formulation and compare the results with our reference solution at the three observation points p1,p2 and p3. 4. from the three graphs, we can clearly see the improvement in the solutions with increasing pml thickness. the main difference, as expected, is given for the pressure at observation point p3, which is the corner point between propagation region and pml region (see fig., the results demonstrate that the reduced pml formulation denoted by rpml just has slightly worse properties than the full pml formulation and the differences vanishes almost completely for a damping layer thickness of /2 (see fig. 4). based on the obtained results we further computed the l2-norm of the error as given in (48). in fig. 5 we show the obtained error introduced by the pml and rpml with a thickness of /8 and /4. again, we observe the decrease in the error over time when increasing the pml thickness. we had to stop the computation of the error at t = 40 s, since at this time the reference solution already showed first reflections back to the propagation region. therefore, in order to investigate the stability, we also computed the overall acoustic energy at each time step tn within the propagation region by the following formula(49)eacoustic(tn)=prop02v(tn)v(tn)+p(tn)220c2dx.in (49) 0 denotes the mean density of the fluid and v the acoustic particle velocity. for this long term stability study, we perform computations for all three cases of layer thicknesses (/8,/4 and /2) and use the two different damping functions constant and inverse distance as introduced in section 2. the acoustic particle velocity v is related to the acoustic pressure p via the linear momentum conservation0vt=-p.therewith, in a time discrete setting using a trapezoidal scheme, we obtainv(tn)=v(tn-1)-t0p(tn),which is inserted into (49). fig. 6 displays the computed acoustic energy within the propagation region for the reference solution and the pml as well as rpml formulations. the first main observation is that for the /8 damping layer the results for both damping functions get instable. canceling the critical term in the pml formulation (denoted by rpml) as revealed by the stability proof, leads to stable results (see fig. the second main observation is that both damping functions results in stable long time computations for layer thicknesses of /4 and /2. furthermore, applying the rpml formulation achieves in all cases a stronger damping behavior of the acoustic energy for increasing time. furthermore, when comparing the energy curves with the reference solution, we may clearly state that the inverse distance damping function (both for pml and rpml) provides the most accurate results. as a practical application of our pml formulation, we consider the computation of flow induced sound by applying lighthill s analogy, which results in an inhomogeneous wave equation(50)1c22pt2-p=(t).here, t denotes lighthill s tensor, which for incompressible flows and isentropic state can be approximated by(51)tfvvwith v the flow velocity and f the mean density of the fluid. for all details about our formulation, our focus is on the application of the pml formulation and so we just briefly discuss the flow computation and then the acoustic results. 7 displays the geometry of the side view mirror and the computed flow field for a characteristic time step. we have computed the flow field by applied a les (large eddy simulation) on a grid with 6.5 million hexahedra applying ansys fluent very similarly as reported in. in order to preserve the acoustic energy, we have first computed the acoustic sources within our fe formulation on the fine flow grid. in a second step, we have performed a conservative interpolation to the coarser acoustic grid. 8 displays the acoustic field around the side view mirror on two slices for a characteristic time step. one can clearly see the propagating waves which are then absorbed within the pml region. we have used a quite coarse acoustic grid with about 340.000 elements having about four elements within the pml thickness and applying an inverse distance damping function. as in our numerical test case (see section 4), we used a fully implicit newmark time discretization scheme and observed no instabilities for our pml formulation. therewith, we can state that also for complex computational setups with a broadband acoustic field the pml formulation works effective. in addition, the few additional auxiliary unknowns within the pml region result in a fast computation. finally, fig. 9 displays the computed sound pressure level (spl)1 over the frequency range at the indicated monitoring point compared to measurements. the setup of our numerical example for investigating the pml formulation is displayed in fig. 1. on the surface of the sphere we set a dirichlet boundary condition for all nodes according to the following functionf(t)=ddte-2(f0t-1)2.we choose the propagation region to be /2 (being the wavelength given by =c / f0 with c the speed of sound) and use pml regions with /8,/4 and /2. the propagation as well as pml region are discretized with tetrahedra finite elements of first order having an average edge length of about /16. thus, the pml is discretized in thickness direction with two finite elements for the /8 layer, with four finite elements for the /4 layer, and with eight finite elements for the /2 layer, respectively. furthermore, we use an implicit newmark time stepping scheme with =0.25 and =0.5 and we choose a time step size of t=1/(80f0). for the first investigations concerning the accuracy, we use the inverse distance damping functions as given in section 2. for this simple setup however, to just measure the error introduced by the pml, we compute a reference solution using the same computational mesh in the propagation region and use instead of the pml region an additional large propagation region. we consider the evolution of the l2-error at each time step tn(48)errorpml(tn)=1npropi=1nproppi(tn)-pnref(tn)2.in (48) pi denotes the solution at each fe node i using the pml formulation, piref the reference solution at fe node i and nprop the number of nodes within the propagation region. in a first investigation, we computed the acoustic pressure over time applying the pml formulation and compare the results with our reference solution at the three observation points p1,p2 and p3. 4. from the three graphs, we can clearly see the improvement in the solutions with increasing pml thickness. the main difference, as expected, is given for the pressure at observation point p3, which is the corner point between propagation region and pml region (see fig., the results demonstrate that the reduced pml formulation denoted by rpml just has slightly worse properties than the full pml formulation and the differences vanishes almost completely for a damping layer thickness of /2 (see fig. 4). based on the obtained results we further computed the l2-norm of the error as given in (48). in fig. 5 we show the obtained error introduced by the pml and rpml with a thickness of /8 and /4. again, we observe the decrease in the error over time when increasing the pml thickness. we had to stop the computation of the error at t = 40 s, since at this time the reference solution already showed first reflections back to the propagation region. therefore, in order to investigate the stability, we also computed the overall acoustic energy at each time step tn within the propagation region by the following formula(49)eacoustic(tn)=prop02v(tn)v(tn)+p(tn)220c2dx.in (49) 0 denotes the mean density of the fluid and v the acoustic particle velocity. for this long term stability study, we perform computations for all three cases of layer thicknesses (/8,/4 and /2) and use the two different damping functions constant and inverse distance as introduced in section 2. the acoustic particle velocity v is related to the acoustic pressure p via the linear momentum conservation0vt=-p.therewith, in a time discrete setting using a trapezoidal scheme, we obtainv(tn)=v(tn-1)-t0p(tn),which is inserted into (49). fig. 6 displays the computed acoustic energy within the propagation region for the reference solution and the pml as well as rpml formulations. the first main observation is that for the /8 damping layer the results for both damping functions get instable. canceling the critical term in the pml formulation (denoted by rpml) as revealed by the stability proof, leads to stable results (see fig. the second main observation is that both damping functions results in stable long time computations for layer thicknesses of /4 and /2. furthermore, applying the rpml formulation achieves in all cases a stronger damping behavior of the acoustic energy for increasing time. furthermore, when comparing the energy curves with the reference solution, we may clearly state that the inverse distance damping function (both for pml and rpml) provides the most accurate results. as a practical application of our pml formulation, we consider the computation of flow induced sound by applying lighthill s analogy, which results in an inhomogeneous wave equation(50)1c22pt2-p=(t).here, t denotes lighthill s tensor, which for incompressible flows and isentropic state can be approximated by(51)tfvvwith v the flow velocity and f the mean density of the fluid. for all details about our formulation we refer to. here, our focus is on the application of the pml formulation and so we just briefly discuss the flow computation and then the acoustic results. 7 displays the geometry of the side view mirror and the computed flow field for a characteristic time step. we have computed the flow field by applied a les (large eddy simulation) on a grid with 6.5 million hexahedra applying ansys fluent very similarly as reported in. in order to preserve the acoustic energy, we have first computed the acoustic sources within our fe formulation on the fine flow grid. in a second step, 8 displays the acoustic field around the side view mirror on two slices for a characteristic time step. one can clearly see the propagating waves which are then absorbed within the pml region. we have used a quite coarse acoustic grid with about 340.000 elements having about four elements within the pml thickness and applying an inverse distance damping function. as in our numerical test case (see section 4), we used a fully implicit newmark time discretization scheme and observed no instabilities for our pml formulation. therewith, we can state that also for complex computational setups with a broadband acoustic field the pml formulation works effective. in addition, the few additional auxiliary unknowns within the pml region result in a fast computation. finally, fig. 9 displays the computed sound pressure level (spl)1 over the frequency range at the indicated monitoring point compared to measurements. we have introduced an efficient pml formulation for the second order wave equation. by using auxiliary variables, we achieve at a time domain formulation without convolution integrals. for our un - split pml formulation we observed a term within our formulation which we could not bound due to its adverse sign, and which may be a source for long time instabilities. the rigorous numerical tests revealed that for layer thicknesses of /4 and /2 the pml formulation for both investigated damping functions (constant and inverse distance) achieves long time stability. omitting the critical term, we arrive at our rpml formulation, which strongly improves the stability as demonstrated by the numerical examples. therewith, we can summarize the three main results as follows : (1) we can prove long time stability of our formulation ; just one critical term containing the mixed products of the damping functions, may disturb the stability ; omitting this term yields long time stability for arbitrary layer thickness, (2) numerical tests show that the full pml formulation is long time stable for thick enough damping layers (in our examples at least /4), and (3) numerical tests demonstrate that for thin damping layers one should use the rpml formulation to achieve long time stability. | we consider the second order wave equation in an unbounded domain and propose an advanced perfectly matched layer (pml) technique for its efficient and reliable simulation. in doing so, we concentrate on the time domain case and use the finite - element (fe) method for the space discretization. our un - split - pml formulation requires four auxiliary variables within the pml region in three space dimensions. for a reduced version (rpml), we present a long time stability proof based on an energy analysis. the numerical case studies and an application example demonstrate the good performance and long time stability of our formulation for treating open domain problems. |
subjects and data collection : the subjects were dogs examined at kakogawa animal hospital, kakogawa, hyogo, japan between march 2005 and april 2010. cases were excluded, if permission of the owner, interictal, hematological and biochemical data from physical and neurologic examinations, csf findings or mri data were unavailable. routine csf analysis included the total nucleated cell count (tncc) and measurement of microprotein concentration using a micro total protein kit (wako pure chemicals, osaka, japan). protein concentration was considered to be elevated if>25 mg / dl, and pleocytosis was judged to be present if tncc in the csf was>5 /l. if csf analysis was abnormal, titers for infectious causes of encephalitis were evaluated, including canine distemper virus, ehrlichia canis, neospora canis, toxoplasma gondii, aspergillus sp. and cryptococcus sp. mri was performed with 0.2 t system (signa, ge medical imaging systems, tokyo, japan), and mri findings were evaluated independently of csf analysis. routine imaging included t2, fluid attenuation inversion recovery and t1-weighted sequences performed before and after administration of intravenous gadolinium (gadoteridol, eizai, tokyo, japan). images were obtained in at least 3 orthogonal planes relative to the region of interest. clinical diagnosis of ie was defined retrospectively by exclusion of cases that with a positive csf titer or fungal or bacterial culture or showed morphological abnormality on mri or based on the results of clinical and neurologic examinations. cases that responded to anticonvulsant therapy (oral phenobarbital or bromide) and required this therapy after a presumptive diagnosis were included in the ie group. se due to intracranial diseases was diagnosed when abnormalities were found in brain mri, csf tap results or clinical course after presentation. normal csf was sampled from a control group of 18 healthy domestic dogs with the owner s consent. csf preparation : csf preparation for extraction and derivatization of low - molecular weight metabolites was performed as described by kuhara. collected csf was immediately centrifuged at 5,000 g for 10 min at 4c, and the supernatant was transferred to a clean tube and stored at 80c until use. to extract low - molecular - weight metabolites, 25 l of csf was mixed with 900 l of 70% ethanol containing 10 ng of 2-isopropyl malic acid and centrifuged at 15,000 g for 10 min at 4c. metabolites in the dried residue were converted to trimethylsilyl derivatives with 100 l of n, o - bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane (10:1) (thermo, tokyo, japan) and analyzed by gc - ms. gc - ms analysis and data processing : gc - ms analysis was carried out using a gc - ms - qp2010plus system (shimadzu, kyoto, japan) with inert cap 5 ms / np (0.25 mm i.d. 30 m, 0.25 m ; gl science, tokyo, japan), as described in kuhara. chromatogram acquisition, detection of mass spectral peaks and waveform processing were performed using shimadzu gc - ms solution software (ver. identification of low - molecular - weight metabolites was carried out using the library of the national institute of standards and technology. semiquantitative assessment was performed using the peak intensity of 2-isopropylmalic acid as an internal standard. multiple classification analysis and statics : a dataset for multiple classification analysis was compiled from the metabolic profiling data. a three - dimensional matrix was constructed based on sample names (observations), metabolite name (variable indices) and normalized peak intensities (variables). partial least square discriminant analysis (pls - da) was performed in metaboanalyst 2.0. subject characteristics and csf analysis : a total of 16 cases met the inclusion criteria for canine ie. there were subtle neurological deficits in some cases, including delayed proprioceptive positioning on one side, in interictal neurological examinations (table 1table 1.characteristics of subjectsbreedsexage at csfcollectionage at first seizure onsetneurologicstatus at restdiagnosiscsf protein(mg / dl)beagles0.80.8nie15.7pomeranianc4.33.9nie46.8chihuahuaf3.03.0nie33.0bernese mountainc4.33.4nie9.9chihuahuam4.92.0nie26.4yorkshire terrierc1.13.4nie8.3toy poodlem2.00.5nie13.2beaglec1.61.4nie15.2miniature dachshundm5.63.5nie17.1shetland sheepdogc14.27.2nie3.6chihuahuam0.50.5nie27.2mongs13.57.3nie39.5welsh corgim8.26.4nie16.8welsh corgic7.27.5nie20.3miniature schnauzerc13.68.2aie16.4yorkshire terriers3.52.8nie16.4chihuahuam2.92.6nse (mue)2.3pugm3.23.2ase (mue)17.7labrador retrieverm8.58.2ase (mue)43.0shih - tzum3.53.5ase (mue)8.2mongf6.75.8ase (mue)194.7mongm13.913.9ase (mue)5.8mongc15.014.9ase (mue)2.4golden retrieverf8.17.3nse (brain tumor)10.0 labrador retrieverf7.87.8ase (brain tumor)80.7welsh corgim8.78.6ase (brain tumor)89.0golden retrieverm10.610.6nse (brain tumor)2.6shibam11.111.0nse (brain tumor)83.7welsh corgic11.811.8ase (brain tumor)56.2shibam12.512.5ase (brain tumor)30.5mongm15.915.8ase (brain tumor)36.3pugm3.53.5ase (brain tumor)64.5beaglef6.16.1nse (meningoencephalitis)29.7french bulldogf6.36.3ase (meningoencephalitis)202.9pugm13.313.2ase (meningoencephalitis)82.9maltesec1.1-ncontrol40.3mongs1.4-ncontrol12.1shibam0.6-ncontrol32.0toy poodlem10.3-ncontrol27.8miniature dachshundc8.3-ncontrol9.2west highland white terriers7.3-ncontrol57.1papillonm0.8-ncontrol21.2shih - tzuc15.2-ncontrol8.6mongc12.0-ncontrol18.2corgif10.4-ncontrol23.2italian greyhoundm7.8-ncontrol20.1welsh corgim2.3-ncontrol17.7maltesef0.6-ncontrol8.9welsh corgim0.5-ncontrol46.3toy poodlem0.5-ncontrol8.1golden retrieverm4.9-ncontrol13.3shibam2.3-ncontrol13.1pomeranianf0.6-ncontrol18.6f, female ; m, male ; c, casted ; s, spayed ; a, abnormal ; n, normal.). the mean age at csf collection was 5.5 4.5 years (median 4.3, range 0.5 to 14.2), and that at first onset of seizure was 3.9 2.6 years (median 3.4, range 0.5 to 8.2). se was diagnosed in 19 cases, including 3 with meningoencephalitis, 7 with meningoencephalomyelitis of undetermined etiology (mue) and 9 with brain tumors. the mean age at csf collection was 8.9 4.0 years (median 8.5, range 2.9 to 15.9), and that at first onset of seizure was 8.8 4.1 years (median 8.2, range 2.6 to 15.8). background data did not differ significantly among the disease categories in the se group. the mean age of the healthy controls was 4.8 4.7 years (median 2.3, range 0.5 to 15.2). age at csf collection in the ie group was slightly lower than that in the se group (p=0.026) and not significantly different from that in the control group. age at csf collection in the se group was significantly higher than that in the control group (p=0.006). the mean protein concentrations in the csf were 22.0 mg / dl (median 18.4, range 8.1 to 57.1 mg / dl) in controls, 20.4 mg / dl (median 16.6, range 3.6 to 46.8 mg / dl) in the ie group and 54.9 mg / dl (median 36.3, range 2.3 to 202.9 mg / dl) in the se group with no significant differences among the groups. f, female ; m, male ; c, casted ; s, spayed ; a, abnormal ; n, normal. evaluation of differences in csf profiles using multiple classification analysis : a total of 60 metabolites identified in the csf were analyzed by pls - da. a. gc - ms data for the control (squares), ie (triangles) and se (circles) groups were analyzed using pls - da, and the results are represented as a score plot. b. loading plot of canine csf metabolites.). the metabolites largely responsible for this separation included ascorbic acid, glyceric acid, threonic acid, glutaric acid, isoleucine, leucine, phosphoric acid, methionine, phenylalanine and pyruvic acid (fig. the levels of 20 metabolites differed significantly among the groups, and 16 metabolites showed significant changes in ie cases compared to controls (fig. 2.levels of metabolites in csf in the control (black), ie (stripes) and se (white) groups. levels are expressed in arbitrary units (see materials and methods).). the levels of glutamic acid, isoleucine, phosphoric acid, malonate, stearic acid, glutaric acid, glutamine, acetic acid, oxanilic acid, glyceric acid, threonic acid and succinic acid increased, and those of ascorbic acid, pyruvic acid and 3-hydroxy-2-butanone decreased in ie. the levels of 13 metabolites differed significantly between ie and se, including higher levels of glutamic acid, isoleucine, methionine and serine in ie. the levels of 9 metabolites were significantly different in se cases compared to controls with glutamic acid, methionine, phenylalanine, ascorbic acid, malic acid, acetone and pyruvic acid all decreasing in se. a. gc - ms data for the control (squares), ie (triangles) and se (circles) groups were analyzed using pls - da, and the results are represented as a score plot. levels of metabolites in csf in the control (black), ie (stripes) and se (white) groups. epileptic seizure is a common neurological disease, but is difficult to diagnose by observation. in most patients, the present study was performed in a population of epileptic dogs encountered commonly in veterinary practice. classification of canine epilepsy as ie or se is currently performed by exclusion of identifiable underlying causes for epileptic seizures, and there is a need to establish a method for rapid diagnosis of these types of epilepsy. recent developments in metabolomics permit mining of metabolic information in disease states and during treatment. the approach is founded on the assumption that a disease signature exists within a given biological sample, but requires no prior knowledge of sample content. suitable body fluids for metabolomics include urine, plasma, serum, saliva and tissue homogenates, all of which carry the signature of biochemical and metabolic responses to a myriad of genetic and environmental influences. metabolomics also holds promise for early diagnosis of disease and for identification of metabolic pathways as targets for disease amelioration. serum metabolomics of cns disorders has been vindicated by identification of blood biomarkers of parkinson s disease. serum is the basic sample for metabolomics, being readily collectable by minimally invasive techniques, but serum metabolic profiles of responders and non - responders to anti - epilepsy drugs may not be clearly distinguishable. this suggests that serum may not always be optimal for evaluation of heterogeneous disorders, such as epilepsy, because of significant blood - brain barrier interference with metabolite exchange between blood and csf. for this reason, we used gc - ms - based canine csf metabolic profiling to detect potential biomarkers applicable for diagnosis of epilepsy. in canine csf, 16 of 60 identified metabolites had significantly different levels in ie cases compared to controls. pls - da separated ie from se and controls, and metabolites with higher loading values in this analysis were identified (fig. the levels of 20 metabolites, including amino acids, fatty acids and organic acids that could be potential diagnostic biomarkers for epilepsy, showed significant differences among the groups (fig. it has been proposed that the balance of excitatory and inhibitory neurotransmitters is a key regulator of seizure. glutamic acid and -aminobutyric acid (gaba) are major excitatory and inhibitory compounds in brain, respectively. in this study, the glutamic acid content in ie increased significantly compared to those in se and in controls. levels of related compounds (glutamine, which is synthesized from glutamic acid and serine) in ie were also high compared to those in controls, whereas gaba levels in ie and se did not differ from that in controls (data not shown). the glutamic acid level in csf is regulated by glutamate transporters located on the surface of epithelial cells of veins, glia and neuronal cells and depends on the concentration in serum. it was also reported that some metabolites in brain are often excluded into extracellular fluid and that the glutamic acid level in csf has been related to the severity of neurological disorder in dog and cat models, although the level in csf has also been found to decrease in canine epilepsy. these findings indicate that a disorder of glutamic acid regulation and metabolism might be related to the pathogenesis of canine ie and that further evaluation of glutamic acid is required as a potential diagnostic biomarker for ie. it was shown that levels of ascorbic acid, pyruvic acid, glyceric acid, threonic acid and glutamine were significantly changed in epilepsy (fig. reactive oxygen species (ros) formation increases during seizures and removal of ros depends on the antioxidant system. the protective effect of antioxidants may partly be due to antioxidant nutrients, such as ascorbic acid and carotenoids. ascorbic acid is a powerful water - soluble antioxidant that has a protective role against oxidative damage in tissues, including in central nervous system. ascorbic acid contents in csf of ie and se cases were significantly lower than that in controls, but there was no significant difference between ie and se. threonic acid, a metabolite of ascorbic acid, was also elevated in both epilepsy groups. these findings indicate that ros - related oxidative stress might be present in the brain in ie and se and that ascorbic acid may be a potent biomarker for epilepsy. further investigation is needed to evaluate these findings in chronic intracranial disease, but it is possible that ascorbic acid and its metabolite in csf could be a marker of epileptic symptoms. the decrease of pyruvic acid and increase of glyceric acid in epilepsy were significant in se (fig. high glycolytic activity is common in tumors known as the warburg effect, and serum levels of metabolites related to it are also known to be changed in tumors. thus, se cases with tumors might have contributed to the differences in the metabolites among the groups in this study. thus, in cases with epileptic symptoms, investigation of metabolic changes in csf in those with brain tumors is needed to distinguish these cases from those with inflammatory disease. in conclusion, gc - ms - based canine csf metabolic profiles in ie differed from those in se and in healthy controls. glutamic acid may be a potent biomarker for ie, and ascorbic acid and threonic acid may be useful for general detection of epilepsy. | abstractepilepsy is a common neurological disorder with seizures, but diagnostic approaches in veterinary clinics remain limited. cerebrospinal fluid (csf) is a body fluid used for diagnosis in veterinary medicine. in this study, we explored canine epilepsy diagnostic biomarkers using gas chromatography - mass spectrometry (gc - ms)-based metabolic profiling of csf and multivariate data analysis. profiles for subjects with idiopathic epilepsy differed significantly from those of healthy controls and subjects with symptomatic epilepsy. among 60 identified metabolites, the levels of 20 differed significantly among the three groups. glutamic acid was significantly increased in idiopathic epilepsy, and some metabolites including ascorbic acid were changed in both forms of epilepsy. these findings show that metabolic profiles of csf differ between idiopathic and symptomatic epilepsy and that metabolites including glutamic acid and ascorbic acid in csf may be useful for diagnosis of canine epilepsy. |
anaemia in patients with chronic kidney disease (ckd) is a complex condition that is associated with morbidity and mortality and a decline in quality of life (qol) [14 ]. erythropoiesis - stimulating agents (esas) have been shown to effectively improve and maintain haemoglobin (hb) levels and reduce the need for red - cell transfusions and have become a standard of care for managing renal anaemia [711 ]. since the introduction of the first esa in 1989, advances in treatment have focused on the needs of patients and healthcare providers, including the development of longer - acting esas and various dosing strategies. guidelines for renal anaemia were introduced to provide a framework for treating patients appropriately, recommending the range in which hb concentrations should be maintained with some discussion of individualized treatment [713 ]. as our knowledge of managing renal anaemia with esas has grown, early observational studies in dialysis and non - dialysis ckd populations described associations between low hb levels and increased risk of mortality and morbidity. furthermore, observational and some prospective studies reported that higher or normalized hb levels in ckd patients were not associated with increased risk of adverse outcomes [2,1618 ] and might improve mortality and morbidity outcomes, particularly cardiovascular outcomes, and qol [14,1823 ]. these and other findings led to a series of randomized controlled trials in dialysis patients and then in non - dialysis patients to assess the efficacy and safety of targeting high hb levels with esas. while it was hypothesized that high hb would provide morbidity and mortality benefits, results from these trials consistently showed that intervention with esas to a high hb target provided no clinical benefit compared with the control treatment [2427 ] and, in some situations, increased morbidity and mortality risk [2730 ]. the publication of data from the trials investigating high hb targets in non - dialysis patients in 2006 led to important changes to renal anaemia guidelines. the 2004 european best practice guidelines (ebpgs) for renal anaemia recommended a hb target of > 11 g / dl for most patients, with an exact target defined by the individual patient s gender, age, ethnicity, activity, comorbid conditions and disease state. the upper hb limit was generally to be maintained below 14 g / dl, particularly for haemodialysis patients, and below 12 g / dl for ckd patients with severe cardiovascular disease or diabetes. in 2009 and 2010, the european renal best practice working group (formerly ebpg) recommended that all ckd patients should be treated to a target hb between 11 and 12 g / dl, with the exception of patients with type-2 diabetes mellitus (t2 dm) and a history of stroke (recommended target of 1012 g / dl). the working group recognized that hb levels for individual patients would probably fall outside this narrow target over the course of treatment but recommended that levels above 13 g / dl should not intentionally be exceeded, and levels above 12 g / dl should not be targeted in patients with t2 dm. similar changes had been made to the kidney disease outcomes quality initiative guidelines in 2007. treatment goals for patients with renal anaemia will continue to be refined as our knowledge broadens. however, the discordance between observational and clinical trial data and the significant changes to guidelines are challenging for clinicians [3235 ] and patients, particularly in view of the recommendation to treat all ckd patients to a narrow hb target. patients differ by disease severity, age, medical history, healthcare behaviours and other factors. there is significant interpatient variability in the response to esas, as the complex interactions among physiological, environmental and medical factors that affect erythropoiesis vary among patients [4043 ]. the individual patient s hb levels may fluctuate (i.e. intrapatient variability). in view of these new challenges, there is a need to reassess individualized treatment for renal anaemia. the subsequent sections will review clinical data regarding high hb targets, esa dose and hb variability, followed by a discussion of individualized anaemia therapy. table 1 summarizes data from four randomized controlled trials that assigned ckd patients to intervention with an esa to achieve a high versus low hb target the normal hematocrit cardiac trial (nhct), the correction of hemoglobin and outcomes in renal insufficiency (choir) trial, the cardiovascular risk reduction by early anemia treatment with epoetin beta (create) trial and the more recent trial to reduce cardiovascular events with aranesp therapy (treat). outcomes in pivotal randomized controlled trials examining low and high hb / hct targets in ckd populations with anaemia hazard or risk ratio (95% confidence interval) unless otherwise noted (1 favours low target). primary study endpoint. cabg, coronary artery bypass grafting ; chf, congestive heart failure ; cvd, cardiovascular disease ; esrd, end - stage renal disease ; hb, haemoglobin ; hct, haematocrit ; hd, haemodialysis ; hf, heart failure ; ihd, ischaemic heart disease ; mi, myocardial infarction ; ptca, percutaneous transluminal coronary angioplasty ; pvd, peripheral vascular disease ; rrt, renal replacement therapy ; t2 dm, type-2 diabetes mellitus ; tia, transient ischaemic attack. the nhct study randomized haemodialysis patients with congestive heart failure (chf) or ischaemic heart disease who had been receiving epoetin alfa to continue treatment to achieve a haematocrit (hct) target of 42% (normalized hct) versus 30%. by 6 months, the mean hct had increased to the target range in the normal hct group, corresponding to a 3-fold increase in the epoetin alfa dose. the study was halted early because of a trend towards increased risk of the composite endpoint of death or first non - fatal myocardial infarction (mi) associated with the normal hct target [hazard ratio (hr) = 1.3 ; 95% confidence interval (95% ci) 0.91.9 ]. there was no difference between treatment arms with regard to secondary endpoints with the exception of transfusion rate, which was significantly lower in the normal hct compared with the low hct group (21 vs 31% ; p 30 000 iu / week do not confer additional harm or benefit in elderly haemodialysis patients. more detailed dosing algorithms for esa therapy would be helpful to clinicians, particularly for patients who do not achieve target hb levels in whom large doses would be ineffective and expensive and might increase risk. data from well - designed, controlled trials are needed to more clearly define whether risk is due to esa dose alone or to underlying conditions that require high dosing to obtain a sufficient response. the phase iii clinical evaluation of the dose of erythropoietins trial (nct00827021) should provide some critical data. this fixed - dose study, initiated in 2009, randomized haemodialysis patients with hb 150 000 patients for each study) to examine the relationship between hb patterns and adverse outcomes for 6-month periods in 2003 and 2004. the first study defined comparison groups by the monthly measured hb concentration (low [12 g / dl with esa treatment should be approached with caution and, as noted earlier, is not recommended by guidelines across the spectrum of ckd. it will also be prudent to minimize the esa dose, as well as hb variability, until more definitive data assessing these markers of risk become available. a practical management strategy is to first conduct a global assessment of the patient to determine the hb threshold at which esa therapy should be initiated. haemoglobin levels consistently < 11 g / dl is a general threshold for initiating therapy, but a lower hb threshold may be advisable for higher - risk dialysis and non - dialysis patients such as those with diabetes or cardiovascular disease unless symptomatic anaemia is present. a patient s iron status should be evaluated and supplementation initiated prior to initiation of esa therapy for patients with iron deficiency. the benefits and risks of esa treatment should be discussed openly with the patient, as well as treatment goals, which are likely to differ according to the lifestyle of the patient (e.g. active vs sedentary). all currently available esas have the same mode of action and have been shown to effectively improve and maintain hb concentration in patients with renal anaemia (reviewed in [5961 ]). however, the pharmacological properties of the various esas differ, which affects dosing frequency options and dosing efficiency (dose required to achieve hb target). thus, selecting the type of esa that best matches the needs of the patient is a relevant consideration for individualized treatment. a patient who is not on dialysis may prefer the convenience of subcutaneous self - administration and a less frequent dosing schedule, while this may not be an advantage to a patient who receives routine dialysis. esa treatment should be initiated in iron - replete patients at a low dose and then titrated incrementally to avoid rapid increases in hb and to achieve the hb target at the lowest possible dose. if increasing the esa dose does not lead to the expected rise in hb, further increases should be contemplated only after careful risk evaluation of the individual patient. hopefully, updates to the treatment guidelines will address the issue of maximum allowable esa dose. the 2004 ebpg defined resistance to esas as the failure to achieve the hb target while receiving more than ~ 20 000 iu / week of epoetin alfa / beta or ~ 100 g / week of darbepoetin alfa or the need for consistently high esa doses to maintain target hb. until more data become available to better understand the benefit - to - risk profile of treating various ckd patient populations to different targets, the hb target will need to remain narrow for all ckd patients. for patients with ckd and significant comorbidities (e.g. cardiovascular disease or diabetes), a cautious approach is warranted with a hb target of 1011 g / dl with levels not exceeding 12 g / dl. on the other hand, a hb target of 1112 g / dl is practical for ckd patients without significant comorbidities with the realization that hb levels may rise above this limit on occasion because of hb variability. variation in hb levels is expected, but large fluctuations and persistently low or high hb levels outside the target should be avoided. if a definite trend of increasing or decreasing hb levels has been determined, esa dose changes should be implemented after other factors that may impact hb levels (e.g. infection) have been addressed. dose changes should be incremental to reduce the risk of hb levels cycling across and outside the target. in view of these parameters, several treatment- and patient - related factors should be considered for the individual patient to minimize the esa dose and to help maintain stable hb levels within target [6367 ]. switching the esa type, route of administration or the dosing frequency may help to improve esa dose efficiency and hb stability in some patients. table 2 summarizes some of the patient - related factors and intercurrent events that are associated with hb variability and resistance to esas. several such factors, including iron status, inflammation and infection, are modifiable, and strategies can be implemented to mitigate their impact [6367 ]. acute infections should be treated with antibiotics, and the presence of occult infections should be evaluated in patients who become hyporesponsive to esa therapy. for dialysis patients, high - quality dialysis water and biocompatible membranes, daily dialysis and protein - energy malnutrition may exacerbate inflammation ; thus, it is important to follow nutritional markers to facilitate early interventions. prior to inpatient procedures, an incremental increase in esa dose may be warranted and should also be considered immediately after a hospitalization to maintain stable hb levels. initiation of certain medications, such as angiotensin - converting enzyme inhibitors, may affect erythropoiesis. in such cases, alternative medications can be considered, the medication dose can be reduced or the esa dose can be increased if appropriate. patient - related factors and intercurrent events that impact hb variability in ckd patients (reviewed in [6367 ]) it is becoming more apparent that a general approach to managing renal anaemia a protocolized, it is affected by the underlying disease, comorbid conditions, the environment and several other factors that differ among patients. selection of the hb target based on the patient s disease state, comorbidities and other characteristics has been an essential part of a treatment strategy. however, the risks associated with high hb targets in recent studies prompted updates to the guidelines to recommend a narrower hb target : 1112 g / dl and not exceeding 13 g / dl for most patients and 1012 g / dl for patients with t2 dm avoiding levels above 12 g / dl, particularly for those at risk of stroke. ultimately, properly designed and powered prospective studies will be needed to better understand the complex relationship between hb concentration, esa dose and underlying disease status. until then, a reasonable strategy is to first discuss the benefits and risks of esas with patients and involve them in the decision - making process. for those electing esa treatment, each patient should be treated to the hb target with the lowest effective esa dose while avoiding large fluctuations in hb levels or prolonged excursions outside the target. this strategy may necessitate changes to the esa dose, dosing frequency and iron supplementation over the course of a patient s treatment and proactive management of conditions that can affect esa responsiveness. while all esas effectively increase hb levels, differences with respect to route of administration, pharmacokinetics and dosing frequency and efficiency should be considered to maximize the benefits of esa treatment for the individual patient. a.l.m.f. contributed to interpretation of data, drafting and revising the article, providing intellectual content of critical importance to the work described and final approval of the version to be published. | individualized strategies for managing renal anaemia with erythropoiesis - stimulating agents (esas) need to be advanced. recent outcomes from clinical studies prompted a narrowing of the guideline - recommended haemoglobin target (1112 g / dl) due to increased mortality and morbidity when targeting higher haemoglobin concentrations. maintaining a narrow target is a clinical challenge, as haemoglobin concentration tends to fluctuate. the goal of individualized treatment is to achieve the haemoglobin target at the lowest esa dose while avoiding significant fluctuations in haemoglobin concentrations and persistently low or high concentrations. this may require changes to the esa dose and dosing frequency over the course of treatment. |
a 43-year - old male patient with cerebral palsy (athetoid type) was diagnosed with cervical disc herniation in addition to a symptom of 1-year history of pain radiating to the upper limb. the patient underwent a laminectomy (c3 - 5), an anterior interbody fusion (c3 - 5), a posterior interbody fusion (c3 - 5), and an artificial bone graft operation 1 year ago. however, he complained of persistent pain mediated by the branch of the left c5 after the operations. he was then referred to the pain clinic of our hospital for inpatient collaborative consultation for treatment. at that time, after due consideration that it was difficult to perform interlaminar epidural block because of the inability to put the patient in the appropriate position and that the stellate ganglion block had no effect, a left c5 selective cervical nerve root block was performed. monitoring devices were set to measure the patient 's electrocardiogram (ekg), blood pressure (bp), and arterial oxygen saturation (spo2). he was placed into the supine position and the puncture area was disinfected with the broad application of betadine solution on the neck and was covered with a sterile gauze dressing. under continuous radiography, while having the neck held to the anterior - superior and adjusting c - arm fluoroscopy to 45 degrees, a needle was advanced via the anterolateral approach. when a 5 cm - block needle reached the superior articular process of the left c5 and was advanced to the neural groove, care was taken to confirm that placement of the needle tip did not across the halfway point of the posterior cervical articulate pillar. after injection of 1.5 ml contrast medium, radicular and epidural contrasts were confirmed, but vascular or intraspinal contrasts were not observed (fig. 1). when the patient complained of paresthesia caused by injection needles, a mixed solution of 3 ml 1% mepivacaine and 40 mg triamcinolone was infused. prior to the infusion, no body fluids such as regurgitated blood or cerebrospinal fluid were observed with aspiration. approximately 2 minutes after administration, the patient felt weak in the right arm as well as the left arm, however when his motor ability was grade 3 of 5 in both upper limbs, all senses regarding pain, temperature, and position disappeared. one minute later, the motor ability of both upper limbs was entirely gone, immediately followed by disappearance of the sensation and motor ability of the lower limbs, and the patient complained of labored respiration. at that time, his bp was 150/90 mmhg, spo2 at pulse oximetry was 98%, and ekg indicated a normal sinus rhythm. as his anxiety and breathing difficulty worsened, midazolam 2 mg was administered and the patient 's respiration was assisted with 100% oxygen. his spontaneous breathing was maintained, so that positive pressure ventilation was not employed. within 45 minutes he awakened, with no signs of labored respiration, and when his motor ability was was measured, it showed grade 3 at the upper limbs, grade 2 at the lower limbs, which revealed signs of mild recovery. 2 hours after the patient was transferred to the postanesthetic care unit (pacu), he regained normal motor ability and sensation completely. when his vital signs were confirmed as normal, he was transferred to the general ward. continuous fluoroscopic - guided cervical nerve root block is used as an non - invasive alternative to surgical treatments and is also used for pre - operative diagnosis to determine dororific branch. there have been various opinions with regards to the safety of selective cervical nerve root block. a study without specific complications recognized potential complications such as penetration to the vital vessels or dural puncture, while there has been an additional report on neurological complications including cerebral and spinal cord infarctions. on selective cervical nerve root block following spine surgery as in the present case, neurological sequela is hard to remove completely even though nerve block is performed with care, and medication is inadvertently injected into the wrong place although radicular and epidural contrasts were successfully achieved. as for the development of quadriplegia during cervical nerve root block, we suspect the following causes : steroid particle - induced anterior spinal artery syndrome, and subdural or intraspinal injection of medicines. anterior spinal artery syndrome is usually seen as a result of infarction along the front of the spinal cord when steroid particles are injected to the vertebral artery or the radicular artery. although bp does not alter and vibration sensation and motion function are retained, the pain and temperature senses are suppressed with complete motor paralysis. even after all sensations are recovered the continuous fluoroscopic images of the present case did not show any angiogram suspicious of intravascular injection or blood aspiration of the needles. however, barker. claimed that infusion of drugs into the radicular artery even in a continuous fluoroscopic - guided operation performed with care may cause anterior spinal artery syndrome. in a study using 0.5 - 2 ml contrast medium, furman. reported that the overall rate of intravascular contrast injections was 19.4% and that observing blood in the needle hub could predict intravascular injection with a specificity of 97%, but with sensitivity of only 45.9%. in addition, hwang. reported the overall intravascular injection rate of 63.4% in a study using 3 ml of contrast medium. therefore, when patients complained of labored respiration, decreased sensation of temperature and pain, and decreased motor function, the possibility of anterior spinal cord infarction caused by the intravascular infusion of steroid particles was initially suspected. in the present case, however, the patient regained normal breathing within 2 hours and recovered motor function as well as sensation. contrast patterns of subdural injection are manifested as an opaque bulging in the front of the vertebral canal and symptoms of a subdural injection (block) include a delayed onset varying from 5 to 30 or more minutes. in the present case, however, contrast images did not hint at subdural injection, while quadriplesia showed up immediately within 2 minutes, unlike the case of subdural injection. complete or partial subarachnoid injection can be hypothesized to cause quadriplegia. for the patient 's clinical presentations, the onset of the symptom occurred within 2 minutes and his sensation and motor function were recovered in 45 minutes, which led us to assume intrathecal injection of medication was the primary cause of transient quadriplegia. during the procedure, however, radiographic findings showed clear images of radicular and epidural contrasts, the placement of the needle did not pass over middle of the facet column, and cerebrospinal fluid (csf) was not detected at aspiration, either. therefore, at the initial stage of the symptom, we did not suspect intrathecal injection of medication. on the contrary to our case, brouwers. mistook the initial symptom of anterior spinal artery syndrome as intrathecal injection of medication. the reasons that subarachnoid injection took place without any abnormal findings on contrast images in the present case may be as follows : first, inserted instruments may have obscured the intrathecal contrasts produced by the injected contrast medium for pilot guide ; second, possible anatomical changes such as adhesion resulting from the operation itself may have caused the needle to be placed subdurally ; third, even though the needle was not placed inside the dura, spread of medication through ripped regions of the dural sleeves induced by the operation might have caused subarachnoid injection. anatomically, the dural sleeve is tethered to the transverse process and dura is surrounded from the nerve root 's take - off point out of the spinal cord to the foraminal lateral boundary. reported that the incidence rate of dural tears after spinal interbody fusion was 3 to 10%. in a study of chen. involving 118 patients, the incidence of dural tears was 3.4%, among which 3/4 of the incidences were found in the axilla of the nerve root. this region has been known for its difficulty of restoration after it is damaged. in the current case, intrathecal injection of contrast medium was not observed. therefore, intrathecal spread of contrast medium through ripped regions of the axilla of the nerve root induced by operation seems to be a more plausible mechanism of transient quadriplegia rather than direct intrathecal injection of the medication. because computed tomography (ct) could not confirm the soft tissue, however, an exact explanation is difficult to compose based on the process of intrathecal injection of medication. a recent publication suggests a guideline of securing the safety of the cervical nerve root blocks : first, facilitate real - time c - arm fluoroscopy ; second, administer a test dose of contrast medium ; third, place the needle toward the posterolateral side of the foramen to avoid inadvertent intravascular injection ; fourth, at performing procedure, use the anterolateral approach to the cervical spine. according to anatomical studies, the radicular artery runs to the front of the vertebral nerves, but the feeding arteries are present anywhere. therefore, it needs real - time monitoring of contrast medium and confirmation of the soft tissue. computed tomography (ct) and ct fluoroscopy are useful in confirming the presence of the soft tissue, but they are not reliable for consistent monitoring. therefore, using digital subtraction should be considered. in light of the study by hwang., it is further recommended that use of more than 3 ml of contrast medium could be helpful in identifying wrong placement of the needles (e.g., unintended intravascular injection). to enhance the safety of the procedures, choice of medications the patient had long - standing post - operative pain, and stellate ganglion block or interlaminal cervical epidural block was difficult to perform. although cervical nerve root block was reluctantly decided upon, the procedure was not easy to perform due to post - operative anatomical alteration., soluble steroid is well known for its capacity of rapid intraspinal clearing effect with short duration. another option would be triamcinolone, which is most frequently used as a topical agent for its anti - inflammatory effects, less sodium retention, and long - duration potency. therefore, we chose triamcinolone. because particulate steroids can induce arachnoiditis when intrathecally injected and they can cause critical complications such as spinal cord infarction or cerebral infarction when intravascularly injected, baker. suggested utilization of a test injection of non - steroidal local anesthetic. numerous reports have recommended particle - free steroids as a potential remedy. in particular, lee. argued that there were no differences between particulate steroids and non - particulate steroids in a study of comparing dexamethasone and triamcinolone. therefore, it should be considerable to use dexamethasone as a non - particulate steroid in order to reduce the risks of performing the procedures. in conclusion, anesthesiologists should keep in mind that complications unidentifiable on contrast images alone are potentially present at cervical nerve root block immediately following spine surgery. therefore, when a procedure for a post - operative patient is necessary, it is critical to administer a test dose of local anesthetic, to use enough amount of contrast medium, to facilitate real - time monitoring devices, and to employ fluoroscopy with digital subtraction to check the soft tissues, while exerting a careful observation of the patient 's symptoms at administration of medication. | selective cervical nerve root block is executed for patients who have symptoms of cervical radiculopathy for diagnostic and therapeutic purposes. however several catastrophic complications caused by this procedure have been reported including neurological complications. a 43-year - old male received a c5 selective cervical nerve root block procedure due to continuous radiating pain even after cervical discectomy and interbody fusion was performed. at the time of the procedure, the contrast outline revealed reflux of the nerve root and epidural space. but after the procedure was performed, the patient experienced decreased sensation in the upper and low extremities as well as motor paralysis of both extremities. our sspecting diagnosis was anterior spinal artery syndrome but both sensory and motor functions were subsequently recovered within a few hours after the procedure was completed. due to the difficult nature of this case, we reported these complications and reviewed current literature related to this study. |
traditional kava is an aqueous extract of the roots of piper methysticum and serves as a ceremonious and daily beverage or an herbal remedy for south pacific islanders. kava had also been used clinically to treat mild and moderate anxiety, based on results of numerous clinical trials. anxiolytic kava was typically prepared as an organic extract of kava root with ethanol or acetone, instead of the traditional aqueous preparation. anxiolytic kava had been banned in europe and a few other countries since 2002 because of its risk to induce hepatotoxicity, and it is listed on the usa fda advisory board, but germany s federal administrative court negated the ban in june 2014. various causes have been proposed for kava s hepatotoxic risk, but none have been validated so far. first of all, in response to high demand, anxiolytic kava may have included nonroot toxic plant parts. it has also been postulated that some kava roots were not properly dried, resulting in hepatotoxin contamination. usage of nontraditional cultivars could be another cause ; different kava cultivars have diverse chemical profiles while traditional kava is prepared from only a few of them. due to preparation difference, traditional and anxiolytic kavas have distinct composition profiles, which may impose different hepatotoxic risks as well. furthermore, 90% of the purported hepatotoxic cases associated with kava usage involved concomitant consumption of other drugs or dietary supplements, suggesting that kava s hepatotoxic risk may be mediated via herb herb or herb drug interactions. in addition to kava s anxiolytic benefit, one epidemiological survey suggested that traditional kava usage may be able to reduce cancer risk, which was supported by results from several laboratory animal tumorigenesis models. moreover, despite its ban and being on usa fda s advisory list, kava consumption has experienced a global resurgence based on the amount of kava exported from the major kava producing nations (the republic of vanuatu, fiji, and tonga) between 2008 and 2013. with the recent overturn of the kava ban in germany our recent metabolomics and cellular cytotoxicity analyses of an array of current commercial kava products revealed that they were diverse in chemical profile and cellular cytotoxicity, and likely distinct in their health benefit and risk. considering the increasing human exposure and the diverse chemical composition of current kava products, the hepatotoxic risk of kava needs to be clarified and the responsible chemicals need to be identified, which is the focus of this study. our results showed that kava was safe when given alone but significantly enhanced acetaminophen (apap)-induced hepatotoxicity in c57bl/6 mice. chalcone - based flavokawains a (fka) and b (fkb) recapitulated kava s potentiation of apap - induced hepatotoxicity while dihydromethysticin (dhm) lacked such a risk. an ethanolic extract of the wild crafted kava root from vanuatu was purchased from gaia herbs, inc. (brevard, nc, standardized to 150 mg / ml total kavalactones). dhm was purified from this kava product using normal phase silica gel chromatography as described earlier. kava and all compounds were completely dried under vacuum to remove any solvent residue. the desired drug formulations were prepared by mixing kava or pure compounds with peg-400 and stored at 4 c until use. all animal studies were performed in compliance with the institutional animal care and use committee at the university of minnesota guidelines. six - week - old female c57bl/6j mice (jackson laboratories, me) were housed at specific pathogen - free animal facilities of research animal resources, university of minnesota, with free access to standard rodent food and water. mice were gavaged with dose formulations at the indicated doses and times, and euthanized by co2 overdosing with necropsy performed by experienced researchers. the long - term study was designed to evaluate the hepatotoxicity of kava alone. mice in the control group were given peg-400 (200 l) on a daily basis via gavage, 6 days a week, for 14 weeks. mice in the kava treatment group were given kava at a dose of 500 mg / kg bodyweight on a daily basis via gavage, 6 days a week, for 14 weeks. the chosen kava dose was based on the recent safety studies of another kava product performed by the national toxicology program. final bodyweight was measured and serum from each mouse was analyzed for alanine aminotransferase (alt) and aspartate aminotransferase (ast), two major biomarkers of liver function. the short - term combination studies were designed to evaluate the potential synergism of kava and its chemicals to apap - induced hepatotoxicity. c57bl/6 mice were randomized (815 mice per group) and were administered with peg-400 (200 l), kava (500 mg / kg bodyweight), dhm or fka, and fkb in peg-400 (200 l) at the indicated doses daily via oral gavage for 2 days. on the third day, mice in the respective groups were coadministered with apap (800 mg / kg bodyweight) in peg-400 (200 l). appropriately fixed tissues were processed into paraffin blocks using standard histological techniques, and 5 m sections were cut and stained with hematoxylin and eosin (h&e). histological slides were examined using light microscopy by an experienced a.c.v.p board certified pathologist (m.g.os.) under blinded conditions, with liver lesions graded on a 0 to 4 scale based on the extent of necrosis (0 = absent, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe). the clinical chemistry data were reported as mean sd (n = 415). for the long - term kava alone study, the two - tailed student t - test was used to compare the means between the control and treatment groups. one - way analysis of variance (anova) was used to compare the means among different groups in the short term combination studies. dunnett s test was used for comparisons of apap and other treatment groups when the one - way anova analysis was statistically significant. at the tested dose (500 mg / kg bodyweight), daily kava treatment did not affect mouse growth (data not shown). there were also no statistically or biologically significant differences between control and kava - treated mice with respect to alt and ast (figure 1a and 1b). effect of 14-week daily kava treatment (500 mg / kg bodyweight) via gavage on mouse serum alt (a) and ast (b). p values were given with comparison between the control group (n = 4) and the kava treatment group (n = 4) using the two - tailed student t - test. since 90% of the human kava hepatotoxic cases involved concurrent consumption of other medications or dietary supplements, herb drug interactions based on this and on a recent report that kava enhanced the toxicity of apap in vitro, this study was designed to evaluate the effect of kava on apap - induced hepatotoxicity in vivo. the treatment regimen was designed to mimic potential scenarios in humans kava was consumed on a daily basis while apap was used occasionally. as expected, kava treatment alone had no effect on alt and ast while apap treatment significantly increased serum alt and ast activities (figure 2a). kava and apap combination caused further increase in serum alt and ast activities (3-fold increase relative to apap alone, figure 2a), and these increases were statistically significant in comparison to apap treatment alone. histopathological analyses of the liver tissues revealed no lesions in control and kava treated mice (figure 2b), confirming the lack of hepatotoxicity by kava treatment alone. the lesions from apap - treated mice evenly distributed among different severity categories (0 being no lesion and 4 being the highest grade lesion) while kava and apap combination markedly increased the number of mice with the highest liver lesion (figure 2b), supporting the notion that the increases in alt and ast activities were biologically significant. these clinical chemistry data and histopathological findings for the first time demonstrate that kava enhanced apap - induced hepatotoxicity in vivo, and may reflect the purported kava hepatotoxicity cases in humans. the histopathological lesion severity also nicely correlated positively with the clinical chemistry results (figure 2c). effect of 3-day daily kava treatment (500 mg / kg bodyweight) via gavage on mouse serum alt and ast and liver lesions with / without apap treatment (800 mg / kg bodyweight) via gavage. (c) the relationships among serum alt, ast, and the grades of liver lesions. for a, comparisons were made with the apap treatment group by dunnett s test when one - way anova was statistically significant (n = 815). this experiment was designed to explore the potential of dhm and fkb (figure 3) to synergize the hepatotoxicity of apap following the same kava and apap cotreatment regimen. thirteen chemicals have been isolated and quantified from the kava product used in this study with no detection of pipermethystine. dhm and fkb were selected for this initial evaluation because they are representatives of kavalactones and chalcones, respectively, two major classes of chemicals in kava. in addition dhm has been recently demonstrated to potently and effectively block nnk - induced lung tumorigenesis in mice while fkb has been identified as the most cytotoxic compound in kava to various cancerous cells. the dosages for dhm (37.5 mg / kg) and fkb (11.5 mg / kg) were based on their abundance (7.5% and 2.3%, respectively) in this kava product at a dose of 500 mg / kg. dhm and fkb individually caused no effect on serum alt and ast (figure 4). dhm had no effect on serum alt and ast as well when combined with apap (figure 4). fkb on the other hand when combined with apap moderately increased the serum levels of alt and ast, and the increase in ast was statistically significant (figure 4), suggesting that fkb contributes to kava s potentiation of apap - induced hepatotoxicity. effect of 3-day daily dhm (37.5 mg / kg) or fkb (11.5 mg / kg) via gavage on mouse serum alt and ast with / without apap treatment. comparisons were made with the apap treatment group by dunnett s test when one - way anova was statistically significant (n = 615). given that the kava product used in this study contains flavokawain a (fka) of similar abundance as fkb (figure 3), this experiment was designed to evaluate the dose response effect of fka and fkb together on apap s hepatotoxicity following the same treatment regimen. the final dosages of fka and fkb were 1, 2, and 4 times their abundance (1.6% and 2.3%, respectively) of a kava dose at 500 mg / kg bodyweight. fka and fkb together did not induce any changes on serum alt and ast at the three tested dosages (figure 5a and b). when combined with apap, fka and fkb dose - dependently potentiated the increase in alt and ast induced by apap (figure 5a and b). of note, one mouse with the treatment of the highest dose of fka and fkb in combination with apap died 0.52 h before necropsy (i.e., 22 to 23.5 h after the combined dose of apap with fka and fkb). its serum alt and ast levels were the highest among all mice (figure 5c), and 23 times higher than the next highest values. histopathological examination revealed multifocal and coalescing acute centrilobular necrosis in the liver of this mouse (figure 5d, panel b), whereas livers from a control mouse (figure 5d, panel a) and a mouse treated with fka and fkb alone (not shown) were histologically within normal limits. response effect of 3-day daily fka and fkb via gavage on mouse serum alt and ast and livers with / without apap treatment. comparisons were made with the apap treatment group by dunnett s test when one - way anova was statistically significant (n = 5). (c) serum level of alt and ast from the dead mouse in the high - dose fka and fkb groups with apap cotreatment. (d) photomicrographs of h- and e - stained livers from a control mouse (panel a) and a mouse treated with fka and fkb plus apap (panel b). note extensive karyorrhexis (arrow) reflecting acute necrosis of hepatocytes (increased eosinophilia) in mouse treated with fka, fkb, and apap (panel b). kava has demonstrated anxiolytic activity in the clinic and potentially reduces cancer risk in humans. on the other hand, kava usage has been speculated to be associated with rare but severe hepatotoxicity. various mechanisms have been proposed and different chemicals have been postulated with no confirmation. given kava s global resurgence and the diverse chemical composition among current kava products, it is urgent and important to recapitulate kava s hepatotoxicity in an in vivo model, which can help identify the responsible chemicals and guide the development of strategies to minimize and ideally eradicate such an adverse potential. the results from this study demonstrated that kava when administered alone via gavage in c57bl/6 mice induced no adverse effect even at a fairly high dose (500 mg / kg bodyweight daily) in a chronic manner, as reflected in mouse growth and serum levels of alt and ast (figure 1). on the other hand, kava significantly potentiated the hepatotoxicity of apap in c57bl/6 mice, as indicated by the increase in serum alt and ast, and the increased severity of liver lesions (figure 2). the treatment regimen was designed to mimic potential circumstances among human kava users that kava would be consumed on a daily basis while other medications, apap in this case, were used occasionally when needed. since the majority of kava - associated hepatotoxic cases consumed other medications or dietary supplements concomitantly, the results from this study may have direct indication to the observed hepatotoxicity among kava users. it remains to be determined whether kava usage can potentiate the hepatotoxic risk of other medications or hepatotoxins, such as alcohol consumption. it also remains to be determined whether other kava treatment regimens, such as prolonged kava usage or in a fasted stage (recommended for traditional kava usage), may potentiate its hepatotoxic risk even at lower kava dosages. with the c57bl/6 mouse model that captures kava s hepatotoxic risk in vivo the results demonstrated that a chalcone - based compound in kava, fkb, moderately potentiated apap s hepatotoxicity while dhm, a representative of kavalactones in kava, lacked such a risk when they were evaluated at a dose equivalent to kava at a dose of 500 mg / kg bodyweight (figure 4). as the kava product contains fka, an analog of fkb, at similar abundance, the combination of fka and fkb was evaluated, which dose - dependently enhanced apap - induced hepatotoxicity (figure 5). indeed, the one mouse that died early, and which had the highest alt and ast levels (figure 5c), reflecting extensive acute hepatocellular necrosis (figure 5d, panel b), was in the apap cotreatment group at the highest dose of fka and fkb. these data overall indicate that fka and fkb are the responsible compounds in kava that potentiate apap - induced hepatotoxicity while dhm is free of this risk. besides fka and fkb, flavokawain c (fkc) has been reported in other kava products but was not detectable in the kava product used in this study. our recent analysis of a set of kava products on the current market demonstrates that the abundance of fka and fkb can vary 20-fold. similarly, a recent study analyzed the abundance of fka, fkb, and fkc in different kava cultivars. cultivars not recommended for traditional use were found to contain higher abundance of fka, fkb, and fkc than the traditionally consumed cultivars. further studies therefore are warranted to evaluate whether cultivars or kava products with higher content of fka, fkb, and fkc would impose a higher hepatotoxic risk. future studies are also needed to elucidate the molecular mechanisms of the observed hepatotoxicity enhancement, such as the depletion of glutathione. such knowledge will help guide the preparation of kava products for human use with higher health benefit and minimal adverse effects. | anxiolytic kava products have been associated with rare but severe hepatotoxicity in humans. this adverse potential has never been captured in animal models, and the responsible compound(s) remains to be determined. the lack of such knowledge greatly hinders the preparation of a safer kava product and limits its beneficial applications. in this study we evaluated the toxicity of kava as a single entity or in combination with acetaminophen (apap) in c57bl/6 mice. kava alone revealed no adverse effects for long - term usage even at a dose of 500 mg / kg bodyweight. on the contrary a three - day kava pretreatment potentiated apap - induced hepatotoxicity, resulted in an increase in serum alt and ast, and increased severity of liver lesions. chalcone - based flavokawains a (fka) and b (fkb) in kava recapitulated its hepatotoxic synergism with apap while dihydromethysticin (dhm, a representative kavalactone and a potential lung cancer chemopreventive agent) had no such effect. these results, for the first time, demonstrate the hepatotoxic risk of kava and its chalcone - based fka and fkb in vivo and suggest that herb drug interaction may account for the rare hepatotoxicity associated with anxiolytic kava usage in humans. |
since 1995, the environmental working group (ewg), a united states - based environmental advocacy organization, has developed an annual list of fruits and vegetables, frequently referred to as the dirty dozen, suspected of having the greatest potential for contamination with residues of pesticides. the ewg cautions consumers to avoid conventional forms of these fruits and vegetables and recommends that consumers purchase organic forms of these commodities to reduce their exposure to pesticide residues. the annual release of the report has traditionally generated newspaper, magazine, radio, and television coverage, and the report is considered to be quite influential in the produce purchasing decisions of millions of americans. in june 2010, the ewg released its most recent dirty dozen list. topping the list as the most contaminated commodity was celery, followed by peaches, strawberries, apples, blueberries, nectarines, bell peppers, spinach, cherries, kale, potatoes, and grapes (imported). according to an ewg news release, consumers can lower their pesticide consumption by nearly four - fifths by avoiding conventionally grown varieties of the 12 most contaminated fruits and vegetables. it is unclear how the ewg could make such a statement since the methodology used to rank the various fruits and vegetables did not specifically quantify consumer exposure to pesticide residues in such foods. instead, the methodology provided six separate indicators of contamination, including (1) percentage of samples tested with detectable residues, (2) percentage of samples with two or more pesticides detected, (3) average number of pesticides found on a single sample, (4) average amount of all pesticides found, (5) maximum number of pesticides found on a single sample, and (6) total number of pesticides found on the commodity. each of these indicators was normalized among the 49 most frequently consumed fruits and vegetables, and a total score was developed to form the basis for the rankings. since none of these indicators specifically considered exposure (the product of food consumption and residue levels), it is difficult to see how the ewg could substantiate the claim that consumers could lower their pesticide consumption by nearly four - fifths by avoiding conventional forms of the dirty dozen commodities. additionally, the toxicological significance of consumer exposure to pesticides in the diet is also not addressed through an appropriate comparison of exposure estimates with toxicological endpoints such as the reference dose (rfd) or the acceptable daily intake (adi). to more accurately assess the potential health impacts from consumer exposure to pesticide residues from the dirty dozen the exposure estimates were then compared with toxicological endpoints to determine the health significance of such exposures. the ewg rankings were derived from the results of residue findings of the united states department of agriculture (usda) pesticide data program (pdp) and the united states food and drug administration (fda) pesticide program residue monitoring from 2000 to 2008 [1, 3, 4 ]. the pdp is more appropriate for risk assessment as it is not developed for enforcement, provides residue findings for produce in ready - to - eat forms (i.e., washed or peeled), includes many more samples than the fda program, and relies upon more sensitive analytical methods. as a result, our study relied entirely upon results from the most recent pdp data collected from 2004 to 2008. to estimate exposures to pesticides from the dirty dozen commodities, pdp data was accessed for each commodity using the most recent year of data collection. table 1 provides a summary of the most recent sample collections for each of the twelve commodities by the pdp and the number of samples taken. pdp data were analyzed to identify the ten most frequently detected pesticides on each of the twelve commodities. a total of 120 separate residue files were generated, corresponding to specific files for each of the ten pesticides on each of the twelve commodities. each residue file consisted of sample - specific findings (both detections and nondetections) for all residue determinations. residue findings considered as nondetections were assigned a value of zero, using the same approach taken by katz and winter, rather than using the much more conservative approach of considering nondetectable residues as being to one - half of the detection limits. exposure estimates were made using lifeline probabilistic modeling software (lifeline software version 5.0, annandale, va, http://www.thelifelinegroup.org/). this software is publicly available and uses probabilistic techniques to model exposure and risks for the general population or selected populations to chemicals in food, water, and in the home environment. the model generates populations of simulated individuals, and daily exposures are calculated for each individual on the basis of food consumption (derived from the 199496 and 1998 usda 's continuing survey of food intakes by individuals) and pesticide residue levels. exposure estimates made in this study used an approach similar to that used by katz and winter to differentiate exposures to pesticide residues in imported and domestic fruits and vegetables. in this present study, individual runs of 2000 composite individuals were made for each of the 120 residue files. estimates of lifetime mean daily exposure for each of the pesticides on each of the commodities were developed. to determine the toxicological significance of such exposures, estimates were compared with chronic rfds developed by the united states environmental protection agency (epa). the chronic rfd represents an estimate of the amount of a chemical a person could be exposed to on a daily basis throughout the person 's lifetime that is likely to be without an appreciable risk of harm. for a handful of pesticides identified for which rfds had not been developed, adi values, which are analogous to rfds, were used as substitutes and are denoted in tables 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. the mean exposures for the top ten pesticides detected on each of the twelve commodities are provided and compared with the rfds in tables 213. results demonstrate that the rfd values for each of the pesticides exceed the mean exposure estimates in all cases and that the rfds were more than 1000 times higher than the exposure estimates in more than 90 percent of the comparisons. such findings suggest that the potential consumer risks from exposure to the most frequently detected pesticides on the dirty dozen list of foods are negligible and cast doubts as to how consumers avoiding conventional forms of such produce items are improving their health status. the highest relative exposure for a pesticide / commodity combination was for the organophosphate insecticide methamidophos on bell peppers. the rfd for methamidophos was still 49.5 times higher than the exposure estimate, indicating a large measure of consumer protection. it should be pointed out that the chronic rfd for methamidophos (0.05 g / kg / day) is far lower than any other pesticide rfd considered in this study, and this low value seems anomalous given the lower cholinesterase - inhibiting potential of methamidophos relative to other organophosphate insecticides. ethyl parathion, for example, is considered to be far more toxic and a much more potent inhibitor of cholinesterase than methamidophos. the epa has not established an rfd for ethyl parathion, but the world health organization has established an adi for ethyl parathion of 5 g / kg / day, or 100 times higher than the rfd for methamidophos. regardless of the unusually low rfd for methamidophos, an exposure of 49.5 times lower than the rfd still represents an exposure 49,500 times lower than exposures to methamidophos in laboratory animals that still have not resulted in any adverse health effects. the rfd for methamidophos uses a 1,000-fold uncertainty factor when extrapolating from the results of the most sensitive animal study (a one - year dog feeding study) to determine acceptable levels for human exposure. for three commodities blueberries, cherries, and kale the rfd was more than 30,000 times higher than the exposure estimates for all of the ten most frequently detected pesticides on those commodities. given these findings, the inclusion of blueberries, cherries, and kale on the dirty dozen findings relating exposure estimates for all pesticide / commodity combinations to rfds are summarized in table 14. only one of the 120 exposure estimates exceeded 1% of the rfd (methamidophos on bell peppers at 2% of the rfd), and only seven exposure estimates (5.8 percent) exceeded 0.1% of the rfd. three quarters of the pesticide / commodity combinations demonstrated exposure estimates below 0.01% of the rfd, and 40.8% had exposure estimates below 0.001% of the rfd. to put this in perspective, exposure at 0.01% (one ten - thousandth) of the rfd represents an exposure one million times lower than the no observable adverse effect level (the highest amount given to the most sensitive animal species on a daily basis), assuming that the typical 100-fold uncertainty factor is used. the methodology used to create the dirty dozen list does not appear to follow any established scientific procedures. only one of the six indicators used by the ewg crudely considers the amount of pesticide residue detected on the various commodities, and that indicator fails to relate exposures to such residues with established health criteria. the remaining four indicators seem related as all appear to focus upon the existence of residues of multiple pesticides (percent of samples with two or more pesticides, average number of pesticides found on a single sample, maximum number of pesticides found on a single sample, and total number of pesticides found on the commodity) which suggests that the commodity rankings are significantly skewed to reflect instances of multiple residues. while research has demonstrated that the toxicity of a single chemical may be modulated by the presence of another chemical, such effects still require exposure to the modulating chemical to be at a level high enough (above a threshold dose) to cause a biological effect. results from this study strongly suggest that consumer exposures to the ten most common pesticides found on the dirty dozen commodities are several orders of magnitude below levels required to cause any biological effect. as a result, the potential for synergistic effects resulting from pesticide combinations is negligible, and the ewg methodology which skews rankings due to the presence of multiple residues is not justified. the ewg methodology also does not appear to be capable of justifying the claim that consumers can lower their pesticide consumption by nearly four - fifths by avoiding conventionally grown varieties of the 12 most contaminated fruits and vegetables since no effort to quantify consumer exposure was made. it should also be mentioned that consumption of organic produce should not be equated with consumption of pesticide - free produce. winter and davis summarized pesticide monitoring results from the pdp, the california department of pesticide regulation, the consumers union, and a study in belgium. while conventional produce was between 2.9 and 4.8 times more likely to contain detectable pesticide residues than organic produce, samples of organic produce frequently contained residues the pdp data, in fact, indicated that 23 percent of organic food samples tested positive for pesticide residues. in summary, findings conclusively demonstrate that consumer exposures to the ten most frequently detected pesticides on ewg 's dirty dozen commodity list are at negligible levels and that the ewg methodology is insufficient to allow any meaningful rankings among commodities. we concur with ewg president kenneth cook who maintains that we recommend that people eat healthy by eating more fruits and vegetables, whether conventional or organic, but our findings do not indicate that substituting organic forms of the dirty dozen commodities for conventional forms will lead to any measurable consumer health benefit. | probabilistic techniques were used to characterize dietary exposure of consumers to pesticides found in twelve commodities implicated as having the greatest potential for pesticide residue contamination by a united states - based environmental advocacy group. estimates of exposures were derived for the ten most frequently detected pesticide residues on each of the twelve commodities based upon residue findings from the united states department of agriculture 's pesticide data program. all pesticide exposure estimates were well below established chronic reference doses (rfds). only one of the 120 exposure estimates exceeded 1% of the rfd (methamidophos on bell peppers at 2% of the rfd), and only seven exposure estimates (5.8 percent) exceeded 0.1% of the rfd. three quarters of the pesticide / commodity combinations demonstrated exposure estimates below 0.01% of the rfd (corresponding to exposures one million times below chronic no observable adverse effect levels from animal toxicology studies), and 40.8% had exposure estimates below 0.001% of the rfd. it is concluded that (1) exposures to the most commonly detected pesticides on the twelve commodities pose negligible risks to consumers, (2) substitution of organic forms of the twelve commodities for conventional forms does not result in any appreciable reduction of consumer risks, and (3) the methodology used by the environmental advocacy group to rank commodities with respect to pesticide risks lacks scientific credibility. |
acute kidney injury (aki) is a major concern in intensive care unit (icu) patients, due to not only high incidence but also as an independent variable for poor outcome in these patients. the prevalence of aki in critically ill patients had been reported to be varying from 1.5% to 70% depending upon the population studied and criteria used. the cause of aki in critically ill patients is often multifactorial ; sepsis being the most common. recent evidence suggests that aki in patients with sepsis may have different pathophysiology including hyperemia, vasodilation, and acute tubular apoptosis instead of ischemia, vasoconstriction, or acute tubular necrosis. in few small studies, clinical profile and outcome of septic aki a large prospective observational cohort study of critically ill patients with aki also showed that there was significant increased risk for death in patients with septic aki. in the literature, there are as many as 35 different definitions of acute renal failure in critically ill patients being used. however, a uniform and precise operational definition of arf was not available till recently. in 2004, the acute dialysis quality initiative (adqi) has developed the rifle classification of aki. the acronym rifle defines 3 grades of increasing severity of arf (risk, injury, and failure, respectively, r, i, and f) and 2 outcome variables (loss and end - stage kidney disease, respectively, l and e). a unique feature of the rifle classification is that, it provides for 3 grades of severity of renal dysfunction on the basis of a change in serum creatinine, reflecting changes in gfr or duration and severity of decline in urine output from the baseline. the rifle criteria have the advantage of providing diagnostic definitions for the stage, at which kidney injury still can be prevented (risk stratum), the one when the kidney has already been damaged (injury), and the one when renal failure is established (failure). there are limited data from india on aki in icu patient population, which include both septic as well as non - septic aki patients. however, there is no available data from india focusing on septic aki in critically ill patients. in view of the limited data on septic aki and its likely importance, we sought to describe the clinical characteristics and predictors of outcome in critically ill patients with septic aki. this retrospective study of critically ill patients with septic aki was performed in the department of critical care medicine at a tertiary care university hospital with a 1000 bed capacity. the department of critical care medicine has 12 beds icu that caters to medical as well as surgical patients. sepsis was defined by the presence of both suspected or confirmed infection and at least 2 criteria of systemic inflammatory response syndrome, which includes abnormalities in temperature, heart rate, respiratory rate, or white blood cell count. septic aki was defined by presence of aki in patient with sepsis syndrome and also absence of other obvious cause that leads to aki. in this retrospective study, medical records of patients with septic aki, who were admitted during the period of january 2006 to december 2008, were reviewed. patients whose age was < 18 years, with end stage renal disease or chronic kidney disease on maintenance hemodialysis, withdrawal of therapy during icu stay, or incomplete data were not included in this study. data were retrieved from individual case records of diagnosed septic aki and were collected on a case report form. the form included age, gender, medical vs. surgical, primary source of infection, apache ii score, rifle criteria, number of co - morbidity, use of mechanical ventilation and vasoactive agents, number of non - renal organ failure (nrof), length of icu stay, and outcomes were recorded. primary outcome defined as survival at icu discharge ; while secondary outcome as renal recovery (i.e., independence from renal replacement therapy at time of icu discharge) in survivors. continuous variables described as median (25 - 75 percentile), and categorical variables are described as n (%). for comparative test on continuous variables, mann- whitney u test was applied. for categorical variables, the pearson chi- square test or fisher 's exact test were used as appropriate. overall, predictors showing a p < 0.05 association with icu mortality in univariate analysis were incorporated in regression analysis. logistic regression analysis was used to assess the multivariate relation between multiple patient characteristics and outcome of septic aki. statistical analysis was done using the spss-17 software, p < 0.05 was considered significant. there were 406 patients admitted in icu during study period. out of 323 icu patients with sepsis, of 160, 34 patients were excluded because of any of the following reason : age < 18 years, with end stage renal disease or chronic kidney disease on maintenance hemodialysis, withdrawal of therapy during icu stay, or incomplete data ; and data from medical records of only 126 septic aki patients were analyzed. out of 126 septic aki patients 75% were male ; 85% were medical vs. 15% surgical. primary source of sepsis was lung (40%), followed by intra - abdominal infection (24%), cns (13%), bsi (8%), uti (7%), and other / unknown (8%). aki distribution of these patients in respect of rifle criteria were : risk group 24%, injury group 34%, and failure group 42%. patients with no nrof were 3%, with one nrof 6%, with two 19%, with three 32%, while with four or more nrof were 40% of total patients. comparison of clinical characteristics among non - survivors (n = 66) and survivors (n = 60) were : mean age 55 15 vs. 49 15 (p < 0.05), gender (m : f) 52 : 14 vs. 42 : 18 (ns), apache ii 24.53 6 vs. 18.3 7.8 (p < 0.05), number of nrof 4.47 0.7 vs. 3.45 1.13 (p < 0.05), number of co - morbid illness 2.59 1.1 vs. 2.4 1.14 (ns), length of icu stay 16 vs. 28 days (p < 0.05) [table 1 ]. outcome based on rifle criteria among non - survivors vs. survivors (in %) were : risk group 40 vs. 60 ; injury group 54 vs. 46 ; and in failure group 58 vs. 42. characteristics of icu patients with septic aki age, number of nrof, and apache ii score were significant predictor of outcome on univariate logistic regression analysis ; while number of nrof was found only significant predictor on multivariate logistic regression analysis [tables 2 and 3 ]. relative risk of mortality with individual organ failure was highest with involvement of cvs system (14.5), followed by respiratory failure (9.7), git failure (6.2), dic (3.1), and cns (2.1) [table 4 ]. renal outcome in survivors at time of icu discharge were : dialysis - independent 88% vs. dialysis - dependent 12% only. based on rifle criteria, renal recovery in patients with risk and injury group was complete (100%), while in failure group, it was 68%. predictors of mortality by univariate logistic regression analysis among patients with septic aki predictors of mortality by multivariate logistic regression analysis among patients with septic aki relative risk of mortality with individual organ failure in patients with septic aki. overall incidence of septic aki among all icu admissions is between 15%-20%. in a large multinational, multicenter (no data from indian subcontinent) prospective study of acute kidney injury in critically ill patients (best kidney investigators), it was found that the most common cause of aki in icu patients is sepsis (47.5%). in their study, clinical characteristics of patients were : mean age 63 years, 60% medical patients, 85% patients required mechanical ventilation, and mean length of stay in icu was 16 days ; while in our study, these were 52 years, 85%, 90%, and 22 days, respectively. in another large analysis of 14,039 septic aki patients from australian icus by bagshaw., the proportion of patients stratified by rifle criteria were 38.5%, 38.8%, and 22.7% for risk, injury, and failure, respectively. in our study, incidence of septic aki was high at 31% with more proportion of patients in the failure group (24%, 34%, and 42% for risk, injury, and failure, respectively). this is probably explained by the fact that the study cohort was in a referral hospital. overall mortality among patients with septic aki was found to be 70% by best kidney investigators, and prognostic factors were age, severity of illness, use of vasoactive drugs, and mechanical ventilation. in contrast, in our study, the overall mortality was 52% and number of non - renal organ failure, age, and apache ii score were found to be predictors of outcome in septic aki patients. in our study, rifle criteria did not reveal significant difference for mortality among the 3 grades, but there was a trend of increasing mortality with the severity of aki increasing from risk to failure. however, rifle criteria was found to be a good predictor of recovery of renal function. the time of development of aki in septic patient in relation to icu admission was also not defined in this study while it is evident by other studies that early septic aki is not only more common but also shows a trend toward higher mortality. in this study, in this cohort of indian patients septic aki was found to have a high incidence and mortality. age, severity of illness as assessed by number of non - renal organ failure and apache ii score were important predictors of mortality. the number of non - renal organ failure was also found to be an independent predictor of outcome. | acute kidney injury (aki) is an independent variable for poor outcome in critically ill patients. the pathophysiology of septic aki is distinct from that of non - septic aki. we studied the clinical profile and outcome of septic aki since such data is sparse in indian patients. in this single - center retrospective, observational, cohort study, septic aki has been found with high incidence (31%) and overall mortality was 52%. age, number of non - renal organ failure, and apache ii score were found as significant predictors of outcome in this population. |
anisotropy or alignment is a critical feature of functional soft materials in living organisms, but it remains a challenge for spontaneously generating anisotropic gel materials. here we report a molecular design that increases intermolecular aromatic aromatic interactions of hydrogelators during enzymatic hydrogelation for spontaneously forming an anisotropic hydrogel. this process, relying on both aromatic aromatic interactions and enzyme catalysis, results in spontaneously aligned supramolecular nanofibers as the matrices of a monodomain hydrogel that exhibits significant birefringence. this work, as the first example of monodomain hydrogels formed via an enzymatic reaction, illustrates a new biomimetic approach for generating aligned anisotropic soft materials. |
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they are classified into 4 grades (grade i ii, low - grade ; grade iii iv, and high - grade) according to the guidelines of the world health organization (who). the accurate grading of gliomas has clinical significance for therapeutic decision - making, the monitoring and administration of chemoradiotherapy, and prognostic evaluation. conventional magnetic resonance imaging (mri) and apparent diffusion coefficient (adc) can provide important discerning information for glioma grading. however, misdiagnosis occur in glioma grading due to the overlap in conventional mri manifestations between low - grade (lggs) and high - grade (hggs) gliomas. radiomics is an emerging area that can transform multidimensional image data into the features amenable to comprehensive quantification by using an automatic feature extraction algorithm [710 ]. radiomics provides a comprehensive quantification of tumors by extracting and mining large numbers of quantitative image features from medical images. compared with conventional mri, this technique is a quantitative method providing more detailed quantification of tumor phenotypic characteristics. currently, radiomics is used for evaluating the prognosis of lung cancer as well as the clinical phenotype [1113 ]. for example, radiomic quantitative features contained a number of important prognostic indicators, including the molecular subtypes of tumors. despite enormous potential, finally, the delineations of rois require clinicians to draw layer - by - layer on imaging, which were too complicated and tedious for clinicians. previous studies using single - sequence imaging and a single feature were reasonably useful for glioma grading [1416 ]. however, a single sequence with a single feature can not comprehensively incorporate all features of gliomas. because heterogeneity is an important characteristic of gliomas, using only 1 sequence can not examine all features of gliomas. for example, an mr image using multi - sequence may display diverse signal characteristic due to the various pathological changes of gliomas, such as hemorrhage, necrosis, and cystic degeneration in tumors. thus, radiomic features from a multi - sequence mr image may be useful for glioma grading as a promising noninvasive biomarker of malignant tumors. in the present study, we investigated the efficacy of using radiomic features from multiple mri sequences (t2-flair, t1wi - ce, and adc map) in glioma grading. first, we aimed to determine the optimal radiomic features of each sequence and then combined those features to acquire better efficiency in glioma grading. radiomic features from multiple sequences and combined features may provide more comprehensive information for glioma grading and supplement conventional mri and other functional mri features. combined features may supply a frame of reference for clinical diagnosis and treatment decision - making, as well as for prognosis prediction. this retrospective study was approved by the local institutional review board. written informed consent was obtained from every patient before participation. a total of 66 patients with gliomas underwent mri between february 2012 and october 2015. inclusion criteria were : (a) mr imaging performed in all patients within 2 weeks prior to surgical resection and/or chemoradiotherapy and (b) a histopathologic diagnosis of cerebral glioma and grouped into lggs and hggs using the who 2007 criteria. finally, a total of 66 patients (33 males ; 2273 years of age ; mean age 51.5 years) with t2-flair and t1wi - ce sequences, and 63 patients with dwi sequences were included. mri examinations were performed with a signa hdx 3-t mr scanner using an 8-channel phase array head coil (ge healthcare, milwaukee, wi). all patients underwent conventional mr sequences axial t1-weighted imaging (t1wi) with a repetition time (ms)/echo time (ms) of 195/4.76 and axial t2-weighted imaging (t2wi) with 4000/98 and t2wi - fluid attenuated inversion recovery (t2wi - flair) with 8000/95 and an inversion time (ti) of 2371.8 ms. axial contrast - enhanced t1wi (t1wi - ce) was repeated after intravenous administration of 0.1 mmol / kg of gadolinium contrast with gadopentetate dimeglumine. a total of 63 patients underwent the axial dwi sequence with tr / te 5000/74. other mr sequence parameters included slice thickness and slice intervals of 6.0/1.2 mm, while field of view (fov) was 240240 mm for all axial sequences. dwi scans used the se / epi sequence, and the diffusion coefficient of sensitivity was selected as 0.1000 s / mm. the original dwi maps were transmitted to adw4.4 (advanced workstation 4.4) to generate axial adc maps using ge software processing. the mr image of the t2wi - flair, t1wi - ce, and adc maps were transmitted from the pacs workstation (sichuang ltd., beijing, china) to a personal computer, and then transferred into processable dicom format images using radiant dicom viewer software (available at http://www.radiantviewer.com). owing to heterogeneity of gliomas, 2-dimensional (2-d) regions of interest (rois) were delineated manually using opening itk - snap software (available at http://www.itksnap.org) by 2-way - blinded, experienced neuroradiologists until they reached an agreement on areas of enhancement in each axial t1 post - contrast mr slice, tumor parenchyma t2-flair, and adc maps layer - by - layer. first, we selected tumor regions on each slice of the mri series, such that the sub - regions were made into a matrix with 0 and 1 over the series, where 0 indicated non - tumor and 1 indicated tumor. all calculation procedures were implemented on the cube generated by the dot production of the image matrix of the series and the 01 matrix of selected regions. relevant radiomic features were calculated and extracted from cubes on the t2wi - flair, t1wi - ce, and adc maps, respectively. gross total resection was performed in 65 gliomas, and only 1 glioma was partially resected. surgical specimens were paraffin - embedded after 4% formalin fixation (buffered in phosphate - buffered saline), and 1-m sections were prepared for hematoxylin - eosin (he) staining. in addition to histological grading of the tumors, the immunohistochemistry (ihc) index for gfap was assessed. the tumor parenchyma underwent corresponding paraffin cuts and conventional dewaxing to water, and abc immunohistochemical staining was performed. main reagents and instruments included gfap monoclonal antibody (dako, copenhagen, denmark), abc kit (sigma - aldrich, st. gfap staining was located in the cytoplasm and cell processes, and aperio digital pathology image analysis system (leica, frankfurt, germany) and software cytoplasmic v2 (leica, frankfurt, germany) using a magnification of 200x were used to select richly stained tumor tissue sections. three standard fields of vision were randomly selected and the average optical density was measured 3 times to compute an average for gfap expression levels of cells. the 2-sample t test was used to compare the values of all radiomic features between lggs and hggs on the t2wi - flair, t1wi - ce, and adc map, respectively. we selected the radiomic features that had significant differences between lggs and hggs for further analysis using 1-way analysis of variance (anova) with a post hoc test to test for differences among grade ii, iii, and iv gliomas. roc curve analysis was conducted to determine the diagnostic power of radiomic features that yielded statistically significant differences between lggs and hggs on each sequence in glioma grading. we normalized the features and combined their values to create a new feature (combined feature) to determine whether the efficiency of glioma classification could be increased. relationships between the radiomic features on each mri sequence and ihc index of glioma gfap were analyzed using the pearson correlation method. the features were normalized for each mri sequence using the z - score normalization method and the conversion function is displayed in equation (1). the symbols and represent the mean and standard deviation, respectively, of all sample data. the normalized features were then fitted to equation (2) and the combined features were calculated. here, auc (area under the roc curve [auc]=0.838) and f correspond to the area under curve and the value of each feature, respectively. this retrospective study was approved by the local institutional review board. written informed consent was obtained from every patient before participation. a total of 66 patients with gliomas underwent mri between february 2012 and october 2015. inclusion criteria were : (a) mr imaging performed in all patients within 2 weeks prior to surgical resection and/or chemoradiotherapy and (b) a histopathologic diagnosis of cerebral glioma and grouped into lggs and hggs using the who 2007 criteria. finally, a total of 66 patients (33 males ; 2273 years of age ; mean age 51.5 years) with t2-flair and t1wi - ce sequences, and 63 patients with dwi sequences were included. mri examinations were performed with a signa hdx 3-t mr scanner using an 8-channel phase array head coil (ge healthcare, milwaukee, wi). all patients underwent conventional mr sequences axial t1-weighted imaging (t1wi) with a repetition time (ms)/echo time (ms) of 195/4.76 and axial t2-weighted imaging (t2wi) with 4000/98 and t2wi - fluid attenuated inversion recovery (t2wi - flair) with 8000/95 and an inversion time (ti) of 2371.8 ms. axial contrast - enhanced t1wi (t1wi - ce) was repeated after intravenous administration of 0.1 mmol / kg of gadolinium contrast with gadopentetate dimeglumine. a total of 63 patients underwent the axial dwi sequence with tr / te 5000/74. other mr sequence parameters included slice thickness and slice intervals of 6.0/1.2 mm, while field of view (fov) was 240240 mm for all axial sequences. dwi scans used the se / epi sequence, and the diffusion coefficient of sensitivity was selected as 0.1000 s / mm. the original dwi maps were transmitted to adw4.4 (advanced workstation 4.4) to generate axial adc maps using ge software processing. the mr image of the t2wi - flair, t1wi - ce, and adc maps were transmitted from the pacs workstation (sichuang ltd., beijing, china) to a personal computer, and then transferred into processable dicom format images using radiant dicom viewer software (available at http://www.radiantviewer.com). owing to heterogeneity of gliomas, 2-dimensional (2-d) regions of interest (rois) were delineated manually using opening itk - snap software (available at http://www.itksnap.org) by 2-way - blinded, experienced neuroradiologists until they reached an agreement on areas of enhancement in each axial t1 post - contrast mr slice, tumor parenchyma t2-flair, and adc maps layer - by - layer. first, we selected tumor regions on each slice of the mri series, such that the sub - regions were made into a matrix with 0 and 1 over the series, where 0 indicated non - tumor and 1 indicated tumor. all calculation procedures were implemented on the cube generated by the dot production of the image matrix of the series and the 01 matrix of selected regions. relevant radiomic features were calculated and extracted from cubes on the t2wi - flair, t1wi - ce, and adc maps, respectively. gross total resection was performed in 65 gliomas, and only 1 glioma was partially resected. surgical specimens were paraffin - embedded after 4% formalin fixation (buffered in phosphate - buffered saline), and 1-m sections were prepared for hematoxylin - eosin (he) staining. in addition to histological grading of the tumors, the immunohistochemistry (ihc) index for gfap was assessed. the tumor parenchyma underwent corresponding paraffin cuts and conventional dewaxing to water, and abc immunohistochemical staining was performed. main reagents and instruments included gfap monoclonal antibody (dako, copenhagen, denmark), abc kit (sigma - aldrich, st. gfap staining was located in the cytoplasm and cell processes, and aperio digital pathology image analysis system (leica, frankfurt, germany) and software cytoplasmic v2 (leica, frankfurt, germany) using a magnification of 200x were used to select richly stained tumor tissue sections. three standard fields of vision were randomly selected and the average optical density was measured 3 times to compute an average for gfap expression levels of cells. the 2-sample t test was used to compare the values of all radiomic features between lggs and hggs on the t2wi - flair, t1wi - ce, and adc map, respectively. we selected the radiomic features that had significant differences between lggs and hggs for further analysis using 1-way analysis of variance (anova) with a post hoc test to test for differences among grade ii, iii, and iv gliomas. roc curve analysis was conducted to determine the diagnostic power of radiomic features that yielded statistically significant differences between lggs and hggs on each sequence in glioma grading. we normalized the features and combined their values to create a new feature (combined feature) to determine whether the efficiency of glioma classification could be increased. relationships between the radiomic features on each mri sequence and ihc index of glioma gfap were analyzed using the pearson correlation method. the features were normalized for each mri sequence using the z - score normalization method and the conversion function is displayed in equation (1). the symbols and represent the mean and standard deviation, respectively, of all sample data. the normalized features were then fitted to equation (2) and the combined features were calculated. here, auc (area under the roc curve [auc]=0.838) and f correspond to the area under curve and the value of each feature, respectively. all the values of radiomic features for different sequences in regions containing gliomas are compared with 2-samples t test. we found 2 statistical differential features on t2wi - flair sequence, 3 features on t1wi - ce sequence, and 3 features on the adc map between lggs and hggs (p<0.05). all of the radiomic features that yielded statistically significant differences between lggs and hggs on 3 mri maps are shown in figure 2. for example, the feature of flair glcm variance was lower in lggs compared to hggs (2.0560.843 vs. 2.6821.229, p=0.027). the radiomic features that displayed statistical differences between lggs and hggs were further compared using 1-way anova among grade ii iv gliomas. t2wi - flair glcm cluster shade differed significantly between grades ii and iii (p<0.001) and between grades ii and iv (p<0.001). only 2 radiomic features, t1wi - ce glcm entropy on the t1wi - ce sequence and adc glcm homogeneity on the adc map, were able to identify gliomas from grades ii to iv. comparisons of the radiomic features among grade ii, iii, and iv gliomas on t2wi - flair are shown in figure 3. the diagnostic efficiency of each feature that yielded a statistical difference between lggs and hggs was compared using roc curves, which are shown in figure 4a4c. (1) the auc value of flair glcm cluster shade (0.838), which had high sensitivity (75%) and specificity (84.6%) at a cut - off value of 10.217 (p<0.05), was significantly better than flair glcm variance (auc=0.654) in differentiating lggs from hggs. (2) the cut - off value of t1-ce glcm entropy (1.176) for distinguishing between lggs and hggs had high sensitivity (97.5%) and specificity (80.8%), and the auc was 0.936 (p<0.05), which was higher than t1-ce mean (auc=0.752) and t1-ce glcm energy (auc=0.748). (3) the auc of adc glcm homogeneity (0.905) which had high sensitivity (97.5%) and specificity (80.8%) at a cut - off value of 1.176 (p<0.05) was significantly better than adc glcm sum average (auc=0.684) and adc glrl sre (auc=0.674) on the adc map in differentiating lggs from hggs. the combined feature further increased the diagnostic power, resulting in the highest value of auc (0.943), higher specificity (89%) compared with t1-ce glcm entropy (80.8%), and higher sensitivity (90%) compared to adc glcm homogeneity (84%). the prediction of cellular differentiation, invasion and metastasis is valuable in estimating tumor cell biological characteristic, response to therapy, and prognosis. the correlations were evaluated using pearson correlation analysis between gfap and each feature, which displayed statistical differences between lggs and hggs. a significant negative correlation was found between gfap and the feature t1-ce glcm entropy (r=0.744, p<0.001), while a positive correlation was found between gfap and the feature adc glcm homogeneity (r=0.728, p<0.001). in addition, the features of flair glcm variance (r=0.309, p=0.011), flair glcm cluster shade (r=0.369, p=0.002), t1-ce glcm energy (r=0.546, p<0.001), and adc glcm sum average (r=0.334, p=0.007) demonstrated negative correlations with gfap. the corresponding correlation coefficients and scatter plots figure 6 shows representative t2wi, t2-flair, t1wi, t1wi - ce, dwi, adc maps, pathological he staining, and ihc gfap expression for glioma grades ii, iii, and iv, respectively. all the values of radiomic features for different sequences in regions containing gliomas are compared with 2-samples t test. we found 2 statistical differential features on t2wi - flair sequence, 3 features on t1wi - ce sequence, and 3 features on the adc map between lggs and hggs (p<0.05). all of the radiomic features that yielded statistically significant differences between lggs and hggs on 3 mri maps are shown in figure 2. for example, the feature of flair glcm variance was lower in lggs compared to hggs (2.0560.843 vs. 2.6821.229, p=0.027). the radiomic features that displayed statistical differences between lggs and hggs were further compared using 1-way anova among grade ii iv gliomas. t2wi - flair glcm cluster shade differed significantly between grades ii and iii (p<0.001) and between grades ii and iv (p<0.001). only 2 radiomic features, t1wi - ce glcm entropy on the t1wi - ce sequence and adc glcm homogeneity on the adc map, were able to identify gliomas from grades ii to iv. comparisons of the radiomic features among grade ii, iii, and iv gliomas on t2wi - flair are shown in figure 3. the diagnostic efficiency of each feature that yielded a statistical difference between lggs and hggs was compared using roc curves, which are shown in figure 4a4c. (1) the auc value of flair glcm cluster shade (0.838), which had high sensitivity (75%) and specificity (84.6%) at a cut - off value of 10.217 (p<0.05), was significantly better than flair glcm variance (auc=0.654) in differentiating lggs from hggs. (2) the cut - off value of t1-ce glcm entropy (1.176) for distinguishing between lggs and hggs had high sensitivity (97.5%) and specificity (80.8%), and the auc was 0.936 (p<0.05), which was higher than t1-ce mean (auc=0.752) and t1-ce glcm energy (auc=0.748). (3) the auc of adc glcm homogeneity (0.905) which had high sensitivity (97.5%) and specificity (80.8%) at a cut - off value of 1.176 (p<0.05) was significantly better than adc glcm sum average (auc=0.684) and adc glrl sre (auc=0.674) on the adc map in differentiating lggs from hggs. the combined feature further increased the diagnostic power, resulting in the highest value of auc (0.943), higher specificity (89%) compared with t1-ce glcm entropy (80.8%), and higher sensitivity (90%) compared to adc glcm homogeneity (84%). the prediction of cellular differentiation, invasion and metastasis is valuable in estimating tumor cell biological characteristic, response to therapy, and prognosis. the correlations were evaluated using pearson correlation analysis between gfap and each feature, which displayed statistical differences between lggs and hggs. a significant negative correlation was found between gfap and the feature t1-ce glcm entropy (r=0.744, p<0.001), while a positive correlation was found between gfap and the feature adc glcm homogeneity (r=0.728, p<0.001). in addition, the features of flair glcm variance (r=0.309, p=0.011), flair glcm cluster shade (r=0.369, p=0.002), t1-ce glcm energy (r=0.546, p<0.001), and adc glcm sum average (r=0.334, p=0.007) demonstrated negative correlations with gfap. the corresponding correlation coefficients and scatter plots are shown in figure 5. figure 6 shows representative t2wi, t2-flair, t1wi, t1wi - ce, dwi, adc maps, pathological he staining, and ihc gfap expression for glioma grades ii, iii, and iv, respectively. in the present study we evaluated the potential of radiomic features based on multiple sequences for use in glioma grading. we then combined features that were able to differentiate between lggs and hggs to determine whether diagnostic efficiency could be improved for glioma grading. our results showed that the features of flair glcm cluster shade, t1-ce glcm entropy, and adc glcm homogeneity had high power in differentiating lggs and hggs on each mri sequence. a significant correlation was found between gfap and the feature t1-ce glcm entropy (r=0.744, p<0.001) and adc glcm homogeneity (r=0.728, p<0.001). thus, combined features from different mri sequences may serve as an optimal radiomic feature for grading gliomas in clinical practice. radiologists can make an accurate diagnosis for the majority of gliomas by using experience, but in some cases different diseases may exhibit the same or similar image features. for instance, the scope of necrosis and signal characteristics of tumors on various mr images may not be visually distinct for radiologists, but after radiomic calculation processes and feature extraction using various algorithms, detailed quantitative feature information may vary to a large extent. radiomics is an emerging field of research, and has recently become an area of great interest. we hoped to apply radiomics to extract quantitative features for the comprehensive assessment of gliomas. we found that glcm entropy from the t1 ce sequence and glcm homogeneity from the adc map were best able to differentiate the differentiate glioma grades. a previous study by ryu. investigating the ability of texture features on the adc map to distinguish between hggs and lggs demonstrated that entropy of adc could be used for differentiation and the value of entropy was significantly higher in hggs than lggs. in our study, however, glcm entropy on the t1 ce sequence displayed high diagnostic efficiency among the different grades of gliomas. the discordance between sequences may be caused by differences between algorithms and the delineation of tumor rois between radiologists. additionally, our finding that entropy value was significantly higher in hggs than lggs is consistent with the findings of the previous study. entropy of texture is an index that has been broadly used for radiomic analysis in previous research [2022 ]. when all of the elements in the co - occurrence matrix have the greatest randomness and the elements of the co - occurrence matrix distributes in the dispersion, the value of entropy is large. therefore, hgls display high heterogeneity due to multiple tissue elements and mri shows complex signal changes. kang. found that the mean adc values of grade ii and iii gliomas were lower than grade iv gliomas because of the inclusion of microscopic necrosis regions and the partial volume - averaging effect in measurement. in our study, glcm homogeneity could reflect the texture homogeneity in images and measure the local variation in texture. the value of glcm homogeneity decreased with increased changes in image texture between the different regions. in this study, adc glcm homogeneity values significantly reduced in the high - grade gliomas. therefore, lower value of adc glcm homogeneity might reflect and explain the internal heterogeneity of malignant gliomas. in the majority of recent studies employing quantitative image feature assessment for gliomas, only 1 category feature from 1 mri sequence, such as texture, was applied, which provided limited information. in this study we investigated the efficiency of radiomic features from multiple mri sequences for gliomas grading, yielding promising results. we demonstrated the use of radiomic quantitative mri features on multiple sequences to differentiate between different grades of gliomas. we found that the features of t1-ce glcm entropy and adc glcm homogeneity had high potential in differentiating between lggs and hggs. in previous studies, the cumulative histogram was used to distinguish between hggs and lggs. however, specific feature values reflect only partial features of the tumor. in our study we normalized and combined the radiomic features of multiple mri sequences that significantly differentiated between lggs and hggs. the combined feature further increased diagnostic power and had the highest auc (0.943) and higher specificity (89%) compared with t1-ce glcm entropy, as well as higher sensitivity (90%) compared with adc glcm homogeneity after roc curve analysis. combining multiple radiomic features improved the ability to differentiate hggs and lggs. one possible explanation is that normalized combined radiomics from multiple mri sequences may take into account the malignance and heterogeneity of gliomas more objectively. thus, we could non - invasively quantify tumor features better, and provide a new research strategy for glioma grading using the radiomics method. temporal and spatial heterogeneity is one of the most significant characteristics of malignant tumors, due to molecular and pathological changes. gfap is an intermediate filament cytoskeleton protein which is expressed specifically by the gliocyte, with a molecular weight of 51 kda. reduced expression of gfap from neuroglia cells can cause changes that make it more easy for tumor cells to infiltrate surrounding structures. reported that decreased gfap expression was associated with increasing malignancy grade in gliomas. in the present study, significant correlations were revealed between gfap expression and the features t1-ce glcm entropy and adc - glcm homogeneity. we found a significant negative correlation between gfap and the feature t1-ce glcm entropy (r=0.744, p<0.001), as well as a positive correlation between gfap and the feature adc glcm homogeneity (r=0.728, p<0.001). the decreased gfap expression correlated with aggressiveness and malignance of gliomas, while entropy reflected spatial irregularity ; these could be potential explanations for the negative correlation between t1-ce glcm entropy and gfap expression, as well as the positive correlation between the feature adc glcm homogeneity and gfap. increasing the heterogeneity and complexity of the microenvironment in gliomas can result in non - homogeneity of the signal on different mri sequences. therefore, cellular karyokinesis, during which cells from gliomas infiltrate surrounding tissue, can be non - invasively evaluated using the features t1-ce clcm entropy and adc glcm homogeneity. second, rois were manually delineated one - by - one on slices, which may lead to variability between radiologists. on the other hand, manually drawing rois is a tedious procedure. finally, our investigation did not involve prognosis analysis, which should be explored in future research. in conclusion, radiomic features of t1-ce glcm entropy and adc glcm homogeneity based on entire tumor volume can be a useful tool for grading gliomas. the greatest diagnostic efficiency was found for the combined feature for distinguishing lggs from hggs. furthermore, we showed that radiomic features are significantly associated with the underlying molecular phenotype patterns of gfap. therefore, our strategy of radiomics provides a noninvasive, convenient, and repeatable method of glioma grading for use in clinical medicine, potentially accelerating the development of personalized medicine. | backgroundgliomas are the most common primary brain neoplasms. misdiagnosis occurs in glioma grading due to an overlap in conventional mri manifestations. the aim of the present study was to evaluate the power of radiomic features based on multiple mri sequences t2-weighted - imaging - flair (flair), t1-weighted - imaging - contrast - enhanced (t1-ce), and apparent diffusion coefficient (adc) map in glioma grading, and to improve the power of glioma grading by combining features.material/methodssixty-six patients with histopathologically proven gliomas underwent t2-flair and t1wi - ce sequence scanning with some patients (n=63) also undergoing dwi scanning. a total of 114 radiomic features were derived with radiomic methods by using in - house software. all radiomic features were compared between high - grade gliomas (hggs) and low - grade gliomas (lggs). features with significant statistical differences were selected for receiver operating characteristic (roc) curve analysis. the relationships between significantly different radiomic features and glial fibrillary acidic protein (gfap) expression were evaluated.resultsa total of 8 radiomic features from 3 mri sequences displayed significant differences between lggs and hggs. flair glcm cluster shade, t1-ce glcm entropy, and adc glcm homogeneity were the best features to use in differentiating lggs and hggs in each mri sequence. the combined feature was best able to differentiate lggs and hggs, which improved the accuracy of glioma grading compared to the above features in each mri sequence. a significant correlation was found between gfap and t1-ce glcm entropy, as well as between gfap and adc glcm homogeneity.conclusionsthe combined radiomic feature had the highest efficacy in distinguishing lggs from hggs. |
the skin wound healing is a dynamic and complex process divided into three complementary stages : inflammatory, proliferative, and maturation. the inflammatory phase comprehends the intense leucocytes recruitment to the wound area, removal of cellular and extracellular matrix debris, and syntheses of regulatory molecules such as cytokines and chemokines [1, 2 ]. the proliferative phase progresses with an intense proliferation and migration of fibroblasts, endothelial cells, and keratinocytes as well as formation of the granulation tissue (rich in type iii collagen) and progressive reepithelialization [13 ]. at the maturation phase, type iii collagen is gradually replaced by type i collagen, which originates more thicker and resistant collagen fibers [24 ]. it has been demonstrated that flaws on the leukocyte recruitment and function can impair the healing process due to reductions in the synthesis of regulatory molecules that drives the extracellular matrix assembly [57 ] and neoangiogenesis. in this way, the development of drugs and alternative treatments that favor the migration and cellular activity during the inflammatory and proliferation phases may enhance the skin wound repair. skin wounds represent a serious health problem worldwide frequently associated with high costs and inefficient treatments [9, 10 ]. the use of herbal drugs is opening a new perspective for the treatment of skin wounds, mainly in developing countries. once herbal strategies represent a simple pharmacological option, 80% of the population uses herbal drugs in their health care. although several plant species are currently used in the popular medicine to treat skin wounds worldwide [1114 ], the scientific evidence that supports this practice is scarce. thus, determining the security and efficiency of herbal drugs is an urgent and challenging task, which is essential to develop new technologies and products potentially applied in wound care. in general, the healing properties of plant products are related to specific secondary metabolites, especially tannins, saponins, flavonoids, and alkaloids [11, 47, 48 ]. plant products present a broad spectrum of biological functions such as astringent, antimicrobial, antioxidant, and anti - inflammatory [4954 ] functions, which has been systematically associated with the beneficial effects in stimulating the healing process [49, 52, 54 ]. before extrapolation to the human condition, preclinical researches using animal models have been useful for testing the toxicological security and biological effects of plant fractions and isolated molecules with potential applicability in the treatment of skin wounds [11, 52 ]. despite the increasing number of experimental trials in the last decade, considering that studies using animal models are conceived to support clinical investigations, there is a clear limitation in translating the findings obtained from animal models of wound healing to the human context. considering that herbal drugs are extensively used in the popular medicine, we still do not know where the gap is that hinders the implementation of experimental findings for the development of innovations and technologies potentially useful in the clinical management of skin wounds. thus, we systematically revised preclinical studies with murine models that investigated the effects of plant fractions and isolated molecules on the treatment of skin wounds. beyond determining the relevance of plant derivatives in the skin repair, we analyzed the methodological quality of all preclinical studies identified, especially considering that the quality of evidence generated from flawed methodological studies could compromise the generalizability of the findings and derail conducting clinical studies. research papers that investigate the action of plant fractions and isolated molecules in murine models of skin wound healing, published until 09/04/2015 (15:05:23), were recovered and independently analyzed by three researchers (fbl, mms, and rvg). the search strategy was constructed by four components : animals (filter), injury (wounds), organ (skin), and plants extract (isolates and fractions). the filters were developed from pubmed database according to the hierarchical distribution of medical subject headings [mesh terms ]. the same search strategy was adapted and used to recover studies in the scopus platform. the complete search strategy is described in table s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4916068. language restrictions were applied to recover only articles in english, spanish, and portuguese. an initial selection based on title and abstract [tiab ] duplicate studies were removed and only studies investigating the effect of fractions and isolated molecules from plant extracts in murine models of skin wound healing were considered. after the initial search, all relevant studies were recovered in full text and evaluated by eligibility criteria. works containing unrefined extracts, commercial isolates, in vitro assays, humans, nontraumatic injuries, other animal models, first intention wounds, metabolic diseases associates, and secondary studies (i.e., letter to the editor, note, review, and editorial) were excluded (figure 1). data were extracted and tabulated in a descriptive way (tables 1(a), 1(b), 2(a), and 2(b)). the characteristics investigated were publication characteristics (author, title, publication year, and country) ; research methods (control group, randomization, experimental procedures, and blind evaluation of the results) ; experimental model (animal, number of animals, sex, age, weight, species, acclimation period, animal 's housing, number of animals per cage and experimental groups, food supply, temperature, and light cycle) ; plants (plant 's species, isolates, fractions, dose, toxicity test, exotic / native plant, popular name, utilized part of the plant, and popular indication) ; wounds description (wound area, measurement interval, and treatment duration) (tables 1(a), 1(b), 2(a), and 2(b)). in a comprehensive approach, ethnobotanical / ethnopharmacological aspects were also investigated as follows : plant 's species investigated (geographic distribution and existence or not of bioprospecting), popular indication, and reports of toxicity tests (figure 2). the articles quality was analyzed by the criteria described on the arrive platform (animal research : reporting of in vivo experiments). these criteria are based on short descriptions that indicate essential characteristics of all studies with animal models, such as theoretical and methodological basis, research objective, refinement of the analytical methods, statistical design, sample calculations, and measure outcomes. recently there has been an increasing interest in the systematic reviews of research involving animals. considering the purpose of the systematic review on evaluating important aspects of the referenced publications, we built a table summarizing all the aspects investigated as well as their relevance, describing positive and negative characteristics of the recovered studies (tables 2(a) and 2(b)). 164 duplicated studies and 489 with thematic inadequacy were excluded after reading the title and abstract. after recovery of 329 articles in full text, 303 studies were excluded for not meeting the eligibility criteria. thus, 26 studies were included in the systematic review. the reference list of all included studies was carefully analyzed to ensure the identification of additional relevant studies. thus, six studies were additionally identified and recovered, completing 32 works added to this review. from these studies, 19 studies utilized fractions, 12 studies utilized plant isolates, and 1 study used both fractions and isolates for the treatment of cutaneous wounds (figure 1). the analyzed studies were conducted in 13 different countries, especially india (40.62%, n = 13), followed by brazil and turkey (12.5%, n = 4 each). the most utilized animal models on the experiments were rats (75%, n = 24), followed by mice (12.5%, n = 4) and both (12.5%, n = 4). considering the animal strain, 65.7% were wistar rats, 17.14% were sprague dawley rats, 11.42% were swiss mice, and 5.71% were hairless mice. half of the experimental models used male animals (n = 16), 15.62% (n = 5) used females, and 18.75% (n = 6) used both sex. the animals ' age ranged from 2 to 5 months for rats and from 8 to 12 weeks for mice ; however 71.8% (n = 23) of the studies did not relate this information. the weight of rats ranged from 150 to 400 g and the mice weighted between 18 and 40 g, and only 2 studies (6.25%) did not report this data. more than half of the studies did not describe the popular name of the plant species investigated (59.37%, n = 19). the first treatments utilized on the control group were as follows : 25% (n = 8) used ointment base (which did not have its formulation described), 15.6% (n = 5) used saline solution, 9.4% (n = 3) used nitrofurazone, and 6.2% (n = 2) utilized distilled water. only 3.1% (n = 1) did not present the treatment for the control group. the other works utilized miconazole and nonionic cream ; gentamicin ; matrigel solution ; soft paraffin (85%), cetostearyl alcohol (5%), hard paraffin (5%), and wool fat (5%) ; framycetin ointment ; pbs ; sodium alginate ; vaseline ; tween 80 ; tragacanth ; povidone iodine ointment ; madecassol and ointment base ; chlorocresol bp 0.1% mentioned only once, representing 40.6% of all included studies (n = 13). 62.5% (n = 20) of the plant species were native and 12.5% (n = 4) were exotic and 25% (n = 8) of the studies did not describe this characteristic. investigated wound area presented a large variation (5 mm to 600 mm), and 9.37% (n = 3) of the studies did not describe this data. the calculations used to measure the wound area were described in only 59.37% (n = 19) of the studies. all the works described the interval in which the wound area was measured, and the most common interval was daily, 31.25% (n = 10), followed by measurements taken each 4 days, 28.12% (n = 9) (tables 1(a) and 1(b)). from the 32 species of plants, 23 different families were reported, and the main ones are asteraceae 18.75% (n = 6), euphorbiaceae 9.37% (n = 3), leguminosae 6.25% (n = 2), and fabaceae 6.25% (n = 2), and the other families, liliaceae, boraginaceae, scrophulariaceae, ranunculaceae, apiaceae, myrsinaceae, mimosae, malpighiaceae, tiliaceae, crassulaceae, martyniaceae, rutaceas, araliaceae, piperaceae, solanaceae, caprifoliaceae, dipterocarpaceae, oleaceae, and combretaceae, were mentioned once and represent 59.37% (n = 19) of the included studies. the most used plant structures were the leaves representing 37.5% (n = 12), followed by the flowers 12.5% (n = 4), bole bark 12.5% (n = 4), and seeds 6.25% (n = 2). the fruit, the whole plant, and the latex were mentioned once, representing 3.12% (n, 21.87% (n = 7) of the studies did not mention this information. considering the popular indication, healing effects were described in 46.87% (n = 15) of the studies, followed by anti - inflammatory effects 34.37% (n = 11), treatment of gastrointestinal diseases 28.12% (n = 9), burns 18.75% (n = 6), and antirheumatic 12.5% (n = 4) and ophthalmological diseases 6.25% (n = 2). 18.75% (n = 6) of the studies did not report the popular indication. only 33.4% (n = 11) of the studies report toxicity tests (figure 2). among the analyzed works, 78.12% presented a title coherent to the text, 90.6% presented abstracts containing the objectives, methods, main results, and conclusions, and 75% presented an introduction with sufficient scientific base. most studies (87.5%) related to the therapeutic dose administered (90.62%) reported the route of administration and (96.87%) the treatment duration. were reported in 84.37% and 93.75% of the works, respectively, but only 31.25% provided information about the age of the animals. 59.37% of the studies provided information about the experimental conditions (temperature, humidity, light cycles, feed, and water). a statistical analysis was conducted by all studies, but only 68.75% of the studies specified the data analyzed. a coherent interpretation of the results and direct relationship between objectives and hypothesis were described in 75% of all included studies (table s2). in general, the animals treated with isolates and fractions of plants presented an elevated closure rate of the wound, representing 72.72% of the studies [17, 18, 20, 2327, 29, 30, 33, 3546 ], increase in tissue reepithelialization (30.3%) [15, 1820, 23, 27, 35, 38, 43 ], increase of the traction strength on the cicatricle tissue (75.75%) [15, 1720, 2225, 27, 31, 3346 ], greater content and organization of the extracellular matrix on fast expansion of the granulation tissue (42.42%) [17, 18, 23, 30, 32, 33, 36, 3840, 42, 4446 ], and stimulation of the activity of endogenous antioxidant enzymes (9.09%) [16, 21, 22 ] (tables 2(a) and 2(b)). the use of plant based strategies is opening a new perspective for the treatment of skin wounds, mainly in developing countries, once it represents a simple, low cost, and affordable therapy [1, 7, 5658 ]. there are several studies indicating beneficial effects of herbal medicines in all phases of the healing process. in fact, most of the studies included in this systematic review reported that plant fractions and isolates were able to improve the skin wound healing. apparently, these medicines were especially favorable in controlling the cutaneous inflammatory and oxidative response and in stimulating the granulation tissue formation, collagen maturation, and reepithelialization. in this review, we did not include studies testing crude plant extracts, since the chemical characterization of the extracts makes it difficult to determine the herbal components responsible for the effects reported. even in case of including only studies with murine models, this aspect makes the generalizability of the results difficult, since the biological variability directly influences the response to the treatments. in addition, among the 32 analyzed studies, there were large methodological variation and discrepancies in the measure outcomes. an evident example was the wide variation in wound area and time of wound closure. these considerations are important because they are directly associated with the tensile force experienced by the tissue, which profoundly affect the speed and quality of skin repair [59, 60 ]. our findings show that 20% of the studies that utilized fractions neglected the analysis of wound closure, an essential piece of information to assess the ability of any intervention to stimulate the healing process. in addition, the interval between measurements of wound area and the used protocols for the calculations were variable, representing methodological flaws that compromise the study reproduction and generalizability of the findings [61, 62 ]. considering that the reepithelization and organization of the granulation tissue are fundamental aspects to understand how chemical substances act to stimulate wound healing, only 60% of all studies analyzed the reepithelialization rate and 75% evaluated the molecular components of the extracellular matrix, especially collagen. these parameters indicate if the wound closure follows a normal process, in which the newly formed tissue gradually develops drastic structural changes to reconstitute the morphofunctional characteristics of the intact skin. works which demonstrated the importance of these analyses assert that the type and quantity of collagen fibers deposited on the tissue can be used as a marker of tissue mechanical resistance [2, 3, 9, 58 ]. the connective and epithelial tissues form a support structure to promote the correct closure of the wound [57, 63 ], reducing the chances of opportunistic infections in the wounded area [38, 64 ]. during the formation of granulation tissue, there is predominance of sulfated molecules which attract water, facilitating the cellular migration, and also serve as a support structure for the first formed collagen (type iii collagen). there are enough evidences that the synthesis and differentiation of cells and matrix components are crucial for a normal wound closure. it is already known that the oxidative stress induces cell damage, lipid, protein, and nucleic acids oxidation [66, 67 ]. it is recognized that cutaneous trauma increases the tissue oxidative stress in the wounded area [6669 ]. although reactive species are able to activate cell signaling pathways and stimulate cell proliferation, differentiation, and neoangiogenesis, excessive production of these molecules inhibits the healing process, especially by inducing cell death and molecular damage in the extracellular matrix [23, 70, 71 ]. thus, there is a notorious importance in analyzing the redox balance during skin repair. this is a surprising finding, since the antioxidant effect is a pivotal mechanism indicated in several studies to support the applicability of plant extracts in the treatment of tissue damage, including skin wounds [6669 ]. another fundamental result on the cutaneous repair process is the restoration of the biomechanical properties, especially the tensile force of the newly formed tissue, which provides functional estimates on the quality of the healing process. in this review, only 35% of all studies investigating plant fractions evaluated the traction resistance of the scar tissue, aspects investigated in 61.53% of the studies with plants isolates. in our findings, we see that the majority of the studies used male animals, an aspect potentially associated with the hormonal stability, which is not observed in female animals due to the estrous cycle. the use of rats as the experimental model was higher (75%), aspect potentially related to the large body area needed to perform experimental wounds (1 to 5) thus, it is possible to construct a larger number of wounds and to use a smaller number of animals in each group. in addition, in rats it is possible to collect enough fragments in order to fully analyze the healing process. another interesting piece of data was the age of the animals, which presented a large variation (rats, 2 to 8 weeks ; mice, 5 to 12 weeks). however, 71.8% of the studies did not report this information, making it difficult to establish a temporal basis to determine the effectivity of the herbal treatments investigated. more than half of the studies (59.37%) did not describe the popular name of the plant species. the large number of works that did not describe important variables such as age of the animals and plant species represents a concerning number, once these characteristics are of great importance to ensure the study reproducibility and to allow the elaboration of broad reports with a critical review of the findings. the orientation cited in the arrive guideline describes the minimum information that all scientific publications using animals should include. this guide also brings items that help to understand the quality of the writing and potential methodological bias that compromise the quality of the evidence. the work title should refer the readers to a brief summary of the article content, providing keywords and terms that could be researched in electronic databases. only 78.12% of the studies presented a coherent title, while 90.6% presented abstracts with clear information relative to the objectives, methods, main results, and conclusions. furthermore, 75% presented introduction with enough scientific base, which can make it harder for the reader to understand the relevance of the study. another observation made through arrive guide refers to the health conditions of the animals during the experiment. thus, aspects such as information about environmental conditions (temperature and humidity), mortality, feeding, randomization, and reactions indicative of systemic or local toxicity were neglected in most studies, demonstrating that the report bias is a serious limitation of these preclinical tests that compromise the reliability of the results and the quality of the evidence. the current evidence indicates that fractions and isolated molecules from plant extracts stimulate the healing process in cutaneous wounds. apparently, the main effects of these herbal medicines are associated with the stimulation of collagen synthesis, expansion of the granulation tissue, reepithelialization, modulation of the inflammatory response, and oxidative stress during tissue repair. together, these effects promote increase of the speed of wound closure and the biomechanical resistance of newly formed tissue. however, the serious methodological flaws and report bias observed in most included studies make the current evidence fragile. thus, the relevance of fractions and isolated molecules from plant extracts in the treatment of skin wound can not be accurately determined. considering these limitations, it seems impossible to use these evidences to construct a rational basis that supports clinical studies. therefore, there is an urgent need to improve research reports in experimental studies with herbal medicines in murine models of skin wound healing. this task requires a collective effort of authors, journal editors, reviewers, and financial organisms, to ensure the reproducibility, reliability, and generalizability of the evidence, fundamental elements to determine to what extent herbal medicines are promising in the treatment of skin wounds. | background and purpose. skin wound healing is a dynamic process driven by molecular events responsible for the morphofunctional repair of the injured tissue. in a systematic review, we analyzed the relevance of plant fractions and isolates on skin wound healing. by revising preclinical investigations with murine models, we investigated if the current evidence could support clinical trials. methods. studies were selected in the medline / pubmed and scopus databases according to the prisma statement. all 32 identified studies were submitted to data extraction and the methodological bias was investigated according to arrive strategy. results. the studies demonstrated that plant fractions and isolates are able to modulate the inflammatory process during skin wound healing, being also effective in attenuating the oxidative tissue damage in the scar tissue and stimulating cell proliferation, neoangiogenesis, collagen synthesis, granulation tissue expansion, reepithelialization, and the wound closure rate. however, we identified serious methodological flaws in all studies, such as the high level of reporting bias and absence of standardized experimental designs, analytical methods, and outcome measures. conclusion. considering these limitations, the current evidence generated from flawed methodological animal studies makes it difficult to determine the relevance of herbal medicines to treat skin wounds and derails conducting clinical studies. |
bone marrow evaluation is essential in the diagnostic evaluation of both hematopoietic and many non - hematopoietic disorders, determining the efficacy of treatment and to monitor the recovery process in patients undergoing bone marrow transplantation or marrow - ablative chemotherapy and is also part of the staging process for newly diagnosed patients with lymphoproliferative diseases and certain non - hematopoietic malignancies. regardless of the diagnostic value of the biopsy procedure, the pain experienced during and after the procedure makes some patients fear the procedure and/or reluctant to undergo follow - up biopsies. attempting to mitigate the procedural pain and discomfort, some providers elect to use conscious sedation during the bone marrow procedures ; but this may expose the patient to additional physical risks, increased liability to the provider, and a requirement for increased patient monitoring during and after the procedure. since 1971 the jamshidi needle has been the device of choice for bone marrow sampling with no substantial advancement in marrow sampling technology thereafter. biopsy procedures facilitated by drill - powered needles have been attempted, but with mixed results. in 1993, ahlstrom and astrom described a 32-patient study in which a makeshift bone biopsy system, that included a power drill, was used to obtain the bone marrow sample. more recently, buckley. describe a 68 patient study in which patients underwent bone biopsy using a black and decker drill to access the iliac crest. oncontrol is a battery - powered bone marrow biopsy system (vidacare corporation, shavano park, tx, usa) which allows operators to quickly and efficiently obtain both bone marrow core and aspirate (figure 1). we recently reported the preclinical comparison of the oncontrol to manual biopsy in swine together with an uncontrolled prospective clinical evaluation for outpatient bone marrow aspiration and biopsy. these findings suggested that the powered system was able to produce specimens of equal or greater quality faster and in a more efficient manner. while prospective analysis has shown a strong correlation between the duration of the procedure and the morbidity - particularly with respect to patient discomfort, there has not yet been a clinical study published comparing the new powered device to traditional manual bone marrow biopsy needles. we report on this study designed to comparatively determine if the powered core biopsy needle has advantages over traditional manual needles in terms of improved bone marrow sample yield, decreased pain, and procedure time. the power driver and biopsy needle components of the oncontrol powered bone marrow sampling system. the powered bone marrow biopsy system. the power driver and biopsy needle components of the oncontrol powered bone marrow sampling system. this single - center, randomized, controlled trial was approved by integreview institutional review board, performed in accordance with the declaration of helsinki and good clinical practice, and conducted in a community - based cancer clinic. after obtaining informed consent, a medical history and demographics were recorded ; followed by complete blood count analysis (cbc) and a physical examination. each subject, serving as his / her own control, received bone marrow biopsy procedures using powered and manual devices. the powered device was the oncontrol bone marrow biopsy system (vidacare corporation), an fda - cleared device consisting of a battery - powered driver and biopsy needle set. the driver resembles a small hand - held drill, and powers a single lumen needle set into the medullary cavity of the adult iliac crest. the needle set consists of two parts : an outer cannula, 11 gauge by 4 inches (102 mm) long ; and a bevel - tip inner stylet - used to penetrate the cortex. richmond, va) bone marrow biopsy needle (11 gauge by 4 inches), which has a two - piece t - handle design, a trocar - tapered stylet point and a triple crown cannula tip. two operators (one private practice and one academic hematologist / oncologist), experienced in the use of both devices, performed the bone marrow procedures using the posterior iliac crest. the order in which the devices were used on the subject, and which side was biopsied first (right or left) was randomized. there was minimal time between the two biopsy procedures ; just enough for the physicians to reposition after the first procedure. five to 7 ml of 1% lidocaine buffered with sodium bicarbonate was injected intradermally, subcutaneously and periosteally as a local anesthetic for each biopsy procedure. to assure that the subjects were not biased towards one type of biopsy needle over the other, they were carefully screened and oriented. they were told that the purpose of the trial was to see if there was a difference in the intensity of pain experienced between the two types of needles. they did not know, nor were they told, if either of the needle types was preferred by the investigators. to minimize the noise caused by the powered device, and to decrease the potential for the noise to compromise the blinding of procedure order, noise - cancelling headphones were placed on the subjects during each procedure. to further ensure that noise was not a clue for the subject, the time, in seconds, from contact of the needle with skin to sample acquisition was recorded. pain was measured for intensity using a 100 mm visual analog scale (vas), with scores ranging from 0 to 100, and higher scores indicating more pain. participants indicated their pain level by placing a mark on a 100 mm line at the instant of cortical penetration, biopsy core acquisition, and needle removal. it was recognized that since these volunteers had never experienced bone marrow sampling procedures, the pain scores for the first procedure might be skewed. therefore, following the second procedure, subjects were given the opportunity to change the overall pain score indicated for the first procedure. vas pain scores were also recorded 30 minutes, 24 hours and 48 hours after the procedure. subjects were also queried concerning complications at those time points, with a final complication evaluation at 7 days post procedure. immediately following the procedures, core biopsy samples were measured and submitted to a blinded pathologist for quantitative and qualitative analysis. one subject was disqualified because the biopsy needle insertions sites were not adequately anesthetized before the start of procedures, and the subject did not inform investigators of the inadequacy until biopsy procedures were completed. the subject subsequently experienced severe pain bilaterally and required multiple doses of narcotic analgesia and repeated visits to medical facilities for pain relief. another subject was obese and, after subcutaneous insertion, the first needle was not long enough to penetrate the iliac crest cortex to complete the procedure. the decision was made to not make a second attempt and the subject was disqualified. of the 24 evaluable subjects, there were 24 manual insertions and 24 powered insertions. for those insertions, biopsy core samples were obtained in 66.7% of the manual insertions and 100% of powered insertions - a significant difference (p=0.002). the mean time to core acquisition was 85.7 seconds for manual device and 46.5 seconds for the powered device, a significant difference (p<0.001). using the second look overall pain score (for those that opted to change their score), the mean score for the manual device was 33.3 and 20.9 for the powered - a significant difference (p=0.039). there was no statistical or clinical difference in other pain scores between the two devices. the initial mean biopsy core length was 11.14.5 mm for the manual device and 17.06.8 mm for the powered device ; a statistical difference (p<0.005). after fixation and processing, pathology assessment for the manual device showed a mean length of 6.15.6 mm, width of 1.00.7 mm, and volume of 11.010.8 mm. measurements for the powered device were mean length of 15.36.1 mm, width of 2.00.3 mm, and volume of 49.121.5 mm. for overall quality, 33.3% of manual samples and 79.2% of powered samples were graded adequate - a statistical difference (p=0.002). results are compared with regard to device used for the procedure.variablemanualpoweredp(n=24)(n=24)successful core acquisition, first attempt (%) 66.7100.00.002mean time to core acquisition (seconds)85.731.046.515.80.000mean biopsy core length (mm) per acquired specimen11.14.517.06.80.004mean vas needle insertion (0100)28.924.420.517.30.177mean vas biopsy acquisition (0100)36.820.938.018.30.134mean vas needle removal (0100)28.826.627.021.00.792mean vas overall (0100)36.124.423.916.90.051mean vas overall after 2 look adjustment (0100)33.323.920.915.60.039mean vas 30 minutes post procedure (0100)7.311.44.48.50.328mean vas 24 hours post procedure (0100)5.88.59.113.80.325mean vas 48 hours post procedure (0100)1.21.93.48.00.201pathology biopsy core length (mm)6.15.615.36.1<0.001pathology biopsy core width (mm)1.00.72.00.3<0.001pathology biopsy core volume (mm)11.010.849.121.5<0.001pathology - graded adequate biopsy core (%) 33.379.20.002denotes statistical significance;vas, visual analog scale. denotes statistical significance ; vas, visual analog scale. between operators, there was a difference for mean time to core acquisition (75.5 seconds vs 48.4 seconds, p=0.003), mean biopsy core length (16.7 mm vs 12.0 mm, p=0.027), and mean needle insertion vas (19.4 vs 33.6, p=0.024). between operators, there was no statistical difference in other vas pain scores, ability to capture a core specimen or proportion of core biopsy specimens graded adequate by pathology (see table 2). results are compared with regard to operator performing the procedure.variableoperator aoperator bp(n=30)(n=20)successful core acquisition, first attempt (%) 76.794.40.113mean time to core acquisition (seconds)75.533.248.417.60.003mean biopsy core length (mm) per acquired specimen16.66.912.05.20.027mean vas needle insertion both needles (0100)19.417.433.624.70.024mean vas biopsy acquisition both needles (0100)28.618.938.720.60.090mean vas needle removal both needles (0100)26.323.530.624.70.550mean vas overall both needles (0100)24.820.134.022.50.327mean vas overall after 2nd look adjustment both needles (0100)27.621.230.922.30.332mean vas 30 minutes post procedure both needles (0100)5.39.96.810.60.607mean vas 24 hours post procedure both needles (0100)9.012.85.08.90.247mean vas 48 hours post procedure both needles (0100)2.67.21.82.60.641pathology biopsy core length (mm)11.08.110.26.30.711pathology biopsy core width (mm)1.40.81.70.60.187pathology biopsy core volume (mm)31.527.827.721.80.629pathology - graded adequate biopsy core (%) 60.050.00.104denotes statistical significance;vas, visual analog scale. denotes statistical significance ; vas, visual analog scale. the only complication noted during the study was intense and extended pain experienced by one subject, which took nearly 4 weeks to subside. in a 2009 article describing a 48-patient study that assessed pain during biopsy procedures, ruegg and associates reported mean biopsy needle insertion pain to be 38.5 when patients were anesthetized with buffered lidocaine. in our study, in which buffered lidocaine was also used, subjects rated their pain at 38.0 on average with the powered device. when given the opportunity to change the score for the first procedure, the mean pain score for the powered insertions was reduced to 20.9. the procedure time (needle - to - skin contact to core biopsy sample acquisition) was substantially shorter when using the powered device. indeed, in kuball s 2004 publication describing 263 patients undergoing bone marrow procedures, investigators reported that the duration of the procedure, which averaged 7 minutes, was identified as the sole independent predictive factor for patients pain intensity. the investigators did not define how the duration of needle insertion was measured, but the finding was quantitatively and qualitatively substantiated in our study. this was particularly true for patients with harder bones in which more time and effort is required for cortical penetration. in our study, 154 seconds were required for one subject using the manual needle, compared to 70 seconds using the powered device. for this subject, the manual procedure resulted in a bent needle (figure 2) and no biopsy core sample. this shows the condition of the manual needle following insertion into a patient with hard bones. this shows the condition of the manual needle following insertion into a patient with hard bones. while clinicians strive to conduct bone marrow sampling procedures with as little pain and discomfort to the patient as possible, the ultimate goal of the procedure is the acquisition of a specimen suitable for diagnosis by pathologists. inadequate specimens have a significant impact on clinicians ability to treat patients whose treatment depend an accurate diagnosis. in a 767 patient study, bishop. reported that only 42% of their biopsy specimens were adequate for accurate diagnosis. an adequate sample must be of sufficient size and relatively free of crush artifact and trabecular distortion. the superior core specimens acquired using the powered device is the key finding of this study. aside from a 100% first attempt core capture rate compared to 67% for the manual device, the powered device delivered core specimens that were 53% longer, and contained 346% more volume (figure 3). these are typical examples of bone marrow core specimens obtained following bilateral bone marrow biopsy procedures. these are typical examples of bone marrow core specimens obtained following bilateral bone marrow biopsy procedures. a limitation of our study was the use of healthy volunteer subjects, rather than actual patients. we found it would be difficult and time - consuming for clinicians to accrue a satisfactory number of patients requiring bi - lateral procedures. a second reason is because pain as a study outcome is generally problematic in actual patients due to the great amount of variability, bias and subjectivity. these confounding factors are multiplied with cancer patients who often have underlying pain and an altered perception of pain. another limitation was the decision not to collect a bone marrow aspirate - a normal component of bone marrow sampling procedures. for many patients, the negative pressure within the medullary space during the syringe aspiration causes the worst pain of the procedure, and the intensity of pain is not a function of needle - type. for this reason one other limitation was our failure to standardize the amount of time between the local anesthetic infiltration and the biopsy procedure. an additional limitation was failure to capture total pain (e.g. intensity of the pain times the duration of the pain). while the difference in the intensity of the pain did not reach statistical significance in several of the parameters tested, there is little doubt that the duration of the pain was substantially longer in the manual arm of the study. several subjects stated that they would much rather have the powered needle because the procedure was faster. results suggest the superior size and overall quality of core specimens delivered by the powered device provide more material for pathological evaluation of hematopoietic and oncological disorders. the capture rate in obtaining a satisfactory sample on first attempt was much higher with the powered device, negating the necessity of conducting a second biopsy. while the powered bone marrow biopsy showed a trend toward decreased pain overall at the time of the procedures, use of the powered bone marrow biopsy device | bone marrow sampling remains essential in the evaluation of hematopoietic and many non - hematopoietic disorders. one common limitation to these procedures is the discomfort experienced by patients. to address whether a powered biopsy system could reduce discomfort while providing equivalent or better results, we performed a randomized trial in adult volunteers. twenty - six subjects underwent bilateral biopsies with each device. core samples were obtained in 66.7% of manual insertions ; 100% of powered insertions (p=0.002). initial mean biopsy core lengths were 11.14.5 mm for the manual device ; 17.06.8 mm for the powered device (p<0.005). pathology assessment for the manual device showed a mean length of 6.15.6 mm, width of 1.00.7 mm, and volume of 11.010.8 mm3. powered device measurements were mean length of 15.36.1 mm, width of 2.00.3 mm, and volume of 49.121.5 mm3 (p<0.001). the mean time to core ejection was 86 seconds for manual device ; 47 seconds for the powered device (p<0.001). the mean second look overall pain score was 33.3 for the manual device ; 20.9 for the powered (p=0.039). we conclude that the powered biopsy device produces superior sized specimens, with less overall pain, in less time. |
zebrafish (danio rerio) is extensively used as a model organism in many fields, including developmental biology, cancer and immunology. there is a clear temporal separation between the innate and adaptive immune responses of this organism. zebrafish relies more on the innate immune system than mammals because it does not have a morphologically and functionally mature adaptive immune system until 4 weeks after fertilization. as a result, the zebrafish model can provide new insights into the function and evolution of innate immune responses. as the first line of host defense, the innate immune system relies on a large family of pattern recognition receptors (prrs) to recognize pathogen - associated molecular patterns (pamps) derived from various microbial pathogens, including viruses, bacteria, fungi, parasites and protozoa, and danger - associated molecular patterns (damps) that are present in aberrant locations or abnormal molecular complexes as the consequence of infection, inflammation or cellular stress. currently, the four best characterized groups of prrs include the toll - like receptors (tlrs), the nucleotide - binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid - inducible gene - i (rig - i)-like receptors (rlrs) and the absent in melanoma-2 (aim-2)-likereceptors (alrs). prrs can be localized at the cell surface (tlrs, clrs), within the cytoplasm (nlrs, rlrs and alrs) or in endosomes (tlrs). the cell surface prrs are responsible for surveying the extracellular environment, whereas the cytoplasmic prrs sense intracellular pathogens or danger signals. on pamp or damp recognition, prrs activate signaling cascades leading to the nf-b and interferon (ifn) response factor (irf) transcription factors, thus leading to the induction of proinflammatory cytokines, chemotactic cytokines and antimicrobial responses. zebrafish rely on cytokine and ifn production, complement activation, innate immune cell activation and cellular cytotoxic stimulation as host defense mechanisms against pathogens. several classical receptor families that are involved in primary immune responses have been identified in the zebrafish genome. counterparts of the majority of vertebrate prrs and downstream signaling components have been identified in zebrafish, and some of these have been functionally characterized (table 1). in this review, we focus on the pattern recognition receptors and their signaling pathways in zebrafish and compare them with their counterparts in mammals, including humans, to reveal their evolutionary relationship and provide new insight into innate immune defense mechanisms. the mammalian tlr family consists of 12 members that are integral glycoproteins possessing an extracellular (or intra - endosomal) ligand - binding domain with a leucine - rich repeat (lrr) motif and a cytoplasmic signaling toll / interleukin-1 (il-1) receptor homology (tir) domain. some tlrs (tlr1, -2, -4, -5, -6 and -10) are expressed at the cell surface, whereas others (tlr3, -7, -8, -9, -11 and -13) are located almost exclusively in intracellular compartments, including endosomes and lysosomes. tlrs are principally responsible for the recognition and response to pathogen ligands such as lipopolysaccharide (lps) from gram - negative bacteria (tlr4 ligand), lipoteichoic acid from gram - positive bacteria (tlr2 ligand) and the flagellin protein (tlr5 ligand). nucleic acids such as dsrna, ssrna and single - stranded unmethylated cpg motif - containing dna are recognized by tlr3, tlr7 and tlr9, respectively, in antiviral or antibacterial pathways. in a typical mammalian tlr signaling cascade, ligand binding to the extracellular leucine - rich repeats region causes tlrs to dimerize and undergo conformational changes and/or oligomerization of their intracellular tir domains, which in turn recruit and activate cytosolic tir domain - containing adaptor molecules such as myd88, myd88 adaptor - like protein (mal / tirap), trif / ticam1 and trif - related adaptor molecule (tram / ticam2) to transduce signals from the membrane surface to the cytosol and then to the nucleus via the activation of downstream transcription factors such as atf, nf-b, ap-1, irf and the stat family. the myd88-dependent signaling pathway recruits downstream iraks and activates traf6 and the ib kinase (ikk), leading to the translocation of nf-b to the nucleus and the secretion of anti - infection molecules and inflammatory cytokines and type - i ifn production in dendritic cells (dcs). in the myd88-independent signaling pathways, the non - typical ikks ikki / ikk and tbk1 mediate the activation of irf3 downstream of trif, which is responsible for type - i ifn responses in non - dcs. the tlr protein family is conserved from insects to mammals, but tlr signaling pathways in fish exhibit different features than those in mammals. tlrs are highly expressed in the skin of zebrafish, which suggests a prominent role in the defense against pathogens. zebrafish has an almost complete set of 20 putative tlr variants. among them, 10 are orthologs of human tlr family members, and tlr22 belongs to a fish - specific subfamily that is closely related to the drosophila melanogaster toll-9 gene. zebrafish tlr9 and tlr21 were found to have similar expression profiles and antimicrobial activities toward cpg - odns. because the genome of teleost fish was duplicated during evolution, zebrafish have two counterparts of some mammalian tlrs, including tlr4ba / tlr4bb for the lps - specific tlr4 and tlr5a / tlr5b for tlr5, and tlr8a / tlr8b. homologs of mammalian tlr6 and tlr10 are absent from fish, but tlr14 and tlr18 are non - mammalian. it has been reported that japanese flounder tlr14 shares some features with tlr1, tlr6 and tlr10, suggesting that tlr14 might be a functional substitute for mammalian tlr6 and tlr10. tlr18 in zebrafish and channel catfish is the homolog of human tlr1 and may correspond to tlr14 in other fish. in addition, peitretti. identified tlr20 in zebrafish and common carp, finding that this protein is similar to tlr11 and tlr12 in mice. it is not clear when such gene duplications or deletions occurred during evolution, suggesting that the innate immune system of fish may be more complex than that of mammals. the ancient jawless vertebrate japanese lamprey has no more than 16 tlr genes, whereas there are up to 20 putative tlr variants in zebrafish. ohno proposed that two rounds of whole - genome duplication have occurred during early vertebrate evolution, with a third fish - specific genome duplication occurring later in a basal teleost. a fourth whole - genome duplication even occurred in some cyprinids 821 million years ago, resulting in the appearance of paralogs of ancestral genes and the development of neofunctionalization. with respect to the tlr repertoire, it is of interest to note that, except for a single tlr21 gene, two tlr23 genes and tlr22-related genes, most tlr genes seem absent from the atlantic cod, which may be the result of positive selection to generate neofunctionalization. however, whether the increase in the number of tlrs in fish is associated with diversification in ligand recognition is still unknown. for example, tlr2 can form heterodimers that are responsible for the recognition of bacterial lipoproteins / lipopeptides or pam3csk4. tlr3, -5 and -9 recognize dsrna, flagellin and unmethylated cpg dna, respectively. however, zebrafish tlr4 is not responsive to lps stimulation, despite the fact that md1 and rp105, which mediate ligand delivery and/or recognition, have been identified. biochemical and functional studies indicate that md1 binds both rp105 and tlr4 in zebrafish, but accessory molecules such as lbp, cd14 and md2 have not been isolated from fish, suggesting that tlr4 ligand specificity is not conserved in zebrafish. the knockdown of tlr4a, tlr4b and myd88 did not disrupt the zebrafish immune response to lps exposure, suggesting other unidentified roles of tlr4 signaling in pamp responses. in addition, zebrafish lacks a clear ortholog of caspase-11, which serves as an intracellular lps receptor in mice. on one hand, zebrafish caspy 2 shows the highest homology to human caspase-5 and a preference for caspase-5-like substrates, and caspy 2 also induced apoptosis in mammalian cells that was inhibited by general caspase inhibitors ; on the other hand, the caspy 2 does not contain a card domain as found at the n - terminal of caspases. actually the n - terminal domain of caspy 2 is most homologous to the n - terminal domain of human nlrp3 (46% similarity), a pyrin - domain - containing nod - family protein, thus it remains to be seen whether fish caspy 2, which is similar to human caspase-4/5 (caspase-11), can interact with lps directly and activate the inflammasome. the above discussion suggests that the mechanism of lps recognition in fish could be different from that in mammals, and it is possible that ancestral or other genes are involved in sensing lps. in addition, the fish - specific tlr22 recognizes dsrna viruses or polyi : c ; this is followed by the recruitment of trif to induce ifn expression. in fact, zebrafish also has tlr3, and tlr3 and tlr22 recruit a common adaptor trif to augment the local ifn response to viral infection. the innate immune signaling molecules downstream of tlrs are conserved in zebrafish as well, and include orthologous myd88, sarm1, tollip, ikap (ikk complex associated protein), nemo (nf-b essential modulator), tirap, trif and the central intermediator irfs, the signal transducers and activators of transcription (stats), ap-1, and all of the traf family members (traf1 to traf7). myd88, the most - studied tlr adaptor in zebrafish, has important roles in host defense against microbial infections. in fact, all adaptor molecules except for ticam2 (tram) have been identified in zebrafish. although mammals have two copies of ticam, ticam2/tram was lost specifically from teleost fish, and the only homologous gene is distantly related to ticam1 or ticam2, indicating that an ancestral gene subsequently diverged to two copies. zebrafish ticam1 localizes to the golgi apparatus and lacks the n - terminal and c - terminal proline - rich domains found in the mammalian protein, despite being able to activate nf-b promoters and irf3- and irf7-mediated pathways. the partial function attenuation of tirap in zebrafish, which may weaken its ability to recruit myd88, leads to low sensitivity to lps. overexpression of the adaptor trif induces ifn production in zebrafish, suggesting that zebrafish trif is a true homolog of the mammalian gene. in conclusion, tlr adaptors are highly conserved between mammals and teleosts, suggesting that the signaling downstream of tlrs is highly conserved between the innate immune responses of mammals and fish. the zebrafish has unique features in the components of its tlr downstream pathways, including some gene duplications or losses. with regard to ifn response factors, all of the nine irf orthologs of mammals have been identified in fish. there are two additional irfs in zebrafish, namely irf10 and irf11, which are absent in mammals. furthermore, zebrafish traf1 differs from mammalian traf1 in that it contains a single zinc finger motif. the zebrafish is now being utilized as a model for infectious disease because of the ease of using particular stages to examine immune responses to infection as well as host microbe interactions. tlrs are the best understood innate immune receptors that respond to infection in fish. in 2002, neely. established a zebrafish model of streptococcus infection their studies suggested that the principles underlying host pathogen relationships in fish are very similar to those in humans. mycobacterial infection of zebrafish results in elevated tlr1, tlr2, tlr5a/5b and tlr18 expression and also induces the expression of fish - specific tlr20a and tlr22. these studies suggest that the microbial pamp recognition mechanism is already established in the common vertebrate ancestor and is conserved in mammals and teleosts. the zebrafish mal orthologs that are downstream of tlrs also show increased expression, whereas the expression levels of other tir domain - containing adaptor genes such as myd88, trif and sarm are not responsive to mycobacterial infection, suggesting that the signaling pathways downstream of fish tlrs are different from those in mammals. of course, infecting the entire animal with a live pathogen induces alterable changes in gene expression due to the presence of multiple pamps. as a result, it is difficult to distinguish the correlation between the upregulation of tlr signaling genes and the recognition of specific pathogen - derived ligands. intracellular monitoring is performed by several families of receptors to detect those pathogens that evade extracellular and endosomal surveillance. these include the nlrs for different pamps and damps, rig - like helicase receptors (rlrs) for viral rna, and the alrs for cytosolic dna. the innate immune signaling pathways associated with the nlrs and rlrs are largely conserved in mammals and teleost fish, but the signaling pathways for the alrs are not. the expression of alrs is restricted to and conserved among mammalian species, and the loss of alrs in fish suggests they have evolved alternate mechanisms to cope with dna viruses and intracellular bacteria. owing to the importance of inflammasomes in the innate immune defense system, numerous studies were extended to vertebrates to uncover their functions. at present, the function of nlrs in lower vertebrates and invertebrates is less well understood than that in mammals. with nearly 421 nlr family members in zebrafish, it is predicted that at least one prototype gene exists in lower organisms. with events such as gene loss, duplication and acquisition in various species, this unique gene family is likely to have formed gradually in vertebrates during evolution. by analyzing the molecular phylogeny and expression of nlr subfamilies in zebrafish, three nlr subfamilies have been identified, with the first subfamily (nlr - a) containing eight genes that resemble mammalian nods, the second subfamily (nlr - b) containing nine genes that resemble mammalian nacht-, lrr- and pyd - containing proteins (nlrp), and the third subfamily (nlr - c) containing 405 nlr genes that are unique to teleost fish. recent reports have indicated that several members of the mammalian nlr family, namely nod1, nod2 and nlrc3, are conserved in zebrafish. nod2 cooperates with the dual oxidase (duox) enzyme to produce bactericidal reactive oxygen species in epithelial cells in mammals. similarly, the morpholino - mediated depletion of zebrafish nod1 or nod2 significantly decreases zebrafish duox expression, which reduces the ability of embryos to control systemic infection in a salmonella infection model and suggests that nod - like receptors are also important for innate antibacterial immunity in teleost fish. in contrast, nlrp3, the most extensively studied mammalian nlr in the inflammasome, is not conserved in zebrafish, and there are no direct nlrp3 orthologs in fish. although boyle identified a gene consisting of an ntpase domain, leucine - rich repeats and a c - terminal pry - spry domain on zebrafish chromosome 17 using human nlrp3 in a blastp search, the lack of an n - terminal effector domain distinguished this gene from mammalian nlrp3. in addition, there are not any other putative nlrp3 orthologs in the genomes of other fish. stein. has suggested that these nlrps in lower vertebrates are not similar to the nlrs in mammals and are not the origin of inflammasome components, as these genes provide diverse responses to infection and injury in different species. such differences between fish and mammals are interesting and may encourage scientists to reconsider the mechanism and evolution of the inflammasome. in brief summary, nlrps are not functionally conserved in fish, and whether other zebrafish - specific nlrs may function through inflammasome - like molecular complexes awaits future investigation. despite these differences, as a key adaptor molecule in the inflammasome pathways, apoptosis - associated speck - like protein containing a card (asc) links upstream receptors (such as nlrs / pyhins) and downstream signaling caspases through homotypic or heterotypic protein protein interactions to form the inflammasome complex, a granular structure formed in the perinuclear region of the cytosol upon inflammasome activation that ultimately leads to the processing of pro - il-1 and pro - il-18. zebrafish has a single ortholog of asc (figure 1, accession number nm_131495) that is also composed of an n - terminal pyd domain and a c - terminal card domain. zasc has been observed to form specks in vitro and in the embryo (li and jin, unpublished data), suggesting a conserved function of asc in inflammasome assembly. in addition to the canonical inflammasomes that activate caspase-1, a non - canonical caspase-11-dependent inflammasome pathway was recently discovered. the n - terminal card domain of caspase-11 serves as a direct receptor for intracellular lps. activated caspase-11 cleaves gasdermin d to induce pyroptosis. in zebrafish, there are two caspase genes, namely caspy and caspy 2. interestingly, their n - terminal regions share higher sequence similarity with the pyd than the card domain of mammalian caspases, both of which belong to the death - fold superfamily. the sequence of the zebrafish caspy pyd domain is similar to the n - terminal domains of caspases from non - mammals such as tetrapod, suggesting their functional similarity. comparative alignment of the pyd or card domains of zebrafish shows that the n - terminal domain of caspy has sequence similarity to the zebrafish asc pyd domain, and the sequence of the zebrafish asc card domain is similar to the card domain from zebrafish nlp3 (nacht, lrr and pyd domains - containing protein 3-like isoform x2 ; figure 2). caspy performs the function of human caspase-1, and caspy 2 shows the highest sequence similarity to human caspase-4/5, both of which have substrate specificity. although the function of these fish caspases is poorly defined, they appear to be essential for the morphogenesis of the jaw and gill - bearing arches. it is not yet clear whether fish caspy 2, which is similar to human caspase-4/5 (mouse caspase-11), can directly interact with lps and activate the inflammasome or cleave the relevant substrates and induce, pyroptosis as its orthologs do in mammals. interestingly, the zebrafish dfna5 gene (ay603655) also contains a gasdermin family domain, a recently identified caspase-4/5 substrate and the cleavage of which induce pyroptosis in mammals. zebrafish il-1 represents a single ancestral gene for the il-1 superfamily in mammals and functions similarly to il-1 in mammals. however, the processing and cleavage mechanism of pro - il-1 is less clear in fish because fish il-1 lacks the conserved caspase-1 cleavage site. with respect to function a study demonstrated the caspase-1-mediated processing of il-1 at an aspartic residue distinct from the cleavage site of mammalian il-1 in the european sea bass, suggesting that fish have a more sophisticated inflammasome activation mechanism than mammals. the mammalian rig - i - like receptor (rlr) family consists of retinoic acid - inducible gene - i (rig - i), melanoma differentiation - associated factor 5 (mda5) and laboratory of genetics and physiology 2 (lgp2), and has a major role in initiating antiviral immunity against rna virus infection. the three rlr members are broadly expressed in most tissues, but are typically maintained at low levels in resting cells. rig - i and mda5 share similar domain structures, including the n - terminal tandem card domains for protein protein interactions, a central dexd / h box rna helicase domain with the ability to hydrolyze atp and bind rna ligands, and a c - terminal domain involved in ligand binding and auto - inhibition. however, lgp2, another member of the rlr family, lacks the n - terminal card domain and is thought to function as a negative regulator of rig - i and mda5 signaling. most of the signaling components of the rlr pathways, including rig - i, mda5, lgp2, mavs and tbk1, have been identified in the zebrafish genome. although zebrafish have two variants of rig - i and mda5, they appear to participate in the immune pathways in a manner similar to that of their mammalian homologs. the analysis of the evolutionary pattern of rig - i and mda5 key domains has indicated that rlrs are conserved from lower organisms to higher mammals. recent reports have shown the presence of sting / mita orthologs in zebrafish, demonstrating that rlr - mediated ifn activation is conserved from fish to mammals. were the first to identify a rig - i homolog from zebrafish that contained the complete rig - i sequence with all of the functional domains (n - terminal tandem card, central dexd / h box, an rna helicase domain, and ctd with rd) and demonstrated the conserved functional involvement of zebrafish rig - i in the ifn - i and nf-b signaling pathways and the conserved regulatory role of trim 25 in the rig - i signaling pathway. mammalian rig - i contains three important lysine residues (k99, k169 and k172) involved in k63-ub binding or card card interaction in ubiquitin - mediated antiviral signaling. two of these three ubiquitination sites (namely k169 and k172) are conserved in zebrafish rig - i (figure 3a), indicating that rig - i - mediated antiviral innate immunity is conserved and may originate as early as teleosts. an mda5 ortholog was also identified in zebrafish, and the length of zebrafish mda5 is comparable to that of mammals, ranging from 987 residues to 1285 residues, suggesting that mda5 might possess a conserved function. zebrafish mda5 has a conserved dexd / h domain and a helix c domain but lacks an n - terminal card domain, which may not be directly involved in downstream signaling. recently, gabor. reported the role of zebrafish mda5 in protection against snakehead rhabdovirus infection, suggesting the conservation of this antiviral signaling pathway in lower vertebrates and the possession of recognition strategies for a wide array of viruses by fish early in evolution. two isoforms of lgp2 have been identified in zebrafish and rainbow trout. because this molecule has both inhibitory and stimulatory effects, the role of lgp2 in the innate antiviral response is still unclear in fish as well as in mammals. zebrafish lgp2 has a domain structure similar to zmda5. in the zebrafish genome, mavs, the adaptor downstream of rlr signaling, is composed of a conserved n - terminal card domain and a functional mitochondrial transmembrane domain but lacks the proline - rich domain and the traf6/traf3 binding motif found in mammalian mavs (figures 3 and 4). the virus - induced ifns in zebrafish are classified into group i and group ii ifns. although fish ifns are not the true homologs of mammalian type - i ifns and their receptors differ from mammalian type - i ifn receptors, their expression is activated through similar mechanisms, such as the stat pathway. in zebrafish, viral rna induces the expression of ifn that is most similar to the mammalian ifn - alpha (type i). it is intriguing to observe that the initial production of ifns in zebrafish induces the expression of irf3 through the stat pathway and activates irf7 through phosphorylation, both of which in turn induce a second wave of ifn. in addition, zebrafish irf10 inhibits irf3-mediated ifn induction, and irf7 is targeted by mavs_tv2 for the negative regulation of type - i ifn expression ; this demonstrates special immune mechanisms to balance ifn responses in fish (figure 4). the overexpression of mavs or rig - i cards in zebrafish leads to the constitutive induction of both ifns and ifn - stimulated genes. this results in the inhibition of viral replication and protection against viral infection, suggesting that zebrafish possess a robust antiviral system and share a common evolutionary origin with mammals. in addition to the above examples, other new prrs such as cytosolic dna sensor dna - dependent activator of ifn - regulatory factors and a cytosolic dna receptor named aim-2 have been described in mammals. the mammalian tlr family consists of 12 members that are integral glycoproteins possessing an extracellular (or intra - endosomal) ligand - binding domain with a leucine - rich repeat (lrr) motif and a cytoplasmic signaling toll / interleukin-1 (il-1) receptor homology (tir) domain. some tlrs (tlr1, -2, -4, -5, -6 and -10) are expressed at the cell surface, whereas others (tlr3, -7, -8, -9, -11 and -13) are located almost exclusively in intracellular compartments, including endosomes and lysosomes. tlrs are principally responsible for the recognition and response to pathogen ligands such as lipopolysaccharide (lps) from gram - negative bacteria (tlr4 ligand), lipoteichoic acid from gram - positive bacteria (tlr2 ligand) and the flagellin protein (tlr5 ligand). nucleic acids such as dsrna, ssrna and single - stranded unmethylated cpg motif - containing dna are recognized by tlr3, tlr7 and tlr9, respectively, in antiviral or antibacterial pathways. in a typical mammalian tlr signaling cascade, ligand binding to the extracellular leucine - rich repeats region causes tlrs to dimerize and undergo conformational changes and/or oligomerization of their intracellular tir domains, which in turn recruit and activate cytosolic tir domain - containing adaptor molecules such as myd88, myd88 adaptor - like protein (mal / tirap), trif / ticam1 and trif - related adaptor molecule (tram / ticam2) to transduce signals from the membrane surface to the cytosol and then to the nucleus via the activation of downstream transcription factors such as atf, nf-b, ap-1, irf and the stat family. the myd88-dependent signaling pathway recruits downstream iraks and activates traf6 and the ib kinase (ikk), leading to the translocation of nf-b to the nucleus and the secretion of anti - infection molecules and inflammatory cytokines and type - i ifn production in dendritic cells (dcs). in the myd88-independent signaling pathways, the non - typical ikks ikki / ikk and tbk1 mediate the activation of irf3 downstream of trif, which is responsible for type - i ifn responses in non - dcs. the tlr protein family is conserved from insects to mammals, but tlr signaling pathways in fish exhibit different features than those in mammals. tlrs are highly expressed in the skin of zebrafish, which suggests a prominent role in the defense against pathogens. zebrafish has an almost complete set of 20 putative tlr variants. among them, 10 are orthologs of human tlr family members, and tlr22 belongs to a fish - specific subfamily that is closely related to the drosophila melanogaster toll-9 gene. zebrafish tlr9 and tlr21 were found to have similar expression profiles and antimicrobial activities toward cpg - odns. because the genome of teleost fish was duplicated during evolution, zebrafish have two counterparts of some mammalian tlrs, including tlr4ba / tlr4bb for the lps - specific tlr4 and tlr5a / tlr5b for tlr5, and tlr8a / tlr8b. homologs of mammalian tlr6 and tlr10 are absent from fish, but tlr14 and tlr18 are non - mammalian. it has been reported that japanese flounder tlr14 shares some features with tlr1, tlr6 and tlr10, suggesting that tlr14 might be a functional substitute for mammalian tlr6 and tlr10. tlr18 in zebrafish and channel catfish is the homolog of human tlr1 and may correspond to tlr14 in other fish. in addition, peitretti. identified tlr20 in zebrafish and common carp, finding that this protein is similar to tlr11 and tlr12 in mice. it is not clear when such gene duplications or deletions occurred during evolution, suggesting that the innate immune system of fish may be more complex than that of mammals. the ancient jawless vertebrate japanese lamprey has no more than 16 tlr genes, whereas there are up to 20 putative tlr variants in zebrafish. ohno proposed that two rounds of whole - genome duplication have occurred during early vertebrate evolution, with a third fish - specific genome duplication occurring later in a basal teleost. a fourth whole - genome duplication even occurred in some cyprinids 821 million years ago, resulting in the appearance of paralogs of ancestral genes and the development of neofunctionalization. with respect to the tlr repertoire, it is of interest to note that, except for a single tlr21 gene, two tlr23 genes and tlr22-related genes, most tlr genes seem absent from the atlantic cod, which may be the result of positive selection to generate neofunctionalization. however, whether the increase in the number of tlrs in fish is associated with diversification in ligand recognition is still unknown. for example, tlr2 can form heterodimers that are responsible for the recognition of bacterial lipoproteins / lipopeptides or pam3csk4. tlr3, -5 and -9 recognize dsrna, flagellin and unmethylated cpg dna, respectively. however, zebrafish tlr4 is not responsive to lps stimulation, despite the fact that md1 and rp105, which mediate ligand delivery and/or recognition, have been identified. biochemical and functional studies indicate that md1 binds both rp105 and tlr4 in zebrafish, but accessory molecules such as lbp, cd14 and md2 have not been isolated from fish, suggesting that tlr4 ligand specificity is not conserved in zebrafish. the knockdown of tlr4a, tlr4b and myd88 did not disrupt the zebrafish immune response to lps exposure, suggesting other unidentified roles of tlr4 signaling in pamp responses. in addition, zebrafish lacks a clear ortholog of caspase-11, which serves as an intracellular lps receptor in mice. on one hand, zebrafish caspy 2 shows the highest homology to human caspase-5 and a preference for caspase-5-like substrates, and caspy 2 also induced apoptosis in mammalian cells that was inhibited by general caspase inhibitors ; on the other hand, the caspy 2 does not contain a card domain as found at the n - terminal of caspases. actually the n - terminal domain of caspy 2 is most homologous to the n - terminal domain of human nlrp3 (46% similarity), a pyrin - domain - containing nod - family protein, thus it remains to be seen whether fish caspy 2, which is similar to human caspase-4/5 (caspase-11), can interact with lps directly and activate the inflammasome. the above discussion suggests that the mechanism of lps recognition in fish could be different from that in mammals, and it is possible that ancestral or other genes are involved in sensing lps. in addition, the fish - specific tlr22 recognizes dsrna viruses or polyi : c ; this is followed by the recruitment of trif to induce ifn expression. in fact, zebrafish also has tlr3, and tlr3 and tlr22 recruit a common adaptor trif to augment the local ifn response to viral infection. the innate immune signaling molecules downstream of tlrs are conserved in zebrafish as well, and include orthologous myd88, sarm1, tollip, ikap (ikk complex associated protein), nemo (nf-b essential modulator), tirap, trif and the central intermediator irfs, the signal transducers and activators of transcription (stats), ap-1, and all of the traf family members (traf1 to traf7). myd88, the most - studied tlr adaptor in zebrafish, has important roles in host defense against microbial infections. in fact, all adaptor molecules except for ticam2 (tram) have been identified in zebrafish. although mammals have two copies of ticam, ticam2/tram was lost specifically from teleost fish, and the only homologous gene is distantly related to ticam1 or ticam2, indicating that an ancestral gene subsequently diverged to two copies. zebrafish ticam1 localizes to the golgi apparatus and lacks the n - terminal and c - terminal proline - rich domains found in the mammalian protein, despite being able to activate nf-b promoters and irf3- and irf7-mediated pathways. the partial function attenuation of tirap in zebrafish, which may weaken its ability to recruit myd88, leads to low sensitivity to lps. overexpression of the adaptor trif induces ifn production in zebrafish, suggesting that zebrafish trif is a true homolog of the mammalian gene. in conclusion, tlr adaptors are highly conserved between mammals and teleosts, suggesting that the signaling downstream of tlrs is highly conserved between the innate immune responses of mammals and fish. the zebrafish has unique features in the components of its tlr downstream pathways, including some gene duplications or losses. with regard to ifn response factors, all of the nine irf orthologs of mammals have been identified in fish. there are two additional irfs in zebrafish, namely irf10 and irf11, which are absent in mammals. furthermore, zebrafish traf1 differs from mammalian traf1 in that it contains a single zinc finger motif. the zebrafish is now being utilized as a model for infectious disease because of the ease of using particular stages to examine immune responses to infection as well as host microbe interactions. tlrs are the best understood innate immune receptors that respond to infection in fish. in 2002, neely. established a zebrafish model of streptococcus infection their studies suggested that the principles underlying host pathogen relationships in fish are very similar to those in humans. mycobacterial infection of zebrafish results in elevated tlr1, tlr2, tlr5a/5b and tlr18 expression and also induces the expression of fish - specific tlr20a and tlr22. these studies suggest that the microbial pamp recognition mechanism is already established in the common vertebrate ancestor and is conserved in mammals and teleosts. the zebrafish mal orthologs that are downstream of tlrs also show increased expression, whereas the expression levels of other tir domain - containing adaptor genes such as myd88, trif and sarm are not responsive to mycobacterial infection, suggesting that the signaling pathways downstream of fish tlrs are different from those in mammals. of course, infecting the entire animal with a live pathogen induces alterable changes in gene expression due to the presence of multiple pamps. as a result, it is difficult to distinguish the correlation between the upregulation of tlr signaling genes and the recognition of specific pathogen - derived ligands. intracellular monitoring is performed by several families of receptors to detect those pathogens that evade extracellular and endosomal surveillance. these include the nlrs for different pamps and damps, rig - like helicase receptors (rlrs) for viral rna, and the alrs for cytosolic dna. the innate immune signaling pathways associated with the nlrs and rlrs are largely conserved in mammals and teleost fish, but the signaling pathways for the alrs are not. the expression of alrs is restricted to and conserved among mammalian species, and the loss of alrs in fish suggests they have evolved alternate mechanisms to cope with dna viruses and intracellular bacteria. owing to the importance of inflammasomes in the innate immune defense system, numerous studies were extended to vertebrates to uncover their functions. at present, the function of nlrs in lower vertebrates and invertebrates is less well understood than that in mammals. with nearly 421 nlr family members in zebrafish, it is predicted that at least one prototype gene exists in lower organisms. with events such as gene loss, duplication and acquisition in various species, this unique gene family is likely to have formed gradually in vertebrates during evolution. by analyzing the molecular phylogeny and expression of nlr subfamilies in zebrafish, three nlr subfamilies have been identified, with the first subfamily (nlr - a) containing eight genes that resemble mammalian nods, the second subfamily (nlr - b) containing nine genes that resemble mammalian nacht-, lrr- and pyd - containing proteins (nlrp), and the third subfamily (nlr - c) containing 405 nlr genes that are unique to teleost fish. recent reports have indicated that several members of the mammalian nlr family, namely nod1, nod2 and nlrc3, are conserved in zebrafish. nod2 cooperates with the dual oxidase (duox) enzyme to produce bactericidal reactive oxygen species in epithelial cells in mammals. similarly, the morpholino - mediated depletion of zebrafish nod1 or nod2 significantly decreases zebrafish duox expression, which reduces the ability of embryos to control systemic infection in a salmonella infection model and suggests that nod - like receptors are also important for innate antibacterial immunity in teleost fish. in contrast, nlrp3, the most extensively studied mammalian nlr in the inflammasome, is not conserved in zebrafish, and there are no direct nlrp3 orthologs in fish. although boyle identified a gene consisting of an ntpase domain, leucine - rich repeats and a c - terminal pry - spry domain on zebrafish chromosome 17 using human nlrp3 in a blastp search, the lack of an n - terminal effector domain distinguished this gene from mammalian nlrp3. in addition, there are not any other putative nlrp3 orthologs in the genomes of other fish. stein. has suggested that these nlrps in lower vertebrates are not similar to the nlrs in mammals and are not the origin of inflammasome components, as these genes provide diverse responses to infection and injury in different species. such differences between fish and mammals are interesting and may encourage scientists to reconsider the mechanism and evolution of the inflammasome. in brief summary, nlrps are not functionally conserved in fish, and whether other zebrafish - specific nlrs may function through inflammasome - like molecular complexes awaits future investigation. despite these differences, some components of innate inflammatory pathways are conserved in zebrafish. as a key adaptor molecule in the inflammasome pathways, apoptosis - associated speck - like protein containing a card (asc) links upstream receptors (such as nlrs / pyhins) and downstream signaling caspases through homotypic or heterotypic protein protein interactions to form the inflammasome complex, a granular structure formed in the perinuclear region of the cytosol upon inflammasome activation that ultimately leads to the processing of pro - il-1 and pro - il-18. zebrafish has a single ortholog of asc (figure 1, accession number nm_131495) that is also composed of an n - terminal pyd domain and a c - terminal card domain. zasc has been observed to form specks in vitro and in the embryo (li and jin, unpublished data), suggesting a conserved function of asc in inflammasome assembly. in addition to the canonical inflammasomes that activate caspase-1, a non - canonical caspase-11-dependent inflammasome pathway was recently discovered. the n - terminal card domain of caspase-11 serves as a direct receptor for intracellular lps. activated caspase-11 cleaves gasdermin d to induce pyroptosis. in zebrafish, there are two caspase genes, namely caspy and caspy 2. interestingly, their n - terminal regions share higher sequence similarity with the pyd than the card domain of mammalian caspases, both of which belong to the death - fold superfamily. the sequence of the zebrafish caspy pyd domain is similar to the n - terminal domains of caspases from non - mammals such as tetrapod, suggesting their functional similarity. comparative alignment of the pyd or card domains of zebrafish shows that the n - terminal domain of caspy has sequence similarity to the zebrafish asc pyd domain, and the sequence of the zebrafish asc card domain is similar to the card domain from zebrafish nlp3 (nacht, lrr and pyd domains - containing protein 3-like isoform x2 ; figure 2). caspy performs the function of human caspase-1, and caspy 2 shows the highest sequence similarity to human caspase-4/5, both of which have substrate specificity. although the function of these fish caspases is poorly defined, they appear to be essential for the morphogenesis of the jaw and gill - bearing arches. it is not yet clear whether fish caspy 2, which is similar to human caspase-4/5 (mouse caspase-11), can directly interact with lps and activate the inflammasome or cleave the relevant substrates and induce, pyroptosis as its orthologs do in mammals. interestingly, the zebrafish dfna5 gene (ay603655) also contains a gasdermin family domain, a recently identified caspase-4/5 substrate and the cleavage of which induce pyroptosis in mammals. zebrafish il-1 represents a single ancestral gene for the il-1 superfamily in mammals and functions similarly to il-1 in mammals. however, the processing and cleavage mechanism of pro - il-1 is less clear in fish because fish il-1 lacks the conserved caspase-1 cleavage site. with respect to function a study demonstrated the caspase-1-mediated processing of il-1 at an aspartic residue distinct from the cleavage site of mammalian il-1 in the european sea bass, suggesting that fish have a more sophisticated inflammasome activation mechanism than mammals. the mammalian rig - i - like receptor (rlr) family consists of retinoic acid - inducible gene - i (rig - i), melanoma differentiation - associated factor 5 (mda5) and laboratory of genetics and physiology 2 (lgp2), and has a major role in initiating antiviral immunity against rna virus infection. the three rlr members are broadly expressed in most tissues, but are typically maintained at low levels in resting cells. rig - i and mda5 share similar domain structures, including the n - terminal tandem card domains for protein protein interactions, a central dexd / h box rna helicase domain with the ability to hydrolyze atp and bind rna ligands, and a c - terminal domain involved in ligand binding and auto - inhibition. however, lgp2, another member of the rlr family, lacks the n - terminal card domain and is thought to function as a negative regulator of rig - i and mda5 signaling. most of the signaling components of the rlr pathways, including rig - i, mda5, lgp2, mavs and tbk1, have been identified in the zebrafish genome. although zebrafish have two variants of rig - i and mda5, they appear to participate in the immune pathways in a manner similar to that of their mammalian homologs. the analysis of the evolutionary pattern of rig - i and mda5 key domains has indicated that rlrs are conserved from lower organisms to higher mammals. recent reports have shown the presence of sting / mita orthologs in zebrafish, demonstrating that rlr - mediated ifn activation is conserved from fish to mammals. were the first to identify a rig - i homolog from zebrafish that contained the complete rig - i sequence with all of the functional domains (n - terminal tandem card, central dexd / h box, an rna helicase domain, and ctd with rd) and demonstrated the conserved functional involvement of zebrafish rig - i in the ifn - i and nf-b signaling pathways and the conserved regulatory role of trim 25 in the rig - i signaling pathway. mammalian rig - i contains three important lysine residues (k99, k169 and k172) involved in k63-ub binding or card card interaction in ubiquitin - mediated antiviral signaling. two of these three ubiquitination sites (namely k169 and k172) are conserved in zebrafish rig - i (figure 3a), indicating that rig - i - mediated antiviral innate immunity is conserved and may originate as early as teleosts. an mda5 ortholog was also identified in zebrafish, and the length of zebrafish mda5 is comparable to that of mammals, ranging from 987 residues to 1285 residues, suggesting that mda5 might possess a conserved function. zebrafish mda5 has a conserved dexd / h domain and a helix c domain but lacks an n - terminal card domain, which may not be directly involved in downstream signaling. recently, gabor. reported the role of zebrafish mda5 in protection against snakehead rhabdovirus infection, suggesting the conservation of this antiviral signaling pathway in lower vertebrates and the possession of recognition strategies for a wide array of viruses by fish early in evolution. two isoforms of lgp2 have been identified in zebrafish and rainbow trout. because this molecule has both inhibitory and stimulatory effects, the role of lgp2 in the innate antiviral response is still unclear in fish as well as in mammals. zebrafish lgp2 has a domain structure similar to zmda5. in the zebrafish genome, mavs, the adaptor downstream of rlr signaling, is composed of a conserved n - terminal card domain and a functional mitochondrial transmembrane domain but lacks the proline - rich domain and the traf6/traf3 binding motif found in mammalian mavs (figures 3 and 4). the virus - induced ifns in zebrafish are classified into group i and group ii ifns. although fish ifns are not the true homologs of mammalian type - i ifns and their receptors differ from mammalian type - i ifn receptors, their expression is activated through similar mechanisms, such as the stat pathway. in zebrafish, viral rna induces the expression of ifn that is most similar to the mammalian ifn - alpha (type i). it is intriguing to observe that the initial production of ifns in zebrafish induces the expression of irf3 through the stat pathway and activates irf7 through phosphorylation, both of which in turn induce a second wave of ifn. in addition, zebrafish irf10 inhibits irf3-mediated ifn induction, and irf7 is targeted by mavs_tv2 for the negative regulation of type - i ifn expression ; this demonstrates special immune mechanisms to balance ifn responses in fish (figure 4). the overexpression of mavs or rig - i cards in zebrafish leads to the constitutive induction of both ifns and ifn - stimulated genes. this results in the inhibition of viral replication and protection against viral infection, suggesting that zebrafish possess a robust antiviral system and share a common evolutionary origin with mammals. in addition to the above examples, other new prrs such as cytosolic dna sensor dna - dependent activator of ifn - regulatory factors and a cytosolic dna receptor named aim-2 have been described in mammals. counterparts of the major vertebrate prrs and their downstream signaling components have been identified in zebrafish ; these counterparts are functionally similar and more diverse compared with their mammalian counterparts. due to their importance in innate immunity, we compared the conservation and diversity of the zebrafish pattern recognition receptors and their signaling components with those in mammals in this review. nearly all of the tlrs, nlrs and rlrs identified in mammals have been described in fish ; however, there are still differences between them. compared with other teleost fish, the zebrafish genome encodes unique tlrs. zebrafish - specific tlr4 appears to be non - responsive to lps, and changes in ligand recognition specificities reflect differences in aquatic and terrestrial pathogens or divergence that occurred during vertebrate evolution. zebrafish tlr5 harbors two isoforms (tlr5a and tlr5b) ; it is not clear how these tandem copies of tlr5 function, such as whether both are involved in immune responses and have similar roles or one functions as a co - receptor or an antagonistic receptor. in contrast, functional rlr - triggered ifn antiviral signaling pathways are highly conserved from lower organisms to mammals. interestingly, although genes closely related to mda5 and mavs have been identified in branchiostoma, the products of these genes could not induce ifn production. additional knowledge of vertebrate evolution will help uncover the evolutionary history of the rlr family. furthermore, although the nlrp receptors in the classic nlrp inflammasome signaling pathway are specific to mammals, other nlr family members may have evolved for inflammasome activation in zebrafish. in conclusion, although mammals diverged from fish 450 million years ago, the prrs in zebrafish provide functional and evolutionary insights into innate immune signaling pathways. additional studies in this field will not only enrich our understanding of the immune response in zebrafish itself but will also provide unique perspectives on the evolution of the immune system. | pattern recognition receptors (prrs) and their signaling pathways have essential roles in recognizing various components of pathogens as well as damaged cells and triggering inflammatory responses that eliminate invading microorganisms and damaged cells. the zebrafish relies heavily on these primary defense mechanisms against pathogens. here, we review the major prr signaling pathways in the zebrafish innate immune system and compare these signaling pathways in zebrafish and humans to reveal their evolutionary relationship and better understand their innate immune defense mechanisms. |
the vitreoretinal interface underlies a significant proportion of retinal pathology that has previously required surgical intervention. the spectrum of these diseases, while previously limited to macular holes, myopic schisis - like retinopathy and vitreomacular traction syndrome, are now suspected to also encompass diabetic retinopathy, retinal vein occlusion, subtypes of cystoid macular edema and even exudative age - related macular degeneration.13 the extracellular matrix comprising the vitreoretinal interface, composed mainly of collagen and the proteoglycans fibronectin and laminin,4 bridges the cortical vitreous to the retina s internal limiting membrane and has most recently been pharmacologically targeted by ocriplasmin (jetrea ; thrombogenics, inc., iselin, nj, usa) a truncated catalytic domain of plasmin.5 following a series of phase ii and iii clinical trials, the most pivotal of which were collectively called the microplasmin for intravitreous injection traction release without surgical treatment (mivi - trust) study,6 the united states food and drug administration (fda) granted approval of ocriplasmin for symptomatic vitreomacular adhesion on october 18, 2012. while initial experiences with ocriplasmin have generally been favorable since its commercial availability in january of 2013,7 surveillance of post - approval drug safety remains critical. toward that end, we report two cases of retinal breaks occurring after intravitreal injection of ocriplasmin. a 66-year - old female was referred to the vitreoretinal surgery clinic for decreased central visual acuity in the right eye. her best corrected visual acuity (bcva) was 20/70 - 1 and 20/20 in the right and left eyes, respectively. posterior segment examination of the right eye was notable for a blunted foveal light reflex and central macula thickening due to vitreomacular traction with no weiss ring seen on examination. spectral - domain optical coherence tomography (sd - oct) of the right eye demonstrated vitreomacular traction yielding foveal distortion as well as intraretinal and subretinal fluid (figure 1). after obtaining informed consent, the patient was treated with 125 g in 100 l of intravitreal ocriplasmin, which was injected into the inferotemporal quadrant of the right eye. routine follow - up 1 week later revealed symptomatic improvement in central vision of the right eye with examination demonstrating a stable bcva of 20/70. sd - oct confirmed posterior hyaloid separation from the central macula with improvement in intraretinal fluid and persistent subretinal fluid (figure 2). the peripheral retinal exam was notable only for a nasal, flame shaped hemorrhage in the right eye with no retinal breaks noted. twelve days after ocriplasmin administration, the patient developed acute onset of flashing lights in the right eye. her bcva was 20/60 with posterior examination now demonstrating a weiss ring, continued subfoveal fluid and release of vitreomacular traction. however, peripheral examination revealed a superotemporal operculated retinal hole with a surrounding cuff of subretinal fluid. the patient was treated with laser retinopexy in the right eye without complication. over the next 2 months the patient s bcva improved to 20/40 though she noted intermittent, though self - limited, flashing lights in her right eye. posterior segment examination at 2 months demonstrated residual subfoveal fluid, laser scars surrounding the superotemporal retinal break and no other retinal breaks. sd - oct confirmed the continued presence of subfoveal fluid, though progressive improvement in the foveal contour of the right eye was noted (figure 3). visual acuity and a 65-year - old female was referred to the vitreoretinal surgery clinic for a decline in central visual acuity in the left eye. her bcva was 20/25 - 1 and 20/80 - 1 in the right and left eyes, respectively. posterior segment examination of the right eye was unremarkable while examination of the left eye demonstrated a full - thickness macular hole with vitreomacular traction. sd - oct (figure 4) of the left eye demonstrated a full - thickness macular hole with vitreomacular traction. after obtaining informed consent, the patient was treated with 125 g in 100 l of intravitreal ocriplasmin which was injected into the inferotemporal quadrant of the left eye. at follow - up examination 1 week later, the patient revealed she had suffered a 4 day history of flashing lights in the left eye starting 1 day after ocriplasmin injection. though this subsided, she then noted a shadow in the nasal and central regions of her left visual field which started 6 days after her ocriplasmin injection. posterior segment examination demonstrated interval macular hole closure, but also revealed three horseshoe retinal tears with an associated macula - involving retinal detachment (figure 5). the patient underwent immediate and successful retinal detachment repair surgery by scleral buckling, pars plana vitrectomy, air - fluid exchange, endolaser and 14% c3f8 gas - injection. at the 6 month follow - up visit, the patient was noted to have a best - corrected visual acuity of 20/50 with (visually significant) nuclear sclerotic and posterior subcapsular cataract. posterior segment examination demonstrated the macula and peripheral retina to be attached with a good encircling buckle effect and without any additional retinal breaks. while ocriplasmin represents a potent new tool in the treatment of vitreomacular traction, careful monitoring of treated patients, as with any novel therapy, remains paramount in assessing both its efficacy and safety. while the above represents the first case series of retinal breaks occurring after intravitreal ocriplasmin administration, their occurrence in and of themselves is not without precedent. the mivi i trial (phase i / ii clinical trial), which enrolled 60 patients, reported a far peripheral retinal tear with an associated retinal detachment noted on post - injection day 1.8 a later phase ii trial, in which 92 patients received varying doses of ocriplasmin, also reported two retinal tears. these tears occurred within 7 days of intravitreal injections but the exact timing of their occurrence is unclear.9 similarly, the mivi - trust studies (phase ii / iii clinical trials) reported two post - injection retinal tears with associated retinal detachments in the 464 patients treated with ocriplasmin though the exact timing of these tears with respect to the injections was again not reported.6 given the timing of each of the prior reported instances, the breaks may have been a result of the intravitreal injection itself rather than the pharmacologic action of ocriplasmin. there is some evidence supporting this theory : the mivi i trial authors reported that during surgical repair of the aforementioned retinal tear and detachment, the vitreous was found still firmly adherent to the retina.8 regardless of the cause of previously reported retinal tears, the retinal break in case 1 can be attributed to the pharmacologic action of ocriplasmin in inducing a posterior vitreous detachment rather than the physical action of an intravitreal injection in yielding vitreoretinal traction. this is supported by the fact that no retinal breaks were noted on dilated fundus examination 1 week after the intravitreal injection and the patient s relevant symptoms began 12 days after the injection. in both cases, ocriplasmin s pharmacologic action, while relieving the patients vitreomacular traction, resulted in an incomplete or pvd : the extent of ocriplasmin - mediated vitreous liquefaction not only released macular vitreoretinal adhesion, but also caused vitreoschisis that left residual vitreous cortex attached to the retina which ultimately contributed to a retinal break.2 our observation diverges from preclinical studies, which note that in addition to inducing vitreous liquefaction, ocriplasmin produces complete separation of the vitreous cortex from the internal limiting membrane.10 this has, in fact, previously been cited as one of the advantages of ocriplasmin s use over surgical pvd induction, which was deemed less favorable in yielding incomplete cortical removal from the retinal surface with the consequent potential for fibrocellular proliferation and vitreoretinal traction.11,12 anomalous pvd formation has, however, previously been implied in ocriplasmin - treated eyes which eventually underwent vitrectomy as surgeons reportedly encountered intraoperative vitreoschisis and continued vitreoretinal adhesion in these eyes.2 while it is not surprising that an anomalous pvd occurs in ocriplasmin treated eyes which suffer treatment failure, our cases are notable in that the vitreomacular traction was successfully relieved, yet both patients still suffered an anomalous pvd, and consequently, retinal breaks. one harbinger of this case series is perhaps phase iii clinical trials of ocriplasmin where 26.5% of ocriplasmin - injected eyes were observed to have resolution of vitreomacular adhesion, yet only 13.4% of those eyes demonstrated a total pvd by ultrasonography.6 in the same vein, it is perhaps unfeasible for early pharmacologic generation of an anomalous pvd to be ameliorated non - surgically more than 24 hours after intravitreal injection, as the second order (autoproteolytic) kinetics describing ocriplasmin s concentration result in its decrease by over 97% during this interval.13,14 thus, in patients treated with ocriplasmin, maintained vigilance for retinal breaks even after resolution of vitreomacular traction is critical with immediate evaluation of all symptomatic patients. | ocriplasmin represents a new treatment option for numerous vitreoretinopathies involving an abnormal vitreomacular interface. while the drug may circumvent the traditional risks of surgical treatment, pharmacologic vitreolysis is not devoid of risk itself. this report presents two cases, one of vitreomacular traction syndrome and the other of a full - thickness macular hole, both of which were treated with an intravitreal injection of ocriplasmin. notably, in both cases, vitreomacular traction of the macula appears to have been alleviated ; however, failure to completely relieve vitreoretinal traction from the peripheral retina generated retinal breaks with one patient eventually developing a macula - involving retinal detachment. thus, even in instances of successful pharmacologic treatment of vitreomacular traction, continued follow - up evaluation is essential. |
common bile duct (cbd) cancer is a relatively rare but increasing malignancy worldwide that arises from biliary epithelium, known as a cancer with chemoresistance and poor prognosis. complete resection is the most effective and the only potentially curative treatment.1 however, at the time of diagnosis most patients are presented with advanced diseases.2 even the patients who had undergone curative resection show high recurrence rates. thus systemic chemotherapy is the mainstay of the treatment but cbd cancer is highly resistant to most chemotherapeutic agents.3 currently, there is no standard chemotherapeutic regimen established for cbd cancer. gemcitabine appears to be the most effective single agent.4 target therapy to avoid multidrug resistance is now under investigation. here, we report a case of unresectable cbd cancer with liver metastasis, which completely responded to combination chemotherapy of gemcitabine and s-1. to our knowledge, this is the first case reporting a patient with advanced cbd cancer which showed pathologic complete remission after chemotherapy. a 65-year - old male was admitted for abdominal pain and fever, which started 2 weeks ago. laboratory finding demonstrated white blood cells 12.310/l, hemoglobin 11.6 g / dl, platelets 30910/l, total bilirubin 0.7 mg / dl, alkaline phosphatase 117 iu / l, aspartate aminotransferase 26 iu / l, alanine aminotransferase 33 iu / l, high sensitivity c - reactive protein 22.1 mg / dl, carcinoembryonic antigen 2.0 ng / ml, carbohydrate antigen 19 - 9 7.6 u / ml, and alphafetoprotein 1.3 ng / ml. hepatitis b surface antigen was negative and hepatitis c virus antibody was positive. abdominal computed tomography (ct) showed about 9 cm lobulating low attenuating mass with peripheral rim enhancement in the left lobe of the liver, which was thought to be mature pyogenic liver abscess (fig. he started antibiotics therapy with ceftriaxone and metronidazole, and underwent percutaneous drainage tube insertion using 10.2 fr pigtail catheter into the liver abscess. two days later fever subsided and a week later he was discharged with oral antibiotics. one month later, follow - up liver ct scan showed 1.5 cm enhancing lesion in distal cbd with progression of biliary dilatation, suggesting distal cbd cancer (fig. additional ct scan (fig. 5) and magnetic resonance imaging (mri) (fig. 6) showed multiple small nodules in right hepatic lobe and metastatic adenocarcinoma was confirmed from frozen slide of sono - guided liver biopsy (fig. since the patient was inoperable, he underwent combination chemotherapy with gemcitabine 1,600 mg intravenous infusion on day 1 and s-1 (tegafur+gimeracil+oteracil) 500 mg twice a day for 14 days with 1-week rest as one course. serial ct scans checked every three cycles showed gradual regression of the metastatic nodules in liver (fig. 8). grossly the resected cbd showed a 11 cm sized gray solid mass infiltrating into adjacent soft tissue. microscopically there were no residual tumor but chronic active cholangitis with multifocal erosion and subepithelial fibrosis in the resected cbd (fig. then, 22 days after the operation he was discharged without any complication. for 3 months of follow - up, he did not develop any signs of recurrence and no evidence of recurrence was found in the 3-month postoperative ct scan (fig. although radical surgery is the most effective therapy for cure in patients with cholangiocarcioma, only 20% of patients are diagnosed with resectable diseases.5 patients with unresectable cholangiocarcinoma have dismal prognosis with median survival of 6 to 12 months6 and 5-year survival rate of less than 5%.7 for these patients palliative therapy is important in terms of quality of life as well as the survival rate. the role of chemotherapy however chemotherapy has been reported to be more beneficial than the best supportive care.8 gemcitabine has shown to be an effective therapy for cholangiocarcinoma in phase ii trials.9,10 fluorouracil also has shown activity in combination with gemcitabine.11 s-1 is a novel oral fluoropyrimidine agent containing tegafur, gimeracil and oteracil potassium. gimeracil is a competitive inhibitor of dihydropyrimidine dehyrogenase, which achieves higher concentrations of 5-fluorouracil in plasma and tumor tissues.12 yoshizawa.13 tested the combination of s-1 with other anticancer drugs (gemcitabine, cisplatin, irinotecan, mitomycin c, adriamycin, and paclitaxel) and reported that synergistic effect was most evident in gemcitabine / s-1 combination. a recent review reported phase ii trials supporting the following combinations : gemcitabine / cisplatin, gemcitabine / oxaliplatin, gemcitabine / capecitabine, and 5-fluorouracil in unresectable or metastatic cholangiocarcinoma.14 also there have been phase ii trials of gemcitabine and s-1 combination chemotherapy showing a promising survival benefit with acceptable toxicity in patients with advanced biliary tract cancer.15,16 in our case, we used gemcitabine combined with s-1. gemcitabine was chosen for its extensively evaluated data supporting effectiveness in advanced cbd cancer. s-1 was selected as an alternative to 5-fluorouracil for its convenience of administration and less toxicity. so far, several cases have been reported, in which advanced cholangiocarcinoma was completely treated with gemcitabine chemotherapy in japan,17 - 20 although only one of them has shown complete remission histopathologically.14 in our case, the metastatic lesion disappeared radiologically and primary tumor was confirmed to have disappeared histopathologically after the chemotherapy. thus there are chances to have radiologically invisible remnant malignant cells in hepatic tissue and potential risk of recurrence. however, the 3-month postoperative follow - up ct scan showed no evidence of recurrence. it is remarkable that the combination chemotherapy intended for palliation played a role of curative treatment. in conclusion, we experienced a case of advanced cbd cancer with liver metastasis completely responded to combination chemotherapy with gemcitabine and s-1. after 12 cycles of chemotherapy, surgical resection for primary tumor was performed because the metastatic lesion had completely disappeared, from which no remnant cancer cells were found. | common bile duct (cbd) cancer is a relatively rare malignancy that arises from the biliary epithelium and is associated with a poor prognosis. here, we report a case of advanced metastatic cbd cancer successfully treated by chemotherapy with gemcitabine combined with s-1 (tegafur+gimeracil+oteracil). a 65-year - old male presented with pyogenic liver abscess. after antibiotic therapy and percutaneous drainage, follow - up computed tomography (ct) showed an enhanced nodule in the cbd. biopsy was performed at the cbd via endoscopic retrograde cholangiopancreatography, which showed adenocarcinoma. additional ct and magnetic resonance imaging showed multiple small nodules in the right hepatic lobe, which were confirmed as metastatic adenocarcinoma by sono - guided liver biopsy. the patient underwent combination chemotherapy with gemcitabine and s-1. after nine courses of chemotherapy, the hepatic lesion disappeared radiologically. pylorus - preserving pancreaticoduodenectomy was performed, and no residual tumor was found in the resected specimen. three weeks after the operation, the patient was discharged with no complications. through 3 months of follow - up, no sign of recurrence was observed on ct scan. gemcitabine combined with s-1 may be a highly effective treatment for advanced cholangiocarcinoma. |
pachymeningitis is a rare feature of wegener 's granulomatosis (wg) and an uncommon initial manifestation of the disease. we demonstrate that pachymeningeal lesions refractory to conventional therapy can resolve completely by the addition of rituximab (rtx) to standard remedies. this case also illustrates that besides global headache, wg - related pachymeningitis may occasionally manifest itself by lateralized pain and cranial nerve involvement [25 ]. in 2004, a 27-year - old caucasian female was admitted for persistent right - sided ear, jaw, and neck pain, with recent advent of tinnitus, hearing loss, and nasal sores. a ct - scan showed mucosal thickening of the maxillary sinuses without bony erosions. a biopsy from nasal mucosa showed vasculitis and granuloma formation. bronchoalveolar lavage (bal) fluid was negative for bacterial growth, mycobacterial pcr, and malignant cells. however, the patient tested positive for pr-3 anca (49 u / ml, < 10). blood cell counts were normal, crp below 15 mg / l, creatinine clearance, and urinalysis were normal. based on these findings, the patient was diagnosed with wegener 's granulomatosis. cerebral magnetic resonance imaging (mri) with gadolinium showed infratentorial, meningeal thickening along the right cerebellar tentorium, the cerebellopontine angle ascending into the right foramen jugulare. besides, a thrombus extending from the right sinus transversus to bulbus venae jugularis was diagnosed. based on these findings, wegener related pachymeningitis was suspected although not histologically confirmed. induction treatment included intravenous methylprednisolone pulses (1 gram / day for 3 days), plasma exchange on 5 consecutive days, and oral cyclophosphamide. low molecular weight heparin and warfarin were added for prophylaxis against progression of cerebral venous thrombosis. however, the cyclophosphamide dosing target at 2 mg / kg could not be achieved due to debilitating nausea and liver toxicity, and she suffered from persistent cervicocranial pain despite normal crp, pr-3 anca seroconversion, and partial regression of meningeal pathology by mri. in december 2006, the patient complained of diplopia. a repeat mri scan showed marked progression of the infratentorial dura - meningeal enhancement (figure 1(a)), whereas partial revascularisation of the transverse sinus was noted. azathioprine 50 mg / daily was added to the current prednisolone medication (50 mg daily), but it had to be discontinued after 3 weeks due to intolerable nausea and elevated liver enzymes. due to similar drug toxicity, methotrexate 15 mg weekly added to daily 40 mg prednisolone medication was abandoned after 2 months treatment. in february 2007, rituximab was added as follows : 4 weekly rituximab infusions (375 mg / m) combined with i.v. cyclophosphamide (500 mg / m) on a separate day. already within 1 week after the first infusion, diplopia and neck pain had vanished. a follow - up mri at 6 months showed partial regression of the meningeal pathology (figure 1(b)), and chest ct revealed complete regression of the lung infiltrate. subsequently, relapsing neck and ear pain with concomitant epistaxis, nasal sores or sinusitis occurring at 810 months intervals have been successfully brought into remission 4 times using the treatment protocol described above. an mri - scan in february 2009 showed complete regression of meningo - dural enhancement (figure 1(c)). global headache is the most frequent manifestation of wg related pachymeningitis although the condition may occasionally manifest itself with lateralized pain and cranial nerve involvement like in the present case [25 ]. signs and symptoms of meningeal involvement in wg may result from different disease processes including vasculitis, bony destruction by invasive inflammatory cell infiltration or intraparenchymal granuloma formation [3, 5 ]. besides, the increased risk of complicating venous thromboembolism reported recently should be considered as illustrated by the sinus thrombosis encountered in this case. the relative contribution of these pathways can not be settled in this case, but the absence of destruction and granulomatous lesions by mri favours a dominant role of vasculitis. although conventional therapy using oral prednisolone and cyclophosphamide was followed by regression of the meningeal lesions seen on mri, a parallel clinical response was not achieved. probably, this is due to the submaximal dosing of these agents due to adverse effects. based on reports on favourable responses of rtx in refractory wg, we decided to add rituximab to i.v. although the exact mechanism behind the response to rtx in wg patients is unknown, it is likely that targeting cd20 + b - cells, which are precursors of anca - producing plasma cells, is important as evidenced by the long - term pr-3 anca seroconversion observed in this patient. successful treatment of wegener 's meningitis has been reported in the past (table 1) [710 ]. this case is unique by demonstrating that multiple rtx cycles can be administered safely and effectively, resulting in long - term complete clinical and imaging remission despite substantially reduced immunosuppressive comedication, glucocorticoids in particular (figure 2). the effect of each single agent applied in the present combination protocol can not be determined. however, as shown in figure 2, a marked reduction of the median pdn and cyc doses was observed during rtx treatment as compared with the pre - rtx period, thereby supporting the notion of a specific effect of rtx on wg disease pathways. this view is further supported by the need for repeat infusions (figure 2). in conclusion, this paper illustrates that wg may present with localized meningeal involvement and that rituximab added to conventional agents has a steroid sparing effect and can lead to clinical and imaging remission in wegener - related pachymeningitis. | a 27-year - old woman was admitted for intractable right - sided neck, ear, and jaw pain with gradual development of tinnitus and hearing loss. a cerebral mri showed meningo - dural enhancement, and additional diagnostic workup revealed a right pulmonary infiltrate and positive pr-3 anca. biopsies from nasal mucosa and lung showed chronic inflammation with granuloma formation. based on these findings the patient was diagnosed with wegener 's granulomatosis with pachymeningitis. there was no clinical response to oral prednisolone and cyclophosphamide, but complete clinical and imaging remission was achieved by adding rituximab. |
our understanding of the mechanisms and patterns of international migration over time are impeded both by the lack of data and by inconsistencies in the measurement and collection of the data that are available. in fact, it is well known that the patterns of migration vary significantly depending on which country is reporting the data (kupiszewska and nowok 2008 ; nowok. considering that international migration is the main factor contributing to population growth in europe, this is very unfortunate. in response to the problem of inconsistent migration data, we have developed a methodology for harmonising the data available to us from countries in europe. more specifically, we make use of doubly counted information obtained from migrant sending and migrant receiving countries to estimate adjustment factors necessary for producing a consistent set of migration flows. harmonisation of migration data is required for the development of policies on immigration (kraier. differences in both the concepts and techniques used to measure migration make any international comparison of migration difficult. there has been a lot of work on data issues and migration definitions, for example, see champion (1994), kelly (1987), kraly and gnanasekaran (1987), poulain (1993), poulain. (2006), raymer and willekens (2008), united nations (2002) and willekens (1994, 1999). several international institutes such as the international labour organization, the organisation for economic co - operation and development, the united nations and the european commission have all invested heavily in the harmonisation of international migration data, but without much success or progression (bilsborrow. 1997 ; herm 2006a ; fassmann 2009). in fact, the situation today in terms of migration definitions and measurement is not much better than it was, say, 20 years ago. recently, some renewed efforts have been made to improve the migration data situation in europe. in 2007, the european parliament adopted a new regulation on migration statistics. this regulation provides clear definitions of immigration and emigration (official journal of the european union 2007), and lists the migration data that must be supplied to eurostat, the statistical office of the european union (eu), by member states. however, this regulation leaves the member states free to decide how they will provide these data, including the use of estimation methods (fassmann 2009). the methodology presented in this paper should help national statistical offices to improve and harmonise the data they currently provide to international organisations, such as eurostat. the migration definition set out in the 2007 regulation corresponds to the definition recommended by the united nations (1998), where an international migrant is defined as a person who moves to a country other than that of his or her usual residence for a period of at least a year. one problem affecting the implementation of this definition is that some countries are unable to identify their nationals who have left (fassmann 2009). furthermore, many european countries exclude the immigration of nationals from the published statistics, as they are not considered to be migrants. another important obstacle has to do with the recommended duration of residence in the country of destination. it may take up to 2 years to identify all persons who have stayed at least 1 year, as they may arrive anytime during the annual time period of interest. this means that the publication of migration statistics based on the actual duration of stay may be delayed for some time. to provide statistics to the user community in a quicker fashion, many countries simply count those migrants who have stayed for at least 3 months, which leads to higher numbers than if the 1-year criterion was applied. other countries use the intended duration of stay as the criterion (fassmann 2009). this is mainly caused by the fact that migrants have little incentive to report their move to the administration of the country they have emigrated from. moreover, it is difficult to count persons leaving the country because they are no longer present in the country collecting the data. in this situation, comparisons of sending country data with receiving country data provide important information on the degree of underestimation found in reported emigration flows (unece 2009). in fact, the analysis of the so - called double - entry matrix of migration flows produced by unece since the early 1970s, and more recently by eurostat, has been found to be very useful and informative. kelly (1987) and poulain (1999), for example, have used the information contained in this matrix to assess the degree of harmonisation amongst reported data. in doing so, the possibility that very narrow or loose definitions of migration may be used for reported immigration statistics must be taken into account, which results in lower or higher levels of migration flows, respectively, in relation to, say, the united nations recommended 1-year definition (unece 2009). the aim of this paper is to illustrate how reliable estimates of harmonised migration statistics may be obtained from a set of origin destination flows, where two reported flows are available for each particular flow, i.e., from receiving and sending countries. the new method that we present is based on earlier efforts by poulain (1993, 1999), and is applied to reported flows between 19 european countries from 2002 to 2007. note, however, that this paper does not consider flows outside the 19 country system, or those that are missing. the reliability of migration statistics can be measured by how well they correspond to a particular country s definition or concept of migration. however, as definitions differ across countries, reliability does not guarantee comparability. moreover, under - registration, under - coverage and accuracy of the collection system also affect the measurement of migration (bilsborrow.. this may be the case if the data depend on declarations by the migrants themselves. the willingness to report changes in places of residence varies both between countries and between groups of migrants. in general, migrants have more incentive to report their arrival than their departure, as there are usually direct benefits in doing so (e.g., access to social services). therefore, immigration statistics are generally considered more reliable than emigration statistics (thierry. 2005 ; unece 2009). this measurement category refers to the non - inclusion of particular migrant groups. here, the differences are most often caused by the absence or inclusion of nationals, students, asylum seekers or irregular (illegal) migrants in the data. in general, asylum seekers are included only when they have been granted refugee status and received a temporary or permanent residence permit. however, in some instances, they are registered at an earlier stage of the asylum process. in other instances, irregular migrants are generally not included in migration statistics, as they are especially difficult to measure (for obvious reasons). in fact, spain is the only eu country that includes irregular migrants in the official statistics. finally, data based on sample surveys may be unreliable due to sampling errors. furthermore, unless the sample size is very large, the data are likely to show irregularities in the patterns across ages or in the distribution of origins or destinations over time, as flows of migrants represent a relatively small proportion of the overall population being surveyed. the main sources of the differences in the definitions used by eu countries to measure migration are the concepts of place of residence and duration of stay (zlotnik 1987 ; bilsborrow. the de jure (legal) approach to residence implies that in order to become a resident, a migrant must comply with certain regulations, which tend to differ between nationals and foreigners, and among foreigners, between eu- and non - eu - nationals. for example, it is not uncommon for emigrants to be registered in their country of citizenship (origin) even after several years of living abroad (thierry. thus, having a place of residence does not necessary imply a presence in that country. the de facto (actual) approach is connected with physical presence in a country, usually for a specified minimum period of time. to prevent the delay caused by measuring actual duration of stay, most european countries use the intended duration of stay instead (nowok. 2006). alternatively, the intended duration of stay may be used to provide provisional statistics, which are updated at a later point with the actual duration of stay statistics. another group of countries measure permanent change of residence only (e.g. poland and slovakia), which is very restrictive and tends to produce flow levels that are much lower relative to other definitions. the duration of stay criterion used by the majority of eu countries is between 3 months and 1 year. only three countries (cyprus, sweden and uk) apply strictly the 1-year criterion for immigration, as well as for emigration and for both nationals and non - nationals (thierry. in fact, some countries do not take duration of stay into account at all. germany is such an example, where everybody taking up a residence is counted as a migrant. because of differences in definition, coverage, registration and accuracy of the collection mechanism, the origin destination matrix of migration flows between european countries based on immigration data reported by the countries of origin tends to differ from the matrix reported by the countries of destination. with respect to definitions, for example, the german definition is wider than the dutch definition which, in turn, is wider than that of sweden. in fact, germany reports higher figures than the netherlands, and the figures of the netherlands are higher than those reported by sweden (kupiszewska and nowok 2008). a comparison of the size of these reported flows provides information on the effects of differences in definition on the size of migration flows (bilsborrow.. however, as mentioned above, not all differences can be explained by differences in definition. in some cases, furthermore, sudden jumps in observations may be caused by changes in definitions or by changes in the registration method. data on immigration and emigration flows by country of origin and destination are usually presented in an origin destination matrix with off diagonal entries containing the number of people moving from any origin i to any destination j in a given calendar year. for this study, we have collected migration data for the 19 countries set out in table 1. as each flow can be reported by both sending and receiving countries, two migration tables may be produced. the average 20022007 values of migration between the 19 european countries set out in table 1 are presented. table 2 contains flows reported by the countries of destination and table 3 contains the flows reported by the countries of origin. clearly, there are large differences between the two sets of reported numbers (see, e.g., spain to the united kingdom or poland to germany).table 1list of european countries reporting both immigration flows by country of origin and emigration flows by country of destination, 20022007countryabbreviationaustriaatcypruscyczech republicczgermanydedenmarkdkspainesfinlandfiicelandisitalyitlithuanialtluxembourglulatvialvnetherlandsnlnorwaynopolandplswedensesloveniasislovakiaskunited kingdomuktable 2reported migration by country of destination, averages 20022007fromtoatcyczdedkesfiisitltat4131014,2573037741093377417cy22132762325231303cz1,3161189,218262833564267224de15,4473321,3624,00115,98292125512,809490dk20325462,6879643651,41326585es700457114,7031,758644682,044252fi27021382,1734148444523543is31042361,665131503510it1,6084925422,1969869,3202507482lt17935474,4961,0342,27473272378lu67322,28216212350272135lv83104132,1554573008793183175nl7917025513,6818644,7622615590541no9814241,3783,1481,69684536416787pl5,2317521,608136,9272,4368,2771872,2299,045120se48988673,3483,3131,8263,50249237991si5569171,79846136693212sk3,19243214,06411,14814978822456904uk1,2223,17050613,2633,48238,6749462284,553875total31,5045,30618,702256,22124,50287,7258,3975,74133,6952,407fromtolulvnlnoplsesiskuktotalat895591111803071002081,39519,496cy025115761222,5333,087cz4155111164516469794,10918,489de4541669,1822,2682,8763,37429944619,03989,701dk11464752,943345,2643211,87416,721es24183,1017681191,30083614,58140,239fi34337979963,204166849,208is067537311462124173,509it67331,811246309599791095,82943,900lt123630292643574052,50713,380lu216118590516823,897lv21252336264051,2275,511nl272071116397912416,79930,436no224453485,0981241,66715,135pl19455,7444,6023,718327636,759217,977se14546964,91711315203,21322,635si1190142421603,064sk444652381811044,58435,961uk391905,8201,6241,1263,1142211678,969total68291330,00020,9215,11128,7235592,311107,897671,315source : eurostattable 3reported migration by country of origin, averages 20022007fromtoatcyczdedkesfiisitltat189376,665166429231271,022111cy62157619120399cz18613560243528211210de17,7872718,1043,09516,8072,37128731,2352,455dk228241792,6121,6693681,347716655es1559572 68615711091 163120fi9723427584006715320321is132172051,800594810564it58866710,2061491,5081361711lt488541,2691586288723204lu31313911997935191754lv1886302451846551138nl6165029810,4935333,774322541,27854no6917437093,093789855412146108pl538156314,41711134120465056se298731041,6343,1591,3483,40341346348si311314589527411861sk177162925541610420uk1,5934,0602,69212,5791,93233,4316821035,2701,074total22,7584,60013,33966,90514,93361,6498,7582,81842,9144,887fromtolulvnlnoplsesiskuktotalat4542426872,4013884021,77890116,076cy21810211113032371724cz3781165832489,53921911,449de1,6861,4949,2932,122100,8273,9742,0049,45617,233230,499dk1383166022,9478335,25331953,88921,898es871986915939820310453 4309 684fi7127233777633,2164101,1757,842is37294948287247825562324,570it2188531121417199151403,50817,879lt18163116199122233352,6385,975lu4971223735111661,760lv62034266712196987nl191337311,020900451387,95328,482no23692872815,0835611,39513,444pl2335571273032105,21922,306se127625224,74635427293,90520,713si2403055386701,319sk201331583691,235uk3623245,9431,9936,5072,66601,05382,264total3,0622,61919,67614,561114,85423,1172,72422,36452,567499,105source : eurostat list of european countries reporting both immigration flows by country of origin and emigration flows by country of destination, 20022007 reported migration by country of destination, averages 20022007 reported migration by country of origin, averages 20022007 the differences between reported immigration and emigration numbers are useful for improving and harmonising the migration data. if reported emigration numbers for a given country turn out to be systematically lower than the corresponding immigration numbers reported by the countries of destination, this suggests that the reported emigration numbers are too low. adjusting these numbers in an upward direction we can estimate one adjustment factor for immigration and one for emigration in such a way that the adjusted immigration and emigration numbers are closer to each other than the reported numbers. to prevent arbitrary judgments biasing the results, we believe the estimation of adjustment factors for immigration and emigration flows should be estimated simultaneously. moreover, it should be noted that immigration is not necessarily recorded more accurately than emigration. in some situations, sending country data may be considered better (nowok. poulain (1993, 1999) was the first to develop a method to adjust reported immigration and emigration numbers for the purpose of obtaining a consistent set of migration flows. correction factors were estimated by minimising the sum of squares \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sum\limits_{i, j } { (\hat{\alpha } _ { j } i_{ij } - } \hat{\beta } _ { i } e_{ij }) ^{2 } $ $ \end{document }, where iij denotes migration from country i to country j reported by the receiving country j, eij denotes the same flow reported by the sending country i, j is the adjustment factor for all immigration to country j and i is the adjustment factor for all emigration from country i. poulain and dal (2008) refined this method by dividing the squared differences by the sum of the reported numbers, i.e.,1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sum\limits_{i, j } { (\hat{\alpha } _ { j } i_{ij } - } \hat{\beta } _ { i } e_{ij }) ^{2 } /(i_{ij } + e_{ij }). $ $ \end{document } this refinement prevents flows from (or to) large countries from biasing the estimates. for instance, following the iterative approach to harmonising migration flows suggested by van der erf and van der gaag (2007), poulain and dal (2008) proposed that the estimates should be normalised to swedish immigration data, as they are generally considered to be highly reliable and in agreement with the un recommended measure, as well as with the new eu regulation (herm 2006b). the parameters j and i may be estimated by solving a system of linear equations, which result from applying the method of lagrange multipliers. multiplying iij by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { j } $ $ \end{document } and eij by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\beta } _ { i } $ $ \end{document } produces two sets of migration flow estimates from country i to country j. the final set of estimates are obtained by simply taking the average of the two, i.e., \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{n}_{ij } = (\hat{\alpha } _ { j } i_{ij } + \hat{\beta } _ { i } e_{ij }) /2 $ $ \end{document }, where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{n}_{ij } $ $ \end{document } denotes the harmonised migration flows. note, poulain and dal (2008) applied their correction method first to countries with relatively reliable data to prevent countries with less reliable data influencing the overall patterns. here, the main concern is that the less reliable data have origin destination patterns that are not consistent with the actual patterns. thus, less reliable flows were adjusted in a hierarchical fashion, i.e., by using the harmonised reliable data as a basis. first, the reported numbers included in the denominator of eq. 1 are known to be incorrect (abel 2009). second, the row and column totals of the two estimated matrices are not equal. as a result, the row and column totals of the average harmonised migration matrix do not correspond to the row and column totals estimated using the adjustment factors. finally, the method can only be applied to a limited set of countries with reasonably reliable data. this implies that the estimates of the adjustment factors depend on the selection of countries, which may not reflect the broader patterns of interest. for these reasons, first, the row - sums and column - sums of the two estimated matrices are set to be equal. second, we introduce additional constraints on individual cells in the migration matrices, so that more countries (with less reliable data) may be included. the adjustment factors for our method can be estimated by solving a system of linear equations and imposing a constraint. if we have a n n receiving country and an equivalent n n sending country matrix, the adjustment factors for receiving country, j, and the adjustment factors for sending country data, i, can be estimated by2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sum\limits_{j } { \hat{\alpha } _ { j } i_{ij } = } \hat{\beta } _ { i } \sum\limits_{j } { e_{ij } } \quad { \text{for } } i = { 1 }, \ldots, \, n;\quad i \, \ne \, j $ $ \end{document}3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { j } \sum\limits_{i } { i_{ij } = } \sum\limits_{i } { \hat{\beta } _ { i } e_{ij } } \quad{\text{for } } j = { 1 }, \ldots, \, n;\quad i \, \ne \, j $ $ \end{document } equation 2 states that for each country the emigration total estimated on the basis of the adjusted matrix of flows reported by receiving countries equals the emigration total estimated on the basis of the adjusted matrix of flows reported by sending countries. equations 2 and 3 can be written as a homogeneous system of 2n linear equations with 2n unknowns, i.e.,4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{array}{l } \hat{\alpha } _ { 2 } i_{12 } + \hat{\alpha } _ { 3 } i_{13 } + \cdots + \hat{\alpha } _ { n } i_{1n } - \hat{\beta } _ { 1 } \sum\limits_{j } { e_{1j } } = 0 \\ \vdots \\ \hat{\alpha } _ { 1 } i_{n1 } + \hat{\alpha } _ { 2 } i_{n2 } + \cdots + \hat{\alpha } _ { n - 1 } i_{nn - 1 } - \hat{\beta } _ { n } \sum\limits_{j } { e_{nj } } = 0 \\ \hat{\alpha } _ { 1 } \sum\limits_{i } { i_{i1 } } - \hat{\beta } _ { 2 } e_{21 } - \hat{\beta } _ { 3 } e_{31 } - \cdots - \hat{\beta } _ { n } e_{n1 } = 0 \\ \vdots \\ \hat{\alpha } _ { n } \sum\limits_{i } { i_{in } } - \hat{\beta } _ { 1 } e_{n1 } - \hat{\beta } _ { 2 } e_{n2 } - \cdots - \hat{\beta } _ { n - 1 } e_{nn - 1 } = 0 \end{array } $ $ \end{document } this system has an infinite number of solutions for j and i. for each set of values of j and i that solve this system, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k\hat{\alpha } _ { j } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ k\hat{\beta } _ { i } $ $ \end{document } are solutions as well. in order to find a unique solution one restriction needs to be imposed. in accordance with poulain and dal (2008), we assume that the adjustment factor for swedish immigration is equal to one, since sweden uses a definition of migration that is consistent with the new eu regulation and the quality of swedish immigration data is considered to be adequate. this also means that the resulting estimates are harmonised in line with the new european regulation. the basic assumption underlying our estimation procedure (as described above) is that the distributions of reported immigration by country of origin and reported emigration by country of destination correspond to the distribution of actual migration flows under the harmonised definition. this implies that the reported emigration of country a is x% higher or lower than the actual number (based on the standard definition) for all countries of destination. however, as we find in the next section, the estimated receiving country flows by country of origin and the estimated sending country flows by country of destination are not always consistent with each other. in a number of cases, specific origin let us assume that the estimated receiving country migration flow from country p to q, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { q } i_{pq } $ $ \end{document }, differs substantially from the estimated sending country flow, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\beta } _ { p } e_{pq } $ $ \end{document}. to make them consistent, we can multiply \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { q } i_{pq } $ $ \end{document } by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\gamma } _ { pq } $ $ \end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\beta } _ { p } e_{pq } $ $ \end{document } by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\delta } _ { pq } $ $ \end{document } so that both estimates of migration are equal. the question whether we should adjust the estimate based on the reported receiving country or the estimate based on the reported sending country depends on our knowledge of the data. given the estimated values of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { q } $ $ \end{document}and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\beta } _ { p } $ $ \end{document}we can calculate the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\gamma } _ { pq } $ $ \end{document } easily from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\gamma } _ { pq } = \hat{\beta } _ { p } e_{pq } /\hat{\alpha } _ { q } i_{pq } $ $ \end{document } or the value of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\delta } _ { pq } $ $ \end{document } from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\delta } _ { pq } = \hat{\alpha } _ { q } i_{pq } /\hat{\beta } _ { p } e_{pq } $ $ \end{document}. however, introducing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\gamma } _ { pq } $ $ \end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\delta } _ { pq } $ $ \end{document } changes the estimates of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { q } $ $ \end{document } or \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\beta } _ { p } $ $ \end{document}. this also means that the row and column totals of both estimated migration matrices no longer tally. therefore, we adjust the system of linear eqs. 2 and 3 by adding constraints on individual cells of the matrices. if we assume that the emigration number reported by country p needs to be adjusted, eqs. 2 and 3 can be rewritten as5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sum\limits_{j } { \hat{\alpha } _ { j } i_{ij } = \, } \hat{\beta } _ { i } \sum\limits_{j } { e_{ij } (1 + \hat{\delta } _ { pq}^ { } d_{ij }) } \quad { \text{for } } i = { 1 }, \ldots, \, n;\quad i \, \ne \, j $ $ \end{document}6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { j } \sum\limits_{i } { i_{ij } = } \sum\limits_{i } { \hat{\beta } _ { i } e_{ij } (1 + \hat{\delta } _ { pq}^ { } d_{ij }) } _ { { } } \quad{\text{for } } j = { 1 }, \ldots, \, n;\quad i \, \ne \, j $ $ \end{document}where dij = 1 if i = p and j = q, dij = 0 otherwise, and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\delta } _ { pq}^ { } = \hat{\delta } _ { pq } - 1 $ $ \end{document}. the equations including ipq and epq in the system of eq. 4 can be rewritten as follows:7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { 1 } i_{p1 } + \cdots + \hat{\alpha } _ { q } i_{pq } + \cdots + \hat{\alpha } _ { n } i_{pn } - \hat{\beta } _ { p } e_{p1 } - \cdots - \hat{\delta } _ { pq } \hat{\beta } _ { p } e_{pq } - \cdots - \hat{\beta } _ { p } e_{pn } = 0 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\alpha } _ { q } \sum\limits_{i } { i_{iq } } - \hat{\beta } _ { 1 } e_{1q } - \cdots - \hat{\delta } _ { pq } \hat{\beta } _ { p } e_{pq } - \cdots - \hat{\beta } _ { n } e_{nq } = 0 $ $ \end{document } in contrast with eq. 4, these are non - linear equations, because they include the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \hat{\delta } _ { pq } \hat{\beta } _ { p } e_{pq } $ $ \end{document}. the values of the coefficients can be estimated by an iterative procedure. the model can be extended in a straightforward way to include additional constraints. however, for any particular country, the number of constraints should not be too high, as this reduces the available information to estimate and. the sending and receiving country migration data have been provided by the national statistical institutes of the eu member states in response to annual rounds of data collection conducted jointly by five international organisations and coordinated by eurostat (kupiszewska and nowok 2008). as concerns europe, eurostat processes and disseminates data received from 37 countries on their website (epp.eurostat.ec.europa.eu). data sources used by eu member states to produce migration statistics are very diverse (kupiszewska and nowok 2008 ; nowok. the major types of sources are population registration systems, statistical forms, other administrative registers related to foreigners (such as alien registers, residence permit registers and registers of asylum seekers), sample surveys and censuses. alien registers and residence permit registers are used in seven countries, sometimes in addition to population registers. cyprus and the uk rely on passenger surveys conducted at the borders, while portugal and ireland rely on household surveys. some countries derive their emigration statistics from data on residence permits by assuming a migrant has left the country when a residence permit has expired. moreover, they often assume that the country of next residence is the country of their citizenship. the result, we believe, is an overestimation of actual emigration to those particular countries. finally, several countries include in their so - called administrative corrections emigration that has not been declared, which can not be disaggregated by country of next residence. data on immigration by country of previous residence or emigration by country of next residence are not always available or complete (nowok. thus, the sending country and receiving country matrices, when combined into a double - entry matrix may be incomplete. for some countries, a large share of emigrants have an unknown country of destination : around 75% in slovenia, 40% in luxembourg, 35% in austria, 31% in the netherlands and 39% in spain, for example. fortunately, the estimation of adjustment factors takes this into account. in the next section, we present our harmonised estimates of migration between 19 european countries that provide data on both immigration by country of origin and emigration by country of destination for the calendar years 20022007. although there are some data for ireland, portugal and romania, these have not been used because they cover only a part of the migration flows (e.g. only foreigners or nationals). for iceland, italy and luxembourg, for these countries, the adjustment factors are estimated for averages over the available years. the results presented in this section are obtained by applying the estimation method described in sect. 3. table 2 shows the average values of migration between 19 european countries reported by receiving countries for the years 20022007 and table 3 shows the corresponding numbers reported by the sending countries. the countries listed in the row headings refer to origins and those listed in the column headings refer to destinations. a comparison of tables 2 and 3 reveals large differences between numbers reported by sending and receiving countries. according to the numbers reported by receiving countries, 671,315 migrants per year moved between these 19 countries, whereas the numbers reported by sending countries total 499,105. for 11 countries, the reported receiving country immigration totals are higher than the corresponding sending country totals. for example, germany reported that 256,221 immigrants arrived from the 18 countries in this study, whereas these countries reported that only 66,905 emigrants moved to germany. poland reported that 22,306 persons emigrated to the other 18 countries which, for their part, reported receiving 217,977 immigrants from poland, suggesting that polish emigration data are around 10 times too low. for 15 of the 19 countries, the emigration total reported by the sending country is lower than the corresponding totals reported by receiving countries. keep in mind that receiving country data should not always be considered better than sending country data. consider, for example, the flows from poland to germany in tables 2 and 3. here, germany received an average of 136,927 migrants from poland, whereas poland reported that they only sent an average of 14,417. this difference could be explained by the duration criteria used by these countries, with germany having a very loose definition (instant) and poland having a very restrictive definition (permanent). so, in comparison with the harmonised definition of a 1 year period, germany s reported number is considered too high and poland s too low. we indicated above that in order to estimate the adjustment factors a restriction was introduced, i.e., the adjustment factor for swedish immigration is set equal to one. for 16 of the 19 countries, the eij adjustment factor exceeds one, indicating that sending country numbers tend to be underestimated. however, table 4 also shows that iij numbers seem to be underestimated in the majority of countries as well. this may seem contradictory since for 11 of the 19 countries the reported immigration totals exceed the corresponding emigration numbers reported by the sending countries. this is because the reported receiving country numbers should be compared with the adjusted sending country numbers rather than the reported numbers. for example, the immigration total reported by the uk (107,897) exceeds the reported emigration from sending countries to the uk (52,567). however, since the reported emigration from poland is too low (the adjustment factor equals 18.31, see table 4) the reported emigration from poland to the uk is adjusted from 5,219 to 55,506. moreover, the adjustment factor for spanish emigration data equals 4.32, so the reported emigration from spain to uk is adjusted from 3,430 to 16,792. for several other countries, emigration to the uk is adjusted upwards as well. as a consequence, the adjusted emigration numbers to the uk exceed the total of immigration reported by the uk and thus the reported immigration is adjusted upwards as well. note that the adjustment factors for immigration for most countries are closer to one than the adjustment factors for emigration, which indicates that the reported immigration numbers are more accurate than the emigration numbers.table 4estimates of adjustment factors for immigration and emigration, 20022007immigrationemigrationaustria1.061.74cyprus1.065.29czech republic2.143.33germany1.030.69denmark0.740.80spain0.824.90finland1.261.22iceland0.570.74italy1.422.92lithuania2.332.45luxembourg5.652.43latvia2.926.22netherlands0.971.25norway0.841.19poland17.8510.64sweden1.001.21slovenia5.182.71slovakia18.9043.69united kingdom1.211.18 estimates of adjustment factors for immigration and emigration, 20022007 multiplying the reported numbers in table 2 by the adjustment factors for receiving country data and the reported numbers in table 3 by the adjustment factors for sending country data results in two tables for which the row and column totals are equal (not presented here for space reasons). the differences between the cells in these two matrices are considerably smaller than those in tables 2 and 3. in fact, the root mean squared error (rmse) is reduced from 8,966 to 2,131. in other words, the differences between the two reported migration flow tables are reduced by 77%. however, we still found some substantial differences in the two estimated migration flow tables. for example, the migration from poland to germany estimated on the basis of german immigration data equals 141,035, whereas the estimate based on polish emigration data is equal to 153,399. these differences reflect the fact that the distribution of reported polish emigration by country of destination is not consistent with the share of immigration from poland in the total reported immigration numbers of other countries. as a result, the estimate of the migration flow from poland to germany based on polish data exceeds that based on german data, whereas for most other countries, the adjusted polish emigration numbers are lower than the corresponding adjusted immigration numbers. this means that one substantial inconsistency in the estimates is likely to influence the estimates of other migration flows. to prevent such inconsistencies from affecting the overall estimates, we have added constraints to individual cells (flows) in the model. the introduction of constraints to individual cells in the matrix allows us to consider special cases, such as the poland to germany flow described above. in total specifically, these flows were poland to germany, poland to uk, germany to poland, germany to uk, czech republic to slovakia and uk to poland. after identifying the flows with large differences, we then had to decide whether the constraint should be applied to the numbers of the receiving country or of the sending country. since we believe that reported emigration numbers are generally considered to be less reliable than reported immigration numbers, we apply the constraints to the sending country data, except for the germany to poland and uk to poland flows (i.e., poland s immigration data is considered to be of lower quality that the corresponding emigration data reported by both germany and the uk). the adjustment factors taking into account the six constraints on individual flows are set out in table 5. the coefficients (lagrange multipliers) for the poland to germany and poland to uk flows are both equal to 0.42. this raises the adjustment factor for emigration from poland from 10.64 (table 4) to 18.31 (table 5), while at the same time, the adjustment factor for polish emigration to germany and the uk falls to 7.69 (i.e., 18.31 0.42). for polish immigration, the high adjustment factor for polish receiving data was mainly a consequence of the big difference between the two figures for migration from germany to poland. including a constraint for this flow raises the adjustment factor for poland s reported flow from germany by a factor of 1.74 (i.e. in contrast, the adjustment factor for poland s reported flow from the uk falls to 10.40 (i.e., 14.25 0.37). for the czech republic, the reported emigration numbers are considerably lower than the corresponding reported immigration numbers with one big exception : the number of emigrants reported to slovakia is relatively large. clearly, the emigration flows from the czech republic to all other countries need to be adjusted by a different factor than the emigration flow to slovakia.table 5estimates of adjustment factors for immigration and emigration, 20022007, including six additional constraints on individual flowsimmigrationemigrationaustria1.171.35cyprus0.884.71czech republic1.978.92germany0.810.71denmark0.720.74spain0.734.32finland1.181.12iceland0.590.69italy1.482.44lithuania2.162.15luxembourg5.452.08latvia2.785.44netherlands1.041.06norway0.811.10poland14.2518.31sweden1.001.10slovenia4.902.33slovakia8.3439.40united kingdom1.090.91coefficients for additional constraints (lagrange multipliers) immigration to poland from germany1.74 immigration to poland from the uk0.37 emigration from poland to germany0.42 emigration from poland to the uk0.42 emigration from germany to the uk1.70 emigration from the czech republic to slovakia0.10 estimates of adjustment factors for immigration and emigration, 20022007, including six additional constraints on individual flows the adjustment factors in table 5 illustrate how substantial improvements in the estimated adjustment factors can be made by introducing constraints on specific for example, the inclusion of a constraint for the migration flow from the czech republic to slovakia lowered the adjustment factor for slovakia s receiving migration data from 18.90 to 8.34. this is mainly explained by the reduction of the estimate of polish emigration to germany. since germany has a wide definition of migration, one would expect the adjustment factor to be below one. thus, the adjustment factors in table 5 appear more plausible than those set out in table 4. the harmonised migration tables that used the additional constraints the introduction of these constraints led to a further strong reduction in the differences between both tables, as indicated by the rmse, which fell from 2131 to 952 or by a further 54%. to obtain a final single set of harmonised flows, we believe it is better to rely on table 6 than on table 7. this table gives more weight to the receiving country data, which we consider more reliable. poulain, on the other hand, advocated taking the average of the two estimated matrices. destination patterns in the reported sending country data are as reliable as those in the reported receiving country data.table 6estimated migration by country of origin and destination, including constraints on six individual flows, 2002/2007, based on numbers reported by receiving countriesfromtoatcyczdedkesfiisitltat3661011,526217563129201,14437cy26252231618281447cz1,5471037,453187606662599251de18,1482912,6792,86411,6311,09215218,9201,057dk23821902,172701432840391183es8223913911,8871,259763403,020544fi31719751,7572966142734792is37081911,19295595122it1,8894350017,9457066,78229744177lt21131923,6357411,65586162558lu79251,84511689591631511lv9791261,74232721810355270378nl9306150211,0606193,465309331,33689no11512471,1142,2541,2341,001216246187pl6,1466593,163110,7011,7446,0242211,32413,361259se574771322,7062,3721,3284,150292560197si6538321,4543399754734sk3,75037927,6589,01210757426261,0209uk1,4362,78099610,7222,49328,1451,1211356,7251,886total37,0154,65436,780207,14517,54263,8419,9503,41149,7725,190fromtolulvnlnoplsesiskuktotalat4426584902,5703074881,7301,52221,642cy145312100618182,7643,408cz234353494644164288,1584,48225,200de2,4754629,5861,84371,5143,3741,4623,71520,770172,034dk601284962,3914805,264151762,04416,124es132493,2376241,6981,3004129715,90741,798fi18119395649883,2044517468,819is217783031624626184553,157it365931 8912004,4025993869066,35943,582lt76563167526105741392,73412,858lu416815699024107443,661lv9131189902640381,3385,367nl146555772,323979593437,41730,305no12684736795,09822011,81814,778pl1011255,9973,7393,718122,29840,102199,695se741497273,9961,608741683,50522,690si649412244212903,079sk2412486193254110205,00148,662uk2135286,0771,3205,9073,11410596874,670total3,7152,54031,32117,00093,22228,7232,73519,264117,708751,530table 7estimated migration by country of origin and destination, including constraints on six individual flows, 2002/2007, based on numbers reported by sending countriesfromtoatcyczdedkesfiisitltat241,2618,973223578311361,376150cy2699266268756018142cz1,6581164,9992133092532199889de12,6161925,7482,19511,9211,68120322,1541,741dk168171321,9241,229271991527482es6693724711,592678475375,020516fi10925478524497546022824is91111421,24341337344it1,4331516424,8773643,6753314126lt102161152,7303391,35218750438lu655261,89520516573403648lv9844301,64024510025027277748nl6565331711,1655674,016342571,36057no7618477803,399867940453160118pl9,8572721,160110,7012,0236,2443698459,247116se327801141,7893,4601,4773,72845350752si7257331,37412631024352sk6,9744624,78410,0281446172001,6420uk1,4463,6852,44411,4181,75330,345619934,784975total37,0154,65436,780207,14517,54263,8419,9503,41149,7725,190fromtolulvnlnoplsesiskuktotalat61575731183,2325235412,3931,21321,642cy108645115205901481,7453,408cz25587211405,203213708,1581,95625,200de1,1951,0606,5911,50571,5142,8181,4216,70720,770172,034dk1012334432,1706133,86822702,86316,124es377833,7496861,7188754219514,80241,798fi8030262873703,6175111,3218,819is25203433360233017391603,157it531201,2942951,015484368968,55143,582lt393512504282635027115,67612,858lu9201264715210223453,661lv331091821413624111,0685,367nl203357771,085958481468,46230,305no26753163095,5875671,53414,778pl4126110,2052,3195,5393418940,102199,695se139685715,19938829314,27722,690si57170111188131643,079sk7204931185843281122,69948,662uk3282945,3951,8095,9072,420095674,670total3,7152,54031,32117,00093,22228,7232,73519,264117,708751,530 estimated migration by country of origin and destination, including constraints on six individual flows, 2002/2007, based on numbers reported by receiving countries estimated migration by country of origin and destination, including constraints on six individual flows, 2002/2007, based on numbers reported by sending countries the average adjustment factors estimated for the period 20022007 (table 5) can be applied to the annual reported migration data to create a time series of harmonised flows. in fig. 1, the estimated total immigration and emigration flows for germany from and to the other 18 countries in this study are compared. as expected, the estimated numbers are lower than the reported numbers because the definition for germany is much wider than the harmonised definition. the figure also shows that estimated emigration increases more gradually over time than the reported numbers. in fig. 2, the immigration and emigration flows for the uk, the average levels of the reported and estimated numbers do not differ much, but the estimated flows show a more gradual pattern over time than the reported flows. one reason for the sharp fluctuations in the reported numbers is that they are based on sample surveys.fig. 1reported and estimated immigration from and emigration to 18 european countries, germany, 20022007fig. 2reported and estimated immigration from and emigration to 18 european countries, united kingdom, 20022007 reported and estimated immigration from and emigration to 18 european countries, germany, 20022007 reported and estimated immigration from and emigration to 18 european countries, united kingdom, 20022007 the aim of this paper has been to obtain a reasonable and consistent set of international migration statistics. for this purpose the method is based on an idea originally proposed by poulain (1993, 1995). first, we have estimated a set of adjustment factors for receiving and sending country data in a way that ensures consistency in the two sets of marginal totals. third, instead of calculating the arithmetic average of the two estimated matrices, we believe it is better to use the matrix giving more weight to the reported immigration numbers (i.e. table 6). in this way we take advantage of the fact that the information on countries of origin in receiving country data tend to be more reliable than the country of destination information in sending country data. finally, our estimates are consistent with the harmonised migration definition based on an (intended) minimum duration of stay of 12 months. due to differences in definition, coverage and registration, the origin destination matrix of migration flows between european countries based on receiving country data tends to differ from the matrix based on sending country data. germany has a wide definition of migration, as it does not include a time constraint and thus the reported number may well include short term migrants. in contrast, poland has a very narrow definition of migration and, as a consequence, the reported numbers are very low. by comparing corresponding reported immigration and emigration flows for 19 european countries, we have assessed to what extent german migration statistics are higher than they would be under a harmonised definition and to what extent polish migration statistics are lower. however, the large differences between european countries can not be explained by differences in definitions alone. first, these differences can not explain why emigration flows are more likely to be underestimated than immigration flows. second, whereas 11 countries employ a duration limit that is shorter than that of the harmonised definition (kupiszewska and wisniowski 2009), only five of these countries have an adjustment factor of immigration below one. the other six countries with durations of 6 months or shorter have adjustment factors for immigration greater than one. these include austria, czech republic, italy, luxembourg, the netherlands and slovenia. thus, to an important extent, the differences must also be caused by problems of coverage. this is confirmed by a study comparing migration statistics between sweden, denmark and belgium which suggests that less than 25% of differences are due to differences in the duration criterion (nowok. the effects of differences in definition and coverage may offset each other to some extent. one would expect the under - registration of short term migrants to exceed that of long - term migrants. a wide definition of migration (i.e. a short duration of stay) would lead to a higher reported number of migrants than would be expected on the basis of the harmonised definition. under - registration this may explain why the adjustment factors for germany are not as low as one might expect from applying the very wide definition. the main reason for the relatively low numbers reported by sending countries is that emigrants do not have strong incentives to report leaving a country. in particular, this applies to eu citizens who can live in another eu country without asking for a residence permit., any person leaving country a would be required to fill in a form to be given to the authorities in country b at arrival. after country b has determined whether or not the person is an international migrant under a harmonised definition, it would then inform country a of the arrival. the nordic countries have such a system and their immigration and emigration statistics are mutually consistent (herm 2006a). however, policy makers tend to be more interested in migrants from outside europe and asylum seekers than intra - european migrants, and therefore such a system is not likely to have a high priority in the future. as long as such a system is lacking, cross - country comparability of migration statistics can only be achieved by comparing statistics from different countries. to the extent that the differences between countries are caused by differences in definitions and coverage, the method developed in this paper aims to assess the size of these systematic differences. table 5 shows that for 10 out of the 19 countries in this study, the adjustment factor for sending country data exceeds two, meaning that reported emigration numbers are underestimated by more than 50% in relation to the 1-year duration definition. as a consequence, in addition to correcting the reported receiving and sending country migration data for differences in definition and coverage, our method contributes to producing estimates that tend to fluctuate less strongly over time. since the uk uses a general purpose passenger survey, the reported flows fluctuate considerably over time. moreover, flows to some (smaller) countries may not be observed in some years. we believe our method produces more stable estimates of migration flows for the uk (and other countries relying on sample data). this implies that the sample survey used for estimating migration to and from the uk provides a reasonably reliable estimate of total migration flows on average, but that the annual estimates are affected by sizeable random fluctuations. the adjustment factors shown in table 5 can be used to adjust migration numbers to and from countries not included in the matrix, so that total immigration and emigration numbers and total net migration can be estimated for the 19 countries in this study. before doing so, one - first has to make sure that the share of unknowns in the migration statistics can be distributed evenly across all origins or destinations. thus, for estimating total immigration and emigration numbers, the adjustment factors should be applied to total migration numbers excluding unknowns. raymer (2008) developed a two - step estimation method for countries with missing data (see also de beer. the first step estimates missing immigration and emigration totals based on harmonised migration flows and covariate information. destination interaction patterns of the harmonised migration flows and covariate information to estimate the missing interaction patterns. this estimation step takes into account the fact that migration is relatively high, for example, between neighbouring countries and countries belonging to a similar language group. finally, work is currently being carried out to integrate harmonisation and estimation of missing data into a single (bayesian) model that also includes measures of uncertainty and expert judgements. the integrated modelling of european migration (imem) project recently funded by new opportunities for research funding agency co - operation in europe (norface) is expected to develop such a model (see http://www.norface.org/migration12.html) over the next couple of years. we hope this study will provide an important foundation for work such as this, and other projects aiming to improve our knowledge and understanding of the complexity of international migration. | due to differences in definitions and measurement methods, cross - country comparisons of international migration patterns are difficult and confusing. emigration numbers reported by sending countries tend to differ from the corresponding immigration numbers reported by receiving countries. in this paper, a methodology is presented to achieve harmonised estimates of migration flows benchmarked to a specific definition of duration. this methodology accounts for both differences in definitions and the effects of measurement error due to, for example, under reporting and sampling fluctuations. more specifically, the differences between the two sets of reported data are overcome by estimating a set of adjustment factors for each country s immigration and emigration data. the adjusted data take into account any special cases where the origin destination patterns do not match the overall patterns. the new method for harmonising migration flows that we present is based on earlier efforts by poulain (european journal of population, 9(4) : 353381 1993, working paper 12, joint ece - eurostat work session on migration statistics, geneva, switzerland 1999) and is illustrated for movements between 19 european countries from 2002 to 2007. the results represent a reliable and consistent set of international migration flows that can be used for understanding recent changes in migration patterns, as inputs into population projections and for developing evidence - based migration policies. |
temporomandibular disorders (tmd) are called to a group of painful conditions with a prevalence rate of 3 - 15%, in which temporomandibular joints (tmjs) and/or masticatory muscles are typically involved.1 - 3 patients with tmd frequently complain about multiple bodily pains outside of the orofacial regions.4,5 tmds are often described based on some signs and symptoms like : tmj sounds, impaired mandibular movement, limitation in mouth opening, preauricular pain, facial pain, headaches, and jaw tenderness on function.6 in cases of persistent and recurrent pains, tmd may demonstrate a chronic course. tmd is not a life - threatening disease, but the quality of life may be reduced.7 generalized joint hypermobility is a hereditary problem defined by the increase in range of motion in multiple joints, which might affect tmj in some cases that is named tmj hypermobility (tmjh).3,8 joint range of motion might be affected by numerous factors including : biochemical changes in the structure of collagen and elastin, loss of resistance to traction, laxity, and increase joint mobility.3 winocur. conducted a study about the prevalence of general joint laxity and tmjh among adolescent girls. they concluded that the prevalence of generalized joint laxity was 43% and tmjh was recognized in 27.3%.8 in another survey, adair and hecht discovered that participants with generalized joint hypermobility may be more likely to demonstrate some signs and symptoms of tmd than ones with normal joint mobility.9 in another view, correlations between dental occlusion and (tmd) have been proved, also mandibular resting position can be affected by different factors such as : occlusal interferences, tmd, the position of the head and body of mandible, and emotional tensions, which may lead to bodily adaptations and realignments of tooth and tmj.1,10,11 due to the fact that generalized joint hypermobility and tmjh are reported as risk factors for tmd,12 the aim of this study was to clarify the etiological factors of tmjh and its relations to habitual modalities. ethics : present article is based on thesis with i d number of udk:616.724 - 08.089.23 ; the survey was executed in medical and surgical department of poltava dental clinic and maxillofacial department of pokb, ukraine ; also a medical consent was filled by each contributors and all procedures were required for treatment plans. this observational / case - control clinical study was conducted on 69 individuals between the ages of 22 and 42 who had the manifestation of tmjh. any severe systemic disease (like rheumatoid arteritis) and un - cooperation were assumed as criteria. after profiling personal information and medical history, the patients were divided into three groups based on their maximum mouth opening (mmo), which was measured by a disposable ruler between upper and lower incisors:9 light : 25 patients with mmo of 50 - 55 mm.moderate : 18 patients with mmo between 55 and 65 mm.severe : 26 patients with mmo > 65 mm. severe : 26 patients with mmo > 65 mm. for subjective observations, patients were asked to fill the prepared questionnaire, with the help of examiner if it was necessary, to reveal following information : discomfort of tmj during mmopain in tmj during mmopain in masticatorstmj clicking or other soundsirradiation of pain in the ear(s), temple or neckusual side of chewingusual side of tmj which was lied on during asleepthe first symptoms of tmjh if they were recalled. discomfort of tmj during mmo pain in tmj during mmo tmj clicking or other sounds irradiation of pain in the ear(s), temple or neck usual side of chewing usual side of tmj which was lied on during asleep the first symptoms of tmjh if they were recalled. then extra oral observation followed as facial asymmetry, condition of the facial musculoskeletal system, functional and morphological changes in the teeth and jaw system and a detailed research of the nature of pathological changes in the tmj, palpation of tmj projection zone, manual assessment of muscle tone, movements of the mandible in the horizontal and vertical planes, malocclusion caused by teeth positions, analysis of dynamic occlusion in order to evaluate the performance of jaw movements, the amount of lateral movements of the mandible, passive movement of the mandible, passive compression by means of bilaminar zone adaptation, and dynamic compression for evaluation of ligamentous apparatus, the joint capsule and articular surfaces. to assess masticatory system specially temporal and medial pterygoid muscles, auscultation was performed using stethoscope which was tightly applied to the joint, but without pressure. it was necessary to listen to the noise in the joint space or separate quadrants of the joints. at the end, consultations with orthopedic, dentists, rheumatologist, and neurologist were performed if necessary. all the data subjected to chi - square test by using spss software version 22 at a significant level of 0.05. this study was conducted on 69 patients (54 women and 15 men), at the age from 22 to 42 years old. the largest number of patients with tmjh was at the age of 31 - 42 years old (70.99%), while 37.67% were at the age from 26 to 35 years old. furthermore, the number of women was three times more (78.2%) than men. distribution of subjective and objective criteria (%) of all the groups alongside with statistical comparisons (p value). medical history of patients revealed the presence of familial tmj diseases in one case of a moderate group. eight patients did not ask for medical treatments, 4 people referred to a dentist, 2 people to otorhinolaryngologist, and 2 others to a neurologist. the main treatment was to assign non - narcotic analgesics and physiotherapy, however, the effects were short - term in 11 people and without success for 5 patients. based on the results of this study, tmjh was more common in women (74.2%), which is in agreement with other studies.13 - 15 pain appears to have significant relationship with impairment of oral health related to the quality of life. pain is induced when an imbalance occurs between the amount of stimuli and the efficacy of modulation mechanisms.7,16,17 as the result showed, the light group had significant differences with other groups in the discomfort of tmj and tmj sound. in a study with the aim of classifying symptomatic patients diagnosed with tmd into homogenous groups based on their clinical presentation, pimenta e silva machado. claimed that localized masticatory muscle pain is recognized as the most common complaint in tmd patients.18 similar results were found in present study in which sever group manifested highest percentage of masticatory pains, significantly. in that study, recently the most popular theories about etiology of tmds are based on the biopsychosocial pattern, which is a combination of biological, psychological, and social factors. quality and pattern of sleeping and stress showed correlation with tmds.7,19 sleeping on affected tmj was the highest in the light group. perhaps, the higher level of pain in moderate and sever groups make some uncomfortable sensation during sleeping on the affected tmj. winocur. found a positive correlation between tmjh and mmo.8 this result is in accordance with pasinato. study about evaluating clinical and psychosocial aspects in tmd patients with or without generalized joint hypermobility, too.3 however, westling and helkimo did not found a significant relationship between mmo and peripheral joint mobility.20 furthermore, hirsch. concluded that patients with hypermobility had a lower risk of having limited mmo.21 present result hypothesis the correlation between pain in masticatory muscles and higher mmo concluded that facial asymmetry due to mandibular lateral displacement is a relatively common problem in tmds.22 however, haghigaht. reported that condylar distance was higher in posterior and superior borders in tmjh.23 that might explain the differences. ogtcen - toller believed that clicking and crepitation may be considered as signs of abnormal joint disorder24 and the present result confirmed it too. in the present study, a few of individuals sought for the tmjh treatments. although hypermobility is relatively common in the general population, but reports about musculoskeletal complaints are infrequent. as most symptoms are mild and self - limiting, so patients may not search for medical attention.25 regarding to the limitations of this study like : low sample size, uneven sample size in groups, limitation of time and etc., it can be concluded that pain in tmj would have a correlation with mmo. | background : joint range of motion might affected by some factors like laxity and increase joint mobility. generalized joint hypermobility and temporomandibular joint hypermobility (tmjh) are reported as risk factors for temporomandibular disorders. the aim of this study was to survey the etiological factors of tmjh and its relations to habitual status.materials and methods : in this cross - sectional descriptive study, 69 patients with tmjh were involved. after profiling personal information and medical history, the patients were divided into three groups based on their maximum mouth opening (mmo) as follow:(light) mmo of 50 - 55 mm, (moderate) : mmo between 55 and 65 mm, (severe) mmo > 65 mm. for subjective observations, patients were asked to fill the prepared questionnaire. the objective evaluations conducted by a specialist. finally, all the data subjected chi - square test by using spss software version 22 at a significant level of 0.05.results:tmjh was more common in women (74.2%). the light group had significant differences with other groups in the discomfort of tmj and tmj sound (p < 0.05). furthermore, sever group manifested highest percentage of masticatory pains, significantly (p < 0.05).conclusion : it can be concluded that pain in tmj would have a correlation with mmo. |
familial hemiplegic migraine (fhm) is a rare form of migraine with aura that involves motor aura (hemiparesis) and an autosomal dominant classical migraine subtype. the prevalence of hemiplegic migraine (hm) was reported as at least 0.002% for the sporadic form and at least 0.003% for the familial form in denmark (1). we present the first korean family with fhm due to mutation in the cacna1a gene. a 43-yr - old right - handed man presented to the department of emergency of kangwon national university hospital with suddenly noticed right hemianopia, dysphasia, and dizziness on march 11, 2010. when he arrived at the emergency room, global aphasia and vomiting developed. four and 5 yr before the admission, he had three episodes of right hemiparesis lasting a few hours. at those times, he was evaluated for a suspicion of transient ischemic attack, but without any risk factors for cerebral ischemia mri showed cerebellar atrophy. thus, he underwent genetic tests for mutations in the sca1, 2, 3, 6, 7, 8, and drpla genes, which were all negative. he had occasional headaches, the nature of which could not be recalled because those were trivial. his family history revealed that his mother had suffered from headache and frequent transient hemiparesis, from her 30s to 50s (fig. conventional mri and diffusion - weighted imaging (dwi) performed when global aphasia developed showed only cerebellar atrophy (fig. ct angiography performed during the coma, 3 hr after the current symptom onset, showed mild vasodilation of the intracranial blood vessels and increased vascularity in the left hemisphere (fig. perfusion - weighted imaging (pwi) showed elevated cerebral blood flow (cbf) (fig. 2c). eeg revealed persistent low - voltage arrhythmic delta activity over the left hemisphere (fig. a genetic study of fhm was performed using the samples from the patient and his mother, after obtaining their informed consents. genomic dna was extracted from the peripheral blood using an instagene matrix kit (bio - rad, ca, usa), according to the manufacturer 's instructions. the entire coding region of the cacna1a gene on chr 19p13 was amplified via polymerase chain reaction using primer sets described previously and a rotor - gene 6000 thermal cycler. direct dna sequencing was performed using a bigdye terminator cycle sequencing kit (applied biosystems, usa) on an abi prism 3130 genetic analyzer (applied biosystems). the gene mutation analysis performed in the patient and his mother led to the identification of a heterozygous point mutation (1997ct, t666 m) in the exon 16 of the cacna1a gene. mild dysarthria and gait disturbance, which had existed before the event, were detected by repeated neurological examinations. eeg gradually improved, but still showed a small amount of arrhythmic slowing in the left hemisphere, even after 10 days. we prescribed verapamil preventively and there have been no further attacks for the past 30 months. fhm1, which accounts for approximately 50% of fhm cases, is caused by mutations in the cacna1a gene, which encodes the alpha-1 subunit of a high - voltage - gated p / q type calcium channel (cav2.1) (2). in familial and sporadic hm, the mutation that causes the t666 m substitution is the most common and accounts for 40% of unrelated fhm1 cases (3). previously, only one korean family with symptoms that were suggestive of hm was reported, but without a genetic study (4). a few korean families with different mutations in cacna1a have been reported ; however, they all exhibited episodic ataxia type 2 (5, 6). cav2.1 channels are located in the presynaptic terminals and in the somatodendritic membranes throughout the brain and play a role in controlling neurotransmitter release. the various mutations found in this calcium channel gene cause a wide spectrum of symptoms. fhm1 is associated with mostly missense mutations, whereas ea2 and sca6 are associated with truncating mutations and expansion of a cag repeat, respectively (7). their effects on channel function were suggested to be a gain of function for the fhm1 mutation and a loss of function for the ea2 mutation (8). however, the phenotypes of fhm1, ea2, and sca6 can coexist in a family member (7) and in single patient (9), suggesting that these allelic disorders form a broad disease spectrum. two thirds of fhm patients experience the cerebellar sign and about 30% of them exhibit cerebellar atrophy (10). this channel varies in type within different brain regions, and the cav2.1 channels are mostly expressed in cerebellar purkinje cells, which account for the progressive cerebellar sign in fhm1, ea2, and sca6 (8). the t666 m mutation is particularly associated with the cerebellar symptom and the high incidence of coma (2). the episode of fhm presented here had distinctive features that did not fulfill the ihs diagnostic criteria (11), as the patient exhibited prolonged coma without hemiplegia and the absence of subsequent headache. a review of published cases showed that 38% of hm cases had no attack of hemiplegia (12). one percent of fhm sufferers had no headache, with otherwise typical attacks (13). our patient showed different symptoms between attacks, but left - hemispheric symptoms were always present. unilateral symptoms may switch between attacks, or the same side may always be affected (3). symptom variability among patients and attacks in a patient may be explained by genetic heterogeneity together with the probable presence of environmental and genetic modifying factors (2, 3). symptom progression in the order of visual, sensory, motor, aphasic, and basilar disturbances is the common process (3), which can be explained by a cortical spreading depression (csd) from the visual cortex (8). eeg during hm attacks usually shows attenuation or slow activities in the symptomatic hemisphere, which differentiates this condition from nonconvulsive seizures. the eeg abnormality outlasting the symptom in this patient could be the evidence of permanent sequelae of migraine, as well as cerebellar atrophy. cerebral hemodynamics in fhm has been reported anecdotally by angiography, pwi, or single photon emission computed tomography (spect). most cases showed vasodilation or hyperperfusion during aura, which can differentiate this condition from ischemia (15 - 18). however, vasoconstriction was reported in a few cases (19, 20). neither the severity of symptoms nor the time point of evaluation explained this discordance. in addition, many modifying factors may be involved in trigeminovascular activation secondary to electrophysiological dysfunction of neurons. this is the first report of fhm in a korean family and the only report of perfusion imaging in a genetically identified case. because fhm is a rare disease, without a suspicion of hm, patients may be misdiagnosed as having transient ischemia, epilepsy, or even conversion disorder. symptom progression exceeding the vascular territory from the initial visual symptom, absence of hypoperfusion, and electrical suppression differentiate hm from other episodic neurological disorders. fhm should be one of the differential diagnoses of episodic neurological dysfunction and cerebellar atrophy. | we report the first korean patient with familial hemiplegic migraine type 1, with clinical and multimodal imaging findings. a 43-yr - old man was admitted for right hemianopia and aphasia, followed by coma. mri showed only cerebellar atrophy. ct angiography showed mild vasodilation of intracranial blood vessels and increased vascularity in the left hemisphere and perfusion - weighted imaging showed elevated cerebral blood flow. gene analysis of the patient and his mother led to the identification of a heterozygous point mutation (1997ct, t666 m) in exon 16 of the cacna1a gene. familial hemiplegic migraine should be considered in patients with episodic neurological dysfunction with cerebellar atrophy. |
from march through november 2015, at the people s liberation army 154 hospital in xinyang city, henan province, china, patients who were acutely symptomatic with fever and had a history of tick bites or animal contact within the past month were screened for sfg rickettsiae infection. at admission, dna was extracted by using a qiaamp dna blood mini kit (qiagen, germantown, md, usa). nested pcrs selective for outer membrane protein a (ompa) and citrate synthase (glta) genes were concurrently performed to detect sfg rickettsial dna (technical appendix table 1). positive amplicons were purified and then sequenced in both directions. acute - phase (14 days after illness onset) serum samples were tested by indirect immunofluorescence assay (ifa) for igg against r. rickettsii by using a commercially available ifa kit (focus diagnostics inc., cypress, ca, usa). positive amplification of ompa and glta genes was found for 5 patients, and the obtained sequences for each of the 2 genes from all 5 patients were identical. ku853020) from each of the 5 patients showed 10-bp differences from that of r. massiliae strain azt80 (genbank accession no. cp003319) and 12-bp differences from that of r. rhipicephali strain hj#5 (genbank accession no. ku853022) from each of the 5 patients differed from that of r. massiliae strain azt80 by 4 bp and from that of r. rhipicephali strain hj#5 by 5 bp (technical appendix table 2). according to phylogenetic analysis, the novel sfg rickettsiae genotype, here designated as rickettsia sp. xy99, seems to represent a distinct lineage and could constitute a new species (figure 1). for all 5 patients, seroconversion or a 4-fold increase of igg against r. rickettsii was found between the acute- and convalescent - phase samples, and the patients were determined to have acute infection with sfg rickettsiae (technical appendix table 3). subsequent testing of the 5 patients for infection with severe fever with thrombocytopenia syndrome virus, anaplasma phagocytophilum, a. capra, and babesia microti by molecular (real - time pcr or nested pcr) and serologic tests (elisa or ifa) produced no positive results. phylogenetic analyses based on nucleotide sequences of the outer member protein a (307-bp) (a) and citrate synthase (1,150-bp) (b) genes of rickettsia. asterisks after taxon names indicate that the sequence of rickettsia species was found in china. neighbor - joining trees were conducted by using the maximum composite likelihood method by means of mega version 5.0 (http://www.megasoftware.net). bootstrap analysis of 1,000 replicates was applied to assess the reliability of the reconstructed phylogenies. patient median age was 65 (range 6280) years, and 3 were male (table). two patients had a history of tick exposure, and the other 3 had had contact with livestock. the median time from illness onset to admission was 4 (range 36) days, and the median duration of hospitalization was 10 (range 812) days. all patients experienced fever (highest 38.4c 40.0c), asthenia, anorexia, and nausea ; 4 had cough, 3 vomiting, 2 myalgia, 1 headache, and 1 dizziness. of note, all 5 patients had lymphadenopathy, but none had rash or eschar. at admission, all 5 patients had leukopenia, thrombocytopenia, and elevated hepatic aminotransferase levels ; 4 had elevated lactate dehydrogenase levels, and 2 had elevated creatine kinase levels (figure 2). dynamic changes of 6 laboratory parameters (with 2-day intervals) during hospitalization of 5 patients with rickettsia sp. xy99 infection, china, 2015. red, patient 1 ; yellow, patient 2 ; green, patient 3 ; purple, patient 4 ; gray, patient 5. alt, alanine aminotransferase, reference range 040 u / l ; ast, aspartate aminotransferase, reference range 040 u / l ; ck, creatine kinase, reference range 25200 u / l ; ldh, lactate dehydrogenase, reference range 109245 u / l ; platelets, reference range 100300 10/l ; leukocytes, reference range 4.010.5 complications included pneumonia (3 patients), hemorrhagic signs (3), hydrothorax (2), and neurologic syndromes (1). for 1 patient, severe complications progressively emerged 6 days after disease onset and included pneumonia and hydrothorax (technical appendix figure), confusion, meningeal irritation sign, ecchymosis, and hematuria. laboratory indicators were substantially more out of range 7 days after disease onset, indicative of severe multiorgan dysfunction (figure 2). the other 4 patients were discharged after 812 days hospitalization ; at that time, all clinical signs and symptoms had resolved, but for certain patients, laboratory values remained out of reference range, suggesting slow recovery of organ dysfunction (figure 2). xy99 dna in blood samples collected during the acute period of illness (days 36 after onset) from 5 patients in the same region of china suggests that this organism was the etiologic agent of the infection. seroconversion or a 4-fold increase in titers of igg against r. rickettsii provided supportive evidence of sfg rickettsia infection. in contrast to humans with r. massiliae infection and many other sfg rickettsioses reported previously (4,10), none of the 5 patients infected with rickettsia sp. xy99 had rash or eschar, and only 1 had headache. in recent years, the concept of the classic triad of fever, rash, and headache suggesting infection with sfg rickettsiae has been increasingly challenged. several emerging sfg rickettsiae species, such as r. slovaca (2), r. raoultii (11), r. africae (12), and r. helvetica (13), can infect humans, but such infections lack these traditional features, which were also lacking in the cases reported here. therefore, absence of rash and tick - bite history should not exclude suspicion of sfg rickettsiae infection. similar to r. slovaca and r. raoultii infections, which can be associated with tickborne lymphadenopathy and dermacentor - borne necrosis - erythema - lymphadenopathy (14), lymphadenopathy was also observed in all 5 patients with rickettsia sp. thus, lymphadenopathy might be a typical sign useful for clinical diagnosis of rickettsia sp. all 5 patients had gastrointestinal syndromes, indicating potential tissue lesions or vascular injury of the gastrointestinal tract. the hydrothorax and multiple hemorrhagic signs in 4 patients is possibly suggestive of vascular invasion or damage caused by this novel rickettsia species. confirmation of the novel rickettsia genotype was achieved only by sequencing the ompa and glta genes. although differences based on 2 gene segments support its designation as a novel species, isolation efforts and characterization of all 5 genes (rrs, glta, ompa, ompb, and gened) are warranted, according to the guidelines for classification of a new rickettsia species (15). physicians in this area of china should be aware of human infections with rickettsia sp. it should be included in differential diagnoses with severe fever with thrombocytopenia syndrome, which causes similar clinical illness and is also endemic to the same area in eastern central china. additional materials and methods used in study of human infection with novel spotted fever group rickettsia genotype, china, 2015. | only 4 species of spotted fever group rickettsiae have been detected in humans in china. however, phylogenetic analysis of samples from 5 ill patients in china indicated infection with a novel spotted fever group rickettsia, designated rickettsia sp. xy99. clinical signs resembled those of severe fever with thrombocytopenia syndrome. |
classically mast cells are considered critical effector cells in allergy by virtue of their potential to secrete a variety of allergic mediators. the number of mast cells is increased at sites of allergic inflammation, and there is a correlation between mast cell density in the tissue and the severity of allergic symptoms. in allergy, plurivalent antigens bind and crosslink ige molecules bound to the high - affinity ige - receptor (fcri) expressed on mast cells, resulting in cell degranulation and release of proinflammatory mediators. three major categories of mast cell mediators have been described : (1) preformed granule - associated mediators such as histamine and serotonin ; (2) newly generated lipid mediators such as leukotrienes and prostaglandins ; (3) de novo synthesized cytokines including chemokines. ige - mediated activation of mast cells initiates the early phase of allergic responses, resulting in pathologies including greater epithelial permeability, mucus production, smooth muscle contraction, vasodilitation and neurogenic inflammation. the immediate response is followed by recruitment of a variety of other immune cells that participate in the late phase of the reaction, further exacerbating allergic pathology. mast cells are derived from hematopoietic progenitors in the bone marrow which migrate via blood to tissues all over the body where they further differentiate and mature into different phenotypes, depending on the local microenvironment. stem cell factor (scf), also known as steel factor, kit ligand, or mast cell growth factor, is found to be the primary growth and differentiation factor for mast cells. the cellular receptor for scf is the product of the c - kit proto - oncogene. in addition to scf, mast cell growth and differentiation can be facilitated by several other cytokines including il-3. immature mouse mast cells can be differentiated in vitro from bone marrow precursor cells in the presence of il-3 without scf. mast cells are enriched in the skin, around blood vessels, and in mucosal membranes such as the respiratory and gastrointestinal tracts. most notably, mast cells are highly enriched in the skin and mucosal barriers of the body, where they serve as a first line of defense. it is noteworthy that mature mast cells are capable of differentiating both phenotypically and functionally as a consequence of tissue - specific stimulation under defined microenvironmental conditions. for example, inflamed lungs are reported to have more tryptase / chymase - producing mast cells compared with non - inflamed lung tissue in which tryptase - producing mast cells are dominant. two major subtypes of rodent mast cells have been characterized, i.e. connective tissue mast cells (ctmc) and mucosal mast cells (mmc), based on their tissue localization. for instance, skin mast cells and mast cells residing in the peritoneal cavity are ctmc, whereas mast cells located in the respiratory or gastrointestinal tracts are usually characterized as mmc. in addition to tissue localization, other properties such as protease and cytokine profiles, membrane receptor distribution, and growth factor requirements also distinguish these two types of mast cells. in addition to residing in connective and serosal tissues, ctmc in mice have been found in the submucosa of the stomach and nasal tissue. in contrast, human mast cells are usually grouped based on the expression pattern of two mast cell - specific proteases, i.e. tryptase and chymase. according to this classification, two major human mast cell subgroups have been proposed. mast cells that contain only tryptase are referred to as mct, whereas those that contain both tryptase and chymase are termed mctc. in terms of correlation to their murine counterparts, mct are found mainly in mucosal tissues, resembling mouse mmc, while mctc, which reside in such sites as the skin and small intestinal submucosa, are more closely related to mouse ctmc, although the tissue localization is less stringent for human ctmc and mmc. similar to mouse mast cells, human mast cells also differ in the requirement for growth and differentiation factors. specifically, scf is needed for the survival of both types, whereas il-4 is indispensable for mctc, but not for mct. in addition to ige- and fcri - mediated cell activation, mast cells can be activated by a variety of other stimulators, such as igg immune complexes, cytokines, complement components, neuropeptides, chemical agents, and physical stimuli, as mast cells express broad - ranging surface receptors including fc receptors, complement receptors, and pathogen - associated molecular patterns (pamp) such as toll - like receptors (tlr). these observations, together with the description of a wide spectrum of mast cell mediators, provide a basis for proposals implicating mast cells in almost all aspects of immune responses. therefore, mast cells have been postulated to be modulators of numerous physiological and pathological responses beyond their classically defined role in allergies mediated mainly through fcri. it has to be pointed out that the overwhelming research findings addressing the roles of mast cells have relied on the use of mast cell - deficient, kit mutant mice which have other phenotypic abnormalities in addition to mast cell deficiency. these data await further experimental verification using the kit - independent mast cell - deficient models to eliminate the confounding elements as a result of kit mutation. in addition to ige- and fcri - mediated cell activation, mast cells can be activated by a variety of other stimulators, such as igg immune complexes, cytokines, complement components, neuropeptides, chemical agents, and physical stimuli, as mast cells express broad - ranging surface receptors including fc receptors, complement receptors, and pathogen - associated molecular patterns (pamp) such as toll - like receptors (tlr). these observations, together with the description of a wide spectrum of mast cell mediators, provide a basis for proposals implicating mast cells in almost all aspects of immune responses. therefore, mast cells have been postulated to be modulators of numerous physiological and pathological responses beyond their classically defined role in allergies mediated mainly through fcri. it has to be pointed out that the overwhelming research findings addressing the roles of mast cells have relied on the use of mast cell - deficient, kit mutant mice which have other phenotypic abnormalities in addition to mast cell deficiency. these data await further experimental verification using the kit - independent mast cell - deficient models to eliminate the confounding elements as a result of kit mutation. the earliest observation of a beneficial role of mast cells is their potential in defense against parasitic infection. both ige and mouse mast cell protease-6 (mmcp-6) are required for chronic immune responses against trichinella spiralis infections. in a helminth infection model, mast cells contribute to pathogen clearance by migrating to the draining lymph nodes and producing il-6 and il-4. interestingly, mast cells have also been described to be critical for th1 response - mediated defence against oral infection with toxoplasma gondii. in addition to defense against helminth infections, mast cells have also been described to be protective in bacterial infections. one of the classic examples of mast cell - dependent anti - bacterial infection is demonstrated by the cecal ligation and puncture (clp) model of acute peritonitis which is dependent on tumour necrosis factor (tnf) and the ability of mast cells to lower neurotensin levels. furthermore, -hexosaminidase, which is abundantly contained in mast cell granules, has recently been reported to have bactericidal activity. the roles and relevance of mast cells in defense against viral and fungal infections have also been suggested. mast cells can be activated, through the equipped tlr, by direct recognition of microbial components such as bacterial lipopolysaccharide (lps) and peptidoglycan resulting in distinct outcomes. furthermore, mast cells can be stimulated by endogenous inflammatory factors such as cytokines and complement components secondary to infection. indirect interaction of mast cells with pathogens can also be achieved through the recognition of pathogen - antibody complexes by fc receptors including fcri and fc receptors expressed on mast cells. fc receptor - mediated mast cell activation may also be triggered in the presence of certain pathogen - derived proteins that can bind immunoglobulins in an antigen - independent manner. a classic example of such a bacteria - derived superantigen is protein a from staphylococcus aureus which can activate human and mouse tissue mast cells, as the fcri molecules on these mast cells are most likely to have already been occupied with ige, resulting in crosslinking of fcri upon protein a binding. however, the pathophysiological roles of such superantigen - mediated mast cell activation in defence against infection await further clarification. similar to mast cell activation in other circumstances, the activation by pathogens is also believed to include both degranulation of pre - formed granular contents and selective de novo mediator production, for example, cytokines and lipid mediators, the patterns of which differ greatly depending on the stimulus encountered. these mast cell - associated products, such as tnf, il-4, ox40 ligand and mmcp-6, are important for the recruitment and stimulation of other innate immune participants, e.g. neutrophils, macrophages, natural killer (nk) cells and eosinophils, contributing to the clearance of pathogens. mast cells not only interact with cells in the immediate vicinity where the infection first takes place but also influence distant targets, e.g. cells in lymph nodes through mediators that they release. it is also reported that mast cells can kill bacteria by producing extracellular traps that contain antimicrobial mediators. in addition to contributing to innate immune responses by virtue of their large spectrum of granular products, mast cells also form a link between innate and adaptive immunity. mast cells modulate the phenotype and function of key players in adaptive immunity, such as dendritic cells (dc), b cells, and t cells. mast cells have been shown to functionally interact with professional antigen presenting cells (apc) such as dc and regulate their function mainly through mast cell - derived granular products. for example, histamine is capable of regulating the chemotaxis of immature dc and cross - presentation of extracellular antigens. tlr7 ligand - mediated mast cell activation is effective for the migration and maturation of langerhans cells. maturation and activation of immature dc by mast cell - dc direct contact results in the activation of t cells that release ifn- and il-17 promoting th1 and th17 responses, respectively. mast cells provide essential signals such as il-6 and cd40l to enhance proliferation of b cells and drive their differentiation toward iga - secreting plasma cells. mast cells can enhance the activation of t cells by providing costimulatory signals and secreting tnf. mast cells also contribute to the recruitment of t cells to sites of viral infection by secreting chemotactic molecules. one of the key processes in achieving successful adaptive immunity is the presentation of microbial antigens to t lymphocytes. whether or not mast cells are capable of acting as antigen - presenting cells is still controversial. this is largely because of the argument that mast cells under steady state do not seem to constitutively express major histocompatibility complex class ii (mhc - ii) or co - stimulatory molecules such as cd86. in contrast, mast cells upregulate expression of mhc - ii and costimulatory molecules following stimulation by inflammatory factors such as ifn- and lps. therefore, mast cells may have the potential to directly present antigens to t cells at least under certain circumstances, for example, in inflamed tissues, to initiate adaptive immune responses. mast cells have also been demonstrated to present antigen to and activate cd8 t cells through mhc - i molecules. alternatively, mast cells are reported as participating in antigen cross - presentation to t cells. cross - presentation refers to a process, most typically following intracellular microbial infection, during which professional apc ingest infected cells and display the antigens of the microbes originally engulfed by the infected cells for recognition by t lymphocytes. this is an efficient mechanism for presenting the antigens of those microbes that have infected host cells that may not produce all the signals, e.g. mhc - ii recognition and costimulation needed to initiate t cell activation. the professional apc that have ingested infected cells may present the microbial antigens to both cd4 and cd8 t lymphocytes depending on the processing and presentation routes. morphological changes of the host cells as a result of, e.g. microbial infections, apoptosis, and tumourigenesis, will facilitate ingestion by apc. in principle, any type of cells that have internalized antigens can participate in cross - presentation upon ingestion by apc. importantly, mast cells have been implicated in the phagocytosis of various types of antigens. indeed, mast cells can serve as an antigen - reservoir and participate in antigen cross - presentation. in vitro cultured bone marrow - derived cultured mast cells (bmmc) can internalize ige - bound chicken ovalbumin (ova) protein, followed by engulfment by dc which process and present the ova peptide to t cells that have specific receptors for the ova peptide. induction of bmmc apoptosis is documented to be critical for efficient presentation by dc to t cells of the antigen originally phagocytosed by mast cells. owing to the fact that mast cells are capable of participating in both innate and adaptive immunity, and that they are enriched at the mucosal and skin barriers between the body and the external environment, mast cells, similar to skin langerhans cells, tissue - resident dc and epithelial cells, are believed to be sentinel cells that are probably the first responders to a threat within seconds. equipped with their immunologic armory of mediators, mast cells may possibly exert a pivotal role in the surveillance and elimination of pathogens by diversified mechanisms. while people have been endowing mast cells with a more positive image in health, new findings also implicate mast cells or their released products negatively in infection. although mast cell - associated tnf has been reported to be critical for a clp model of acute peritonitis, it has to be pointed out that mast cell - derived tnf is not always protective in acute peritonitis, especially in models of severe clp. the detrimental effects of mast cells in severe peritonitis have also been ascribed to the release of il-4 that inhibits the phagocytic potential of macrophages. even the potential of mast cells to recruit other immune effector cells during an infection is not always protective as this has been found to promote chlamydia pneumoniae infection. interestingly, mmcp-4, the mouse counterpart of human mast cell chymase, can degrade tnf, thus dampening the severity of inflammation associated with sepsis and limiting the damage caused by tnf, suggesting antagonism between mast cell mediators, thus favouring protection. therefore, the implication and relevance of mast cells in host defense is a complex issue and the net outcome may depend on many antagonistic factors. a vaccine is a biological preparation that stimulates an immune response against specific antigens that either are derived from the pathogen itself or resemble the structure of the pathogen. ever since the first documented vaccination attempt by edward jenner for the prevention of small pox in 1796, vaccines have played a crucial role in protecting people against many infectious diseases. the eradication of smallpox and the effective control of polio represent two classic success stories of how vaccines can play a major role in improving global health. nevertheless, the demand for better and more effective vaccines against many infectious diseases is still growing, especially when infections such as tuberculosis, hiv, dengue fever and malaria still present enormous global problems. from a societal point of view, vaccination remains the most effective intervention in the control of infectious diseases and for the improvement of global health. there are two principal forms of vaccines : those that are live attenuated vaccines and those that are killed whole pathogens or subcomponent vaccines. an advantage of live attenuated vaccines is that they usually stimulate long - term immune responses similar to natural infection. however, live attenuated vaccines always come with a risk of reversion into more virulent organisms that could cause adverse reactions or more severe infections. in contrast, killed vaccines or subcomponent vaccines are more predictable and, therefore, safer. furthermore, another concern that makes live attenuated vaccines less practical is the demand for a cold - chain for storing or transporting these vaccines. therefore, killed vaccines are still much in use, even though they are weaker and usually do not promote as strong long - term memory responses. to make killed vaccines more effective, we need adjuvants which are substances that enhance immune responses and stimulate long - lasting, robust protective immunity. an adjuvant that is included in the vaccine contributes greatly to the efficacy of the vaccination by affecting the immune responses both quantitatively and qualitatively. importantly, protective immunity following vaccination may be generated with lower amounts of antigen and a reduced dosing frequency after addition of an adjuvant. of all currently available adjuvants, aluminium salts (alum) have the longest history in practical vaccination. alum - based vaccines have a good safety record and are capable of inducing early, high - titer, long - lasting protective immunity. at present, alum is still the most widely used adjuvant in both veterinary and human vaccines. the mechanism of action has been proposed to depend on a depot effect, enabling physical adsorption of antigen onto the alum depots. interestingly, mast cells are found to respond to alum stimulation by releasing histamine and a panel of cytokines including il-5 and il-1. although by using the mast cell - deficient kit mice it is demonstrated that mast cells are not required for the priming of endogenous cd4 and cd8 t cells, this does not formally exclude the contribution of mast cells to the adjuvant activity of alum in the wild - type mice as redundant pathways may exist. however, alum does not seem to be effective for mucosal immunisation, a route that has appreciable advantages compared with routes that require needle injections, i.e. intramuscular or subcutaneous delivery of vaccines. needle - free mucosal vaccination can be achieved via oral, intranasal, sublingual, or intravaginal routes. the obvious benefits of mucosal immunisation include avoidance of blood - borne contamination through re - use of syringes and needles as well as the fact that no trained professional personnel are required for vaccine delivery. strikingly, mucosal immunisation can generate effective secretory iga even at mucosal sites distant from where the vaccine is delivered. for example, nasal immunisation can generate protective mucosal antibodies in the genital tract mucosa, which signifies the advantage of nasal vaccination. as most pathogens enter the body through mucosal surfaces, local mucosal immune responses are critically important in defense against invading pathogens. therefore, how to achieve strong local protection has become one of the major goals of vaccine development. as the mucosal route of vaccination, as opposed to the parenteral route, often results in immune tolerance development, potent adjuvants are much warranted. therefore, the selection of a strong mucosal adjuvant for effective vaccination is vital and possibly as important as the vaccine antigens themselves. tlr agonists have been tested and these include tlr4 ligand monophosphoryl lipid a, tlr9 ligand cpg oligodeoxynucleotides (odn) and the tlr5 ligand flagellin. bacterial enterotoxins which include cholera toxin (ct) and escherichia coli heatlabile toxin (lt) constitute another major group of experimental mucosal adjuvants. both ct and lt are composed of five b - subunits (ctb and ltb) and a single copy of the a subunit (cta or lta). the cta subunit is produced as a single polypeptide chain that is post - translationally modified through the action of a vibrio cholerae protease to form two chains, cta1 and cta2, which remain linked by a disulphide bond. cta1 is enzymatically active by adp - ribosylating the cell membrane bound gs-protein, whereas ctb binds to gm1-gangliosides present on virtually all nucleated cells. dc are believed to play a central role in the presentation of antigens to nave t cells, which is a critical process for the development of adaptive immunity following natural infection. as adjuvants are expected to mediate the same consequences as natural infections, quite a number of adjuvant studies are focused on the interaction of adjuvant with dc. for example, b cells, macrophages, nk cells and nkt cells have also been implicated as targets for vaccine adjuvants. given the accumulating evidence suggesting a functional interplay between mast cells and other immune cells such as dc, t cells and b cells in adaptive immune responses, also mast cells have been implicated in adjuvant functions. indeed, mast cell activators such as c48/80 have been reported as exerting a mucosal adjuvant function. more specifically, c48/80 is demonstrated to be an efficient adjuvant by mobilizing dc to the draining lymph nodes through production of tnf. successful vaccinations of several animal infection models using c48/80 as adjuvant have now been reported. retention of c48/80 and antigen on mucosal surfaces by chitosan - based nanoparticles can further promote mucosal immunisation. the il-1 family cytokines such as il-1, il-18 and il-33 have been shown to exert adjuvant function capable of augmenting protection against influenza virus infection. interestingly, the effect of il-18 and il-33 is suggested to be mast cell - dependent, which is not surprising as both cytokines can activate mast cells resulting in proinflammatory cytokine production. il-18 together with il-2 is potent in expanding the mucosal mast cell pool and the production of mmcp-1, which is critical for parasite expulsion. il-33 is described as a danger signal that can alert mast cells and keratinocytederived il-33 can stimulate mast cells to produce tnf and il-6, cytokines critical for defence against herpes simplex virus infection. polymyxins which are clinically approved antibiotics can activate mast cells and boost immunisation. in a quila - adjuvanted cattle vaccination model for protection against nematode infection, mast cells are most likely to be involved in the mechanism of adjuvanticity through the production of granzyme b and granulysin. synthetic particles harboring tnf, mimicking mast cell granules, have been reported to be powerful adjuvant in a mouse model of influenza. furthermore, it is suggested that the gold standard mucosal adjuvant ct may stimulate the release of il-6 from mast cells boosting humoral immune responses. although the bacterial enterotoxins have been demonstrated to be powerful mucosal adjuvants experimentally, these substances are precluded from clinical use because of their toxicity and, hence, they have very limited applicability in human vaccines. extensive studies have, however, focused on the detoxification of these molecules using various approaches. for example, site - directed mutagenesis has generated detoxified mutants, such as ct112k, ltg192, ltr72, or ltk63, with little or no enzymatic activity, but with retained adjuvant function in experimental models. however, a drastically different approach was applied by lycke and co - workers who developed an adjuvant based on the intact cta1 molecule without the b - subunit. the cta1 is linked genetically to a dimer of the d - fragment of staphylococcus aureus protein a forming the cta1-dd adjuvant. thus, cta1-dd has retained the adjuvant function while the molecule can not bind to gm1-ganglioside, rendering the molecule nontoxic. in contrast to ct, intranasal administration of cta1-dd results in neither inflammation nor accumulation in nervous tissues as is found with ct or lt. the adjuvanticity of cta1-dd has been well documented in various infectious disease models, which include chlamydia trachomatis, influenza, hiv, mycobacterium tuberculosis, and helicobacter pylori. in addition, mechanistic studies have identified several mechanisms of action that may explain the adjuvanticity of cta1-dd in vivo. as the dd domain binds to all immunoglobulins, cta1-dd can target b cells through the b cell receptor, i.e. surface bound immunoglobulins, and promote b cell activation and germinal center development. importantly, cta1-dd stimulates germinal center formation effectively generating long - lived plasma cells and long - lived b memory cells. furthermore, also follicular dc and complement activation have been found to be essential elements for the function of this adjuvant. in contrast to intact staphylococcus aureus protein a, which can activate mast cells, cta1-dd fails to activate mast cells. however, as the double d domains derived from protein a have binding sites for immunoglobulins, cta1-dd can bind to all immunoglobulins including igg. we demonstrated that cta1-dd and igg may form complexes that are able to activate mast cells through fc receptors, resulting in degranulation and the production of tnf and il-6. intranasal immunisation in the presence of cta1-dd and igg as an adjuvant can enhance antigen - specific immune responses compared with cta1-dd alone. furthermore, we demonstrated that only ctmc, but not mmc, can be activated by immune complexes composed of cta1-dd and igg. this effect is mediated by fcriiia, an activating receptor that is confirmed to be only expressed on ctmc. indeed, ctmc are found in the nasal submucosa and these cells are demonstrated to express fcriiia. as mmc are not activated in response to stimulation by igg immune complexes because of the lack of fcriiia, it was intriguing to investigate whether or not mmc could contribute to adaptive immune responses somehow, perhaps using another mechanism. we have recently reported that igg immune complex - primed mmc can mediate enhanced antigen - specific activation of t cells, possibly providing a cross - presentation mechanism to boost mucosal vaccination. in practical immunisation the development of adjuvants that enhance the potency of subunit vaccines formulated for administration through the mucosal routes is much desired. dissecting and revealing the molecular mechanisms, through which mast cells precisely control adaptive immune responses to combat microbial infections, may have implications for rationally designing mucosal vaccine formulations. we propose that igg immune complex - induced mast cell activation may be considered as one of the components for mucosal vaccine adjuvants. 1 summarizes the current knowledge regarding the strategies for the selection of vaccine formulations that target mast cells for enhancing immune responses. mast cells can be activated by various factors including compound 48/80 (c48/80), il-33, il-18, cholera toxin (ct), quila, alum and igg immune complexes (igg ic). upon activation, mast cells release tnf-, il-6, il-5, il-1, histamine, mouse mast cell protease (mmcp), granzyme and granulysin that are critical in mobilizing immune responses. however, these are just examples of functional consequences of mast cell activation specific to the findings of those studies addressing roles of mast cells in vaccination cited in this review ; the actual spectrum of mediators released by mast cells can be much more dynamic and complex. one of the challenges associated with mast cell - mediated immune enhancement, of course, lies in overcoming the complexity of safety issues for the clinical development of the vaccines. the constant threats posed by infectious diseases over millions of years may have driven evolutionary pressure to keep mast cells, despite their adverse properties, e.g. in causing allergy, in humans to exploit these cells beneficial functions in host defense. our immune system has evolved mechanisms to balance the positive and negative contributions of mast cells to health. it is worth exploring strategies to make use of the adjuvant properties of mast cells to provide high - quality vaccination while minimizing any health - compromising factors. | abstractin addition to their well - established role in allergy mast cells have been described as contributing to functional regulation of both innate and adaptive immune responses in host defense. mast cells are of hematopoietic origin but typically complete their differentiation in tissues where they express immune regulatory functions by releasing diverse mediators and cytokines. mast cells are abundant at mucosal tissues which are portals of entry for common infectious agents in addition to allergens. here, we review the current understanding of the participation of mast cells in defense against infection. we also discuss possibilities of exploiting mast cell activation to provide adequate adjuvant activity that is needed in high - quality vaccination against infectious diseases. |
a 40-year - old patient was admitted to the hospital on january 29, 2004, with fever, generalized weakness, diarrhea, and vomiting. initial blood tests showed hemoglobin level of 8.5 g / dl, leukocyte count of 4.9 x 10/l, and platelet count of 110 x 10/l. erythrocyte sedimentation rate was elevated at 88 mm / h. her serum glucose was 8.5 mmol / l, and urea and electrolytes were normal. she was started on intravenous ciprofloxacin, but her fever persisted, and she became increasingly confused. her old hospital records became available shortly after her death, and we noted that she had been diagnosed with systemic lupus erythematosus (sle) in 1994. when she last attended the outpatient department 3 months before her hospital admission, she was prescribed 50 mg azathioprine and 5 mg prednisolone daily. she was a housewife and lived in cit la cure, a poor suburb of the capital city port - louis., her home becomes very muddy after heavy rainfall, and her feet were often in mud while performing her household duties. after 5 days of incubation, it produced colonies that appeared dry and rugose on the plates after 48 h and was identified as b. pseudomallei by using api 20ne (biomrieux, marcy letoile, france) with the profile 1156577. antimicrobial susceptibility testing by disc diffusion showed the organism to be resistant to colistin, ampicillin, cephalexin, gentamicin, and ciprofloxacin and susceptible to co - amoxiclav, tetracycline, cefotaxime, ceftriaxone, ceftazidime, piperacillin, and meropenem. a large zone of inhibition was seen around the co - trimoxazole disc, within which a thin film of growth was observed. we are not aware of any study looking for the organism in soil in this country. groodoyal, pers. comm.). whether human cases of melioidosis have been missed in the past is not known, and cases may be missed currently. recognizing the disease depends on awareness on the part of clinicians and on the ability of microbiology laboratories to identify the causative organism (1,8). before 1998, oxidase - positive, gram - negative bacilli other than p. aeruginosa were not identified to species level in laboratories in mauritius. since then, at our laboratory, which receives specimens for bacteriologic investigations from all government healthcare institutions, such organisms are routinely identified by api 20ne when isolated in pure culture from blood, but only occasionally when isolated from nonsterile sites such as sputum and pus swabs. some clinicians routinely request blood cultures from patients with high fever before starting antimicrobial drugs, although in practice, the specimen is often collected by nursing staff after the first dose has already been administered. other clinicians only request blood cultures if fever persists after a few days of empiric antimicrobial therapy. in the case reported here, prior administration of cefotaxime may have delayed b. pseudomallei culture from blood until 5 days of incubation, when the median time to obtain a positive blood culture result is typically 48 hours (1). an association between rainfall and melioidosis has long been recognized ; most cases in thailand (9) and northern australia (10) occur during the wet season. the increased number of cases noted during the rainy season may be caused by the movement of b. pseudomallei from deeper layers toward the surface when dry topsoil is moistened by rainfall (2). in mauritius 196 mm rainfall was recorded in port - louis, which is 37% higher than the 19712000 mean rainfall for the region during this month. january 2004 was the sixth wettest january of the past 30 years in port - louis. recent reviews have suggested a predominant role for percutaneous b. pseudomallei infection in the pathogenesis of melioidosis (11). studies carried out in regions where melioidosis is endemic have shown that exposure to wet soil and water are associated with increased risk for disease (9). the feet of our patient were regularly exposed to wet soil during rainy periods. in melioidosis - endemic areas, although a large percentage of the population has been exposed to b. pseudomallei, as determined by seroprevalence studies, only a few develop melioidosis (12). most cases occur in patients with underlying illnesses, such as diabetes mellitus, renal disease, and alcoholism (9,10) or in those who are immunosuppressed (1). this first case of melioidosis in mauritius occurred in an immunosuppressed patient who had a history of prolonged and regular exposure to mud during a year when rainfall was higher than average. this combination of 3 risk factors does not occur regularly, and it is possible that few additional cases will be recorded in mauritius in the future. however, clinicians and laboratory staff must remain aware of this disease, particularly because in a noncommunicable disease survey carried out in 1998, almost 20% of the mauritian population > 20 years of age were found to have type 2 diabetes mellitus (14), the most common predisposing condition for melioidosis (1). determining the distribution of b. pseudomallei in soil in mauritius by conducting environmental investigations will also be useful. | we report the first case of human melioidosis from mauritius, where burkholderia pseudomallei has never been isolated. the patient was immunocompromised, had never traveled abroad, and had a history of regular exposure to mud. she became ill at a time when rainfall was higher than the monthly average. |
oxidatively modified lipoproteins, such as oxidized low - density lipoprotein (oldl) have been implicated in the pathogenesis of atherosclerosis and neurodegenerative diseases such as alzheimer 's disease (ad) [1 - 5 ]. this peroxidation of the low - density lipoprotein (ldl) molecule renders it immunogenic and causes monocyte recruitment, foam cell formation and cytotoxicity to various cells including neurons [6 - 8 ]. in contrast, epidemiological data has suggested a strong inverse correlation between plasma hdl concentrations and the incidence of coronary and cerebral atherosclerosis. traditionally, this relationship has been proposed to be due in part to the involvement of hdl in reverse cholesterol transport, however, recent studies have shown that hdl can prevent the oxidation of ldl. this prevention of ldl oxidation by hdl contributes to a decrease in the formation of lipid peroxides, foam cell formation and cytotoxicity otherwise caused by oldl [12 - 14 ]. recent evidence suggests that hdl associated enzymes such as paroxonase, may play a critical role in this protective effect. however, hdl itself can get oxidized and the ability of oxidized hdl (ohdl) in reverse cholesterol transport is impaired. furthermore, ohdl is neurotoxic and has been postulated to play a role in the genesis of coronary artery spasm that contributes to the process of chd. epidemiological studies have indicated that premenopausal women have a decreased risk for the development of atherosclerosis when compared to that of age - matched males, however, this decreased risk diminishes following menopause. it has therefore been suggested that ovarian hormones, such as estrogen, play a role in the decreased risk observed in premenopausal women. during menopause, plasma ldl and hdl levels increase and decrease respectively, however studies have shown that estrogen replacement therapy (ert) and hormone replacement therapy (hrt, estrogen plus progestin) alter these levels, in that serum total cholesterol and ldl cholesterol decreases while hdl cholesterol increases. until recently the modification of the plasma ldl : hdl ratio was thought to be the main mechanism for the cardioprotective effects of estrogen observed, however, recent evidence indicates that these play a minor role. more recently, it has been observed that various equine estrogens can also differentially inhibit the oxidation of the ldl and can attenuate the cytotoxicity of oldl on neuronal cells. in the present study, the effects of various equine estrogens on the oxidation of hdl and the combined effect of estrogen and hdl on ldl oxidation was assessed. the equine estrogens tested in this study were : estrone (e1), 17-estradiol (17-e2), 17-estradiol (17-e2), equilin (eq), 17-dihydroequilin (17-eq), 17-dihydroequilin (17-eq), equilenin (eqn), 17-dihydroequilenin (17-eqn), 17-dihydroequilenin (17-eqn), -estrone (-e1), and,17-estradiol (,17-e2). all estrogens tested, with the exception of,17-e2 (a metabolite of -e1), in their sulfate - conjugated form, are components of the conjugated equine estrogens (cee ; premarin, wyeth pharmaceuticals, philadelphia, pa), commonly used by postmenopausal women for ert and hrt. all estrogens in their sulfated form are components present in the estrogen replacement drug cee (conjugated equine estrogens ; premarin, wyeth pharmaceuticals, pa, usa). the effects of various concentrations of equine estrogens on the kinetics of hdl oxidation induced by cuwere determined as described in the methods. representative examples of dose - response curves obtained with the various estrogens are shown in figure 2. three phases of diene formation were observed in all control samples : an initial induction phase (lag phase), a propagation phase and a decomposition phase (plateau). during the induction phase, the formation of dienes progressed slowly. in the propagation phase, a sharp, linear increase in diene formation was noted and the plateau phase was followed by a slow increase due to decomposition of products. the lag time for control was 65 4 min, and in presence of estrogens, it ranged from 75 to 244 min. representative concentration - response curves of conjugated - diene formation for various estrogen concentrations against hdl oxidation. (a) 17-e2, (b) 17-eqn (c) e1 (d) -e1. all 11 estrogens tested protected the hdl from oxidation and increased the lag phase in a concentration dependant manner. the results obtained for each of the highest concentration of estrogen tested were expressed as percent of 17-e2 antioxidant activity (lag time for the highest 17-e2 concentration used was set to 100%, figure 3). in the presence of 17-e2 (0.5 g, 610 nm) the lag phase increased from a control lag phase of 65 4 min for hdl alone, to 212 4 min (p 17-eqn >,17-e2 = -e1 > 17-eq = 17-eq = 17-e2 > e1 = eq > 17-e2. antioxidant activity of various estrogens measured against the antioxidant potential of 17-e2. for comparison purposes the 17-e2 value the ring b unsaturated equine estrogens, eqn, 17-eqn and 17-eqn, were found to be the most potent inhibitors of hdl oxidation having two- to four - fold higher activity than 17-e2. similarly, -e1 and,17-e2 were found to have 150% greater antioxidant activity than control. 17-eq and 17-eq were found to be equipotent with 17-e2, while eq, e1 and 17-e2 were found to be less potent, with activities ranging from approximately 60 to 70% that of 17-e2 (figure 3). the minimum concentration of estrogen required to double the lag phase from a mean (control) lag time of 65 4 min are listed in table 1. eqn, 17-eqn, 17-eqn and,17-e2 all required approximately 50 to 80 nm to double the lag phase. in contrast, 17-eq, 17-eq, eq, 17-e2, e1, and 17-e2 all required approximately 210 to 360 nm concentrations to double the lag phase. dose of estrogen required to double the lag phase of hdl oxidation the mean control lag phase for oxidation of ldl alone was observed to be 73 1.3 min. when ldl was subjected to oxidation in the presence of hdl the lag phase was prolonged to 104.5 7 min (p 17-eqn >,17-e2 = -e1 > 17-eq = 17-eq = 17-e2 > e1 = eq > 17-e2. antioxidant activity of various estrogens measured against the antioxidant potential of 17-e2. for comparison purposes the 17-e2 value the ring b unsaturated equine estrogens, eqn, 17-eqn and 17-eqn, were found to be the most potent inhibitors of hdl oxidation having two- to four - fold higher activity than 17-e2. similarly, -e1 and,17-e2 were found to have 150% greater antioxidant activity than control. 17-eq and 17-eq were found to be equipotent with 17-e2, while eq, e1 and 17-e2 were found to be less potent, with activities ranging from approximately 60 to 70% that of 17-e2 (figure 3). the minimum concentration of estrogen required to double the lag phase from a mean (control) lag time of 65 4 min are listed in table 1. eqn, 17-eqn, 17-eqn and,17-e2 all required approximately 50 to 80 nm to double the lag phase. in contrast, 17-eq, 17-eq, eq, 17-e2, e1, and 17-e2 all required approximately 210 to 360 nm concentrations to double the lag phase. the mean control lag phase for oxidation of ldl alone was observed to be 73 1.3 min. when ldl was subjected to oxidation in the presence of hdl the lag phase was prolonged to 104.5 7 min (p 70 ng / hr / ml, and thus daily pharmacological doses of cee during ert or hrt can provide sufficient plasma estrogen levels needed to prevent the oxidation of hdl to the extent we have observed in our in vitro study. under our experimental conditions, esterification of estrogens was not possible, indicating that inhibition of hdl oxidation by estrogens does not require prior esterification of estrogens. for example, eq, 17-eq, and 17-eq all displayed antioxidant activities similar to 17-e2, whereas the and -estrogens had two- to four - fold greater activities. this difference is most likely due to the extra double bonds in the b ring, thus making the molecule more labile and therefore able to scavenge any free radicals produced more readily. for example, the ring b unsaturated estrogens were more potent antioxidants than the classical estrogens tested. previous reports have demonstrated that the free radical scavenging activity of estrogen depends upon the phenolic structure of the molecule [30 - 32 ]. in addition to this, our observations indicate that the presence of a -napthol ring, such as that in the estrogens, may play a larger role in the antioxidant potential observed in these types of estrogens as they were found to be the most potent antioxidants. thus, our results further extend the observation that the presence of the phenolic a ring and the 3-hydroxyl group is required for steroid antioxidant activity, but unsaturation in the b ring of the steroid moiety greatly enhances estrogens ' antioxidant potency. the order of oxidative protection for estrogens tested does not appear to involve estrogen receptors as the relative binding affinities to estrogen receptor (er) and do not correlate with their antioxidant activity. for instance, compared to 17-e2, the relative binding affinities for the ring b unsaturated estrogens : eq, eqn, -e1 and 17-eqn are 2 to 8 times lower for both ers, whereas these same estrogens with the exception of eq displayed the highest antioxidant potentials. moreover, this order of antioxidant activity does not correlate with their uterotropic activities as reported earlier. in the current study we have demonstrated that hdl can prolong the oxidation lag time of ldl using the conjugated diene formation method. this increase in oxidation lag time is most likely due to the 10-fold higher concentration of hdl over ldl as lower concentrations of hdl did not provide any protection (data not shown). similarly, a study by huang showed hdl protection of ldl is concentration dependant. one possible mechanism for this protective effect is that hdl is preferentially oxidized over ldl and can act as a sink for harmful lipid peroxides. however, a more current hypothesis is that hdl associated enzymes such as paroxonase, and even the apolipoprotein apo - a1, provide hdl with its antioxidant activity toward ldl oxidation. in support of this hypothesis, equine estrogens have recently been shown to stimulate the expression of apo - a1 mrna and protein levels by an estrogen receptor independent mechanism. moreover, in these studies eqn was also the most potent stimulator of apo - a1 gene expression in addition to the protective effect of hdl on ldl oxidation, we have also observed that the prolongation of ldl oxidation with hdl can be enhanced with the addition of estrogen. treatment with 17-e2 plus hdl was more protective than 17-e2 or hdl alone against ldl oxidation. similarly, treatment with eqn plus hdl displayed a greater protection against ldl oxidation than either of the constituents alone. in line with the previously defined order of protection, the eqn plus hdl combination was observed to have a greater antioxidant effect than the 17-e2 plus hdl combination against ldl oxidation. also, the increased protection by the estrogen plus hdl treatment on ldl oxidation is not simply the sum of lag times with hdl or estrogen alone on ldl oxidation, and thus is suggestive of an interaction between estrogen and hdl. this estrogen enhancement of hdl 's antioxidant activity is to our knowledge the first reported interaction of estrogen with hdl that causes a delay or inhibition of ldl oxidation. following ert, the main hdl subfraction that is found to increase, is hdl2. more studies are needed to determine the role of each hdl subfraction in presence of estrogen on the inhibition of ldl oxidation. although the mechanisms involved in the antioxidant properties of estrogen have not been fully defined, a number of hypotheses have been put forth. estrogens acting as free radical scavengers are able to break the free radical chain formation produced from membrane oxidation processes and hence inhibit lipid peroxidation. in support of this, it has been postulated that estrogen may be able to sequester metal ions or donate a proton to reduce peroxy - free radicals. furthermore, the stimulation of apo - a1 by estrogen may also be of importance. thus acting as antioxidants directly and by the up - regulation of antioxidant factors may be two additional mechanisms that may be involved in the cardio- and neuroprotective effects of estrogen. in conclusion, these results indicate that hdl oxidation can be differentially inhibited by equine estrogens with the three ring b unsaturated equine estrogens (figure 1, figure 3) being three to four times more potent than 17-e2. furthermore, the protection of ldl oxidation by hdl is enhanced in the presence of 17-e2 and more so by eqn. the inhibition of hdl oxidation and the enhancement provided by estrogen toward the inhibition of ldl oxidation may therefore be another mechanism by which estrogens reduce the risk of chd and neurodegenerative diseases in healthy young postmenopausal women. blood (10 ml) was drawn by venipuncture into vacutainer tubes containing ethylenediaminetetraacetic acid (edta ; 1 mg / ml) from fasting (overnight), postmenopausal women and healthy men. the plasma was stabilized by addition of 6 mg / ml sucrose and either used immediately for lipoprotein isolation or stored at -80c for up to four weeks as described previously. hdl (d = 1.063 - 1.21 g / ml) and ldl (d = 1.019 - 1.063 g / ml) were isolated simultaneously by a three gradient, single - step ultracentrifugation as described previously. following ultracentrifugation the lipoprotein containing fractions (yellow - orange / colour) were collected by aspiration. assessment of absorbance at 200 to 300 nm of the crude and purified preparations of hdl, indicated that the crude preparation was essentially similar to the purified preparation and thus the single step method of isolation was used for the experiments described. the ldl isolated by this method had properties similar to those previously described. the freshly isolated lipoproteins were stored at 4c under argon and used within 1 week of preparation. prior to oxidation, edta was removed from the lipoprotein isolate by rapid gel filtration through disposable desalting columns (econo - pac 10 dg, bio - rad laboratories). the concentration of protein in each lipoprotein isolate was measured using the bradford method (bio - rad protein assay kit, catalog number 5000006, mississauga, on, canada). oxidation of 50 g hdl was induced by addition of 1.67 m cuso4 in the presence or absence of 0.025 g, 0.05 g, 0.25 g, 0.5 g, 1 g of the 11 estrogens. the kinetics of lipoprotein oxidation were assessed by measuring the rate of formation of conjugated dienes measured at 234 nm over a period of 6 hours at 32c. absorbance was measured in a beckman du 640 spectrophotometer equipped with a six - position cuvette changer and a peltier temperature controller (beckman instruments, mississauga, on, canada) essentially as previously described. the extent of lipoprotein oxidation was confirmed by assessing the electrophoretic mobility of ohdl and oldl on a 1% agarose gel in 0.1 m tris, ph 8.6 compared to the native form of hdl and ldl. oxidation of 20 g ldl was induced in a similar manner, however, in this case the effect of 200 g hdl and the combination of 17-e2 plus 200 g hdl or eqn plus 200 g hdl on ldl oxidation were assessed. the oxidation lag phase, defined as the point at which the initial slope representing the initiation rate and the point at which the propagation slope intersect, was calculated from the kinetic plot of the change in absorbance versus time as described previously. from the dose response curves, the concentration of each estrogen (nm) required to double the length of the control lag time of oxidation, was calculated. for comparison purposes, all values obtained were adjusted to percent activity of 17-e2. blood (10 ml) was drawn by venipuncture into vacutainer tubes containing ethylenediaminetetraacetic acid (edta ; 1 mg / ml) from fasting (overnight), postmenopausal women and healthy men. the plasma was stabilized by addition of 6 mg / ml sucrose and either used immediately for lipoprotein isolation or stored at -80c for up to four weeks as described previously. hdl (d = 1.063 - 1.21 g / ml) and ldl (d = 1.019 - 1.063 g / ml) were isolated simultaneously by a three gradient, single - step ultracentrifugation as described previously. following ultracentrifugation the lipoprotein containing fractions (yellow - orange / colour) were collected by aspiration. assessment of absorbance at 200 to 300 nm of the crude and purified preparations of hdl, indicated that the crude preparation was essentially similar to the purified preparation and thus the single step method of isolation was used for the experiments described. the ldl isolated by this method had properties similar to those previously described. the freshly isolated lipoproteins were stored at 4c under argon and used within 1 week of preparation. prior to oxidation, edta was removed from the lipoprotein isolate by rapid gel filtration through disposable desalting columns (econo - pac 10 dg, bio - rad laboratories). the concentration of protein in each lipoprotein isolate was measured using the bradford method (bio - rad protein assay kit, catalog number 5000006, mississauga, on, canada). oxidation of 50 g hdl was induced by addition of 1.67 m cuso4 in the presence or absence of 0.025 g, 0.05 g, 0.25 g, 0.5 g, 1 g of the 11 estrogens. the kinetics of lipoprotein oxidation were assessed by measuring the rate of formation of conjugated dienes measured at 234 nm over a period of 6 hours at 32c. absorbance was measured in a beckman du 640 spectrophotometer equipped with a six - position cuvette changer and a peltier temperature controller (beckman instruments, mississauga, on, canada) essentially as previously described. the extent of lipoprotein oxidation was confirmed by assessing the electrophoretic mobility of ohdl and oldl on a 1% agarose gel in 0.1 m tris, ph 8.6 compared to the native form of hdl and ldl. oxidation of 20 g ldl was induced in a similar manner, however, in this case the effect of 200 g hdl and the combination of 17-e2 plus 200 g hdl or eqn plus 200 g hdl on ldl oxidation were assessed. the oxidation lag phase, defined as the point at which the initial slope representing the initiation rate and the point at which the propagation slope intersect, was calculated from the kinetic plot of the change in absorbance versus time as described previously. from the dose response curves, the concentration of each estrogen (nm) required to double the length of the control lag time of oxidation, was calculated. for comparison purposes, all values obtained jp is a graduate student who participated in the development of the hypothesis, study design and carried out most of the experimental work and preparation of the manuscript. mb is a research associate who took part in the development of the hypothesis, study design and carried out the initial experimental work during the developmental phase of the study. ac is a physician who participated in the study design and recruitment of subjects and their clinical evaluation and obtained the consent and blood samples from the subjects. ag is also another physician who participated in the study design and recruitment of subjects and their clinical evaluation and obtained the consent and blood samples from the subjects. bb conceived the study and participated in the development of the hypothesis, the study design, and overall direction of the study and preparation of the manuscript. all authors have read and approved the final preparation of the manuscript and its submission to lipids in health and disease this work was supported by the medical research council of canada grant mt-11929 and a basic research grant from women 's health care research, wyeth pharmaceuticals, philadelphia, pa, usa. | backgroundan inverse relationship between the level of high - density lipoprotein (hdl) and coronary heart disease (chd) has been reported. in contrast, oxidized hdl (ohdl) has been shown to induce neuronal death and may play an important role in the pathogenesis of chd. in the present study we have investigated a : the effect of various equine estrogens on hdl oxidation, b : the inhibition of ldl oxidation by hdl and c : the effect of these estrogens on ldl oxidation in the presence of hdl.resultsall 11 equine estrogens tested protected the hdl from oxidation in a concentration dependant manner. equilenin, 17-dihydroequilenin, and 17-dihydroequilenin (68-estrogens) were found to be the most potent inhibitors of hdl oxidation. some of the novel ring b unsaturated estrogens were 2.5 to 4 times more potent inhibitors of hdl oxidation than 17-estradiol. hdl was found to delay ldl oxidation. the protection of ldl oxidation by hdl is enhanced by the addition of estrogen, with equilenin being again more potent than 17-estradiol.conclusionsequine estrogens can differentially inhibit the oxidation of hdl with the 68-estrogens being the most potent antioxidants. the ability of estrogens to enhance hdl 's antioxidant activity is to our knowledge the first report of an interaction of estrogen with hdl that results in the delay or inhibition of ldl oxidation. this may be another mechanism by which estrogens may reduce the risk of chd and neurodegenerative diseases in healthy and younger postmenopausal women. |
the greater part of human mortality and morbidity (certainly in the developed world) focuses on cardiovascular disease and its risk factors, cancer, and connective tissue disease. the pathophysiology of cardiovascular disease (endothelial damage leading to hypertension and thrombosis) is established, whilst those subjects with connective tissue disease (generally inflammatory) are also at risk of possible life - terminating atherothrombosis. a relationship between cancer and thrombosis has been recognised for almost 150 years, and each year brings additional data that confirms this association [4, 5 ]. furthermore, the large proportion of terminal events in neoplastic diseases are thrombotic, leading to the hypothesis that cancer is a prothrombotic disease [69 ]. however, such thromboses may occur in arteries and/or veins, and many authorities fail to differentiate between these two circulations, generally focusing on venous thromboembolism (vte). nevertheless, of pubmed citations of works on arterial and venous thrombosis, approximately 25% are to arterial thrombosis. an additional aspect of thrombosis and cancer is the inevitably adverse effect of chemotherapy (often anti - neoplastic and cytotoxic) in promoting thrombosis [8, 9 ]. although there are isolated reports where hypercoagulability and thrombosis closely follows chemotherapy [10, 11 ], guidelines advocating prophylactic anticoagulation therapy in the para - antineoplastic drug setting are becoming available. indeed, one study named thromboembolism and infection as leading precise causes of death in cancer patients on chemotherapy the objective of this paper is to summarise potential pathophysiological mechanisms to explain this relationship, and to update facets of both arterial and venous thrombosis in various cancers. to achieve this aim, on - line search engines such as pubmed and medline were probed using key words cancer, arterial thrombosis, venous thrombosis, and anticoagulation. whilst acknowledging advances brought by tissue culture and animal models perhaps the oldest and most dominant theory of the pathophysiology of thrombosis is that of virchow, which has three separate but overlapping parts : the contents of the blood, the blood vessel wall, and blood flow. we currently interpret this triad in terms of, respectively, platelets and coagulation factors (with minor roles for red and white blood cells), the endothelium, and blood turbulence (as may be present at valves and at bifurcations) and venostasis. certainly, however, these principles are as equally applicable to arterial thromboses and to venous thromboses, and also to cancer (table 1). indeed, patients with various cancers frequently demonstrate abnormalities in each component of virchow 's triad, leading to a prothrombotic or hypercoagulable state. for example, tumour cells may be directly prothrombotic, inducing thrombin generation, whilst normal host tissues may stimulate (or be stimulated to) prothrombotic activity as a secondary response to the cancer. however, dissecting the exact mechanisms is frustrated by comorbidity and treatment effects, such as bed rest, infection, surgery, and drugs. nevertheless, virchow 's triad also gives us the opportunity to dissect and identify different aspects of the causes of thrombosis. the former clearly centre on the platelet, although there are potential roles (if only minor) for white blood cells and red blood cells, the latter as adp donors [15, 16 ]. examples of platelet (hyper)activity in cancer include reduced life span in myeloma, reduced sensitivity to prostacyclin in endometrial, and cervical cancer [17, 18 ], with increased aggregability and higher levels of platelet specific products soluble p - selectin, platelet factor 4, thrombospondin and beta - thromboglobulin in lung, breast, prostate, ovarian, and other cancers [1926 ]. these and other findings support the general hypothesis of altered platelet activity in cancer, especially in metastatic disease [2729 ]. furthermore, it has been suggested that inappropriate platelet activity promotes metastases [30, 31 ]. cancer cells may activate platelets in vitro by contact, by releasing platelet stimulators such as adp and thromboxane a2, and by generating thrombin through the activity of the tumour - associated procoagulants. increased von willebrand factor, ristocetin cofactor, and enhanced adp - induced platelet aggregation have all been demonstrated [3236 ], and all these mechanism may promote thrombosis. however, in at least one clinical setting, the true value of this has been questioned, as canobbio. found that hypercoagulability was more likely to be related to coagulation than to the expected increase in platelet aggregability. nevertheless, there are several calls for antiplatelet therapy in cancer [3840 ], although concerns have been aired. increased numbers of platelet microparticles have been described in gastric, colon, and breast cancer [4446 ]. interest in platelet microparticles follows a number of themes ; in the promotion of angiogenesis and metastases [4648 ], in the promotion of thrombosis (such as by bearing tissue factor and phospholipids, thus providing a platform for thrombosis) [45, 49, 50 ], and in the promotion of invasiveness. the tumour itself may also shed microparticles, and these too may be prothrombotic [5254 ]. early work suggested a role in an immunological response to neoplasia [56, 57 ], and although this persists, a more recent view linking monocytes to coagulation has been introduced, based on their ability to express tissue factor [5861 ]. however, other mechanisms may also be important, such as the delivery of cytokines and a role in angiogenesis [62, 63 ] and there are also reports of the procoagulant activity of monocyte microparticles, and also of cross - talk between the monocyte and the platelet, all of which have the potential to promote thrombosis. there is also considerable evidence which implicates soluble coagulation factors in cancer - related thrombosis. the normal clotting - fibrinolytic system of haemostasis involves a fine balance between the activation and inhibition of platelets, procoagulant factors, anticoagulant factors, and fibrinolytic factors. this can easily be disrupted as tumour cells can, for example, activate the coagulation system directly through interactions with the clotting and fibrinolytic systems to generate thrombin. the delicate balance between the coagulation and fibrinolytic systems can easily shift to induce a prothrombotic state, perhaps via an excess of procoagulant proteins such as tissue factor, fibrinogen and plasminogen activator inhibitor (pai-1), and/or deficiencies in other molecules, such as anti - thrombin, proteins c and s, and tissue plasminogen activator (tpa) [67, 69 ]. however, perhaps the greater part of the literature in this area considers tissue factor and cancer procoagulant. forming a complex with factor vii to activate factors x and ix, it is produced by monocytes and the endothelium and is functional both at the surface of the cell and as a soluble component of plasma [70, 71 ]. thus in vivo expression of tissue factor, either by tumour and/or normal cells, and soluble tissue factor has been implicated in intratumoral and systemic activation of blood coagulation via the extrinsic pathway, as well as in tumour growth and dissemination [7275 ]. cancer procoagulant is a cysteine proteinase growth factor that is a calcium - dependent, mn stimulated enzyme. unlike tissue factor, cancer procoagulant is a direct activator of factor x without the need for factor vii and is found in malignant and fetal tissue, but not in normally differentiated tissue. concerns that at least some part of cancer procoagulant activity could be accounted for by contaminating tissue factors seem to have been dispelled. in the presence of factor v, increased cancer procoagulant levels have been reported in patients with acute promyelocytic leukemia, malignant melanoma, and cancers of the colon, breast, lung, and kidney, and there is in vitro evidence that it may also be involved in metastatic potential [7982 ] numerous other proteins within the coagulation cascade have been shown to be abnormally elevated, often in association with a fall in the activity of anticoagulant factors. for example, reduced levels of t - pa activity have been described in patients with gastrointestinal cancer, whilst that of pai-1 and other inhibitory factors of the fibrinolytic pathway can be elevated. in addition, there are decreases in other anticoagulant factors, including anti - thrombin and resistance to activated protein c. the evidence of this on - going underlying state of heightened coagulation in cancer is clear from the numerous studies looking at levels of fibrinogen and other indices of fibrin turnover including d - dimers [71, 8589 ]. increased levels of the latter reflect increased clot turnover (thrombolysis) which in turn implies increased thrombotic load, as is present in actual venothromboembolic disease, and levels of d - dimers are part of the clinical assessment of subjects with suspected venothromboembolism. however, there is evidence of very high d - dimer levels in patients with cancer who do not have vte. this suggests that elevated d - dimer levels in patients with vte and malignancy are not solely due to presence of thrombus. knowlson. suggest that high d - dimer levels in malignancy are likely to reflect the biology of the underlying tumour, with higher levels observed in breast, prostate, and bowel cancers. the role of endothelium in mediating the prothrombotic or hypercoagulable state is well - known, and disturbances in vascular function may be assessed by changes in plasma levels of certain molecules such as von willebrand factor (vwf), soluble thrombomodulin, and soluble e - selectin. increased vwf has long been described in cancer [93, 94 ], and this may not only indicate endothelial damage but also seem likely to contribute to thrombosis by promoting platelet - platelet and platelet - subendothelium adhesion, as may increased fibrinogen [9294 ]. similarly, increased soluble (i.e., plasma) thrombomodulin (as in present in cancer) may account for the loss of anticoagulant membrane thrombomodulin at the endothelial surface. thus changes in vwf and thrombomodulin are likely to promote coagulopathy and thus provide a paradigm for thrombosis in cancer. increased soluble e selectin in cancer may simply be a marker of endothelial disturbance with no direct implications for hameostasis endothelial cells may become prothrombotic under the influence of inflammatory cytokines such as tumour necrosis factor (tnf) and interleukin (il)-1. such cytokines suppress endothelial fibrinolytic activity, increase endothelial cell production of il-1 and vwf, and downregulate thrombomodulin expression that diminishes the activation of the anticoagulant protein c. cytokines such as tnf and il-1, often increased in cancer, also increase the endothelial expression of e selectin, platelet activating factors, and tissue factor [98, 100 ]. thus hypoxia and/or cytokine - mediated endothelial cell damage or dysfunction has the potential to further contribute to hypercoagulable state. a damaged endothelium may also present less of a challenge to a metastatic tumour cell seeking to penetrate the vessel wall. solid tumours growing outside of the blood vasculature may also increase the permeability of the microvasculature, allowing fibrinogen and other plasma - clotting proteins to leak into the extravascular space where procoagulants associated with tumour cells or with benign stromal cells can initiate clotting and subsequent fibrin deposition. one of the oldest provides a rationale for a role for platelet derived growth factor (pdgf), and platelet derived endothelial growth factor, possibly shed from para - neoplastic thrombus, in neovascularisation [101, 102 ]. others have localised tissue factor expression in the vascular endothelium of breast cancer tissue, which strongly correlate with the initiation of angiogenesis, hence suggesting a further possible link between the prothrombotic states and angiogenesis in these patients [103, 104 ]. tissue factor expression has also been shown to correlate positively with microvessel density and the expression of the angiogenic modulator, vascular endothelial growth factor (vegf). interestingly, vegf induces hyperpermeability by a direct action on the endothelium and (unlike basic fibroblast growth factor) promotes platelet activation and adhesion although both in vitro. an additional factor is the possibility that platelet - derived vegf and other angiogenic molecules may be important in malignancy [108110 ]. there seems to little firm in vivo data directly implicating this third aspect of virchow 's triad in the pathogenesis of human cancer, although imaging studies of blood flow may be a useful investigation. what would seem to be a likely mechanism in cardiovascular disease may not be the case in neoplasia, although there are in vitro data. nevertheless, blood viscosity at both high and low rates of shear, and a yield stress index measured preoperatively in cancer patients, has been correlated with the incidence of post - operative dvt. the possibility arises that abnormal blood vessel formation (perhaps related to cancer angiogenesis and factors promoting this) may cause flow disturbance. pathophysiology, as exemplified by virchow 's triad, many interventions to treat cancer are prothrombotic. there is ample evidence that numerous chemotherapeutic agents, including methotrexate, cisplatin and etoposide, as well as hormonal therapies used in cancer, such as medroxyprogesterone acetate and thalidomide, have all been implicated as risk factors for thromboembolism [116121 ]. other interventions such as central venous catheter placements, surgery, sepsis, and venous stasis from immobility also contribute to risk of thromboembolism, although some of this extra risk may be due to local inflammation and/or infection. furthermore, the general principle that chemotherapy induces thrombosis extends to nonmalignant diseases such as lupus. an exact position for a role of radiotherapy in promoting thrombosis is marred in many studies by combination chemotherapy. nevertheless, there are instances where radiotherapy does indeed increase the risk of thrombosis but may also damage the endothelium, and collectively, these issues prompt the need for pro - active anticoagulant therapy in high risk groups [121, 125128 ]. levels of plasma markers of thrombin and plasmin generation have been related to staging, prognosis and intervention in patients with various cancers, including those of the cervix, lung, ovary, prostate, and breast. for example, in the study by gadducci., levels of prothrombotic indices were significantly raised in patients with cervical cancer, and related to surgical - pathological stage and tumour size, but not to histologic type. similarly, several studies have shown an association between activation of blood coagulation and fibrinolysis with distant metastasis, histologic type of tumour and response to chemotherapy ; indeed, gross abnormalities of prothrombotic indices might even be a sign of unfavourable prognosis in certain patients [129131 ]. others failed to note any changes in preoperative or sequential measurements up to 9 months postoperatively of fibrinopeptide a, fibrin fragment b beta 1542, fibrinogen and serum fibrin degradation products that correlated with early recurrent breast cancer, although some markers were higher in patients with oestrogen receptor positive tumours, or increased postoperatively, largely because of an increase in patients with oestrogen receptor negative tumours. as discussed, there is considerably more data on venous thrombosis than for arterial thrombosis in cancer. nevertheless, thrombosis in arteries has long been recognised, although the exact mechanisms, in many cases, remain obscure [133135 ]. however, increased levels of coagulation molecules, concurrent disease (such as endocarditis), use of growth factors, and cytotoxic chemotherapy may all precipitate thrombosis [136140 ]. a potential mechanism for the latter may be endothelial damage with the loss of its natural anticoagulant nature and acquisition of a procoagulant profile. however, the fact that a large proportion of these studies are case reports underlines the rarity of arterial thrombosis in cancer [138141 ]. ross. provided additional possible pathogenic mechanisms related to atherosclerosis whilst lowe summarised common risk factors for arterial and venous thrombosis [142, 143 ]. a detailed discussion of the prevention and treatment of thromboembolism in cancer is beyond the scope of this paper, although at least one commentator refers to treatment of arterial cancer with anticoagulants. nevertheless, primary prevention of vte, possibly by vitamin k antagonists (vkas) should be considered for high - risk cancer patients during and immediately after chemotherapy, when long - term indwelling central venous catheters are placed, during prolonged immobilization from any cause, and following surgical interventions [118, 127 ]. for example, in cancer patients going for general surgery without prophylaxis, the incidence of deep vein thrombosis is approximately 29% as compared to 19% of patients without cancer. thus a possible route for the prevention of vte in high - risk cancer patients undergoing surgery or adjuvant chemotherapy may be the use of long - term low - intensity anticoagulation. however, there are concerns of the safety of vka anticoagulants in cancer as these may cause an increase in all bleeding, major bleeding, and minor bleeding compared to patients free of cancer. a meta - analysis found no evidence that warrants treatment with vkas with the aim of improving survival. these and other data suggest that, for many, low molecular weight heparins (lmwhs) will be the therapy of choice, not merely because of a reduced rate of complications such as haemorrhage, but also because it improves overall survival. lee. reported a rate of recurrent vte of 17% in patients on vka anticoagulants compared to 9% in those on an lmwh, and later noted that the use of a lmwh relative to coumarin derivatives was associated with improved survival in patients with solid tumours who did not have metastatic disease at the time of an acute vte. indeed, a recent international guideline on the use of anticoagulants in cancer for prophylaxis and treatment is dominated by lmwhs (table 2). although use of lmwh compared to unfractionated heparin confers a survival advantage, the precise role of lmwhs in survival in those free of vte is unclear but demands careful prospective analysis. but whatever treatment mode is adopted, its use is likely to be long - term in high risk groups [148, 149, 160 ]. it has been long been recognised that cancer confers a prothrombotic or hypercoagulable state, most likely through an altered balance between the coagulation and fibrinolytic systems. more recently it is clear that this risk can be related to long - term prognosis and treatment [162164 ], and hospitalisation for a vte is a risk factor for a second cancer. whilst most thromboses in cancer are venous, arterial thrombosis is common, possible because the two share many risk factors. indeed, patients with an unprovoked vte are at increased risk of an arterial thrombosis. procoagulants such as tissue factor are expressed by many tumours [73, 75, 104 ], and although platelet turnover and activity are also increased, it is unclear whether or not platelets themselves and/or their products actively promote thrombosis [167, 168 ], although a high platelet count is a risk factor for chemotherapy - associated thrombosis. risk factors for vte whilst on chemotherapy include presence of metastases, high leukocyte count and platin - based chemotherapy. there is also a growing view that some cancers are more prothrombotic than others (table 3). although a flawed analysis, a crude summation of this table suggests that pancreatic cancer and ovarian cancer are most likely to provoke a vte. although guidelines and a consensus statement for anticoagulant treatment are available [148150, 171 ], further work is needed to elucidate the mechanism (s) leading to the prothrombotic state in cancer, the potential prognostic and treatment implications, and the possible value of quantifying indices of hypercoagulability in routine clinical practice. carefully designed studies with the appropriate methodology to establish the predictive value of various abnormalities of the prothrombotic or hypercoagulable state are needed. in this respect the development of a risk factor score, which includes leukocyte count, platelet count, and levels of tissue factor, soluble p - selectin and d - dimer, points a possible way forward in identifying those at greatest risk of thrombosis, possibly due to chemotherapy, and who therefore warrant treatment [172174 ]. in support of this hypothesis is data showed the value of adding soluble p - selectin and d - dimer to a risk calculator. | the most frequent ultimate cause of death is myocardial arrest. in many cases this is due to myocardial hypoxia, generally arising from failure of the coronary macro- and microcirculation to deliver enough oxygenated red cells to the cardiomyocytes. the principle reason for this is occlusive thrombosis, either by isolated circulating thrombi, or by rupture of upstream plaque. however, an additionally serious pathology causing potentially fatal stress to the heart is extra - cardiac disease, such as pulmonary hypertension. a primary cause of the latter is pulmonary embolus, considered to be a venous thromboembolism. whilst the thrombotic scenario has for decades been the dominating paradigm in cardiovascular disease, these issues have, until recently, been infrequently considered in cancer. however, there is now a developing view that cancer is also a thrombotic disease, and notably a disease predominantly of the venous circulation, manifesting as deep vein thrombosis and pulmonary embolism. indeed, for many, a venous thromboembolism is one of the first symptoms of a developing cancer. furthermore, many of the standard chemotherapies in cancer are prothrombotic. accordingly, thromboprophylaxis in cancer with heparins or oral anticoagulation (such as warfarin), especially in high risk groups (such as those who are immobile and on high dose chemotherapy), may be an important therapy. the objective of this communication is to summarise current views on the epidemiology and pathophysiology of arterial and venous thrombosis in cancer. |
liver transplantation is the only treatment option for patients with end stage liver disease or advanced hepatocellular carcinoma. for the last 25 years, the number of waiting list candidates has gradually increased and exceeded the number of available grafts. the number of heart beating donors has decreased over the last decade. at the same time, numbers of marginal grafts, such as donation after cardiac death (dcd), as well as old and fatty livers have increased. marginal grafts are often declined for liver transplantation because of the higher chance of primary graft non- or delayed function. in dcd grafts, development of ischemic type biliary strictures (itbs) is of special concern. with the conventional static cold preservation technique, itbs occur in about 10 - 40% of dcd grafts. in the majority of patients, itbs leads to re - transplantation or patient death. especially prolonged warm and cold ischemic times are risk factors for itbs. donor age, genetic predispositions (such as ccr5 delta 32), and the choice of preservation solution have also been discussed as additional risk factors. partial microthrombosis of the peribiliary vessels has been suggested as potential mechanism for itbs after liver transplantation with dcd grafts. prior to the clinical introduction of liver transplantation, ex vivo liver perfusions have been used to study hepatic metabolism and physiology. after liver transplantation found its way into the clinical setting in the 1960s, innumerable attempts have been made to use ex vivo liver perfusion as a preservation method by mimicking physiological nutrition and oxygenation conditions. its utility for preservation of marginal grafts has been investigated in the last decade, but it did not reach standard clinical care. we recently described a reduction in bile duct injury in dcd liver transplantation by ex vivo perfused preservation. the selection ranges from cellular solutions like whole blood from the donor animal or packed red cells in combination with human plasma, to acellular approaches like machine university of wisconsin solution, igl solution, or steen solution. all different techniques, solutions, and temperature settings aim at 1) stable perfusion conditions, 2) sufficient oxygenation, and 3) re - establishment of organ function. an enhanced preservation capacity as well as the ability of organ assessment and treatment during normothermic and subnormothermic perfusion faces higher technical complexity and costs compared to hypothermic perfusion. we have developed a subnormothermic ex vivo liver perfusion system over the last 4 years. the system can be used to 1) recharge the hepatic energy content, 2) to assess the graft s quality, and to 3) repair marginal livers prior to transplantation. the porcine study design of liver injury is based on a donation after cardiac death (dcd) model. after dissection of all liver vessels, cardiac death is induced followed by 45 min of warm graft ischemia. to simulate a graft transport in between the donor and recipient hospitals in a clinical setting, the graft is stored on ice for 4 hr after cold, dual flush. after cold storage, the organ is subnormothermic perfused for 6 hr in order to assess the perfusion stability. in a transplant model, the perfusion time could be shorter in order to recharge energy storage and to assess the organ viability. note : male yorkshire pigs, 30 - 35 kg, were utilized for this study. all animals received humane care in compliance with the principles of laboratory animal care formulated by the national society for medical research and the guide for the care of laboratory animals published by the national institutes of health. house male yorkshire pigs in research facilities for 1 week before perfusion / transplantation to reduce the level of stress and to accustom the animals to the housing conditions. less than 2 days of housing inside the facility will lead to a stress - induced physical reaction, which can alter the perfusion s outcome. injection of a mixture of ketamine (25 mg / kg), atropine (0.04 mg / kg), and midazolam (0.15 mg / kg). prior to intubation, ensure the pig breathes spontaneously 2 l of o2 dosed with 5% isoflurane. spray the vocal chords with 2% lidocaine 2 min before intubation to avoid vocal cord spasms. lower the isoflurane gas to 2%. set the ventilator to 14 - 16 breaths / min and a tidal volume of 10 ml / kg bodyweight. catheter in one of the ear veins to allow infusion of ringer s lactate solution (200 ml per hr). then scrub the pig and cover it with sterile drapes. use a towel to cover large and small bowels and move them to the left side. separate inferior vena cava (ivc) and distal aorta from each other ; ligate aorta branches to the back ; isolate and free renal arteries from adherent tissue. divide the falciforme ligament and the triangular ligament using cautery. release the portal vein by an incision of the peritoneum between pancreas and portal vein. tie off veins draining from the pancreas to the portal vein. dissect the coeliac trunk below the portal vein and follow it backwards to the aorta. surround the mesenteric artery with a 2 - 0 tie ; surround the splenic and left gastric arteries, which branch posteriorly to the coeliac trunk. dissect the aorta behind the diaphragm between heart and coeliac trunk ; place a 2 - 0 tie around the aorta. release the liver from the lower cava on the right side using electro cautery ; use scissors for the upper part between cava and liver. remove the gallbladder and cauterize bleeders from gallbladder bed. induce cardiac arrest by intracardial injection of 40 mval kcl 3 min after heparin administration. for the perfusion, collect 1.6 l of pig blood in cpda bags (citrate, phosphate, dextrose, adenosine) immediately after cardiac death. perform a soft spin (2,000 x g without brake). remove the plasma and the buffy coat under sterile condition (biosafety cabinet class ii) and store the erythrocytes in cpda bags for transfusion. tie off the previously set ties around femoral, renal, splenic, mesenteric, and left gastric arteries as well as the upper aorta. for a heart beating donor (hbd) model, perform the cannulation of aorta and portal vein under heart beating conditions. after 45 min warm ischemia, flush the liver with university of wisconsin (uw) solution using dual perfusion via aorta (pressure bag) and portal vein (gravity driven). cut the liver out of the pig, leaving all remaining vessels long. during back - table preparation, clamp the upper ivc using a satinsky clamp and flush the liver a second time with about 0.5 l of uw solution retrogradely via lower ivc until the portal vein outflow is clear. perform an arterial back - table pressure perfusion with about 0.5 l of uw solution. cannulate the upper and lower parts of the ivc using 1/2 x 3/8 reducers with luer lock ; cannulate the portal vein and liver artery using 3/8 x 1/4 and 1/4 x 3/8 reducers with luer lock. place the liver in an organ bag, close the organ bag, and store the liver on ice until the perfusion has started. prepare the perfusion solution containing 2,000 ml steen solution, 400 ml washed erythrocytes, 550 mg sodium pyruvate, 100 ml amino acid solution (10% travasol), 10 mg calcium gluconate, 1,000 ie rapid acting insulin, 1 g cefazolin, 500 mg metronidazol, and 10,000 iu heparin. add other molecules for vasodilatation, immunosuppression, scavenging of reactive oxygen species, or liver cell treatment based on the particular study protocol. for the dialysis component, use standard dialysate containing 3.5 mm potassium, 25 mm bicarbonate, 27 mm glucose, as well as 275 mg / l pyruvate. set up the perfusion circuit (schema see figure 2). coming from the main reservoir as the starting and end point, the perfusion solution is driven by a centrifugal pump through an oxygenator. right after oxygenation of the solution, the circuit splits into a smaller line running to a dialyzer unit for electrolyte homeostasis and a bigger line running to a leukocyte filter for reduction of remnant white blood cells (wbc). the solution that runs through the dialyzer returns to the main reservoir. after the leukocyte filter, the circuit splits up again into 2 equal lines. one line runs at high pressure (around 60 mmhg) directly into the aorta to perfuse the hepatic artery. the pressure of the portal perfusion depends on the elevation energy of the reservoir s solution level (around 2 - 6 mmhg). all fluids are drained via the infra- and supra - hepatic cava back into the main reservoir. for gravity reduction and homogeneous perfusion, the liver is placed in a pool filled with temperature regulated water. it is separated from the water by an impermeable membrane and it swims in a perfusion suspension. collect all fluids from the circuit in a 3 l reservoir (main reservoir) and clamp the outflow.connect the outflow to a centrifugal pump followed by a commercial oxygenator.behind the oxygenator, split up the tubing into 2 lines. connect the second line to a leukocyte reduction filter.split up the line after the leukocyte reduction filter into an arterial line, which supplies perfusion solution to the hepatic artery, and a portal venous line delivering perfusion solution to a second reservoir, which drains into the portal vein by gravity outflow. clamp the portal inflow.connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection.for collection of ascites or leakage of perfusion solution from the liver, prepare a suction line connected to the main reservoir. collect all fluids from the circuit in a 3 l reservoir (main reservoir) and clamp the outflow. connect the outflow to a centrifugal pump followed by a commercial oxygenator. behind the oxygenator, split up the tubing into 2 lines. split up the line after the leukocyte reduction filter into an arterial line, which supplies perfusion solution to the hepatic artery, and a portal venous line delivering perfusion solution to a second reservoir, which drains into the portal vein by gravity outflow. connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection. for collection of ascites or leakage of perfusion solution from the liver, prepare a suction line connected to the main reservoir. release the outflow clamp from the main reservoir and fill the circuit with the perfusion solution. the perfusion solution will drive through the arterial line into the vena cava line back into the main reservoir. place the liver, with its convex side down to facilitate the access to the vessels, ideally in a gravity - free environment to avoid organ compression at the contact surface. cover the water bath with an impermeable membrane and place the liver onto that membrane. reduce gravity driven compression by submerging the liver with perfusion solution. reduce the speed of the centrifugal pump to 1,000 rounds / min and place two clamps at the connection of arterial and vena cava lines. release the clamp from the arterial line, pour perfusion solution into the arterial cannula to get rid of bubbles, and connect the line to the cannula. connect pressure lines to the luer locks of the arterial, portal, and vena cava cannulas. to mimic physiological conditions, apply treatments into the right vessel. inject glucose into the portal vein and not into the arterial line in order to establish a gradient mimicking an increased portal venous glucose gradient and inducing glycogen synthesis. after connecting the liver to the circuit, raise the temperature to 33 c within 60 min. aim for an arterial starting flow at about 250 ml / min at 40 mmhg. this may reach 700 ml / min during perfusion once the pressure is increased up to 70 mmhg. at starting temperature, aim for a portal vein flow of 500 - 600 ml / min at 3 - 5 mmhg. after raising the temperature, monitor the portal venous flow, which will increase up to 1,100 ml / min at 4 - 6 mmhg. avoid exceeding portal pressure above physiological values (around 8 mmhg) to protect sinusoidal fenestrations. avoid exceeding total flow above 2,000 ml / min in order to prevent damaging the organ. set the outflow to -2 mmhg by lowering the main reservoir to prevent liver congestion by functional outflow obstruction. add the dialysis component to the circuit in order to equilibrate the perfusion solution to predetermined values. set the dialysate flow to 500 ml / hr. take special attention to adjust the dialysis outflow so that the perfusion solution is neither diluted nor concentrated. within the first hour of perfusion ensure homogeneous oxygenation of the tissue to recover and maintain organ function by using a main gas mixture component of o2 (95 - 98%) and co2 (2 - 5%). use variable gas during perfusion since the liver changes its metabolism and its ph demand during perfusion. maintain a low ph during the start of perfusion to protect the organ using the paradox ph concept and avoid severe tissue damage that can result from fast connections to physiological ph under reoxygenation, since after storage in uw solution, the organ has an acidotic ph below 7. adjust the partial pressure of co2 continuously down to 25 - 30 mmhg so that the ph will reach a physiologic level within 1 hr. add sodium- or potassium bicarbonate to the circuit to achieve a physiological concentration of standard bicarbonate in the perfusion solution. monitor vascular flow and pressure and note a stable perfusion by a constant vascular resistance. keep the perfusion system stable for up to 8 hr. at the end of the ex vivo perfusion period, cool down the perfusion system to 20 c and, after disconnecting the circuit s tubing from the liver, flush the perfusion solution out the liver dually with ice cold uw solution. store the liver once again placed on ice in a sterile organ bag note : male yorkshire pigs, 30 - 35 kg, were utilized for this study. all animals received humane care in compliance with the principles of laboratory animal care formulated by the national society for medical research and the guide for the care of laboratory animals published by the national institutes of health. house male yorkshire pigs in research facilities for 1 week before perfusion / transplantation to reduce the level of stress and to accustom the animals to the housing conditions. less than 2 days of housing inside the facility will lead to a stress - induced physical reaction, which can alter the perfusion s outcome. injection of a mixture of ketamine (25 mg / kg), atropine (0.04 mg / kg), and midazolam (0.15 mg / kg). prior to intubation, ensure the pig breathes spontaneously 2 l of o2 dosed with 5% isoflurane. spray the vocal chords with 2% lidocaine 2 min before intubation to avoid vocal cord spasms. lower the isoflurane gas to 2%. set the ventilator to 14 - 16 breaths / min and a tidal volume of 10 ml / kg bodyweight. catheter in one of the ear veins to allow infusion of ringer s lactate solution (200 ml per hr). then scrub the pig and cover it with sterile drapes. use a towel to cover large and small bowels and move them to the left side. separate inferior vena cava (ivc) and distal aorta from each other ; ligate aorta branches to the back ; isolate and free renal arteries from adherent tissue. divide the falciforme ligament and the triangular ligament using cautery. release the portal vein by an incision of the peritoneum between pancreas and portal vein. tie off veins draining from the pancreas to the portal vein. dissect the coeliac trunk below the portal vein and follow it backwards to the aorta. surround the mesenteric artery with a 2 - 0 tie ; surround the splenic and left gastric arteries, which branch posteriorly to the coeliac trunk. dissect the aorta behind the diaphragm between heart and coeliac trunk ; place a 2 - 0 tie around the aorta. release the liver from the lower cava on the right side using electro cautery ; use scissors for the upper part between cava and liver. induce cardiac arrest by intracardial injection of 40 mval kcl 3 min after heparin administration. set cardiac arrest as the starting point of warm ischemia. for the perfusion, collect 1.6 l of pig blood in cpda bags (citrate, phosphate, dextrose, adenosine) immediately after cardiac death. remove the plasma and the buffy coat under sterile condition (biosafety cabinet class ii) and store the erythrocytes in cpda bags for transfusion. tie off the previously set ties around femoral, renal, splenic, mesenteric, and left gastric arteries as well as the upper aorta. for a heart beating donor (hbd) model, perform the cannulation of aorta and portal vein under heart beating conditions. after 45 min warm ischemia, flush the liver with university of wisconsin (uw) solution using dual perfusion via aorta (pressure bag) and portal vein (gravity driven). cut the liver out of the pig, leaving all remaining vessels long. during back - table preparation, clamp the upper ivc using a satinsky clamp and flush the liver a second time with about 0.5 l of uw solution retrogradely via lower ivc until the portal vein outflow is clear. perform an arterial back - table pressure perfusion with about 0.5 l of uw solution. cannulate the upper and lower parts of the ivc using 1/2 x 3/8 reducers with luer lock ; cannulate the portal vein and liver artery using 3/8 x 1/4 and 1/4 x 3/8 reducers with luer lock. place the liver in an organ bag, close the organ bag, and store the liver on ice until the perfusion has started. prepare the perfusion solution containing 2,000 ml steen solution, 400 ml washed erythrocytes, 550 mg sodium pyruvate, 100 ml amino acid solution (10% travasol), 10 mg calcium gluconate, 1,000 ie rapid acting insulin, 1 g cefazolin, 500 mg metronidazol, and 10,000 iu heparin. add other molecules for vasodilatation, immunosuppression, scavenging of reactive oxygen species, or liver cell treatment based on the particular study protocol. for the dialysis component, use standard dialysate containing 3.5 mm potassium, 25 mm bicarbonate, 27 mm glucose, as well as 275 mg / l pyruvate. set up the perfusion circuit (schema see figure 2). coming from the main reservoir as the starting and end point, the perfusion solution is driven by a centrifugal pump through an oxygenator. right after oxygenation of the solution, the circuit splits into a smaller line running to a dialyzer unit for electrolyte homeostasis and a bigger line running to a leukocyte filter for reduction of remnant white blood cells (wbc). the solution that runs through the dialyzer returns to the main reservoir. after the leukocyte filter, one line runs at high pressure (around 60 mmhg) directly into the aorta to perfuse the hepatic artery. the pressure of the portal perfusion depends on the elevation energy of the reservoir s solution level (around 2 - 6 mmhg). all fluids are drained via the infra- and supra - hepatic cava back into the main reservoir. for gravity reduction and homogeneous perfusion, it is separated from the water by an impermeable membrane and it swims in a perfusion suspension. collect all fluids from the circuit in a 3 l reservoir (main reservoir) and clamp the outflow.connect the outflow to a centrifugal pump followed by a commercial oxygenator.behind the oxygenator, split up the tubing into 2 lines. connect the second line to a leukocyte reduction filter.split up the line after the leukocyte reduction filter into an arterial line, which supplies perfusion solution to the hepatic artery, and a portal venous line delivering perfusion solution to a second reservoir, which drains into the portal vein by gravity outflow. clamp the portal inflow.connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection.for collection of ascites or leakage of perfusion solution from the liver, prepare a suction line connected to the main reservoir. collect all fluids from the circuit in a 3 l reservoir (main reservoir) and clamp the outflow. connect the outflow to a centrifugal pump followed by a commercial oxygenator. behind the oxygenator, split up the tubing into 2 lines. split up the line after the leukocyte reduction filter into an arterial line, which supplies perfusion solution to the hepatic artery, and a portal venous line delivering perfusion solution to a second reservoir, which drains into the portal vein by gravity outflow. connect the arterial line to a vena cava line draining into the main reservoir for fluid recollection. for collection of ascites or leakage of perfusion solution from the liver, prepare a suction line connected to the main reservoir. release the outflow clamp from the main reservoir and fill the circuit with the perfusion solution. the perfusion solution will drive through the arterial line into the vena cava line back into the main reservoir. place the liver, with its convex side down to facilitate the access to the vessels, ideally in a gravity - free environment to avoid organ compression at the contact surface. use a heat- and coolable water bath. set the starting temperature of the circuit and water bath to 20 c. cover the water bath with an impermeable membrane and place the liver onto that membrane. reduce gravity driven compression by submerging the liver with perfusion solution. reduce the speed of the centrifugal pump to 1,000 rounds / min and place two clamps at the connection of arterial and vena cava lines. then, cut the tubing in between the clamps. using a 3-way connector, release the clamp from the arterial line, pour perfusion solution into the arterial cannula to get rid of bubbles, and connect the line to the cannula. connect pressure lines to the luer locks of the arterial, portal, and vena cava cannulas. to mimic physiological conditions, apply treatments into the right vessel. inject glucose into the portal vein and not into the arterial line in order to establish a gradient mimicking an increased portal venous glucose gradient and inducing glycogen synthesis. after connecting the liver to the circuit, raise the temperature to 33 c within 60 min. aim for an arterial starting flow at about 250 ml / min at 40 mmhg. this may reach 700 ml / min during perfusion once the pressure is increased up to 70 mmhg. at starting temperature, aim for a portal vein flow of 500 - 600 ml / min at 3 - 5 mmhg. after raising the temperature, monitor the portal venous flow, which will increase up to 1,100 ml / min at 4 - 6 mmhg. avoid exceeding portal pressure above physiological values (around 8 mmhg) to protect sinusoidal fenestrations. avoid exceeding total flow above 2,000 ml / min in order to prevent damaging the organ. set the outflow to -2 mmhg by lowering the main reservoir to prevent liver congestion by functional outflow obstruction. add the dialysis component to the circuit in order to equilibrate the perfusion solution to predetermined values. set the dialysate flow to 500 ml / hr. take special attention to adjust the dialysis outflow so that the perfusion solution is neither diluted nor concentrated. ensure homogeneous oxygenation of the tissue to recover and maintain organ function by using a main gas mixture component of o2 (95 - 98%) and co2 (2 - 5%). use variable gas during perfusion since the liver changes its metabolism and its ph demand during perfusion. maintain a low ph during the start of perfusion to protect the organ using the paradox ph concept and avoid severe tissue damage that can result from fast connections to physiological ph under reoxygenation, since after storage in uw solution, the organ has an acidotic ph below 7. adjust the partial pressure of co2 continuously down to 25 - 30 mmhg so that the ph will reach a physiologic level within 1 hr. add sodium- or potassium bicarbonate to the circuit to achieve a physiological concentration of standard bicarbonate in the perfusion solution. monitor vascular flow and pressure and note a stable perfusion by a constant vascular resistance. keep the perfusion system stable for up to 8 hr. at the end of the ex vivo perfusion period, cool down the perfusion system to 20 c and, after disconnecting the circuit s tubing from the liver, flush the perfusion solution out the liver dually with ice cold uw solution. store the liver once again placed on ice in a sterile organ bag below, we present the results of 5 perfusion experiments with dcd - grafts after 45 min warm- and 4 hr cold ischemia prior to the start of the subnormothermic ex vivo perfusion. the main goal for an ex vivo liver perfusion is to ensure a sufficient oxygen supply to the organ. achieving constant vascular flows with stable pressures is a good indicator of adequate oxygenation. during an induction period of 1 - 2 hr the perfusion solution and the organ are warmed up to 33 c, which deceases the vascular resistance of the liver. once the target temperature of 33 c is achieved, flow values level at a constant, nearly physiological range for the rest of the 6 hr perfusion time (figures 3a-3d). at the same time, the organ becomes metabolically active. figure 4a shows the venous po2, a marker of oxygen consumption. within the initial 2 hr the venous po2 declines to a constant plateau. at this metabolically active state, h&e staining after 6 hr of perfusion reveals hepatocyte necrosis < 5 % with an intact lobular and sinusoidal structure (figure 6). pas staining at the same time point shows replenished cellular glycogen storage compared to exhausted storage in cold preserved dcd - grafts (figure 7). perfusion flows and pressure (n = 5, error bars show standard deviation). (a, b) hepatic artery (ha) flow and pressure : during the warming phase in the first 1 - 2 hr, the flow increases at stable pressures and is constant afterwards. looking at the decreasing portal venous pressure (c), the increase of ha flow towards the end of the perfusion might be an autoregulatory reaction of the liver. (c, d) the portal venous (pv) flow increases corresponding to the ha flow during the first 2 hr of warming. figure 4. monitoring parameters (n = 5, error bars show standard deviation). (a) the venous po2 as a marker of oxygen demand and metabolic activity decreases within the initial phase of warming due to activated cellular metabolism ; it remains stable afterwards. (b) bile production as a marker of metabolic activity starts at temperatures around 30 c and, thus, between the first and second hour of perfusion. (c, d) the dialyzer assures electrolyte homeostasis ; an initial hyperkalemia is quickly balanced. ast is a sensitive marker of hepatocellular injury ; the shallow increase suggests no significant injury during ex vivo perfusion. (a) sham liver sample before warm ischemia, one representative liver lobule with intact architecture. (b) liver sample after 45 min of warm ischemia, 4 hr of cold ischemia, and 6 hr of subnormothermic perfusion, the lobular architecture is intact without necrosis and only minimal cell swelling, the sinusoidal spaces are mildly dilated in comparison to the sham sample. in a pig model that mimics dcd liver transplantation, we demonstrated that subnormothermic liver perfusion with a cellular perfusion solution results in stable perfusion parameters, minimal hepatocyte injury, and active hepatic metabolism. ex vivo liver perfusion as preservation technique offers for the first time the opportunity to assess markers of graft function and injury during organ preservation and prior to transplantation. beside the macroscopic evaluation of the graft perfusion homogeneity, flow values provide a good indicator of the graft s viability and the extent of the ischemic injury it had suffered earlier. levels of hepatic enzymes like ast can be used to assess the degree and dynamics of hepatocellular injury. this thorough graft assessment may allow a reliable discrimination between transplantable and non - transplantable marginal organs. we chose a subnormothermic temperature of 33 c in our perfusion system because the temperature is sufficient to allow metabolism as well as atp and glycogen synthesis. at the same time, it provides a decreased oxygen demand in comparison to normothermic perfusion settings which provides additional safety against ischemic injury. in general, perfusion temperatures above 30 c have shown to minimize cold ischemic injury and provide sufficient metabolic activity. contrary to other groups, we did not use whole blood as perfusate, but a normo - osmotic albumin solution (steen) with washed and filtered red blood cells. by excluding the plasma components as well as thrombocytes and leukocytes, the perfusion solution is designed to minimize pro - inflammatory signaling during the ex vivo perfusion. in addition to the graft assessment, stable perfusion conditions over several hours allow graft treatment. however, almost no treatment regime has made its way into clinical practice, yet. one reason seems to be the lack of opportunity to apply those treatments during cold storage. a metabolically active liver on an ex vivo perfusion system is optimal for applying any kind of treatment. in this regard, not only treatments to ameliorate reperfusion conditions like attenuation of kupffer cell activity or scavenging of reactive oxygen species are conceivable but also treatments like gene therapy to condition the graft, e.g., against hepatitis c recurrence. other potential strategies could include reduction on steatosis during the ex vivo perfusion period. in summary, ex vivo liver perfusion is a novel strategy to minimize cold ischemic injury and to assess marginal liver grafts prior to liver transplantation. the ex vivo perfusion setting provides unique opportunity to repair and condition grafts prior to transplantation. | the success of liver transplantation has resulted in a dramatic organ shortage. in most transplant regions 20 - 30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. one strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (dcd). the current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. in addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.these shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. the optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.in spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.this paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. we will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs. |
accumulating epidemiologic evidence suggests that the effect of obesity on chronic disease risk differs by ethnicity. asians experience a higher risk for hypertension, diabetes, and cardiovascular disease at lower levels of body mass index (bmi) compared to caucasians [13 ]. for example, japanese americans experience a 2-fold higher diabetes risk than caucasians, with higher risk at all bmi levels. ethnic differences in body composition and adipose tissue distribution may explain the significant interaction of ethnicity and bmi on chronic disease risk. particularly, a higher proportion of abdominal visceral relative to subcutaneous adiposity among asians than caucasians [4, 5 ] may be responsible for insulin resistance and other adverse metabolic health effects [6, 7 ]. biomarkers linking adiposity, inflammation, and disease, such as c - reactive protein (crp) and interleukin-6 (il-6), have been investigated ; however, no biomarker has been identified that is associated with the ethnic heterogeneity in chronic disease risk. adipocyte - derived hormones, leptin and adiponectin, are likely candidates for elucidating the biological mechanisms underlying ethnic differences in disease risk. leptin and adiponectin respond to increasing adiposity in a reciprocal manner, and the plasma leptin to adiponectin ratio may be a potential measure of insulin resistance. it is unclear whether leptin and adiponectin are specific biomarkers for adipose tissue distribution ; however, it appears that adiponectin concentrations are predominantly determined by visceral and leptin by subcutaneous adipose tissue. released by adipose tissue after insulin stimulation, leptin may serve as a possible link between nutritional status and immune function. adiponectin possesses strong anti - inflammatory, antiatherogenic, and insulin - sensitizing properties [14, 15 ]. low adiponectin is an independent predictor of incident type 2 diabetes and cardiovascular disease. furthermore, lower adiponectin levels have been observed in chinese, japanese, korean, and south asians compared to caucasians [1821 ]. the objective of this study was to determine whether there are ethnic differences in serum leptin and adiponectin levels relative to bmi and their impact on inflammatory markers as predictive factors for future chronic disease among premenopausal women of asian american, caucasian, and native hawaiian background. the university of hawaii committee on human studies and the kaiser permanente institutional review board approved the study protocol. all participants were recruited through mailed invitations from mammographic screening clinics on the island of oahu, hawaii. all subjects signed an informed consent form and gave written permission to use frozen samples for future analyses. in the nutritional intervention trial, 220 premenopausal women aged 3547 years were randomized to a soy diet or to the control group as described previously. women were excluded for use of oral contraceptives or other sex hormones, diagnosis of cancer, hysterectomy, no intact ovary or no regular menstrual periods, or high soy intake. data obtained from baseline serum samples were used in the current investigation ; bmi and biomarkers were available for 183 women. ethnic background was self - reported and when possible women of mixed ancestry were assigned to one ethnic category with native hawaiian having the highest priority. serum levels of leptin, adiponectin, and il-6 were assessed by double - antibody enzyme - linked - immunosorbent - assay (elisa) (r&d systems, minneapolis, mn, usa) according to the manufacturer 's specifications. the crp assay was based on a latex particle - enhanced immunoturbidimetric method using a kit (pointe scientific, inc, lincoln park, mi, usa) and a cobas mira plus clinical autoanalyzer. the assay quality was assessed by 49 blinded controls from a pooled sample donated by 10 premenopausal volunteers. the mean intrabatch coefficient of variation (cv) for leptin, adiponectin, il-6, and crp were 4.6%, 14.0%, 6.8%, and 6.1%, whereas interbatch cvs were 9.5%, 24.9%, 17.8%, and 18.2%, respectively. the higher intra- and interbatch cvs with adiponectin were attributable to less precise measures because the studies samples contained a lower range of the standard curve for the assay. the primary goal of these analyses was to determine any ethnic differences in measured biomarkers (leptin, adiponectin, il-6, and crp) for women with similar bmi. secondary analyses included evaluating ethnic - specific models to examine the relation of leptin and adiponectin with crp. leptin, adiponectin, crp, and il-6 were log transformed to normalize their distributions. general linear models were applied to estimate mean levels for biomarkers by ethnicity or bmi category and differences in mean values were made using tukey 's test for multiple comparisons. our ethnic - specific analyses focused on differences between asians and caucasian women due to the small sample size for native hawaiian / other women. models were adjusted for bmi (continuous) and/or ethnicity (asian, caucasian, native hawaiian / other) as appropriate. statistical computing was conducted using sas version 9.2 (sas institute inc., cary, nc, usa). the participants were of diverse ethnicity : 67 caucasian, 23 native hawaiian, 49 japanese, 14 chinese, 11 filipino, and 19 mixed / other ethnicities. japanese, chinese, and filipino women were combined into one group (asians) due to small sample sizes. the mean age of the study population was 43.0 2.9 years and did not differ across ethnic groups (p =.26). close to half of the women (49%) were overweight or obese, with substantial ethnic differences in bmi and measures of adiponectin, leptin, il-6, and crp (table 1). bmi was lower among asians than in the two other ethnic groups (p 35 for example, the few caucasians in the lower - normal range of bmi and the few asians in the high bmi categories may explain the similar adiponectin levels in these extreme bmi categories. as with any cross - sectional design, also, we did not have measures of abdominal fat distribution (visceral versus subcutaneous) or total body fat and were unable to assess whether the addition of measures for visceral adiposity attenuated ethnic - specific associations. it is possible that leptin, crp, or il-6 levels may be confounded by ethnic differences in inflammatory or autoimmune conditions ; however, this is unlikely given that the women in this study were apparently healthy and relatively young. additional outcome measures of interest that may have further elucidated the significance of lower adiponectin in asian women with respect to metabolic markers include plasma glucose, insulin and lipid profiles, and visfatin, an adipocyte - derived protein predominantly found in visceral adipose tissue. our findings contribute to the limited data available on apparently healthy premenopausal women of normal bmi. additional strengths of our study include an ethnically diverse study population, well - controlled blood collection, and reliable assays. in conclusion, we propose that lower adiponectin levels in asian compared to caucasian women may be attributed to higher amounts of visceral adipose tissue at any given level of total adiposity. leptin is a stronger predictor of crp ; however, in overweight and obese women, asian women showed a stronger link between circulating crp with both leptin and adiponectin. as crp is a predictive factor for future chronic disease, adipokines may be important mediators of risk for asian women. nevertheless, studies are needed that measure visceral, subcutaneous, and total adipose tissue (e.g., magnetic resonance imaging or computed tomography dual energy x - ray absorptiometry study of adiposity distribution) among a weight - diverse population to clarify the association between adipokines and distribution of adiposity tissue. additionally, future studies that include outcome measures such as insulin resistance, diabetes, and metabolic syndrome as well as heart disease and rheumatological conditions are needed to further elucidate whether adiponectin is a key link to explain the ethnic differences in disease risk in asians and caucasians. | ethnic differences in adipose tissue distribution may contribute to different chronic disease risks across ethnic groups, and adipokines may mediate the risk. in a cross - sectional study, we examined ethnic differences in adipokines and inflammatory markers as related to body mass index (bmi) among 183 premenopausal women with caucasian and asian ancestry. general linear models were used to estimate adjusted mean levels of leptin, adiponectin, interleukin-6, and c - reactive protein (crp). asian women had significantly lower serum levels of leptin, adiponectin, and crp than caucasian participants (p.01) across all levels of bmi. among overweight and obese women, asians showed a stronger association of crp with leptin (= 1.34 versus = 0.64) and with adiponectin (= 0.95 versus = 0.75) than caucasians. compared to caucasians of similar bmi, asians may experience a higher chronic disease risk due to lower levels of adiponectin despite their lower levels of leptin. |
3h2o) and tq (2-isopropyl-5-methyl-1,4-benzoquinone) were purchased from sigma - aldrich chemical co. (st. louis, mo, usa). healthy adult (4-month - old) male wistar rats, weighing 200230 g, obtained from the tunisian society of pharmaceutical industries, were used in this study. the animals were housed in plastic cages (free from any source of chemical contamination) with free access to tap water (free from pb) and standard diet. the rats were kept at 223c, in a natural light / dark cycle, with 55% humidity and a ventilation system. the experiments were started after the animals were allowed to adapt to the laboratory conditions for a week. all experimental procedures in this study were in full compliance with the european council directive (86/609/eec) and approved by the institutional bioethics committee. after the acclimation period, the rats were randomly divided into four groups of eight animals each and were treated for 5 weeks as follows : the control group received tap water ; the pb group received an aqueous solution containing 2,000 ppm of pb acetate (0.2% w / v) (2022) ; the pb - tq group was cotreated with pb (as in the pb group) plus tq (5 mg / kg bw / day) ; and the tq group received tap water and was given tq (5 mg / kg bw / day) (2325). tq was administered by gastric tube daily between 8:00 and 9:00 am. at the end of the treatment period the kidneys were removed quickly from the rats, cleared of adhesive tissue, washed in ice - cold 0.9% (w / v) nacl solution, and frozen at 80c until assayed. the kidney tissue was homogenized in 10 volumes of ice - cold phosphate - buffered saline (136.75 mm nacl, 2.68 mm kcl, 10.14 mm na2hpo4, 1.76 mm kh2po4, ph 7.4) and the homogenates were centrifuged at 3,500g for 15 min at 4c. sod (26), gpx (27), and gr (28) activities were determined in the kidney homogenates using commercial kits (randox laboratories ltd., the gsh level was determined spectrophotometrically using the method previously described by ellman (30). 3h2o) and tq (2-isopropyl-5-methyl-1,4-benzoquinone) were purchased from sigma - aldrich chemical co. (st. louis, mo, usa). healthy adult (4-month - old) male wistar rats, weighing 200230 g, obtained from the tunisian society of pharmaceutical industries, were used in this study. the animals were housed in plastic cages (free from any source of chemical contamination) with free access to tap water (free from pb) and standard diet. the rats were kept at 223c, in a natural light / dark cycle, with 55% humidity and a ventilation system. the experiments were started after the animals were allowed to adapt to the laboratory conditions for a week. all experimental procedures in this study were in full compliance with the european council directive (86/609/eec) and approved by the institutional bioethics committee. after the acclimation period, the rats were randomly divided into four groups of eight animals each and were treated for 5 weeks as follows : the control group received tap water ; the pb group received an aqueous solution containing 2,000 ppm of pb acetate (0.2% w / v) (2022) ; the pb - tq group was cotreated with pb (as in the pb group) plus tq (5 mg / kg bw / day) ; and the tq group received tap water and was given tq (5 mg / kg bw / day) (2325). tq was administered by gastric tube daily between 8:00 and 9:00 am. at the end of the treatment period the kidneys were removed quickly from the rats, cleared of adhesive tissue, washed in ice - cold 0.9% (w / v) nacl solution, and frozen at 80c until assayed. the kidney tissue was homogenized in 10 volumes of ice - cold phosphate - buffered saline (136.75 mm nacl, 2.68 mm kcl, 10.14 mm na2hpo4, 1.76 mm kh2po4, ph 7.4) and the homogenates were centrifuged at 3,500g for 15 min at 4c. sod (26), gpx (27), and gr (28) activities were determined in the kidney homogenates using commercial kits (randox laboratories ltd., the gsh level was determined spectrophotometrically using the method previously described by ellman (30). 1a through d, the kidney activities of sod, gpx, cat, and gr in the rats receiving tq alone were not significantly different (p>0.05) from those of the control group ; following pb treatment, the activities of these enzymes were significantly decreased (p0.05) on kidney gsh levels compared to those of the control rats. by contrast, pb exposure caused a significant decrease (p0.05) from those of the control group ; following pb treatment, the activities of these enzymes were significantly decreased (p0.05) on kidney gsh levels compared to those of the control rats. by contrast, pb exposure caused a significant decrease (p<0.05) of about 61.8% in the concentration of this non - enzymatic antioxidant in relation to the control rats. this effect was significantly mitigated (p<0.05) by 55.16% when the pb - treated animals simultaneously received tq. effects of lead (pb), thymoquinone (tq), and their coadministration on the kidney level of reduced glutathione (gsh) in rats after five weeks. student 's t - test : p<0.05 versus control ; p<0.05 versus tq - treated rats ; p<0.05 versus pb - treated rats. pb is a pervasive environmental and industrial pollutant with no beneficial biological role, and its toxicity continues to be a major public health problem throughout the world. recent studies point to the potential involvement of the cell 's antioxidant capacity failure in the pathogenesis of pb poisoning, suggesting that exogenous antioxidants may play an effective protective effect. in the present study, we adopted an in vivo experimental animal model to investigate whether tq could maintain renal intracellular antioxidant reserves in pb subchronic treatment. sod catalyzes the dismutation of o2 to h2o2 and o2. because h2o2 is still harmful to cells, cat and gpx further catalyze the decomposition of h2o2 to water. in the reaction catalyzed by gpx, gsh is oxidized to gssg, which can then be reduced back to gsh by gr. in the present study, we found that treatment with pb for 5 weeks significantly decreased the activities of sod, gpx, cat, and gr in the rat kidney. these results are in concordance with previous findings (3, 31, 32). it has been shown that pb directly alters antioxidant activities by irreversible direct binding to functional sulfhydryl (sh) groups of several enzymes such as sod, gpx, cat, and gr (33). because pb interferes with the metabolism of essential trace elements such as copper, zinc, selenium, and iron needed for proper molecular structure and enzymatic activity (2) the decrease in antioxidant enzyme activities may be explained by the downregulation of antioxidant enzyme mrna expression (34). gsh is a tripeptide - containing cysteine that has a reactive sh group with reductive potency. accordingly, gsh plays a vital role in the protection of cells against oxidative stress. it can act as a non - enzymatic antioxidant by direct interaction of sh groups with reactive oxygen species, or it can be involved in the enzymatic detoxification reactions for reactive oxygen species, as a cofactor or a coenzyme. in agreement with recent investigations studying the effect of pb in the kidneys of rats and mice (35, 36), our data show that pb treatment significantly lowered the renal gsh level. as for antioxidant enzymes, the reduction in concentration of gsh may be due to the high affinity of pb to the sh groups of this tripeptide, thereby interfering with its antioxidant activity (33). pb can also decrease the level of gsh by inhibiting the activities of gsh metabolizing enzymes, such as gr, gst, and glucose-6-phosphate dehydrogenase, by blocking their sh groups (37). further, the reduction of gsh synthesis can be proposed as another explanation. despite extensive research now focusing on herbal products as alternative medicines, no evidence has been reported in the literature regarding the role of tq against pb - induced renal toxicity. in the present study, cotreatment of pb - exposed rats with tq significantly improved the altered antioxidant defense system in the kidneys. our results are in consonance with recent literature data indicating that oral supplementation of tq (10 mg / kg / day, 15 days) protects rat kidneys against sodium arsenite induced depletion of antioxidant enzyme activities (sod, gpx, and cat) (38). furthermore, farag. (39) reported that tq (10 mg / kg / day, 28 days) prevented reduction in kidney sod activity and gsh levels provoked by chronic treatment with cyclosporine a, an immunosuppressant drug, and by acute renal ischemia / reperfusion in rats. (40) reported that tq enhances the declined renal antioxidant status in gentamicin - treated rats. tq (10 mg / kg / day, 10 days, po) also reversed a renal decrease in cat activity and gsh concentration in rats receiving methotrexate, an anticancer drug (41). the restoration of tissue antioxidant function by tq clearly demonstrated in the current work could be attributed to its ability to upregulate antioxidant gene expression (42, 43). the ability of tq to correct the disrupted antioxidant system, as demonstrated in the present research, does not precisely mean that kidney oxidative stress can be decreased. numerous previous data showed that this component has powerful free radical scavenging activity (12, 14, 4446), which may be related to the redox properties of the quinone structure of tq molecule and to its unrestricted crossing of morphophysiological barriers to access subcellular compartments (46). thus, in the current work we are limited to some antioxidant markers. in conclusion, our results clearly indicate that tq oral supplementation, at a safe dose, protects against pb - induced cellular antioxidant defense system depletion in rat kidneys. am and hbc collected the data, and am also analyzed the data, designed the study, and wrote the paper. this work was supported by funds allocated to the genetic, genotoxicity and childhood illness research unit (ur12es10) by the tunisian ministry of higher education and scientific research. | objectivealteration of the antioxidant status in the kidneys may be related to lead (pb) intoxication. the present study aimed to investigate the possible beneficial effect of thymoquinone (tq), the major active ingredient of the volatile oil of nigella sativa seeds, on pb - induced renal antioxidant defense system impairment.methodsa total of thirty two healthy adult male wistar rats were randomly divided into four equal groups as follows : a control group, which received no treatment ; a pb group, which was exposed to 2,000 ppm of pb acetate in drinking water ; a pb - tq group, which was cotreated with pb plus tq (5 mg / kg / day, per os) ; and a tq group receiving only tq. all treatments were applied for five weeks.resultstq alone did not induce any significant changes in the antioxidant defense system. by contrast, pb exposure significantly decreased reduced glutathione level and superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase activities in the renal tissue. interestingly, supplementation with tq significantly improved the affected antioxidant parameters.conclusionour data are the first to provide evidence on the protective effect of tq against pb - induced renal antioxidant capacity impairment and suggest that this component might be a clinically promising alternative in pb nephrotoxicity. |
lipoid proteinosis (lp) is a very rare autosomal recessive genodermatoses whose true incidence is not known. it was first described by siebenmann in 1908 and the first case series as lipoidosis cutis et mucosae was presented by a viennese dermatologist, urbach and an otorhinolaryngologist, weithe in 1929. later was renamed by urbach in 1930 as lp cutis et mucosae. it has been known by several terms such as urbach weithe disease, lipoglycoproteinosis, the disease is characterized by the deposition of an amorphous hyaline material in the skin, mucosa, and viscera, classically manifesting as hoarseness of voice during infancy and consequently involving skin, mucosa, and any organ. recently, loss of function mutations in the gene encoding extracellular matrix protein 1 (ecm1) on band 1q21 has been identified as the cause of lp. histopathological examination shows epidermis with hyperkeratosis, focal parakeratosis, acanthosis, papillomatosis, and elongated rete ridges overlying. superficial dermis showed deposition of dense eosinophilic material oriented perpendicular to epidermis and forming thick perivascular mantles around blood vessels. previous studies have attributed the prevalence of lp to consanguineous parents. here, we report oral and clinical manifestations of two young adults diagnosed with lp but without consanguinity among parents along with a concise review of the literature on the disease. a 19-year - old male reported to our department with a complaint of multiple large ulcerations on his tongue since 1-week with associated burning sensation and fever. he gave the history of hoarseness of voice, alopecia, cracked skin over soles of the feet, repeated vomiting, and loose stools since childhood. intraoral examination revealed multiple nontender ulcerations covered with slough measuring about 11.5 cm [figure 1 ]. entire floor of the mouth, palate and bilateral buccal mucosa were coated with scrapable curdy white patches. all second premolars were missing along with retained 53, 82, 83, and supernumerary tooth between 14 and 16 were noticed. multiple ulcerations on the dorsum of the tongue blood investigations revealed leukocytosis with increased monocytes (12.8%). serum iron level was slightly low (36 g / dl), with raised total iron binding capacity (471 g / dl). patient was a known case of lp, the facial and oral findings were considered to be due to the same condition. the treatment plan for the patient included debridement with hydrogen peroxide and betadine solution under topical la. patient was instructed to maintain good oral hygiene and follow liquid diet till the ulcers heal. debridement was carried out for the next 4 days, and healing of tongue ulcers was appreciated on the last visit. an 11-year - old boy reported with a complaint of multiple ulcers on the tongue and hoarseness of voice since 6 months of age with similar ulcerations on bilateral buccal mucosa in the past and was associated with recurrent throat pain and dysphagia from past 2 to 3 years. he also had recurrent episodes of cough, cold and fever occurring once every month. on general examination, multiple shiny skin - colored papules were present over forehead, nose and malar region in a background of thickened waxy skin with few comedones and multiple hyperpigmented papules bilaterally over both the eyelid margins [figure 2 ]. hyperpigmentation and verrucous surface skin were seen over both the elbows, knuckles, sides of fingers, and over the palmar surface of hands and digits [figure 3 ]. on intraoral examination, whitish plaques were seen over the bilateral buccal mucosa, lips, sublingual mucosa, and palate. erosions, few fissures, and whitish plaques were noticed on the tongue. based on the clinical findings and considering the patient being a known case of lp, multiple hyperpigmented papules on the face hyperpigmented and verrucous surface skin over both the elbows, knuckles, sides of fingers x - ray skull showed focal areas of increased density, projected over the dorsum sellae. histopathologic examination revealed a diagnosis of lp [figure 4 ] showing hyperkeratosis, acanthosis, and elongated rete ridges along with deposition of dense eosinophilic material in the superficial dermis. histopathological picture showing hyperkeratosis, acanthosis, and elongated rete ridges with deposition of dense eosinophilic material in the superficial dermis patient was instructed to maintain oral hygiene and to be under liquid diet. patient was recalled after 1-week during which there was a considerable improvement in the healing of the intraoral lesions. a 19-year - old male reported to our department with a complaint of multiple large ulcerations on his tongue since 1-week with associated burning sensation and fever. he gave the history of hoarseness of voice, alopecia, cracked skin over soles of the feet, repeated vomiting, and loose stools since childhood. intraoral examination revealed multiple nontender ulcerations covered with slough measuring about 11.5 cm [figure 1 ]. entire floor of the mouth, palate and bilateral buccal mucosa were coated with scrapable curdy white patches. all second premolars were missing along with retained 53, 82, 83, and supernumerary tooth between 14 and 16 were noticed. multiple ulcerations on the dorsum of the tongue blood investigations revealed leukocytosis with increased monocytes (12.8%). serum iron level was slightly low (36 g / dl), with raised total iron binding capacity (471 g / dl). patient was a known case of lp, the facial and oral findings were considered to be due to the same condition. the treatment plan for the patient included debridement with hydrogen peroxide and betadine solution under topical la. patient was instructed to maintain good oral hygiene and follow liquid diet till the ulcers heal. debridement was carried out for the next 4 days, and healing of tongue ulcers was appreciated on the last visit. an 11-year - old boy reported with a complaint of multiple ulcers on the tongue and hoarseness of voice since 6 months of age with similar ulcerations on bilateral buccal mucosa in the past and was associated with recurrent throat pain and dysphagia from past 2 to 3 years. he also had recurrent episodes of cough, cold and fever occurring once every month. on general examination, multiple shiny skin - colored papules were present over forehead, nose and malar region in a background of thickened waxy skin with few comedones and multiple hyperpigmented papules bilaterally over both the eyelid margins [figure 2 ]. hyperpigmentation and verrucous surface skin were seen over both the elbows, knuckles, sides of fingers, and over the palmar surface of hands and digits [figure 3 ]. on intraoral examination, whitish plaques were seen over the bilateral buccal mucosa, lips, sublingual mucosa, and palate. erosions, few fissures, and whitish plaques were noticed on the tongue. based on the clinical findings and considering the patient being a known case of lp, multiple hyperpigmented papules on the face hyperpigmented and verrucous surface skin over both the elbows, knuckles, sides of fingers x - ray skull showed focal areas of increased density, projected over the dorsum sellae histopathologic examination revealed a diagnosis of lp [figure 4 ] showing hyperkeratosis, acanthosis, and elongated rete ridges along with deposition of dense eosinophilic material in the superficial dermis. histopathological picture showing hyperkeratosis, acanthosis, and elongated rete ridges with deposition of dense eosinophilic material in the superficial dermis patient was instructed to maintain oral hygiene and to be under liquid diet. patient was recalled after 1-week during which there was a considerable improvement in the healing of the intraoral lesions. lipoid proteinosis is a rare collagen disorder with a genetic predisposition, with a higher prevalence in south africa and sweden, where consanguineous marriages are very common. it is characterized by the deposition of an amorphous hyaline material with a glycoprotein constitution in the skin, mucous membranes, and internal organs. the etiopathogenesis of the lp is not still completely understood, but it seems to be related to an alteration in the synthesis and metabolism of the collagen, leading to an increase in the synthesis of types iv and v collagen by the endothelial cells of the blood vessels, a glycoprotein substance by the fibroblasts, and a decrease in the production of collagen types i and ii. lipoid proteinosis usually exhibits in early infancy with hoarseness of voice due to the vocal cord infiltration. it is then followed by collects of recurrent blood or pus - filled vesicles, bullae, macules, papules, and skin - colored nodules, which are often pruritic and may coalesce resulting in diffuse thickening of the skin and mucous membrane. mucocutaneous lymphatic system plays a major role in this disease because the lesions are usually seen in those areas having greater mobility demanding for high plasticity for example : the flexion and extension of knees, elbows, antecubital fossa ; oral mucosa as a result of eating and speaking. frequent involvement of salivary glands, usually submandibular and parotid gland can cause hyposalivation or xerostomia, leading to poor oral hygiene. whether the oral manifestation or the cutaneous manifestations appear first is not known due to its variable presentation. moreover, the respiratory system, upper gastrointestinal tract, central nervous system, blood vessels, and lymph nodes may also be involved. lp are known to have calcification of the temporal lobes or hippocampi in the brain as in our second case radiographic investigation revealed suprasellar calcifications. epilepsy, memory loss, schizophrenic behavior, mental retardation, emotional changes, and other neuropsychiatric abnormalities may be seen in some patients. hyaline deposits have also been described in the conjunctiva, cornea, trabeculum, and retina. corneal opacities or secondary glaucoma the presence of beadlike papules on the margins of both upper and lower palpebral conjunctiva (moniliform blepharosis) is the universal sign of this disease. nail dystrophy with hemorrhagic blisters on the wrists, fingers, and nailbed is a common finding. histological appearance shows disruption and/or duplication of the basement membrane along with deposition of hyaline material at the dermo - epidermal junction, papillary dermis, surrounding capillaries, and around adnexal epithelia with appearance of sweat coils. immunofluorescence labeling with anti - collagen antibody shows types iv and v collagen accumulation and reduction of types i and iii collagen around blood vessels. it has a role in the structural organization of the dermis (binding to perlecan, matrix metalloproteinase-9 and fibulin) as well as being targeted as an autoantigen. this disease compromises the quality of life due to its disfiguring scars and multisystem involvement. the disease is not life - threatening except for the respiratory obstruction in severe cases. if the lesions start at an early age, it is found to be self - limiting. however, for esthetic consideration, dermabrasion can be done, as it can become a boon to the patient who is psychosocially affected due to his / her appearance. treatment with dimethyl sulfoxide, d - penicillamine, etretinate, intralesional heparin has also been tried. laser microlaryngoscopy, mucosal stripping, or dissection of the vocal cords and excision of deposits may be performed to preserve or improve the quality of voice. in very severe cases, if there is diffuse infiltration of the pharynx and larynx causing respiratory distress, tracheostomy is required. as lp extensively involves the oral mucosa and indirectly, the dentition as well, it is important that the dentist be familiar about it. furthermore, the oral mucosa exhibits clinical features resembling the oral manifestations of several other systemic diseases. thus, careful history taking and detailed clinical examination followed by necessary investigations is a must in establishing the diagnosis and whenever such cases are encountered all measures of alleviating patient symptoms should be undertaken so that oral complications of the systemic condition do not further deteriorate the quality - of - life. it is also important that parents of affected children be taken for genetic counseling concerning the risks of having other affected offspring. | lipoid proteinosis (lp) is a rare autosomal recessive genodermatoses characterized by deposition of amorphous hyaline material in different parts of the body, especially the skin, mucous membranes of the upper aerodigestive tract, and internal organs. oral cavity is most extensively affected area by the disease. this paper reports two classic cases of lp with oral manifestations but without a history of consanguinity along with a concise review of the literature on the disease. |
nearly half of all dental restorations fail within 10 years, and replacement of these failed restorations accounts for 50%70% of all procedures in restorative dentistry. composites are popular filling materials because of their esthetics and direct - filling capabilities. one main problem, however, is that composite tends to accumulate more biofilms than other restorative materials in vivo. biofilms at the restoration margins could produce acids and cause secondary caries, the main reason for restoration failure. from restorative dentistry, the use of bonding agents is known to improve the adhesion of composite resins. bonding agents create a micromechanical interlock between the dentin collagen and resin by forming hybrid layers. bonding agents adhere the composite restoration to the tooth structure to form a functional and durable interface. antibacterial adhesives are promising for combating bacterial infection and reducing recurrent caries at tooth - restoration margins. nanosized fillers, such as nanosized aerosol silica filler, were introduced to the field of bonding agents by means of nanotechnology. nanofiller technology is claimed to increase adhesion to both the enamel and dentin and improves marginal integrity. recently, quaternary ammonium dimethacrylate was synthesized and incorporated into resins to inhibit biofilm growth. traditionally, micro / nanofillers have been introduced into epoxy resins to improve their mechanical performance, for example, silicon, titanium, and aluminum oxides. the use of nanosized aluminum trioxide (al2o3) particles is one approach to improve the mechanical performance of adhesive materials. in these particulate - filled systems, binding at the inorganic filler / epoxy matrix interface has a great effect on the mechanical properties of the adhesive material. dudkin. demonstrated that the strength of the epoxy matrix was increased when reinforced by al2o3 because of interactions between the active surface groups of the oxide nanoparticles and the functional groups of the epoxy matrix. however, whether the addition of filler particles improves the mechanical behavior of these adhesives still remains unclear since their mechanical properties rely on other factors that can not be studied in isolation using commercial adhesive systems. the purpose of this study was to evaluate the effect of adding nanosized al2o3 particles at a concentration of 2% to nano - bond adhesive on the microshear bond strength of nanocomposite resin to dentin. one available type of adhesive system was used as the control (nano - bond adhesive ; pentron clinical technologies, usa ; lot # 183421). nanosized al2o3 particles at a 2% concentration were added to nano - bond adhesive ; this and one type of nanofilled composite resin (artiste nanocomposite, pentron clinical technologies ; lot # 182066 - 185215) were used in this study. twenty caries - free freshly extracted human molar teeth were collected to be used in this study. the teeth were cleaned by an ultrasonic scaler and stored in distilled water at 37c before testing. a dentin slice approximately 1.0-mm thick was cut perpendicular to the long axis of each tooth from the upper middle coronal portion region using a low - speed diamond saw (isomet, buehler, lake bluff, il, usa) under water coolant. the occlusal surfaces of the slices were ground with up to 600-grit silicon carbide paper to expose a flat dentin surface. a dentin slices were divided into two main groups (ten each) according to the bonding agent containing nanosized al2o3 particles at a 2% concentration. group a was tested using the nano - bond adhesive system without additives, and group b was tested using the nano - bond adhesive system containing nanosized al2o3 particles at a 2% concentration. each dentin slice was acid etched using 37% phosphoric acid gel (eco - etch ; ivoclar vivadent, schaan, liechtenstein, swiss) for 15 s. then, the dentin slices were rinsed with water spray and dried in an oil - free stream of air for 5 s. the adhesives were applied according to manufacturers instructions. the adhesives were applied to the entire dentin surface and air - dried for 15 s. a gentle stream of dry air was applied to disperse the material into a thin, uniform, shiny surface, and before irradiation, three or four cylinders (internal diameter : 0.7 mm, height : 1.0 mm) of microbore tygon tubing (r-3603, norton performance plastic co., cleveland, oh, usa) were placed on the flat dentin at different locations. the adhesive was then light cured for 10 s with light - emitting diodes (bg light ltd. after irradiation, the tubing was filled with nanofilled composite resin and then light cured for 40 s with the tip as close to the surface as possible. curing radiometer equipment (li-189 li - cor inc., lincoln, ne, usa) was used to ensure steady light intensity throughout the polymerization of all specimens. the specimens were stored under moist conditions at room temperature (23c) for 1 h before removing the tygon tubing. the specimens were immersed in water at 37c for 24 h, then subjected to thermocycling to simulate clinical thermal stress conditions before testing according to the guidelines set by the american national standards institute / american dental association and international organization for standardization for direct filling resins and dental adhesion. all specimens were subjected to thermocycling by storing them alternately in water reservoirs at 5c and 55c, with the specimen staying in each reservoir for 30 s. this procedure was carried out for 500 cycles and controlled by a computer to simulate thermal stress. a diagram of the microshear bond test setup is shown in figure 1. each dentin slice with the resin cylinders was placed in the lower attachment of a lloyd universal testing machine (model lrx plus ii, fareham, england) for microshear bond testing. diagram of the microshear bond test setup a thin wire (diameter, 0.20 mm) was looped around each resin cylinder, making contact with half of the cylinder base, and was placed as close as possible to the resin - dentin interface. a shear force was applied to each specimen at a crosshead speed of 0.5 mm / min until failure occurred. the resin - dentin interfaces of the specimens and the wire loops were aligned as straight as possible to ensure that the same shear orientation was maintained. the loads at failure were recorded, and the data were analyzed by one - way analysis of variance and tukey 's tests were used to test the significance of differences between the means of the tested materials, which were considered statistically significant when the p 0.05. the mean percentages for the tested nano - bond adhesive system without additives and nano - bond adhesive system containing nanosized al2o3 particles at a 2% concentration are presented in table 1 and figure 2. comparison between mean microshear bond strength in (mpa) of the tested nano - bond adhesive system groups a bar chart of the mean microshear bond strengths (mpa) of the tested nano - bond adhesive system groups the nano - bond adhesive system containing nanosized al2o3 particles at a 2% concentration (group b) showed a statistically significantly greater mean microshear bond strength (23.15 mpa) than that of the nano - bond adhesive system without additives (15.03 mpa, group a). the results of the microshear bond strength test showed a significant difference (p < 0.05) between group b and group a. the microshear bond strength was increased in the specimens containing nanoparticles of al2o3. the major goals of using dentin bonding systems are to enhance the bonding strength between the resin and the tooth structure, increase the retention of the restoration, reduce microleakage across the dentin - resin interface, and dissipate occlusal stress. the adhesive layer acts as an elastic intermediate layer (elastic cavity wall) between the cavity walls and the adjacent composite. this layer could resist polymerization shrinkage stress from resin composites and absorbs shocks produced by occlusal loads and thermal cycling. according to many investigators, the use of filled adhesive resin increases its mechanical properties and improves the marginal and internal seals of composite restorations. these adhesives may be categorized as mild or strong adhesives depending on their ph and therefore their etching potential. if the adhesive 's capacity dissolves the smear layer is limited, the bond strength to dentin with a thick smear layer may be reduced. the shear bond strength test has been widely used, mainly because of its relative simplicity when compared with the tensile bond strength test, in which it is difficult to align the specimen in the testing machine without creating a deleterious stress distribution. a new test method using specimens with reduced dimensions has been advocated by some authors as a substitute for the conventional shear test : this called microbond or microshear bond strength test. according to these authors, this test would allow for the testing of small areas of material, thus permitting a regional mapping or depth profiling of different substrates and preparing multiple specimens from the same tooth. the present study used nanosized al2o3 particles at a 2% concentration in the adhesive because the antimicrobial activity of aluminum nanoparticles is due to the release of metal ions and because aluminum ion nanoparticles attach to the surface of bacteria because of their surface charge ; the charge of the bacterial surface is negative while that of the aluminum nanoparticles is positive at the ph studied. the present study showed that the microshear bond strength of nano - bond adhesive containing nanosized al2o3 particles at a 2% concentration was greater than that of the nano - bond adhesive system without additives. factors contributing to this effect may include the fact that the number of metal oxygen (me - o) bonds increases with the release of residual water and organic solvent during the early stages of drying. therefore, a further increase in cross - linking and me - o bonding occurred during the curing regimen because the release of water and solvent from the adhesive is controlled by the cure time and thus increased the bond strength to dentin. the addition of nanosized al2o3 particles at a concentration of 2% to nano - bond adhesive increased the microshear bond strength. | objectives : the present study was conducted to evaluate the effect of nanosized aluminum trioxide (al2o3) particles when added to the nano - bond adhesive system and its effect on the microshear bond strength of nanocomposite resin to dentin.materials and methods : a newly developed adhesive (nano - bond) and one type of light - cured resin restorative material (nanocomposite resin) were used in this study. the occlusal surfaces of extracted human molar teeth were ground perpendicular to the long axis of each tooth to expose a flat dentin surface. the adhesives were applied to the dentin surfaces according to manufacturers instructions. the nanocomposite resin was then placed and light cured for 40 s. after immersion in water at 37c for 24 h, the specimens were subjected to thermocycling before testing, and a microshear bond test was carried out. the recorded bond strengths (mpa) were collected, tabulated, and statistically analyzed. a one - way analysis of variance and tukey 's tests were used to test for significance between the means of the groups ; statistical significance was assumed when the p 0.05.results:the mean microshear bond strength of the nano - bond adhesive system containing nanosized al2o3 at a concentration of 2% was 23.15 mpa (group b), which was significantly greater than that of the nano - bond adhesive system without additives (15.03 mpa, group a).conclusions : these results indicate that nanosized al2o3 added to the nano - bond adhesive system at a concentration of 2% increases the microshear bond strength. |
ovarian cancer is the most frequent cause of death from gynaecological cancer and the fourth most frequent cause of cancer - related death in women in europe and the united states. it has the highest fatality - to - case ratio of all gynaecological malignancies, mainly due to the fact that it is characterized by early widespread metastasis and high - grade malignancy at diagnosis. the five - year survival proportion is about 8090% for patients with stage i disease and only 1520% for patients with stage iii or iv disease. although survival has improved with the use of maximal cytoreduction surgery along with platinum- and taxane - based chemotherapy, nearly 80% of ovarian cancers relapse and patients inevitably succumb to the development of chemotherapy - resistant disease. clinicopathological features known to be prognostic variables for ovarian cancer are surgical stage (figo stage), histological grade, lymph node involvement, residual tumour size after cytoreductive surgery, histological subtype, ascites, and age. according to the three - year analysis of the figo annual report on the results of treatment in gynaecological cancer, stage, grade, and residual tumour size have the greatest prognostic value. however, these factors provide an insufficient picture of the biology of ovarian cancer and they are frequently interrelated. the identification of new serological biological markers that predict the outcome of the disease would be extremely useful for developing individually tailored and possibly more effective treatments. serum analysis is a noninvasive technique feasible in cases where no tissue is available and it can also be performed during followup. it is well established that angiogenesis, the formation of new blood vessels, is necessary for the growth and metastatic spread of solid tumours [47 ]. a high degree of tumour angiogenesis has been shown to correlate with poor survival in women with ovarian cancer [710 ]. vascular endothelial growth factor (vegf) plays an essential role in angiogenesis in many tumour types [1115 ]. it is a heparin - binding dimeric glycoprotein involved in angiogenic, mitotic, and microvascular permeability - inducing activities, leading to extravasation of plasma proteins and proangiogenic stromal changes. several studies have found vegf levels to be significantly higher in the tissues and biological fluids of women with ovarian cancer compared with healthy controls [1720 ], whereas its association with tumour progression and/or patient survival is still controversial. we performed a review of the literature to elucidate the prognostic role of serum vegf (svegf) levels in ovarian cancer, both alone and in comparison with established clinicopathological factors. eligible studies in english and italian were identified in medline (pubmed version) from vegf discovery to october 2011 using the terms vegf, vascular endothelial growth factor and synonyms, ovarian cancer, ovary cancer, and synonyms. we searched studies that used these terms in title and abstract and that was indexed by bibliographic database with the mesh term relevant papers were independently selected by two of the reviewers (e. bandiera and r. franceschini) based on the following inclusion criteria : studies that evaluated (i) svegf levels before any surgical and chemotherapeutic treatment ; (ii) the association of svegf levels with the established clinicopathological prognostic factors (figo stage, tumour grade, residual tumour size, lymph node involvement, histological type, ascites, age) ; (iii) the value of svegf levels in predicting patients ' outcomes (overall survival (os), disease - free survival (dfs), progression free survival (pfs)). any disagreement in the inclusion of one study between two reviewers was solved by discussion. two of the reviewers (e. bandiera and r. franceschini) independently reviewed each study and abstracted data on first author, country of study, study characteristics (study design, followup duration, therapy), clinical and pathological variables, and study outcomes. our search strategy identified 758 journal abstracts. from these, we retrieved for further evaluation 15 full - text articles focused on the relationship between circulating preoperative vegf and prognosis in ovarian cancer. of these 15 articles, nine [2129 ] studies met the inclusion criteria and were used for this review. hefler. gathered together some cases from trials by tempfer., gadducci., chen., and cooper. because hefler. added a series of new patients, we reported these five [2123, 25, 28 ] studies independently. the paper by manenti. was excluded because these authors analysed plasma vegf levels. since vegf is secreted also by platelets, manenti. boss., bamias., yamamoto., and rudlowski. were excluded because they did not directly evaluate the prognosis of patients with abnormal svegf levels and/or the association between svegf levels and clinicopathological characteristics. finally, dirix. evaluated patients with different cancer types and did not report independent results for ovarian cancer. the nine selected studies were published between 1996 and 2010 and included 529 patients from seven countries. six of these nine studies reported details on the duration of followup (median : 42 months, 34 months, 29 months or mean : 39 months, or length : 60 months, 24 months). all studies analysed the association between svegf levels and the established prognostic variables and evaluated the association between svegf levels and os. three studies [21, 23, 27 ] analysed the association between svegf and dfs whereas only one analysed the association between svegf and pfs. all studies enrolled women with newly diagnosed and histopathologically confirmed ovarian cancer except the one by cooper., which included also a small group of women with peritoneal and fallopian tube malignancies (table 1). the mean (or median) ages of the studied women ranged from 52.5 to 64 years. most cancers (> 95%) were epithelial and the predominant histological type was serous carcinoma. most patients were diagnosed with poorly differentiated ovarian cancer, advanced figo stage, and ascites. with the exception of gadducci., who followed only 27 patients with advanced disease receiving chemotherapy, all other studies monitored all patients enrolled. surgery for optimal tumour debulking included hysterectomy, bilateral salpingo - oophorectomy, omentectomy [21, 23, 28, 29 ], pelvic and para - aortic lymphadenectomy [21, 23, 28, 29 ], and appendectomy [23, 28, 29 ]. five studies [22, 2427 ] did not describe the surgical approach, but according to the reported data (residual tumour size [22, 2427 ], omental metastasis, lymph node involvement), we may presume that maximal cytoreductive surgery was performed. surgery was followed by chemotherapy consisting of platinum analogues alone [2124, 28 ] or in combination with taxane [25, 29 ]. early stages of disease were treated according to the standards established by the respective institutions : patients with stage ia - ib, i - ii, ia, and ia - ib excluding clear cell histology did not receive any chemotherapy or were treated like patients with advanced disease [23, 24, 29 ]. although postoperative chemotherapy is the accepted standard treatment, two studies [26, 27 ] omitted any information about it. seven studies [2125, 27, 28 ] used the same quantikine sandwich elisa kit (r&d systems minneapolis, usa). 's study used a home - made indirect elisa kit, whereas mahner. 's study used vegf-165 elisa kit (siemens healthcare diagnostic, tarrytown, usa). data on the precision of svegf assays were reported in three studies [21, 24, 28 ], and in all studies, the intra / inter - assay coefficient of variation was < 10%. median values of svegf reported by authors were : 466 pg / ml, 229 pg / ml, 458 pg / ml, 440 pg / ml, 379 pg / ml, 387 pg / ml, 407 pg / ml, and 171 pg / ml. the association between svegf concentrations and figo stage, tumour grade, residual tumour size, lymph node involvement, histological type, ascites, and age was analysed by 89%, 89%, 89%, 44%, 67%, 56%, and 78% of studies respectively (table 2). when the median (or mean) of svegf values was evaluated in relation to clinicopathological features, a statistically significant association between the level of svegf and figo stage, tumour grade, residual tumour size, lymph node involvement, and presence of ascites was found in at least one study. by contrast, no statistically significant association was found between svegf levels and histological type or age. tempfer., chen., and li. demonstrated that elevated svegf levels were associated with a high malignant potential of tumours (g1 versus g2-g3 [21, 23 ], g1-g2 versus g3). gadducci., cooper., and li. reported a positive association between svegf concentrations and ascites volume (gadducci. selected patients with stages iii - iv). found that patients with suboptimally debulked cancer had higher svegf values than patient in whom tumour debulking was optimal. finally, only the study by li. showed that svegf values were higher in patients with advanced figo stages and lymph node involvement. in evaluating its association with outcome variables, the levels of svegf were dichotomised using different cut - offs : 75th percentile [21, 23 ] or median [24, 29 ] in ovarian cancer patients, mean in healthy subjects, and 95th percentile in patients with benign disease. finally, only hefler and coworkers considered svegf as a continuous variable. in the univariate analyses, seven [21, 23, 24, 2629 ] studies used the kaplan - meier product - limit method to estimate how svegf and other clinicopathological variables might predict os and dfs. cooper. did not explicitly report the method of univariate analysis. in the multivariate analyses, all studies claim to have used the cox proportional hazards regression model to assess the independent role of different, simultaneously evaluated prognostic factors in determining outcomes. estimates are reported in terms of relative risk (rr) and hazard ratio (hr). the results of univariate and multivariate analyses were considered statistically significant when the p values were < 0.05. all studies analysed the association between svegf levels and os. with the exception of gadducci. and mahner. moreover, five [21, 23, 25, 27, 28 ] of these seven studies found svegf to be an independent prognostic factor. as expected, clinicopathological features known to be prognostic variables for eoc such as figo stage, tumour grade, residual tumour size after cytoreductive surgery, lymph node involvement, and age have been shown as independent prognostic factors in at least one study. notably, svegf, in comparison with others prognostic variables, was reported as independent prognostic factors by the majority of studies. chen. selected a subset of 40 patients with residual tumour size less than 2 cm. univariate analysis, performed only for svegf, showed that elevated svegf was associated with shorter os. li. demonstrated that there was no significant difference in cumulative survival probability between stage i / ii patients with high values of svegf and stage i / ii patients with low levels of svegf. by contrast, the cumulative survival probability of stage iii / iv patients with high values of svegf was lower than that of stage iii / iv patients with low levels of svegf. a planned subgroup analysis was performed for 56 patients with figo stage i in the study by hefler.. only three [21, 23, 27 ] of the nine included studies considered dfs as an end point. in univariate analysis, a significant association between dfs and svegf level was found by 2 [21, 23 ] out of 3 [21, 23, 27 ] studies. in multivariate analysis, svegf levels were shown to be independent prognostic factors by 2 [21, 23 ] out of 3 [21, 23, 27 ] studies. chen. further evaluated dfs for 40 ovarian carcinoma patients with residual tumour size less than 2 cm, and they found that elevated svegf levels were significantly associated with lower dfs in univariate analysis and svegf levels, figo stage and grade were independent prognostic factors for dfs in multivariate analysis. considered pfs as an end point and he did not find a significant association between pfs and svegf level. the management of patients with ovarian cancer is based on established prognostic factors such as tumour stage, histological grade, and residual tumour size after cytoreductive surgery. recently, the concept of standard chemotherapeutic treatment with platinum / taxane combination, the necessity of adjuvant chemotherapy in early stages of disease, the use of neoadjuvant chemotherapy for patients expected not to be optimally debulked at primary cytoreductive surgery and the use of consolidation chemotherapy for patients at high risk of recurrence have all been questioned. the need for additional prognostic data to calibrate therapeutic tools on an individual basis in women with ovarian cancer seems obvious. in contrast to other malignancies, no serological prognostic parameter other than ca125 has been shown to have clinical value in ovarian cancer, even though ca125 serum levels at diagnosis are not associated with os and dfs [36, 37 ]. from vegf discovery till 2011, structured data extraction was performed on the articles to compare study populations, svegf assays, associations between svegf and clinicopathological characteristics, patient management, and outcome evaluation. unfortunately, because of the heterogeneity of the studies and missing or incomplete information, it is not possible to pool data and to perform a meta - analysis in order to obtain univocal indications about svegf 's prognostic value. the data reported in tables 1, 3, and 4 show evident differences among studies. all studies included only epithelial ovarian carcinoma patients except those by cooper. and li., where other ovarian, peritoneal, and tubal malignancies were included. patients underwent different chemotherapy regimens based on platinum analogues alone [2124, 28 ] or in combination with taxane [25, 29 ]. patients with early - stage disease were treated differently or were not treated, depending on the standards of the respective institutions. none of the studies exhaustively described followup (time, lost patients, events). although seven [2125, 27, 28 ] out of nine studies used the same svegf assay, one of these measured significantly lower svegf values in ovarian cancer patients. finally, widely differing svegf cut - off values (ranging from 100 to 826 pg / ml) were chosen for univariate and multivariate analysis, depending on the statistical methods chosen for the analysis. in order to find out how svegf influences ovarian cancer biology, all studies analysed the association between svegf and the clinicopathological characteristics of the patients. the results seem to confirm that vegf plays an important biological role in the pathogenesis of ascites [38, 39 ]. vegf increases vessel permeability for circulating macromolecules, thus facilitating extravasation of a plasma - rich exudate into the peritoneal cavity. moreover, seven out of eight studies, concerning the relationship between svegf and figo stage, showed that vegf concentrations measured in sera were not associated with figo stage. this may indicate that the effects promoted by vegf are a continuous process and are independent of the clinical progression of the disease. interestingly, in our review of literature, svegf appears to be the best prognostic marker for os in comparison with the established prognostic variables, since it stands out as an independent prognostic factor in most of the studies considered. the scarcity of the data on the relationship between svegf levels and dfs makes it difficult to draw any firm conclusions in this regard. however, it is worth noting that svegf appears to be an independent prognostic factor for dfs in 2 out of 3 studies, as well as tumor stage and grade. analyzed the prognostic value of svegf in a selected low - risk group of patients. chen. showed that svegf, figo stage and tumour grade were independent prognostic factors for os and dfs in 40 patients with size tumour less than 2 cm. in a cohort of 56 patients with figo stage i found that svegf and tumour grade were independent prognostic factor for os. the value of these results is conspicuous in those situations where the usefulness of adjuvant chemotherapy or the advisability of more chemotherapy cycles for certain categories of patients is under discussion. in conclusion, almost all of the studies analysed in the present review, including the largest one by hefler. it is worth noting that multiple phase iii studies, icon 7, gog218, and oceans, have recently showed that the use of bevacizumab, a humanized antibody against vegf, provides a clinically meaningful benefit in eoc patients outcome [40, 41 ]. thus, from analysis of the literature reported in this review, as well as from results reported by recent clinical trials, svegf appears to be a promising prognostic factor in ovarian cancer that could identify a subgroup of patients with poor survival and higher risk of death that could benefit of bevacizumab therapy to improve their outcome. | introduction. we performed a review of the literature to elucidate the potential prognostic significance of serum vascular endothelial growth factor (svegf) levels in ovarian cancer. methods. eligible studies in english and italian were identified in medline / pubmed from vegf discovery to october 2011. all studies evaluating : (i) svegf levels before any surgical and chemotherapeutic treatment ; (ii) the association between svegf levels and the established prognostic variables ; (iii) the value of svegf levels in predicting patients ' outcomes, were selected for this review. results. the search resulted in 758 titles. nine studies met the inclusion criteria. a statistically significant association between the level of svegf and figo stage, tumour grade, residual tumour size, lymph node involvement, and presence of ascites was found in at least one study. svegf, in comparison with the established prognostic factors, appears to be the best prognostic marker for overall survival, since it stands out as an independent prognostic factor in most of the studies considered. moreover, svegf levels were shown to be independent prognostic factors by 2 out of the 3 studies that considered dfs as an end point. conclusion. high levels of svegf identify a subgroup of patients with higher risk of death and/or recurrence. these patients should be eligible for individually tailored therapeutic interventions. |
failure to grasp the rationale behind cleaning and shaping concepts can increase the incidence of procedural complications such as blockage, ledge formation, apical transportation and perforations. according to the glossary of endodontic terms of the american association of endodontists, canal transportation is defined as follows : removal of canal wall structure on the outside curve in the apical half of the canal due to the tendency of files to restore themselves to their original linear shape during canal preparation ; may lead to ledge formation and possible perforation. various undesirable apical preparation outcomes such as damage to the apical foramen, elbow formation, zip formation and perforation have been described as possible results of canal transportation. perforation represents a communication between the root canal space and the external root surface, causing irritation of the periradicular tissues. similar terms describing the shape of a zipped apical part of the root canal are an hourglass shape, a teardrop or a foraminal rip. it can result in a poorly cleaned root canal, which fails to provide a resistance form to compact gutta percha, and leads to obturation which is vertically overextended but internally under - filled. apical transportation can be categorized into : type 1 : represents a minor movement of the position of the physiologic foramen, resulting in its iatrogenic relocation. type ii : represents a moderate movement of the physiologic position of the foramen, resulting in a considerable iatrogenic relocation on the external root surface. in this type, a larger communication with the periapical space exists, and attempt to create a more coronal shape may weaken or perforate the root. type iii : represents a severe movement of the physiologic position of the canal, resulting in a significant iatrogenic relocation of the physiologic foramen. canals exhibiting type i transportation can be cleaned and obturated, if sufficient residual dentin is maintained and shape created above the foramen. type ii cases are managed by placing a barrier to control bleeding and provide a backstop to pack against during subsequent obturation procedures. in type iii situations, a barrier technique is usually not feasible ; it requires obturation as best as possible followed by corrective surgery. a healthy 21-year - old young male presented with the intention of seeking relief from discomfort and swelling associated with a painful endodontically retreated maxillary central incisor. five months ago, he had undergone root canal treatment for the resolution of a lesion of endodontic origin associated with 11 and 12. clinical examination revealed non - carious, discoloured, tender 11 and incisally fractured non - tender 12. softness to palpation indicated loss of buccal cortical plate over root apices of 11 and 12. iopa radiograph, obtained at the time of visit, exhibited periradicular radiolucency involving insufficiently obturated 11 and overfilled 12. periradicular curettage and removal of extruded gutta percha, after raising a mucoperiosteal flap under local anesthesia, exposed the apical canal transportation of 11. repair of this canal defect by packing a wet sand consistency mix of white mineral trioxide aggregate (mta) (angelus, brazil), with distilled water, restored it. cold compaction by a ball burnisher, after removal of overextended gutta - percha, sealed the root apex of 12. a decrease in the size of radiolucency in iopa radiograph supports the clinical finding [figures 1a g ] and [figures 2a and b ]. (a) previous occlusal radiograph, (b) preoperative iopa radiograph, (c) exposed lesion, (d) exposed apical transportation, (e) defect repaired by mta, (f) third month recall, (g) recall radiograph (a) preoperative radiograph, (b) fourth month recall radiograph it is important to understand the objectives for shaping and cleaning the root canal system. the factors associated with an increased risk of canal transportation include insufficiently designed access cavities, use of inflexible instruments, instrumentation technique, insufficient irrigation during mechanical enlargement, degree and radius of a canal curvature, unseen canal curvatures in two dimensional radiography and experience of operator. canal transportation may result in inadequately cleaned root canals, over - reduction of sound dentin and destruction of the integrity of the root. mineral trioxide aggregate is the material of choice for managing various undesirable apical preparation outcomes. the sealing ability of this non - absorbable material is not adversely affected by blood contamination. cementum grows adjacent and onto this radiopaque material, thus allowing for a normal periodontal attachment apparatus. in this case, mta was used to repair apical transportation defect, as it has above - mentioned advantages over other materials. apicoectomy or root - end preparation was avoided as it would have further removed the sound dentin. many endodontic complications, including canal transportation, can be prevented while cleaning and shaping. apical transportation may lead to post - operative flare - up, surgery and extraction. however, depending upon the extent of occurrence, it can be managed. in the present case, the outcome of this case, indicate that the result of these procedures, is predictable and successful. | procedural accidents leading to complications such as canal transportation have been ascribed to inapt cleaning and shaping concepts. canal transportation is an undesirable deviation from the natural canal path. herewith a case of apical transportation of root canal resulting in endodontic retreatment failure and its management is presented. a healthy 21-year - old young male presented discomfort and swelling associated with painful endodontically retreated maxillary incisor. radiograph revealed periradicular radiolucency involving underfilled 11 and overfilled 12. insufficiently obturated 11 exhibited apical transportation of canal. this type iii transportation was treated by periradicular surgery and repair using white mineral trioxide aggregate (mta). comfortable asymptomatic patient presented uneventful healing at third and fourth month recall visits. a decrease in the size of radiolucency in radiograph supported the clinical finding. in the present case, mta is useful in repairing the transportation defect. the result of these procedures is predictable and successful. |
gastrointestinal basidiobolomycosis is an emerging infection, with fewer than 80 cases reported in the english literature. also, a few cases of gastrointestinal basidiobolomycosis, accompanied by liver involvement as part of a disseminated disease, have been reported. this is the first case report of an isolated liver involvement of this fungal infection in a 2-year - old girl, who presented with a liver mass resembling a hepatic abscess. zygomycosis includes 2 orders, one of which causes fungal infections in an immunocompromised host (mucorales) and the other in an immunocompetent host (entomophthorales) (1). basidiobolus ranarum belongs to the second group and is a saprophyte found mostly in soil and decaying vegetable material (2). it is a low virulent fungus, and the first human case of this fungal infection was reported in 1956 in subcutaneous tissue (3). however, the gastrointestinal (gi) involvement of this fungus is an emerging infection and has rarely been reported (4). recently, a few cases of gi basidiobolomycosis, accompanied by liver involvement as part of a disseminated disease, have been reported (5). to the best of our knowledge, no case has been reported in the english literature with an isolated liver mass caused by basidiobolomycosis without the involvement of any other organ. accordingly, herein we report our experience with a 2-year - old girl, who presented with a liver mass subsequently identified as basidiobolomycosis. a 2-year - old well - nourished and well - developed girl from the iranian city of kangan (bushehr province) presented with vague and generalized abdominal pain. she was the first child of the family, born via normal vaginal delivery without any specific disorder. she had had a normal infancy until 2 months prior to her referral, when she developed abdominal pain with no response to routine treatment. laboratory tests showed microcytic hypochromic anemia (hemoglobin level = 7.8 gr / dl). white blood cell count was high (about 1112000/cc) with significant eosinophilia (25% - 35%). immunoserology tests revealed high c - reactive protein (crp = 13 mg / l, normal < 6) and erythrocyte sedimentation rate (esr = 83, normal for the patient s age < 10). liver function tests showed high aspartate aminotransferase and alanine aminotransferase (57 and 45 iu / l respectively, normal < 31) and alkaline phosphatase (4030 abdominal ultrasonography demonstrated a prominent liver with a well - defined mass lesion measuring 40 35 cm. the other parts of the gi tract, including the stomach and intestine walls, were normal. upper abdominal magnetic resonance imaging (mri) showed normal thickness of the gi tract with no mass, but there were multiple masses in the liver. the first impression of both sonography and computed tomography scan was liver or biliary abscesses (figure 1). tru - cut needle biopsy displayed a mainly normal liver with foci of eosinophilic infiltration, which was nondiagnostic. therefore, the patient underwent surgery, which showed multiple nonencapsulated liver masses with ill - defined borders, the main one in the parenchyma and the other in the hilar area. during surgery, precise search was made to find any accompanied gi mass, but no mass was identified. also, the omentum was completely free of any tumor or mass lesion. the pathology sections showed splendore - hoeppli bodies and many eosinophils as well as radiating eosinophilic granular material surrounding the fungal elements within the liver parenchyma and in the hilar mass within the lymph node tissue (figures 2a and2b). the fungal elements exhibited broad hyphae with thin walls with no septae or sparse septation. according to the characteristic pathologic features the immune system, cellular and humoral, of the patient was thoroughly investigated, even for the possibility of chronic granulomatous disease. subsequently, antifungal therapy was started, mainly composed of amphotericin b (1 mg / kg / d) for at least 6 months. now after 3 months, the patient is well, the abdominal pain has been relieved, and also esr and eosinophil counts have returned to normal level. basidiobolomycosis is an uncommon fungal infection caused by basidiobolus ranarum, which is an environmental saprophyte (6). the most common reported site of involvement is subcutaneous tissue, caused by insect bite or contamination of wounds by soil and dust (7). however, gi involvement of this fungus is rare and seems to be secondary to the ingestion of contaminated food and water (8). fewer than 80 cases of gi basidiobolomycosis have been reported in the english literature (9), and a small number of them have been accompanied by a liver mass. the cases reported from iran were from shiraz, tehran, and mashhad (9). in our previous 14 cases, there was a wide variation of lifestyles, both from rural and urban areas (10). it means that the reported liver masses caused by basidiobolomycosis have always been part of a disseminated disease (10 - 13). to the best of our knowledge, there has been no report of an isolated liver mass caused by this pathogen without the involvement of the gi tract. the probable theory for the pathogenesis in isolated liver involvement can be the acquisition of this infection after the ingestion of contaminated material and dissemination of the fungus through small lymphatics in the gi tract without creating a mass in the intestine the clinical presentation of hepatic basidiobolomycosis is very similar to tubular gi inasmuch as abdominal pain is the most common presenting symptom in the gi tract. our patient underwent liver biopsy and then laparotomy with the clinical impression of malignancy, which is the exact usual preoperative diagnosis of gi basidiobolomycosis in the previous reports (13). the most important laboratory findings in our case were high esr and crp, with significant eosinophilia. also aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were significantly high, which were all indicative of parenchymal liver injury most probably secondary to an inflammatory or neoplastic process. imaging studies play a key role in the diagnosis of liver masses before surgery or biopsy. all masses in the solid organs such as liver and kidney tend to show an inflammatory component with adjacent soft tissue stranding, with or without abscess formation (14). in this case, sonography and mri were in favor of a liver mass ; however, the possibility of a hepatic abscess was also considered. in most of the previous cases of gi basidiobolomycosis, the final diagnosis was made after surgery and resection of the liver mass (9). nonetheless, the gold standard for the diagnosis of every fungal infection is culture. in the majority of the previously reported cases of gi basidiobolomycosis, culture was either negative or was not performed because of the unavailability of the proper tissue (5). in our case, cultures turned out to be negative. the pathologic characteristics of this fungal infection are the presence of splendore - hoeppli bodies with many eosinophils and degenerated fungal hyphae (13). it can cause liver granuloma with heavy infiltration of eosinophilic liver granuloma and should be considered in the differential diagnosis of hepatic granulomas (15). the best treatment in this pathogen is the resection of the mass, accompanied by antifungal therapy including itraconazole or amphotericin b. our patient showed a dramatic response to amphotericin b. in less than a week, eosinophilia disappeared and esr returned to normal and within 2 weeks, she resumed weight gain and her abdominal pain subsided. she is still under treatment, and the plan is to continue the antifungal therapy for at least 6 months, because our previous experience showed the high possibility of recurrence after an early discontinuance of the treatment. in conclusion, basidiobolomycosis should be considered as a differential diagnosis of hepatic abscess with or without gi involvement to prevent delayed treatment. | introduction : gastrointestinal basidiobolomycosis is an emerging infection, with fewer than 80 cases reported in the english literature.case presentation : also, a few cases of gastrointestinal basidiobolomycosis, accompanied by liver involvement as part of a disseminated disease, have been reported.conclusions:this is the first case report of an isolated liver involvement of this fungal infection in a 2-year - old girl, who presented with a liver mass resembling a hepatic abscess. |
although this concept implies that every tumor is defective in one or more aspects of the cell cycle control, it clearly does not mean that oncogenesis targets only oncogenes and the cell cycle clock. development of malignancy appears to require aberrations in the cell death machinery, cell - cell and/or cell - matrix interactions that co - operate with cell cycle defects. many of the processes in which adhesion molecules play central role like anchorage dependent growth, apoptosis, differentiation and migration are those that are characteristically dysregulated in malignancy. squamous cell carcinoma (scc) is the most common malignant tumor of the oral cavity, accounting for over 90% of the malignant neoplasms. nearly 60% of the patients present with an advanced stage of disease and its prognosis is influenced by a variety of factors, the most important being tumor stage (tumor size), tumor site, tumor thickness (depth of invasion), tumor grade (broder 's histological grading) and nodal status.. cd44 is the major human cell surface receptor for hyaluronate and functions in a diverse range of physiological processes. cd44 may play a role in stimulating in vivo aggressiveness of tumors through hyaluronate - rich stroma. expression of cd44 has been described to correlate with metastasis in various tumors, although evidence in oral cancers is inconclusive. the purpose of the present study was to examine cd44s expression in oscc and to investigate its correlation with a number of established prognostic parameters such as tumor stage (tumor size), tumor site, tumor thickness (depth of invasion), tumor grade (broder 's histological grading) and nodal status. thirty cases of oscc were retrieved from the archives of our college which included 10 cases of well differentiated (wd scc), 10 cases of moderately differentiated (md scc) and 10 cases of poorly differentiated squamous cell carcinomas (pd scc). the avidin - biotin - peroxidase method was performed using the primary monoclonal antibodies against cd44s, standard isoform (anti - cd44 antigen, clone bgx- 297). briefly, the sections were deparaffinized and washed in phosphate - buffered saline (pbs). endogenous peroxidase activity was blocked using 0.3% solution of hydrogen peroxidase at room temperature for 5 minutes. after microwave treatment for antigen retrieval, primary antibodies were applied for 60 minutes at room temperature and washed in pbs. linking antibody and hr - peroxidase complex (biogenex super sensitive polymer - hrp detection kit) were added consecutively for 20 minutes at room temperature and washed in pbs. the peroxidase activity was visualized with diaminobenzidine (dab), applied for 5 minutes. the degree of positive staining for cd44s antibody was evaluated by a well - established semiquantitative scoring on a scale of 1 to 4 for intensity (i) such as none, mild, moderate and strong ; and for distribution (d) such as none, focal, patchy and diffuse. tissues with i d less than or equal to four were considered weakly positive and those with i d greater than four were designated strongly positive. tumor thickness or histological depth of invasion was assessed objectively using oculometer placed in the microscope. measurements excluded layers of surface keratin and parakeratin. the cases were separated into three groups : tumors with depth of invasion equal or less than 4 mm, tumors with depth of invasion greater than 4 mm but less than 8 mm and tumors with depth of invasion greater than 8 mm. the data so obtained for wdscc, mdscc0 and pdscc are listed in table 1. the degree of positive staining for cd44s antibody was evaluated by a well - established semiquantitative scoring on a scale of 1 to 4 for intensity (i) such as none, mild, moderate and strong ; and for distribution (d) such as none, focal, patchy and diffuse. tissues with i d less than or equal to four were considered weakly positive and those with i d greater than four were designated strongly positive. tumor thickness or histological depth of invasion measurements excluded layers of surface keratin and parakeratin. the cases were separated into three groups : tumors with depth of invasion equal or less than 4 mm, tumors with depth of invasion greater than 4 mm but less than 8 mm and tumors with depth of invasion greater than 8 mm. the data so obtained for wdscc, mdscc0 and pdscc are listed in table 1. using the above criteria following results were obtained : cd44s immunoexpression in the three grades of oral scc : higher mean cd44s immunoexpression is observed in wd scc [figures 1, 4 and 8 ] group showed the highest mean 10.80, followed by md scc [figures 2, 7 and 9 ] group with a mean 5.90 and the pd scc [figures 3, 5 and 6 ] group showed the least cd44s immunoexpression of 3.70. the difference in mean expression between the groups is found to be statistically significant (p value = 0.002) [table 2, graph 1]from the above anova table, we conclude that there is a significant difference between the three groups with respect to the mean cd44s immunoexpression (p 0.05) [table 3]association between cd44s immunoexpression and tumor site : the association between site and cd44s immunoexpression is not found to be statistically significant (p > 0.05). both weakly positive and strongly positive cases were seen in higher numbers in buccal mucosal samples [table 4, graph 2]association between tumor stage and cd44s immunoexpression : the association between tumor stage and cd44s immunoexpression is not found to be statistically significant (p > 0.05). except in stage t2, almost all the samples showed strongly + ve cd44s immunoexpression. in stage t3, the samples are equally present in both weakly + ve and strongly + ve cd44s immunoexpression [table 5, graph 3]association between nodal status and cd44s immunoexpression : the association between nodal status and cd44s immunoexpression is not statistically significant (p > 0.05). higher number of n0 samples as well as n1 samples showed strongly + ve cd44s expression category compared to weakly + ve cd44s immunoexpression category [table 6, graph 4]association between tumor thickness and cd44s immunoexpression : the association between thickness and cd44s immunoexpression category is not statistically significant (p > 0.05). samples 0.05) [table 3 ] association between cd44s immunoexpression and tumor site : the association between site and cd44s immunoexpression is not found to be statistically significant (p > 0.05). both weakly positive and strongly positive cases were seen in higher numbers in buccal mucosal samples [table 4, graph 2 ] association between tumor stage and cd44s immunoexpression : the association between tumor stage and cd44s immunoexpression is not found to be statistically significant (p > 0.05). except in stage t2, almost all the samples showed strongly + ve cd44s immunoexpression. in stage t3, the samples are equally present in both weakly + ve and strongly + ve cd44s immunoexpression [table 5, graph 3 ] association between nodal status and cd44s immunoexpression : the association between nodal status and cd44s immunoexpression is not statistically significant (p > 0.05). higher number of n0 samples as well as n1 samples showed strongly + ve cd44s expression category compared to weakly + ve cd44s immunoexpression category [table 6, graph 4 ] association between tumor thickness and cd44s immunoexpression : the association between thickness and cd44s immunoexpression category is not statistically significant (p > 0.05). samples < 4 mm tumor thickness showed high cd44s immunoreactivity in strongly positive category when compared with weakly positive cd44s immunoexpression category [table 7, graph 5 ]. (a) photomicrograph of well - differentiated sqamous cell carcinoma (h&e stain, 100). (ihc stain, 100) (a) photomicrograph of moderately differentiated squamous cell carcinoma (h&e stain, 100). (ihc stain, 100) (a) photomicrograph of poorly differentiated squamous cell carcinoma (h&e stain, 400). (b) cd44s expression of the same (ihc stain, 400) cd44 expression in well differentiated squamous cell carcinoma. (ihc stain, 100) analysis of the cd44s immunoexpression in the three grades of oscc intensity of cd44s expression in the three grades of oscc multiple comparisons test using bonferroni method. dependent variable : immunoexpression bonferroni evaluation of the association between cd44s immunoexpression (weakly + ve and strongly + ve) and tumor site cd44s expression in tumor site evaluation of the association between cd44s immunoexpression category (weakly + ve and strongly + ve) and tumor stage cd44s expression in the tumor stages evaluation of the association between cd44s immunoexpression category (weakly + ve and strongly + ve) and nodal status cd44s expression in nodal status evaluation of the association between cd44s immunoexpression category (weakly + ve and strongly + ve) and tumor thickness cd44s expression according to tumor thickness cd44 is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines and growth factors to the corresponding receptors, docking of proteases at the cell membrane, as well as in signaling for cell survival. all these biological properties are essential to the physiological activities of normal cells, but they are also associated with the pathologic activities of cancer cells. cd44 is known to be a receptor of hyaluronate ; functions in lymphocyte homing, cell adhesion and leukocyte activation. genomic analysis indicated that the cd44 gene has 20 exons and the region encoding the insertion is composed of 10 exons that are alternatively spliced to produce variable isoforms carrying different membrane proximal inserts. expression of the variable exons has been correlated with tumor progression and metastasis in a range of cell types. multiple cd44 isoforms are expressed by normal stratified squamous epithelia, such as the epidermis and the lining of the oral cavity. numerous studies on histopathologic features of tumor and host response parameters in oscc have shown variable prognostic significance. the main prognostic determinate in carcinomas of the oral cavity is stage of the disease. the tnm classification of cancers arising in oral cavity, based upon extent and size of primary tumor, absence / presence and extent of regional lymph node metastasis, is a generally useful and widely applied method for estimating prognosis and planning therapy. several studies have indicated that the down regulation of cd44 variants, including cd44v9, v4/5 and v6 is associated with tumor metastasis in oscc. it has also reported that there is a positive correlation between the reduced immunoexpression of cd44v9, metastasis to lymph nodes and poor survival in oscc of tongue. keeping the above - discussed points in mind, we aimed at studying the relationship of cd44s expression in oscc with the prognostic parameters. in the present study, cd44s expression is observed in wd scc group followed by md scc group and pd scc group. the difference in mean cd44s expression between the groups is found to be statistically significant (p < 0.01). the staining pattern and intensity varied according to the degree of dysplasia and to the degree of differentiation of the scc., fonseca. (2001), and ue., (2007). the decrease in the intensity of the cd44s levels with the increase in the grade of the tumor suggests reduced cell - to - cell adhesion, resulting in easy detachment of the cells from a rigid constitution. low expression of cd44s in oscc tissues may be an indicator of high metastatic potential and may be related to lymph node metastasis. no correlation was found between cd44 expression and tumor stage, tumor site, tumor thickness or nodal status. the pattern and the thickness of tumor and presence of lymphatic - vascular invasion are important indicators in prediction of cervical lymph node metastasis. the pattern of invasion characterized by infiltrative single cell is associated with a higher frequency of regional lymph node metastasis than the pattern composed of invasive cords of tumor. most of the cases in our study were less than 4-mm tumor thickness and greater than 4 mm but less than 8 mm in thickness cases. the reason being, majority of patients had their tumors diagnosed at an early stage. cd44s expression also did not correlate with the nodal status as the tumors were diagnosed early. based on these findings, we conclude that cd44s immunoexpression might be of prognostic value in assessing the stage of the tumor, invasive capacity and metastatic potential of the tumor. but, further studies on a larger samples are needed to establish the prognostic value of cd44 and its variant isoforms. | background and objectives : the study aims at the observation of the immunohistochemical expression of cd44s in oral squamous cell carcinoma (oscc) and to correlate its expression with prognostic parameters.materials and methods : a total of 30 cases of oscc, - 10 cases of each well differentiated (wd scc), moderately differentiated (md scc) and poorly differentiated squamous cell carcinomas (pd scc) were included in the study. the sections were subjected to immunohistochemical study using cd44s antigen marker. the degree of intensity and distribution of cd44s immunostaining was assessed and correlated with prognostic markers such as tumor stage (tumor size), tumor grade (broder 's histological grading), tumor site, tumor thickness (histological depth of invasion) and nodal status.results:cd44s expression by tumor cells in osccs is statistically correlated with tumor grade i.e. higher mean of cd44s immunoexpression was observed in wd scc group (10.80 3.97), followed by md scc group (5.90 3.38) and pd scc group showed least cd44s immunoexpression (3.70 4.64). there was no statistical significance observed with respect to the other prognostic markers.conclusion:based on these observations it can be suggested that the decrease in expression of cd44s in oscc cells may be due to the reduced cell - to - cell and cell - to - matrix adhesion, resulting in easy detachment from the rigid constitution. low expression of cd44s in oscc tissues may be an indicator of tumor invasion and high metastatic potential. |
small airways are considered the major sites of airflow limitation in patients with chronic obstructive pulmonary disease (copd).1 structural and inflammatory changes in distal airways increase with more severe bronchial obstruction in copd.1 furthermore, association of distal airway impairment with mortality has been also observed in patients with copd.2 however, the understanding of the exact role of small airway dysfunction in the clinical features and progression of the disease is still limited.3 one of the reasons for this fact is that, though pulmonary function testing is the gold standard to diagnose and manage copd, there is little agreement on the most useful lung function parameter to assess the small airways.3 the ability of spirometry to indicate small airway obstruction is still debated.4 on the other hand, the forced oscillation technique has been successfully used as a measure of the airway resistance heterogeneity and gas trapping.5 notably, the impulse oscillometry system (ios) has been used to exhaustively investigate small airway dysfunction in patients with asthma.69 in patients with copd, ios has been recently used to measure both proximal and peripheral airway resistance1013 and its relationship with the health status of and dyspnea in patients,10 as well as to detect copd.12 response to a bronchodilator is considered to be a crucial finding to diagnose copd and to distinguish copd from asthma because copd is characterized by progressive airflow obstruction that is only partly reversible,14 whereas asthma is associated with widespread, but variable, airflow obstruction within the lung that is often reversible either spontaneously or with treatment.15 in spite of the not entirely reversible airflow obstruction, patients with copd may show significant bronchodilator responsiveness,1620 defined as : a 12% increase in either forced expiratory volume in 1 second (fev1) or forced vital capacity (fvc), calculated from the prebronchodilator value, and a 0.2 l increase in fev1 or fvc21after an adequate dose of an inhaled bronchodilator, ie, 400 g salbutamol. changes in fev1 or fvc characterize, respectively, flow or volume response after bronchodilator administration. a 12% increase in either forced expiratory volume in 1 second (fev1) or forced vital capacity (fvc), calculated from the prebronchodilator value, and a 0.2 l increase in fev1 or fvc21 the aim of the present study was to ascertain whether a relationship between small airway dysfunction and bronchodilator responsiveness may be demonstrated in patients with copd. small airway dysfunction was assessed by means of ios, and bronchodilator responsiveness was expressed as changes in both fev1 and fvc. female and male patients with stable copd, as defined by the global initiative for chronic obstructive lung disease (gold) criteria,22 were studied and were consecutively recruited from our outpatient clinic at the parma university hospital, italy. the eligibility criteria included the following : 1) smoking history of > 20 pack - years ; 2) fev1/fvc ratio of 20 pack - years ; 2) fev1/fvc ratio of 10,000 patients with copd, which reported a paradoxical response to bronchodilators in nearly 5% of participants.27 compared with white patients, a paradoxical response was twice as common in african american patients (7% vs 3%). in the multivariate analyses, african american ethnic origin, less emphysema, airway wall thickness, worse dyspnea, reduced exercise capacity, and a greater frequency of exacerbations were independently associated with a paradoxical response.27 bronchial hyperreactivity and adverse reactions to bronchodilators and their excipients are considered to be the potential mechanisms underlying the paradoxical response.27 the present study has some limitations. first, our study is a cross - sectional study and we can only infer and not establish small airway dysfunction as a contributor of bronchodilator responsiveness in patients with copd. second, following the standard procedure, we used the forced maneuver to assess bronchial reversibility with salbutamol. vc tends to be underestimated by the forced maneuver compared to the slow one, especially in subjects with airflow obstruction. thus, a greater number of volume responders might have been found with the slow vc maneuver. third, salbutamol inhalation testing is currently being used to assess the bronchodilator responsiveness of the entire bronchial tree in both clinical and research settings. however, the formulation of salbutamol metered - dose inhaler used in the current study is nonextrafine and deposition in the small airways is modest.32 notably, experimental monodisperse aerosols with small - particle nebulized salbutamol or terbutaline showed a greater peripheral lung deposition and greater effects on lung volumes.32,33 we can not, therefore, exclude that the administration of an extrafine aerosol of a beta2-agonist could increase the effects on volume response in patients with copd. however, we administered salbutamol with a spacer and it was demonstrated that the use of a spacer device can reduce the particle size,34 thus increasing the peripheral lung deposition.32,33 in summary, this study shows that stable patients with copd and small airway dysfunction, as assessed by ios, when compared to patients without small airway dysfunction, show more severe airflow obstruction and lung hyperinflation, as well as significant bronchodilator responsiveness in terms of volume, but not of flow. the results of our study confirm the significant role of small airways in the pathophysiology of copd. | backgroundwe investigated whether a relationship between small airways dysfunction and bronchodilator responsiveness exists in patients with chronic obstructive pulmonary disease (copd).methodswe studied 100 (20 female ; mean age : 6810 years) patients with copd (forced expiratory volume in 1 second [fev1 ] : 55% pred 21% ; fev1/forced vital capacity [fvc ] : 53%10%) by impulse oscillometry system. resistance at 5 hz and 20 hz (r5 and r20, in kpasl1) and the fall in resistance from 5 hz to 20 hz (r5 r20) were used as indices of total, proximal, and peripheral airway resistance ; reactance at 5 hz (x5, in kpasl1) was also measured. significant response to bronchodilator (salbutamol 400 g) was expressed as absolute (0.2 l) and percentage (12%) change relative to the prebronchodilator value of fev1 (flow responders, frs) and fvc (volume responders, vrs).resultseighty out of 100 participants had r5 r20 > 0.03 kpasl1 (> upper normal limit) and, compared to patients with r5 r20 0.030 kpasl1, showed a poorer health status, lower values of fev1, fvc, fev1/fvc, and x5, along with higher values of residual volume / total lung capacity and r5 (p<0.05 for all comparisons). compared to the 69 nonresponders and the 8 frs, the 16 vrs had significantly higher r5 and r5 r20 values (p<0.05), lower x5 values (p<0.05), and greater airflow obstruction and lung hyperinflation.conclusionthis study shows that peripheral airway resistance is increased in the vast majority of patients with copd, who showed worse respiratory reactance, worse spirometry results, more severe lung hyperinflation, and poorer health status. small airway dysfunction was also associated with the bronchodilator responsiveness in terms of fvc, but not in terms of fev1. |
lipase plays a key role in dietary fat absorption by hydrolyzing triglycerides to monoglycerides and free fatty acids. as such orlistat is a potent lipase inhibitor that is sold as a weight loss aid in prescription and over - the - counter formulations. it has been demonstrated that orlistat is an irreversible inhibitor that covalently binds to serine 152 residue of lipase, which causes a pronounced in vitro as well as in vivo inhibition of gastric and pancreatic lipases. previous studies on this time dependent inhibition have used two - phase emulsion monitoring activity with a ph stat, which limits sample throughput. several studies have also revealed the significant effect of modifying the formulation of the orlistat on lipase inhibitory activity. it was found in one study that nano - sized particles of orlistat can be used for enhanced in vitro dissolution rate and lipase inhibition. however, to optimize the formulation for pharmacodynamic effect would require different time - consuming and costly clinical studies. hence, there is a need for a simplified in vitro method to guide the selection of a suitable composition of formulation to optimize the release profile for obtaining better pharmacodynamic effect of orlistat products. to address this need, a dynamic in vitro single - phase system this system was able to provide a simulation of the in vivo lipase activity and is capable of directly testing dissolution or gastrointestinal (gi) fluid samples to assess lipase inhibition by orlistat. to validate the developed assay, two different formulations of orlistat porcine pancreatic lipase (type ii) (ec 3.1.1.3), sodium deoxycholate, sodium phosphate monobasic, isopropanol and p - nitrophenyl palmitate (pnpp) were purchased from sigma aldrich (usa). uv - transparent 96-well plates were used for colorimetric quantification on a tecan infinite m200 spectrophotometer. since the majority of triglyceride hydrolysis is done by pancreatic lipase, porcine pancreatic lipase was used as a model enzyme. crude lipase was dissolved in reaction buffer (10 mg / ml) and centrifuged at 7000 g for 10 minutes to remove insolubles. lipase assays were performed in a 96-well, clear, flat bottomed plate with 200 l reaction volume. pnpp was used as a substrate with a reaction buffer of 50 mm sodium phosphate, 5 mm sodium deoxycholate, and 10% isopropanol at ph 8.0 [9 - 11 ]. lipase assays used a 200 l reaction volume and substrate conversion was monitored with a tecan infinite m200 spectrophotometer at 410 nm. all assays were run at 37c and reported results are the average of six replicates that were blank subtracted. to characterize the system, enzyme linearity of was shown by finding the initial rate for increasing enzyme concentrations. for determination of the kinetic constants, enzyme concentration was fixed at 0.01 mg / ml (the lipase concentration was determined per an established calibration curve, data not shown) and the substrate concentration was varied. minimization of the sum of squares gave a fit to the michalis - menten equation. pre - incubation and co - treatment studies were performed to verify single phase irreversible binding. for pre - incubation, the lipase and orlistat were added together and incubated at 37c for varying times before substrate was added and the initial rate (0.99), which provided linear decreases in the activity with time. lipase activity was completely negated with 20 ng / ml orlistat in a sample with 0.1 mg / ml lipase. two different formulations were able to be distinguished in the dissolution test under identical conditions, with formulation 1 showing a faster initial release rate. the effects were most notable with the formulation 2, where small differences in the initial rate of release correspond to significantly higher inhibition (figure 5). the enzyme demonstrated a high degree of linearity for the range of concentrations considered (0 - 2.5 mg / ml) as shown in figure 1. lipase - pnpp single phase catalysis exhibits michalis - menten kinetics with a km=2.7 0.2 m and kcat=0.019 s as shown in figure 2. lipase inhibition assays showed that orlistat is a potent, time dependent inhibitor in single phase solutions. in the pre - incubation study, an exponential decrease in the observed initial rate of the enzyme co - treatment inhibition kinetics showed a linear decrease in the enzyme activity with time (figure 4). for the incubation results, all 2 order fits had a very good fit (r>0.99), which provided linear decreases in the activity with time. lipase activity was completely negated with 20 ng / ml orlistat in a sample with 0.1 mg / ml lipase. two different formulations were able to be distinguished in the dissolution test under identical conditions, with formulation 1 showing a faster initial release rate. the effects were most notable with the formulation 2, where small differences in the initial rate of release correspond to significantly higher inhibition (figure 5). the study demonstrated the utility of an in vitro single phase assay that would be able to directly test gi and/or dissolution samples to determine lipase activity and inhibition by orlistat. the results confirmed and expanded the findings of previous two - phase studies that orlistat is an irreversible, time - dependent inhibitor of lipase [1,3,5,12 - 14 ], despite using a single - phase reaction. in two phase emulsion studies, coalescence of emulsion particles may gave rise to reduced surface area to the point where reaction rates may become limited in part by substrate. in addition, the resulting heterogeneity caused by creaming of the emulsion may compromise the ability to procure representative subsamples for conducting the lipase assay. therefore, the developed single phase assay is arguably better method to evaluate lipase activity. enzyme linearity over a range of physiologically relevant concentrations and adherence to michalis - menten kinetics provide a simple and robust system for testing dissolution or gi samples. additionally, the resolution that this assay provides can distinguish between minor differences in enzyme inhibition. the ability to directly evaluate dissolution samples is validated by the correlation with the hplc release profile. in combination with bio - dissolution system, the method could be developed as a bio - method to determine the suitability of formulation (such ir or sr) for orlistat - type drugs. a potential example of this is sucrose ester, which alters the surface tension of micelles in an emulsion. as such, its effects would not be able to be seen in a single phase assay. optimizing excipient formulations may allow for lower dosages of the active, but will require improved in vitro - in vivo correlations (ivivc) to predict pharmacodynamic behavior before human trials. future studies using this assay with a biorelevant dissolution method and human lipase or gi fluid will verify the sensitivity of the assay in addition to providing a more accurate ivivc. we have developed a very sensitive, single - phase assay for orlistat that mimics the time - dependent inhibition seen in emulsions. additionally, we have shown a successful proof of concept for using dissolution samples in an in vitro assay as a method of estimating in vivo efficacy. | purposeto develop a bio - assay that would be able to directly test gastrointestinal and/or dissolution samples to determine lipase activity and inhibition by orlistat.methodsenzyme assays were performed with porcine pancreatic lipase and para - nitrophenyl palmitate (pnpp) in ph 8.0 reaction buffer at 37c. substrate hydrolysis was monitored by absorbance changes at 410 nm. the dissolution of two orlistat formulations was tested with a usp ii apparatus. samples were hplc analyzed to determine release profile in addition to being diluted and directly assayed for inhibitory effect.resultsthe lipase - pnpp system demonstrates linearity and michalis - menten kinetics with a km=2.7 0.2 m and kcat = 0.019 s1. orlistat showed highly potent and time dependent inhibition with 5 ng / ml effecting 50% activity after 5 minutes in the lipase - pnpp system. dissolution studies showed a correlation of the drug release profile to the inhibitory effect of dissolution samples in the assay.conclusionsthe lipase - pnpp method can be used as an in vitro assay to monitor orlistat inhibition from drug release or dissolution samples. |
reports of constitutional ring chromosome 22, r(22) are rare. the majority of cases with r(22) are formed de novo, but there are a few reports of familial transmission1). a breakpoint resulting in the loss of the short arm and satellite material has few clinical consequences, whereas a breakpoint on the long arm, which can vary in size, can affect phenotypic expression depending on the size of the deletion2). the phenotypes of individuals with r(22) can further be affected by the continuously evolving mosaicism, caused by the mitotic instability of the ring chromosome. sister chromatid exchanges during mitosis can lead to the formation of dicentric or interlocked rings and subsequent aneuploidy or rearrangements within the chromosome4,5). central nervous system (cns) atypical teratoid / rhabdoid tumors (atrt) are rare, highly malignant tumors primarily occurring in young children under 3 years of age. the majority of atrt cases display genetic alterations of smarcb1 (ini1/hsnf5), a tumor suppressor gene located on 22q11.2, resulting in loss of ini1 protein. ini1 protein is ubiquitously expressed in the nuclei of all normal cells and can be identified using immunohistochemistry. subsequent loss of ini1 protein expression comprises a relatively specific and sensitive diagnostic marker for atrt6). here we present a 4 month - old - boy with 46,xy, r(22)(p13q13.3). high - resolution microarray analysis revealed a 3.5-mb deletion at 22q13.31q13.33. at 11 months of age, an atrt in the cerebellar vermis was detected after brain magnetic resonance imaging (mri). to our knowledge, this is the first reported case of a patient with an atrt and r(22) in korea. a 4-month - old boy was referred to department of pediatrics, daegu catholic university of medicine center because of delayed development. he was the first child of healthy, unrelated korean parents without any known disorders in their family histories. the mother was 22 years old and the father was 27 years old at the patient 's birth. the patient was born at 37 weeks of gestational age by uncomplicated spontaneous vaginal delivery, and he weighed 2,500 g. the physical examination revealed unremarkable findings, except generalized hypotonia with decreased deep tendon reflexes of both knee joint was found. the patient weighed 7.4 kg (50th percentile), and was 65 cm tall (50th percentile). cytogenetic analysis on peripheral blood lymphocytes revealed an r(22) in all analyzed cells, specifically 46,xy, r(22)(p13q13.3) (fig. 1). to specify the breakpoint, high - resolution microarray analysis was performed. upon analyses of the genomic dna using an affymetrix cytoscan 750k array analysis (santa clara, ca however, the smarcb1 (ini1/hsnf5) gene at 22q11.2, which lies proximal to the break point, was not deleted. thus, this chromosomal alteration of the proband was possibly de novo. at 11 months of age, the patient was brought to the emergency department with altered consciousness after falling out of his bed at home. however, it 's unfortunate that we obtained small biopsy specimens. on histopathologic examination, the tumor cells, which had some cytoplasm and vesicular nuclei, were larger than cells of medulloblastoma. the typical rhabdoid tumor cells with eosinophilic cytoplasm were not appeared, and there were no organoid arrangements such as rosette or palisading pattern in the obtained tumor tissue. immunohistochemical staining for expression of the ini1 protein, showed loss of nuclear expression in the tumor cells (fig., the patient 's condition deteriorated with status epilepticus. a ventriculoperitoneal shunt for hydrocephalus underwent in the referred hospital. the chemotherapy (vincristine, cisplatin, doxorubicin, and cyclophosphamide) has been done until now. ring chromosomes are usually resulted from two terminal breaks in both chromosome arms, followed by fusion of the broken ends, or from the union of one broken chromosome end with the opposite telomere region, leading to the loss of genetic material. they can also be formed by fusion of subtelomeric sequences or telomere - telomere fusion with no deletion4). in a ring chromosome, the primary deletion associated with ring formation may be accompanied by a secondary loss or gain of material, and the instability in the ring chromosome contributes to the variable phenotypes observed4). although the phenotypic spectrum of r(22) is broad and can range from mild to severe2,7,8), carriers of r(22) present with most features of 22q13.3 deletion syndrome2,3). in both r(22) and 22q13.3 deletion syndrome, shank3 is suggested to be the most likely candidate gene influencing neurobehavioral features3). shank3 codes for a scaffolding protein that lies at the core of the postsynaptic density in glutamatergic synapses. the 22q13.3 deletion syndrome (or phelan - mcdermid syndrome) typically results from the loss of the distal long arm of chromosome 22. this may result from simple deletions, an unbalanced translocation, or other structural rearrangements involving chromosome 229). major clinical features include neonatal hypotonia, moderate to severe intellectual impairment, severe delayed or absent expressive language, normal to accelerated growth, autism - associated characteristics, and minor dysmorphic features9). generally speaking, individuals with the 22q13.3 deletion syndrome have no life threatening organic malformations. carriers of r(22) may also present with the features of nurofibromatosis type 2-associated tumors such as vestibular schwannomas, multiple meningiomas, and neurofibromas. in these cases, the pathogenesis of these tumors was explained by the loss of both alleles of the nf2 gene (neurofibromin 2), a tumor suppressor gene located on chromosome 22q12.210,11). although the nf2 gene was usually intact within the ring, the ring itself was prone to loss during somatic mitosis, and a pathogenic mutation at the nf2 gene on the remaining chromosome was thought to result in tumor development10,11). smarcb1 (ini1/hsnf5) is a tumor suppressor gene located on 22q11.2, proximal to the nf2 gene. in the majority of at / rt cases, genetic alterations affecting the smarcb1 (ini1/hsnf5) include homozygous deletions, heterozygous deletions as well as copy - number neutral loss of heterozygosity and mutations affecting each of all nine exons of smarcb16). immunohistochemistry using an antibody directed against smarcb1 has evolved as a convenient first line diagnostic tool for the diagnosis of atrt. this is especially relevant in small biopsy specimens, where rhabdoid tumor cells can be missed6). in the past, cns atrts were often misclassified as a medulloblastoma, primitive neuroectodermal tumors, or a different malignant brain tumor, because of their clinical, histological, and radiographic similarities6,12,13). however, they are separated from other embryonal tumors by the presence of rhabdoid cells and specific immunohistochemistry6,13). the coexistence of a cns atrt in a child with an r(22) is rare. a previous case reported a cns atrt in a 4-year - old girl with r(22)14). in addition, a 2-year - old girl with 22q13.3 deletion syndrome and a cns atrt was reported15). in this case, although the array comparative genomic hybridization analysis of the patient 's blood revealed only a de novo subtelomeric 7.2-mb deletion of chromosome 22q13.2-q13.33, the results from the frozen tumor tissue demonstrated an acquired somatic frameshift mutation of the ini1 gene and the loss of the de novo germline 22q13 deleted chromosome15). the net effect was the homogeneous inactivation of the ini1 gene, leading to the development of the atrt15). although we could not perform a genetic analysis of the tumor in our patient, we think that the atrt may have resulted from the combined loss of the r(22) and a pathogenic ini1 mutation on the remaining chromosome 22. therefore, monitoring for not only neurofibromatosis type 2-associated tumors, but also for atrts should be performed in all carriers with an r(22). the familial transmission of r(22) from a normal woman to her clinically affected daughter was also reported. the benign r(22) in a parent may undergo further rearrangements not only during mitosis, but also during meiosis to produce an unbalanced chromosome associated with developmental abnormalities2). therefore, both the gene deletion associated with ring formation, and the secondary genetic imbalance, should be considered. | reports of constitutional ring chromosome 22, r(22) are rare. individuals with r(22) present similar features as those with the 22q13 deletion syndrome. the instability in the ring chromosome contributes to the development of variable phenotypes. central nervous system (cns) atypical teratoid rhabdoid tumors (atrts) are rare, highly malignant tumors, primarily occurring in young children below 3 years of age. the majority of atrt cases display genetic alterations of smarcb1 (ini1/hsnf5), a tumor suppressor gene located on 22q11.2. the coexistence of a cns atrt in a child with a r(22) is rare. we present a case of a 4-month - old boy with 46,xy, r(22)(p13q13.3), generalized hypotonia and delayed development. high - resolution microarray analysis revealed a 3.5-mb deletion at 22q13.31q13.33. at 11 months, the patient had an atrt (5.6 cm5.0 cm7.6 cm) in the cerebellar vermis, which was detected in the brain via magnetic resonance imaging. |
it is well known that the anterior mandible contains several anatomical landmarks such as intra bony vascular canal, which is named the mandibular incisive canal (mic) and lingual foramen. the lingual foramen is situated in the midline of the mandible, at the level of, superior or inferior to the mental spines. these anatomical landmarks in the anterior jaw have regained attention for optimizing surgical planning and avoiding complications. the mandibular intermental foramen region is generally considered as a safe area, involving few risks of damage to vital anatomic structures during surgical procedure. however, these safety recommendations are not based on knowledge of the position and course of some anatomical landmarks. the descriptions of lingual foramen and their bony canals dimensions and locations are important to consider during anterior dental surgery (implant placement, genioplastic, or grafting procedures) for avoiding various complications. some of these complications are as follow : intraoperative bleeding, nerve injury, pulp canal obliteration, and neuropraxia of the mandibular incisive nerve. the reported short- and long - term neurosensory disturbances include alteration or loss of pulp sensitivity in the lower front teeth. nowadays, dental implants are regarded as a standard option for the prosthetic rehabilitation of edentulous patients. in most cases, life - threatening hemorrhagic episodes may occur, due to perforation of the lingual cortex while placing dental implants in the anterior third of the mandible. several studies indicated that if the lingual periosteum is ruptured an extensive hematoma develops within this region and progressive swelling of the floor of the mouth may cause upper airway obstruction. cone - beam computed tomography (cbct) has been shown to be superior to panoramic radiographs in displaying the mandibular lingual foramen and their bony canals variations. image quality of cbct systems and their relatively lower dose and cost when compared to conventional computed tomography have allowed more accessible three - dimensional assessment of craniofacial structures in dental practice. cbct allows comparatively higher resolution (spatial resolution of 0.1 mm homogeneous voxel) than spiral ct, which liang. performed an assessment of the superior and inferior genial spinal foramina and canals in a study. the purpose of this study was to assess the course and anatomical variations of lingual foramen and its bony canals with cbct imaging in iranian population. this was a cross - sectional study and cbct images were obtained from patients who were referred to esfahan school of dentistry for preoperative implant placement planning between 2010 and 2011. the sampling method was consecutive and the sample size was 102 (d = 0.25, = 0.05, and = 0.2). inclusion criteria include age above 18 years old and exclusion criteria include bone pathology in the mandible region and syndromic patients. all patients had informed consent for participation in this study. all cbct (galileos, version 1.7) images were taken using a standard exposure and patient positioning protocol. the acquisition parameters were as follows : tube volume, 85 kv ; tube current, 1042 ma ; acquisition period, 14 s ; effective radiation time was between 2 and 6 s ; reformatted imaging time 2.5 min ; and voxel size was 0.3 0.3 0.3 mm. basic observations consisted of the number of lingual foramina and characteristics of its bony canal including the buccal and lingual canal diameters, the canal length, and canal slope. the distance between the terminal end of lingual canal at the buccal and lingual sides from the inferior border of the mandible and alveolar crest were measured [figures 1 and 2 ]. dimensional measurements on a reformatted cross - sectional image of mandibular cbct scatterplot diagram between age and lower lingual foramen distance to crest. this plot shows a negative linear effect between age and distance of lower lingual foramen to crest (p = 0.031, r = 0.248) in this study, we divided participants into three age groups for statistical analysis : under 35, 3555, and above 55 years. afterward, we evaluated the effect of patient age and gender on the dimensional measurements of the anatomical landmark mentioned above. all data were gathered and statistically analyzed by spss version 16. a five percent level of significance the t test and analysis of variance (anova) were used to determine the effect of age and sex on gathered data and pearson correlation was applied to obtain the relation between age and measured dimensions. cbct imaging of the mandible, from 102 patients which included 57 males (54%) and 55 females (46%), were investigated. the mean age was 52.37 (sd : 13.33) years, range 2191 years. 73.5% of patients showed a dentate anterior mandible, and 26.5% of the patients who were referred for cbct imaging were edentulous. from the 102 mandibles investigated, 102 (100%) had at least one lingual foramen. 54 mandibles (52.9%) had two foramina at the lingual side of the mandibular midline and 20 mandibles (19.6%) showed three foramina in the mandibular midline and three mandibles (2.9%) showed four foramina in the mandibular midline. the average length of the superior lingual canals was 7.83 (sd 2.25) mm. the mean diameter of the opening of the superior lingual canals at the lingual side (superior lingual foramen) was 1.12 (sd 0.31) mm and at the labial side, it was 0.68 (sd 0.17) mm. the mean distances between the openings of the superior lingual canals at the labial side and lower border of the mandible, mandibular crest, and buccal plate were 10.08 (sd 2.06), 18 (sd 5.63), and 4.73 (sd 1.85) mm, respectively. the average length of the inferior lingual canals was 6.33 (sd 1.65) mm. the mean diameter of the opening of the inferior lingual canals at the lingual side was 0.9 (sd 0.39) mm and at the labial side, it was 0.57 (sd 0.18) mm. the mean distances between the opening of the inferior lingual canals at the labial side and lower border of the mandible, mandibular crest, and buccal plate were 6.43 (sd 2.07), 21.89 (sd 4.12), and 4.8 (sd 1.87) mm, respectively. the mean distances between the superior and inferior lingual foramina from lower border of the mandible were 14.12 (sd 2.49) and 4.27 (sd 2.65) mm, respectively. also the mean distances between the superior and inferior lingual foramina from mandibular crest were 14.39 (sd 4.82) and 24.27 (sd 5) mm. in our study, 96% courses of the superior lingual canals were running downward to the labial and 3% of the canals were running horizontally and 1% of the canals were directed upward to the labial side. from all the inferior lingual canals, 21.47% of them had courses running downward to the labial, 2.68% of the canals were running horizontally, and 77.8% of the canals tables 1 and 2 show the distinctive dimensional measurements of the superior and inferior lingual foramina and their bony canals regarding to age and sex of the patients. as it can be seen in table 1, the distance between lingual foramen and alveolar crest was significantly larger in less than 35 years old group. also males had significantly larger distances between buccal end of lingual canal from inferior and buccal plate [table 2 ]. the differences between dimensional characteristics of lingual foramen and canal with age groups the differences between dimensional characteristics of lingual foramen and canal with sex furthermore, we evaluated the correlation between the mentioned measurements and patient 's age. as a result, we did not find any significant correlation rather than a correlation between age and lower lingual foramen distance to crest with p value = 0.031 and a0.248 pearson correlation [figure 3 ]. with the increasing use of implants and grafting procedures for anterior jaw bone, the number of reported postoperative complaints has been rising. however, the lingual foramen is well identified on oral radiographs and thus clearly described in textbooks related to radiographic anatomy. knowledge of lingual foramen could be important for presurgical considerations of implant installation in the midline of the mandible. some studies assume a vascular content, its being an anastomosis of the sublingual branch of the right and left lingual arteries. the artery could be of sufficient size to provoke a hemorrhage intraosseously or in the connective soft tissue, which might be difficult to control. previous studies have been performed about frequency, diameter, and other anatomical features of lingual foramen and its canals. the purpose of the present study was to investigate the prevalence and anatomical variations of lingual foramen among iranian population. in our study, 102 mandibles were investigated and all of the images had at least one lingual foramen. study, in which lingual foramen was seen in 82% of the spiral ct images. one possible explanation for this discrepancy is that the reformatting procedure with some ct scans lacking a reformatted cross - sectional slice exactly at the mandibular midline. furthermore, it is possible that the 1-mm slice thickness may have masked smaller diameter structure on the mandibular midline. recently, cbct, which has an approach different from spiral ct, has come to be used widely. cbct allows comparatively less radiation and higher resolution (spatial resolution of 0.3 mm homogeneous voxel) than spiral ct. therefore, the cbct measurements for the mandibular lingual foramen and canal are considered to be reliable. the superior and inferior lingual foramen frequencies in present study were 99% and 74.5%, respectively. yet, our results provide no evidence for those of kawai., which reported that the superior and inferior lingual foramen occurred with frequencies of 86.8% and 83.8%, respectively. the location of lingual foramen and canal is important to avoid complications during surgery in that region. for instance, the mean distances between the superior and inferior lingual foramina from lower border of the mandible were 14.12 (sd 2.49) and 4.27 (sd 2.65) mm, respectively. while kawai. on japanese mandibles showed that distances of the superior and inferior lingual foramina from the inferior mandibular plane were 11.43 (sd 1.56) and 4.42 (sd 2.64) mm, respectively. as it can be obtained, the mean distance between the superior and inferior lingual foramina from lower border of the mandible was greater in iranian population rather than other populations in previous studies. the vertical distance from the alveolar crest to the opening of the superior lingual canal at labial side was 18 (sd 5.63) mm. this means that a long implant is needed to injure the blood vessels. in type a and b ridges, lekcholm. recommended placements of implants no longer than 13 mm in atrophied mandibles (types c and d), the vertical and horizontal dimensions are shorter, and implant length should be considered carefully. also, the mean height of the opening of the superior and inferior lingual canals at the labial side from lower border of the mandible were 10.08 (sd 2.06) and 6.43 (sd 2.07) mm, respectively, in order to their appearance, which is similar to liang., which reported that the mean height of the superior and inferior lingual canals from the lower cortical border were 11.5 (sd 2.8) and 7.4 (sd 2.4) mm, respectively. according to the results of the study, the mean length of the superior lingual canals was 7.83 (sd 2.25) mm and the mean length of the inferior lingual canals was 6.33 (sd 1.65) mm. from the 50 dry mandibles investigated by liang., they showed that the mean length of the superior and inferior lingual canal was 6.8 (sd 2.3) and 6.1 (sd 2.6) mm, respectively. in a previous study performed by liang., they showed that 72% of the canals had courses running downwards to the labial side and 28% of the canals were directed upward to the labial side. in the study we conducted, the majority of the superior lingual canals were running downward to the labial side. and most of the inferior lingual canals were directed upward to the labial side, which is similar to kawai. study. these canal directions may explain the slightly vertical - oval morphology of midline lingual foramina. thus, it is probable to realize in intraoral radiographs depending on differences in the angle of the projected x - rays. in images of the anterior region of the mandible obtain using the intra oral bisecting method, the mental spine is generally observed as a radio opaque region and occasionally a radio lucent pit can be observed near the spine. in our result, the course of the superior and inferior lingual canal was approximately constant. we recommend that one can confirm the mandibular lingual canal from intra oral bisecting images when the course of the mandibular lingual canal parallels the projection angle of the x - rays. regarding the vertical angulations of the x - ray beam in anterior bisecting images (15), the probability projection of the superior lingual canal section and its distal end (inferior lingual foramen) is more than inferior lingual canal, because the x - ray beam is parallel to cortical border of canal. the mean diameter of the superior and inferior lingual foramen in our observation were 1.12 (sd 0.31) and 0.9 (sd 0.39) mm, respectively, while in a previous study they were 0.9 (sd 0.4) mm and 0.8 (sd 0.4) mm, respectively. although smaller canals with a diameter of less than 1 mm are rare in causing a major hematoma, larger canals could be mentioned in the radiologic reports and considered during the preoperative planning procedure. our results showed that the mean diameter of the superior lingual foramen was more than 1 mm. so the superior lingual foramen must be encountered with greater caution during operations to avoid bleeding complication. in our study, two lingual foramens were more frequent (52.9%), this has also been shown in the previous studies. however, the results are in disagreement with those of liang. and tepper. studies, in that, they found single foramen was most frequent. only those patients with a single lingual foramen (24.5% occurrence in our study) will benefit from the inferior location of this foramen, allowing deeper flap surgery or implant placement without risk of damage to the canal. our study demonstrated that up to four lingual foramens have been detected, which support those of katakami. the data indicate that when there was only a single midline lingual foramen (24.5%), it was normally above the genial spine. from a clinical view, the location, not the number, of the midline lingual foramina is important to avoid complications. in this study the mean diameter of the opening of the superior and inferior lingual canals at the labial side was 0.68 (sd 0.17) and 0.57 (sd 0.18) mm, respectively. a previous study demonstrated that the mean diameter of the opening of the superior and inferior lingual canals at the labial side was 0.4 (sd 0.3) and 0.5(sd 0.3) mm, respectively. from these data, we can suggest that may be there is no significance difference of the mean diameter of the opening of the superior and inferior lingual canals at the labial side between various studies. of 389 consecutively taken cone - beam computed tomograms of the mandible by arx., there was no statistically significant influence on the vertical diameter of the lingual foramina by gender (p = 0.34) or age (p = 0.45). also according to the results of our study, there were no significant difference on the diameter of lingual foramen by sex and gender. but we demonstrated that there is greater distance between the inferior lingual foramina to the alveolar crest and also superior lingual foramina to the inferior cortex in male population. furthermore, the results indicated that the distance of buccal end of lingual canal to inferior and buccal plate was also greater in size in male population. we showed that there is a significant difference between gender and distance of lingual foramen to alveolar crest. the results indicated that we did not find any moderate or strong correlations between age of the participants and the obtained measurements. but we only found a weak negative correlation between age of the participants and the distance of lower lingual foramen to crest in our study. this indicates that with increase in age the distance of lower lingual foramen to crest decreases. the most probable explanation for this correlation is that the crest has atrophied due to aging. in this present study, we found some variations in mentioned anatomical landmarks in isfahan population in comparison with previous studies. due to these findings, we suggest that according to different anatomical positions and measurement for lingual foramen and its bony canal in every individual, it is important to consider this point during preoperative planning for surgery and especially, for implant placement in the anterior mandible. furthermore, cbct imaging was able to show the anatomical features of the lingual foramen and its bony canal, to avoid post operative complications. | background : some studies have been performed on assessing the anatomical variations of lingual foramen and its bony canals, in many different countries but no study has been performed in iran yet. the purpose of this study is to assess the anatomical variations of lingual foramen and its bony canals with cone - beam computed tomography (cbct) imaging in isfahan.materials and methods : this was a cross - sectional study in which cbct images taken from 102 patients referred to the radiology department of head and neck in esfahan (iran) university between 2010 and 2011. the presence of the lingual foramen and its bony canals, the locations, sizes, and length were assessed. the distances between the terminal end of lingual canal at the buccal and lingual side from the inferior border of the mandible and alveolar crest were measured. we also evaluated the effect of patient age and gender on the dimensional measurements of the anatomical landmark mentioned above t test, analysis of variance (anova), and pearson 's correlation were used for statistical analysis and p value lower than 0.05 was considered significant.result:all of the cbct images taken showed the presence of lingual foramen. of all the participants, 52% of them had two foramens in their images. the mean diameters of the upper and lower lingual foramen were 1.12 and 0.9 mm, respectively.conclusion:these anatomical landmarks in isfahan population vary from previous studies. all of the images had at least one lingual foramen which demonstrates high prevalence of this anatomy among isfehanian population. therefore, it is recommended to use cbct imaging for preoperative evaluation prior to installing dental implants. |
immunocompromised patients are susceptible to bacterial, fungal, and viral infections that healthy immune systems usually conquer. chemotherapy and radiation treatments can harm or destroy white blood cells such as t cells, b cells, and neutrophils key components of the immune system. the patient is immunocompromised because of powerful treatments for cancer or blood disorders that leave the immune system severely weakened. skin healing is a complex process that involves inflammation, reepithelization, angiogenesis, granulation tissue formation, and deposition of interstitial matrix, besides other events carried out by different types of cells, such as keratinocytes, fibroblasts, and inflammatory and endothelial cells. these phenomena are influenced by the interstitial matrix, growth factors, and other mediators. it is believed that glucocorticoids hinder the cicatrisation process, likely causing a decrease in cellular proliferation, in neovascularization, and in matrix production. in animals, it is possible that the chronic use of corticoids influences negatively the reepithelization, neovascularization, and collagen synthesis. the effect of prolonged use of corticotherapy on surgical wound healing shows conflicting results in the literature. several factors participate in this controversy, depending on the type and dosage of corticosteroids used, species of animals studied, duration of treatment, and methods of evaluation of healing efficacy. hence, the present study is undertaken to study the healing efficacy of a very effective and multifarious medicinal plant of north east india in various types of wounds like immunocompromised model after priming the animals with hydrocortisone therapy. the plant alternanthera brasiliana kuntze (l.) (amaranthaceae) is a herbaceous plant commonly known in brazil as penicillin or joy weed due to its diverse medicinal properties. after validation of its wound healing activity in normal excision and incision wound, angiogenesis, and antioxidant property, we have carried out studies on its healing efficacy in diabetic or burn wound models (communicated). in this paper immunocompromised animals were used as model as this condition also needs urgent attention of the clinician and researchers for the reasons mentioned above. the plant is used against inflammation, cough, and diarrhea in brazilian popular medicine. the extracts of a. brasiliana exhibited antinociceptive effects in mice, antimicrobial effect, and also anti - herpes - simplex - virus activity. aqueous or ethanolic extracts of a. brasiliana were able to block human mitogen - induced lymphocyte proliferation without any toxic effect. in our previous study, we have reported antinociceptive activity of this plant in its methanol extract. leaves of a. brasiliana were collected from the medicinal garden of the department of pharmacology & toxicology, college of veterinary science, khanapara during the months of february june, 2010. i. c. barua, department of agronomy aau, jorhat identified the plant and a voucher specimen (aau / cvsc / pht/02) was deposited in the herbarium. the leaves were washed with water, air - dried, and powdered in an electric blender. about 250 g of powdered leaves was soaked in 1000 ml methanol for 72 hours in a beaker, and the mixture was stirred every 18 hours using a sterile glass rod. filtrate was obtained after passing through a fine muslin cloth and then by filter paper (whatman no 1) three times. it was then concentrated in rotary evaporator (equitron, roteva) at 5060c under reduced pressure. a dark brown methanol extract obtained was stored in air tight container at 4c till further use. preliminary qualitative phytochemical screening of the plant extract was done for the presence of various active principles. ointment of three different concentrations was prepared by mixing 2.5, 5.0, and 7.5 g of meab with 97.5, 95, and 92.5 g of white soft petroleum jelly (s. d. fine chemicals, india) to prepare 2.5, 5.0, and 7.5% ointment (w / w) of meab. effcorlin (hydrocortisone sodium succinate, gsk, mumbai, india) was used to produce immunocompromised status. healthy adult swiss albino mice of either sex, approximately of the same age, weighing 2530 g, and adult sprague dawley rats of either sex weighing 180200 g were used for the study. they were housed under controlled conditions of temperature (25 3c), humidity (50 5%), and 12-hour light - dark cycles with food and water ad lib. the experiments were performed as per guidelines of the institutional animal ethical committee (770/03/ac / cpcsea / fvscaau/ iaec/06/21) and conform to the national guidelines on the care and use of laboratory animals, india. all the animals were closely observed for any infection and those which showed signs of infection were separated and excluded from the study. the ld50 of a. brasiliana was estimated by following up - and - down stair case method in mice using oecd tg-425 guidelines. the parameters for motor activity and gross effect were determined after administration of a. brasiliana orally at a dose of 2.0 g / kg body weight. immunocompromised state was induced by pretreatment with hydrocortisone (hc) at 40 mg / kg body weight (i.m.) in male rats. following one - week pretreatment with hc, the animals were anesthetized by intraperitoneal (i.p.) the dorsal surface of the rat was shaved and the underlying skin was cleaned with 70% ethanol. the excision wounds were made by excising the full thickness circular skin (approximately 60 mm) on the preshaved dorsal surface of the rats. a preliminary study was conducted for selection of the most effective concentration of meab ointment by using 2.5, 5.0, and 7.5% (w / w) ointment for topical application. as 5% (w / w) ointment showed optimum wound healing activity, the experimental animals (rats) were randomly allocated into three groups of 6 animals each. group i served as control and the rats received topical application of the vehicle, that is, soft white petroleum jelly, twice daily for 7 days. animals of groups ii and iii received topical application of 5% (w / w) ointment of a. brasiliana and positive control drug, that is, himax ointment, respectively, twice daily for 7 days. the wound surface area was measured by tracing its contours using a transparent paper on the 8th day, following wounding before wound excision to determine wound contraction. the percent wound contraction was calculated using the following formula : (1)(initial wound sizespecific day wound size)initial wound size100. the granulation tissue was excised on the 8th following wounding day to analyze prohealing biochemical parameters, namely, hydroxyproline and total protein contents. a 10% homogenate of granulation tissue was prepared in 0.15 m kcl containing 5 mm edta. after homogenization, samples were sonicated (labsonic p, germany) ten bursts of 5 sec each at 5 sec intervals and an aliquot was withdrawn for estimation of reduced glutathione (gsh). in the remaining homogenate, triton x-100 was added at 0.1% (v / v). then, the samples were incubated at 4c for 2.5 hours and centrifuged at 4226 g. the supernatant was used for estimation of superoxide dismutase (sod), catalase (cat), and vitamin c content. for histological studies, granulation tissues collected on the eighth day were fixed in 10% neutral formalin solution and dehydrated with a sequence of ethanol - xylene series of solution. microtome sections were prepared at 6 thicknesses, mounted on glass slides, and stained with hematoxylin and eosin and van geison 's stain, followed by observation for histopathological changes under light microscope. data were expressed as mean se, and statistical significance between experimental and control values was analyzed by one - way anova followed by dunnett 's test using graph pad prism 2.01 (graph pad software inc. a. brasiliana showed the presence of alkaloid by wagner 's and dragendorff 's test, steroid by salkowski 's and liebermann - burchard 's test, and triterpenes by salkowski 's and liebermann - burchard 's test. in acute toxicity study, there was no change in motor activity and gross behaviour during 24 h of observation and the extract was found to be safe up to 2000 mg / kg body weight, p.o. the low toxicity of the plant observed in this study suggests that the plant extract is safe and did not affect any of the parameters studied. significant wound healing activity was observed in animals treated with 5% (w / w) ointment of a. brasiliana in the immunocompromised wound. percent wound contraction on the 8th day in the extract - treated group was 77.10%, which was significantly (p < 0.05) higher compared to the control (39.25%) and standard (60.00%) groups (table 1). the level of various enzymatic and nonenzymatic antioxidants increased significantly (p < 0.05) in the granulation tissue of a. brasiliana - treated group as compared to the vehicle - treated control group. the level of reduced glutathione (gsh) was recorded to be 2.38 0.11 g / mg protein in the a. brasiliana - treated group compared to 1.04 0.06 g / mg protein in the control group (figure 1). similarly, antioxidant activities of sod and cat in granulation tissue of extract - treated animals (figures 2 and 3) also increased significantly (p < 0.05) in comparison to the vehicle - treated control animals. protein content of the granulation tissues on the 8th day following wounding in a. brasiliana - treated group was 129.50 2.17 mg / g tissue, whereas, in the control group, it was 85.17 2.83 mg / g tissue (figure 4). the hydroxyproline and vitamin c content of the granulation tissues were significantly higher (p < 0.05) in the group treated with 5% (w / w) of meab than the control and himax treated standard group (figures 5 and 6). histopathological changes of the granulation tissues of control, a. brasiliana - treated, and standard groups in immunocompromised wounds are shown in figures 7(a)7(e). the control group showed necrosis and polymorphonuclear cell infiltration (h&e 400) (figure 7(a)), whereas granulation tissue of a. brasiliana - treated group showed abundance of collagen fibers (van gieson 's 100) (figure 7(b)), fibroblast proliferation, angiogenesis (h&e 100) (figure 7(c)), and development of basement membrane below the necrotic debris (figure 7(d)) indicating wound healing activity of a. brasiliana. the granulation tissue of himax - treated standard group on the 8th day following wounding revealed proliferation of collagen fibers and angiogenesis (h&e 100) (figure 7(e)). the results of the present study clearly demonstrated that meab possessed a definite prohealing action in immunocompromised wound healing as observed by significant increase in the rate of wound contraction, augmented enzymatic and nonenzymatic antioxidant levels, and total protein contents in the granulation tissue, which was also supported by histopathological study. triterpenes are known to promote the wound healing process mainly due to their astringent and antimicrobial property, which seems to be responsible for wound contraction and increased rate of epithelialization. possibly, the constituents like triterpenes and alkaloids of a. brasiliana may play major role in the process of wound healing. however, wound contraction and period of epithelization in immunocompromised animals were lesser than excision wound in normal animals and healing by epithelization was also delayed in this study as it was an impaired wound. myofibroblasts are believed to play a key role in wound contraction by exerting tension on the surrounding extracellular matrix (ecm) and secreting ecm proteins such as collagen to stabilize the contraction. a. brasiliana promoted wound healing, which might be due to fibroblast proliferation and deposition of abundance of collagen fibers as evidenced by histopathological examination of granulation tissue. angiogenesis is a critical component of wound healing. delayed or aberrant revascularization at the wound sites contributes to the etiology of chronic wounds. in our previous study on wound healing activity of meab, by in vivo (excision and incision wound) as well as in vitro chick chorioallantoic membrane model in normal wound, significantly higher rate of wound contraction, tensile strength, and increased angiogenesis were reported as compared to the control group. in the present study also, histopathological studies of granulation tissues of a. brasiliana - treated group revealed angiogenesis, thus corroborating the previous findings. preventive antioxidants, such as super oxide dismutase (sod), glutathione peroxidase, and catalase (cat), are the first line of defence against ross. the constituents present in the a. brasiliana extract might be responsible for promoting the collagen formation at the proliferative stage of wound healing. histopathological study showed better proliferation of collagen fibers in the extract - treated group compared to the standard drug (himax). in the present study, significant increase in the hydroxyproline content of the granulation tissue of the animals treated with a. brasiliana indicates enhanced collagen maturation by increased cross - linking. the skin is a biological interface with the environment and is frequently and directly exposed to prooxidative stimuli including chemical oxidants, ultraviolet and visible irradiation, which are known to promote the generation of reactive oxygen species (ros) and lipid peroxides. antioxidants like super oxide dismutase (sod), catalase (cat), and glutathione (gsh) in granulation tissues hasten the process of wound healing by destroying the free radicals. these findings suggest that decreased oxidative injury in the wound tissue could be due to increased quenching or scavenging of oxygen free radicals by the elevated levels of antioxidants. sod-1 is a key enzyme in the dismutation of the potentially toxic super oxide radicals into hydrogen peroxide and dioxygen. the effects of ascorbic acid on collagen synthesis, antioxidant status, and immunomodulation make it an appropriate supplement for wound repair protocols. the increased ascorbic acid in the extract treated group might be responsible for protecting the cells from oxidative stress leading to better wound healing. the levels of enzymatic and nonenzymatic antioxidant enzymes were augmented in the treated group ; however, another study conducted by us in diabetic wound model revealed significantly (p < 0.05) increased level of antioxidant enzymes, that is, sod, gsh, vitamin c, and catalase in the treated group compared to the control group and level of catalase was even more compared to the standard- drug- (himax-) treated group indicating promising wound healing activity of meab in diabetic wound model (communicated). again in burn wound model antioxidant enzymes, that is, sod, gsh, vitamin c, and catalase, were found to be significantly (p < 0.05) increased in the meab - treated group compared to the control group and all these antioxidant enzymes except vitamin c were significantly (p < 0.05) increased compared to the standard drug also (communicated). in conclusion, it can be interpreted that topical application of a. brasiliana exhibited significant wound healing activity in immunocompromised wound as evidenced by augmented endogenous antioxidants and increased angiogenesis. further pharmacodynamic investigations are required to identify the active fractions responsible for its wound healing activity for formulation of a plant - based herbal product preserving the indigenous heritage with enormous beneficial value to the society. | alternanthera brasiliana (l.) kuntze (amaranthaceae) is a herbaceous plant used against inflammation, cough, and diarrhea in brazilian popular medicine. in our preliminary study, promising wound healing activity of methanol extract of leaves of a. brasiliana (meab) was observed in normal excision and incision wound models. therefore, the present study was designed to investigate the wound healing activity along with the antioxidant enzyme profile during cutaneous excision immunocompromised wound after topical application of 5% w / w ointment of meab in rats. immunocompromised state was induced by pretreatment with hydrocortisone (hc) at 40 mg / kg body weight (i.m.) in male rats. following one - week pretreatment with hc, wounds were created. the vehicle, 5% (w / w) ointment of meab, or standard drug (himax) was applied topically twice daily. healing potential was evaluated by the rate of wound contraction, estimation of enzymatic and nonenzymatic antioxidants like catalase, sod, gsh, protein, vitamin c, and hydroxyproline content, which was supported by histopathological study on the 8th day following wounding. there was significant increase in the enzymatic and nonenzymatic antioxidant parameters in the extract - reated group as compared to control group. histopathological study revealed collagen deposition, fibroblast proliferation, angiogenesis, and development of basement membrane in a. brasiliana group. the results of the present investigation revealed significant wound healing activity of meab. |
breast cancer is the leading cause of cancer deaths among women in developed countries [1, 2 ]. the use of natural dietary compounds to block or delay the onset of cancer is a promising chemoprevention strategy [37 ]. the purpose of these chemopreventive agents is to suppress cancer cell proliferation, induce cancer cell differentiation, or initiate apoptosis in cancer cells. studies have demonstrated that natural phytochemicals containing phenolic compounds possess anticancer properties [810 ]. polyphenols have anticancer functions both in vitro and in vivo [1116 ]. tannic acid (ta) belongs to the class of hydrolysable tannins and is comprised of a pentagalloylglucose core esterified at all functional hydroxyl groups with gallic acid molecules. apoptotic activity is increased in breast cancer and prostate cancer cells in response to exposure to tannin extracts [1820 ]. type i collagen is a common tissue engineering scaffold due to its intrinsically bioactive and biodegradable qualities. collagen is a naturally derived material and, when uncross - linked, is enzymatically degraded. if used as a biomaterial for tissue engineering purposes the ta - crosslinked collagen type i would not only serve as an attachment scaffold for cells but also function as an extended release anticancer treatment. ta - crosslinked collagen sheets enhance wound healing of the skin in rats and we have previously demonstrated that ta - crosslinked collagen sheets promote adipocyte survival while inducing apoptosis in estrogen receptor - positive (er) breast cancer cells. since ta cross - links collagen and has antitumor properties, the combination could prove to be an effective agent to induce localized apoptosis when introduced in tumor environments. if used for tissue reconstruction, ta - crosslinked collagen could provide increased protection against localized tumor recurrence in reconstructed breasts following mastectomy or lumpectomy. the focus of this work is twofold : (1) ta - crosslinked collagen type i can assume the form of small beads that will, in the long - term, form the basis of an injectable tissue reconstruction scaffold ; (2) it investigates the effects of ta on normal human breast epithelial cells and different types of human breast cancer cells lines in vitro. a 1 mg / ml collagen type i solution was prepared from a stock solution of 3.1 mg / ml (advanced biomatrix, poway, ca) as described elsewhere. the ta cross - linked beads were prepared using a nisco encapsulation unit var v1 electrostatic syringe pump loaded with a 60 : 40 ratio of 1 mg / ml collagen : 1.2% alginate solution in milliq water (sigma - aldrich, st. louis, mo) ; the syringe pump was programmed to pump at 10 ml / h into a 1.5% wt / v cacl2 solution (fisher, pittsburgh, pa) in water. one hour after formation, the beads were filtered out using a mesh strainer and placed in ta crosslinking solution comprising ta (10%, 1.0%, or 0.1% wt / v ta / milliq water), 1.5% cacl2, 0.15 m nacl (sigma - aldrich), and 1.1% wt / v n - cyclohexyl-2-aminoethanesulfonic acid (sigma - aldrich) buffer overnight. twelve hours later the ta - crosslinked beads were placed in 50 mm sodium citrate (fisher) for 3 h, washed with deionized (di)h2o, and stored in phosphate buffered saline (pbs) at 4c. a sample of each concentration of the ta - crosslinked beads was immersed in commercially available blue food coloring for 12 h at room temperature. dyed beads were placed individually in dih2o and the temperature was gradually increased until the bead morphology was distorted. the normal human breast epithelial cell line mcf10a and cell lines isolated from human breast cancers, mcf7 and mda - mb-231 (all from atcc ; manassas, va), were grown in dulbecco 's modified eagle medium (dmem) with mammary epithelial cell growth medium (megm) supplements (epidermal growth factor, insulin, bovine pituitary extract, and gentamicin ; lonza ; walkersville, md) with 10% fetal bovine serum (atlanta biologicals ; atlanta, ga) and 1% antibiotic / antimycotic (life technologies, grand island, ny). the cultures were maintained at 37c with 5% co2. for studies using 0.4 m transwell inserts (corning ; corning, ny), the cells were seeded into the individual wells at 30,000 cells / well of 12-well cell culture plates and grown for 24 h. collagen beads crosslinked with tannic acid at concentrations of 10%, 1.0%, or 0.1% were placed on the transwell inserts. each concentration of ta crosslinked collagen beads was placed in three transwell inserts per plate (400 l / transwell insert). one 12-well plate was used for each timepoint (24, 48 h) examined. cultures of treated and untreated breast cells were washed once with pbs, fixed for 10 min with neutral buffered formalin, washed twice with pbs, and stained with hematoxylin and eosin. all fixation and staining were performed in wells of culture plates. apoptotic cells were visualized using click - it tunel alexa fluor imaging kits from life technologies (grand island, ny) according to manufacturer instructions. images were acquired using a zeiss camera (thornwood, ny) and the cells were manually counted. at least 1500 cells per cell line and treatment (i.e., ta concentration) were counted in random fields in each culture well. the sr flica caspase 9 kit and fam caspase 3/7 kit from immunochemistry technologies, llc (bloomington, mi), and the guava caspase 8 fam kit from millipore (hayward, ca) were used. cultures of mcf10a, mcf7, and mda - mb-231 cells were treated as outlined above. protein lysates were collected using m - per mammalian protein extraction reagent (thermo scientific ; rockford, il) with halt phosphatase inhibitor cocktail (thermo scientific) added. protein concentration was determined using a pierce bca protein assay kit (thermo scientific). lysates were combined with laemmli 's sds sample buffer (boston bioproducts ; boston, ma) and boiled for 5 min. proteins were separated using a 415% criterion tgx gel (bio - rad), transferred to nitrocellulose, and blocked for 1 h with 1% casein in pbs (bio - rad). membranes were probed with anti - caspase 7 and anti--actin (1 : 1000 ; cell signaling technology ; danvers, ma) overnight at 4c. membranes were washed thrice with 0.1% tween-20 in pbs and incubated with hrp - conjugated secondary antibodies (1 : 2500 ; cell signaling technology) for 2 h at rt. chemiluminescence was provided by lumiglo reagent (cell signaling technology) and detected using a fluorchem m (cell biosciences ; santa clara, ca). each experiment was performed a minimum of three times, with at least three replicates performed within each experiment. samples of the three concentrations of ta - cross - linking collagen type i beads underwent denaturation. once dyed, individual beads were placed in 25 ml of dih2o and subjected to increasing temperatures until noticeable shrinkage occurred (figure 1). the average temperature required for denaturation of the 0.1% ta beads was 58.4c 1.5c, while for 1.0% ta beads the denaturation temperature was 61.2c 1.3c and for 10% ta beads the denaturation temperature was 63.8c 1.8c (table 1). the denaturation temperature of the 0.1% ta beads was significantly lower than that of both the 1.0% and 10% ta beads (p < 0.05). these results are in agreement with previous reports demonstrating that uncross - linked collagen scaffolds have a denaturation temperature of 55c, while collagen scaffolds crosslinked with 1 mg / ml ta elevated the denaturation temperature to 68c. the ta - crosslinked collagen beads are stable at human body temperature and therefore have the potential to serve as a growth scaffold for cells during tissue reconstruction. the normal human breast epithelial cell line mcf10a and the human breast cancer cell lines mcf7 and mda - mb-231 were grown in conventional 2d cultures. mcf10a cells are immortalized normal breast epithelial cells ; mcf7 cells are er, while the mda - mb-231 cells are triple negative breast cancer cells (er negative, progesterone receptor (pr) negative, and her2 negative) [2729 ]. collagen type i beads crosslinked by various concentrations (10, 1.0, and 0.1%) of ta were added to transwell inserts placed above the growing cell lines. figure 2 illustrates the different effects of the various concentrations of ta on the three cell lines. in cultures of mcf10a cells treated with ta, a change in morphology of the cells treated with the two higher doses of ta (10% and 1.0%) was observed after 24 h, while it was not until 48 h of treatment that a noticeable change in phenotype was observed in the cells treated with the lowest concentration of ta (figure 2(a)). the changes in observed morphology include rounder cells with fewer protrusions, an increase in the number of detached cells, and an increase in the interstitial space between cells in the cultures. all three concentrations of ta inhibited the growth of the mcf10a cells after 48 h of exposure (figure 3(a)). the highest concentration of ta used to cross - link the collagen beads had a dramatic effect on the morphology of mda - mb-231 cells within 24 h of exposure (figure 2(b)). the lower two concentrations of ta had noticeable effects on the mda - mb-231 cells after 48 h of treatment. the changes in cellular morphology of the mda - mb-231 cells were similar to those observed in mcf10a cells, with the addition of brown precipitate seen in the cells exposed to the high concentration of ta. the proliferation of the mda - mb-231 cultures was inhibited for 24 h after initial exposure to the ta ; the cell numbers increased after 48 h but not to that of untreated levels (figure 3(b)). the effects of ta on mcf7 cells were similar to those seen on mcf10a cells. there was a change in cell morphology, from spindle shaped to more round cells (figure 2(c)). at the highest concentration of ta, brown precipitate if ta reduces cell numbers and alters cell morphology of breast cells by inducing apoptosis, ta - treated cultures of breast cell lines were subjected to tunel assays. previous studies have demonstrated that mcf7 cells are sensitive to the effects of ta [24, 30 ]. only the highest concentration of ta, 10%, significantly induced apoptosis in the normal mcf10a cells after 24 h of exposure (figure 4(a)). two concentrations of ta induced significant levels of apoptosis in mda - mb-231 cells compared to untreated mda - mb-231 cells (figure 4(b)). mcf7 cells were more sensitive to ta exposure, as all three concentrations induced significant apoptosis within 24 h of exposure and the percentage of apoptotic cells remained significantly higher after 48 h in response to the two higher concentrations of ta, unlike in the other two cell lines (figure 4(c)). the addition of the highest dose of ta initiated apoptosis in all three cell lines within 24 h of treatment, with the breast cancer cells having a greater number of apoptotic cells when exposed to the lower levels of ta compared to the normal mcf10a cells. the levels of activated caspase 9 found in mcf7 cells were significantly higher than the levels observed in mcf10a and mda - mb-231 cells, for the two highest ta concentrations used after 24 h of exposure (figure 5(a)). levels of activated caspase 9 in mcf10a and mda - mb-231 cells were only significantly elevated in response to the highest concentration of ta. all ta - induced elevated levels of caspase 9 remained elevated after 48 h of exposure. levels of activated caspase 3/7 were elevated in response to ta exposure in all three cell lines. mcf7 cells are most sensitive to the proapoptotic effects of ta, as significant elevated levels of caspase 3/7 were found in response to all concentrations of ta (figure 5(b)). the levels of activated caspase 3/7 were also elevated in mda - mb-231 cells and in mcf10a but not to the extent observed in mcf7 cells. the triple negative mda - mb-231 cells had significantly higher levels of activated caspase 3/7 in response to the highest dose of ta compared to the mcf10a cells. the flow cytometry - based assay does not discriminate between caspases 3 and 7. caspases 3 and 7 share 57% sequence homology and have similar substrate preference [31, 32 ]. the results of the flow cytometry - based caspase activity assays were confirmed using western blotting. we found levels of cleaved caspase 7 elevated in a concentration - dependent manner in mcf10a cells after 24 h of ta exposure (figure 6(a)). the levels of cleaved caspase 7 remained elevated after 48 h of ta treatment. a similar pattern of elevated cleaved caspase 7 was also observed in cell lysates from mda - mb-231 cells (figure 6(b)). additional forms of cleaved caspase 7 were found in mcf7 cell lysates following ta exposure. the smaller cleaved caspase 7 isoforms were only found in er mcf7 cell lysates, not in lysates from the normal mcf10a cells or the triple negative mda - mb-231 cells. in accordance with the flow cytometry results, the mcf7 cells demonstrated the highest levels of caspase 7 activity in response to ta exposure. according to the protein determination assay, equal amounts of protein were loaded in all lanes. however the -actin loading control demonstrates that this did occur in the lanes labeled ta 10, the highest concentration of ta used. we theorize that the high levels of ta crosslinked additional proteins thereby interfered with the protein concentration determination and protein separation. this did not interfere with flow cytometry as intact cells were analyzed in that assay. the er mcf7 cells were more sensitive to the effects of ta, based on activated caspases 9 and 3/7, as lower concentrations of ta were able to induce elevated levels of the caspases compared to the normal mcf10a cells and the triple negative mda - mb-231 breast cancer cells. in all conditions analyzed, the number of cells expressing caspase 8 was less than 1.5%, equal to untreated cells, indicating that caspase 8 is not playing a major role in ta - induced apoptosis (data not shown). ta - crosslinked collagen type i beads are stable at human body temperature and reduce er breast cancer cell numbers at a higher rate than normal breast epithelial cells and triple negative breast cancer cells. the reduction in cell number is via induction of apoptosis through activation of caspases 3/7 and 9. in this study the effects of ta on normal breast epithelial and breast cancer cells were investigated. previous studies have investigated the effects of ta on one type of breast cancer cell and colon cancer cells. two breast cancer cell lines, mcf7 and mda - mb-231, were used in order to determine if the ta affected different forms of breast cancer differently. the two breast cancer cell lines were compared to the mcf10a cell line, representing normal breast epithelial cells. the mcf7 cell line is er, while the mda - mb-231 cell line is triple negative (erprher2). our results indicate that the er mcf7 cells were more susceptible to the apoptotic - inducing effects of the ta. type i collagen is used in a variety of tissue engineering methods, including breast reconstruction following trauma or mastectomy, due to its intrinsic bioactivity, its biodegradability, and its availability. uncross - linked collagen type i is unstable over a period of months, but when a crosslinking agent is added the altered mechanics result in decreased enzymatic and thermal degradability. a leading factor in breast - cancer - related death is the recurrence of a tumor following mastectomy [35, 36 ]. if an anticancer treatment could be incorporated into a tissue - engineered product, the recurrence of cancer might be reduced. the ta - crosslinked beads used in this study have the potential to be such a tool for breast tissue engineering. the ta - crosslinked beads are stable at body temperature (table 1) and the released ta possesses anticancer properties. ta - crosslinked collagen sheets are biocompatible and biodurable and have demonstrated a capacity to decrease wound healing time. taken together, these properties make ta - crosslinked collagen type i, either as sheets or small injectable beads, a biomaterial with great potential in the fight against breast cancer recurrence. multiple epidemiological studies have indicated that diets rich in vegetables and fruits possess anticancer properties. chemicals present in fruits and vegetables trigger apoptosis in cancer cells and have the potential to be effective in cancer prevention and treatment [312 ]. in most cases, the caspase (cysteinyl - directed aspartate - specific proteases) family of cysteine proteases are central regulators of apoptosis [37, 38 ]. apoptosis can be initiated by numerous methods, including activation of cell surface receptors, such as tnf receptor and the fas receptor, or by extracellular stress that induces dna damage or mitochondrial damage [39, 40 ]. each of these pro - apoptotic pathways uses different members of the caspase family. caspases 8 and 9 are initiator caspases, while caspases 3 and 7 are involved in cellular breakdown. in this study, caspase 8 is usually regulated by cell surface receptors such as tnfr and the fas receptor / cd95 ; as such, this indicates that ta is not activating a cell surface death receptor. activation of caspases 3, 7, and 9 can occur due to extracellular stress. caspase 9 is an initiator caspase that, once activated, cleaves and activates downstream caspases such as 3 and 7. the results show that ta exposure facilitated increased caspase 3/7 and caspase 9 but did not increase caspase 8. one possible explanation for the increased sensitivity of the er mcf7 cells to the effects of the ta may be due to the lack of expression of caspase 3 by mcf7 cells. both caspase 3 and caspase 7 are downstream of caspase 9 and caspase 3 can act as a feedback regulator of caspase 9. the lack of caspase 3 within the mcf7 cells may allow unchecked activation of the executioner caspase 7 by the initiator caspase 9. we found levels of both activated caspase 9 and caspase 7 elevated in response to ta. in conclusion, our results demonstrate a previously undocumented mechanism where ta induces apoptosis in breast epithelial cells via activation of caspases 9 and 7. we demonstrated that the er cell line mcf7 was more susceptible to the effects of ta exposure compared to normal mcf10a cells and triple negative breast cancer cells mda - mb-231. ta crosslinks collagen and collagen is a biomaterial used in breast reconstruction surgeries following mastectomies ; thus ta - crosslinked collagen represents a new type of biomaterials that possess anticancer properties. from a clinical perspective this represents a new avenue in the fight against er breast cancer recurrence in patients. | research efforts investigating the potential of natural compounds in the fight against cancer are growing. tannic acid (ta) belongs to the class of hydrolysable tannins and is found in numerous plants and foods. ta is a potent collagen cross - linking agent ; the purpose of this study was to generate ta - cross - linked beads and assess the effects on breast cancer cell growth. collagen beads were stable at body temperature following crosslinking. exposure to collagen beads with higher levels of ta inhibited proliferation and induced apoptosis in normal and cancer cells. ta - induced apoptosis involved activation of caspase 3/7 and caspase 9 but not caspase 8. breast cancer cells expressing the estrogen receptor were more susceptible to the effects of ta. taken together the results suggest that ta has the potential to become an anti - er+ breast cancer treatment or preventative agent. |
circumscribed palmo - plantar hypokeratosis (cph) is a rare dermatosis, first described by perez. in 2002. only about 50 cases have been described in the literature so far, of which two were with a unique plantar localization. the disorder usually affects women (with a female : male ratio of 4:1), with a mean age of presentation of 61 years. it presents clinically with annular erythematous depressed patch rimmed by a slightly hyperkeratotic border ; the lesion is usually unique and localized over the thenar or hypothenar eminence of the palms, rarely on the soles. cph presents clinical stereotypical features even if sometimes it is misdiagnosed as palmar porokeratosis or bowen 's disease. its main histopathologic feature shows a characteristic epidermal depression with an abrupt decrement in the thickness of the stratum corneum, with a sharp stair a 68-year - old woman presented with an acquired, asymptomatic, slowly enlarging erythematous patch on the thenar eminence of her right hand. her medical history included a mammary adeno - carcinoma treated with surgery and radio - therapy 10 years before. examination revealed over the right eminence thenar an approximately 15 10 mm, well - circumscribed circular erythematous area, depressed, with sharp step between involved and uninvolved skin [figure 1 ]. histopathology showed a sharply demarcated depressed area with markedly thinned stratum corneum with normal basal layers. slight dilatation of the dermal superficial vessels was noted, with poor lymphocytic infiltration and edema. there was no evidence of cornoid lamella excluding a diagnosis of porokeratosis. corneocytes of the upper layers showed signs of vacuolization and they became eosinophilic at the edge [figure 2 ]. based on these findings the patient is currently on clinical follow - up and in 7 years there has been no evidence of enlargement or evolution. a circular and well - circumscribed area of depressed and erythematous skin on the right thenar eminence skin biopsy specimen from border of the lesion : sharp step - off from the thick stratum corneum of normal acral skin to the thin stratum corneum of the involved skin (hematoxylin eosin staining ; original magnification 200) cph is a benign dermatosis of unknown origin predominantly seen in adult women (female : male ratio 4:1 ; mean age 61 years). argued that it is a localized acquired epidermal malformation because of the presence of the lesions for many years and the absence of a trauma at the site of the lesions. using transmission electron microscopy, showed a reduction in keratin bundles and keratohyaline granules, and increased lipid in the horny layer, suggesting a primary disorder of keratinization. in contrast to these studies, resnik. suggested that circumscribed palmar or plantar hypokeratosis represents a localized defect in the maturation of keratin, which could follow subclinical episodes of trauma. in interestingly, in this case the lesion had been present for an unusual short time of 6 months. cph is chronic dermatosis with a benign evolution ; the lesions slowly progress or remain stable over years as in our case. only in one recently reported case the lesions typically involve the thenar and hypothenar eminences of the palm or the medial side of the sole. clinically they appear as circular and well - circumscribed areas of depressed and erythematous skin with slightly elevated scaly borders. in most cases, histopathologic features are characteristic and they consist of a localized depression of the epidermis, with a sharp stair between normal and involved skin. the stratum corneum is thinner and orthokeratotic, with a stratum granulosum slightly decreased compared to the adjacent non - involved skin., a biopsy should be performed at the border 's lesion to compare the horny layer of the involved skin with that of the non - involved adjacent skin. in literature, various topical treatments are proposed, including corticosteroids, salicylic acid and retinoids, but they all have been disappointing. urbina. reported resolution in one case after 4 years of topical therapy with calcipotriol. boffa and de gaetano reported a case of resolution after treatment with liquid nitrogen cryotherapy. no improvement was evident after treatment with a topical steroid for 3 months. in summary as has been described in literature, we did not find evidence of trauma as an inciting factor in our case. unlike another reported case, we did not identify evidence of hpv within lesions by immunohistochemistry. we consider this disorder as an acquired non - inflammatory defect in keratinization on acral sites with a remote potential of malignant transformation ; for this reason, we consider a prolonged clinical follow - up essential, as in our case. | circumscribed palmar or plantar hypokeratosis is a rare benign epidermal malformation of the skin. clinically it shows asymptomatic, well - circumscribed, and depressed erythema persisting for many years on the palms or soles. its main histopathologic feature shows a characteristic epidermal depression with an abrupt decrement in the thickness of the stratum corneum, with a sharp stair between normal and involved skin. we describe a case of a 68-year - old woman who presented with an erythematous, asymptomatic, well - circumscribed, depressed patch, on the right thenar eminence which had been present for years. |
it contains various enzymes of which some are involved in milk biosynthesis pathway in the mammary gland while the others are specific for the digestion of proteins, fats or carbohydrates, facilitating the infant 's ability to exploit these hm major constituents. hm also contains biologically active compounds, hormones, vitamins, cytokines and antibodies, as well as polyamines such as spermine (sp), spermidine (spd) and putrescine (put) [25 ]. among the other things, it has been shown that polyamines have important role in gastrointestinal tract functional maturation during the neonatal period preventing the food allergies in sucking babies by decreasing mucosal permeability to antigenic proteins [68 ]. polyamine oxidase (pao ; ec 1.5.3.11) enzyme, which is present in all vertebrate tissues and biological fluids represent one of the key enzymes in catabolic pathways of polyamines. pao catalyzes the oxidative deamination of sp or spd, producing spermidine or put, depending on the substrate nature [911 ]. diamine oxidase (dao), histaminase (ec, 1.4.3.6), a copper - containing enzyme, catalyses the biodegradation of put, producing -aminobutyric acid (gaba) or malondialdehyde (mda) [12, 13 ]. the study sought to elucidate polyamine metabolism in colostrum (1st and 2nd day) and mature human milk, 30th day of lactation, through the investigation of pao and dao activities, as well as by measuring the levels of mda, the end product of polyamine biodegradation. the study group consisted of 60 healthy nursing mothers, admitted for delivery at a clinic of obstetrics and gynecology in clinical center, faculty of medicine nis, serbia, between may 2010 and july 2010. all the mothers recruited belonged to middle / higher social classes, had to have termed delivery, practiced exclusive breastfeeding, had no treatment during pregnancy except folic acid, and had no history of infection since the beginning of parturition. exclusion criteria were the presence of any clinical sign of lactating mastitis in nursing mothers or failure to meet the inclusion criteria as outlined. the study was approved by the ethical committee for medical research, clinical center, faculty of medicine, university of nis (protocol number 01 - 3565 - 1). the morning samples (between 8 a.m. and 10 a.m.) of human milk had been collected with a manual breast pump (ginevri, milan, italy) on the 1st and 2nd day of lactation (colostrum) as well as 30th day of lactation (mature milk) being stored at 20c, until analyzed. to measure the pao activity, spermine - tetrahydrochloride sigma and to measure dao activity, put - dihydrochloride sigma was used as a substrate. the measurements were done by a spectrophotometric method of bacharach and reaches, modified by quash [14, 15 ]. one unit of the enzyme activity was defined as an increase in optical density of 0.100 at 660 nm. the amount of mda, reflecting the level of lipid peroxidation in human milk, was determined by spectrophotometric method, using thiobarbituric acid (tba) purchased from all milk samples for enzymatic analysis had been dissolved (1:10) in bidistilled water. all statistics were done using the spss computer package version 13 (spss, chicago, il, usa). a total of 60 healthy nursing mothers of term infants, mean age 246,3 years (age range 18 - 36 years) were included in the study. 120 colostrum and 60 mature milk samples were analyzed for enzymes activity. we have demonstrated the significant increase of pao activity as well as marked decrease of dao activity, throughout the first lactation month. we also found persistent drop of mda levels during the same period. the pao activity was significantly lower on the 1st (0.170.02 u / ml) as well as 2nd lactation day (0.180.03 u / ml) compared to pao activity in mature milk, on the 30 lactation day (0.240.06 u / ml, p<0.01) (fig. 1). measured values of pao activity (units / ml) in human colostrum and mature milk on the 1st, 2nd and 30th lactation day ; 1 - 1st lactation day, 2 - 2nd lactation day and 3 - 30th lactation day. dao activities were found to be significantly higher on the 1st (0.1860.02 units / l) as well as 2nd lactation day (0.1750.02) compared to dao activity measured on the 30th lactation day (0.1390.026 units / ml, p<0.01) (fig 2). measured values of dao activity (units / ml) in human colostrum and mature milk on the 1st, 2nd and 30th lactation day ; 1- st lactation day, 2 - 2nd lactation day and 3 - 30th lactation day. mda levels, were higher in colostrum 1st and 2nd days of lactation (2.480.4 mol / ml ; 2.470.38 mol / ml respectively), vs mda level measured on the 30th day of lactation (1.70.26 mol / ml, p<0.001) (fig. 3). measured values of mda levels (mol / ml) in human colostrum and mature milk on the 1st, 2nd and 30th lactation day ; 1 - 1st lactation day, 2 - 2nd lactation day and 3 - 30th lactation day. we have found the significant increase of human milk pao activity during first month of lactation. on the other hand we have demonstrated marked decrease of dao activity and mda levels during the same period. to investigate the effect of stage of lactation on the concentration of sp, spd, and put in sow colostrum and milk, cheng 1997, observed the highest concentration of sp in sow milk on day 7, with levels gradually declining to day 28 of lactation. the decreasing levels of sp was explained as a consequence of increased pao activity, the same was observed in our investigation. as an integral component of the polyamine inter - conversion pathway, pao, has an important place in regulating cellular polyamine levels [7, 11, 18 ]. our results could be explained as a reflection of pao metabolic activity of lactating mammary gland on polyamines synthesis rate. in spite of the fact that the mechanism of polyamines action is not completely elucidated, it was shown that polyamines, mainly sp, spd and much less put have essential role in proliferation, differentiation and migration of mammalian cells. recent data also suggest their important regulatory role on intestinal epithelial cell membrane structure, by means of nucleic acid and protein synthesis promotion, thus improving gut maturation by decreasing mucosal permeability to antigenic proteins. from the clinical viewpoint this could prevent the development of food allergies in suckling babies [23, 24 ], considering our results, demonstrating significant increase in the activity of human milk pao during first month of lactation, it could be possible that pao via its metabolites (spd or put) plays important role in gut maturation, enhancing the maternal milk polyamine supply in the first days of lactation. polyamine oxidase catalyzes the oxidative deamination of sp and spd, producing put as well as corresponding aminoaldehydes, amonia (nh3), and h2o2. these amino aldehydes and h2o2. thus, the increasing polyamine oxidase activity during the first month of lactation, observed in our investigation, may be interpreted as a body tendency to raise the levels of aminoaldehide as a potent antimicrobial agent [25, 26 ]. the naturally substrate for dao is put. put levels in human milk remained very low and varied little during the first week postpartum. dao is also a histamine - degrading enzyme which is produced in high amounts in the placenta and has been supposed to act as a metabolic barrier to prevent excessive entry of bioactive histamine from the placenta into the maternal or fetal circulation. the highest dao activity in colostrum as well as decreased dao activity in mature human milk (30 day of lactation), what we found, may be related in some way with the need of histamine degradation during early lactation [28, 29 ] the importance of mda in milk is even more difficult to evaluate. it was demonstrated that mda from the blood might be excreted through the mammary gland to the colostrum before the parturition. while investigating mda levels in the first weeks of lactation, smith 1976, found progressive mda decrease during this period. the authors stated that dynamic changes of mda level in their study, reflects the intensity of the metabolic changes, under endocrine regulation, that occur in the onset of lactation. our results are in agreement with this observation. to summarize, mature human milk possesses a detectable activity of pao and dao, as well as considerable amount of mda. the malondialdehyde (mda) level, reflecting the level of lipid peroxidation and the final product of polyamine catabolism, also decreases. it is most likely that the early changes in pao and dao activity as well as mda level reflect merely the metabolic activity of the nursing mother mammary glands. it is likely that human milk pao throughout the lactation period, contributes to the protective effects of human milk via amino aldehydes and hydrogen peroxide (h2o2) which induce the oxidative stress. it is to be investigated however, whether these milk constituents have other possible health effects and benefits in various puerperal pathological clinical scenarios. our studies had limitations because the sample size was relatively small and the percentage of mothers with cesarean delivery was relatively high (13%), which may potentially influence the colostrum or mature milk composition. since the products of pao activity such as, amino aldehydes and hydrogen peroxide (h2o2) might have potential antimicrobial effects, promoting the oxidative stress, it is likely that human milk pao throughout the lactation period, contributes to the protective effects of human milk. | objectivehuman milk (hm) is the ideal food for all newborns and infants. apart from various bioactive compounds, including cytokines, antibodies, hormones, vitamines, it also contains polyamines, such as spermine (sp), spermidine (spd) and putrescine (put).aimthe present study investigated polyamine metabolism in colostrum and mature human milk by measuring the polyamine oxidase (pao) and diamine oxidase (dao) enzyme activities, which are necessary for polyamine catabolism, as well as by determining the malondialdehyde (mda) levels, the final product of polyamine biodegradation.methodsthe pao, dao activity and mda levels were quantified in colostrum (1st and 2nd day) as well as in mature human milk, 30th day of lactation.findingswe found the steady increase of pao activity and steady decrease of dao activity and mda levels during first month of lactation.conclusionsince the products of pao activity such as, amino aldehydes and hydrogen peroxide (h2o2) might have potential antimicrobial effects, promoting the oxidative stress, it is likely that human milk pao throughout the lactation period, contributes to the protective effects of human milk. |
the online version of this article doi:10.1007/s10886 - 007 - 9404 - 0 contains supplementary material, which is available to authorized users. plants respond to herbivore attack with an array of changes in plant chemistry, morphology, and physiology, which frequently result in the increased resistance of plants to further attack (karban and baldwin 1997). induced resistance may be caused by direct effects on the herbivore through plant - derived toxic metabolites or anti - digestive and anti - nutritive compounds. furthermore, plants may utilize indirect defenses that facilitate top - down control of herbivore populations by the herbivore s predators and parasitoids (price. one way a plant may attract beneficial arthropods is by providing suitable food sources such as extrafloral nectar (efn ; koptur 1992). efn is an aqueous solution, with sugars and amino acids being the most abundant solutes (ruffner and clark 1986 ; galetto and bernardello 1992 ; heil. in addition, plants may increase the emission rate of volatile organic compounds (vocs) in response to herbivore attack. vocs can serve as a cue that guides foraging parasitoids and predators to the feeding herbivore (turlings. the composition of the herbivore - induced volatile blend depends not only on the plant species or cultivar but also varies with the species and even the larval stage of the herbivore (for review, see arimura. 2005), thus providing highly reliable signals to the members of the third trophic level. in many cases, plants do not rely on a single defense strategy but employ a complex array of different defensive mechanisms. for example, several plant species feature both voc emission and efn secretion (arimura. the presence of two indirect defenses within one individual plant gives rise to the questions whether both defenses contribute to plant fitness and how they interact in attracting beneficial arthropods (price. studies on the benefit of indirect defense traits in nature are generally rare (but see thaler 1999 and kessler and baldwin 2001). no study to date has tried to study the defensive roles of two indirect defenses within one plant species simultaneously. the lima bean (phaseolus lunatus) is a common model system in studies on induced indirect defenses. heil (2004b) showed that plants growing in the wild, which had been induced by exogenous application of the phytohormone jasmonic acid (ja), responded by increasing both their voc emission and efn secretion. the application of ja repeatedly (i.e., every 3 days) resulted in benefits for the treated plants as reflected by a decreased herbivory rate and an increased seed set. however, ja also affects fitness - relevant processes such as fruit development and ripening (creelman and mullet 1997). thus, the multiple effects constrain the interpretation of the observed morphological changes as being exclusively attributable to induced defenses. furthermore, kost and heil (2005) demonstrated that an artificial increase of the amount of available efn benefits lima bean in nature by attracting predacious and parasitoid arthropods. these two pilot studies represented a starting point for unraveling whether both induced defense traits contribute to plant defense or whether efn secretion solely is responsible for lima bean protection. we chose an experimental approach similar to the one used in heil (2004b) : groups of five tendrils were used as experimental units (fig. 1 ; table 1). the inclusion of two additional bean tendrils with artificially increased amounts of either vocs or efn enabled us to study the protective effect of the two defenses. five groups of lima bean tendrils (p. lunatus) served as experimental unit with c no treatment, tc application of lanolin paste, ja spraying with jasmonic acid, v application of a synthetic volatile blend dissolved in lanolin paste, and n application of a synthetic mixture of efn. see table 1 for detailstable 1five chemical treatments and their effect on treated tendrilsgroup name (abbreviation)treatmentdefensive traits (i = induced, e = experimentally increased)effectcontrol (c)untreatednoneuntreated controltreatment control (tc)lanolin pastenonelanolin paste onlyja - treatment (ja)javocs (i) efn (i) putative direct resistance (i)induction of vocs, efn, and putative ja - dependent direct defenses (heil 2004b)volatile treatment (v)volatile mixture dissolved in lanolin pastevocs (e) efn (i)induction of efn, attraction of arthropods to vocs and efn (kost and heil 2006)nectar treatment (n)synthetic mixture of efnefn (e)attraction of arthropods to efn (kost and heil 2005)ja jasmonic acid, efn extrafloral nectar, voc volatile organic compound experimental design. five groups of lima bean tendrils (p. lunatus) served as experimental unit with c no treatment, tc application of lanolin paste, ja spraying with jasmonic acid, v application of a synthetic volatile blend dissolved in lanolin paste, and n application of a synthetic mixture of efn. see table 1 for details five chemical treatments and their effect on treated tendrils ja jasmonic acid, efn extrafloral nectar, voc volatile organic compound we repeatedly applied these five treatments to address the following questions : (1) what is the relative contribution of the two indirect defenses volatile emission and extrafloral nectar secretion to the overall herbivore defense of the lima bean ? (2) is there an additional influence of a putative ja - dependent direct defense ? and (3) what kind of arthropods (including putative defenders) are attracted to the treated tendrils ? study site and species this study was conducted in the coastal area near puerto escondido in the state of oaxaca, mexico. the climate in the study area is characterized by one main rainy season from june to october, which follows a bimodal distribution peaking in july and september. annual rainfall averages between 1 and 1.4 m, and the mean annual temperature is 28c (strssne 1999). the two sites were used in previous studies (heil 2004b ; kost and heil 2005) and located 15 km northwest of puerto escondido and about 3 km apart (1555.596n/09709.118w and 1555.357n/9708.336w). in this study, lima bean grows naturally along dirt roads that lead to extensively used pastures or plantations. all experiments were performed on this native population of lima bean plants in 2003 and 2004, during the transition from wet to dry season (october to december). design of the long - term experiment to reduce environmental and genotypic variability, 23 groups of five lima bean tendrils were selected as experimental units. within a unit, the maximum distance between the two outer tendrils was less than 3 m, and tendrils forming one group usually were part of the same plant individual, although this could not always be ensured because of the tangled growth of the lima bean. all selected tendrils were trained along supporting ropes, and the tendrils of each group assigned randomly to one of five treatments (for details, see the next section) : tendrils were either left untreated (control group) or treated every 34 days with lanolin paste only (treatment control group), sprayed with an aqueous solution of ja (ja group), or supplied with a synthetic blend of vocs (volatile group) or efn (efn group ; fig. 1 ; table 1). application of pure lanolin served as a procedural control to test for any effect caused by lanolin alone. starting 27th october 2003, the experiment lasted 25 days and comprised six applications. during this time, the initial number of tendril groups was reduced to a final sample size of 17 because of cattle and human impact. treatment of tendril groups tendrils of the ja group were sprayed with an aqueous solution of 1 mmol ja until runoff (i.e., approximately 10 l per cm leaf area). levels of ja were adjusted to those of previous experiments in which similar concentrations were applied without phytotoxic effects becoming apparent (heil 2004b). the synthetic volatile blend and the synthetic mixture of efn were adjusted to mimic their natural models within 3 days post - induction according to previous experiments (kost and heil 2005, 2006). however, the amount of vocs and efn cumulatively applied ranged largely below the physiological limit of a plant for producing the two indirect defenses within this period (kost and heil 2005, 2006). the synthetic volatile blend consisted of 0.12 g (r)-()-linalool, 0.13 g -caryophyllene, 0.19 g methyl salicylate, 0.26 g (z)-jasmone (all purchased from sigma - aldrich), 0.02 g (3z)-hexen-1-yl acetate (avocado research chemicals ltd., leysham, lancaster, uk), 0.85 g (e, z)--ocimene (mixture of e / z - isomers approximately 70:30 ; kindly provided by roger snowden, firmenich, geneva, switzerland), 0.63 g (3e)-4,8-dimethyl - nona-1,3,7-triene (dmnt), and 0.9 g (3e,7e)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (tmtt ; synthesized by standard methods ; pattenden and weedon 1968) per microliter lanolin. pure lanolin and lanolin paste that contained volatiles were spotted on green plastic strips attached to tendrils to prevent any diffusion of compounds into the treated plant. a total of 120 l of paste were applied per five leaves of either the volatile- or the treatment - control group. the synthetic efn consisted of an aqueous solution of 4.01 g l sucrose and 24.24 g l of each fructose and glucose. of this blend, 40 l were applied with an eppendorf pipette directly to the extrafloral nectaries of every trifoliate leaf of the efn group. comparison of treatments to ensure comparability between experimental groups of the long - term experiment, we quantified voc and efn levels of differently treated tendrils in preceding experiments. the ability of our voc mixture to mimic the volatile emission of herbivore - damaged and ja - induced tendrils was examined by quantifying the vocs present in the headspace of tendrils that had received one of five treatments. tendrils were either left untreated or treated with lanolin paste, infested for 48 h with a representative mixture of natural herbivores of lima bean (six ensifera or caelifera and ten beetles of various species, i.e., cerotoma ruficornis, gynandrobrotica guerreroensis, epilachna varivestis, and a curculionid species ; for details, see kost and heil 2006), treated with a blend of synthetic vocs, or sprayed with a 1-mmol aqueous solution of ja. groups of five tendrils that had received the different treatments originated from plants that were spaced apart at a maximum distance of approximately 3 m, had five leaves, matched each other in terms of leaf age and visual appearance, and were collected at the same time for simultaneous volatile collection. treated tendrils were detached, immediately supplied with a water reservoir, and bagged in a pet foil (bratenschlauch, toppits, minden, germany) that does not emit detectable amounts of volatiles by itself. emitted vocs were collected continuously on charcoal traps (1.5 mg charcoal, clsa filters, le ruisseau de montbrun, france) by using air circulation, as described by donath and boland (1995). after 24 h, volatiles were eluted from the carbon trap with dichloromethane (40 l) that contained 1-bromodecane (200 ng l) as an internal standard. samples were transferred to glass capillaries, sealed by melting the open end, and stored at 0.05). 2effect of different treatments on the secretion rate of extrafloral nectar (efn) given in milligrams soluble solids per gram leaf dry mass per 24 h. comparisons between tendrils treated with lanolin paste (tc), the synthetic volatile blend dissolved in lanolin (v), and jasmonic acid (ja) are shown. different letters indicate significant differences among treatments (univariate anova, p 0.05). community composition of arthropods visiting lima bean in total, 899 arthropods were caught on sticky traps, and > 94% were identified to the order or family level. among them, diptera (55%) and hymenoptera (26%) were the most abundant (table 1 ; fig. other groups trapped included coleoptera (6%), araneida (5%), and thysanoptera (3%). based on information derived from the literature, the trapped arthropods were assigned to feeding guilds, whereas multiple affiliations per taxon were allowed. this analysis indicated that 73% of all trapped arthropods were characterized by parasitoid or predacious life habits and may, thus, be classified as potentially beneficial to the lima bean (fig. 5). on the other hand, only 16% of the trapped arthropods were assigned to herbivorous or flower - feeding groups, which potentially could have had detrimental effects on the plant. other plant - derived food sources, such as efn, pollen, or honeydew, are known to be used by 67% of all trapped arthropod groups, and 19% of all trapped taxa additionally rely on other food sources such as fungi, fruits, or detritus. tourists and are likely to have a neutral effect on the lima bean (fig. 5 ; table s1). insert affiliation of trapped taxa to different guilds : predator / entomophaga (r) ; parasitoid (p) ; utilization of plant - derived resources including floral or extrafloral nectar, pollen and honeydew (s) ; frugivore (f) ; herbivore and flower feeder (h) ; detrivore including phytosaprophage and zoosaprophage (d) ; blood - sucking and ectoparasitic (b) ; and fungivore (m). arthropod groups were assigned to guilds according to nutritional or functional aspects whenever larval or adult stages feature the respective trait. sample size was two traps per 14 tendril groups arthropods taxa trapped on sticky traps. insert affiliation of trapped taxa to different guilds : predator / entomophaga (r) ; parasitoid (p) ; utilization of plant - derived resources including floral or extrafloral nectar, pollen and honeydew (s) ; frugivore (f) ; herbivore and flower feeder (h) ; detrivore including phytosaprophage and zoosaprophage (d) ; blood - sucking and ectoparasitic (b) ; and fungivore (m). arthropod groups were assigned to guilds according to nutritional or functional aspects whenever larval or adult stages feature the respective trait. sample size was two traps per 14 tendril groups a closer look at the trapped arthropod taxa revealed that dolichopodidae (34% of all trapped diptera), phoridae (24%), and chloropidae (7%) were the most abundantly trapped dipterans (table s1). all three groups share parasitoid and predacious life habits and are occasionally known to feed on efn (table s1). the individuals trapped by this superfamily of parasitoid wasps belonged to 16 different families, with eulopidae (13% of all trapped hymenoptera), encyrtidae (12%), and pteromalidae (8%) being most frequently trapped. among the hymenopterans, formicidae (12%) and braconidae (6%) were the most often captured non - chalcid families. comparison of treatments the aim of the present study was to study simultaneously the protective effect of the two indirect defenses, efn secretion and voc emission, on the lima bean under field conditions. the performance of plants that were induced by spraying with ja and, thus, had increased amounts of vocs and efn, was monitored and compared to plants of which the amount of either vocs or efn was increased experimentally. an important prerequisite for any conclusion that can be drawn from such an experimental design is knowledge of the performance of the two indirect defenses under investigation. a quantitative and qualitative comparison of the voc blends emitted from herbivore - damaged, volatile - treated, and ja - sprayed lima bean tendrils revealed besides subtle differences a pronounced similarity among these three groups with respect to the 13 dominant emitted compounds (table 2). however, the volatile blends emitted from these three groups were characterized by a high degree of quantitative variation, an observation well known from literature (kessler and baldwin 2001 ; fritsche - hoballah. 2002 ; rse and tumlinson 2004). the question whether such differences hamper the ability of predators and parasitoids to locate the voc - emitting plant has been studied intensively under laboratory conditions, where it has been shown that carnivorous arthropods can discriminate even minor differences in volatile blends offered (e.g., changes in the enantiomer ratios ; dicke. 1990). a species of curculionid beetles, for example, which also was observed feeding on lima bean in this study (c. kost, personal observation), in laboratory experiments, used vocs of slightly induced plants as a host - location cue, yet avoided plants with high induction levels (heil 2004a). in the field, plants generally show a higher quantitative and qualitative variability of their emitted vocs than under constant laboratory conditions (gouinguene. foraging predators should respond to this situation by relaxing the specificity of their search patterns (dicke. 2003). indeed, field studies on the attractiveness of vocs suggest that predatory and predacious insects are also attracted to jasmonate - induced plants (thaler 1999 ; kessler and baldwin 2001), even if the emitted volatiles differ substantially from blends emitted by herbivore - damaged plants (farag and par 2002). moreover, field experiments indicate that the presence of single compounds can be sufficient to attract carnivorous insects (james 2003, 2005 ; james and price 2004), which then act as indirect plant defenders (kessler and baldwin 2001). consequently, under our experimental conditions, the vocs of both ja- and volatile - treated tendrils likely have attracted plant defenders. 2), thus, confirming recent results obtained for the same plant species under laboratory (choh. 2006) and field conditions (heil and kost 2006 ; kost and heil 2006). hence, the tendrils of the volatile group experienced the combined defensive effect of both vocs and efn to an extent comparable to the tendrils of the ja group. these two defenses are not only connected by the shared signaling molecule ja (heil 2004b) but are also airborne vocs implicated in the induction of efn secretion within one (heil and silva bueno 2007) or between two conspecific plant individuals (kost and heil 2006). consequently, this physiological linkage precludes an experimental separation of the two indirect defenses. treatment effects on the plant tendrils of the ja, the volatile, and the efn group generally benefited from the respective treatments, as these tendrils suffered less herbivory by leaf - chewing herbivores and bore fewer dead shoot tips than the two control groups (fig. the picture, however, changes when other fitness - relevant parameters are considered : the numbers of living shoot tips, leaves, and inflorescences differed significantly from the controls only in tendrils treated with vocs and efn (fig. a possible explanation for the weaker effect experienced by the ja - treated tendrils could be that induction with ja incurred allocation costs to the treated tendrils, which were greater than costs imposed by the voc - induced efn secretion and absent in efn - treated tendrils (heil 2002 ; strauss. 2002). beyond eliciting stress - related responses, such as insect and disease resistance, ja is known to be involved in various physiological or morphological changes not necessarily related to resistance (creelman and mullet 1997). given that these are costly in terms of metabolic resources, the induction of such processes may have affected the measured fitness parameters and could explain the weaker growth and reproductive status of ja - treated tendrils (agrawal. ja may also directly affect processes such as fruit development and ripening (creelman and mullet 1997). an increased seed set of ja - treated plants, therefore, does not necessarily reflect an enhancement of the plant s defense status. furthermore, external application of ja in elevated concentrations can, in principle, have detrimental effects on a plant and cause chlorosis, necrosis, or abscission of leaves (husain. in contrast, the leaves of ja - treated tendrils looked even healthier than those of both control tendrils. additionally, ja application may have induced putative direct defenses (halitschke and baldwin 2004). however, no such alternative defense strategy has yet been described for lima bean. in its close relative, p. vulgaris, english - loeb and karban (1991) did not find evidence for induced direct resistance to spider mites. however, the protective effect of a direct defense, which in our long - term experiment could have been induced after ja application, can not be excluded. in this case, the direct defense did not significantly contribute to plant protection because tendrils with increased amounts of efn only (efn group) or volatiles and efn combined (volatile group) performed better than tendrils with volatiles, efn, and the putative direct defense (ja group ; fig. the development of the fitness - relevant plant parameters measured in the ja - treated group and the two controls confirmed a preceding study (heil 2004b). in both studies, ja treatment increased the number of newly produced leaves and decreased both the number of dead shoot tips, as well as the herbivory rate. the induction of these two indirect defenses was shown to benefit lima bean plants in two independent studies performed in two consecutive years. by applying synthetic vocs and efn externally, we could trace the observed effects back to these two kinds of defensive metabolites and corroborate the importance of efn secretion and voc emission for lima bean defense in nature. treatment effects on the insect community ants were by far the dominant insect group observed on experimental tendrils, and they were significantly attracted to tendrils that experienced the ja, volatile, and efn treatment (fig. although chemical cues are generally important for ants (vander meer. 1998 ; keeling. 2004), little is known about whether plant - derived volatiles also influence ant behavior. some reports are available on the role of yet unidentified plant chemicals that orient ants to their host plant (fiala and maschwitz 1990 ; agrawal and dubin - thaler 1999 ; djieto - lordon and dejean 1999a, b) or facilitate within - host plant patrolling behavior (brouat. 2000). in lima bean ants could use herbivore - induced volatiles as long - distance cues to detect patches of increased availability of efn, which simultaneously are characterized by the increased presence of herbivores (i.e., potential prey). however, preliminary experiments with camponotus novogranadensis, one of the three dominant ant species that visit lima bean at the two study sites (kost and heil 2005) did not support this hypothesis. given the choice in a y - olfactometer, workers of c. novogranadensis neither preferred lanolin paste that contained volatiles over pure lanolin paste. they also did not discriminate between ja - induced vs water - sprayed control plants. however, in previous trials they significantly chose an arm with mashed banana (c. kost, unpublished data). more experiments of this kind will clarify the role of plant volatiles in ant foraging behavior. in contrast to vocs, efn is a known attractant to ants (koptur 1992) that has been reported to translate into enhanced plant protection in the vast majority of studies (for review, see bentley 1977 ; koptur 1992 ; heil and mckey 2003 ; oliveira and freitas 2004), although some studies did not detect a defensive effect (odowd and catchpole 1983 ; boecklen 1984 ; becerra and venable 1989 ; rashbrook. also, wasps were more attracted to the tendrils of the ja, volatile, and efn groups than to the control tendrils, yet in much smaller numbers than ants. the majority of wasps trapped on experimental tendrils belonged to predacious or parasitoid families (table s1). these observations suggest that not only ants but also wasps likely contributed to the protection of the treated tendrils. because of the intrinsic physiological linkage between volatiles and efn, however, it remains unclear which indirect defense was mainly responsible for wasp attraction. as the emission of vocs from a lima bean plant is correlated with its amount of efn secreted, it appears reasonable to assume that wasps may have used vocs as long - distance cues to detect patches with an increased herbivore density and/or efn availability. comprehensive evidence for the volatile - mediated attraction of wasp is available from many laboratory - based studies (e.g., turlings. 1990 ; takabayashi. 1996), yet relatively few field studies. among them, james (2005) identified methyl salicylate as an attractant for parasitic wasps such as encyrtidae and mymaridae. furthermore, braconid wasps were attracted to (3z)-hexen-1-yl acetate and (z)-jasmone (james 2005). these three compounds were also constituents of the synthetic volatile blend used in our study, and the above - mentioned parasitic wasps were also trapped on our experimental tendrils. in another study, dmnt (another constituent of the lima bean s induced voc blend) emitted from molasses grass (melinis minutiflora) significantly attracted parasitoid wasps in the y - tube olfactometer and was likely involved in increasing the parasitation rate of stem - borer larvae that were feeding on nearby growing maize plants (khan. wasps feeding on efn have been reported for several different plant species (for review, see koptur 1992), yet so far, only one study has also demonstrated a fitness - benefit for the efn - secreting plant (cuautle and rico - gray 2003). a more detailed analysis of the attractive effects of vocs and efn on wasps 4), suggesting that flies were more attracted to synthetic efn than to airborne vocs. the community of trapped diptera covered a diverse spectrum of feeding habits ranging from predacious or parasitoid over herbivorous to detritus- or fungi - feeding (table s1). this heterogeneous composition complicates a clear functional assignment of the caught flies. in most cases, the offered efn seems to have been exploited by the flies as an additional food source rather than having contributed to a large extent to plant protection. in this case, the consumption of efn without providing plant protection would cause ecological costs because the efn - producing plant would be less protected against herbivores (heil 2002 ; heil. 2004). in summary, ecological studies on the benefit of indirect defenses have generally focused on the protective role of a single defensive trait. while this simplification is easy to understand from the viewpoint of experimental feasibility, such univariate approaches may be inappropriate because they do not appreciate the complex interplay of several plant defenses that can co - occur within one plant species (duffey and stout 1996). the mere application of synthetic efn (nectar group) resulted in a fitness benefit that was always stronger or quantitatively similar to that experienced by tendrils of the ja and voc treatments (fig. moreover, the number of ants observed on the experimental tendrils (i.e., typical efn feeders but less known to respond to vocs) overwhelmingly exceeded the number of all other arthropod groups (fig. these observations suggest that, under our experimental conditions, the presence of efn was more important for plant defense than was the voc - mediated attraction of arthropods. however, the inducing effect of vocs on efn impeded an experimental separation of vocs and efn and, thus, the exclusion of an attractive effect of airborne vocs on flying or crawling arthropods. this finding underlines the necessity of studying different plant traits such as indirect defenses simultaneously in their ecological context. studying them separately may provide a distorted picture of their true function and likely cause an under- or overestimation of their true effect this issue needs to be addressed in future studies, which should focus especially on the role of volatiles and efn for short- and long - distance attraction of herbivores and plant defenders. several of the arthropod taxa that were identified in this study could serve as possible targets for such analyses. further laboratory and field - based experimentation is needed to study whether inductive situations exist, in which either the volatile emission or the efn secretion is differentially up- or down - regulated or if both defenses always respond similarly to herbivore attack. below is the link to the electronic supplementary material : arthropod taxa trapped on the experimental tendrils with sticky traps (doc 168 kb). | lima bean (phaseolus lunatus) features two indirect anti - herbivore defenses emission of volatile organic compounds (vocs) and secretion of extrafloral nectar (efn)which are both inducible upon herbivore damage. in a previous field study, lima bean benefited from the simultaneous induction of the two defenses, yet it remained unclear whether both had contributed to plant protection. our experimental approach aimed at studying the defensive role of both indirect defenses simultaneously. tendrils were sprayed with jasmonic acid (ja) to induce both defenses, and performance was compared to that of others that were treated with a synthetic blend of either efn or vocs. confirming earlier results, ja treatment and application of the voc mixture induced efn secretion in treated tendrils in quantitatively similar amounts. the composition of the applied synthetic blend of efn was adjusted to match the concentration of efn secreted from ja- and voc - treated tendrils. repeated application of either enhanced the performance of several fitness - relevant plant parameters such as growth rate and flower production. tendrils treated with ja showed a similar trend, yet some fitness - related parameters responded less to this treatment. this suggests a minor importance of any putative ja - dependent direct defense traits or higher costs of ja - elicited responses as compared to vocs and efn, as otherwise ja - treated tendrils should have outperformed voc- and efn - treated tendrils. moreover, the beneficial effect of applying synthetic efn alone equaled or exceeded that of vocs and ja. ants were by far the dominant group among the arthropods that was attracted to ja-, voc-, or efn - treated tendrils. the results suggest that efn plays a more important role as an indirect defense of lima bean than vocs or any other ja - responsive trait.electronic supplementary materialthe online version of this article doi:10.1007/s10886 - 007 - 9404 - 0 contains supplementary material, which is available to authorized users. |
aquatic insects are a major group of arthropods which at least one stage of their life cycle occurs in water. most of them live in water in primary stages that fallowed by terrestrial adult (eg, ephemeroptera, odonata, plecoptera, trichoptera, megaloptera). semi aquatic insect are only associated with aquatic and semi aquatic vegetation, the water s surface, or the margins of water habitats (merritt and cummins 1996). some species of aquatic insect are medically important vectors that transmit diseases such as malaria, dengue, filariasis, yellow fever, and some other main arboviruses (foil 1989). furthermore few numberof them have a painful bite that cause dermatological effect on human and animal host (villiers 1987). some of them act as a host of termatods such as dragonflyand damselfly (chae. 2000). in some countries dragonfly are considered as a threat to the poultry industry because they transmit a parasitc flatworm of prosthogonimus spp (angel 1973). water quality is evaluated by comparing the number of tolerant species (some midge larva) to the number of intolerant species (ephemeroptera, plecoptera, and trichoptera orders) (voshell 2002). furthermore some of these insects are used in toxicological researches in primary stages (merritt and cummins 1996). aquatic insect are found in a wide variety of aquatic habitats from pond, spring, stream to rivers which are different in salinity, ph and other characteristics. apart from medically importance of aquatic insects, they play an important role in the ecosystem. for instance they serve as food for fish, amphibians, and water birds. some of the aquatic insects are responsible for breaking down the dead leaves and plant parts that fall on the water surface. some of them filter suspended particles in water and cause light reach to bottom of streams where algae grow. another kind of aquatic insects mix soft sediment of bottom while searching for food and this makes bottom appropriate for organisms and this phenomen is due to oxygen enreachment of the bottom. additionally, predator quatic insects reduce the numbers of other invertebrates and help keep to have a balance among different organism and food reservoir (voshell 2002). apart from researches that have been conducted on culicidae family members, there is a few studies on aquatic insects. the isfahan province located in the center of iran and situated 340 kilometers far away of iran s capital. isfahan region has generally arid climate with hot summer with maximum temperature around 36 c and cool winter. it has an average annual rainfall of 150 millimeter zayandeh roud river in this region provides a suitable habitat for aquatic insect. isfahan province is surrounded by qoum, semnan and markazi provinces to the north, fars and kohkiloiye province to the south, yazd province to the east, and lorestan, khuzestan and chaharmahal to the west. with a total area of around 105, 937 square kilometers (6.57 % total area of iran). it lies at an altitude of 1575 meters above sea level at a latitude of 30 42 n to 34 30 n and a longitude 49 36 e to 55 32 e. the climate is temperate. it has the warm and semi - humid climate in north and east parts and cold climate in south. the studied areas were selected by clustered random sampling consist of several localities in the study area. 1684 m (32 22 n, 51 22 e) to baghbahadoran city, ca. 1). map of study area in isfahan province, iran aquatic insects collected in different habitats. the sampling was carried out from rifles, under stones, aquatic vegetation, over hanging terrestrial vegetation, within burrows, leaf packs and fine sediment. the specimens collected by d frame net - collector, plastic pipette and forceps. after collection, all specimens were preserved in 70% alcohol, date and time of sampling and place of collection were recorded on each container. the samples were transferred to the laboratory of medical entomology department, tehran university of medical sciences. then the samples were identified using stereo - typed microscope, and valid identification keys (clifford 1991, borror and white 1998, epler 2001, bouchard 2004, sangradub and boonsoong 2006, subramanian and sivaramakrishnan 2007, azari - hamidian and harbach 2009, mullen and durden 2009). the isfahan province located in the center of iran and situated 340 kilometers far away of iran s capital. isfahan region has generally arid climate with hot summer with maximum temperature around 36 c and cool winter. it has an average annual rainfall of 150 millimeter zayandeh roud river in this region provides a suitable habitat for aquatic insect. isfahan province is surrounded by qoum, semnan and markazi provinces to the north, fars and kohkiloiye province to the south, yazd province to the east, and lorestan, khuzestan and chaharmahal to the west. with a total area of around 105, 937 square kilometers (6.57 % total area of iran). it lies at an altitude of 1575 meters above sea level at a latitude of 30 42 n to 34 30 n and a longitude 49 36 e to 55 32 e. the climate is temperate. it has the warm and semi - humid climate in north and east parts and cold climate in south. the studied areas were selected by clustered random sampling consist of several localities in the study area. 1684 m (32 22 n, 51 22 e) to baghbahadoran city, ca. aquatic insects collected in different habitats. the sampling was carried out from rifles, under stones, aquatic vegetation, over hanging terrestrial vegetation, within burrows, leaf packs and fine sediment. the specimens collected by d frame net - collector, plastic pipette and forceps. after collection, all specimens were preserved in 70% alcohol, date and time of sampling and place of collection were recorded on each container. the samples were transferred to the laboratory of medical entomology department, tehran university of medical sciences. then the samples were identified using stereo - typed microscope, and valid identification keys (clifford 1991, borror and white 1998, epler 2001, bouchard 2004, sangradub and boonsoong 2006, subramanian and sivaramakrishnan 2007, azari - hamidian and harbach 2009, mullen and durden 2009). during several times sampling in the study area a total of 741 aquatic insects were collected that including : 2 orders, 7 families and 12 genera witch summarized in table 1. in the diptera order there are 3 families : culicidae (n=384, 51.82%), syrphidae (n=4, 0.54%) and chironomidae (n=296, 39.95%) and coleoptera order was including 4 families : gyrinidae (n= 6, 0.81%), dytiscidae (n=24, 3.23%), haliplidae (n=7, 0.94%), hydrophilidae (n=20, 2.7%) and culex theileri belong to culicidae family was the most frequent (51.82%) moreover peltodytes in the haliplidae family with 0.40% of all collected samples was the least frequent (table 1 and fig. 2, 3). the prevalence of some aquatic insects in the study area aquatic insect genus and family composition in study area families of collected aquatic insects, a : hydrophilidae b : haliplidae c : gyrinidae d : dytiscidae e : culicidae f : chironomidae g : syrphidae total number of 741 samples belongs to 2 orders and 7 families and 12 genera were identified using stereo - typed microscope. culex theileri with 51.82% of collected sample was the most abundance frequent (table 1 and fig. 2). in the same study that conducted in the our study area by shayeghi. in 2011, their result were compatible with our result, for the study on the aquatic insects of isfahan province and also their probable use of biological control, coleoptera order was one of the abundance collected aquatic insect and this result was likely to our result (shayeghi. theileri was the most abundance frequency and their results were agree with our results (mousa - kazemi. this species is the more prevalent species at higher altitudes in rural areas of zanjan province (ghavami and ladonni 2005) and east azerbaijan province (abai. (2007) conducted a study for studying fauna of aquatic insects in sewage maturation ponds of kashan. the families of chironomidae and hydrophilidae were prevalent. in the other study that conducted by vafaei. 2007 for surveying of the aquatic beetles (coleoptera : polyphaga) of markazi province (central iran) after investigation in freshwater habitats of study area, 24 species (coleoptera : hydrophilidae, helophoridae, hydraenidae, elmidae, and dryopidae) belonging to 13 genera and five families were identified and in this study hydrophilidae family was one of the collected samples like present study (vafaei. some aquatic insect are an important for biological control of larvae and adults of mosquitoes in the breeding places also some of these insects play an important role in transmission of some human and animal diseases, for example in iran several species belong to anophelinae sub family including anopheles culicifacies s.l. 2012a, 2012b, 2012c, vatandoost and abai 2012a, soleimani - ahmadi. therefore the ecological specifications of these insects could provide a clue for further arthropod - borne disease control. according to the results it could be concluded that there are several species of insects in the study area. they also could be considered as biological control agent for vectors as well as bio indicators. | background : aquatic insects are the major groups of arthropods that spend some parts of their life cycle in the water. these insects play an important role for transmission of some human and animal diseases. there is few information about the aquatic insects fauna of iran.methods:to study the aquatic insects fauna, adult, nymphal and larval collections were carried out from different habitats using the standard technique in zayandeh roud river, isfahan province, central iran, during summer 2011.results:in total, 741 speimens of aquatic insects were collected and morphologically identified. they include 7 families and 12 genera representing 2 orders. the order of diptera (92.31%) and coleoptera (7.69%). the families culicidae, syrphidae and chironomidae from diptera order, gyrinidae, dytiscidae, haliplidae, hydrophilidae from coleoptera order were identified.conclusion:some aquatic insects play an important role for transmission of human and animal diseases. these insects also are important for biological control. therefore ecological study on aquatic insects can provide information about ecology of insects in an area for any decision making. |
acute coronary syndromes (acs) continue to be a major cause of morbidity and mortality. data from the united states and europe have reported a decrease in the incidence of stelevation myocardial infarction (stemi) with an increase in nonstelevation myocardial infarction (nstemi) in the past decade. current 2012 american college of cardiology / american heart association (acc / aha) guidelines recommend early invasive strategy (eis) for management of patients with nstemi and refractory angina or electrical or hemodynamic insufficiency (class ib), initially stabilized patients with nstemi, and high risk of clinical events (class ia). the early invasive approach (within 12 to 24 hours of presentation) to reduce ischemic complications is also recommended in initially stabilized highrisk patients with nstemi (class iia). the optimal timing of eis has been debated and undergone a paradigm shift in the last decade. the transition from 48 hours to within 24 hours occurred after the publication of the timing of intervention in acute coronary syndromes (timacs) trial in 2009. however, there is a paucity of information with regard to the secular trends in utilization of eis in patients with nstemi and how this may vary among different nstemi patient populations. using a large nationwide administrative database, the present study aimed to analyze and describe age, sex, and race / ethnicityspecific trends in the incidence, utilization of eis, and inhospital outcomes for nstemi in the united states from 2002 to 2011. data were obtained from the 2002 to 2011 nationwide inpatient sample (nis) databases. the nis, sponsored by the agency for healthcare research and quality as a part of the healthcare cost and utilization project (hcup), is the largest publicly available allpayer inpatient care database in the united states. it contains dischargelevel data from 8 million hospital stays from approximately 1000 hospitals designed to approximate a 20% stratified sample of all community hospitals in the united states. criteria used for stratified sampling of hospitals include hospital ownership, bed size, teaching status, urban or rural location, and geographic region. discharge weights are provided for each patient discharge record, which were used to obtain national estimates. discharge weights are calculated for nis data by first stratifying the nis hospitals on the same variables used for creating the sample. a weight is then calculated for each stratum by dividing the number of universe discharges in that stratum (obtained from american hospital association data) by the number of nis discharges in the stratum. weighted estimates are calculated by uniformly applying stratum weights to the discharges according to the stratum from which the discharge was drawn. weights are assigned to each discharge and are stored in each record in the data element, discwt. when the discharge weights are applied to the unweighted nis data, the result is an estimate of the number of discharges for all inpatient discharges from community hospitals in the united states. this study was deemed exempt by the new york medical college institutional review board (valhalla, ny) because hcupnis is a public database with no personal identifying information. we used the international classification of diseases, ninth edition, clinical modification (icd9cm), codes 410.xx to identify all patients 40 years of age with the principal diagnosis of acute myocardial infarction (ami ; n=6 512 372). patients with the principal diagnosis of nstemi were then identified using icd9cm codes 410.7x (n=3 981 119). we chose the principal diagnosis because it is considered the primary reason for hospital admission. eis was defined as coronary angiography (icd9cm procedure codes 88.55, 88.66, 37.22, or 37.23) with or without revascularization (ie, percutaneous coronary intervention (pci ; icd9cm procedure codes 00.66, 36.01, 36.02, 36.05, 36.06 and 36.07) or coronary artery bypass graft surgery (cabg ; icd9cm procedure code 36.1x) on day 0 (considered within 24 hours of admission) or on days 0 or 1 (considered within 48 hours of admission). we initially studied the overall age, sex, and race / ethnicityspecific trends in the incidence of nstemi. our primary outcome of interest was allcause, inhospital mortality, defined as death during the hospitalization encounter in the nis database. we analyzed the overall age, sex, and race / ethnicityspecific trends in utilization of the eis, riskadjusted inhospital mortality, average length of stay, and total hospital cost in patients with nstemi. the nis provides total charges, which reflect the amount a hospital billed for services, rather than actual costs or the amount a hospital received in payment. in this study, we used the hcup costtocharge ratio file developed by the agency for healthcare research and quality (ahrq) to translate total charges into costs. each file contains hospitalspecific costtocharge ratios based on allpayer inpatient cost for nearly every hospital in the nis databases. cost information is obtained from the hospital accounting reports collected by the centers for medicare and medicaid services. baseline patient characteristics used included demographics (age, sex, race / ethnicity, primary expected payer, weekday versus weekend admission), 29 elixhauser comorbidities as defined by the ahrq and other clinically relevant comorbidities (smoking, dyslipidemia, known coronary artery disease [cad ], family history of cad, previous myocardial infarction [mi ], previous transient ischemic attack or stroke, previous pci, previous cabg, carotid artery disease, dementia, and atrial fibrillation). a list of icd9cm and clinical classifications software codes used to identify comorbidities and inhospital procedures is provided in table 1. hospital characteristics, such as hospital region (northeast, midwest, south, and west), bed size (small, medium, and large), location (rural or urban), and teaching status were also included. international classification of diseases, ninth edition, clinical modification (icd9cm) and clinical classifications software (ccs) codes used to identify comorbidities weighted data were used for all statistical analyses. for trend analysis, we used mantelhaenszel 's chisquare () test of linear association for categorical variables and linear regression for continuous variables. to assess whether the incidence of nstemi, utilization of eis, or inhospital mortality has changed over time, unadjusted and multivariable adjusted logistic regression models were constructed. our independent variable, calendar year, was initially entered as a continuous variable in the regression models to obtain unadjusted and adjusted odds ratios (ors ; per year) for the temporal trends. to determine whether there was a temporal variability from year to year in the incidence of nstemi, utilization of eis, or inhospital mortality, we also evaluated calendar year as a categorical variable, with 2002 as the reference year. the regression models adjusted for all demographics (except sex and race / ethnicity for sex and race / ethnicityspecific trends, respectively), hospital characteristics, and all elixhauser and other clinically relevant comorbidities. we graphically displayed the unadjusted and adjusted ors and 95% confidence intervals (cis) for incidence of nstemi, utilization of eis for nstemi, or inhospital mortality over time. statistical analysis was performed using ibm spss statistics 20.0 (ibm corp., armonk, ny). all p values were 2 sided with a significance threshold of p<0.001 (a lowerthanusual pvalue threshold was selected to correct for the effects of a large sample size as well as inflation of type i error because of repeated testing using a large number of variables). categorical variables are expressed as percentage and continuous variables as mean sem. or, and data were obtained from the 2002 to 2011 nationwide inpatient sample (nis) databases. the nis, sponsored by the agency for healthcare research and quality as a part of the healthcare cost and utilization project (hcup), is the largest publicly available allpayer inpatient care database in the united states. it contains dischargelevel data from 8 million hospital stays from approximately 1000 hospitals designed to approximate a 20% stratified sample of all community hospitals in the united states. criteria used for stratified sampling of hospitals include hospital ownership, bed size, teaching status, urban or rural location, and geographic region. discharge weights are provided for each patient discharge record, which were used to obtain national estimates. discharge weights are calculated for nis data by first stratifying the nis hospitals on the same variables used for creating the sample. a weight is then calculated for each stratum by dividing the number of universe discharges in that stratum (obtained from american hospital association data) by the number of nis discharges in the stratum. weighted estimates are calculated by uniformly applying stratum weights to the discharges according to the stratum from which the discharge was drawn. weights are assigned to each discharge and are stored in each record in the data element, discwt. when the discharge weights are applied to the unweighted nis data, the result is an estimate of the number of discharges for all inpatient discharges from community hospitals in the united states. this study was deemed exempt by the new york medical college institutional review board (valhalla, ny) because hcupnis is a public database with no personal identifying information. we used the international classification of diseases, ninth edition, clinical modification (icd9cm), codes 410.xx to identify all patients 40 years of age with the principal diagnosis of acute myocardial infarction (ami ; n=6 512 372). patients with the principal diagnosis of nstemi were then identified using icd9cm codes 410.7x (n=3 981 119). we chose the principal diagnosis because it is considered the primary reason for hospital admission. eis was defined as coronary angiography (icd9cm procedure codes 88.55, 88.66, 37.22, or 37.23) with or without revascularization (ie, percutaneous coronary intervention (pci ; icd9cm procedure codes 00.66, 36.01, 36.02, 36.05, 36.06 and 36.07) or coronary artery bypass graft surgery (cabg ; icd9cm procedure code 36.1x) on day 0 (considered within 24 hours of admission) or on days 0 or 1 (considered within 48 hours of admission). we initially studied the overall age, sex, and race / ethnicityspecific trends in the incidence of nstemi. our primary outcome of interest was allcause, inhospital mortality, defined as death during the hospitalization encounter in the nis database. we analyzed the overall age, sex, and race / ethnicityspecific trends in utilization of the eis, riskadjusted inhospital mortality, average length of stay, and total hospital cost in patients with nstemi. the nis provides total charges, which reflect the amount a hospital billed for services, rather than actual costs or the amount a hospital received in payment. in this study, we used the hcup costtocharge ratio file developed by the agency for healthcare research and quality (ahrq) to translate total charges into costs. each file contains hospitalspecific costtocharge ratios based on allpayer inpatient cost for nearly every hospital in the nis databases. cost information is obtained from the hospital accounting reports collected by the centers for medicare and medicaid services. baseline patient characteristics used included demographics (age, sex, race / ethnicity, primary expected payer, weekday versus weekend admission), 29 elixhauser comorbidities as defined by the ahrq and other clinically relevant comorbidities (smoking, dyslipidemia, known coronary artery disease [cad ], family history of cad, previous myocardial infarction [mi ], previous transient ischemic attack or stroke, previous pci, previous cabg, carotid artery disease, dementia, and atrial fibrillation). a list of icd9cm and clinical classifications software codes used to identify comorbidities and inhospital procedures is provided in table 1. hospital characteristics, such as hospital region (northeast, midwest, south, and west), bed size (small, medium, and large), location (rural or urban), and teaching status were also included. international classification of diseases, ninth edition, clinical modification (icd9cm) and clinical classifications software (ccs) codes used to identify comorbidities weighted data were used for all statistical analyses. for trend analysis, we used mantelhaenszel 's chisquare () test of linear association for categorical variables and linear regression for continuous variables. to assess whether the incidence of nstemi, utilization of eis, or inhospital mortality has changed over time, unadjusted and multivariable adjusted logistic regression models were constructed. our independent variable, calendar year, was initially entered as a continuous variable in the regression models to obtain unadjusted and adjusted odds ratios (ors ; per year) for the temporal trends. to determine whether there was a temporal variability from year to year in the incidence of nstemi, utilization of eis, or inhospital mortality, we also evaluated calendar year as a categorical variable, with 2002 as the reference year. the regression models adjusted for all demographics (except sex and race / ethnicity for sex and race / ethnicityspecific trends, respectively), hospital characteristics, and all elixhauser and other clinically relevant comorbidities. we graphically displayed the unadjusted and adjusted ors and 95% confidence intervals (cis) for incidence of nstemi, utilization of eis for nstemi, or inhospital mortality over time. statistical analysis was performed using ibm spss statistics 20.0 (ibm corp., armonk, ny). all p values were 2 sided with a significance threshold of p<0.001 (a lowerthanusual pvalue threshold was selected to correct for the effects of a large sample size as well as inflation of type i error because of repeated testing using a large number of variables). categorical variables are expressed as percentage and continuous variables as mean sem. or, and from 2002 to 2011, we identified 6 512 372 patients 40 years of age admitted with ami. of these, 3 981 119 (61.1%) had nstemi. among patients with ami, the proportion of those presenting with nstemi increased from 52.8% in 2002 to 68.6% in 2011 (ptrend<0.001). after adjusting for baseline demographics, hospital characteristics, and comorbidities, we observed an increasing trend in the proportion of patients hospitalized with nstemi from 2002 to 2011 (adjusted or [per year ], 1.055 ; 95% ci, 1.054 to 1.056 ; p<0.001 ; figure 1 ; table 2). adjusted temporal trends of proportion of nonstelevation myocardial infarction : overall cohort, age, sex, and race / ethnicity stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend were determined using logistic regression models evaluating calendar year as a continuous variable. numbers in parenthesis represent 95% confidence interval. a, nstemi (%) was calculated as the number of patients with nstemi divided by the number of patients with acute myocardial infarction (ami) per year100 ; ptrend<0.001. b, trends in nstemi presented as unadjusted and adjusted odds ratio and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. 95% cis are depicted, but are too narrow to be visualized outside the marker width. the increase in proportion of nstemi, it increased from 45.4% to 60.2% in those 40 to 64 years of age (adjusted or [per year ], 1.045 ; 95% ci, 1.044 to 1.046 ; p<0.001), from 54.5% to 70.7% in those 65 to 74 (adjusted or [per year ], 1.056 ; 95% ci, 1.055 to 1.058 ; p<0.001), and from 59% to 77.1% in patients 75 (adjusted or [per year ], 1.066 ; 95% ci, 1.065 to 1.068 ; p<0.001). when stratified according to sex, women (56.1% to 73.6% ; adjusted or [per year ], 1.070 ; 95% ci, 1.069 to 1.071 ; p<0.001) had a steeper proportional increase, compared to men (50.4% to 65.3% ; adjusted or [per year ], 1.046 ; 95% ci, 1.045 to 1.047 ; p<0.001). age, sex, and race / ethnicity specific trends in incidence rates of nonstelevation myocardial infarction. a, age stratified ; ptrend<0.001, (b) sex stratified ; ptrend<0.001, and (c) race / ethnicity stratified ; ptrend<0.001. table 3 depicts the baseline demographic, hospital, and clinical characteristics of patients admitted with nstemi from 2002 to 2011. there was a slight decrease in mean age at admission during the study period (70.3613.11 years in 2002 versus 69.8713.53 in 2011 ; p<0.001). more specifically, there was an increase in the proportion of nstemi patients aged 40 to 64 years and decrease in those aged 75 years from 2002 to 2011 (ptrend<0.001). similarly, there was a small, but statistically significant, increase in the proportion of men (56% in 2002 to 57.5% in 2011 ; ptrend<0.001) and a consequent decrease in the proportion of women (44.0% in 2002 to 42.5% in 2011 ; ptrend<0.001) with nstemi during the study period. the prevalence of smoking, dyslipidemia, cad, previous mi, carotid artery disease, diabetes mellitus, hypertension, obesity, peripheral vascular disease, chronic renal failure, alcohol abuse, deficiency anemias, coagulopathy, fluid / electrolyte disorders, and previous pci increased from 2002 to 2011 (ptrend<0.001 for all). the prevalence of heart failure and chronic pulmonary diseases remained relatively constant throughout the study period. baseline demographics, hospital characteristics, comorbidities, utilization of early invasive strategy, and overall inhospital mortality in patients with nonstelevation myocardial infarction aids indicates acquired immunodeficiency syndrome ; cabg, coronary artery bypass graft surgery ; cad, coronary artery disease ; h / o, history of ; pci, percutaneous coronary intervention ; ra, rheumatoid arthritis ; tia, transient ischemic attack. utilization of eis on day 0 of presentation increased from 14.9% in 2002 to 21.8% in 2011 (adjusted or [per year ], 1.065 ; 95% ci, 1.064 to 1.066 ; p<0.001). the adjusted temporal trends for utilization of eis were similar across all age, sex, and racial / ethnic groups. patients 75 years of age had the highest proportional increase in utilization of eis (adjusted or [per year ], 1.072 ; 95% ci, 1.069 to 1.074 ; p<0.001 ; figures 3 and 4 ; table 4). adjusted temporal trends of nonstelevation myocardial infarction with utilization of the early invasive strategy at day 0 : overall cohort, age, sex, and race / ethnicity stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend were determined using logistic regression models evaluating calendar year as a continuous variable. numbers in parenthesis represent 95% confidence interval. trends in utilization of early invasive strategy for nonstelevation b, trends in early invasive strategy at day 0 presented as unadjusted and adjusted odds ratio and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. d, trends in early invasive strategy at day 0 or 1 presented as unadjusted and adjusted odds ratio and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. 95% cis are depicted, but are too narrow to be visualized outside the marker width. age, sex, and race / ethnicity specific trends in utilization of early invasive strategy (eis) for nonstelevation myocardial infarction. a, age stratified, eis day 0 ; ptrend<0.001, (b) sex stratified, eis day 0 ; ptrend<0.001, (c) race / ethnicity stratified, eis day 0 ; ptrend<0.001, (d) age stratified, eis day 0 or 1 ; ptrend<0.001, (e) sex stratified, eis day 0 or 1 ; ptrend<0.001, and (f) race / ethnicity stratified, eis day 0 or 1 ; ptrend<0.001. results showed a temporal increase in the overall as well as the age, race / ethnicity, and sexstratified cohorts (figures 3 and 4 ; table 5). adjusted temporal trends of nonstelevation myocardial infarction with utilization of the early invasive strategy at day 0 to 1 : overall cohort, age, sex, and race / ethnicity stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend were determined using logistic regression models evaluating calendar year as a continuous variable. inhospital mortality decreased from 5.5% in 2002 to 3.9% in 2011 in all patients with nstemi (adjusted or [per year ], 0.976 ; 95% ci, 0.974 to 0.978 ; p<0.001). on agestratified analysis, those 75 years of age had the greatest decrease in inhospital mortality (adjusted or [per year ], 0.968 ; 95% ci, 0.966 to 0.971 ; p<0.001). women had a steeper temporal decline in inhospital mortality, compared to men, from 2002 to 2011 (adjusted or [per year ], 0.965 ; 95% ci, 0.962 to 0.968 ; p<0.001 in women and adjusted or [per year ], 0.986 ; 95% ci, 0.984 to 0.989 ; p<0.001 in men). statistically significant decline in mortality was also observed in all racial / ethnic groups. in patients receiving eis on day 0 of presentation, however, the overall trend in this decrease was not statistically significant (adjusted or [per year ], 0.998 ; 95% ci, 0.991 to 1.004 ; p=0.471). a temporal decline in inhospital mortality was observed in patients treated with invasive strategy on day 0 or 1 (2.4% in 2002 to 1.8% in 2011 ; adjusted or [per year ], 0.988 ; 95% ci, 0.983 to 0.993 ; p<0.001 ; figures 5 and 6 ; table 6). in patients not receiving eis, inhospital mortality decreased from 6.7% in 2002 to 5.4% in 2011 (adjusted or [per year ], 0.977 ; 95% ci, 0.975 to 0.979 ; p<0.001 ; figure 6 ; table 6). adjusted temporal trends of riskadjusted inhospital mortality in patients with nonstelevation myocardial infarction : overall cohort, age, sex, race / ethnicity, and early invasive strategy stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend a, inhospital mortality (%) was calculated as the number of patients who died during the hospitalization divided by the number of patients with nstemi per year100 ; ptrend<0.001. b, trends in inhospital mortality presented as unadjusted and adjusted odds ratio (or) and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. age, sex, race / ethnicity, and utilization of invasive strategyspecific trends in inhospital mortality in patients with nonstelevation myocardial infarction. a, age stratified, (b) sex stratified, (c) race / ethnicity stratified, and (d) stratified according to utilization of invasive strategy (ptrend<0.001 for all). no eis was defined as patients who did not receive coronary angiography with intent to revascularize within day 0 or 1. detailed results of the length and cost of stay temporal trends stratified by age, sex, and race / ethnicity are shown in tables 7 and 8, respectively. the mean (sem, in days) length of stay decreased in the overall cohort of patients during the study period (5.70.01 in 2002 to 4.80.01 in 2011 ; ptrend<0.001). patients with nstemi undergoing eis within day 0 had the shortest length of stay, and the length of stay declined in all patients undergoing eis, irrespective of timing (from 4.40.03 in 2002 to 3.90.02 in 2011 for those with eis at day 0 and from 4.80.02 in 2002 to 4.10.01 in 2011 for those with eis within day 0 or 1 ; ptrend<0.001 for all ; figure 7 ; table 7). temporal trends in length of stay (meanse) in days temporal trends in total hospital cost (meanse) in u.s. a, overall, (b) age, (c) sex, (d) race / ethnicity, and (e) timing of invasive strategy (ptrend<0.001 for all). mean (sem, in dollars) cost of stay in the overall patient population admitted with nstemi increased from $ 14 77430 from 2002 to $ 20 26935 in 2011 (ptrend<0.001). the mean (sem, in dollars) costs accrued by those undergoing eis were higher and showed a similar increasing trend (from $ 17 38580 in 2002 to $ 24 34576 in 2011 for those with eis at day 0 and from $ 17 53459 in 2002 to $ 23 97155 in 2011 for those with eis within day 0 or 1 ; ptrend<0.001 for all ; figure 8 ; table 8). (a) overall, (b) age, (c) sex, (d) race / ethnicity, and (e) timing of invasive strategy (ptrend<0.001 for all). from 2002 to 2011, we identified 6 512 372 patients 40 years of age admitted with ami. of these, 3 981 119 (61.1%) had nstemi. among patients with ami, the proportion of those presenting with nstemi increased from 52.8% in 2002 to 68.6% in 2011 (ptrend<0.001). after adjusting for baseline demographics, hospital characteristics, and comorbidities, we observed an increasing trend in the proportion of patients hospitalized with nstemi from 2002 to 2011 (adjusted or [per year ], 1.055 ; 95% ci, 1.054 to 1.056 ; p<0.001 ; figure 1 ; table 2). adjusted temporal trends of proportion of nonstelevation myocardial infarction : overall cohort, age, sex, and race / ethnicity stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend were determined using logistic regression models evaluating calendar year as a continuous variable. numbers in parenthesis represent 95% confidence interval. a, nstemi (%) was calculated as the number of patients with nstemi divided by the number of patients with acute myocardial infarction (ami) per year100 ; ptrend<0.001. b, trends in nstemi presented as unadjusted and adjusted odds ratio and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. 95% cis are depicted, but are too narrow to be visualized outside the marker width. the increase in proportion of nstemi, it increased from 45.4% to 60.2% in those 40 to 64 years of age (adjusted or [per year ], 1.045 ; 95% ci, 1.044 to 1.046 ; p<0.001), from 54.5% to 70.7% in those 65 to 74 (adjusted or [per year ], 1.056 ; 95% ci, 1.055 to 1.058 ; p<0.001), and from 59% to 77.1% in patients 75 (adjusted or [per year ], 1.066 ; 95% ci, 1.065 to 1.068 ; p<0.001). when stratified according to sex, women (56.1% to 73.6% ; adjusted or [per year ], 1.070 ; 95% ci, 1.069 to 1.071 ; p<0.001) had a steeper proportional increase, compared to men (50.4% to 65.3% ; adjusted or [per year ], 1.046 ; 95% ci, 1.045 to 1.047 ; p<0.001). age, sex, and race / ethnicity specific trends in incidence rates of nonstelevation myocardial infarction. a, age stratified ; ptrend<0.001, (b) sex stratified ; ptrend<0.001, and (c) race / ethnicity stratified ; ptrend<0.001. table 3 depicts the baseline demographic, hospital, and clinical characteristics of patients admitted with nstemi from 2002 to 2011. there was a slight decrease in mean age at admission during the study period (70.3613.11 years in 2002 versus 69.8713.53 in 2011 ; p<0.001). more specifically, there was an increase in the proportion of nstemi patients aged 40 to 64 years and decrease in those aged 75 years from 2002 to 2011 (ptrend<0.001). similarly, there was a small, but statistically significant, increase in the proportion of men (56% in 2002 to 57.5% in 2011 ; ptrend<0.001) and a consequent decrease in the proportion of women (44.0% in 2002 to 42.5% in 2011 ; ptrend<0.001) with nstemi during the study period. the prevalence of smoking, dyslipidemia, cad, previous mi, carotid artery disease, diabetes mellitus, hypertension, obesity, peripheral vascular disease, chronic renal failure, alcohol abuse, deficiency anemias, coagulopathy, fluid / electrolyte disorders, and previous pci increased from 2002 to 2011 (ptrend<0.001 for all). the prevalence of heart failure and chronic pulmonary diseases remained relatively constant throughout the study period. baseline demographics, hospital characteristics, comorbidities, utilization of early invasive strategy, and overall inhospital mortality in patients with nonstelevation myocardial infarction aids indicates acquired immunodeficiency syndrome ; cabg, coronary artery bypass graft surgery ; cad, coronary artery disease ; h / o, history of ; pci, percutaneous coronary intervention ; ra, rheumatoid arthritis ; tia, transient ischemic attack. utilization of eis on day 0 of presentation increased from 14.9% in 2002 to 21.8% in 2011 (adjusted or [per year ], 1.065 ; 95% ci, 1.064 to 1.066 ; p<0.001). the adjusted temporal trends for utilization of eis were similar across all age, sex, and racial / ethnic groups. patients 75 years of age had the highest proportional increase in utilization of eis (adjusted or [per year ], 1.072 ; 95% ci, 1.069 to 1.074 ; p<0.001 ; figures 3 and 4 ; table 4). adjusted temporal trends of nonstelevation myocardial infarction with utilization of the early invasive strategy at day 0 : overall cohort, age, sex, and race / ethnicity stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend numbers in parenthesis represent 95% confidence interval. trends in utilization of early invasive strategy for nonstelevation myocardial infarction. b, trends in early invasive strategy at day 0 presented as unadjusted and adjusted odds ratio and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. d, trends in early invasive strategy at day 0 or 1 presented as unadjusted and adjusted odds ratio and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. 95% cis are depicted, but are too narrow to be visualized outside the marker width. age, sex, and race / ethnicity specific trends in utilization of early invasive strategy (eis) for nonstelevation myocardial infarction. a, age stratified, eis day 0 ; ptrend<0.001, (b) sex stratified, eis day 0 ; ptrend<0.001, (c) race / ethnicity stratified, eis day 0 ; ptrend<0.001, (d) age stratified, eis day 0 or 1 ; ptrend<0.001, (e) sex stratified, eis day 0 or 1 ; ptrend<0.001, and (f) race / ethnicity stratified, eis day 0 or 1 ; ptrend<0.001. results showed a temporal increase in the overall as well as the age, race / ethnicity, and sexstratified cohorts (figures 3 and 4 ; table 5). adjusted temporal trends of nonstelevation myocardial infarction with utilization of the early invasive strategy at day 0 to 1 : overall cohort, age, sex, and race / ethnicity stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend were determined using logistic regression models evaluating calendar year as a continuous variable. inhospital mortality decreased from 5.5% in 2002 to 3.9% in 2011 in all patients with nstemi (adjusted or [per year ], 0.976 ; 95% ci, 0.974 to 0.978 ; p<0.001). on agestratified analysis, those 75 years of age had the greatest decrease in inhospital mortality (adjusted or [per year ], 0.968 ; 95% ci, 0.966 to 0.971 ; p<0.001). women had a steeper temporal decline in inhospital mortality, compared to men, from 2002 to 2011 (adjusted or [per year ], 0.965 ; 95% ci, 0.962 to 0.968 ; p<0.001 in women and adjusted or [per year ], 0.986 ; 95% ci, 0.984 to 0.989 ; p<0.001 in men). statistically significant decline in mortality was also observed in all racial / ethnic groups. in patients receiving eis on day 0 of presentation, however, the overall trend in this decrease was not statistically significant (adjusted or [per year ], 0.998 ; 95% ci, 0.991 to 1.004 ; p=0.471). a temporal decline in inhospital mortality was observed in patients treated with invasive strategy on day 0 or 1 (2.4% in 2002 to 1.8% in 2011 ; adjusted or [per year ], 0.988 ; 95% ci, 0.983 to 0.993 ; p<0.001 ; figures 5 and 6 ; table 6). in patients not receiving eis, inhospital mortality decreased from 6.7% in 2002 to 5.4% in 2011 (adjusted or [per year ], 0.977 ; 95% ci, 0.975 to 0.979 ; p<0.001 ; figure 6 ; table 6). adjusted temporal trends of riskadjusted inhospital mortality in patients with nonstelevation myocardial infarction : overall cohort, age, sex, race / ethnicity, and early invasive strategy stratified multivariable adjustment for baseline demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities as outlined in the methodology. adjusted odds ratios (or ; per year) for trend were determined using logistic regression models evaluating calendar year as a continuous variable. a, inhospital mortality (%) was calculated as the number of patients who died during the hospitalization divided by the number of patients with nstemi per year100 ; ptrend<0.001. b, trends in inhospital mortality presented as unadjusted and adjusted odds ratio (or) and 95% confidence interval (ci) for each year relative to 2002 (reference year). regression model adjusted for demographics, hospital characteristics, and 29 elixhauser and other clinically relevant comorbidities. age, sex, race / ethnicity, and utilization of invasive strategyspecific trends in inhospital mortality in patients with nonstelevation myocardial infarction. a, age stratified, (b) sex stratified, (c) race / ethnicity stratified, and (d) stratified according to utilization of invasive strategy (ptrend<0.001 for all). no eis was defined as patients who did not receive coronary angiography with intent to revascularize within day 0 or 1. for secondary outcome analysis, we studied the temporal trends in cost and length of stay. detailed results of the length and cost of stay temporal trends stratified by age, sex, and race / ethnicity are shown in tables 7 and 8, respectively. the mean (sem, in days) length of stay decreased in the overall cohort of patients during the study period (5.70.01 in 2002 to 4.80.01 in 2011 ; ptrend<0.001). patients with nstemi undergoing eis within day 0 had the shortest length of stay, and the length of stay declined in all patients undergoing eis, irrespective of timing (from 4.40.03 in 2002 to 3.90.02 in 2011 for those with eis at day 0 and from 4.80.02 in 2002 to 4.10.01 in 2011 for those with eis within day 0 or 1 ; ptrend<0.001 for all ; figure 7 ; table 7). temporal trends in length of stay (meanse) in days temporal trends in total hospital cost (meanse) in u.s. dollars temporal trends in length of stay, in days. a, overall, (b) age, (c) sex, (d) race / ethnicity, and (e) timing of invasive strategy (ptrend<0.001 for all). mean (sem, in dollars) cost of stay in the overall patient population admitted with nstemi increased from $ 14 77430 from 2002 to $ 20 26935 in 2011 (ptrend<0.001). the mean (sem, in dollars) costs accrued by those undergoing eis were higher and showed a similar increasing trend (from $ 17 38580 in 2002 to $ 24 34576 in 2011 for those with eis at day 0 and from $ 17 53459 in 2002 to $ 23 97155 in 2011 for those with eis within day 0 or 1 ; ptrend<0.001 for all ; figure 8 ; table 8). (a) overall, (b) age, (c) sex, (d) race / ethnicity, and (e) timing of invasive strategy (ptrend<0.001 for all). we report an increasing trend in the proportion of nstemi admissions for the overall cohort and in the age, sex, and race / ethnicitystratified subgroups from 2002 to 2011. this is associated with an increase in utilization of eis, decreased inhospital mortality, and decreased length of stay during the same period. analysis of the national registry of myocardial infarction from 1990 to 2006 reported an increase in the proportion of nstemi from 14.2% in 1990 to 59.1% in 2006 (p<0.001). the proportional increase in nstemi may be the result of an aging population, increasing chronic comorbidities, and reduction in smoking. part of the increase in the proportion of nstemi in our study may be attributable to increased utilization and higher sensitivity of cardiac biomarkers (troponins). in the age and sexstratified analysis older patients are more likely to be women and have significant comorbidities that predispose them to nstemi. on trend analysis of baseline characteristics, we report an increase in the proportion of patients in the 40 to 64 years of age group. this is in concordance with the analysis of get with the guidelines coronary artery disease registry from 2003 to 2008, with that age group representing the fastestgrowing segment of our population over the years. importantly, there was a temporal increase in utilization of the eis in the nstemi patient population. metaanalysis of randomized, control trials has consistently shown a benefit of eis in the setting of nstemi, especially in the highrisk patient population. definition of optimal timing of invasive management has undergone a paradigm shift in the last decade. the 2002 acc / aha guidelines for management of nstemi and the 2007 acc / aha guidelines for management of nstemi in the elderly patient population defined the eis as routine cardiac catheterization within 48 hours of presentation. the more recent timacs trial compared the routine early intervention (24 hours of presentation ; median time, 14 hours) to delayed intervention (36 hours after presentation ; median time, 50 hours). there was no significant difference in the primary endpoint, other than in the prespecified subgroup with high global registry of acute coronary events scores. after 6 months, they reported a 28% relative reduction in secondary outcome of death, mi, and refractory ischemia in the early intervention group (or, 0.72 ; 95% ci, 0.58 to 0.89 ; p=0.003). the results of the crusade (can rapid risk stratification of unstable angina patients suppress adverse outcomes with early implementation of the acc / aha guidelines) quality improvement initiative from 2000 to 2002 indicated that the eis (within 48 hours) was not utilized in a majority of highrisk patients with nstemi. they reported male sex, ischemic electrocardiographic changes, younger age, positive biomarkers, lack of congestive heart failure, lack of renal insufficiency, and white race as predictors of early invasive management. we analyzed the trends in both day 0 and day 0 or 1 of presentation. utilization of eis at day 0 increased from 14.9% in 2002 to 21.8% in 2011 (p<0.001), and utilization of eis at day 0 or 1 increased from 27.8% in 2002 to 41.4% in 2011 (p<0.001). the increase in utilization was consistent among all age, race / ethnicity, and sexstratified subgroups after multivariable adjustment. men had a higher proportional increase in eis at both day 0 and day 0 or 1 of presentation. previous studies have discussed the sex differences in the invasive management of acs, with women being managed less aggressively than men. interestingly, patients 75 years of age had a higher proportional increase in eis at day 0 and patients 40 to 64 years of age had an overall higher proportional increase at day 0 or 1. using the linked crusade registry and medicare and medicaid claims data, reported an increased utilization of evidence based nstemi care and decline in inhospital mortality in 2006, compared with 2003 (or, 0.82 ; 95% ci, 0.67 to 1.00 ; p=0.045) in patients 65 years of age with nstemi. recent trend analysis from denmark also reported an increase in initiation of coronary angiography for firsttime nstemi admissions at 3 days from 18.2% in 2001 to 55.7% to 2009. we also report a decrease in riskadjusted inhospital mortality and length of stay for patients admitted with nstemi. the decline in inhospital mortality was observed in patients receiving eis as well as those not receiving eis. this observation is consistent with improvement in, or better adherence to, medical therapy, independent of eis utilization. the lower inhospital mortality observed in the eismanaged patients is likely the result of patient selection bias, as previously demonstrated by analysis of the crusade registry, and no causal association between eis and inhospital mortality can be inferred from the results of our study. the decline in overall inhospital mortality was most prominent in those 75 years of age. we have previously reported a 24% relative reduction of inhospital mortality in octogenarians treated with the early invasive approach versus the initial conservative strategy (or, 0.76 ; 95% ci, 0.74 to 0.78 ; p<0.001). however, it is not possible to conclusively establish a causal relationship between increasing utilization of eis and decreasing inhospital mortality from this study. the possibility of selection bias and residual measured and unmeasured confounding can not be completely eliminated. nis is an administrative database ; there is the potential for unrecognized miscoding of diagnostic and procedure codes. there is a possibility that widespread troponin utilization in the last decade may have contributed to the increase in nstemi diagnosis and also potentially decreased case severity of those classified as nstemi. also, conditions other than acs may have led to troponin elevations and hence miscoding of nstemi exists. clinical presentation and it is our assumption that day 0 corresponds to 24 hours and days 0 or 1 corresponds to 48 hours, because the database does not provide the timing of procedure in hours. for trends in total hospital costs the nis databases do not include nstemi hospitalizations for veterans hospitalized in veterans affairs hospitals. last, outcomes in the nis database are limited to inhospital events and causes of death are not differentiated. however, these potential limitations may be partially compensated by the large size of the database and the ability to obtain nationwide estimates using the discharge weights provided. the possibility of selection bias and residual measured and unmeasured confounding can not be completely eliminated. nis is an administrative database ; there is the potential for unrecognized miscoding of diagnostic and procedure codes. there is a possibility that widespread troponin utilization in the last decade may have contributed to the increase in nstemi diagnosis and also potentially decreased case severity of those classified as nstemi. also, conditions other than acs may have led to troponin elevations and hence miscoding of nstemi exists. clinical presentation and it is our assumption that day 0 corresponds to 24 hours and days 0 or 1 corresponds to 48 hours, because the database does not provide the timing of procedure in hours. for trends in total hospital costs the nis databases do not include nstemi hospitalizations for veterans hospitalized in veterans affairs hospitals. last, outcomes in the nis database are limited to inhospital events and causes of death are not differentiated. however, these potential limitations may be partially compensated by the large size of the database and the ability to obtain nationwide estimates using the discharge weights provided. in this large, nationwide analysis of patients with mi, we report an increase in proportion of nstemi with increasing utilization of eis in the united states. we also report a decrease in inhospital mortality and decrease in length of stay during the study period. nevertheless, age, sex, and race / ethnicityspecific differences in the management and outcomes of nstemi were observed, and further studies are needed to develop strategies to ensure more equitable care for nstemi. | backgroundthere has been a paradigm shift in the definition of timing of early invasive strategy (eis) for patients admitted with nonstelevation myocardial infarction (nstemi) in the last decade. data on trends of eis for nstemi and associated inhospital outcomes are limited. our aim is to analyze temporal trends in the incidence, utilization of early invasive strategy, and inhospital outcomes of nstemi in the united states.methods and resultswe analyzed the 20022011 nationwide inpatient sample databases to identify all patients 40 years of age with the principal diagnosis of acute myocardial infarction (ami) and nstemi. logistic regression was used for overall, age, sex, and race / ethnicitystratified trend analysis. from 2002 to 2011, we identified 6 512 372 patients with ami. of these, 3 981 119 (61.1%) had nstemi. the proportion of patients with nstemi increased from 52.8% in 2002 to 68.6% in 2011 (adjusted odds ratio [or ; per year ], 1.055 ; 95% confidence interval [ci ], 1.054 to 1.056) in the overall cohort. similar trends were observed in age, sex, and race / ethnicitystratified groups. from 2002 to 2011, utilization of eis at day 0 increased from 14.9% to 21.8% (ptrend<0.001) and utilization of eis at day 0 or 1 increased from 27.8% to 41.4% (ptrend<0.001). riskadjusted inhospital mortality in the overall cohort decreased during the study period (adjusted or [per year ], 0.976 ; 95% ci, 0.974 to 0.978).conclusionsthere have been temporal increases in the proportion of nstemi and, consistent with guidelines, greater utilization of eis. this has been accompanied by temporal decreases in inhospital mortality and length of stay. |
menarche, an important milestone of sexual development, signals the end of puberty and the beginning of reproductive life in females [1, 2 ]. both early and delayed menarche have been associated with increased cardiovascular disease (cvd) risk factors (including metabolic syndrome) and disease in adolescent girls and young women [1, 37 ]. further more, women with long and irregular menstrual cycles have been shown to have higher risk for cvd and type 2 diabetes [5, 7, 8 ]. preeclampsia, a multisystem pregnancy disorder, is characterized by hypertension and proteinuria that develop after 20 weeks of gestation [914 ]. the precise pathophysiologic mechanisms of preeclampsia remain unknown and are subjects of extensive research [912, 1520 ]. since previously identified risk factors of preeclampsia (including maternal obesity, insulin resistance, and other hormonal factors) [15, 18 ] are potentially associated with early menarche, delayed menarche and/or menstrual irregularities [4, 5 ], age at menarche, and menstrual characteristics may be associated with risk of preeclampsia. previous reports on associations of menstrual characteristics with preeclampsia were based on case - control studies. among a well - characterized pregnancy cohort, we investigated relationships between age at menarche and menstrual characteristics with risk of preeclampsia. we also examined whether prepregnancy body mass index (bmi), adult weight gain, or maternal birth weight, a marker of intrauterine development that has been related to reproductive outcomes [2124 ], modified these associations. study participants were drawn from participants of the omega study [9, 25 ]. briefly, the study population comprised of women attending prenatal care clinics affiliated with swedish medical center (smc) in seattle, wa, and tacoma general hospital (tgh) in tacoma, wa. women were eligible if they initiated prenatal care before 20 weeks of pregnancy, were > 18 years old, were able to speak and read english, plan to carry the pregnancy to term, or planned to deliver at either of the two research hospitals. during 19962008, 5,063 eligible women were approached and 4,000 (79%) participants were enrolled in the study. of these, participants who moved or delivered elsewhere (n = 151), delivered before 20 weeks (n = 43), had prior chronic hypertension (n = 167), and had no available information on menstrual age (n = 274) or menstrual characteristics were excluded from current analyses (n = 3, 365). all participants provided written informed consent. using standardized questionnaires administered by a trained interviewer at or near the time of enrollment, we gathered information on age at menarche, menstrual characteristics, and other covariates including maternal sociodemographic factors, medical / reproductive histories, prepregnancy bmi, adult weight gain (differences in weight at age 18 and prepregnancy weight), and maternal birth weight. after delivery, maternal and infant medical records were reviewed for information on the course and outcomes of pregnancy. preeclampsia was defined using american college of obstetricians and gynecologists (acog) guidelines as sustained pregnancy - induced hypertension with proteinuria. hypertension was defined as sustained elevated blood pressure (bp) readings of 140/90 mm hg (with readings taking place 6 hours apart) after 20 weeks of gestation. proteinuria was defined as protein concentration of 30 mg / dl on 2 random urine specimen collected 4 hours apart. we gathered information on menstrual cycle characteristics including (a) age at menarche (the interviewer asked at what age did you have your first period ?), (b) irregular menses after menarche (the interviewer asked during the first year after starting your menstrual periods, did your periods become regular ? that is, could you predict within one week when your next menstrual period would begin ? and have your period ever been regular without using birth control pills, injection, or implants ?), and (c) usual cycle length (the interviewer asked on average, how often did you have your menstrual period ? that is, how many days were there between the first day of one menstrual period and the first day of the next ?). we examined general characteristics of the study population using mean (standard deviation) for continuous variables and numbers (%) for categorical variables. we used unadjusted and multivariable adjusted logistic regression analyses to compute odds ratio (or) and 95% confidence interval (95% ci). we used a priori identified indicator variables to categorize age at menarche (11, 12 13, 14, and 15 years) and cycle length (24, 2530, 3135, and 36 days). categories of age 13 years and 2530 days were used as references for age at menarche and cycle length categories, respectively. potential confounders that resulted in > 10% difference in estimated ors (comparing unadjusted and adjusted regression coefficients) were considered as confounders and were retained in multivariable models. the first adjusted model included variables for maternal age, race / ethnicity, parity, maternal birth weight, and pregestational diabetes. the second adjusted model included variables for prepregnancy bmi in addition to variables included in model 1. we also evaluated potential effect modification of the relationship between early age at menarche and menstrual cycle length with preeclampsia risk by prepregnancy overweight / obesity status (bmi 25 kg / m), adult weight gain, and maternal birth weight. for these analyses, we defined early menarche as age at menarche 11 years and long menstrual length as menstrual cycle 36 days. we used both interaction terms and stratified analyses to evaluate the presence of effect modification. participants, 32.6 years old on average, were predominantly white (86.4%) (table 1). among 3,365 participants of the cohort, 80 (2.4%) we observed a statistically nonsignificant higher risk of preeclampsia (unadjusted or : 1.56, 95% ci : 0.763.19) among participants with long cycle length (36 days) compared with participants with normal cycle length (2530 days) (table 2). there was a significant inverse association between age at menarche and risk of preeclampsia (p value for trend 0.05). women who were overweight and had early menarche (11 years) had a 4-fold increase in risk of preeclampsia compared with women who were not overweight and had menarche after age 11 (95% ci : 2.248.66). women who gained 5.0 kg in adulthood and did not have long cycles or early menarche had 4- to 5-fold higher risks of preeclampsia that were statistically significant compared with women who gained 0.05). we observed associations of early age at menarche with increased risk of preeclampsia and evidence of effect modification of the cycle length and risk of preeclampsia association by maternal prepregnancy body mass index. we also noted potential effect modifications of the early menarche and risk of preeclampsia association by maternal birth weight. similarly, adult weight gain appeared to modify the associations of age at menarche with risk of preeclampsia and long menstrual cycles with risk of preeclampsia, though these interactions were not statistically significant. previously, early menarche (12 years) has been associated with increased risk of cvd events ; investigators have hypothesized that observed associations are potentially mediated by increased adiposity associated with early menarche. in the current study, we found an inverse relationship between age at menarche and increased risk of preeclampsia. in a case - control study, also conducted in seattle, wa, rudra and colleagues reported nonsignificant 3-fold increase in risk of preeclampsia among overweight women with longer menstrual cycle length (or : 3.11, 95% ci : 0.6215), similar to our findings. the evidence from the current study is stronger since it is based on a study population from a prospective cohort study where maternal recall of age at menarche and menstrual characteristics is not influenced by the outcomes of the pregnancy. previously, adult weight gain of 10 kg has also been associated with a 5-fold increased risk of preeclampsia (or : 5.1, 95% ci : 2.212.2). however, its role in the relationships of age at menarche and cycle length with risk of preeclampsia has not been well investigated. in the current study girls who mature early (have early age at menarche) tend to be heavier (overweight or obese) as adults [1, 6, 28 ]. it has been suggested that early maturing girls may have a longer period of positive energy balance or other endocrine factors that influence both the rate of sexual maturation and the accumulation of body fat. obesity is associated with cardiometabolic abnormalities, including inflammation, and oxidative stress insulin resistance, which have been related to preeclampsia and other pregnancy complications. a number of biomarkers (e.g., c - reactive protein, tumor necrosis alpha) or pathomechanisms (e.g., endothelial dysfunction and dyslipidemia) that have been shown to be associated with both overweight / obesity status and preeclampsia support this evidence [4, 9, 16, 19, 2830 ]. birth weight is an indicator of intrauterine growth, a period of critical growth and programming [2124 ]. thus, maternal birth weight may be directly related to pathophysiologic changes that occur during pregnancy, including those that relate to risk of preeclampsia [21, 22 ]. in addition, maternal birth weight may play an indirect role in preeclampsia risk through its influence on other risk factors of preeclampsia (e.g., obesity). some limitations of our study deserve mention. while we collected and had access to information about maternal characteristics and other covariates, we are aware of the fact that self - reported medical histories may be subject to recall bias and misclassification. however, our prospective study design would assure that the potential misclassification is not conditional on the diagnosis of preeclampsia. although we adjusted for several potential confounders, we can not exclude the possibility of residual confounding. finally, we can not comment on the sequential influence of childhood and pubertal obesity on menstrual characteristics and eventual risk of preeclampsia. we have shown that early onset of menarche is associated with an increased risk of preeclampsia and prepregnancy weight modifies associations of cycle length with risk of preeclampsia. prepregnancy weight, adult weight gain, and maternal birth weight all appear to influence preeclampsia risk associated with early age at menarche and longer menstrual cycles. future larger studies that investigate this research area may create opportunities for prevention and early intervention of preeclampsia. | we examined associations of age at menarche and menstrual characteristics with the risk of preeclampsia among participants (n = 3,365) of a pregnancy cohort study. data were collected using in - person interviews and medical record abstraction. logistic regression was used to estimate adjusted odds ratio (or) and 95% confidence interval (95% ci). there was a significant inverse association between age at menarche and risk of preeclampsia (p value for trend 36 days) with higher risk of preeclampsia was present only among women who had prepregnancy body mass index < 25 kg / m2 (interaction p value = 0.04). early menarche is associated with higher risk of preeclampsia. prepregnancy weight may modify associations of long menstrual cycles with risk of preeclampsia. |
with the rapid development of dna microarray technology, we can now get the expression levels of thousands of genes via one single experiment with a relative cheap cost 1., 2.. these microarray data are essential in analyzing health situation of the human body and recognizing symptoms of human illnesses. mathematically, they can be expressed as a gene expression matrix x = (xij)nm, where each row represents a gene, while each column represents a sample or a patient for tumor diagnosis. that is, the numerical value xij denotes the expression level of a specific gene i at a particular sample j. as a matter of fact, there are many microarray datasets available on the web. for medical diagnosis and treatment, it is very important to select or discover informative genes of a tumor via the analysis of microarray data, since the informative genes can not only provide valuable information for discovering the crucial reasons of the tumor as well as the treatment methods, but also support to construct an efficient tumor diagnosis system from their expression levels directly without any influence of the other irrelevant genes. however, most of them are based on ranking genes according to a kind of criterion, such as t, f, rank sum and test statistics 3. recently, the up - and down - regulation probabilities for each gene were defined and then the informative genes can be successfully selected according to the decrease rank of the absolute difference values between the two regulation probabilities 9., 10.. generally, these statistical and information methods just select a number of top genes (the number is fixed or determined by the threshold given to the criterion). in this way, informative genes are selected through individual gene evaluations and thus the relations among the genes are neglected, which may lead to an incomplete selection of informative genes for tumor analysis and diagnosis. the relations or structures of genes can be discovered through unsupervised classification or clustering. in fact, some typical clustering methods have been already applied to the analysis of informative genes and tumor diagnosis, such as hierarchical clustering 4., k - means (13) and self - organizing map (som) (3). however, as the number of genes is large, hierarchical clustering is very difficult to be implemented and can not determine the informative genes by itself. moreover, the other classical clustering methods like k - means and som require predetermination of the number of gene clusters. however, we usually do not know the number of gene clusters since it depends on the structures of genes that are implied in the microarray data. in 1993, a new kind of unsupervised clustering method, called the rival penalized competitive learning (rpcl) algorithm 14., 15., was proposed to automatically determine the number of clusters during the clustering or competitive learning on the sample data. for each input sample, the basic idea is that not only the weight vector of the winner unit is modified to adapt to the input, but also the weight vector of its rival (the 2nd winner) is de - learned by a smaller learning rate. in this way, as the learning and de - learning rates are properly selected and the number of units or weight vectors is larger than the number of actual clusters in the sample data, the rpcl algorithm can automatically allocate an appropriate number of weight vectors for a sample dataset, with the other extra weight vectors being driven far away from the sample data. recently, the rpcl algorithm was generalized to the distance sensitive rival penalized competitive learning (dsrpcl) algorithm through a cost function theory (16). actually, the implementation of the dsrpcl algorithm becomes more efficient and easier since the learning rate can be easily set. thus, we can use the dsrpcl algorithm to analyze gene clusters from microarray data without knowing the actual number of gene clusters. in fact, the rpcl and dsrpcl algorithms have already been used to analyze microarray data 17. (17), the rpcl algorithm was applied to classify the genes into a number of clusters that could have certain functional meanings. moreover, the dsrpcl algorithm was utilized to classify the genes into a number of clusters from which a compact set of informative genes could be established via a post - filtering gene selection method (19). similarly to the dsrpcl algorithm, the cooperative competition clustering algorithm (20) was established to classify the genes into an appropriate number of clusters and then the informative genes can be selected from each of the obtained clusters. as the dsrpcl algorithm can automatically divide the genes into a number of functional clusters, we may wonder whether there exists a cluster that can be served as a set of informative genes directly. actually, we can utilize support vector machine (svm) (21) to check which gene cluster contributes best to the tumor diagnosis and whether it can be used as an informative gene set for a tumor. that is, we can use svm to train a tumor diagnosis system with the m expression profiles on the genes in each cluster and to see which tumor diagnosis system gets the best prediction accuracy. if a tumor diagnosis system gets the best prediction accuracy that is high enough, we consider the corresponding gene cluster is just the set of informative genes for the tumor. moreover, since the distributions of gene expression levels on the samples should have the similar structures at the critical or powerful informative genes, that is, the informative genes that can strongly discriminate the tumor from the normal via their expression levels on the samples, we can consider these critical or powerful informative genes belong to a sub - cluster in this informative gene set. thus, we can find these critical or powerful informative genes of the tumor by further clustering and checking on the sub - clusters of this informative gene cluster or set. as for the critical informative gene selection, guyon. have already proposed an svm - based method (22). they used a linear svm to train a tumor diagnosis system and selected the genes with higher weights in the final discriminate function as the critical or powerful genes. recently, zhang. improved this svm - based method by adding the smoothly clipped absolute deviation penalty on the original objective function of the svm (23). however, the major disadvantage of the svm - based method is that the number of informative genes is still determined by the pre - assumed threshold value to the weights, not by the structure of the genes. in this paper, in light of the above ideas, we propose a dsrpcl - svm approach where the dsrpcl algorithm is firstly utilized to classify the genes expressed through the microarray data into a number of clusters and the informative gene cluster or set is then detected with the help of svm. moreover, the critical or powerful informative genes is found through further classifications and detections on the informative gene clusters. the performance of this method is demonstrated by experiments on the colon, leukemia, and breast cancer datasets. given a microarray dataset x = (xij)nm with n genes and m samples, we let s={xi}i=1n, where x = [x1, x2,, xm ] represents the -th gene through its expression levels over all the m samples. suppose that x is just an input to a simple competitive learning network, that is, a layer of k competitive units. these competitive units are dominated by the corresponding weight vectors wi = [wi1, wi2,, wim ] for i = 1, 2,, k. all the weight vectors can be represented by a big vector w = vec[w1, w2,, wk ]. for each input x, the basic idea of the dsrpcl algorithm is that not only the weight vector of the winner unit (the closest weight vector to the sample) is modified to adapt to the input, but also the weight vectors of the rivals or losers (the other weight vectors) are punished to keep away from the input. as a weight vector diverges to infinity, the corresponding cluster becomes empty and can be canceled. therefore, we can automatically obtain the number of gene clusters as well as the centers of these clusters assuming k is larger than the true number of the actual gene clusters. as a result, the genes are automatically divided into several clusters by classifying each gene into the cluster whose center is closest to it. theoretically, the dsrpcl algorithm can be realized by minimizing the cost function in equation 1, where c() is the index of the winner unit for the -th gene, wc() is the nearest weight vector for x, and p is a positive constant. ma and wang (16) obtained the derivatives of e(w) with respect to wij as in equation 2, where i, j is the kronecker function. with these derivatives, table 1 summarizes the details of the dsrpcl algorithm and its variants, where we denote it as the batch dsrpcl algorithm. the dsrpcl1 algorithm is the adaptive dsrpcl algorithm, and the dsrpcl2 algorithm modifies only the rival weight vector (the second winner) so that e2(w) is only affected by the largest term with r(), which is consistent with the original rpcl algorithm 14., 15.. the other variant of the dsrpcl algorithm is the simulated annealing rival penalized competitive learning (sarpcl) by applying the simulated annealing mechanism to the dsrpcl1 algorithm. parameters k0, k1, c0 and c1 are positive constant numbers that can be selected by experience. since these dsrpcl algorithms have the similar functions in clustering analysis, we typically use the dsrpcl1 algorithm in our experiments.(1)e(w)=e1(w)+e2(w)=12xwc()2 + 2p,ic()xwip(2)e(w)ij=(i, c()xjij)+,i(1i, c()xwip2(xjij) according to the properties of the dsrpcl algorithm shown in ma and wang (16), when k is selected to be large enough, the dsrpcl algorithm can detect the number of gene clusters during the clustering. from the obtained gene clusters, we can check them with svm and find the informative gene cluster or set. we further consider the informative gene analysis through the dsrpcl algorithm and svm. in order to do so, we can implement the dsrpcl algorithm directly on the sample data s={x}=1n from the microarray data with respect to a tumor. usually, we can overestimate the number of the clusters in s and set it to be k. as a result of the implementation, the dsrpcl algorithm divides the n genes (represented by x) into a number of functional gene clusters among which there is an informative gene cluster contributing the best to the recognition or diagnosis of the tumor. if we think this informative gene cluster is too large, that is, it contains too many genes for tumor analysis, or we just want to get some more critical or powerful informative genes for the tumor, we can implement the dsrpcl algorithm on the previously obtained informative gene cluster or set (the obtained subset of s). again, the dsrpcl algorithm divides the selected informative genes into a number of gene clusters, among which we can find the most powerful sub - cluster and genes. in such a way, we can finally find a small number of critical or powerful genes for the tumor. on the other hand, the above informative gene search can be only done with the help of a supervised learning system for checking which gene cluster or sub - cluster contributes the best to the recognition or diagnosis of the tumor. actually, suppose gs={gi1,,gip } is a divided gene cluster or sub - cluster obtained by the dsrpcl algorithm, if the genes in this cluster (or sub - cluster) are informative (or powerful informative) to the tumor, then a supervised learning system on the sample expression profiles of these genes, x^j=[xi1,j,,xip, j]t(j=1,,m), and their corresponding diagnosis results (if the sample is tumorous, the diagnosis result is 1 ; otherwise the diagnosis result is 0) will lead to the highest prediction accuracy or the lowest average error. in this way, we can find the informative or powerful informative genes through a supervised learning system. svm (21) is a powerful learning machine for the supervised learning or classification. in fact, many researchers have used it to analyze microarray data and demonstrated its advantages 24., 25., 26.. therefore, we also utilize svm to check the informative or powerful informative gene cluster. for comparison, we use the matlab toolbox osusvm 3.0 to set up three kinds of svms, including linear svm (no kernel), 3-poly svm (cubic polynomial kernel), and radial basis function svm (rbf kernel). to test the effectiveness of our proposed dsrpcl - svm approach for informative gene analysis, we conducted experiments on three real - world microarray datasets : colon cancer dataset. it contains expression profiles of 2, 000 genes in 22 normal tissues and 40 colon tumor tissues, which can be retrieved from the web at http://microarray.princeton.edu/oncology/affydata/index.html. in our experiment, we used the training set (22 normal and 22 tumorous tissues) and the test set (18 tumorous tissues) provided at the website. it contains expression profiles of 7, 129 genes in 47 acute lymphoblastic leukemia (all) and 25 acute myeloid leukemia (aml) samples, which can be retrieved from the web at http://www.genome.wi.mit.edu/mpr/data_set_all_aml.htm. in our experiments, we used the training set (27 all and 11 aml) and the test set (20 all and 14 aml) provided at the website. it contains expression profiles of 5, 776 genes in 14 normal tissues and 13 breast tumorous tissues, which can be retrieved from the web at http://genome-www.stanford.edu/breast_cancer/sbcmp/data.shtml. in our experiment, we used the 14 normal and 13 tumorous samples provided at the website as both the training set and the test set since the number of the samples is very limited. for the three kinds of svms, there are two parameters in the last two svms, and c, and their selection affects the performance of svm. in our experiments, by experience we set = 0.02, c = 0.05 on colon cancer data and = 0.002, c = 10 on leukemia data and breast cancer data. to improve the efficiency of the dsrpcl1 algorithm, we set the de - learning rate in the update rule, that is, the corresponding learning rate for the opposite or minus direction learning, to attenuate to zero with the number of iterations. in this way, the algorithm could make the convergent weight vectors converge to the centers of the actual clusters in the sample data without any deviation, keeping the extra weight vectors being driven far away from the sample data. for the convenience of implementation of the two algorithms, we filtered out 10% obviously irrelevant genes using the cosine method. that is, we computed the cosine value of the vector of the expressions of each gene at all the samples with the reference vector in which each element is 1, and then ranked these genes with the decrease of the cosine value and finally filtered out 10% genes from the last. first, we ran the dsrpcl1 algorithm on the three microarray datasets and obtained five, four, and nine gene clusters (except the empty clusters represented by the weight vectors of the dsrpcl1 algorithm), respectively, where the number of the weight vectors or clusters was always set initially by 10 (the number of clusters in each microarray data was assumed to be no more than 10). according to these gene clusters obtained by the dsrpcl1 algorithm, we trained the three kinds of svms with the training data on each gene cluster of the three datasets. then we obtained the prediction or classification accuracy of each trained svm with the test data. the experiment results on the three datasets are summarized in table 2. from table 2, we can find that the dsrpcl1 algorithm not only discovers a good information gene set to a tumor, but also improves the accuracy of tumor diagnosis or classification. actually, in some cases, the svm on the selected informative gene set (cluster) can even reach 100% prediction accuracy. this means that the optimal gene cluster obtained from a dsrpcl - svm procedure on the microarray data is just the informative gene set to the tumor. next, we repeated the above dsrpcl - svm procedure (dsrpcl clustering and svm checking) on the optimal or informative gene cluster. that is, we just considered the genes in the optimal cluster and neglected all the other genes. in this way, we clearly obtained a smaller optimal gene cluster or sub - cluster. if the svm on this smaller optimal gene cluster still has a high prediction accuracy, we could consider the genes in this cluster are more powerful. in other words, they are powerful informative genes to the tumor. from the second dsrpcl - svm procedure, we also obtained a number of sub - clusters of genes and their prediction accuracies on the three datasets (table 3). from table 3, we can find that, after the second dsrpcl - svm procedure on each microarray dataset, a smaller informative gene cluster was obtained, while the best prediction accuracy of svm on this gene cluster was still rather high. in this way, we could repeat the procedures and ultimately find a set of most powerful genes to the tumor. table 4 gives the size (the number of genes) of the optimal gene cluster and the corresponding highest prediction accuracy of svm after each round of the dsrpcl - svm procedure. from table 4, we can find that the division of the optimal gene cluster would stop after several dsrpcl - svm procedures. as we successively divided the optimal gene cluster, the prediction accuracy of svm would drop slightly. this means that some informative genes were left, while the remaining genes became more powerful. as the division of the dsrpcl - svm procedure on the optimal gene cluster finally stopped, we obtained the most powerful informative genes to the tumor in this indivisible cluster table 5 is a list of the identity numbers of the powerful genes obtained from the experiments on the three datasets, respectively. although we do not know the biological meanings of these genes, we are sure that they are critical to the corresponding tumors for the medical diagnosis and treatment. from the further experiments, we can find that the dsrpcl - svm procedure can always find a set of several powerful genes to a tumor or some biological phenotypes. that is, the powerful genes may be changed greatly with the different initial values of the parameters in the dsrpcl algorithm. we think that the reasons of the instability or sensitivity to the initial parameter values may be two - fold. first, the dsrpcl algorithm may be sensitive with the initial values of the parameters when there are just a small number of samples in the dataset. second, the real powerful genes for a tumor my be dependent and our dsrpcl - svm method can only find a set of powerful genes on which an svm diagnosis system can still be made efficiently. given a microarray dataset x = (xij)nm with n genes and m samples, we let s={xi}i=1n, where x = [x1, x2,, xm ] represents the -th gene through its expression levels over all the m samples. suppose that x is just an input to a simple competitive learning network, that is, a layer of k competitive units. these competitive units are dominated by the corresponding weight vectors wi = [wi1, wi2,, wim ] for i = 1, 2,, k. all the weight vectors can be represented by a big vector w = vec[w1, w2,, wk ]. for each input x, the basic idea of the dsrpcl algorithm is that not only the weight vector of the winner unit (the closest weight vector to the sample) is modified to adapt to the input, but also the weight vectors of the rivals or losers (the other weight vectors) are punished to keep away from the input. as a weight vector diverges to infinity, the corresponding cluster becomes empty and can be canceled. therefore, we can automatically obtain the number of gene clusters as well as the centers of these clusters assuming k is larger than the true number of the actual gene clusters. as a result, the genes are automatically divided into several clusters by classifying each gene into the cluster whose center is closest to it. theoretically, the dsrpcl algorithm can be realized by minimizing the cost function in equation 1, where c() is the index of the winner unit for the -th gene, wc() is the nearest weight vector for x, and p is a positive constant. ma and wang (16) obtained the derivatives of e(w) with respect to wij as in equation 2, where i, j is the kronecker function. with these derivatives, table 1 summarizes the details of the dsrpcl algorithm and its variants, where we denote it as the batch dsrpcl algorithm. the dsrpcl1 algorithm is the adaptive dsrpcl algorithm, and the dsrpcl2 algorithm modifies only the rival weight vector (the second winner) so that e2(w) is only affected by the largest term with r(), which is consistent with the original rpcl algorithm 14., 15.. the other variant of the dsrpcl algorithm is the simulated annealing rival penalized competitive learning (sarpcl) by applying the simulated annealing mechanism to the dsrpcl1 algorithm. parameters k0, k1, c0 and c1 are positive constant numbers that can be selected by experience. since these dsrpcl algorithms have the similar functions in clustering analysis, we typically use the dsrpcl1 algorithm in our experiments.(1)e(w)=e1(w)+e2(w)=12xwc()2 + 2p,ic()xwip(2)e(w)ij=(i, c()xjij)+,i(1i, c()xwip2(xjij) according to the properties of the dsrpcl algorithm shown in ma and wang (16), when k is selected to be large enough, the dsrpcl algorithm can detect the number of gene clusters during the clustering. from the obtained gene clusters, we can check them with svm and find the informative gene cluster or set. we further consider the informative gene analysis through the dsrpcl algorithm and svm. in order to do so, we can implement the dsrpcl algorithm directly on the sample data s={x}=1n from the microarray data with respect to a tumor. usually, we can overestimate the number of the clusters in s and set it to be k. as a result of the implementation, the dsrpcl algorithm divides the n genes (represented by x) into a number of functional gene clusters among which there is an informative gene cluster contributing the best to the recognition or diagnosis of the tumor. if we think this informative gene cluster is too large, that is, it contains too many genes for tumor analysis, or we just want to get some more critical or powerful informative genes for the tumor, we can implement the dsrpcl algorithm on the previously obtained informative gene cluster or set (the obtained subset of s). again, the dsrpcl algorithm divides the selected informative genes into a number of gene clusters, among which we can find the most powerful sub - cluster and genes. in such a way, we can finally find a small number of critical or powerful genes for the tumor. on the other hand, the above informative gene search can be only done with the help of a supervised learning system for checking which gene cluster or sub - cluster contributes the best to the recognition or diagnosis of the tumor. actually, suppose gs={gi1,,gip } is a divided gene cluster or sub - cluster obtained by the dsrpcl algorithm, if the genes in this cluster (or sub - cluster) are informative (or powerful informative) to the tumor, then a supervised learning system on the sample expression profiles of these genes, x^j=[xi1,j,,xip, j]t(j=1,,m), and their corresponding diagnosis results (if the sample is tumorous, the diagnosis result is 1 ; otherwise the diagnosis result is 0) will lead to the highest prediction accuracy or the lowest average error. in this way, we can find the informative or powerful informative genes through a supervised learning system. svm (21) is a powerful learning machine for the supervised learning or classification. in fact, many researchers have used it to analyze microarray data and demonstrated its advantages 24., 25., 26.. therefore, we also utilize svm to check the informative or powerful informative gene cluster. for comparison, we use the matlab toolbox osusvm 3.0 to set up three kinds of svms, including linear svm (no kernel), 3-poly svm (cubic polynomial kernel), and radial basis function svm (rbf kernel). to test the effectiveness of our proposed dsrpcl - svm approach for informative gene analysis, we conducted experiments on three real - world microarray datasets : colon cancer dataset. it contains expression profiles of 2, 000 genes in 22 normal tissues and 40 colon tumor tissues, which can be retrieved from the web at http://microarray.princeton.edu/oncology/affydata/index.html. in our experiment, we used the training set (22 normal and 22 tumorous tissues) and the test set (18 tumorous tissues) provided at the website. it contains expression profiles of 7, 129 genes in 47 acute lymphoblastic leukemia (all) and 25 acute myeloid leukemia (aml) samples, which can be retrieved from the web at http://www.genome.wi.mit.edu/mpr/data_set_all_aml.htm. in our experiments, we used the training set (27 all and 11 aml) and the test set (20 all and 14 aml) provided at the website. it contains expression profiles of 5, 776 genes in 14 normal tissues and 13 breast tumorous tissues, which can be retrieved from the web at http://genome-www.stanford.edu/breast_cancer/sbcmp/data.shtml. in our experiment, we used the 14 normal and 13 tumorous samples provided at the website as both the training set and the test set since the number of the samples is very limited. for the three kinds of svms, there are two parameters in the last two svms, and c, and their selection affects the performance of svm. in our experiments, by experience we set = 0.02, c = 0.05 on colon cancer data and = 0.002, c = 10 on leukemia data and breast cancer data. to improve the efficiency of the dsrpcl1 algorithm, we set the de - learning rate in the update rule, that is, the corresponding learning rate for the opposite or minus direction learning, to attenuate to zero with the number of iterations. in this way, the algorithm could make the convergent weight vectors converge to the centers of the actual clusters in the sample data without any deviation, keeping the extra weight vectors being driven far away from the sample data. for the convenience of implementation of the two algorithms, we filtered out 10% obviously irrelevant genes using the cosine method. that is, we computed the cosine value of the vector of the expressions of each gene at all the samples with the reference vector in which each element is 1, and then ranked these genes with the decrease of the cosine value and finally filtered out 10% genes from the last. first, we ran the dsrpcl1 algorithm on the three microarray datasets and obtained five, four, and nine gene clusters (except the empty clusters represented by the weight vectors of the dsrpcl1 algorithm), respectively, where the number of the weight vectors or clusters was always set initially by 10 (the number of clusters in each microarray data was assumed to be no more than 10). according to these gene clusters obtained by the dsrpcl1 algorithm, we trained the three kinds of svms with the training data on each gene cluster of the three datasets. then we obtained the prediction or classification accuracy of each trained svm with the test data. the experiment results on the three datasets are summarized in table 2. from table 2, we can find that the dsrpcl1 algorithm not only discovers a good information gene set to a tumor, but also improves the accuracy of tumor diagnosis or classification. actually, in some cases, the svm on the selected informative gene set (cluster) can even reach 100% prediction accuracy. this means that the optimal gene cluster obtained from a dsrpcl - svm procedure on the microarray data is just the informative gene set to the tumor. next, we repeated the above dsrpcl - svm procedure (dsrpcl clustering and svm checking) on the optimal or informative gene cluster. that is, we just considered the genes in the optimal cluster and neglected all the other genes. in this way, we clearly obtained a smaller optimal gene cluster or sub - cluster. if the svm on this smaller optimal gene cluster still has a high prediction accuracy, we could consider the genes in this cluster are more powerful. in other words, they are powerful informative genes to the tumor. from the second dsrpcl - svm procedure, we also obtained a number of sub - clusters of genes and their prediction accuracies on the three datasets (table 3). from table 3, we can find that, after the second dsrpcl - svm procedure on each microarray dataset, a smaller informative gene cluster was obtained, while the best prediction accuracy of svm on this gene cluster was still rather high. in this way, we could repeat the procedures and ultimately find a set of most powerful genes to the tumor. table 4 gives the size (the number of genes) of the optimal gene cluster and the corresponding highest prediction accuracy of svm after each round of the dsrpcl - svm procedure. from table 4, we can find that the division of the optimal gene cluster would stop after several dsrpcl - svm procedures. as we successively divided the optimal gene cluster, the prediction accuracy of svm would drop slightly. this means that some informative genes were left, while the remaining genes became more powerful. as the division of the dsrpcl - svm procedure on the optimal gene cluster finally stopped, we obtained the most powerful informative genes to the tumor in this indivisible cluster. table 5 is a list of the identity numbers of the powerful genes obtained from the experiments on the three datasets, respectively. although we do not know the biological meanings of these genes, we are sure that they are critical to the corresponding tumors for the medical diagnosis and treatment. from the further experiments, we can find that the dsrpcl - svm procedure can always find a set of several powerful genes to a tumor or some biological phenotypes. that is, the powerful genes may be changed greatly with the different initial values of the parameters in the dsrpcl algorithm. we think that the reasons of the instability or sensitivity to the initial parameter values may be two - fold. first, the dsrpcl algorithm may be sensitive with the initial values of the parameters when there are just a small number of samples in the dataset. second, the real powerful genes for a tumor my be dependent and our dsrpcl - svm method can only find a set of powerful genes on which an svm diagnosis system can still be made efficiently. we investigated the problem of informative gene discovery and analysis from the perspective of the newly established unsupervised clustering method dsrpcl algorithm. since the dsrpcl algorithm can detect the number of clusters automatically in a dataset, we apply it to dividing the genes expressed through microarray data into a number of functional gene clusters and use svm to check which cluster is the set of informative genes to the tumor. moreover, this dsrpcl - svm procedure can be further implemented on the informative gene cluster successively and find out the critical or powerful informative genes of the tumor. our experiments on the colon, leukemia, and breast cancer datasets demonstrated that this dsrpcl - svm method is really efficient for discovering the informative gene set as well as the powerful informative genes for a tumor through the microarray data. wx and zc participated in the method design, performed the experimental work and drafted the manuscript. jm conceived the idea of using this approach, guided the research and completed the manuscript. wx and zc participated in the method design, performed the experimental work and drafted the manuscript. jm conceived the idea of using this approach, guided the research and completed the manuscript. | microarray data based tumor diagnosis is a very interesting topic in bioinformatics. one of the key problems is the discovery and analysis of informative genes of a tumor. although there are many elaborate approaches to this problem, it is still difficult to select a reasonable set of informative genes for tumor diagnosis only with microarray data. in this paper, we classify the genes expressed through microarray data into a number of clusters via the distance sensitive rival penalized competitive learning (dsrpcl) algorithm and then detect the informative gene cluster or set with the help of support vector machine (svm). moreover, the critical or powerful informative genes can be found through further classifications and detections on the obtained informative gene clusters. it is well demonstrated by experiments on the colon, leukemia, and breast cancer datasets that our proposed dsrpcl - svm approach leads to a reasonable selection of informative genes for tumor diagnosis. |
a devastating consequence of the worldwide obesity epidemic is a steady rise in the number of patients with type 2 diabetes (t2d). the global prevalence of t2d is estimated at 347 million currently, and this number is projected to increase to 552 million by 2030. in the united states alone, 29.1 million individuals (9.3% of the population) are diabetic, with t2d accounting for 90% to 95% of all diagnosed cases. the risk of diabetes complications increases as a function of rising plasma glucose levels ; currently, adequate glycemic control is achieved by only approximately 40% of patients with t2d. these observations collectively highlight the need for new strategies to more effectively treat t2d and thereby reduce the enormous toll on human health taken by this disease. historically, the primary focus of the diabetes research community has been on pancreatic islet cells and insulin - sensitive tissues, and this work has revealed an enormous amount with respect to the contribution made by defective insulin secretion or action (or both) to the development of impaired glucose tolerance (igt) and t2d. however, an increasing body of evidence supports the existence of specific neurocircuits that sense and integrate a variety of afferent signals and in response trigger adaptive changes of glucose metabolism in peripheral tissues, collectively referred to as the brain - centered glucoregulatory system (bcgs). the bcgs appears to act coordinately with pancreatic islets to regulate blood glucose levels via both insulin - dependent and insulin - independent mechanisms. an improved understanding of how bcgs - islet interactions participate in normal and abnormal glucose homeostasis may ultimately facilitate the development of more successful therapeutic regimens. a role for the brain in glucose homeostasis was first proposed by the french physiologist claude bernard on the basis of his observations that glycosuria could be induced in rabbits by lesions directed to the floor of the fourth ventricle. nearly a century later, oomura and colleagues and anand and colleagues confirmed the presence of specialized glucose - sensing neurons within the hypothalamus that could be either excited or inhibited by fluctuations of ambient glucose levels. subsequent studies identified distinct subsets of these neurons as being either glucose - excited or glucose - inhibited, and new technologies in mouse genetics, viral tracing, optogenetics, and dreadds (designer receptor exclusively activated by designer drugs) are rapidly accelerating research progress. the goals of this review are to highlight recent advances in our understanding of neural and hormonal connections linking the brain and gastrointestinal tract in the control of glucose homeostasis and how they interact with the islet - centered glucoregulatory system. after ingestion of a meal, the presence of nutrients in the gastrointestinal tract initiates complex neural and hormonal responses that inform the brain of ongoing changes in nutritional status. the gut is richly supplied with primary visceral afferent nerve fibers from both sympathetic and parasympathetic branches of the autonomic nervous system. neural signals from the gut are integrated by hindbrain nuclei that project in turn to forebrain regions, including the hypothalamus. the gut also informs the brain of current nutritional status by secreting a host of gut peptides from both enteroendocrine cells ghrelin, cholecystokinin (cck), and glucagon - like peptide 1 (glp-1)and enterocytes (fibroblast growth factor 19, or fgf19). some of these hormones communicate with the central nervous system (cns) primarily via effects on nearby afferent nerve fibers supplying the gut, whereas others are secreted from the gut into the circulation, whereupon they enter the brain to mediate their central effects. an emerging picture suggests that some of these gut signals are involved a gut - brain - liver axis that participates in glucose homeostasis. increased levels of circulating nutrients (glucose, fatty acids, and amino acids) following a meal generate signals to the cns regarding short - term energy status and act within the hypothalamus and hindbrain to regulate both food intake and glucose homeostasis. neurons in these brain areas can be either excited or inhibited in response to glucose, lactate, or other nutrients (for example, oleic acid or leucine), and central infusion of many of these nutrients has been shown to lower circulating glucose levels by inhibiting hepatic glucose production (hgp) [1619 ]. the intestinal neuronal network has also been identified as a target for nutrient regulation of glucose homeostasis. using an intra - intestinal nutrient infusion paradigm in rats, lam and colleagues report that infusion of lipids into either the duodenum or jejunum reduces hgp, with the specific mechanisms underlying this effect depending on the intestinal segment being targeted. the metabolic response to intra - intestinal lipid infusion appears to involve increased concentrations of long - chain fatty acids (lcfas) within enterocytes and is abolished by either hepatic vagotomy or blockade of hindbrain glutamatergic receptors (vagal afferents communicate with hindbrain neurons via glutamate release). intraduodenal lipids also stimulate the release of cck from duodenal i cells, and activation of cck receptors on vagal afferents is implicated in the mechanism that lowers hgp. the same group reported that, in the jejunum, administration of either glucose or lipids also reduces hgp, and this effect can not be explained by entry of nutrients into the hepatic portal vein, since equimolar infusion of glucose directly into the portal vein was without effect. the gut hormone glp-1 is also secreted postprandially from the distal small intestine and acts through a specific g - protein - coupled receptor (glp1r) expressed on pancreatic and cells, cns neurons, afferent and efferent enteric neurons, vagal and dorsal root ganglia, vascular smooth muscle, and gastric antrum and pylorus among other sites. incretin on the basis of its ability to enhance glucose - induced insulin secretion. when administered peripherally, glp-1 also inhibits glucagon secretion, slows gastric emptying, reduces food intake, and increases insulin - independent glucose disposal. accordingly, long - acting glp-1 analogues are widely used in the treatment of t2d, but they also have documented benefit in patients with type 1 diabetes (t1d), despite having no ability to increase insulin secretion in this setting. the mechanisms underlying the insulin - independent action of glp-1 are unknown, but both peripheral and central sites of action likely contribute. in the periphery, glp-1 infusion into the hepatic portal vein both increases the firing rate of the vagus nerve and enhances glucose disposal. similarly, centrally administered glp-1 improves glucose tolerance, whereas intracerebroventricular (icv) infusion of a pharmacological inhibitor of glp-1 receptors impairs glucose tolerance. together, these findings suggest that intact neuronal glp-1 signaling is required for normal glucose homeostasis, which implies that the glucose - lowering action of glp-1 involves central as well as peripheral mechanisms. however, the role of cns or vagal glp-1 signaling (or both) in glucose homeostasis remains controversial, as the glucose - lowering effects of the long - acting glp-1 agonist liraglutide are maintained in mice with either vagal- or brain - specific deletion of glp1r. the enterocyte hormone fgf19 is a member of the fgf hormone family (which includes fgf21 and fgf23) that was discovered in a screen of genes induced by activation of the bile acid receptor, farnesoid x receptor (fxr). in response to a meal, increased release of bile acids into the intestine is hypothesized to increase fxr signaling in enterocytes and thereby induce fgf19 synthesis and secretion. fgf19 was initially identified as a key regulator of hepatic bile acid homeostasis via its effect to inhibit cyp7a1, the rate - limiting enzyme in hepatic bile acid synthesis, and growing evidence suggests that fgf19 may participate in glucose homeostasis as well. when administered peripherally, fgf19 improves glucose tolerance in both genetically obese (for example, leptin - deficient ob / ob mice) and diet - induced obese (dio) mice. in the liver, fgf19 reduces hgp via actions that closely resemble those of insulin but are insulin - independent. moreover, these hepatic actions may be mediated centrally, since icv fgf19 administration improves glucose tolerance in dio rats, whereas icv administration of an fgf - receptor inhibitor has the opposite effect. as for glp1, available evidence suggests that intact neuronal fgf signaling is required for normal glucose homeostasis. the fact that insulin inhibits hgp via a direct action on hepatic insulin receptors is well established. binding of insulin to its receptor on hepatocytes activates the canonical insulin receptor substrate (irs)-phosphatidylinositol 3-oh (pi3k)-akt pathway. among many intracellular targets of the serine - threonine kinase akt is forkhead box protein o1 (foxo1), a transcription factor that potently stimulates hepatic gluconeogenesis. phosphorylation of foxo1 by akt inhibits its transcriptional activity, and this effect is required for insulin to suppress hgp. a role for hepatic insulin action in the physiological control of hgp was established with the demonstration of severe glucose intolerance and insulin resistance in mice with liver - specific knockout of the insulin receptor (lirko mice), and a similar phenotype is seen in mice with liver - specific deletion of the two hepatic akt isoforms (dlko mice). not surprisingly, activation of foxo1 in hepatocytes (owing to reduced akt signaling) is implicated in the increased hgp characteristic of these mouse models. in addition to its direct hepatic effect, however, growing evidence suggests that hgp can be inhibited by insulin action at a remote site. buettner and colleagues demonstrated in mice that although knockdown of hepatic insulin receptors (using insulin receptor anti - sense oligonucleotides) has the expected effect of markedly attenuating hepatic insulin signaling, hgp can nevertheless be inhibited by hyperinsulinemia. subsequent evidence of an indirect pathway for insulin regulation of hgp comes from comparison of dlko mice with mice with liver - specific deletion of foxo1 as well as of the two akt isoforms (tlko mice). unlike glucose intolerance of lirko mice, tlko mice exhibit normal glucose homeostasis and intact inhibition of hgp in response to hyperinsulinemia, although hepatocytes of these mice are insulin - insensitive. these data suggest that (a) insulin can inhibit hgp via both direct and indirect pathways, (b) insulin action via the indirect pathway is sufficient to maintain normal glucose homeostasis, (c) excessive hepatic foxo1 signaling (as occurs in lirko and dlko mice) can block both direct and indirect pathways. although the mechanisms mediating the indirect control of hgp by insulin remain to be elucidated, insulin action on neuronal insulin receptors likely plays a role. in humans, intranasal application of insulin lowers postprandial blood insulin levels and improves whole - body insulin sensitivity in lean patients as assessed by the hyperinsulinemic euglycemic clamp. in rodents, low - dose icv insulin is reported to increase hepatic insulin sensitivity, whereas infusion into the third ventricle of insulin receptor antibodies or an inhibitor of pi3k decreases hepatic insulin sensitivity and impairs the ability of systemic insulin to suppress hgp. these findings suggest that neuronal insulin signaling is required to maintain hgp within normal limits. a signal transduction mechanism forwarded to explain this insulin effect involves activation of neuronal atp - sensitive potassium (katp) channels via the irs - pi3k pathway. katp channels are expressed by neurons in the hypothalamus and are composed of an inwardly rectifying potassium ion channel (kir6.2) subunit and sulfonylurea receptor subunit. when the channel is activated (or opened) (for example, in response to the drug diazoxide), potassium ions diffuse down the concentration gradient from the intracellular to the extracellular space, thereby reducing resting membrane potential and the likelihood of firing. a similar effect is observed following icv administration of insulin, which activates katp channels and thereby decreases the firing rate of insulin - responsive hypothalamic neurons. these neuronal effects of insulin are blocked by icv co - infusion of either the katp channel inhibitor tolbutamide or the pi3k inhibitor wortmannin, and these interventions also negate the effect of icv insulin to lower hgp. interestingly, icv infusion of diazoxide mimics the glucose - lowering effects of icv insulin in rats, and in humans, systemic administration of diazoxide reduces hgp even when circulating insulin levels are clamped, implying an insulin - independent mechanism. extra - hypothalamic sites such as the dorsal vagal complex of the hindbrain are also implicated in the indirect control of hgp by insulin, apparently via insulin receptor - mediated activation of extracellular signal - related kinase (erk) 1/2 rather than pi3k. a distributed network of insulin - responsive neurons may therefore participate in the indirect control of hgp by insulin. the efferent mechanism(s) linking the brain to indirect control of hgp by insulin may involve vagal efferent fibers, as ablation of efferent, but not afferent, vagal input to the liver prevents the effect of centrally administered insulin to inhibit hgp. additional mechanisms are likely, however, and going forward, studies to definitively identify mechanisms linking the brain to indirect control of hgp are an important scientific priority. although there is little doubt that insulin receptor signaling in peripheral tissues plays a key role in glucose homeostasis (especially in the postprandial state), growing evidence suggests that both hgp and tissue glucose uptake can also be controlled via insulin - independent mechanisms. evidence that the brain can potently induce insulin - independent glucose lowering stems in part from studies of rodents with uncontrolled diabetes. unger and colleagues demonstrated that, when administered systemically at pharmacological doses, the adipocyte hormone leptin normalizes elevated blood glucose levels in rats and mice with uncontrolled diabetes, whether induced by a cell toxin streptozotocin (stz)or by autoimmune destruction (in non - obese diabetic mice). a role for the brain in this anti - diabetic leptin action was identified initially on the basis of the finding that, in rats with stz - diabetes, continuous icv infusion of leptin normalized markedly elevated blood glucose levels at low doses that are ineffective when administered peripherally. subsequent work in the stz - diabetic rat model by morton and colleagues demonstrated that icv leptin infusion both increases tissue glucose uptake and normalizes hgp despite persistent severe insulin deficiency. one implication of these findings is that, since insulin levels were too low to explain the observed normalization of hgp, the effect of central leptin can not be attributed to increased hepatic insulin sensitivity. this conclusion implies that the brain has the capacity to normalize hgp and restore euglycemia to diabetic animals even in the absence of insulin. although the mechanisms whereby central leptin administration mediates glucose lowering in the absence of insulin remain uncertain, the effect involves both decreased gluconeogenic gene expression in liver and increased whole - body glucose turnover and glucose uptake in peripheral tissues, including skeletal muscle, heart, and brown adipose tissue. work from the shulman laboratory recently suggested that the primary mechanism mediating the central action of leptin in diabetic rats involves inhibition of the hypothalamic - pituitary - adrenal (hpa) axis and associated lowering of elevated circulating glucocorticoid levels. such an effect is plausible since the hpa axis is clearly activated in uncontrolled diabetes and since icv leptin reverses this effect. other findings, however, suggest that normalizing hpa axis activity is insufficient to reverse diabetic hyperglycemia, suggesting that further work is needed for a comprehensive understanding of the mechanisms by which central leptin signaling normalizes blood glucose in the absence of insulin. glucose tolerance measures the efficiency with which glucose is cleared from the bloodstream after a glucose load, and it is determined by both insulin - dependent and -independent mechanisms. the insulin - independent component of glucose tolerance, termed glucose effectiveness (ge), can be calculated in humans and rodents on the basis of minimal model analysis of glucose and insulin kinetics during a frequently sampled intravenous glucose tolerance test developed by bergman and colleagues. our recent work in ob / ob mice investigated whether the glucose - lowering action of fgf19 in the brain involves an insulin - independent mechanism. the rationale underlying this work lies in part in the fact that insulin - independent mechanisms contribute as much to glucose tolerance as insulin does and that in ob / ob mice ge is reduced by approximately 75%. it was therefore difficult to envision a scenario in which fgf19 could normalize glucose tolerance in these animals (as reported by fu and colleagues in 2004) unless ge were to increase. consistent with this hypothesis, we found that icv fgf19 markedly improves glucose tolerance in ob / ob mice by potently, rapidly, and selectively increasing ge. these observations support the overarching concept that, in response to neural and hormonal signals generated by nutrient signaling in the gut, the brain can influence glucose homeostasis via both insulin - dependent and -independent mechanisms (figure 1). glucose homeostasis is hypothesized to involve cooperative and coordinated interactions between brain- and islet - centered regulatory systems. after a meal, nutrients in the gastrointestinal tract rising blood glucose levels trigger pancreatic cells to secrete insulin, which lowers blood glucose levels both by inhibiting hepatic glucose production and by stimulating glucose uptake into insulin - sensitive tissues (primarily muscle and adipose tissue). like the islet, the brain - centered glucoregulatory system (bcgs) senses and integrates multiple humoral nutrients, glucose, leptin, and fibroblast growth factor 19 (fgf19)and neural (vagal afferent) signals of nutritional status and in response promotes glucose disposal by both insulin - dependent and -independent mechanisms. in this way, coordinated activation of both islet- and brain - centered systems by incoming nutrients serves to maintain blood glucose levels within a narrow physiologic range. the ineffectiveness of medical therapy for the treatment of obesity has driven the development of multiple bariatric surgical procedures, including roux - en - y gastric bypass (rygb) and sleeve gastrectomy. in 1952, henrikson performed the first surgical intervention for the treatment of obesity, and with subsequent refinement bariatric surgery has emerged as a relatively safe and effective therapeutic option for some obese patients. unexpectedly, some bariatric surgical procedures also produce an almost immediate glucose - lowering effect that appears to involve mechanisms that are at least partly independent of weight loss. consequently, although remission of t2d is rare with conventional treatment, it is common (typically, in more than 50% of cases) following rygb surgery. although the physiological and molecular mechanisms underlying the metabolic benefits of bariatric surgery remain incompletely understood, a marked increase in the rapidity with which ingested nutrients enter the small intestine appears to be key. in response, a number of profound intestinal adaptations are described, including hypertrophy of intestinal mucosa, increased postprandial secretion of gut hormones (including glp-1, pyy, and fgf-19), increased serum bile acid levels, reprogramming of intestinal glucose metabolism, and alterations of gut microbiota [6568 ]. a growing literature has begun to unravel the contributions made by each of these adaptations to improvements of glucose homeostasis after bariatric surgery, and it seems reasonable to anticipate that metabolic benefit is conferred by a constellation of many of these effects (rather than by a single, overarching mechanism). nonetheless, seeley and colleagues recently reported that metabolic benefit arising from vertical sleeve gastrectomy is linked to increased circulating bile acids and is prevented in mice lacking the bile acid receptor, fxr. interestingly, activation of fxr by bile acids is required for intestinal secretion of fgf19 (fgf15 in rodents), raising the untested possibility that this hormone plays a key role in the glucose - lowering effect of the procedure. does the brain play a role in the response to bariatric surgery ? in humans, changes in neural activity in brain reward centers in response to food cues can be measured by functional magnetic resonance imaging, and this brain response is modified after rygb in a manner dissimilar to that observed in patients who underwent gastric banding for obesity. (unlike rygb, gastric banding reduces, rather than increases, the rate of intestinal nutrient delivery.) in a rat model, jiao and colleagues demonstrated that the effect of duodenal - jejunal bypass (djb) to improve glucose tolerance is prevented by hepatic vagotomy. moreover, breen and colleagues reported that djb is effective in lowering blood glucose levels in rats with stz - induced uncontrolled diabetes, implicating insulin - independent mechanisms in the effect, and paranjape and colleagues reported that intact insulin signaling in the ventromedial hypothalamic nucleus (vmn) is necessary for the ability of rygb to improve hepatic insulin sensitivity in a rat dio model. the hypothesis that the vmn is involved in the improvement of glucose homeostasis after bariatric surgery is of interest given its known role in the neural control of hepatic glucose metabolism by efferent sympathetic nervous system fibers that reach the liver via the splanchnic nerve [7375 ]. whether the brain plays a major role in the anti - diabetic effect of bariatric surgery in humans is an important unanswered question ; if so, interventions that target brain, intestine, and islet together may achieve improvements in glucose homeostasis greater than those that can be achieved by insulin - based therapies alone. a growing body of evidence in animal models and humans supports a key role for the brain in the control of glucose homeostasis. although the focus of the diabetes research community has traditionally been placed on the islet, clear evidence now indicates that the brain can exert potent and rapid effects on both hgp and glucose uptake and that, in some cases, the effect involves insulin - independent mechanisms. historically, the delivery of therapeutics to the brain has been hindered both by the challenge imposed by drug delivery across the blood - brain barrier (bbb) and by the potential for worrisome off - target effects. however, the development of novel drug delivery systems designed to circumvent the bbb, including intranasal preparations, nanocarriers, and structural manipulation and targeted use of endogenous transport / carrier systems, holds promising for the future of brain - directed therapies. similarly, the use of biologicals may reduce unwanted side effects associated with small - molecule drugs, many of which have significant off - target effects. moving forward, it will be important to better understand how brain and islet interact on a day - to - day basis in the normal and abnormal control of glucose homeostasis. also important are studies to clarify how the brain promotes insulin - independent glucose disposal and whether such an effect contributes to the improvement of glucose homeostasis in patients with t2d after bariatric surgery. studies along these lines have important potential to advance our understanding of diabetes pathogenesis and to identify new treatments for diabetes and related disorders. michael w. schwartz has served as a consultant to novo nordisk (bagsvrd, denmark) and janssen (beerse, belgium) within the past year. | our current understanding of glucose homeostasis is centered on glucose - induced secretion of insulin from pancreatic islets and insulin action on glucose metabolism in peripheral tissues. in addition, however, recent evidence suggests that neurocircuits located within a brain - centered glucoregulatory system work cooperatively with pancreatic islets to promote glucose homeostasis. among key observations is evidence that, in addition to insulin - dependent mechanisms, the brain has the capacity to potently lower blood glucose levels via mechanisms that are insulin - independent, some of which are activated by signals emanating from the gastrointestinal tract. this review highlights evidence supporting a key role for a gut - brain - liver axis in control of glucose homeostasis by the brain - centered glucoregulatory system and the implications of this regulatory system for diabetes pathogenesis and treatment. |
the incidence of b - cell neoplasms in europe has been estimated at approximately 21 per 100,000. optimization of conventional cytostatic regimens through addition of tumor - specific anti - cd20 monoclonal antibodies (mabs) or dose intensification followed by autologous / allogeneic stem cell transplantation has significantly improved treatment outcome of b - cell neoplasms over the last years. however, many patients eventually succumb either to treatment - refractory disease or to severe treatment - related side effects [3, 4 ]. this necessitates the development of target - directed anticancer therapies with increased antitumor efficacy, yet acceptable systemic toxicity. antibody - drug conjugates (adcs) harness the targeting function of monoclonal antibodies towards tumor - associated antigens (taa) to deliver potent cytotoxic drugs. adcs have progressed to phase iii trials and the first such compounds approved were gemtuzumab ozogamicin and brentuximab vedotin for the treatment of acute myeloid leukemia and relapsed hodgkin and anaplastic large cell lymphoma, respectively. with only modest complete remission rates of 30% and unexpectedly severe postapproval toxicity that in part outweighed its clinical benefit more recently trastuzumab emtansine (t - dm1) has been approved for the treatment of metastasized her2-positive breast cancer. for the treatment of hematologic malignancies several other adcs, targeting cd79b, cd74, cd33, cd30, cd22, and cd19, are currently in clinical development. prerequisite for the antitumoral activity of adcs is sufficient cellular internalization of the compound upon taa - binding, followed by the intracellular release of the carried payload. the b - cell lineage restricted receptor cd22, being overexpressed in the majority of b - cell non - hodgkin lymphomas (b - nhl), as well as in b - cell precursor acute lymphoblastic leukemia (bcp - all), is a particularly attractive target for adc approaches. this is due to the very rapid and sustained internalization of the targeted receptor [11, 12 ] and its absence on hematopoietic stem cells. inotuzumab ozogamicin (cmc-544), an anti - cd22-calicheamicin adc, has been extensively studied in patients with both indolent and aggressive b - cell nhl as well as acute leukemias. several phase i and ii studies conducted with inotuzumab ozogamicin demonstrated in part highly significant clinical activity across all explored entities. however, in 2013 an ongoing phase iii study in patients with aggressive b - nhl was discontinued since an interim analysis of overall survival demonstrated no statistically significant superiority of cmc-544 in combination with rituximab over the comparator arm. the press release reporting on the study termination concluded that hematologic cancers are a complex group of diseases, with more than 70 different types of lymphomas, leukemias or myelomas that require unique treatment options. therefore, clinical development of anti - cd22 adcs with alternative payloads remains of utmost importance. the murine anti - cd22 igg1 mab rfb4 and a disulfide antibody fragment derivative, dsfv - rfb4, have been covalently linked to plant toxins or genetically fused to bacterial toxins, respectively [1519 ]. from these compounds however, administration of bl22 was associated with severe adverse effects such as immunogenic reactions and in a few cases development of capillary leak syndrome. as a consequence, a higher affinity antibody fragment derivative for linkage to the bacterial toxin has been developed and the compound (ha22, cat 8015) exhibited a more favorable toxicity profile, yet similar potent activity as its predecessor in a phase i trial in patients with chemotherapy - resistant hairy cell leukemia. valuable payload alternatives to bacterial toxins are ribonucleases from the pancreatic ribonuclease (rnase) a superfamily with near absence of immunogenicity. onconase (onc, ranpirnase), a 12 kda basic single - chain protein, originally isolated from oocytes of rana pipiens, kills tumor cells with an ld50 of 10 m which is comparable to the potency of maytansinoids and auristatins. the antitumor effects of onc can be ascribed to trna- [24, 25 ], dsrna-, and mirna - cleavage [26, 27 ], as well as to transcriptional gene regulation interactions. in phase i / ii clinical trials for treatment of various solid tumors and malignant mesothelioma onc was immunologically well tolerated and displayed acceptable and reversible systemic toxicity [29, 30 ]. after conjugation to the murine anti - cd22 igg2a - mab ll2, onc caused only mild off - target toxicity in lymphoma xenografted mice, and the toxic total cumulative dose (tcd) was reached only at concentrations of > 300 mg / kg body weight. in comparison, a pseudomonas exotoxin - ll2 immunoconjugate caused 100% lethality in mice at a tcd of 7 mg / kg. thus, onc seems to present a promising payload for anti - cd22 immunoconjugates by combining high antitumor potency and low systemic toxicity. in this study we report on the generation, purification, and in vitro characterization of different onc - based anti - cd22 immunoconjugates. as antibody targeting moiety we reengineered the previously humanized rfb4 scfv into a humanized igg1. we tested two different chemical conjugation strategies using various crosslinkers for noncleavable and thiol - cleavable linkage and different onc payload formats. sophisticated purification procedures were used for obtaining active adcs with distinct molar drug to antibody ratios (dars). we show that immuno - rnases were able to kill targeted lymphoma and leukemia cells in a dar - dependent manner. all tumor cell lines were purchased from atcc (manassas, va, usa). onconase (ranpirnase) was a kind gift from kuslima shogen (alfacell corporation, new jersey, usa). the rnase substrate poly - ru was acquired from amersham biosciences (little chalfont, uk) ; the fluorogenic substrate 6-fam - darudgda - bhq-1 (6-carboxyfluorescein - darudgda - black - hole - quencher-1) was from biomers.net (ulm, germany). fitc- (fluorescein isothiocyanate-) conjugated secondary antibodies were obtained from jackson immunoresearch (laboratories, west grove, pa, usa) ; the control antibody rfb4 was from santa cruz biotechnology (dallas, texas, usa). benchmark prestained protein ladder, dmem (dulbecco 's modified eagle 's medium), 2-mercaptoethanol, nupage lds sample buffer (4x), novex himark pre - stained protein standard, novex tris - acetate sds running buffer, and novex nupage 38% tris - acetate gels were purchased from life technologies gmbh (darmstadt, germany). runblue lds sample buffer, runblue rapid sds run buffer, and runblue 12% sds gels were acquired from expedeon (harston, uk). fbs (fetal bovine serum), pbs (phosphate buffered saline), penicillin, rpmi-1640 (roswell park memorial institute) medium, sodium azide, and streptomycin were obtained from sigma - aldrich (st. louis, mo, usa). alamarblue, 2-it (2-iminothiolane), smcc (succinimidyl 4-(n - maleimidomethyl) cyclohexane-1-carboxylate), spdp (n - succinimidyl 3-(2-pyridyldithio) propionate), and spectra multicolor broad range protein ladder were procured from thermo fisher scientific (waltham, ma, usa). aps (ammonium persulfate), coomassie brilliant blue r-250, dmso (dimethyl sulfoxide), dtt (dithiothreitol), g418 (geneticin disulfate), glycerol, isopropanol, mes (2-(n - morpholino) ethanesulfonic acid), nacl (sodium chloride), rotiphorese gel 30 acrylamide / bisacrylamide solution, sds (sodium dodecyl sulfate), toluidine blue, tris - hcl (tris(hydroxymethyl)aminomethane hydrochloride), and zellutrans / roth dialysis membranes were provided by carl roth gmbh (karlsruhe, germany). cdm hd (chemically defined medium high density) serum replacement and the hollow fiber cell culture bioreactor were purchased from fibercell systems (frederick, usa). amicon ultra centrifugal filter units, edta (ethylenediaminetetraacetic acid, disodium salt dihydrate), and millex - gv sterile filter units were purchased from merck kgaa (darmstadt, germany). n, n, n - tetramethylethylenediamine) was obtained from fluka biochemika (buchs, switzerland). cell culture plates were purchased from greiner bio - one (kremsmnster, austria). all columns used for purification were provided by ge healthcare (little chalfont, uk). for generation of a humanized anti - cd22 igg1, variable domain genes of the previously humanized scfv sgiii were synthesized (entelechon gmbh, bad abbach, germany) with splice donor and acceptor signal sequences and cloned into eukaryotic expression vectors containing regulatory elements of the immunoglobulin locus, a human constant heavy 1 chain, and a human constant chain, respectively. heavy chain and light chain plasmids of the humanized antibody construct were linearized with ahdi and sfii, respectively, and transfected into sp2/0-ag14 mouse myeloma cells by electroporation (230 v, 975 f). cells were grown by limiting dilution in selective media (dmem, 10% fbs, 50 m 2-mercaptoethanol, and 1 mg / ml g418) for 2 - 3 weeks to obtain single - cell clones. positive clones were identified by flow cytometric analysis and monitored for igg secretion rates by dot blot using a purified mab as reference standard. the highest producing clone for hurfb4 igg was expanded to t-175 flasks, adapted to high glucose dmem culture media supplemented with 10% cdm hd serum replacement, and subsequently inoculated into a hollow fiber cell culture bioreactor. iggs were purified from cell culture supernatants by protein a chromatography using a hitrap rprotein a ff column and dialyzed against pbs (ph 7.4). purity was assessed by analytical size exclusion chromatography on a superdex 200 10/300 gl column as 95%. intermolecular protein conjugation of onc and mab hurfb4 was performed using as heterobifunctional cross - linking agents either succinimidyl 4-(n - maleimidomethyl) cyclohexane-1-carboxylate (smcc) or n - succinimidyl 3-(2-pyridyldithio) propionate (spdp) for noncleavable and thiol - cleavable linkage, respectively. smcc and spdp solutions were freshly prepared in dmso and diluted 1 : 10 in pbs (ph 7.4) shortly before use. to generate smcc - based immunoconjugates purified hurfb4 igg antibody (1 mg / ml) was incubated with 40-fold molar excess of smcc in pbs (ph 7.4) containing 5 mm edta for 30 min at room temperature. onc (2 mg / ml) was simultaneously reduced to des(30 - 75)-onc in pbs (ph 7.2) in the presence of 5 mm dtt and 0.2 nm edta for 120 min at 15c. smcc and dtt were removed by dialysis against pbs (ph 7.4) containing 5 mm edta. the maleimide activated antibody was added to a 4.0-fold molar excess of des(30 - 75)-onc and the reaction mixture was incubated for 30 min at room temperature. in order to prepare spdp - based immunoconjugates, purified hurfb4 igg antibody (1 mg / ml) was incubated with a 40-fold molar excess of 2-iminothiolane (2-it) in pbs (ph 8.0) in the presence of 5 mm edta for 60 min at room temperature. onc (2 mg / ml) was simultaneously incubated with a 2.0-fold molar excess of spdp in pbs (ph 7.4) in the presence of 5 mm edta for 60 min at room temperature. excess reagents were removed using pd - minitrap g-25 columns equilibrated with pbs (ph 7.4) containing 5 mm edta. both modified proteins were combined and incubated overnight at 4c for conjugation using a 10-fold molar excess of pyridyldithiol - activated onc. the smcc - based immunoconjugate preparations were subjected to preparative size exclusion chromatography (sec) on a superdex 200 10/300 gl column equilibrated and eluted with pbs (ph 7.4) at a flow rate of 0.5 ml / min. preparative sec of the spdp - based immunoconjugate preparations was performed on a hiload 16/60 superdex 200 pg column with a flow rate of 0.3 ml / min using 20 mm mes, 50 mm nacl at ph 6.0 as elution buffer. antibody - onc conjugate populations isolated by sec were subjected to preparative ion exchange chromatography (iex) on a mono - s 5/50 gl column at a flow rate of 1 ml / min. spdp immunoconjugates with different onc - mab ratios were separated using 20 mm mes (ph 6.0) with a linear nacl gradient (50 mm1 m) as elution buffer. purified protein samples were concentrated using centrifugal filter units, sterile - filtered, and stored at 4c. purity and identity of concentrated immunoconjugates were analyzed by calibrated analytical sec using a superdex 200 10/300 gl column. concentrations of homogenously purified proteins were calculated from the absorbance at 280 nm measured on a nanodrop nd-1000 spectrophotometer (thermo fisher scientific, waltham, ma, usa) using the beer - lambert law with the molar absorption coefficient and the molecular weight calculated for each individual compound. the calculated value for the unmodified igg was 203,900 mcm, for onc 10,010 mcm, and for the chemically linked immuno - rnases carrying one, two, or three onc payloads 213,910 mcm, 223,920 mcm, and 233,930 mcm, respectively. the molecular weight was set at 150,000 g / mol for the unmodified igg, 11,840 g / mol for onc, and 161,840 g / mol (oar 1 : 1), 173,680 g / mol (oar 2 : 1), and 185,520 g / mol (oar 3 : 1) for the immunoconjugates. the extinction coefficient and the molecular weight for immunoconjugates with determined sizes of 250290 kda were calculated for an adc with an average payload of 10 rnases and set at 304,000 mcm and 268,400 g / mol, respectively. to compare antigen binding activity, cytotoxicity, and ribonucleolytic activity of generated immunoconjugates, igg and onc determined protein concentrations protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) performed under nonreducing conditions on precast 38% tris - acetate gels using tris - acetate buffer and under reducing conditions on precast 12% bis - tris gels using tris - mops buffer followed by detection by staining with coomassie brilliant blue. novex himark pre - stained protein standard and spectra multicolor broad range protein ladder were used as protein standards. coomassie stained protein bands on reducing sds - page gels of spdp - based adcs were captured on an epson perfection v750 pro scanner (seiko epson corporation, nagano, japan) and analyzed by photoshop elements 10 software (adobe systems incorporated, san jose, usa). after converting to greyscale and applying the invert filter, mean densities of onc protein bands specific binding of hurfb4 igg was determined by flow cytometry using the human cd22-positive b - cell lines daudi, raji, and ramos and the human cd22-negative t - cell line jurkat. bound hurfb4 igg and chemically linked immuno - rnases were detected using a fitc - conjugated rabbit antibody specific for the fc region of human igg. the mouse monoclonal control antibody rfb4 was detected by staining with an fitc - conjugated goat anti - mouse igg. fluorescence recordings were made on a facscanto ii flow cytometer (bd biosciences, san jose, ca, usa) and median fluorescence intensity (mfi) was calculated using the facsdiva software (bd biosciences, san jose, ca, usa). background fluorescence was determined using cells incubated with fitc - conjugated secondary antibody under the same conditions. equilibrium - binding curves were determined by incubating 5 10 raji cells in triplicate with serial dilutions of either murine rfb4, hurfb4 igg, or chemically linked immuno - rnases for 2 h at room temperature in 100 l pbs - facs buffer containing 2% fbs and 0.1% sodium azide. after two washes with 200 l facs buffer bound antibodies were detected as described above. background fluorescence was subtracted from measured median fluorescence and relative affinities were determined according to the levenberg - marquardt algorithm for nonlinear regression using graphpad prism 5.0 (graphpad software, la jolla, ca, usa). ribonucleolytic activity of smcc - based immunoconjugates was investigated by zymogram gel electrophoresis as previously described. briefly, samples were prepared in zymogram loading buffer containing final concentrations of 62.5 mm tris - hcl, ph 6.8, 10% glycerol, and 2.5% sds and separated under nonreducing conditions on 12% polyacrylamide gels containing 0.3 mg / ml poly - ru in the separating gel as a substrate for onc. after electrophoresis the gel was washed twice using 10 mm tris - hcl, ph 8.0, containing 20% isopropanol to remove sds, and placed in renaturating buffer containing 100 mm tris - hcl, ph 8.0 for 12 h (4c). rnase activity was detected by staining with 0.2% (w / v) toluidine blue in 10 mm tris - hcl, ph 8.0 for 10 min, followed by destaining with 10 mm tris - hcl, ph 8.0, until visualization of the ribonucleolytic activity bands was obtained. ribonucleolytic activity of spdp - based immuno - rnases and onc was quantified using the fluorogenic substrate 6-carboxyfluorescein - darudgda - black - hole - quencher-1 (6-fam - darudgda - bhq-1). increase in fluorescence after cleavage of substrate was monitored over time using an infinite f200 pro microplate reader (tecan group, mnnedorf, switzerland) with a 485/535 nm (excitation / emission) filter set. reactions were carried out in black 96-well plates in 100 mm mes - naoh buffer (ph 6.0) containing 100 mm nacl and 6-fam - darudgda - bhq-1 (5 nm) at (25 2)c in a total reaction volume of 200 l per well. buffer served as negative control and an excess concentration of rnase a was used as positive control. values of kcat / km were calculated using(1)kcatkm=f/tfmaxf0e, where f/t represents the initial reaction velocity, f0 the initial fluorescence intensity before addition of rnase, fmax the fluorescence intensity after complete cleavage of the substrate by excess rnase a, and [e ] the rnase concentration. human lymphoma and leukemia cells were maintained in rpmi 1640 supplemented with 10% fetal bovine serum, 100 u / ml penicillin, and 100 g / ml streptomycin. daudi were seeded at a density of 2 10/100 l, while nalm6 and jurkat were seeded at a density of 1 10/100 l into 96-well flat - bottom plates and incubated with various concentrations of protein or buffer as control at 37c, 5% co2 for 72 h in a total volume of 110 l. in order to determine the viability, cells were incubated with 10 l alamarblue per well at 37c, 5% co2 for 4 h. absorbance was measured at 570 nm (reference : 620 nm) using an infinite f200 pro microplate reader (tecan group, mnnedorf, switzerland). cell viability was expressed as percentage of viable cells treated with protein related to buffer control. the concentration required to inhibit cell viability by 50% relative to buffer - treated control cells was defined as ic50 (half maximal inhibitory concentration) and was determined from semilogarithmic plots. at least two independent assays with each assay containing triplicates were performed. we have previously grafted the specificity of the murine anti - cd22 monoclonal antibody (mab) rfb4 into human vh and vl frameworks preselected for stability from a human antibody phage display library. the resulting humanized hurfb4 scfv (originally designated sgiii) displayed excellent antigen binding and stability properties. to generate a humanized igg1 derivative (mab hurfb4) from the scfv the variable domain encoding genes of the humanized vl chain and vh chain were cloned into immunoglobulin expression vectors containing a human constant heavy 1 chain and a human constant chain, respectively. the humanized antibody was produced from stably transfected sp2/0 mouse myeloma cell lines under serum - free conditions in a hollow - fiber culture system and purified from culture supernatants to homogeneity by protein a chromatography. the purified hurfb4 igg demonstrated specific binding to the human cd22-positive b - cell lines daudi, raji, and ramos, but no binding to the human cd22-negative t - cell line jurkat (data not shown). flow cytometric affinity measurements confirmed that hurfb4 igg retained the same high apparent affinity of 0.27 0.02 nm as its murine ancestor mab rfb4 (figure 1). bivalent binding of the humanized igg increased the apparent binding affinity by 36-fold when compared to the parental monovalent scfv hurfb4. for conjugating onc to hurfb4 igg we first employed the membrane permeable crosslinker succinimidyl 4-(n - maleimidomethyl) cyclohexane-1-carboxylate (smcc). we therefore reduced onc under mild conditions and simultaneously modified the -amino groups of igg - lysine residues via the n - hydroxysuccinimide (nhs) ester reactive group of smcc. the smcc - modified igg was reacted with free accessible sulfhydryl groups of des(30 - 75)-onc via the maleimide group of smcc, generating nonreducible thioether bonds. this conjugation approach yielded 41% nonconjugated igg (150 kda) and 22% multimeric immuno - rnase conjugates (300 kda) (data not shown). although reductive unfolding of onc into a single stable intermediate des(30 - 75)-onc has been described [34, 36 ], reduction of the 30 - 75 disulfide bond in onc for chemical conjugation has not yet been investigated. to assess if this site of conjugation interferes with the ribonucleolytic activity of onc the 300 kda immunoconjugates were subjected to zymography. although multimeric immunoconjugates were enzymatically active (figure 2(a)) and retained specific antigen binding (figure 2(b)) they were not cytotoxic (figure 2(c)). therefore we next used the cleavable n - succinimidyl 3-(2-pyridyldithio) propionate (spdp) crosslinker reacting with amino groups of onc. lysine residues of the hurfb4 igg were modified by traut 's reagent (2-iminothiolane, 2-it) to provide an additional sulfhydryl group for subsequent immunoconjugation. preparative size exclusion chromatography (sec) of the spdp - immunoconjugate preparations yielded three distinct peaks (figure 3(a)) corresponding to 17.3% adc multimers (58.6 ml), 70.2% immuno - rnase adcs (67.9 ml), and 12.5% pyridyldithiol - activated onc excess (106.4 ml) as confirmed by sds - page under nonreducing and reducing conditions (figures 3(b) and 3(c)). multimerization (300 kda adcs) was most likely attributed to small amounts of onc molecules carrying two pyridyldithiol reactive groups, resulting in the cross - linking of two 2-it - modified igg molecules. sizes of immuno - rnase adcs were between 162 and 186 kda (figure 3(b)), corresponding to onc to antibody ratios (oars) between 1 : 1 and 3 : 1. the small differences of the molecular weights of immuno - rnase adcs with distinct oars did not allow a preparative separation by size exclusion chromatography (figure 3(a)). to separate immuno - rnase adcs by their number of cytotoxic payloads we thus took advantage of the highly basic nature (pi > 9.5) of onc and subjected the conjugate pool eluting at 67.9 ml in sec (figure 3(a)) to a polishing step by cation exchange chromatography. by increasing the ionic strength of the elution buffer slowly from 140 to 300 mm nacl we were able to separate intact mab species with low onc payloads (figures 4(a)4(c)). immuno - rnase conjugates eluting at 142174 mm nacl, 194221 mm nacl, and 248270 in ion exchange chromatography migrated under nonreducing conditions on sds - page with estimated sizes of 162 kda, 174 kda, and 186 kda, respectively (figure 4(a)). the average molecular weights of 162 kda, 174 kda, and 186 kda were confirmed by subsequent calibrated sec and correspond to distinct immuno - rnase adcs with oar of 1 : 1, 2 : 1, and 3 : 1, respectively. minor adc multimer contaminants with sizes > 300 kda could be separated by sec (figure 4(b)). analysis of immuno - rnase conjugates (defined amount of 1 g each) on reducing sds - page gels showed an oar depending increase of the staining intensities of onc protein bands (12 kda), while staining of the protein bands of the respective igg heavy and light chains remained unchanged (figure 4(c)). when compared to the 162 kda immunoconjugate measured densities of onc bands increased 1.9-fold for the 174 kda adc and 2.5-fold for the 186 kda adc, which corresponds very well to the relative amounts of one, two, or three onc loads per adc of 6.2 pmol, 11.5 pmol, and 16.2 pmol, respectively. it has been shown that conjugation procedures requiring pretreatment of onc with reducing agents such as dithiothreitol (dtt) can diminish ribonucleolytic activity up to 60%. by employing a conjugation strategy that avoids reduction of onc we were able to fully maintain the catalytic activity of the ribonuclease in the adc format (table 1). as a consequence, the 162 kda immuno - rnase adc with an oar of 1 : 1 showed exactly the same ribonucleolytic activity as onc alone. the 174 kda immuno - rnase adc exhibited 1.9-fold increased ribonucleolytic activity according to two onc payloads. the 186 kda immuno - rnase adc carrying three onc moieties exhibited a 2.9-fold higher catalytic efficiency than onc alone. thus, determination of ribonucleolytic activity additionally confirmed the separation of immuno - rnases with oars of 1 : 1, 2 : 1, and 3 : 1. immunoconjugation had no significant impact on the biological activity of the antibody since antigen binding affinities of the immuno - rnase adcs (kds 0.40.6 nm) were comparable to the affinity of the native hurfb4 igg (table 1). specific antitumor activity of the spdp - based immunoconjugates with oars of 1 : 1, 2 : 1, and 3 : 1 was tested on human burkitt 's lymphoma daudi and pre - b acute lymphoblastic leukemia nalm6 cells in comparison with cd22-negative human acute t - cell leukemia cell line jurkat. the spdp - based immuno - rnases meditated a dose - dependent cytotoxicity towards targeted cd22-positive tumor cells (figures 5(a) and 5(b)) but not towards the cd22-negative leukemia cells (figure 5(c)). incubation of daudi and nalm6 cells with hurfb4 igg alone did not result in any cytotoxicity (figures 5(a) and 5(b)). notably, a direct correlation between the cytotoxicity of the immuno - rnase adcs and the number of attached cytotoxic payloads became apparent. as anticipated, cytotoxicity successively increased with the number of conjugated payloads (table 2). in comparison with onc alone, displaying ic50 values of 1.5 m on daudi cells and 0.5 m on nalm6 cells, targeted delivery of three onc moieties per antibody by the most potent 186 kda adc resulted in enhanced cytotoxicity of 18,400-fold on daudi cells (ic50 80 pm) and 3,600-fold on nalm6 cells (ic50 140 pm) (table 2). spdp conjugation generated also conjugates with onc multiplicities eluting in gel filtration chromatography on a calibrated superdex 200 column at retention times correlating to sizes between 250 and 290 kda and most likely representing rnase loads of eight to twelve (data not shown). although this mixture of immunoconjugate species could not be further separated, the effect of higher onc loading on in vitro potency was evaluated and compared with the immunoconjugates carrying low onc payloads (figure 5(d)). at equal molar doses of 10 nm, immunoconjugates with one, two, and three onc payloads reduced the mean viability of daudi cells by 38%, 64%, and 84%, respectively. although cytotoxic activity correlated with drug loading levels for immunoconjugates with low oar, immuno - rnase adcs with higher onc loadings displayed a significantly decreased cytotoxic activity of only 17%. cytotoxic payloads currently under clinical evaluation in adcs are antimicrotubule agents, dna minor groove binding agents, and alkylating agents. with biological activities in the ng / kg range these compounds represent a major safety challenge both in clinical product manufacturing and in systemic application to cancer patients. the safety and therapeutic index of adcs significantly depend not only on the reproducibility of exact attachment sites and number of attached payloads but also on the linker technology and conjugate homogeneity. improvements in linker stability have in fact accelerated the clinical development of new generation adcs and resulted in the recent approvals of brentuximab vedotin and ado - trastuzumab emtansine, respectively. despite implementation of sophisticated downstream purification protocols conjugation via lysines or cysteines of the antibody results in inherent heterogeneity of the final clinical product with 08 drug payloads per antibody on average. to decrease heterogeneity and achieve uniform drug - loading site - specific drug attachment employment of these technologies has resulted in adcs with defined dar of either two or four payloads per antibody [3840 ]. in this study we employed amphibian rnase onc as effector moiety for creating a novel protein - protein adc to target cd22-positive leukemia and lymphoma cells. prerequisite for the full enzymatic activity of onc is the formation of pyroglutamate at its n - terminus through hydrogen bonding with k9 [41, 42 ]. it is therefore important that crosslinking of onc to the antibody moiety must not occur via k9 of the enzyme to preserve the catalytic activity of the ribonuclease. consequently, we explored two different conjugation strategies for analyzing the impact of the nature of the cross - linking bonds, the stoichiometry, and the efficiency of purification procedures for retaining enzymatic activity and cytotoxicity of the immuno - rnase conjugates. studies on the unfolding pathways of onc have shown that onc reductively unfolds via a single stable intermediate des(30 - 75)-onc [34, 36 ]. it has been further shown that an onc variant lacking the disulfide bond 30/75 mimics the unfolding intermediate des(30 - 75)-onc and exhibits comparable cytotoxic properties as wild - type onc. mild reduction of the solvent - accessible c30/c75 disulfide bond of onc was therefore considered a feasible approach for generating active immuno - rnase adcs. although amine - to - sulfhydryl cross - linking with smcc resulted in enzymatically active adcs with sufficient binding activity, a major drawback of this conjugation approach was the uncontrolled formation of higher molecular weight immunoconjugates with impaired matrix - binding on iex. des(30 - 75)-onc carrying two sulfhydryl groups per molecule most likely caused multimerization by cross - linking smcc - modified igg molecules that resulted in abolishment of cytotoxic activity. in contrast, formation of spdp - linked immuno - rnase adcs via lysine residues seemed primarily to be a result of well - controllable 2-it and spdp cross - linking reactions, allowing for a preferential formation of immuno - rnase adcs with favorably low dars. high binding affinity to cd22-positive cells, well - preserved ribonucleolytic activity, and high cd22-specific cytotoxicity in vitro indicate no significant alterations of the molecules in critical regions, namely, the cdrs of the igg, as well as lysine residues k9 and k31 of onc. empirical evidence of random conjugation approaches has shown that the number of attached drugs has a significant impact on target antigen binding, systemic clearance, and antitumor efficacy of immunoconjugates [4448 ]. separation of isolated drug load species is at present only possible for dipeptide - linked adcs, such as anti - cd30 brentuximab vedotin through hydrophobic interaction chromatography (hic) [44, 4951 ]. we have shown in the present study that iex matrix - binding of spdp - linked immuno - rnase adcs not only allowed for successful purification from nonreacted igg but also yielded homogenous adc species with one, two, and three rnase moieties per antibody molecule. moreover, our data revealed that the number of attached onc payloads is crucial for achieving significant in vitro cytotoxicity towards cd22-positive lymphoma and leukemia cell lines. in this respect, at least two onc payloads were required for achieving significant in vitro cytotoxicity and the spdp - linked immuno - rnase adc with a dar of 3 : 1 was most effective. although reasons for the significantly decreased in vitro potency of immunoconjugates containing eight to twelve onc payloads per anti - cd22 mab were not examined in closer detail our data are in line with data from other antibody - drug conjugates showing that higher drug loads increase the risk of reduced cytotoxic activity most likely due to conjugational involvement of lysine residues within the complementarity determining regions (cdrs) of the igg. in addition, it has been observed in mouse xenograft models that higher drug loading levels (eight drugs per antibody) can cause an accelerated systemic clearance of adcs which leads to a decreased therapeutic index when compared to conjugates with only four or even only two drugs per antibody. thus, similar to other optimized non - rnase - based adcs, it is apparent that the conjugational design of chemically linked immuno - rnases should aim at a low dar. another option for successfully obtaining immuno - rnases with stoichiometrically defined number of onc cargos through employment of the dock - and - lock method has recently been reported. preferably in a head - to - head comparison advantages and disadvantages of both methodologies should be addressed in future studies. current clinical and preclinical development strategies for cd22-targeting adcs and immunotoxins focus on the use of traditional cytotoxic payloads, such as calicheamicins, auristatins, maytansinoids, and truncated pseudomonas exotoxin. all of these cytotoxic agents have been associated with significant systemic toxicity either due to their off - target release through destabilization of the cross - linking bonds or because of immunogenicity, as in case of pseudomonas exotoxin. payload - dependent hemato- and hepatotoxicities, ranging from mild, reversible transaminasemia to even fatal venoocclusive disease (vod), have been reported for the anti - cd22 calicheamicin immunoconjugate inotuzumab ozogamicin (cmc-544) used in clinical phases i iii for the treatment of patients with relapsed / refractory b - cell malignancies [53, 5658 ]. substantial postapproval safety risks attributed to vod led to the refusal of the marketing authorization in europe for gemtuzumab ozogamicin [5, 59, 60 ] in 2008 and its voluntary withdrawal from the us in 2010. despite efforts to obtain a maximal adc stability through introduction of noncleavable thioether bonds or intracellularly cleavable dipeptide bonds preterm payload release with potentially increased toxicity remains a major clinical problem. as with other dipeptide - linked auristatin - based immunoconjugates [6163 ], preliminary results of a phase ii trial of the anti - cd22-mmae immunoconjugate pinatuzumab vedotin in combination with rituximab in patients with relapsed / refractory non - hodgkin lymphoma reported on significant payload - related systemic toxicities including neutropenia and peripheral neuropathy. since similar toxicities were also common among patients within phase i / ii trials of naked auristatin payloads [65, 66 ], deconjugation of the payload from the antibody with systemic release of the neurotoxic microtubule inhibitors can thus be deduced. payload release from presumably noncleavable thioether bonds has been also demonstrated : during the thioether fragmentation reaction the cytotoxic moieties were shown to be transferred to the unpaired c34 cysteine residue of serum albumin. identification of albumin as a covariate affecting the pharmacokinetics of the recently fda - approved smcc - linked anti - her2 trastuzumab emtansine (t - dm1) might explain at least in part payload - related systemic toxicities observed in late clinical development. lately, application of self - hydrolyzing maleimide drug linkers has been shown to reduce off - target bone marrow toxicities in rats through enhanced adc stability, which holds promise for future clinical development of such adcs. while adcs under current development represent a unique treatment option they create a series of challenges in engineering, chemistry, and safety. alternative payloads with reversible, easily manageable systemic toxicities, high antitumoral efficacy, such as onc in the present study, represent valuable alternatives. in summary, we have developed novel spdp - linked immuno - rnase adcs with stoichiometrically defined number of cytotoxic onc payloads for the targeted therapy of cd22 malignancies. we have shown for the first time that the number of attached onc payloads well correlates with the tumor - specific cytotoxicity of the adc. because of their highly specific toxicity towards targeted tumor cells, a fairly well - controllable conjugation and purification procedure, and expected favorable safety and immunogenicity profile we believe that further preclinical development of the 3 : 1 dar spdp - linked immuno - rnase adc is warranted. | antibody - drug conjugates (adcs) have evolved as a new class of potent cancer therapeutics. we here report on the development of adcs with specificity for the b - cell lineage specific (surface) antigen cd22 being expressed in the majority of hematological malignancies. as targeting moiety a previously generated humanized anti - cd22 single - chain variable fragment (scfv) derivative from the monoclonal antibody rfb4 was reengineered into a humanized igg1 antibody format (hurfb4). onconase (ranpirnase), a clinically active pancreatic - type ribonuclease, was employed as cytotoxic payload moiety. chemical conjugation via thiol - cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the adc. development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of onconase cargos. a minimum of two onconase molecules per igg was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. antibody - drug conjugates with an onconase to antibody ratio of 3 : 1 exhibited an ic50 of 0.08 nm, corresponding to more than 18,400-fold increased cytotoxicity of the adc when compared with unconjugated onconase. these results justify further development of this adc as a promising first - in - class compound for the treatment of cd22-positive malignancies. |
maxillofacial defects may have a profound psychological impact on the patient. with the rehabilitation of these defects, we not only restore normalcy to the patient 's face, but also restore his self - image and the ability to function and interact in a social environment, thus giving him the confidence needed to live in society. a maxillofacial prosthodontist must undertake the task of head and neck rehabilitation posttrauma in cases where a surgical approach is no longer possible. burns can leave a patient with severely debilitating and deforming contractures, which can lead to significant disability even after treatment. the objectives of burn rehabilitation are to minimize the adverse effects caused by the injury in terms of maintaining a range of movement, minimizing contracture development, maximizing functional ability, maximizing psychological wellbeing, maximizing social integration. maxillofacial rehabilitation is advantageous for such patients because it allows for early rehabilitation, shortening surgery and hospitalization time, lowering treatment cost, and allowing the patient early psychosocial reintegration. the use of implants in maxillofacial prosthetics provides patients with a predictable esthetics, improved retention and stability of their prostheses in comparison with other retention methods. traditional methods include the use of medical - grade skin adhesives, spectacles, and tissue undercuts. these modalities are associated with difficulties related to retention reliability, stability, adverse tissue reactions, and accelerated discoloration and prosthesis deterioration, discomfort, and reduced acceptance. implant retained prostheses are a suitable option for their enhanced retentive property and are preferable to surgical reconstruction which may have unpredictable results. the construction of the missing auricle puts a test on the skill of the prosthodontist to reproduce the form, texture and tone of the existing contralateral ear but its successful rehabilitation is a rewarding experience for the dentist and patient alike. this clinical report describes the rehabilitation of a patient who had received substantial burns to the face with loss of the right auricle by overcoming challenges including lack of conventional retentive modalities and the compromised condition of surrounding burn tissues. a 47-year - old male patient was referred to the department of maxillofacial prosthodontics and maxillofacial prosthetics with the chief complaint of facial disfigurement along with the loss of the right ear after severe burns received to the face. the patient had received these burns several years ago when scalding hot water fell on the right side of the face [figure 1 ]. preoperative frontal and profile view of the patient the patient was extremely concerned about his facial disfigurement and requested for an economic solution for replacing the missing ear in order to regain some normalcy of appearance. a thorough evaluation of the affected area, medical history, and physicians consent was taken. external examination revealed scarring of tissue with gross discoloration, complete loss of hearing from the right ear. a dermatologic evaluation was conducted which revealed that the patient had scarred tissue, decreased blood supply to the area, increased contracture formation ; and reduced epithelialization and collagen formation. a radiographic evaluation conducted showed suitable bone quality, adequate thickness of the temporal bone and density of the mastoid air cells to receive implants. three - dimensional (3d) lateral cephalography were recorded of the affected and normal side along with soft tissue reconstruction. a stereolithographic model was obtained of the patient 's temporal bone and temporomandibular joint [figure 2 ]. sites for the placement of implants were located and marked and used as a guide to fabricate a surgical stent for use during surgery. surgical stent fabricated on the stereolithographic model of temporal bone a single stage implant placement surgery was carried out. the surgical site was isolated, and the surgical stent was fastened in place with surgical tape and used as a guide. two (3.75 mm 10 mm) branemark mark ii implants were placed in the region of the missing right auricle, in the mastoid bone of the temporal bone of the patient [figure 3 ]. surgical procedure for implant placement the higher placed implant showed adequate stability and osseointegration 4 months postoperatively while the lower placed implant showed repeated signs of infection and was subsequently submerged and left as a sleeping implant. the opposing left ear was normal and healthy, and was used as a guide for the fabrication of the wax pattern for the missing auricle. to make an impression of the healthy ear, an unused casting ring of adequate dimensions was used to hold the irreversible hydrocolloid impression material (algitex ; dental products of india, mumbai, india). the surrounding hair was coated with petroleum jelly, and the ear canal was blocked with cotton. the alginate was first coated into the folds of the ear in a thin consistency, followed by application in bulk. the impression was beaded and boxed and poured in dental stone (kalastone ; kalabhai pvt ltd. a wax pattern of the right ear was carved out as a mirror image of the opposite side using modeling wax (hindustan dental products, hyderabad, india) [figure 4 ]. wax up of mirror image of contralateral ear the implant abutment was blocked out with carding wax. light body consistency of polyvinyl siloxane elastomeric impression material (aquasil, dentsply, caulk, milford, del) was applied to the implant and surrounding tissues, followed by a base of putty consistency polyvinyl siloxane elastomeric material (aquasil, dentsply, caulk, milford, del). the impression was boxed and poured in die stone (elite rock - extra hard, zhermack, germany). the retrieved cast was used as a template to fabricate a shim of clear autopolymerizing acrylic resin (dpi cold cure ; dental products of india ltd. a triangular shaped acrylic shim was fabricated which was attached to an acrylic cap to be cemented on the implant abutment. samarium cobalt magnets of 4 mm diameter were embedded into the three angles of the acrylic triangle using an autopolymerizing acrylic resin [figure 5 ]. acrylic shim trial on patient face another acrylic triangle of similar dimensions as the original was fabricated to support the magnets of the opposing poles to be embedded into the final prosthesis. it was ensured that the magnets were of differing polarities such that the two triangular shims fit together in only one orientation. care was taken to ensure proper incorporation of the magnets with the autopolymerizing resin to avoid abrasion during the final polishing. the sculpted wax model of the missing right ear was tried on the patient 's face, and its size, orientation and position was confirmed to ensure symmetry with the opposing side [figure 6 ]. the acrylic shim with cap was cemented in position onto the implant abutment using glass ionomer cement in luting consistency. the opposing acrylic shim was embedded into the sculpted auricular model, and the position was finalized chair side. trial of waxed up ear on patient 's face - frontal and profile view after satisfactory positioning, the acrylic shim was sealed into the wax pattern, and the entire framework was flasked and invested using a combination of dental stone and die stone forming a three piece mold. after wax elimination, the die stone component was separated out of the flask to aid in layered packing of the maxillofacial silicone. gold primer (a-330-gold, factor ii, lakeside, az) was applied to the acrylic to assist bonding with the silicone. color matching was done using intrinsic colors (principality medical, uk) to blend with the patient 's skin tone and the medical grade silicone (cosmesil m511 - part a and b, cosmesil prosthetic system, south wales, uk) was packed into the mold and left to cure for 1 h at 100c. extrinsic tinting principality medical, uk) was performed to further blend with the patient 's skin tone. the excess silicone was cut out. the light weight of the prosthesis ensured easy support by the magnets. the final fitting of the prosthesis was done, and the patient was instructed on the placement and removal as well as the home care instructions of the same [figure 7 ]. deformity in the burned ear may be characterized by various combinations such as : (i) the presence of scarred skin at the site of, and surrounding the ear, with dramatic loss of skin elasticity ; (ii) the presence of longitudinal scars of the pinna due to previous drainage of the perichondritis as an initial trial for saving the ear ; (iii) absence of different components of the framework of the ear, mostly the helix / antihelix complex (the cartilage - containing part) with or without the ear lobule. patients with missing ears deserve comprehensive care that could be of the following types. surgical treatment including the autogenous ear reconstructive surgery, use of osseointegrated implants and bone anchored hearing aid. prosthetic replacement is a more suitable option for patients who are not surgical candidates, e.g. those with high operative risk, failed previous reconstructions and severely compromised conditions like burned tissues. extraoral implant retained prosthesis have been proven to be a predictable treatment option for maxillofacial rehabilitation. implant retained auricular prosthesis provide multiple advantages for the patient : convenience, security, consistent retention and positioning, elimination of the need for adhesives, and maintenance of marginal integrity and longevity. the densely corticated bone of the auricular region makes it easy to stabilize the implant at surgery, and the vasculature in this region ensures the maintenance of a bone / implant interface adequate to support the functional loads. despite the inability to use both implants, in this case, the suitable bone surrounding the implant, and successful osseointegration enabled the use of the single implant supporting a light weight prosthesis. specifically, they eliminate disengagement caused by surrounding soft tissue movement or perspiration, which can result in loss of contact of the silicone prosthesis margins. the implant - retained auricular prosthesis has become a viable treatment alternative for auricular deformed patients because of its predicable results. extra - oral implants differ from intra - oral in terms of length and prosthetic platform. ideally, small length implants are placed in the auricular region. however, in this case, there was sufficient bone length ; hence 10 mm intra - oral implants were placed in the mastoid air cells. though the inside bone was porous with spaces, the 3 mm cortical bone at the surface gave good primary stability for the implant. similar case reports suggest the use of a magnet and bar clip retained prosthesis as the primary source of retention for an implant supported prosthesis. in the present case report due to the inability to use both implants, the use of metal components would have increased the weight on the single implant, so the use of autopolymerizing acrylic resin was preferred. chung. made use of a composite bar to eliminate the costly and technique - sensitive casting procedures. in the present study, an alternative of autopolymerizing acrylic resin was used to retain the prosthesis with the use of gold primer to bond the acrylic resin to the silicone structure. the use of magnets is advantageous over other attachments because metal clips may fracture over time making revision and repair difficult. furthermore, they are easily available, economical, hygienic, esthetic and convenient to use and maintain and replacement is easy. the patient 's financial condition also suited the use of magnets which are indigenously acquired and economical. furthermore, the use of autopolymerizing resin for the framework is more economical and less technique sensitive than a cast framework which would increase the overall cost and weight of the prosthesis. use of a triangular framework provides an increased area for placement of larger magnets and the use of an increased number of magnets. this also helped increase the surface area for support and retention of the prosthesis on a single implant. because the flexural strength of autopolymerizing resin is lower than precious alloys, the thickness of each bar must be increased for adequate stiffness and accommodation of keepers. the thickness of the bar itself also provides additional support for the ear prosthesis against gravitational and lateral dislodgement forces. lemon and chamber gave an active, passive engagement of a slant - lock system to improve the retention of the prosthesis which is unlike the breakaway force required to disengage the prosthesis as in magnets. however, a large amount of vertical space is required to incorporate the attachment which can be problematic. intrinsic coloring of silicone is difficult to master for proper matching with the adjacent tissues and the use of extrinsic stains are useful in providing the additional effect to further improve the esthetic appeal of the prosthesis. the intrinsic coloring was done based on the silicone shade guide reported by guttal. in the present situation, the patients burnt half of the face showed substantial color difference in comparison with the normal side. after consulting with the patient, it was decided that the color tone of the replacement ear would be matched to the burned skin to help blend better for a more natural appearance. the severe skin contractures and reduction of malar prominence on the affected side of the patients face due to burns, and further during reconstructive grafting, resulted in difficulty in accurate symmetric placement of the ear. the future trends in auricular cartilage engineering include stem cell, biomaterial, and bio - molecules. researchers have demonstrated that neo - cartilage can be constituted in a predetermined shape and in complex 3d structures, such as a human ear, using cell transplantation on polymer constructs. however, these are still in the trial phase, and many unsolved problems exist. no perfect materials and methods have been found to substitute the shapely elastic cartilage normally present in the ear, and the current constructs have not proven to be durable over time. further in vitro and in vivo studies are required before these become a clinical norm. the magnets on the acrylic shim helped in proper orientation and fixation of the silicone ear. the symmetric placement and size ensured a good fit with the help of the implant providing for a natural looking replacement albeit with a slight discrepancy in color match. the patient was satisfied with the end result of the prosthesis. at follow - up visits, he informed us of increased social interaction due to acceptance of the prosthesis by his peers. it is important to remember that each patient is different, and the technique for rehabilitation and handling will differ with each case. the treatment plan must be customized for every individual to ensure a tailor made prosthesis that becomes a part of the patient 's body. although challenging, maxillofacial prosthesis can be an excellent mode of rehabilitation of patients if successful. this article describes the reconstruction of a missing right auricle of the patient using an implant supported silicone prosthesis which is retained using indigenously acquired magnets. it is a simple technique which was suitable for the patient and prosthodontist with its simplified clinical and laboratory procedures along with reduced cost of the prosthesis. | burns can leave a patient with a severely debilitating disability even after treatment. the objectives of burn rehabilitation are to minimize the adverse effects caused by the injury while rehabilitating the patient 's physical and psychological well - being, maximizing social integration. long - term success of maxillofacial prostheses mainly depends on the retention. extra oral implant retained prostheses have proved to be a predictable treatment option for maxillofacial rehabilitation. replacement of a severely deformed external ear with burned tissues may be satisfactorily accomplished by a cosmetic prosthesis anchored by implants integrated in the skull. the use of such implants is now a well - recognized method for creating a stable result in maxillofacial rehabilitation. this case report describes a safe, simple and economical method for the rehabilitation of a patient with missing right auricle using an implant supported silicone prosthesis. the implant was placed in the mastoid region of the temporal bone. reconstruction of the ear was done with auricular silicone prosthesis, retained using magnets incorporated in an autopolymerizing resin shim to decrease the weight of the prosthesis on a single implant. this method eliminates the need of tedious laboratory procedures and exact casting and fitting requirements of a metal substructure while minimizing the overall weight and cost of the prosthesis while maintaining adequate support, esthetics and retention of the prosthesis. |
the all of isolates of g. umbellata and companion fungus used in this study were isolated by guo (2002) in shanxi province of china. they were maintained in petri plates on a wba medium (g / l : wheat bran 30, glucose 20, kh2po4 3, mgso4 1.5, agar 15, adjust ph to 6.0) at 22 to 24 under dark condition. the interaction between hypha of g. umbellata and companion fungus was studied according to the following procedure. for short time culture, each plate (9 cm) containing 20 ml of wba was used. at the same time, 250 ml flasks containing 100 ml wba were also prepared for long time culture. mycelial plugs (5 mm diameter) collected from actively growing agar colonies of both fungi were placed 3.0 cm apart each other on the surface of the agar and allowed to grow at 23 under dark condition. both hypha grew toward each other, and hyphal contact occurred by 10 to 12 days after inoculation. ten samples were collected from interface region 25 and 35 days, respectively after inoculation were fixed with faa (formalin : glacial acetic acid : 50% ethanol 1 : 1 : 18), embedded in wax following dehydration through a graded alcohol and sectioned with a rotary microtome at 7 m. embedding and staining with safranin and fast green followed the protocol of johansen (1940). preparations were observed and photographed under microscope (olympus co., japan). for each sample, ten samples collected from interface region on 25 and 35 days, respectively after inoculation were immediately fixed by immersion in 2.5% glutaraldehyde in 0.1 mol / l sodium cacodylate buffer, ph 7.2, for 2 h at room temperature. samples were post fixed with 1% osmium tetroxide in the same buffer for 1 h at 4 and dehydrated in a graded ethanol series prior to being embedded in epon 812. thin sections (0.7 m) cut from the epon - embedded material with glass knifes were mounted on glass slides and stained with 1% aqueous toluidine blue prior to examination with a olympus microscope. ultra thin sections (0.1 m) collected on nickel grids were examined with a jeol 100s transmission electron microscope. for each sample about 10 ultra thin sections were examined under the electron microscope (jeol co, japan). the all of isolates of g. umbellata and companion fungus used in this study were isolated by guo (2002) in shanxi province of china. they were maintained in petri plates on a wba medium (g / l : wheat bran 30, glucose 20, kh2po4 3, mgso4 1.5, agar 15, adjust ph to 6.0) at 22 to 24 under dark condition. the interaction between hypha of g. umbellata and companion fungus was studied according to the following procedure. for short time culture, each plate (9 cm) containing 20 ml of wba was used. at the same time, 250 ml flasks containing 100 ml wba were also prepared for long time culture. mycelial plugs (5 mm diameter) collected from actively growing agar colonies of both fungi were placed 3.0 cm apart each other on the surface of the agar and allowed to grow at 23 under dark condition. both hypha grew toward each other, and hyphal contact occurred by 10 to 12 days after inoculation. ten samples were collected from interface region 25 and 35 days, respectively after inoculation were fixed with faa (formalin : glacial acetic acid : 50% ethanol 1 : 1 : 18), embedded in wax following dehydration through a graded alcohol and sectioned with a rotary microtome at 7 m. embedding and staining with safranin and fast green followed the protocol of johansen (1940). preparations were observed and photographed under microscope (olympus co., japan). for each sample, ten samples collected from interface region on 25 and 35 days, respectively after inoculation were immediately fixed by immersion in 2.5% glutaraldehyde in 0.1 mol / l sodium cacodylate buffer, ph 7.2, for 2 h at room temperature. samples were post fixed with 1% osmium tetroxide in the same buffer for 1 h at 4 and dehydrated in a graded ethanol series prior to being embedded in epon 812. thin sections (0.7 m) cut from the epon - embedded material with glass knifes were mounted on glass slides and stained with 1% aqueous toluidine blue prior to examination with a olympus microscope. ultra thin sections (0.1 m) collected on nickel grids were examined with a jeol 100s transmission electron microscope. for each sample about 10 ultra thin sections were examined under the electron microscope (jeol co, japan). the grifola umbellata strain was differentiated white villiform - like colonies that grew vigorously, covering the whole petri dish in 24 days of incubation in monoculture of strains. it formed aerial mycelia and one month old colonies exudated brown oil - like liquid on the colony surface when the color of the media turned yellow. the companion fungus strain differentiated white to yellow colonies that grew slightly slower than the g. umbelleta, covering the whole petri dish on 28 days after incubation. the color of medium turned to filemot, caused by exudation of fungus. when grown together, g. umbellata and companion fungus colonies established contact around 10 days after inoculation. twenty - five days later of dual cultures, an evident antagonism line was formed at the interface region (fig. prolonged, the antagonism line became more developing and maturing., and matured 35 days after inoculation. the mature antagonism line reached 0.4 cm high like a wall in the middle between two colonies (fig. the color of media turned brown gradually. on the surface of g. umbellata colony, many hyphal strands emerged and crossed each other. initial sclerotia was formed from where hyphal strands crossed (fig. 2 arrow). however, in monocultures of g. umbellata, hyphal strands and sclerotia initials could hardly be seen. the size of these sclerotia initials which obtained in petri plates cultures no more enlarged probably because of nutrition exhaustion in the media. the further development and maturation of sclerotia in 250 ml flasks prepared were observed at the same time as petri plates (figs. 3, 4). with the enlargement of sclerotia size, the differentiation of rind in some sclerotia initials 4 showed the mature sclerotia of g. umbellata resulted from dual cultured with companion fungus (arrow). at the light microscope level, the whole process of antagonism formation was observed. the first mycelial contact between both fungal colonies was established after 10 days of incubation and both colonies hypha interwove at the interface region (fig. 5). then, an evident antagonism line formed 25 days after inoculation (fig. 6). for this time after dual staining with safranin and fast green, only fast green presents positive reaction. examination of the mature antagonism line showed that this structure was composed of three main layers : the rind, which was composed of closely arranged cells, the rind underlayer, in which the cells arranged more loosely than in the rind, and the hypha layer, which was just the active hyphal interaction region of both fungus (fig. after dual staining, epidermis was red, both epidermal underlayer and hypha layer were green. the rind underlayer was composed of compact hypha, looked like a transitional state of epidermis. however, these observations by light microscopy could not clearly differentiate hyphae of g.umbellata and companion fungus in the antagonism line. a more detailed pictures of the hyphal interaction between g. umbellata and companion fungus were obtained through tem observations of ultrathin sections. examination of samples collected at 25 days of incubation revealed that hyphal interaction actively occurred on the interface of both colonies. the adherence between the mycelia of g. umbellata and companion fungus occurred after their contact. electron - dense materials were observed in the adherence region, maybe related to the presence of viscous matrix or to the hyperplastic cell wall (fig. 8 arrow). after contact with companion fungus, a series of reactions occurred in g. umbellata, such as rupture of cell wall, outflow of cytoplasm, formation of septa at both sides of cracked section (fig. a series reactions also occurred in g. umbellata include retraction of cytoplasm and formation of septa and degradation of cell wall, the degraded cell wall existed in the hypha (fig. adherence also occurred in hypha of companion fungus, the viscous matrix derived from the outflowing cytoplasm of cracked g. umbellata hypha (fig. after dual culturing with g. umbellata, clamp connections of the companion fungus hyphae were easily observed (fig. 11), but few were seen in its monocultures. examination of samples collected on 35 days on incubation showed that the center of the antagonism line was composed of dead cells with thickened wall (figs. hyphal cells of g. umbellata and companion fungus which located on the two sides of antagonism line respectively could be clearly differentiated. cells of g. umbellata adjacent to the antagonism line were usually large and hollow, with unilateral thickened wall (fig. 14), whereas those of companion fungus were empty, with thin or thick walls (fig. a little apart from antagonism line, the hypha of companion fungus branched irregularly (fig. 15), and those of g. umbellata were made of hypha with both thin and thick wall (fig. the branches connected each other by confluence of tips, and a dense hyphal network was differentiated (fig. most of the studies dealing with fungus - fungus relation focus on the interaction between mycoparasites and their host fungi. these interactions are usually accompanied by haustoria formation such as appressoria, hyphal coils or hook - like bodies (dragt., 1996 ; inbar., 1996 ; li and shen, 1996). with the g. umbellata and companion fungus, no haustoria type structures could be observed. the nutritional relationship concerning sclerotia formation, therefore between g. umbellata and its companion fungus is unclear. the interactions resulted in dense antagonism line formed at the interface region of colonies, which role is expected to protect the fungus against invasion by antagonists. recognition of both fungus may be made through chemical mechanisms of chemotaxis which induce the hyphae of companion fungus to adhere to g. umbellata. after companion fungus adhered to the hyphae of g. umbellata, a series of reaction occurred in both of them, such as rupture of cell walls and outflow of cytoplasms in g. umbellata, and collapse of cell walls, retraction of cytoplasm and formation of septa in companion fungus. a specific lectin was found to be involved in the recognition of a fungus - fungus interaction (barak, 1985). a series of reaction always induced by recognition in both host fungi and mycoparasites. it is then hypothesized that a lectin may also be involved in the grifola - companion fungus recognition, too. the hyphal behavior of g. umbellata in sclerotial production was similar to the hyphal behavior of sclerotium rofsii during sclerotia formation (townsend and willetts 1957). chet and henis (1975) have reviewed the factors which affected the sclerotial formation of filamentous fungi in detail. it was also noted that sclerotial production usually occurred when the mycelium reached the petri plate walls and its linear growth was thus restricted (henis., 1965 ; wheeler and sharan, 1965). when g. umbellata and companion fungus grew in dual cultures and established contact, the linear growth was restricted essentially to the zone of antagonism and sclerotia were differentiated in abundance, but g. umbellata in monoculture, no sclerotia differentiation occured at the edges of the petri plates. this indicates that sclerotia induction not attribute to g. umbellata hyphal linear growth was restricted but the presence of the companion fungus. morphogenetic processes in fungi are often stimulated by microorganisms or by their products (bitancourt, 1951 ; manning and crossan, 1966). bedi (1958) also discovered that the number of sclerotia formed by s. sclerotiorum significantly increased when staling products of other cultures or the same fungus were stored to the growth medium. the hyphal interaction between g. umbellata and companion fungus resulted in a dual organism recognition. in spite of the incapacity of the hyphae of companion fungus to invade g. umbellata cells, the diffusion of a chemical agent all over the growing media or interaction at the interface may affected the capacity of g. umbellata strain to differentiate sclerotia. this study dealed essentially with morphological changes of hyphae in the hyphal interaction between g. umbellata and its companion fungus. companion fungus seemed to be acting as a bio - controller capable to regulate the sclerotia differentiation of g. umbellata. the mechanism of sclerotial formation and the interaction with companion fungus certainly implied diverse process, which complicated the comprehension of the phenomenon. | morphological characteristics of hyphal interaction between grifola umbellata (pers. ex fr.) pilat and its companion fungus which related to sclerotia formation from hyphae were investigated by external observations, light microscopy and transmission electron microscopy (tem). external observations showed that a dense antagonism line was formed by both g. umbellata and companion fungus after their hyphae contacted each other in dual culture. many hyphal strands emerged on the colony of g. umbellata and differentiated to sclerotia from where hyphal strands crossed. light microscope observations revealed the process of antagonism line formation. mature antagonism with structural differentiation, was composed of three main layers : the rind, the rind underlayer and the hypha layer. tem observations showed that after colonies hyphal contact, a series of reactions always occurred in both g. umbellata and companion fungus. cells in the center of antagonism line were dead. cells of g. umbellata adjacent to the antagonism line were usually large and hollow, with unilateral thickened wall, whereas those of companion fungus were empty, with thin or thick wall. both hyphal interaction at the antagonism line may be one of the main reasons for sclerotia of g. umbellata differentiation from hypha. |
the cranial bones are separated by sutures and fontanelle which will accommodate the growing brain. premature closure of one or more of these sutures results in cessation of skull bone growth perpendicular to the suture with compensatory expansion of the skull parallel to the closed suture. this will result in deformity of the skull vault along with raised intra cranial pressure and the resulting neurocognitive and ophthalmological complications. the present case report is about an 11-month - old male child born as the third offspring of 3 degree consanguineous parents was brought for evaluation and management of abnormal skull shape noted since birth. he was delivered by cesarean section at full term and had normal cry at birth. he could not recognize his mother, had not attained stable head control and was unable to sit even with support at 11 months of age. on examination, there was microcephaly with a head circumference of 40.5 cm (4 sds below mean for age and sex). the other features noted on examination included coarse facial features with bilateral proptosis, upturned tip of nose with depressed nasal bridge, micrognathia, gum hypertrophy, low set ears, narrow chest, joint contractures and bent forearms, thighs and legs with folds of the overlying skin [figure 1 ]. computed tomography (ct) of the head showed fusion of the anterior sagittal suture, bicoronal sutures, bilateral lambdoid sutures and temporo - parietal sutures. we explained to the parents regarding the progressive nature of disease, risk of increased intra cranial pressure and cosmetic deformity. before contemplating corrective surgery,. skeletal survey [figure 2 ] showed significant dysostosis with broad oak shaped ribs, inferior beaking of vertebrae and diaphyseal and epiphyseal dysplasia with periosteal cloaking. as these clinical features and skeletal features were suggestive of i - cell disease, fluorometric assays for three lysosomal enzymes (iduronate 2 sulfatase, total hexosaminidase and hexosaminidase a) were performed in the plasma sample. the plasma levels of all three enzymes were significantly elevated (more than 10 times above normal range). based on the enzyme levels and the clinical features, the diagnosis of i - cell disease with craniosynostosis was established in the child. the parents were counseled about the underlying genetic basis and prognosis of the condition and in addition, about the autosomal recessive inheritance pattern and the 25% risk of recurrence in future offspring. on being explained about the co - existing skeletal dysplastic changes, intellectual disability, respiratory problems and the limited survival associated with the disorder, the parents opted not to get surgery done for the craniosynostosis. (a) clinical photograph of the child showing coarse facial features, bilateral proptosis, upturned tip of nose, micrognathia and low set ears. (b) computed tomography (ct) head with 3d reconstruction image anterior view showing fused sagittal, bicoronal sutures. (d) ct axial bony window showing sutural synostosis skeletal survey of the child. (a) x - ray chest anteroposterior (ap) view showing broad oak shaped ribs, (b) x - ray dorso lumbar spine lateral view showing inferior beaking of vertebrae, x - ray lower limb lateral view (c) and x - ray pelvis ap view (d), showing diaphyseal, metaphyseal and epiphyseal dysplasia, (e) clinical photograph showing bent limbs with skin folds craniosynostosis occurs in 1 in 2500 births, with the non - syndromic subtype present in 0.4 - 1 in 1000 births. craniosynostosis is classified as simple if it involves a single suture and named according to the suture involved and the deformity of the skull vault. craniosynostosis is classified as syndromic craniosynostosis if it is associated with well - described genetic syndromes, or non - syndromic, when there are no associated anomalies. most common syndromic associations with craniosynostosis are crouzon, apert, pfeiffer, muenke and saethre - chotzen syndromes. patients with syndromic craniosynostoses are much more complicated to care for, requiring a multidisciplinary approach to address all of their needs effectively. non - syndromic craniosynostosis is also believed to have a strong genetic component with possible gene gene or gene environment interactions that remain to be identified. our patient presented mainly with the complaints of global developmental delay and craniosynostosis. however, on thorough clinical genetic evaluation he was found to have numerous dysmorphic features including coarse facies and bent limb bones in addition to hepatomegaly. skeletal survey revealed features of dysostosis multiplex and typical diaphyseal and epiphyseal changes consistent with the diagnosis of i - cell disease. i - cell disease, also known as mucolipidosis type ii, is a rare genetic inborn error of metabolism. it is associated with limited postnatal growth, joint contractures, thickening of the skin, coarsening of facial features and hypertrophic gingiva. all cases have dysostosis multiplex and typically have small epiphyses with delayed ossification and shortening and widening of diaphyses with osteopenia, coarse trabeculations and periosteal cloaking. craniosynostosis has been reported in some cases of i - cell disease, but may not be present in all cases. orthopedic abnormalities are present at birth and may include thoracic deformity, kyphosis, clubfeet, deformed long bones and/or hip dislocation. cardiac involvements in the form of thickening and insufficiency of the mitral valve and sometimes of the aortic valve occur in almost all cases. the most common cause of death is respiratory insufficiency, which results from progressive mucosal thickening and narrowing of the airways and stiffening of the thoracic cage. i - cell disease is caused by mutations in the gnptab gene, which codes for the alpha and beta subunits of the n - acetylglucosamine-1-phosphotransferase protein. mutations in the gene result in impairment of receptor - mediated transport of lysosomal enzymes through the golgi network into the lysosomal compartment. as these enzymes are unable to enter the lysosomes, they accumulate in the plasma, which is why plasma shows several fold elevation of the lysosomal enzymes in this disorder. the risk of recurrence of the disease in each offspring of carrier parents is 25%. our patient had primarily presented with craniosynostosis but before contemplating surgery the child was sent for genetic evaluation and was found to have i - cell disease, which significantly changed the prognosis and management plan. until now, only few cases of i - cell disease associated with craniosynostosis have been reported. out of these, to the best of our knowledge, only one case presented primarily with craniosynostosis as was the scenario with our patient. it can be treated by skull bone reconstruction. however, before contemplating surgery, evaluation of syndromic craniosynostosis is advisable as it will significantly affect the prognosis and management. in addition, diagnosis of the exact underlying genetic syndrome is also important for appropriate genetic counseling of the family, in terms of recurrence risk in future pregnancies and prenatal diagnosis to prevent recurrence. we are hereby reporting a rare case of i - cell disease presenting with craniosynostosis. | we are reporting a rare case of i - cell disease presenting with craniosynostosis. an 11-month - old child presented with abnormal head shape, developmental delay and bent bones. we planned for corrective surgery for craniosynostosis, but on genetic analysis i - cell disease was confirmed. after explaining the prognosis of i - cell disease, parents denied surgery. this case report emphasizes the fact that syndromic evaluation of craniosynostosis is very much essential before proceeding for corrective surgery. |
adequate polymerization is a crucial factor in obtaining the optimal physical performance of resin composite. the polymerization depends on chemical composition filler content, photoinitiators, shade, and thickness of composites ; it also correlated with the irradiation time and intensity, as well as the distance of the light tip from the tooth - restorative material. depth of cure is getting importance as the composite cure through the tooth structure in many clinical cases. the intensity of the curing light is attenuated by the tooth structure through which the light passes. inadequate polymerization results in poor resistance to wear and color stability, increased rates of water sorption and solubility, decreased dynamic elasticity modulus, as well as early restoration failure. furthermore, there may be greater deterioration at the margins of restoration, decreased bond strength between the tooth and the restoration, greater cytotoxicity and pulpal irritation, postoperative sensitivity, and reduced hardness. further, the unreacted monomers could be eluted from restoration due to hydrolysis over time which in turn leads to the decline in the physical properties. several studies demonstrated these poor mechanical properties of composite while being cured through the tooth structure. subsequently, some researchers have suggested increasing the light intensity and exposure time to compensate for reduced light intensity in these situations. however, the results have been controversial and the threshold thickness of tooth substance, in which a reduction in intensity becomes clinically important, remains unclear. to the best of our knowledge, there has not been a study done on irradiation through tooth structure on primary teeth. with regard to structural differences of primary and permanent teeth and existing controversial results in studies done on permanent teeth, the aim of this study was to assess the effect of increased exposure time and light intensity on microhardness of cured composite through different thicknesses of tooth structures in primary teeth. this study was approved by the research and ethics committee, school of dentistry, mashhad university of medical science, mashhad, iran, and all experiments were carried out at the pediatric department of the mashhad dental school. in this study, intact extracted human primary second molars, free of any type of decay, cracks, hypocalcification, fractures, abrasions, previous restorations, or structural deformities were used to prepare different thicknesses of sound tooth structure slices (1, 2, and 3 mm). written informed consent was obtained from the parent of each patient to sanction the use of the extracted teeth for the abovementioned purpose. the teeth were disinfected with thymol 0.1% immediately after extraction, cleaned with slurry of pumice, and stored in normal saline before the experiment. different slices were prepared using a grinder (metaserv 2000 grinder / polisher, buehler uk ltd., coventry, england) with 240, 400, and 600 grit sic papers. they were prepared from lingual surfaces of the teeth (along the mesiodistal axis) ; they had 1 mm enamel and zero, 1, and 2 mm dentin thicknesses, respectively. the slices were stored in distilled water before and during the experiment to prevent desiccation. they were dried with air before light application and put back into distilled water after testing. one hundred - seventy specimens of the hybrid resin composite a2 z-250 (3 m dental products, st. paul, mn, usa) were prepared (3 mm in diameter and 2 mm in thickness) using teflon molds. the mold was filled in a single increment while covered by mylar strips (kerrhawe striproll 8 mm/0.05 mm, switzerland) at the top and bottom using glass slabs and finger pressure to remove any excess material. the light guide tip of a light - emitting diode (led) light curing unit (lcu) (bluephase style, ivoclar vivadent, schaan, liechtenstein) was positioned on the surface of the composite specimen with the light passing through the mylar strip (direct light curing) or tooth slices (indirect light curing : 1, 2, and 3 mm thicknesses). specimens were located on a vertically adjustable holding device to obtain an accurate distance and position in relation to light guide tip. a dentinal table was used under the specimens to simulate the light reflection. before light application to each specimen, the intensity of lcu was calibrated with a radiometer (coltolux light meter, model no. all specimens were divided into 16 experimental subgroups (n = 10) according to light intensity and curing time and the thickness of the tooth slices and one control group (n = 10) (400 mw / cm intensity and 40 s of exposure time using a quartz - tungsten - halogen [qth ] lcu [astralis 7, ivoclar vivadent, schaan, liechtenstein ] applied in control group). irradiation protocols used in the study all specimens were stored in 100% humidity in a light proof container at 37c for 24 h, followed by a careful polish of the bottom and top surfaces of the each specimen with 600, 1000, 2000, and 2500 grits sic papers. the vickers hardness test under a 300 g load for 20 s (300 g/20 s) was carried out on the top and bottom surfaces of each specimen with a microhardness tester (matsuzawa seiki co., ltd., the average of these values was considered the mean vickers hardness number (vhn) of the specimen 's surface. the hardness ratio of the specimens was then calculated using the formula : if this ratio at the both, top and bottom, surfaces of the specimen was at least 90%, that specimen was considered clinically acceptable. statistical analysis was performed with anova and independent t - test at a significance level of 5%. the absolute frequency distributions of composite specimens according to intensity, curing time, and method of light curing (direct or indirect) are shown in figure 1. absolute frequency distribution of composite specimens in experimental groups according to intensity, curing time, and direct or indirect light curing through different tooth structure thicknesses a multifactor anova revealed double and triple interactions between independent variables (p = 0.001) of the tooth structure thicknesses, the exposure time and light intensity ; therefore, the variables were analyzed distinctly. regarding indirect curing (curing through tooth structure), as the tooth slices increased in thickness, the vhn of the cured resins decreased in all groups [figure 2 ]. mean vickers hardness number of composite resins polymerized through different tooth thicknesses in studied subgroups the means and standard deviations of the vhn of the specimens which polymerized directly or through tooth structure under the experimental conditions used in this study are presented in [tables 2 and 3 ]. mean (standard deviation) of vickers hardness number of the specimens polymerized directly or through tooth structure : effect of increased intensity mean (standard deviation) of vickers hardness number of the samples polymerized directly or through tooth structure : effect of increased exposure time as shown in table 2, with increasing light curing intensity, the means of the vhn of directly cured specimens and those that were cured through 1.0 mm thick tooth structure were significantly decreased (p < 0.05), whereas the means of the vhn of just the top surfaces of the specimens that were cured through 2.0 mm thick tooth structure were statistically increased (p < 0.05). furthermore, the obtained results showed that there were no statistical differences for microhardness while the composite specimens cured through 3 mm of tooth structure thickness. table 3 shows the results after increasing the exposure times. indirectly cured specimens and those that were cured through 1.0 mm thick tooth structure, increasing curing times along with high intensity resulted in significantly improved hardness. whereas, the values of the specimens that were cured through 2 and 3 mm thick tooth structures were mostly significantly higher only at the top surfaces. only in the specimens of the directly cured group and the groups that were cured through 1 mm thickness of primary tooth structure, the microhardness of the top and the bottom surfaces was near clinically acceptable microhardness [figure 3 ]. due to the structural differences between permanent and primary teeth, the results of the former can not generalized to the latter. studies have shown that the density and diameter of dentinal tubules and also mineral contents in primary teeth are less than permanent teeth. all of these factors could affect the light distribution during passing through tooth structure, the primary teeth. in the present study, microhardness of the composite resin as the thickness of tooth structure was increased, more reduction in microhardness was observed. however, the hardness of the specimens cured through 1 mm tooth thickness was still in a clinically acceptable limit. it has been demonstrated that the light - attenuating effect of enamel and dentin reduces the degree of polymerization, resulting in poor mechanical properties. to compensate for light energy loss while passing through the tooth structure, some researchers have reported that the use of high - intensity lcus negatively affects the integrity of restoration - cavity interface ; increases the incidence of restorative margin fracture, enamel margin fracture, and marginal openings ; increases shrinkage stresses ; and results in higher microleakage values. high intensity may cause rapid formation of a highly crosslinked polymeric network in surface layers of composite resin ; subsequently, the extent of the light passing through the bulk of composite may be reduced. thus, high intensity can lead to rapid polymerization and the formation of a low polymeric chain and frequency of crosslinking, reducing the modulus of elasticity, and decreasing the hardness of composite resin. hence, while light curing is applying directly, the use of high - intensity lcus is not recommended. the data were not in agreement with the results of da silva. and the two studies indicated that using higher intensities would improve the curing efficiency of composite resins. these contradictory findings might be explained by differences among studies in lcus, wavelengths, characteristics of composite resins, and light intensities they used. in the cases that light curing was carried out through the tooth structure the exception was in the specimens cured through 1 mm depth, in which the microhardness decreased as the light intensity increased. in these cases, it seems that despite the decrease in power density during passage through the teeth, it is still high enough to cause a drop in the microhardness of the composite for reasons mentioned earlier. however, in thicker slices (2 and 3 mm) which consist of dentin besides enamel, it is assumed that the light absorption and scattering are so great that even doubling the power density can not improve the hardness of composite resin except for the top surfaces in the 2 mm thickness group. with regard to the fact that surface hardness is not an adequate indicator for complete material polymerization and the hardness of the bottom surface should be also closed to the hardness of the top surface, it seems that further increasing in intensity might be required. in this study, the results for the exposure time revealed that in specimens cured directly or through 1 mm thickness slices, increasing the exposure time in cases with high intensities, resulted in higher values of hardness ; however, at low intensities in most cases, it did not cause a change in microhardness. perhaps, this is because differences in exposure time do not result in differences in degree of conversion of optimally cured resin composite material (at low intensities) ; however, at high intensities that existing hardness has been decreased, increasing exposure time improved hardness significantly but not at the clinically acceptable limit. as the thickness of the tooth which the light passes through increases, the numbers of the photons available to raise camphorquinone to an activated state is limited by absorption and scattering factors. decreased light intensity, the exposure time can be increased, providing enhanced opportunity for an excited camphorquinone molecule to collide with an amine, thus creating a free radical. it seems to be the reason for increasing hardness in high - intensity groups after increasing exposure time. increasing exposure time had a significant effect just on the top surfaces of the specimens cured through 2 or 3 mm thicknesses ; therefore, it is not a clinically helpful result. consequently, in previous researches on adequate polymerization, the ratio between microhardness of the bottom and top surfaces is designated as a standard. some researchers advise that the optimal ratio should be 90% while others recommend that it should be 80%. the composite resins can be polymerized almost ideally in vitro, in the clinical situations ; however, in most of the time, it is not possible to polymerize them ideally. hence, to generalize the results with more confidence, level of 90% was selected at the present study. however, it must be taken into consideration that the results not to be misinterpreted. in studies which aimed at assessing new lcus or irradiation modes, there is a possibility of specimens being poorly cured throughout and if the bottom surface is compared to the top surface of the same specimen, the ratio still could exceed 80 or 90% and be misinterpreted as a sufficiently cured material. on this note, the hardness of both surfaces must be compared to standard cured surface (control). in this study, the vhn values obtained with 40 s curing at 400 mw / cm using a qth lcu were used as the control. the microhardness ratio reached 90% in approximately all the specimens that were directly light cured and in the groups that the specimens were light cured through 1 mm thick tooth structure. accordingly, when there were 2 or 3 mm tooth thicknesses between the light guide and composite resin, the used led - lcu (bluephase) could not establish the acceptable clinical microhardness, even after doubling the manufacturer 's recommended exposure times or power densities or both. the further studies will be needed to determine the effect of other lcus such as plasma arc and laser systems on microhardness of the composites cured through thicker tooth structure, as well as similar clinical investigations should be performed to confirm this in vitro results. with the limitations of this study, we concluded that the presence of different tooth thicknesses of primary teeth between the light guide and composite resin could cause reduction of microhardness of cured composite. with the led device, bluephase is applied for curing composite with the light guide most possible close to the resin or 1.0 mm thickness of tooth between them, increasing light intensity when there are 2 or 3 mm thicknesses of tooth, increasing light intensity or curing time (doubling) caused no helpful changes in microhardness. perhaps, because it may require a greater increase in power density and exposure time to increase the hardness to the acceptable clinical level. just in the cases of direct light curing or curing through 1 mm tooth thicknesses, the microhardness of the specimens was at the clinically acceptable rate. | objective : the aim of this study was to evaluate the effect of increased exposure time and light intensity on microhardness of cured composite through different thicknesses of tooth structure in primary teeth.materials and methods : one hundred and seventy cylindrical resin composite specimens were prepared. all specimens were divided into 17 experimental and control groups. light - emitting diode light curing unit (lcu) applied directly or through 1, 2, and 3 mm thicknesses tooth slices for experimental groups. the irradiation protocols were 25 and 50 s at 650 mw / cm2 and 15 and 30 s at 1100 mw / cm2. the quartz - tungsten - halogen lcu (400 mw / cm2) for 40 s was used in control group. microhardness was measured by the vickers hardness test.results:indirectly cured specimens and those cured through a 1 mm thick tooth structure, an increase in intensity caused hardness drop. in the specimens cured through 2 and 3 mm thick tooth structures, increased intensity and/or exposure time did not show any appropriate changes on microhardness.conclusion:irradiation through a 1.0 mm thick tooth slice resulted in reduced microhardness although it was still within the clinically acceptable level. the hardness values of the specimens cured through 2 or 3 mm thick tooth slices fell below the clinically acceptable level even after doubling the exposure time and/or light intensity. |
after the first description of tunnel widening (tw) following anterior cruciate ligament (acl) reconstruction in the 1990s,1 the condition has been reported in many other studies.2345678 the correlation between tw and the clinical outcomes of acl reconstruction remains unclear ; however, many researchers have investigated the causes of tw and methods of preventing it because tw can be a factor in graft failure after acl reconstruction9 and makes revision acl reconstruction difficult. the etiology of tw is still unclear as both mechanical and biological factors have been suggested to play roles.10 the biological factors proposed include an antigenic immune response,11 a toxic effect,1 a nonspecific inflammatory response,12 and cellular necrosis from drilling and graft remodeling,131415 and the mechanical factors proposed include local stress deprivation of the tunnel wall,15 graft - tunnel motion,12 aggressive rehabilitation,16 and increased graft forces due to improper graft placement. many recent studies have tried to prevent tw by modifying the surgical techniques used ; strategies include the use of a bone plug application with press - fit,1718 bone impaction using a dilator,19 proper tunnel positioning,8 and a periosteal envelope.5 it has been reported that these surgical procedures can effectively reduce the extent of tw ; however, there is still controversy regarding the most effective method of preventing tw. this study suggests surgical techniques to prevent tw by using an allo - achilles tendon graft and to evaluate tw after acl reconstruction with these techniques. we hypothesized that acl reconstruction using an allo - achilles graft with the suggested surgical tips would lead to less tw. 85 patients who underwent acl reconstruction by a single surgeon (dws) between september 2011 and june 2013 were included in this retrospectively study. twenty three patients were excluded based on the following exclusion criteria : age under 18 (n = 5), revisional acl reconstruction (n = 13), and associated bony surgery such as high tibial osteotomy (n = 5). among the remaining 62 patients, 29 patients with thirty knees (one patient underwent bilateral acl reconstruction within a 2-month interval) were followed for more than 1 year. ethical approval for the current study was obtained from the public institutional review board of country. all patients underwent single - bundle acl reconstruction by a transtibial technique with careful targeting of the femoral insertion of the native acl. after assessing the amount of remaining fiber and the tension of the injured acl, the surgeon chose to perform an acl reconstruction. because of its advantages in ligament healing and proprioception, we preferred remnant - preserving acl reconstruction with internal sutures between the remnant and reconstructed graft.2021 to prevent tw after acl reconstruction, we modified a few steps of procedure : first, we preferred bone - to - bone healing to bone - to - tendon healing.22 second, we used gradual reaming with bone impaction with a dilator to minimize bone loss during tunnel reaming. third, to prevent undesired tibial tunnel reaming during femoral reaming, we applied a tibial tunnel - independent guide pin during the femoral reaming procedure. fourth, to obtain bone - to - bone healing of the tibial tunnel, the tibial tunnel was fixed with a bone plug with a small interference screw. we designed the allo - achilles graft with a 10 mm diameter that preserved the bone - tendon junction as much as possible [figure 1 ]. first, the bone block was cut and prepared into a cylindrical shape, 10 mm in diameter and 20 mm in length using a bone saw. to preserve the bone - tendon junction as much as possible, the bone block of the allo - achilles tendon was prepared along the direction of the tendon fiber as shown in figure 1, not perpendicular to the junction. we also prepared a free bone block from remaining allo - calcaneus bone to be used as a bone plug for the tibial tunnel, which was 5 mm wide and 25 mm long. (a) for anterior cruciate ligament reconstruction, the allo - achilles graft is created to pass through the 10 mm diameter tunnel and to preserve the bone - tendon junction. (b) at the bone - tendon junction, a cylindrical tunnel 10 mm in diameter and 20 mm in length in the bone block is made along the direction of the tendon fiber and the tendon portion is prepared to pass through the 10 mm diameter tunnel. (c) an additional bone plug for the tibial tunnel is made using the remaining calcaneal bone ; the tunnel is 5 mm in width and 25 mm in length during tunnel reaming, reamers can cause bone debris or thermal injury to the tunnel wall. these are known to cause tw. to prevent tw and to increase the compactness of the bone around the tunnel, a previous researcher had used a dilator with bone impaction.19 however, bone impaction using a dilator could result in a cortical bone fracture on the articular side. therefore, we modified the previous bone impaction technique by gradually reaming from 7 to 9 mm, then carrying out bone impaction with a dilator beneath the articular cortex, and finally reaming the articular cortical wall using a 10 mm reamer. for femoral tunnel reaming, a guide pin was passed through the tibial tunnel and fixed on the distal femur. if the 10 mm diameter reamer passes through the same 10 mm diameter tibial tunnel within the rigid guide pin, the reamer can injure the tibial tunnel if there is a mismatch between the tibial tunnel and the fixed guide pin. to prevent tunnel damage via this mechanism, we recommend intraarticular reamer application [figure 2 ]. before the reamer was used, the guide pin was pulled proximally until the tip of the guide pin which was located in the intraarticular space. then, the reamer was passed through the tibial tunnel freely and attached to the tip. before reaming, the guide pin was pushed distally about 2 cm to prevent axis mismatch between the guide pin and the reamer. after reaming, the guide pin was pulled again to prevent injury that can occur while the reamer is passed through the tibial tunnel for separating. after these procedures, the guide pin was pushed in again until the tip passed the tibial tunnel. (a and b) after targeting the anterior cruciate ligament femoral insertion, the guide pin is applied. because the targeted anterior cruciate ligament femoral footprint is not equal to the advanced point of the tibial tunnel, axis of the guide pin for the femoral tunnel can be different to the tibial tunnel. (c) with these different axes, the reamer can violate and injure the tibial tunnel. (d and e) to prevent this injury, the guide pin is pulled proximally until the tip is located in the articular space. this allows the reamer to pass the tibial tunnel freely, without damaging the tibial tunnel. (f) on reaming, if the distance between the guide pin and the reamer is too short, then the direction of the reamer may be off causing improper and damaging tunnel reaming. (g) by using the 10 mm head reamer with a narrow shaft, femoral tunnel reaming can be completed without injuring the tibial tunnel. (h and i) to prevent similar tibial tunnel injury during reamer detachment, the guide pin is pulled proximally again, and the reamer is separated from the guide pin and knee joint. after that the guide pin is passed through the tibial tunnel using the cannulated guide the bony portion of the allo - achilles tendon graft is applied to the femoral tunnel and that an 8 mm metal interference is used for fixation. with this fixation, in addition, it provides a strong bone - to - bone union between the graft and the tunnel. on the tibial side, the previous studies used the bone plug to reduce tw.1718 to obtain a strong bone - to - bone healing on the tibial side, we also recommended a bone plug. in our cases, a bone plug 5 mm wide and 25 mm long was prepared by using the remaining calcaneal bone of the allo - achilles tendon graft. for stable fixation, dual fixation was recommended ; the graft was fixed by a screw with a spike washer on the extratunnel part, and then intratunnel fixation was accomplished via the prepared bone plug. to prevent motion of the bone plug and to obtain a greater compression force on the graft - tunnel junction, we added a 7 mm bioabsorbable interference screw between the bone plug and the tibial tunnel [figure 3 ]. after the graft passes through, it is fixed on the femoral tunnel and is fixed with a spike washer and screw, a bone plug and a bioabsorbable screw are applied for dual fixation. (a - c) a bone plug 5 mm in diameter and 25 mm in length from the bony portion of the allo - achilles graft is passed between the allo - achilles graft and tibial tunnel. (d - f) a guide pin is applied and a 7 mm diameter bioabsorbable screw is fixed after tapping between the bone plug and tibial tunnel on postoperative day 1 (pod1) and during the followup visit, patients underwent simple radiographs in both the anteroposterior (ap) and lateral views. the diameters of the femoral and tibial tunnels were measured on three levels (proximal, middle, and distal) as was described in previous studies.2324 the automatic distance measurement tool of the piview star program (infinitt healthcare, seoul, korea), a type of picture archiving and communication system, was used to export all images and perform all measurements. between the data obtained on pod1 and during followup, a difference of up to 1 mm was considered to be clinically relevant. whitney u - test and data on tibial tunnels were analyzed by paired t - tests. all statistical analyses were performed by the statistical package for the social sciences (spss) software version 12.0 (spss, chicago, il, usa) and a p value under 0.05 was considered statistically significant. all patients underwent single - bundle acl reconstruction by a transtibial technique with careful targeting of the femoral insertion of the native acl. after assessing the amount of remaining fiber and the tension of the injured acl, the surgeon chose to perform an acl reconstruction. because of its advantages in ligament healing and proprioception, we preferred remnant - preserving acl reconstruction with internal sutures between the remnant and reconstructed graft.2021 to prevent tw after acl reconstruction, we modified a few steps of procedure : first, we preferred bone - to - bone healing to bone - to - tendon healing.22 second, we used gradual reaming with bone impaction with a dilator to minimize bone loss during tunnel reaming. third, to prevent undesired tibial tunnel reaming during femoral reaming, we applied a tibial tunnel - independent guide pin during the femoral reaming procedure. fourth, to obtain bone - to - bone healing of the tibial tunnel, the tibial tunnel was fixed with a bone plug with a small interference screw. we designed the allo - achilles graft with a 10 mm diameter that preserved the bone - tendon junction as much as possible [figure 1 ]. first, the bone block was cut and prepared into a cylindrical shape, 10 mm in diameter and 20 mm in length using a bone saw. to preserve the bone - tendon junction as much as possible, the bone block of the allo - achilles tendon was prepared along the direction of the tendon fiber as shown in figure 1, not perpendicular to the junction. we also prepared a free bone block from remaining allo - calcaneus bone to be used as a bone plug for the tibial tunnel, which was 5 mm wide and 25 mm long. (a) for anterior cruciate ligament reconstruction, the allo - achilles graft is created to pass through the 10 mm diameter tunnel and to preserve the bone - tendon junction. (b) at the bone - tendon junction, a cylindrical tunnel 10 mm in diameter and 20 mm in length in the bone block is made along the direction of the tendon fiber and the tendon portion is prepared to pass through the 10 mm diameter tunnel. (c) an additional bone plug for the tibial tunnel is made using the remaining calcaneal bone ; the tunnel is 5 mm in width and 25 mm in length during tunnel reaming, reamers can cause bone debris or thermal injury to the tunnel wall. these are known to cause tw. to prevent tw and to increase the compactness of the bone around the tunnel, a previous researcher had used a dilator with bone impaction.19 however, bone impaction using a dilator could result in a cortical bone fracture on the articular side. therefore, we modified the previous bone impaction technique by gradually reaming from 7 to 9 mm, then carrying out bone impaction with a dilator beneath the articular cortex, and finally reaming the articular cortical wall using a 10 mm reamer. for femoral tunnel reaming, a guide pin was passed through the tibial tunnel and fixed on the distal femur. if the 10 mm diameter reamer passes through the same 10 mm diameter tibial tunnel within the rigid guide pin, the reamer can injure the tibial tunnel if there is a mismatch between the tibial tunnel and the fixed guide pin. to prevent tunnel damage via this mechanism, we recommend intraarticular reamer application [figure 2 ]. before the reamer was used, the guide pin was pulled proximally until the tip of the guide pin which was located in the intraarticular space. then, the reamer was passed through the tibial tunnel freely and attached to the tip. before reaming, the guide pin was pushed distally about 2 cm to prevent axis mismatch between the guide pin and the reamer. after reaming, the guide pin was pulled again to prevent injury that can occur while the reamer is passed through the tibial tunnel for separating. after these procedures, the guide pin was pushed in again until the tip passed the tibial tunnel. (a and b) after targeting the anterior cruciate ligament femoral insertion, the guide pin is applied. because the targeted anterior cruciate ligament femoral footprint is not equal to the advanced point of the tibial tunnel, axis of the guide pin for the femoral tunnel can be different to the tibial tunnel. (c) with these different axes, the reamer can violate and injure the tibial tunnel. (d and e) to prevent this injury, the guide pin is pulled proximally until the tip is located in the articular space. this allows the reamer to pass the tibial tunnel freely, without damaging the tibial tunnel. (f) on reaming, if the distance between the guide pin and the reamer is too short, then the direction of the reamer may be off causing improper and damaging tunnel reaming. (g) by using the 10 mm head reamer with a narrow shaft, femoral tunnel reaming can be completed without injuring the tibial tunnel. (h and i) to prevent similar tibial tunnel injury during reamer detachment, the guide pin is pulled proximally again, and the reamer is separated from the guide pin and knee joint. the bony portion of the allo - achilles tendon graft is applied to the femoral tunnel and that an 8 mm metal interference is used for fixation. with this fixation, in addition, it provides a strong bone - to - bone union between the graft and the tunnel. on the tibial side, the previous studies used the bone plug to reduce tw.1718 to obtain a strong bone - to - bone healing on the tibial side, we also recommended a bone plug. in our cases, a bone plug 5 mm wide and 25 mm long was prepared by using the remaining calcaneal bone of the allo - achilles tendon graft. for stable fixation, dual fixation was recommended ; the graft was fixed by a screw with a spike washer on the extratunnel part, and then intratunnel fixation was accomplished via the prepared bone plug. to prevent motion of the bone plug and to obtain a greater compression force on the graft - tunnel junction, we added a 7 mm bioabsorbable interference screw between the bone plug and the tibial tunnel [figure 3 ]. after the graft passes through, it is fixed on the femoral tunnel and is fixed with a spike washer and screw, a bone plug and a bioabsorbable screw are applied for dual fixation. (a - c) a bone plug 5 mm in diameter and 25 mm in length from the bony portion of the allo - achilles graft is passed between the allo - achilles graft and tibial tunnel. (d - f) a guide pin is applied and a 7 mm diameter bioabsorbable screw is fixed after tapping between the bone plug and tibial tunnel on postoperative day 1 (pod1) and during the followup visit, patients underwent simple radiographs in both the anteroposterior (ap) and lateral views. the diameters of the femoral and tibial tunnels were measured on three levels (proximal, middle, and distal) as was described in previous studies.2324 the automatic distance measurement tool of the piview star program (infinitt healthcare, seoul, korea), a type of picture archiving and communication system, was used to export all images and perform all measurements. between the data obtained on pod1 and during followup, whitney u - test and data on tibial tunnels were analyzed by paired t - tests. all statistical analyses were performed by the statistical package for the social sciences (spss) software version 12.0 (spss, chicago, il, usa) and a p value under 0.05 was considered statistically significant. the mean followup duration until the simple radiographs were taken was 16.2 months (range 12 to 31 months), and patients demographics are shown in table 1. because the bone portion of the allo - achilles graft was placed in the femoral tunnel, we could not identify the tunnel margin of the femoral tunnel in most of the simple radiographs. in eighteen of the thirty knees (60%), we could not see the femoral tunnel margin on either the pod1 or in the followup radiographs either in anteroposterior or lateral views [figure 4 ]. in four cases, the radiographs taken on pod1 had a line showing the femoral tunnel margin, and in only one case, the visible femoral tunnel margin was available in the ap view alone. that case showed an increase of tunnel diameter in the followup radiographs [figure 5 ]. it was also the case which had the largest femoral tunnel width in the present study. in eight other cases, the margins of the femoral tunnel could only be identified on the followup radiographs ; however, the diameters at the three levels in both ap and lateral view were smaller than 10 mm, the reamer diameter for the femoral tunnel. the mean diameter of the femoral tunnels that could be identified on followup radiographs was 8.6 mm. according to the results of the mann whitney u - test, there were no differences in tunnel diameter between pod1 and followup radiographs [table 2 ]. a 31-year - old male patient underwent anterior cruciate ligament reconstruction in our institution using the allo - achilles graft. (a and b) x - ray of knee joint anteroposterior and lateral views showing the margins of the femoral tunnel could not be identified on postoperative day 1. the tibial tunnel diameters were 10.6, 10.4, and 10.0 mm (proximal, middle, and distal, respectively), and 10.0, 10.2, and 11.1 mm. (c and d) x - ray of knee joint anteroposterior and lateral views showing after 31 months, we could again not find the femoral tunnel margin and the diameters of the tibial tunnel had decreased (8.3, 8.4, 8.6, 8.9, 8.9, and 9.6 mm, respectively) a case of femoral tunnel widening. a 29-year - old female patient underwent anterior cruciate ligament reconstruction with our surgical techniques (a) x - ray of knee joint anteroposterior view on postoperative day 1, the femoral tunnel margin was visible in the anteroposterior view and its diameters were 10.3, 9.0, and 7.6 mm (distal, middle, and proximal, respectively). (b) x - ray knee joint lateral view, the margin was not visible. (c) x - ray knee joint anteroposterior view showing nineteen months postoperatively, the femoral tunnel had increased in size to 12.1, 11.0, and 10.1 mm in the anteroposterior view (distal, middle, and proximal, respectively), and (d) x - ray knee joint lateral view showing the margin was invisible in the lateral view mean diameter of tunnels (mm) the mean diameters of the tibial tunnel in pod1 were 10.2, 10.7, and 11.3 mm in the ap view and 10.4, 11.0, and 11.6 mm in the lateral view (proximal, middle, and distal levels, respectively). on the followup radiographs, the mean diameters were decreased to 9.9, 10.5, and 11.1 mm in the ap view and 10.3, 10.6, and 11.1 mm in the lateral view (proximal, middle, and distal levels, respectively) [table 2 and figure 4 ]. of the thirty knees, half (n = 15) showed an increase in mean tibial tunnel diameter and 4 (13.3%) had more than a 1 mm increment in mean tibial tunnel diameter ; the increments were 1.4, 1.5, 2.0, and 2.8 mm [figure 6 ]. between pod1 and the followup, the paired t - tests showed no statistically significant differences in all three levels in both views. a 44-year - old female patient underwent anterior cruciate ligament reconstruction and was followed up for 21 months. (a and b) x - ray knee joint anteroposterior and lateral views on postoperative day 1 showing the diameters of the tibial tunnel were measured as 9.0, 10.8, 12.2, 9.0, 10.4, and 11.0 mm (distal, middle, and proximal, respectively). (c and d) x - ray knee joint anteroposterior and lateral views after 21 months, showing radiographs revealed that the tibial tunnel had widened as the tunnel 's diameters increased to 10.3, 12.2, 14.3, 13.5, 13.9, and 15.0 mm. the mean diameter was 10.4 mm on postoperative day 1 and 13.2 mm at followup, which is a change of 2.8 mm because the bone portion of the allo - achilles graft was placed in the femoral tunnel, we could not identify the tunnel margin of the femoral tunnel in most of the simple radiographs. in eighteen of the thirty knees (60%), we could not see the femoral tunnel margin on either the pod1 or in the followup radiographs either in anteroposterior or lateral views [figure 4 ]. in four cases, the radiographs taken on pod1 had a line showing the femoral tunnel margin, and in only one case, the visible femoral tunnel margin was available in the ap view alone. that case showed an increase of tunnel diameter in the followup radiographs [figure 5 ]. it was also the case which had the largest femoral tunnel width in the present study. in eight other cases, the margins of the femoral tunnel could only be identified on the followup radiographs ; however, the diameters at the three levels in both ap and lateral view were smaller than 10 mm, the reamer diameter for the femoral tunnel. the mean diameter of the femoral tunnels that could be identified on followup radiographs was 8.6 mm. according to the results of the mann whitney u - test, there were no differences in tunnel diameter between pod1 and followup radiographs [table 2 ]. a 31-year - old male patient underwent anterior cruciate ligament reconstruction in our institution using the allo - achilles graft. (a and b) x - ray of knee joint anteroposterior and lateral views showing the margins of the femoral tunnel could not be identified on postoperative day 1. the tibial tunnel diameters were 10.6, 10.4, and 10.0 mm (proximal, middle, and distal, respectively), and 10.0, 10.2, and 11.1 mm. (c and d) x - ray of knee joint anteroposterior and lateral views showing after 31 months, we could again not find the femoral tunnel margin and the diameters of the tibial tunnel had decreased (8.3, 8.4, 8.6, 8.9, 8.9, and 9.6 mm, respectively) a case of femoral tunnel widening. a 29-year - old female patient underwent anterior cruciate ligament reconstruction with our surgical techniques (a) x - ray of knee joint anteroposterior view on postoperative day 1, the femoral tunnel margin was visible in the anteroposterior view and its diameters were 10.3, 9.0, and 7.6 mm (distal, middle, and proximal, respectively). (b) x - ray knee joint lateral view, the margin was not visible. (c) x - ray knee joint anteroposterior view showing nineteen months postoperatively, the femoral tunnel had increased in size to 12.1, 11.0, and 10.1 mm in the anteroposterior view (distal, middle, and proximal, respectively), and (d) x - ray knee joint lateral view showing the margin was invisible in the lateral view mean diameter of tunnels (mm) the mean diameters of the tibial tunnel in pod1 were 10.2, 10.7, and 11.3 mm in the ap view and 10.4, 11.0, and 11.6 mm in the lateral view (proximal, middle, and distal levels, respectively). on the followup radiographs, the mean diameters were decreased to 9.9, 10.5, and 11.1 mm in the ap view and 10.3, 10.6, and 11.1 mm in the lateral view (proximal, middle, and distal levels, respectively) [table 2 and figure 4 ]. of the thirty knees, half (n = 15) showed an increase in mean tibial tunnel diameter and 4 (13.3%) had more than a 1 mm increment in mean tibial tunnel diameter ; the increments were 1.4, 1.5, 2.0, and 2.8 mm [figure 6 ]. between pod1 and the followup, the paired t - tests showed no statistically significant differences in all three levels in both views. a 44-year - old female patient underwent anterior cruciate ligament reconstruction and was followed up for 21 months. (a and b) x - ray knee joint anteroposterior and lateral views on postoperative day 1 showing the diameters of the tibial tunnel were measured as 9.0, 10.8, 12.2, 9.0, 10.4, and 11.0 mm (distal, middle, and proximal, respectively). (c and d) x - ray knee joint anteroposterior and lateral views after 21 months, showing radiographs revealed that the tibial tunnel had widened as the tunnel 's diameters increased to 10.3, 12.2, 14.3, 13.5, 13.9, and 15.0 mm. the mean diameter was 10.4 mm on postoperative day 1 and 13.2 mm at followup, which is a change of 2.8 mm with our surgical tips using an allo - achilles tendon graft, tw at least 1 year after surgery was effectively prevented in this study. although the clinical relevance of tw is not clear, the risk of revision after acl reconstruction and the problems associated with revision acl reconstruction from tw would be reduced by our surgical techniques. many studies have evaluated tw after acl reconstruction based on the type of graft, fixation methods, or surgical techniques.234678252627 the mean extent of tw was 1030% in those studies. according to the results of a previous study which used the same measurement methods as the present study, the mean amount of tw was about 7 mm.23 on the other hand, the mean amount of tw was found not to increase significantly in the present study. furthermore, there were only four cases (13.3%) of tw > 1 mm. given the results of this and other studies, we suggest that our surgical techniques are good options to prevent tw after acl reconstruction. as various types of grafts can be used for acl reconstruction, many researchers have focused on which graft has the best clinical outcomes and the lowest morbidity rate. in particular, autograft versus allograft has been an important topic in orthopedic research.282930 regarding tw, a previous study reported that a significantly higher amount of tw was observed in the allograft group compared to the autograft group,26 but some studies have reported that there are no significant differences between autograft and allograft groups.252731 in the current study, though achilles tendon allografts were used for acl reconstruction, the results revealed no increase in tunnel diameter, which is better than the results of previous studies.252627 these results imply that the allo - achilles tendon graft is a good option in acl reconstruction to prevent tw. for the junction between the graft and the tunnel, bone - to - bone healing may be better than bone - to - tendon healing. a previous study, which compared patellar and hamstring tendons, reported that tw occurred less in the patellar tendon, which allows for bone - to - bone healing on both the femoral and tibial sides.32 another study used the periosteal envelope to overcome the limitation of bone - to - tendon healing and reported minimal tw.5 to obtain bone - to - bone healing in our acl reconstructions without complications, we used the allo - achilles tendon graft, which can provide bone - to - bone healing on the femoral tunnel. for the tibial tunnel, we used a bone plug from the remaining calcaneal bone of the previous allo - achilles tendon graft. for fixation, we used an 8 mm metal interference screw for the femoral side and dual fixation for the tibial side, which was composed of the press fit by a bone plug and bioabsorbable interference screw, and the postfixation by a spike washer. the results of the present study showed no change after the 1 year, meaning that these techniques can be good options for obtaining stability after acl reconstruction and to prevent tw. during tunnel reaming, thermal injury or mechanical injury caused by the reamer and/or bone debris can occur. a previous study used a dilator to create more compact bone on the tunnel wall and reported less tw than with reaming alone.19 we also used a bone dilator on the tibial tunnel to prevent tw, and our results also showed a successful reduction in tw. however, there is a risk that the dilator will negatively impact the articular cortical bone, possibly resulting in the development of a fracture on the tunnel aperture. therefore, when performing bone impaction using a dilator, we recommend impacting just beneath the articular cortex and finalizing on the articular cortex with a 10 mm reamer. using this technique, no fracture of the aperture occurred in our cases. during transtibial acl reconstruction, guide pin application and tunnel reaming for the femoral tunnel were performed through the tibial tunnel. ideally, the guide pin and reamer for the femoral tunnel would be smaller or of an equal diameter to the tibial tunnel so that no injury would occur when constructing the femoral tunnel. however, the femur and tibia are not fixed to each other, and the axis of the femur and tibia can change during acl reconstruction. thus, an axis mismatch between the femoral and tibial tunnels can develop, potentially leading to an injury on the tibial tunnel wall. this can minimize tibial tunnel injury during tibial tunnel - dependent femoral tunnel reaming acl reconstruction. the previous studies have had success using a bone plug in the tibial tunnel to improve bone - to - tendon healing and to prevent tw in acl reconstruction.1718 jagodzinski. reported that press - fit bone plug fixation decreases the amount of tw.17 another study used an autogenous bone plug for tibial press - fit fixation and also reported that autogenous bone plugs reduce tibial tw compared with bioabsorbable interference screws.18 we also used a bone plug for the tibial tunnel ; however, we were concerned about the limited expansive force of the cylindrical bone plug compared to the cone - shaped interference screw. therefore, we added a smaller bioabsorbable interference screw between the bone plug and tunnel to increase the compression force between the graft and bone plug. with this method there is a probable complication inherent in our surgical procedures due to a mismatch between the femoral tunnel and the metal interference screw. after passing the allo - achilles graft through both tunnels, therefore, we applied the screw through the anteromedial portal after full flexion. with this modification, almost no cases had mismatches however, among the 63 knees in the present study, the postoperative magnetic resonance images of eight cases (12.7%) showed mismatch [figure 7 ]. although there was no difference in tw or revision rate according to the occurrence of the mismatch, more studies on the modified methods, such as transportal tunnel reaming, and the clinical results of long term followup are needed to further bolster our present results. a case of mismatch between the femoral tunnel and the metal interference screw. a postoperative magnetic resonance image of a 20-year - old male patient showing the same aperture between the femoral tunnel and the metal interference screw ; however, the axes of the two were different there are certain limitations to the current study. first, there was no untreated control group, and it was not a prospective, randomized study. third, the followup period was relatively short. in the current study, some patients followup radiographs were taken only 1 year after the operation. fourth, we can not prove which of our tips had the greatest effect in preventing tw. our surgical techniques using an allo - achilles tendon graft effectively prevented tw after acl reconstruction in our case series. | background : tunnel widening (tw) after anterior cruciate ligament (acl) reconstruction can be a serious complication, and there is controversy over how to prevent it. this study aimed to suggest surgical approaches to prevent tw using an allo - achilles tendon graft, and then to evaluate tw after these surgical tips were applied.materials and methods : sixty two patients underwent acl reconstruction with an allo - achilles tendon graft. four surgical approaches were used : making a tibial tunnel by bone impaction, intraarticular reamer application, bone portion application for the femoral tunnel, and an additional bone plug application for the tibial tunnel. after more than 1-year, followup radiographs including anteroposterior and lateral views were taken in 29 patients encompassing thirty knees. the diameter of the tunnels at postoperation day 1 (pod1) and at followup was measured and compared.results:in 18 knees (60%), there were no visible femoral tunnel margins on the radiographs at pod1 or followup. in the other 12 cases, which had visible femoral tunnel margins on followup radiographs, the mean femoral tunnel diameter was 8.6 mm. in the tibial tunnel, the mean diameters did not increase on all three levels (proximal, middle, and distal), and there was no statistically significant difference between the diameters at pod1 and followup.conclusion:the suggested tips for surgery involving an allo - achilles tendon graft can effectively prevent tw after acl reconstruction according to this case series. these surgical tips can prevent tw. |
pleomorphic xanthoastrocytoma (pxa), first described by kepes.14), is a rare neoplasm that accounts for 1% of all astrocytic tumors. pxas are supratentorial and involve the temporal lobe, so the most common presentation is seizure3,6,12 - 14). the histologic features of the tumor are marked cellular pleomorphism, variable lipidization and abundant reticulin. features such as necrosis, nuclear atypia and mitotic figures are suggestive of a high grade tumor7,11,14,17,18,21). despite this, pxas possess a relatively favorable prognosis with a 10-year survival of 70%, and are classified as grade ii in the world health organization (who) classification6,20). factors such as mitotic activity and surgical extent are known to be related to prognosis6). however, the clinical course and prognostic factors are still unknown. here we describe the clinicopathologic and radiologic features, treatment outcomes and prognostic factors of 22 patients with pxa in a single institution. between january 2000 and march 2012, 22 consecutive patients were pathologically diagnosed with pxa in our institute. a comprehensive analysis was conducted on these patients with regard to the clinical, radiologic and pathologic features at diagnosis as well as treatments and outcomes. all patients underwent diagnostic and serial follow - up computed tomography (ct) or magnetic resonance (mr) scans on a regular basis. the images were evaluated with respect to the following factors : location, degree of enhancement, size, demarcation, presence of cystic degeneration, peritumoral edema, hemorrhage, calcification, leptomeningeal involvement and bone remodeling. peritumoral edema was defined as a maximum extent of increased t2 signal intensity on the tumor margin. peritumoral edema was classified20) as mild, 1 cm ; severe, > 2 cm. an atypical tumor site was defined as any location other than the supratentorial cortex area. a diagnosis of pxa was established based on the pathologic features described in the who classification of tumors of the central nervous system. the presence of necrosis, mitotic figures and ki-67 were carefully evaluated by two pathologists. the objective of surgery in all patients was to achieve gross total resection (gtr). the extent of resection was defined as follows : gtr, no surgical or imaging evidence of residual disease ; near total resection, 5 mitoses per 10 high - power fields and suggested that the poor outcome group with > 5 mitoses should be referred to as pxa with anaplastic features, although this remains controversial6,10). in our study, mitoses were seen in 12 patients, and most of those had mitotic indices of 5 mitoses per 10 high - power fields and suggested that the poor outcome group with > 5 mitoses should be referred to as pxa with anaplastic features, although this remains controversial6,10). in our study, mitoses were seen in 12 patients, and most of those had mitotic indices of < 2. just 4 patients were diagnosed as suffering from pxas with anaplastic features and their outcomes were diverse. two of 3 patients suffering from pxas with anaplastic features initially suffered rapid disease progression. however, the remaining patient and a patient with a tumor that became malignant remained free from recurrence. necrosis was present in 5 of our patients and was not correlated with disease progression. usually the ki-67 index in pxas is very low (0 - 1%)4). in our study, six tumors were found to have ki-67 indices above 5% and of those, 4 (67%) recurred (p=0.064). 11 showed no mitoses, a ki-67 index of less than 1% and no necrosis. 20 had malignant biopsy results including 6 mitoses and a ki-67 index of 12.6% with necrosis but was stable without adjuvant therapy for 21 months. there is a previous report of a patient with no mitoses, necrosis, or endothelial proliferation whose disease progressed, with spinal metastasis and malignant transformation to glioblastoma23). since the pathologic factors determining prognosis are not well understood, it is difficult to identify anaplastic pxas. macaulay.21) reported no difference in overall survival between patients who had undergone gtr and those in which gtr was not achieved. however, many studies such as those of pahapill.24) and giannini.6) found that gtr conferred a significant overall survival advantage. giannini.6) reported that extent of resection was the single most significant predictor of recurrence - free survival in a multivariate analysis. although gtr was not a significant factor in our study because of the small sample, it may promote disease control and is the recommended treatment for patients with pxa like other glioma. the findings regarding the use of adjuvant therapy such as radiation or chemotherapy are controversial. giannini.6) did not specifically address this question in their report, because only a small number of patients received adjuvant radiotherapy. pahapill.24) noted that the survival curves for those receiving adjuvant radiotherapy were not significantly different from those who did not. adjuvant therapy was not administered after gtr, even for pxas with anaplastic features ; in those cases the patients were followed up using mr. however, koga.15) reported that stereotactic irradiation was helpful for disease control of pxa as shown in the case (patient 6) of our study. the trial of various radiation therapeutic modalities is needed for treating recurrent tumors when gtr is not achieved and metastasis occurs. the frequency of recurrence of pxa has been reported to be 30%, and that of malignant transformation 10 - 20%16,23,34). in our study, disease progression occurred in 7 patients (33%) similar to other reports, and malignant transformation occurred in one patient (5%). marton.22) noted that surgery is also the therapy of choice for recurrent tumors without malignant transformation and radiotherapy and chemotherapy did not seem to play a significant role. fouladi.4) also noted that further surgery is recommended in the case of recurrent disease. re - operation and gtr is the most important treatment for recurrent tumors ; adjuvant therapy can be administered provided pathological markers and clinical progression are taken into account. the tumor morphology was a solid dominant, well - enhanced and well - demarcated cystic mass and was located in the left temporal lobe. a good prognosis was predicted as the patient showed low peritumoral edema and typical supratentorial cortical location on mr images, no mitoses, low ki-67 index and no necrosis on the biopsy. adjuvant therapies can be administered to cases where recurrent tumors are shown to have metastasized or have aggressive extensions on mr images or new proliferative pathological markers are seen. peritumoral edema, atypical location and large tumor size were poor prognostic factors in our study. although the difference between the effects of gtr and str on prognosis was not significant, we suggest that gtr is the treatment of choice for primary and recurrent tumors. adjuvant radiotherapy, especially stereotactic irradiation can be an option for recurrent, residual or metastatic tumors. | objectivepleomorphic xanthoastrocytoma (pxa) is a rare primary low - grade astrocytic tumor classified as who ii. it is generally benign, but disease progression and malignant transformation have been reported. prognostic factors for pxa and optimal therapies are not well known.methodsthe study period was january 2000 to march 2012. data on mr findings, histology, surgical extents and adjuvant therapies were reviewed in twenty - two patients diagnosed with pxa.resultsthe frequent symptoms of pxa included seizures, headaches and neurologic deficits. tumors were most common in the temporal lobe followed by frontal, parietal and occipital lobes. one patient who died from immediate post - operative complications was excluded from the statistical analysis. of the remaining 21 patients, 3 (14%) died and 7 (33%) showed disease progression. atypical tumor location (p<0.001), peritumoral edema (p=0.022) and large tumor size (p=0.048) were correlated with disease progression, however, ki-67 index and necrosis were not statistically significant. disease progression occurred in three (21%) of 14 patients who underwent gtr, compared with 4 (57%) of 7 patients who did not undergo gtr, however, it was not statistically significant. ten patients received adjuvant radiotherapy and the tumors were controlled in 5 of these patients.conclusionthe prognosis for pxa is good ; in our patients overall survival was 84%, and event - free survival was 59% at 3 years. atypical tumor location, peritumoral edema and large tumor size are significantly correlated with disease progression. gtr may provide prolonged disease control, and adjuvant radiotherapy may be beneficial, but further study is needed. |
cancer patients showed an increased risk of pulmonary embolism (pe) compared with the general population.1,2 these patients are subject, during the follow - ups, to imaging studies, including total body computer tomography (ct) with contrast and positron emission tomography ct, to assess the extent of the malignancy and the response to the treatment. in these patients, the detection of unsuspected pulmonary embolism (upe) related to asymptomatic pulmonary embolism (ape) has become increasingly common,3,4 with an increase in health cost.5,6 in most cases, these patients are treated with anticoagulant drugs (eg, warfarin) for at least 3 months, since the american college of chest physicians recommends to consider these patients as patients with symptomatic pe.7 warfarin is commonly used prophylactically in patients with a high risk of thromboembolic events. however, it has several potential adverse effects (eg, bleeding) and drug interactions;8 therefore, it requires frequent monitoring and dose adjustments with a negative impact on the quality of life.9 in contrast, low - molecular - weight heparins (lmwhs) have a predictable pharmacokinetic profile, few drug interactions, and good safety.10 fondaparinux, a synthesized pentasaccharide with antifactor xa activity, is the newest agent with venous thromboembolism (vte) prophylaxis activity.11 here, we evaluated the efficacy and safety of fondaparinux vs warfarin in the prevention of upe in patients with active cancer. a randomized, prospective, single - blind, and parallel group study was performed in the department of clinical medicine and surgery, federico ii university of naples, between march 2013 and june 2015. the single - blind study was chosen in place of the double - blind due to the different methods of administration (eg, warfarin orally and fondaparinux subcutaneously). in order to exclude any risk for the patients, the physicians who evaluated the patients knew the protocol and the group that was being treated, whereas physicians who evaluated the data were not aware of the treatment. the study was conducted according to the ethical principles of the declaration of helsinki, and the protocol was approved by the institutional review board independent ethics committee of interuniversity center of phlebolymphology (cifl). international research and educational program in clinical and experimental biotechnology with the following approval number : er.na.2013.34. before beginning the study we enrolled 64 patients (29 males and 35 females) of both sexes who were diagnosed with active primary cancer and were in anticancer treatment, with occasional finding of ape (table 1). exclusion criteria consisted of the following : history of heart failure or stroke, drug allergies, psychiatric diseases, creatinine clearance < 30 ml / min, or platelet count < 100,000/mm. patients treated with dextran, thrombolytic agents, anticoagulants, or heparin or heparinoids for topical use were also excluded. ape was documented most commonly using chest ct during the follow - up for restaging after therapy (34 patients, 53%) and during the baseline cancer staging (21 patients, 33%), whereas in nine patients (14%) it was documented for the assessment of extrathoracic diseases. moreover, the ct scan detected thrombi in all tracts of the pulmonary artery (table 2). patients with primary active cancer regardless of age, sex, or medical history were randomized to receive either warfarin (coumadin ; dose - adjusted to achieve an international normalized ratio of 2.03.0) or fondaparinux (arixtra, 7.5 mg subcutaneously) once daily for 90 days (t1). the randomization was performed in agreement with our previous studies.1216 we used a computer program to generate a sequence of treatment allocations by block randomization using a random number generator. investigators were not aware of the block size to avoid selection bias. at the time of admission (t0) and during the follow - ups (90 days = t1, end of the treatment ; 1 year = t2, end of the observation), the patients underwent clinical evaluation, chemical blood findings (complete blood count with platelet count, activated partial thromboplastin time, and erythrocyte sedimentation rate), urine analysis, and a chest x - ray. moreover, at the same time (t0t2) in agreement with 2014 esc guidelines, we used the multidetector ct (mdct) angiography to determine the presence of upe. all the mdct angiography examinations were performed with a 64-slice scanner mdct (toshiba aquilion, tokyo, japan) using 5070 ml of 350 mg nonionic isoosmolar contrast (fonexal and omnipaque). a hypodense intraluminal filling defect causing partial or total obliteration of vascular lumen in segmental and subsegmental arteries with or without corresponding increase in the diameter of the affected vessel was taken as a positive result for pe on mdct. considering the high diagnostic power of this scan, lung scintigraphy was performed only in patients with contraindications to ct scanning (eg, critically ill patients or patients with multiorgan dysfunction) or with diagnostic doubts. during the follow - ups (t1 and t2), the safety of the study medications was assessed by monitoring for any adverse drug reactions for severity and causality. the relationship between adverse drug reactions and drug treatment was evaluated using the naranjo adverse probability scale in agreement with our previous studies.1719 the bleeding related to the treatments was evaluated as previously described.20 the primary end point was defined as a statistically significant difference (p<0.01) in the thrombotic pulmonary ct findings between the two groups of treatment. the secondary end point was the recurrence / appearance of thrombotic events in other sites during the follow - ups (t1 and t2). the primary end point was defined as a statistically significant difference (p<0.01) in the development of major bleeding between the two groups. major bleeding was defined as hemorrhage occurring at a critical site (eg, intracranial hemorrhage), resulting in a major therapeutic intervention (eg, surgery), causing hemodynamic compromise, requiring at least one unit of red cell concentrates or resulting in death. minor bleeding was defined as bleeding that did not fulfill the criteria for major bleeding. the study analysis was based upon the intention to treat, therefore, all consented patients were included in the analysis. the student s t - test and the test were used when appropriate to test the significance of the differences. once we determined that differences existed, a bonferroni test was used to determine which means differed. spss software version 21 (ibm corporation, armonk, ny, usa) was used for statistical analyses. a randomized, prospective, single - blind, and parallel group study was performed in the department of clinical medicine and surgery, federico ii university of naples, between march 2013 and june 2015. the single - blind study was chosen in place of the double - blind due to the different methods of administration (eg, warfarin orally and fondaparinux subcutaneously). in order to exclude any risk for the patients, the physicians who evaluated the patients knew the protocol and the group that was being treated, whereas physicians who evaluated the data were not aware of the treatment. the study was conducted according to the ethical principles of the declaration of helsinki, and the protocol was approved by the institutional review board independent ethics committee of interuniversity center of phlebolymphology (cifl). international research and educational program in clinical and experimental biotechnology with the following approval number : er.na.2013.34. before beginning the study we enrolled 64 patients (29 males and 35 females) of both sexes who were diagnosed with active primary cancer and were in anticancer treatment, with occasional finding of ape (table 1). exclusion criteria consisted of the following : history of heart failure or stroke, drug allergies, psychiatric diseases, creatinine clearance < 30 ml / min, or platelet count < 100,000/mm. patients treated with dextran, thrombolytic agents, anticoagulants, or heparin or heparinoids for topical use were also excluded. ape was documented most commonly using chest ct during the follow - up for restaging after therapy (34 patients, 53%) and during the baseline cancer staging (21 patients, 33%), whereas in nine patients (14%) it was documented for the assessment of extrathoracic diseases. moreover, the ct scan detected thrombi in all tracts of the pulmonary artery (table 2). patients with primary active cancer regardless of age, sex, or medical history were randomized to receive either warfarin (coumadin ; dose - adjusted to achieve an international normalized ratio of 2.03.0) or fondaparinux (arixtra, 7.5 mg subcutaneously) once daily for 90 days (t1). the randomization was performed in agreement with our previous studies.1216 we used a computer program to generate a sequence of treatment allocations by block randomization using a random number generator. investigators were not aware of the block size to avoid selection bias. at the time of admission (t0) and during the follow - ups (90 days = t1, end of the treatment ; 1 year = t2, end of the observation), the patients underwent clinical evaluation, chemical blood findings (complete blood count with platelet count, activated partial thromboplastin time, and erythrocyte sedimentation rate), urine analysis, and a chest x - ray. moreover, at the same time (t0t2) in agreement with 2014 esc guidelines, we used the multidetector ct (mdct) angiography to determine the presence of upe. all the mdct angiography examinations were performed with a 64-slice scanner mdct (toshiba aquilion, tokyo, japan) using 5070 ml of 350 mg nonionic isoosmolar contrast (fonexal and omnipaque). a hypodense intraluminal filling defect causing partial or total obliteration of vascular lumen in segmental and subsegmental arteries with or without corresponding increase in the diameter of the affected vessel was taken as a positive result for pe on mdct. considering the high diagnostic power of this scan, lung scintigraphy was performed only in patients with contraindications to ct scanning (eg, critically ill patients or patients with multiorgan dysfunction) or with diagnostic doubts. during the follow - ups (t1 and t2), the persistence, reduction, or disappearance of the thrombotic pulmonary findings was evaluated. the safety of the study medications was assessed by monitoring for any adverse drug reactions for severity and causality. the relationship between adverse drug reactions and drug treatment was evaluated using the naranjo adverse probability scale in agreement with our previous studies.1719 the bleeding related to the treatments was evaluated as previously described.20 the primary end point was defined as a statistically significant difference (p<0.01) in the thrombotic pulmonary ct findings between the two groups of treatment. the secondary end point was the recurrence / appearance of thrombotic events in other sites during the follow - ups (t1 and t2). the primary end point was defined as a statistically significant difference (p<0.01) in the development of major bleeding between the two groups. major bleeding was defined as hemorrhage occurring at a critical site (eg, intracranial hemorrhage), resulting in a major therapeutic intervention (eg, surgery), causing hemodynamic compromise, requiring at least one unit of red cell concentrates or resulting in death. minor bleeding was defined as bleeding that did not fulfill the criteria for major bleeding. the study analysis was based upon the intention to treat, therefore, all consented patients were included in the analysis. once we determined that differences existed, a bonferroni test was used to determine which means differed. spss software version 21 (ibm corporation, armonk, ny, usa) was used for statistical analyses. after a detailed clinical history and radiological examination, 64 patients (29 males and 35 females) were recruited and randomized into two groups : group a : 32 patients (13 males and 19 females) were treated with warfarin.group b : 32 patients (16 males and 16 females) were treated with fondaparinux. group a : 32 patients (13 males and 19 females) were treated with warfarin. group b : 32 patients (16 males and 16 females) were treated with fondaparinux. all the drugs were efficacious in the treatment of pe, even if the primary efficacy outcome was statistically significant (p<0.01) in group b. in particular, persistence of thrombus occurred 14 times in group a and four times in group b ; a reduction in thrombus was found in eight patients treated with warfarin (group a) and in 12 treated with fondaparinux (group b) ; and a complete disappearance of ct findings occurred in eleven group a patients and in 16 group b patients (table 3). we did not find statistically significant differences in recurrence (group a : 7 ; group b : 6 ; p=0.32), appearance of symptoms (group a : 5 ; group b : 4 ; p=0.12), and thrombotic events in other locations (group a : 6 ; group b : 6 ; p=0.46 ; table 3). patients treated with fondaparinux experienced a lower incidence of major bleeding (n=3) compared with patients treated with warfarin (n=6 ; p<0.01) and we did not find statistically significant differences in minor bleeding (group a : 5 ; group b : 4 ; p=0.12) and in overall mortality (group a : 4 ; group b : 3 ; p=0.47 ; table 4). after a detailed clinical history and radiological examination, 64 patients (29 males and 35 females) were recruited and randomized into two groups : group a : 32 patients (13 males and 19 females) were treated with warfarin.group b : 32 patients (16 males and 16 females) were treated with fondaparinux. group a : 32 patients (13 males and 19 females) were treated with warfarin. group b : 32 patients (16 males and 16 females) were treated with fondaparinux. all the drugs were efficacious in the treatment of pe, even if the primary efficacy outcome was statistically significant (p<0.01) in group b. in particular, persistence of thrombus occurred 14 times in group a and four times in group b ; a reduction in thrombus was found in eight patients treated with warfarin (group a) and in 12 treated with fondaparinux (group b) ; and a complete disappearance of ct findings occurred in eleven group a patients and in 16 group b patients (table 3). we did not find statistically significant differences in recurrence (group a : 7 ; group b : 6 ; p=0.32), appearance of symptoms (group a : 5 ; group b : 4 ; p=0.12), and thrombotic events in other locations (group a : 6 ; group b : 6 ; p=0.46 ; table 3). patients treated with fondaparinux experienced a lower incidence of major bleeding (n=3) compared with patients treated with warfarin (n=6 ; p<0.01) and we did not find statistically significant differences in minor bleeding (group a : 5 ; group b : 4 ; p=0.12) and in overall mortality (group a : 4 ; group b : 3 ; p=0.47 ; table 4). in this study, we evaluated the effects of fondaparinux and of warfarin in the treatment of ape in cancer patients. a previous study documented that lmwh reduce the recurrent vte in patients with active cancer in a similar manner to warfarin, but with low bleeding.21 moreover, the comparison of low molecular weight heparin versus oral anticoagulant therapy for long term anticoagulation in cancer patients with venous thromboembolism (clot) study showed that dalteparin is the most effective therapy vs vitamin k antagonists (vkas), and the current guidelines recommend the treatment with dalteparin, enoxaparin, or tinzaparin in cancer patients with symptomatic vte.22 unfortunately, these recommendations are based on a single study, and the patients enrolled in the clot study came mostly from north america and do not represent the global population. the lack of diversity and the other bias in the clot study partly explain why the vkas are still widely used in several countries in patients with thrombosis associated with cancer. a multinational, phase iii, open - label, randomized, controlled trial documented the efficacy and safety of long - term tinzaparin vs warfarin for the treatment of acute vte in cancer patients.21 however, more recently lee,23 in a randomized, open - label, multicenter study performed in patients with active cancer and acute symptomatic vte, documented that the use of a daily full - dose tinzaparin (175 iu / kg) compared with warfarin for 6 months did not significantly reduce the composite measure of recurrent vte and was not associated with reductions in overall mortality or major bleeding. in our study, we documented that fondaparinux induced a significant decrease in the development of thrombosis in cancer patients. in our study, the overall mortality in the two groups was comparable and in our opinion acceptable, considering the disease. there was no difference between the two groups in the long - term outcome (1-year follow - up), but a better response in the primary efficacy outcome was found in patients treated with fondaparinux. baseline characteristics between either groups (fondaparinux and warfarin) were not statistically significant, therefore, we can conclude that these could play a role in the difference in response to the drugs. moreover, it is widely known that several factors (genetic, food, and drugs) are able to modify clinical efficacy of warfarin. however, to date the clinical efficacy is reported for value of international normalized ratio between 2.0 and 3.0. in our study, chemical blood evaluation documented that international normalized ratio was in a normal range in all patients throughout the study. cho reported a patient with metastatic breast cancer, mobile right atrial thrombus, and pe who developed thrombocytopenia after heparin and warfarin treatment. thrombocytopenia may represent a side effect during the treatment with heparin,25,26 and several authors have reported that fondaparinux can be used in patients who developed thrombocytopenia during heparin treatment.12,27,28 shetty,29 in a single - arm observational cohort study of fondaparinux in 30 patients with intolerance to vka therapy (16 had a history of recurrent vte, eleven had idiopathic vte, and three had cancer), did not record episodes of recurrent vte or major bleeding. moreover, pesavento,30 reviewing the experience of the riete investigators with subacute fondaparinux therapy for vte (47,378 patients enrolled in the riete study, 263 treated for at least 3 months with fondaparinux, and 78 of these patients had cancer), documented that there was no difference in recurrent pe in patients taking fondaparinux and vka or lmwh. moreover, they also documented that major bleeding was similar between cancer patients taking fondaparinux and lmwh. in agreement with these authors, in our study we did not record the development of thrombocytopenia, whereas we recorded a lower incidence of major bleeding in patients treated with fondaparinux compared with those treated with warfarin, without significant difference in minor bleeding. our data suggest that a standard dose of fondaparinux may be used in cancer patients with ape until the next ct lung control (3 months). however, the lack of randomized clinical trials that include a larger patient cohort does not allow formulation of final recommendations in these patients, but it is desirable to carry out a broader study, with the involvement of a larger number of patients and a longer follow - up. | introductionin cancer patients, the chest computer tomography (ct) can be used to identify asymptomatic pulmonary embolism (ape). in most cases, these patients are treated with anticoagulant drugs for at least 3 months. the american college of physicians recommend treatment of these patients as patients with symptomatic pulmonary embolism. in this study, we evaluated and compared the efficacy and safety of fondaparinux vs warfarin in the prevention of unsuspected pulmonary embolism in patients with active cancer.materials and methodsa prospective and parallel group study was performed on 64 cancer patients (29 males and 35 females) with ape. a multidetector ct angiography with high spatial and temporal resolution and quality of arterial opacification was used to make the diagnosis. lung scintigraphy was reserved to selected patients only. patients were randomized to either the warfarin (group a) or the fondaparinux (group b) for 90 days. the first end point of efficacy was the persistence, reduction, or disappearance of thrombosis after 90 days. the second end point was the reappearance of thrombosis after 1 year. the first end point of safety was the development of major bleeding.resultswe enrolled 32 patients into each treatment group. we reached the first end point of efficacy and safety in group b which showed that fondaparinux was able to induce the disappearance of thrombotic pulmonary with a lower incidence of major bleeding events compared with warfarin. no difference in the secondary end point was recorded.conclusionwe suggest that the treatment of cancer patients with ape can be oriented with the administration of a standard dose of fondaparinux until the next ct lung control (3 months). however, the lack of a randomized clinical trial, including a larger patient cohort, does not allow formulation of final recommendations in these patients. a broader study would be desirable, involving a larger number of patients and a longer follow - up period. |
prior to this study, the affected index patient in family 1 was prescreened for smn1 mutations. whole exome sequencing (wes) was performed by axeq technologies (seoul, south korea) using the illumina (san diego, ca) trueseq kit to generate sequencing data. annotated wes data were examined for variants in genes selected for relevance to the phenotype. preceding sanger sequencing analysis of setx, als2, sod1, lmna, mfn2, and trpv4 did not identify any pathogenic mutations in the affected individual of family 2 ; subsequently wes was carried out at the university of washington genome center. data from vcf files were analyzed using the in - house seave analysis pipeline (kinghorn centre for clinical genomics, sydney, australia) under homozygous and potentially compound heterozygote models. the effect of amino acid substitutions for sequence variants was assessed using the software sift and polyphen2. sanger sequencing was performed to confirm the mutations in the probands and for segregation analysis in family members. two affected siblings were from a nonconsanguineous family of ashkenazi jewish origin (figure 1). in retrospect, she had difficulties performing sporting activities from the age of 15 years. she presented to medical attention at the age of 35 years with progressive lower limb weakness requiring the use of a walking stick. the impairment of motor function was progressive and at age 56 years she required a scooter and long arm crutch to mobilize. there were no significant ocular, bulbar, or sensory symptoms seen over the course of the illness and she had normal intellectual function. (b) schematic graph of the vrk1 coding region and the corresponding vrk1 protein shows the position of mutations identified in family 1 (blue) and 2 (green). clinical examination was consistent with a profound symmetric distal muscle wasting affecting the upper and lower limbs symmetrically (figure 2). there was a predominantly distal pattern of weakness of greater severity in the lower than upper limbs, with relative preservation of proximal strength. neurophysiology showed preservation of sensory responses but severe and progressive reduction in compound motor action potential amplitudes with relative preservation of conduction velocities. motor evoked potentials from the upper limbs were prolonged with a central component suggesting upper motor neuron involvement in the disease, which is consistent with mri findings of atrophy of the spinal cord (figure 3a). pontocerebellar hypoplasia or other developmental anomalies as reported in other patients previously described with vrk1 mutations were absent (figure 3). respiratory muscle weakness resulted in reduced mean inspiratory and expiratory pressures and a nonprogressive elevation of blood gas co2. other respiratory parameters had been stable for 5 years with no requirement for assisted ventilation. serum creatine kinase was not elevated and an mri of the lower limbs showed profound proximal and distal muscle atrophy with fat replacement. there was relative preservation of the iliopsoas and adductor compartments bilaterally (figure 3). (a) mri of proband in family 1 (sma). (a.aa.c) t2 images and (a.d) sagittal t1 image demonstrate mild nonspecific generalized atrophy and absence of pontocerebellar hypoplasia. (a.e) t2 image sagittal and (a.f) transverse spinal image demonstrate cord atrophy. (a.ga.i) t2 images of upper thigh, calf, and stir sequence of thigh demonstrate diffuse fatty replacement of muscle and relative sparing of adductor compartments. (b.ab.c) t2 images and (b.d) sagittal t1 image demonstrate absence of pontocerebellar hypoplasia. the proband has a similarly affected older brother with a slightly later onset of progressive motor weakness in the late teenage years, progression to requiring a walking stick in his early 30s, and the requirement for an articulated walking device and wheelchair in his late 40s. the patient was a 3-year - old girl with short stature and microcephaly who presented with initial distal lower limb muscle weakness and amyotrophy combined with pathologically brisk deep tendon reflexes and normal sensation. weakness progressed in a caudalocephalic direction, with loss of ambulation by 10 years of age and complete dependence for activities of daily living at 18 years. respiratory dysfunction developed with dyspnea and weak cough accompanied by reduction of forced vital capacity to below 40% predicted. neurophysiology studies at age 11 years demonstrated severe reduction in compound muscle action potential amplitudes combined with chronic and active denervation, consistent with motor neuropathy / neuronopathy. sensory responses were initially preserved but subsequently were reduced on repeat testing at age 19 years. the clinical possibility of juvenile als, with the presence of upper and lower motor neuron signs in more than 2 spinal regions and clinical progression, prompted sanger sequencing as described above. participating individuals were enrolled through the neurogenetics clinic concord hospital and the paediatric neuromuscular clinic at sydney children 's hospital randwick. these procedures were performed with informed consent according to protocols approved by the human ethics committees of sydney local health district, concord hospital (hrec/11/crgh/105), and south eastern sydney local health district (hrec/13/powh/203), australia. prior to this study, the affected index patient in family 1 was prescreened for smn1 mutations. whole exome sequencing (wes) was performed by axeq technologies (seoul, south korea) using the illumina (san diego, ca) trueseq kit to generate sequencing data. annotated wes data were examined for variants in genes selected for relevance to the phenotype. preceding sanger sequencing analysis of setx, als2, sod1, lmna, mfn2, and trpv4 did not identify any pathogenic mutations in the affected individual of family 2 ; subsequently wes was carried out at the university of washington genome center. data from vcf files were analyzed using the in - house seave analysis pipeline (kinghorn centre for clinical genomics, sydney, australia) under homozygous and potentially compound heterozygote models. the effect of amino acid substitutions for sequence variants was assessed using the software sift and polyphen2. sanger sequencing was performed to confirm the mutations in the probands and for segregation analysis in family members. two affected siblings were from a nonconsanguineous family of ashkenazi jewish origin (figure 1). in retrospect, she had difficulties performing sporting activities from the age of 15 years. she presented to medical attention at the age of 35 years with progressive lower limb weakness requiring the use of a walking stick. the impairment of motor function was progressive and at age 56 years she required a scooter and long arm crutch to mobilize. there were no significant ocular, bulbar, or sensory symptoms seen over the course of the illness and she had normal intellectual function. (b) schematic graph of the vrk1 coding region and the corresponding vrk1 protein shows the position of mutations identified in family 1 (blue) and 2 (green). clinical examination was consistent with a profound symmetric distal muscle wasting affecting the upper and lower limbs symmetrically (figure 2). there was a predominantly distal pattern of weakness of greater severity in the lower than upper limbs, with relative preservation of proximal strength. neurophysiology showed preservation of sensory responses but severe and progressive reduction in compound motor action potential amplitudes with relative preservation of conduction velocities. motor evoked potentials from the upper limbs were prolonged with a central component suggesting upper motor neuron involvement in the disease, which is consistent with mri findings of atrophy of the spinal cord (figure 3a). pontocerebellar hypoplasia or other developmental anomalies as reported in other patients previously described with vrk1 mutations were absent (figure 3). respiratory muscle weakness resulted in reduced mean inspiratory and expiratory pressures and a nonprogressive elevation of blood gas co2. other respiratory parameters had been stable for 5 years with no requirement for assisted ventilation. serum creatine kinase was not elevated and an mri of the lower limbs showed profound proximal and distal muscle atrophy with fat replacement. there was relative preservation of the iliopsoas and adductor compartments bilaterally (figure 3). (a.aa.c) t2 images and (a.d) sagittal t1 image demonstrate mild nonspecific generalized atrophy and absence of pontocerebellar hypoplasia. (a.e) t2 image sagittal and (a.f) transverse spinal image demonstrate cord atrophy. (a.ga.i) t2 images of upper thigh, calf, and stir sequence of thigh demonstrate diffuse fatty replacement of muscle and relative sparing of adductor compartments. (b.ab.c) t2 images and (b.d) sagittal t1 image demonstrate absence of pontocerebellar hypoplasia. the proband has a similarly affected older brother with a slightly later onset of progressive motor weakness in the late teenage years, progression to requiring a walking stick in his early 30s, and the requirement for an articulated walking device and wheelchair in his late 40s. the patient was a 3-year - old girl with short stature and microcephaly who presented with initial distal lower limb muscle weakness and amyotrophy combined with pathologically brisk deep tendon reflexes and normal sensation. weakness progressed in a caudalocephalic direction, with loss of ambulation by 10 years of age and complete dependence for activities of daily living at 18 years. severe thoracolumbar scoliosis required surgical rod insertion at age 14 years. respiratory dysfunction developed with dyspnea and weak neurophysiology studies at age 11 years demonstrated severe reduction in compound muscle action potential amplitudes combined with chronic and active denervation, consistent with motor neuropathy / neuronopathy. sensory responses were initially preserved but subsequently were reduced on repeat testing at age 19 years. the clinical possibility of juvenile als, with the presence of upper and lower motor neuron signs in more than 2 spinal regions and clinical progression, prompted sanger sequencing as described above. participating individuals were enrolled through the neurogenetics clinic concord hospital and the paediatric neuromuscular clinic at sydney children 's hospital randwick. these procedures were performed with informed consent according to protocols approved by the human ethics committees of sydney local health district, concord hospital (hrec/11/crgh/105), and south eastern sydney local health district (hrec/13/powh/203), australia. testing identified compound heterozygous vrk1 mutations nm_003384.2 (vrk1):c.(356a > g) ; (1072c > t) (p.[h119r];[r358 ]) in the individuals with sma in family 1 and nm_003384.2 (vrk1):c.(403 g > a) ; (583t > g) (p.[g135r];[l195v ]) in the patient with juvenile als in family 2. no pathogenic variants were identified in other genes associated with sma or als in either patient or genes associated with short stature. the present cases with adult - onset distal sma and childhood motor neuron disease broaden the clinical spectrum of patients with vrk1 mutations. the identification of vrk1 mutations among these various motor phenotypes, with clinical overlap among juvenile als, dhmn + ps, and sma, may serve to further unite concepts of pathogenesis among motor neuron disorders. the results for family 1 are consistent with an autosomal recessive mode of inheritance with segregation of both vrk1 variants with the disease (figure 1a). the c.1072c > t mutation is well - characterized and described as pathogenic in the ashkenazi jewish population when in a homozygous state. the c.356a > g variant (esp frequency g missense substitution was reported in one recent case of a 32-year - old man with a 5-year history of als without cerebellar hypoplasia. this sequence variant is most likely to be pathogenic as it is present in both the affected members of family 1 and a reported sporadic als case. in family 2, both substituted amino acids are highly conserved across multiple species and the frequency of the novel c.583t > g mutation in public databases was 2 in 121382. online database tools supported pathogenicity for c.403 g > a and c.583t > g mutations (polyphen2 scores 1 and 0.997, probably damaging ; sift score 0 and 0, deleterious ; and cadd score 22.6 and 19.05, respectively). the mutations were sanger sequenced for confirmation and each parent was found to carry one of the variants, supporting autosomal recessive inheritance (figure 1). three - dimensional structural protein analysis of the p.gly135arg vrk1 variant with the hope protein structure analysis suite database suggested a difference in charge, size, and hydrophobicity resulting in disturbances in local structure and protein misfolding. similarly, the p.leu195val vrk1 variant altered the amino acid size, causing an empty space in the core of the protein. gonzaga - jauregui. described childhood - onset distal sensory motor axonal neuropathy with microcephaly with a simplified gyral pattern and in a second case there was a similar motor and sensory axonal neuropathy with an underdeveloped cerebellar vermis. the latter case and this report suggest that vrk1 is not only important in development of motor system architecture, but are also important in the longer term in maintenance of motor neurons. vrk1 mutations should therefore be considered in the differential diagnosis of patients presenting with adult - onset sma and childhood motor neuron disease. marion stoll : organized and performed genetic testing on proband and family members (family 1), performed bioinformatics analysis and interpretation of wes data, writing of the manuscript. hooi ling teoh : collected data of the proband (family 2), writing of the manuscript. lee : clinically examined and interviewed the proband (family 1), writing of the manuscript. stephen reddel : clinically examined and performed neurophysiology on proband (family 1), critical revision of manuscript. ying zhu : supervised the creation of the bioinformatic pipeline for genomic analysis, assisted with bioinformatics analysis and interpretation of whole exome data, critical revision of manuscript. michael buckley : organized and performed genetic testing on proband and family members (family 2), interpretation of variant results, critical revision of manuscript. hugo sampaio : clinically examined and interviewed the proband (family 2), critical revision of manuscript. tony roscioli : obtained research funding for genomic sequencing, developed genomic consent forms for patient enrolment, supervised the creation of the bioinformatic pipeline for genomic analysis, performed bioinformatics analysis and interpretation of wes data, critical revision of manuscript. michelle farrar : provided clinical and scientific direction for the manuscript, drafted, reviewed and approved the final manuscript. garth nicholson : provided clinical and scientific direction for the manuscript, planned, drafted, reviewed, and finalized the manuscript, clinically examined, characterized, and interviewed the proband (family 1). supported by motor neurone disease research institute of australia. no industry, governmental, or institutional funding j. lee, s. reddel, y. zhu, m. buckley, h. sampaio, t. roscioli, m. farrar, and g. nicholson report no disclosures relevant to the manuscript. | objective : to describe the phenotypes in 2 families with vaccinia - related kinase 1 (vrk1) mutations including one novel vrk1 mutation.methods:vrk1 mutations were found by whole exome sequencing in patients presenting with motor neuron disorders.results:we identified pathogenic mutations in the vrk1 gene in the affected members of 2 families. in family 1, compound heterozygous mutations were identified in vrk1, c.356a > g ; p.h119r, and c.1072c > t ; p.r358, in 2 siblings with adult onset distal spinal muscular atrophy (sma). in family 2, a novel vrk1 mutation, c.403g > a ; p.g135r and c.583t > g ; p.l195v, were identified in a child with motor neuron disease.conclusions:vrk1 mutations can produce adult - onset sma and motor neuron disease in children without pontocerebellar hypoplasia. |
noncancerous causes of bronchoesophageal fistula (bef) are rare and in a majority of the cases are due to trauma or infection, the most common being granulomatous disease. the combination of mediastinal lymphadenopathy and cough following intake of food should alert the treating clinicians about possibility of tuberculous bronchoesophageal fistula. however, a few case reports have suggested that tuberculous bef can be effectively treated with medical management alone. a 25 year old woman, with no premorbid illnesses presented with a history of cough during eating for 3 months duration and mucoid, non blood tinged sputum production for 1 month. there was also no history of foreign body aspiration, ingestion of toxic or corrosive substances or any surgical procedures in past. her younger sister was detected to have pulmonary tuberculosis 1 yr back and completed antituberculous treatment. she had been married for 9 months, had regular menstrual cycles and no history of high risk behaviour. on examination she exhibited mild pallor. there was no icterus, cyanosis, clubbing or lymph node enlargement. pulse rate 86/min, regular ; blood pressure 110/70 mmhg ; respiratory rate 16/min ; and she was afebrile. cardiovascular, respiratory, gastrointestinal, nervous system examination were within normal limits. investigations showed wbc 11,200/cmm, with 64% neutrophils and 31% lymphocytes, hgb 10 g / dl, platelet count 230,000/cmm and esr was 55 mm / h. renal and liver function tests were within normal limits.retroviral screening and autoimmune markers were negative.sputum afb was negative.tuberculin skin test showed an induration measuring 15 15 mm. esophagoscopy was performed which revealed a 30 mm ulcer with irregular borders with communication into respiratory tract, 25 cm from the oral cavity. computed tomography scan of thorax with three dimensional reconstruction was done which showed mediastinal lymphadenopathy, with erosion of posterior wall of left main bronchus, with a fistulous tract into anterolateral wall of esophagus (fig. bronchoscopy revealed inflamed mucosa which revealed granulomatous inflammation on biopsy.afb staining of bronchial secretions was negative, but tested positive for m.tuberculosis by pcr. (b & c) 3d reconstruction of computed tomography thorax showing fistula between left bronchus and esophagus.fig. (b & c) 3d reconstruction of computed tomography thorax showing fistula between left bronchus and esophagus. treatment comprised of an intensive phase for the first two months in which four antituberculous drugs (isoniazid, rifampicin, pyrazinamide, ethambutol) were given. this was followed by a continuation phase for next four months consisting of two drugs (isoniazid and rifampicin). bronchoscopy was done after completing 4 months of treatment and showed normal bronchial lumen with disappearance of fistulous tract.computed tomography of thorax showed resolution of lung lesions without any fistula. bef poses a challenge to the clinician for accurate diagnosis which if confirmed can offer the patient a potential cure from repeated pulmonary infections. some patients are able to avoid the paroxysms of cough by swallowing in the supine position (ono 's sign). braimbridge and keith classified congenital bef into four types depending on the site of the fistulous tract. 49% of acquired bef are malignant in etiology and the rest are secondary to benign causes such as trauma, tuberculosis, actinomycosis and esophageal diverticulosis. trauma foreign body ingestion instrumentation crushing trauma operative trauma chemical burns causes of acquired esophagobronchial fistula. the development of bef in tuberculosis and other granulomatous diseases are related to mediastinal lymph node involvement. inflammation in and around these enlarged lymph nodes lead to involvement of neighboring structures or organs particularly the esophagus and the trachea near its bifurcation resulting in periesophagitis and peritracheitis. if, however necrosis and caseation occur in the lymph nodes with local abscess formation, secondary rupture into the esophagus, trachea or main stem bronchi results in fistula. there was evidence of numerous necrotic mediastinal lymphnodes in the computed tomography performed in our patient. henceforth, fistula would have been caused by the erosion of lymphnode rather than a primary bronchial tuberculosis. the therapy of esophagobronchial communication is usually surgical, performed by division of the fistulous tract and resection of any portion of the lung irreversibly damaged by the suppurative process. if the fistulous tract originates from lymph nodes with no parenchymal complication, simple ligation and resection of the fistula can be performed. however, in our case surgical treatment was not required. in a similar study in 3 patients infected with human immunodeficiency virus presenting with tuberculous bronchoesophageal fistula, thus, tuberculous bef if diagnosed early, both the causative process and the complicating fistula may be effectively treated with antituberculous chemotherapy without the need of surgical intervention. we, authors here by declare that we have no conflict of interest regarding the publication of this article. this article has not been published elsewhere or has not been sent to another journal. | we report a case of bronchoesophageal fistula associated with tuberculosis. a 25 year old woman presented to us with 3 month history of cough worsening with deglutition. radiological examination revealed mediastinal lymphadenopathy and bronchoscopy with esophagoscopy confirmed the presence of fistulous communication with features of endobronchial tuberculosis. histological examination of bronchial biopsy specimen showed non necrotic granuloma with the pcr positive for mycobacterium tuberculosis in her bronchial secretions. she was begun on antituberculous treatment and became asymptomatic after 2 months. bronchoscopy done during follow up after 4 months showed normal bronchial lumen with disappearance of fistulous tract. imaging showed resolution of lung lesions. |
the energy detector (ed) has been widely proposed for spectrum sensing (ss) in cognitive radio (cr) owing to its design simplicity, fast sensing periodicity, and ability to detect primary user (pu) signal without a priori knowledge of its waveform structure (except for the knowledge of noise statistics) [111 ]. however, the demand for fast sensing and high detection accuracy in cr has revealed particular limitations with the ed. in fast sensing, the ed tends to obtain fewer samples for spectral estimation which leads to reduced resolution and invariably poor detection performance [1215 ]. also, it is known that the ed performs poorly in low signal to noise ratio (snr) conditions and fluctuating noise environments [1214 ] which results in increased false alarm rate. these challenges have compelled the need for new improved ed techniques capable of providing better local sensing results at acceptable detection and false alarm rates in both low and high snr conditions. several usable spectrum estimation techniques are well - known and these can be broadly divided into parametric and nonparametric techniques. some examples of these nonparametric techniques include the simple periodogram (sp), welch periodogram (wp), and the multitaper (mt), while parametric techniques include the yule - walker (yw), burg (bg), and covariance (cv) techniques. generally, the ed has been widely assumed to be based on the periodogram approach (developed either in time or in frequency domain) and this could be presumably responsible for its poor performance owing to known limitations of the periodogram, for example, large noise variance [13, 14 ]. on the other hand, few works have been identified on the use of parametric techniques, particularly the autoregressive (ar) technique, in cr [1622 ]. also, these few works have focused more on its use in spectrum hole prediction rather than as an ed for cr application. in this work, the focus is not on developing a new spectrum estimation technique but rather on proposing the use of a real valued neural network (rvnn) based autoregressive (ar) spectrum estimation technique for ed application in cr. the choice of the rvnn based ar technique was based on its advantage over the popular bg technique in solving the challenge of spectral line splitting at high snr and large model order conditions. consequently, it was observed here that such an advantage would lead to an improved ed with less probability of false alarms for cr application. also, while authors in developed the rvnn technique and applied it in voice activity detection, in our own work, we propose necessary additions to standardize the approach as a fully functional ed using an empirical threshold estimation technique for cr application. the rvnn based ar model coefficient estimator developed in has never been applied as an ed for cr application ; hence, its application was considered here to exploit its inherent advantage. therefore, once developed as a functional ed, it was tested using simulated data particularly in low and high snr conditions and varying model order values as described in section 4 and results obtained with corresponding implications are presented in section 5. energy detectors (ed) sense the presence or absence of pu signals by estimating the energy content of a specified band using the power spectral density (psd) measurements and comparing with predetermined thresholds [12, 14 ]. this aids the cr device in vacating pu occupied bands to avoid interference and use vacant bands to improve spectrum utilization. figures 1(a) and 1(b) provide a representation of this process. towards this goal, signals above the threshold typically signify pu presence, h1, and below threshold as noise, h0. these hypotheses h0 and h1 are typically represented as (1)h0:x(n)=w(n)(2)h1:x(n)=s(n)+w(n), where w(n) represents samples of additive white gaussian noise (awgn), s(n) denotes samples of pu signal, and x(n) denotes samples of received signal at the cr input considering a perfect channel. eds are typically developed to decide on these hypotheses either in time or in frequency domain. in time domain, it consists of an input noise prefilter, a magnitude square law device, an integrator, and a detector as shown in figure 2. this approach has been used in several works [1215 ] and well - known for its simplicity and low design and development cost. however, it is based on the super heterodyne technique which is limited by its slow voltage ramping response time another approach is to realize the ed in the frequency domain using the well - known periodogram as shown in figure 3. this provides an opportunity to achieve full digitization of the detection process ; however, this approach constrains the sensing bandwidth owing to known limitations of typical fast fourier transform (fft) realizations. another method identified for the ed in cr is the welch periodogram (wp) approach. particularly, in, performance analysis was carried out on the use of wp in rayleigh fading channels while [26, 27 ] used wp for ofdm systems in cr. these authors observed that wp operates well for narrowband sensing ; however, they conclude that the approach is limited by the excessive variance around the true mean psd value. another approach using the multitaper (mt) technique was proposed in with basic descriptions provided from filter - bank theory point of view. this technique was extended in for signal detection in cr and results were compared with the time domain based ed. it was observed that mt performed better in low snr conditions under raleigh fading channels. this was based on the technique 's dependence on both magnitude and phase of received samples. also, provided an improved mt method for cr application in which a recursive method to improve the accuracy of estimates while keeping real - time properties uncompromised was proposed. authors compared their technique with the sp and ordinary mt and observed better performance for their improved mt. on the other hand, the use of autoregressive (ar) methods has been sparsely pursued, especially for energy detection in cr. though not widely used in cr, we observed that [1921 ] proposed ar for spectrum hole detection in cr using time series forecasting. authors reported on the performance of the proposed approach ; however, it remains unclear how well such an approach would perform in typical deployment conditions. also, authors in [21, 22 ] used particle and kalman filtering for spectrum availability forecasting. in, authors presented a comparison of sp and yule - walker (yw) ar techniques and argued that better performance could be achieved by the yw if large model orders are considered. however, most of these applications have not considered ar application for ed in cr, and hence, it remains unknown how well such techniques would perform if employed. consequently, we considered the use of a real valued neural network (rvnn) ar model estimator to develop a detector for cr application. this approach provides data reported in which was leveraged upon here to develop an improved ed for cr. consequently, it is the focus here to develop, evaluate, and analyze its performance for cr application. the working principles of the real valued neural network (rvvn) autoregressive (ar) coefficient estimation technique were based on. thus, we present here only specific details needed to develop the ed for cr application ; however, for further details, we refer to. it is known that an ar system driven by white noise x(n) produces an output sequence y(n) given as (3)y(n)=k=1paky(nk)+b0x(n), where ak, 1 k p and b0 denotes the model coefficients and p is the model order. by taking the z - transform of (3) and rearranging the terms therein, the transfer function h(z) of the ar - system can be obtained as (4)h(z)=y(z)x(z)=b01+k=1pakzk. by assigning b0 = 1, z = e and taking the square of (4), we obtain the snr as (5)snr=|y(jw)x(jw)|2=1|1+k=1pakejwk|2. to estimate the model coefficient ak, we used a two - layered rvnn as shown in figure 4. by observing figure 4, the output sequence of the rvnn system can be obtained as (6)y(n)=f(l=1mwl1l+h01), where f denotes the representation of a linear transfer function, m is the number of neurons in the hidden layer, wl1 represents the weight connecting node l in the hidden to output layer, h01 is the bias term of the output neuron, l denotes the output of the hidden node l, and is the adaptive coefficient of the linear output activation function. the hidden node output l is obtained as (7)l=lf(k=1pvkly(nk)+g0l), where vkl is the weight connecting input nodes k to hidden node l, g0l is the bias of the hidden node, and l is the adaptive coefficient of the hidden node linear transfer function. by using linear transfer activation functions in both hidden and output layer, that is, f() is linear, and substituting (7) into (4), the output sequence is obtained as (8)y(n) = f(l=1mwl1(lf(k=1pvkly(nk)+g0l))+h01). by rearranging terms in (8) and comparing with (4), thus, the ar model coefficients can be estimated from the synaptic weights and coefficients of the adaptive activation function of a properly trained two - layered rvnn. furthermore, by assuming that the model order p is known a priori, then the number of neurons in the hidden layer can be determined by using the priori data length information at the input. to formulate the necessary constraints for proper evaluation of the system, the solution to a set of linear equations that gives the best performance over the a priori fixed model order was obtained. we obtained this by considering a data set of length n, for which the required number of training data set l was obtained as (10)l = np+1. the required number of training equations neq is expressed as (11)neq = pl and for m hidden nodes, the number of unknowns for the network was obtained as (12)nun=(p+1)m+m+1. it is known from that the case of neq nun produces better results with respect to the unseen data. therefore, by putting (11) and (12) into the inequality (neq nun), we obtained the optimum number of neurons m to satisfy (13)m(pl)1p+2. with these necessary parameters well established, the algorithm used for the rvnn system is as follows.(1)we normalized the input data sequence to band - limit the acquired signal using the z - score normalization technique. other techniques that could be used are the min max, sigmoidal, or unitary data normalization techniques.(2)the data were formatted using frame blocking and appropriate windowing of data sequencing.(3)the optimum number of neurons was determined using (13).(4)the model coefficient was estimated using (9).(5)finally, training of the rvnn system was done using the back propagation (bp) supervised learning algorithm. we normalized the input data sequence to band - limit the acquired signal using the z - score normalization technique. other techniques that could be used are the min max, sigmoidal, or unitary data normalization techniques. finally, training of the rvnn system was done using the back propagation (bp) supervised learning algorithm. with the rvnn model estimator in place this was achieved by generating the power spectral density (psd) of the system using (5) and introducing a well - designed detector based on an empirical threshold estimation technique (details of method in section 4.2). an overview of the design process is as follows.(1)a simple analogue - digital - converter (adc) was used for digitization. here the number of samples was determined using the known nyquist rate of 8 khz for a maximum bandspan of 4 khz for wideband simulation and 100 hz for narrowband simulation.(2)the fast fourier transform (fft) was used for the frequency response estimation. appropriate zero padding was employed for cases of fewer samples for fft operation.(3)the transfer function estimator was realized using the frequency response vector obtained from appropriate adaptive filtering of the fft samples.(4)a simple square law device was introduced for performing the squaring operation.(5)the detector analyzer was realized using empirically deduced thresholds (details in section 4.2).(6)our proposed detector of figure 5 can be used for cr application and details of methods used for its analysis are presented in the next section. a simple analogue - digital - converter (adc) was used for digitization. here the number of samples was determined using the known nyquist rate of 8 khz for a maximum bandspan of 4 khz for wideband simulation and 100 hz for narrowband simulation. the fast fourier transform (fft) the transfer function estimator was realized using the frequency response vector obtained from appropriate adaptive filtering of the fft samples. the detector analyzer was realized using empirically deduced thresholds (details in section 4.2). our proposed detector of figure 5 can be used for cr application and details of methods used for its analysis are presented in the next section. in this section, we provide details of simulation parameters and analytical models used for evaluating the performance of our proposed detector and other methods for comparison. additive white gaussian noise (awgn) with zero mean and unit variance was used and the probability of false alarm pfa conditioned for the null hypothesis (1) was obtained using the expression for large time - bandwidth product given in. the pfa for noise samples distributed according to a gaussian variate n(2tw, 4tw) is given as (14)pfa=18twvtexp[(y2tw)28tw]dy(15)=12erfc[vt2tw22tw ], where t denotes the observation interval, w the bandwidth being sensed, vt the threshold, and tw the time - bandwidth product. these were translated appropriately as follows : tw is known as the total number of samples n (at nyquist rate), 2tw is the sample mean ^ of the noise psd, and 4tw is the sample variance ^2 of the noise psd (the noise power). we note that the particular use of (15) according to was based on the condition of a large time - bandwidth product, meaning large sample number. next, to estimate the probability of detection pd, we considered a modulated discrete signal s(nt) given as (17)s(nt)=a(t)cos(2fcnt) n=0,1,,n1, where a(t) represents the expression for the modulating signal, fc the carrier frequency, n the total number of samples, and t the sampling time. for examination of the effect of wideband sensing, we considered two pu signals with equal amplitudes a1 = a2 = 10 db, and frequencies fc1 = 600 hz, fc2 = 1500 hz estimated over a relatively wide sensing span of 4 hz using a wideband occupancy of 10% as shown in figure 6. we note that though low frequencies were used, the setup could easily apply to high frequency bands with the same concept being applicable. for narrowband sensing, a typical pu signal of bandwidth w = 60 hz was simulated for a narrowband sweep of 100 hz as shown in figure 7 using the rvnn based ed. these input data were kept constant for all simulation conditions and other eds compared herein. then the pd was estimated using (18)pd=12erfc[vt2tw22tw+ ], where denotes the snr for the estimated spectrum. an empirical threshold selection method was employed here and pfa for each threshold was estimated using (16). this was done by varying the threshold values vt over the noise psd and figure 8 provides the result obtained using the rvnn based ed in wideband sensing. by comparing the threshold line in figure 6 with figure 8, it can be easily observed that at a threshold of 35 db / hz, less noise samples crossed the threshold (figure 6) resulting in pfa 0.9 at pfa = 0.1) along with other techniques. in addition, it is shown in figure 17 that the proposed technique provides a 10% average reduction in pfa at thresholds below 20 dbm than yw, bg, and cv. this revealed a possible 1 db reduction in threshold level to achieve pfa = 0.1 as compared to other parametric techniques studied here. this means that the rvnn based ed provides better sensitivity than yw, bg, and cv based approaches. also, we note that yw, bg, and cv have tendencies to overestimate the magnitude response of a signal which resulted in the observed better roc performance of yw, bg, and cv over the proposed technique in figures 15 and 16. nevertheless, higher snr estimation and consequently improved roc can be achieved for the rvnn based ed by including appropriate amplifiers after the square law device in figure 5. on the other hand, false alarms are more difficult to address and can not be solved by simple amplification as proposed because both noise and signals will be amplified accordingly, except if noise reduction techniques are used. consequently, reducing pfa could depend more on threshold selection or on the underlying nature of the detector. therefore, figure 17 emphasizes a critical advantage of the rvnn based ed over other parametric techniques especially in providing an inherent capacity to reduce the pfa level and prevent possible interference to primary user (pu) signals. finally, the rvnn based ed was compared with nonparametric techniques such as the simple periodogram (sp), welch periodogram (wp), and multitaper (mt). for narrowband sensing, figure 18 revealed that the rvnn based ed performed better than the nonparametric techniques compared here except for wp which performed slightly better. this was due to the high variance estimation obtained in the wp which made more noise samples to cross the threshold than in the rvnn based ed. in wideband sensing, figure 19 revealed that the rvnn based ed performed better than other nonparametric techniques. in this paper, a real valued neural network (rvnn) based energy detector (ed) has been proposed, simulated, and analyzed. the choice of the rvnn method for estimating the coefficients of an autoregressive (ar) system was based on its inherent ability to solve the challenge of line splitting and spectral shifting as reported in. thus, our developed rvnn based ed was examined using different simulation parameters for different sensing conditions. we observed that, for model order p = 50, the proposed detector provided a high detection performance in narrowband sensing and similar performance in wideband sensing for p = 20. furthermore, the detector was compared with other parametric based techniques like the yule - walker (yw), burg (bg), and covariance (cv) methods. a close performance was achieved between our proposed ed and others with less than 5% drop in roc performance observed with respect to yw, bg, and cv. however, a 10% reduction in false alarm was observed for the rvnn based ed over other parametric techniques. this result presents an important advantage in using the proposed technique for cr application because techniques with less false alarms are highly desirable. finally, it was compared with some nonparametric techniques like the simple periodogram (sp), welch periodogram (wp), and the multitaper (mt). our proposed detector provided a 10% detection performance gain over these techniques except in narrowband and fast sensing case where wp provided about 2% detection increase than the rvnn based ed. this was attributed to the high estimated noise variance level which consequently resulted in increased false alarm rates in wp. the study reported here indicates that the rvnn based ed provides a new option for spectrum sensing in cr. however, we note that attention was not given here to the degree of design complexity with respect to choice of higher model order. though it has been shown in that computational demand in terms of number of multiplications is less than the periodogram, likewise for the storage demand, no closed form model exists to describe complexity for higher model order. this might not necessarily affect detection or false alarm performance but might affect timing performance. however, this remains to be studied and provides an opportunity for future research investigations in the field of ar application in cr. | a real valued neural network (rvnn) based energy detector (ed) is proposed and analyzed for cognitive radio (cr) application. this was developed using a known two - layered rvnn model to estimate the model coefficients of an autoregressive (ar) system. by using appropriate modules and a well - designed detector, the power spectral density (psd) of the ar system transfer function was estimated and subsequent receiver operating characteristic (roc) curves of the detector generated and analyzed. a high detection performance with low false alarm rate was observed for varying signal to noise ratio (snr), sample number, and model order conditions. the proposed rvnn based ed was then compared to the simple periodogram (sp), welch periodogram (wp), multitaper (mt), yule - walker (yw), burg (bg), and covariance (cv) based ed techniques. the proposed detector showed better performance than the sp, wp, and mt while providing better false alarm performance than the yw, bg, and cv. data provided here support the effectiveness of the proposed rvnn based ed for cr application. |
multiple sclerosis (ms) is an autoimmune disease of the central nervous system that causes myelin destruction and axonal damage in the brain and spinal cord. it is the leading cause of nontraumatic neurological disability among young adults and is associated with a variety of debilitating symptoms, including fatigue. fatigue is the most common symptom experienced by persons with ms, affecting up to 90% of patients at some point in their disease course [13 ]. fatigue imposes significant socioeconomic consequences, including loss of work hours and employment, and is a prominent cause of diminished quality of life among individuals with ms. despite its prevalence in ms as well as other medical conditions, there is no unified definition for fatigue. consequently, there is potential for considerable overlap between fatigue and other subjective terms commonly used by ms patients to describe lack of energy or alertness, including sleepiness. however, many subjects in the general population who have sleep disorders such as obstructive sleep apnea report that problems with fatigue, tiredness, or lack of energy supersede problems with sleepiness and experience improvement in these symptoms when their apnea is treated. nonetheless, little is known about the most common symptoms experienced by ms patients with obstructive sleep apnea and other sleep disorders, or ways in which ms patients may differ from non - ms patients in how they describe symptoms that could be attributable to sleep disorders. research to clarify the relationship between sleep disorders, fatigue, and related symptoms in ms could help clinicians identify which ms patients are most likely to benefit from sleep evaluations and facilitate early treatment for common underlying sleep disorders. the purpose of this study was to assess the relationship between polysomnographic findings and the frequency of self - reported daytime symptoms (fatigue, tiredness, lack of energy, and sleepiness), among ms patients referred for clinical polysomnography (psg), compared to referred controls without ms. this retrospective data analysis was approved by the university of michigan institutional review board. demographic, clinical, and polysomnographic (psg) data were assembled from the university of michigan (u - m) sleep disorders center database and medical records for n = 48 patients, 18 years or older, who had an established diagnosis of ms based on the mcdonald diagnostic criteria and had completed a clinical overnight psg between march 1999 and june 2010. only patients who had completed the university of michigan sleepiness impact assessment (umsia) at the time of psg (n = 30) were considered eligible for the analyses. control subjects without ms (n = 30) who had also completed at least 2 out of 4 categorical responses of interest on the umsia were selected from more than 8,000 adult patients referred for diagnostic sleep studies. controls were matched to each ms patient in a 1 : 1 ratio for paired analyses, based on age (5 years), gender, and body mass index (2 kg / m). matched controls were also selected based on date of study, after or before january 1st, 2008, to control for minor changes in psg scoring criteria that took place following january 1st, 2008. the following variables were extracted from the sleep database : psg date, gender, age, body mass index (bmi), psg diagnosis, apnea - hypopnea index (ahi or rate of apneas and hypopneas per hour of sleep), percentage of sleep time spent in stage 1 sleep (n1%), percentage in stage 2 sleep (n2%), percentage in stage 3 (slow wave) sleep (n3%), percentage in rem sleep (rem%), arousal index (number of arousals per hour of sleep), number of sleep stage shifts, sleep latency, sleep efficiency (se, ratio of time spent asleep to the amount of time spent in bed), the periodic leg movement index (plmi), and epworth sleepiness scale (ess) scores. the epworth sleepiness scale is an eight - item questionnaire that asks the patient to rate, on a likert scale, the likelihood of dozing in a variety of sedentary situations. medical records were reviewed to confirm eligibility and extract additional data regarding ms - specific variables. individuals with concomitant diseases that could increase the risk of sleep apnea or influence fatigue level, including cancer, severe cardiopulmonary disease, pregnancy, major depressive disorder (within 6 months of psg), or neurologic diseases other than ms, were excluded. for ms subjects, additional variables recorded included ms subtype (relapsing - remitting versus progressive), disease duration at time of psg (years), use of disease modifying therapy (dmt, defined as glatiramer acetate or beta - interferon use at the time of psg), and estimates of disability (defined as an edss score of less than 6.0 (low disability) or greater than or equal to 6.0 (high disability)). developed in 1996 by one of the investigators (rc), the university of michigan sleepiness impact assessment (umsia) is a self - administered questionnaire that allows patients to rate, in a parallel manner, the frequency that sleepiness and associated symptoms affect their daily life using 5-point likert scales. for our study, categorical responses regarding the frequency of problematic sleepiness, fatigue, tiredness, and lack of energy (rated as never = 1, seldom = 2, occasionally full, laboratory - based polysomnography (psg) and scoring followed existing standards before 2008, and then slightly different, newly published standards from that point forward. the main relevant change concerned use of nasal pressure to identify hypopneas and rules employed to score them. our laboratory had already been using thoracic or abdominal excursion changes, in addition to thermocouple airflow changes (when any of these were followed by awakenings, arousals, or 4% oxygen desaturations) to identify hypopneas in a sensitive manner before the aasm 2007 standards were published. the apnea - hypopnea index (ahi) was calculated as the number of obstructive apneas, central apneas, or hypopneas per hour of sleep. the presence of obstructive sleep apnea was defined by an apnea - hypopnea index of at least five episodes per hour of sleep. umsia responses for each symptom (fatigue, tiredness, lack of energy, and sleepiness) were dichotomized into 2 categories : frequent (for responses of often or almost always) and infrequent (for responses of never, seldom, or occasionally) and compared between ms patients and controls using chi - square tests. two - sample t tests (for normal continuous data), chi - square tests (for dichotomized data), and wilcoxon rank - sum tests (for nonnormal continuous data) were used to compare baseline characteristics among ms patients and controls. spearman correlation tests were used to examine associations between categorical umsia responses (reported frequencies of problematic fatigue, tiredness, lack of energy, and sleepiness), epworth scores, demographic information, and psg parameters of interest (ahi, sleep efficiency, n1%, n2%, n3%, rem%, sleep latency, arousal index, number of sleep stage shifts, and plmi) separately within ms and control groups. chi - square tests, t tests, and multiple linear regression models were also used to compare dichotomized umsia responses and psg variables between ms subgroups of interest (relapsing - remitting versus progressive ms, use of dmt and disability status, as defined in section 2.1.3). baseline characteristics for n = 30 ms patients and n = 30 matched controls are shown in table 1. no significant differences between ms subjects and matched controls emerged for matched variables (bmi, gender, or age) or the other comparators listed, except for number of sleep stage shifts (p = 0.017). for ms patients, seventy percent of ms subjects and seventy percent of matched controls were female, consistent with the gender distributions seen in ms in the united states. for those with available data, 21 (70%) ms subjects had relapsing - remitting disease, and nine (30%) had progressive disease. twenty (67%) of the ms patients were on disease modifying therapy (dmt), and ten (33%) were not. eighty percent of ms patients and 63% of controls met diagnostic criteria for obstructive sleep apnea (see section 2.3). response rates to categorical umsia items and distribution of symptom frequencies for ms patients and matched controls are shown in figure 1. when responses were dichotomized, significantly more ms patients than controls reported fatigue, tiredness, and lack of energy to occur often or almost always (90% versus 18%, 77% versus 0%, and 90% versus 7% ; chi - square p < 0.0001 for each). conversely, frequent sleepiness as per the umsia was reported similarly by both groups (53% versus 65% ; p = 0.3409). there were strong correlations between fatigue, tiredness, and lack of energy in both groups (table 2). among ms patients, tiredness correlated with sleepiness, (rho = 0.50 ; p = 0.005), and a trend emerged toward a significant correlation between fatigue and sleepiness (rho = 0.33 ; p = 0.076). among controls, fatigue correlated with sleepiness (rho = 0.45 ; p = 0.034). decreased nocturnal sleep efficiency correlated with fatigue, tiredness, and lack of energy in ms patients (rho = 0.55, p = 0.002 ; rho = 0.40, p = 0.029 ; and rho = 0.36, p = 0.048, resp.), but not with sleepiness in ms patients or with any symptom among controls. no significant relationship emerged between mean epworth scores and umsia reports of sleepiness among ms patients or controls, although trends suggested a positive correlation between epworth score and fatigue, and between ess score and tiredness in the ms group (table 2). a trend toward a significant increase in ahi among ms patients compared to controls emerged in regression models, after adjustment for age and bmi (p = 0.0647, not shown). no significant difference was noted in mean n1%, n3%, rem%, arousal index, sleep latency, or sleep efficiency among ms patients compared to controls, though a trend toward decreased n2% emerged in the ms group. there was also a nonsignificant increase in mean plmi among ms patients compared to controls. dichotomized frequencies of fatigue, tiredness, lack of energy, and sleepiness (often or always versus less commonly) did not significantly vary by ms subtype or disability status. sleep efficiency and rem% were significantly decreased in progressive ms subtypes compared to relapsing - remitting ms, after adjusting for ahi and age in regression models (p = 0.0442 and 0.0373, resp.) and in subjects with high disability (edss 6.0) compared to those with low (edss < 6.0) disability status (p = 0.0048 and 0.0061, resp.). the association between progressive ms and apnea severity (ahi) approached significance in regression models, after adjustment for age and bmi (p = 0.0596). the presence or absence of disease modifying therapy (dmt) also did not influence the frequency of any symptom. epworth scores, sleep efficiency, sleep stages, and the plmi were not influenced by dmt use, but use of dmt was associated with decreased ahi (t - test p = 0.0419). details regarding the relationships between dmt, disease subtype, and ahi in all n = 48 ms subjects with available psg data (regardless of umsia completion), after adjustment for additional variables known to influence ahi, have been described previously (see section 2.1). this study of symptoms and psg findings among ms patients and non - ms controls shows that ms patients with suspected sleep problems are more likely than controls to emphasize problematic fatigue, tiredness, and lack of energy, as opposed to sleepiness. furthermore, decreased sleep efficiency correlated with increased symptoms of tiredness, fatigue, and lack of energy in patients with ms. our findings are particularly surprising given that sleep efficiency itself, objectively assessed on polysomnography, was essentially identical between ms patients and controls. these data raise important considerations for understanding fatigue and related symptoms among persons with ms. in particular, our results suggest that patients with ms may exhibit an increased sensitivity to the downstream effects of diminished sleep efficiency compared to patients without ms. our findings raise the possibility that measures to increase sleep efficiency in ms could improve daytime fatigue, regardless of whether concomitant sleep disorders are present. while fatigue has long been recognized for its impact on the health and quality of life of persons with ms, separation of treatable from untreatable causes can be challenging, given the wide range of potential etiologies. in addition to contributions from cytokine dysregulation, ms - related fatigue may also be influenced by cns lesion burden, medication effects, and other conditions concomitant with ms, including depression, pain, and sleep disturbances. while validated instruments to quantify fatigue or assess its impact on the function of persons with ms are commonly employed in both clinical and research settings, most of these instruments do not include items to define fatigue or describe it in the context of similar symptoms such as sleepiness. subsequently, subjects are left to quantify their fatigue on the basis of their own subjective interpretation, which may vary by education, disease - specific biases, or cultural backgrounds. this ambiguity increases the potential for overlap among terms used to describe symptoms experienced by ms patients with concomitant sleep disorders. consequently, ms patients suffering from sleep disorders who describe symptoms other than sleepiness may escape formal sleep evaluations, as fatigue and related symptoms are expected consequences of ms itself. growing evidence suggests that sleep disturbances may be closely linked to fatigue in persons with ms. a recent prospective psg study in ms subjects and controls suggests that severe obstructive sleep apnea (defined as an ahi 30) is independently associated with fatigue in ms, as defined by the fatigue severity scale. the nine - response fatigue severity scale (fss) assesses the impact of fatigue on multiple outcomes, with a physical focus, using a 7-point likert scale. a separate group found that fatigue, quantified using a french version of the fatigue impact scale (fis), correlated strongly with sleep disturbances as measured by the pittsburgh sleep quality index among individuals with ms. a similar association was also demonstrated with the fss as the primary fatigue outcome measure. using home psg, veauthier and colleagues reported an increase in the prevalence of clinically relevant sleep disorders, including sleep disordered breathing, in fatigued ms patients compared to nonfatigued patients as defined by the modified fatigue impact scale. whereas the above studies highlight fatigue as a potential consequence of sleep disturbances in ms, these and other studies of relationships between fatigue and sleep disorders in ms have relied on instruments that do not allow a direct comparison between sleepiness, fatigue, and related symptoms in a systematic manner. our study is the first to address each of these symptoms separately using parallel phrasing, while relating them to objective psg measures. while the umsia questionnaire has yet to be validated in patients with ms, our data suggest that ms patients with sleep disorders may share a propensity for terms other than sleepiness to describe their symptoms. furthermore, use of these terms may signal decreased sleep efficiency in this population. one possible explanation for the strong correlation between fatigue level and sleep efficiency in ms may relate to what is known about the role of cytokine dysregulation in fatigue. proinflammatory cytokines, produced by autoimmune effector cells, are proposed to contribute to fatigue in ms [20, 21 ]. it is noteworthy that two of these cytokines, il-6 and tnf - alpha, are also elevated in the serum of individuals with obstructive sleep apnea and sleep deprivation and correlate with fatigue and sleepiness in general population studies [2225 ]. while conclusions about a causal relationship between cytokine dysregulation and reported symptoms can not be drawn from our retrospective cross - sectional study, our findings highlight a common immunological pathway between ms and sleep disturbances, which may synergistically amplify fatigue when sleep efficiency is reduced. while the lack of correlation between sleepiness and sleep efficiency in both groups may appear counterintuitive, it is consistent with previous reports. despite the traditional clinician emphasis on sleepiness, many subjects in the general population who have sleep disorders prefer to use terms other than sleepiness to describe their daytime symptoms. furthermore, previous data from our group have shown little or no association between measured sleep efficiency and either subjective or objective daytime sleepiness. data on potential variables that could influence fatigue and related symptoms in the ms group were not available in a consistent format that could be useful in the analyses. these include detailed information on depressive symptoms, disease coping strategies, psychosocial factors, and medications (such as antispasmodics, narcotics, or sleep aids) that are often used by patients with ms, as well as mri lesion burden and respiratory function, which could influence apnea scores. subjects whose records indicated severe depression in medical charts were excluded from analyses, but milder depressive symptoms that may not warrant a diagnosis of major depression could not be reliably studied from this retrospective chart review. finally, the number of completed umsia responses for fatigue frequency was by chance slightly lower in the control group, which could theoretically explain the differences in correlation between fatigue level and sleep efficiency among ms patients versus controls. this is thought to be less likely, however, given the strong differences in correlation between tiredness / lack of energy and sleep efficiency among ms patients versus controls, where the number of completed umsia responses was similar for both groups. despite these limitations, our data suggest that ms patients referred to a sleep laboratory are more likely than referred controls to complain of fatigue and related symptoms other than sleepiness, a more widely recognized consequence of sleep disturbances. our study highlights the importance of considering physiologic sleep disturbance in ms patients who complain of fatigue, tiredness, lack of energy, or sleepiness. our data suggest that ms patients, in comparison to matched controls also referred for polysomnography, report more fatigue, tiredness, and lack energy, but not sleepiness. fatigue and significant symptoms other than sleepiness may arise from ms itself or in relation to sleep disturbance as reflected by reduced sleep efficiency in particular, which could offer opportunities for effective intervention. | objectives. to assess the relationship between nocturnal polysomnographic (psg) findings and a group of key self - reported symptoms fatigue, tiredness, lack of energy, and sleepiness among sleep - laboratory referred patients with and without multiple sclerosis (ms). methods. psg and questionnaire data from n = 30 ms patients and n = 30 matched controls were analyzed retrospectively. associations between symptoms of fatigue, tiredness, lack of energy, sleepiness, and psg variables of interest were examined among ms patients and controls. results. more ms patients than controls reported fatigue, tiredness, and lack of energy to occur often or almost always (chi - square p < 0.0001 for each), but sleepiness was reported similarly by both groups (p = 0.3409). among ms patients, tiredness correlated with sleepiness (spearman correlation p = 0.005), and a trend emerged toward correlation between fatigue and sleepiness (spearman correlation p = 0.076). decreased sleep efficiency on psgs correlated with fatigue, tiredness, and lack of energy in ms patients (spearman correlation p = 0.002, 0.029, and 0.048, resp.), but not sleepiness or any symptom among controls. conclusion. in comparison to controls, ms patients report more fatigue, tiredness, and lack energy, but not sleepiness. fatigue and related symptoms may arise from ms itself or in relation to reduced sleep efficiency. |
intensity - modulated radiation therapy (imrt) is one of the treatment choices for localized prostate tumor irradiations. imrt is often delivered with low - energy photons, viz., below 10 mv. these deep - seated prostate tumors were earlier treated with 3d - conformal radiation therapy (3d - crt) using high - energy photons (15 to 25 mv). utilization of low - energy photons in imrt is known to have certain advantages, like high conformity, negligible neutron contamination, etc. but, there are indications provided by certain investigations, that these low - energy photons may deliver high doses in regions far from the) target. also, there are some indications in literature of better target coverage and very low risk of induction of secondary malignancies due to neutron contamination in high - energy beams.[14 ] hence, a complete analysis of the influence of the photon energy on the quality of the imrt plans is necessary. for this study, a cohort of 8 prostate patients was selected, and 6-mv and 15-mv step and shoot imrt plans were compared by calculating relative efficacies of several physical indices, including primitive h - index, modified h - index, primitive hi - index, modified hi - index, s - index, conformity index and mean dose volume histogram (dvh). two mean dvh 's were generated and were considered as completely representative for 6-mv and 15-mv plans, respectively. at our center, the radiation therapy is delivered using elekta precise high - energy linear accelerator. inverse imrt planning is done using segmental inverse optimizer of elekta preciseplan planning system, version 2.03. sample imrt plans were created for all 8 prostate patients, and the dose prescription was 80 gy in 40 fractions with 6-mv photons. five co - planar fields at gantry angles of 255, 315, 45, 105, 180 degrees were used. gross tumor volume (gtv), planning target volume (ptv), rectum, bladder and femoral heads were contoured. the dose constraints used for these plans were in accordance with radiation therapy oncology group (rtog) protocol and are shown in table 1a. thus, the advantage is that, segments too can be kept constant between 6-mv and 15-mv plans. in order to study the effect of energy on the quality of the plans, competing plans were also carried out using 15-mv beams for all patients. to ensure that the similarity or difference was due to energy alone, all other parameters like beam angles, number of beams, number of segments, shapes of the segments, dose constraints, etc. all the patients image sets were chosen such that, there was not much variation in their anatomy. all the patients antero - posterior (ap) and lateral dimensions were very close. the ap diameters were between 22 and 23 cm, and the lateral diameters were between 33 and 34 cm. ptv was drawn by expanding gtv by 1 cm in all directions except in the posterior direction, where 0.6 cm was used. rect_imrt (a portion of rectum) was derived from rectum such that it extended only 3 mm beyond ptv in superior and inferior directions. for the coplanar beams used, this definition of rect_imrt was chosen, as it is more sensitive to the optimization and less sensitive to the anatomy. the following parameters were evaluated for comparison : mean dvh, primitive h - index, modified h - index, primitive hi - index, modified hi - index, s - index and conformity index. dose volume histograms (dvh - s) are two - dimensional representations of complex 3d dose distributions inside the patient. evaluation of homogeneity of dose inside the ptv using dvh - s is of paramount importance, as the cold spots can negatively affect the tumor control probability (tcp). all the 6-mv plans were compared with corresponding 15-mv plans on a one - to - one basis. to facilitate dvh comparison, all the dvh - s were normalized such that 95% volume of the ptv was covered by the prescribed dose of 80 gy. in addition, a mean dvh for each contoured organ (ptv, rect_imrt, bladder, rt. mean dvh can be found by averaging the volumes in each dose bin and re - plotting the data as a function of dose. bioplan software developed by nahum., was used to generate the mean dvh - s. but, many of them have their limitations and fail to predict the homogeneity correctly. some of them used in this study are defined below. the conventionally used homogeneity index (h - index) is defined as the ratio of the maximum dose (dmax) in the ptv to the prescription dose (dp), with a value closer to 1 indicating better homogeneity. the h - index can be made more robust by using d5 instead of dmax (modified h - index). as an improvement to h - index and modified h - index, another index called hi - index has been defined as where d2 and d98 represent doses to 2% and 98% volume of ptv, respectively. this hi - index can be further improved by using d5 and d95 instead of d2 and d98 (modified hi - index). this improvisation can be realized, as these new substitutions clip the usually small - volume high and low dose extremities in the cumulative dvh of the ptv and get only the steeper portion of the curve into the calculation. hence, other parameters were also used in an attempt to find out the appropriate energy. a new index called sigma index (s - index), which gives better representation of homogeneity, was introduced by them. this index uses the differential dvh, unlike other indices, which use cumulative dvh. s - index is a measure of the standard deviation of the doses (entire curve) about the mean dose. dmean is the mean dose of the target (ptv in this study) curve. conformity index is defined as the ratio of the volume of the body receiving the prescription dose to the volume of the ptv receiving the same dose. as stated above, all physical indices were evaluated for the two mean dvh - s of 6 mv and 15 mv, respectively. in figure 1, 6-mv and 15-mv figure 1a shows the mean dvh - s of ptv for both 6 mv and 15 mv. as mentioned earlier, the ptv dvh - s were normalized, to facilitate comparison, thus resulting in very similar curves for both 6 mv and 15 mv. in the mean dvh - s of rectum, shown in figure 1b, for doses greater than 80% (approximately > 64 gy), 2% to 4% more volume received higher dose in 6 mv in comparison to 15 mv. there was no significant difference between 6-mv and 15-mv for bladder, as clearly evident from figure 1c. hence, the graphs were magnified and the values were measured. a : mean dose volume histogram of planning target volume b : mean dose volume histogram of rectum c : mean dose volume histogram of bladder d : mean dose volume histogram of body e : mean dose volume histogram of left femoral head f : mean dose volume histogram of right femoral head in figure 1d, the mean dvh - s of the body are shown. for 20 % to 60 % of doses, 1% to 1.5% more volume was found to receive high dose with 6-mv beam than with 15-mv beam. for 15% to 20% of doses, approximately 0.5% more volume was found to receive high dose with 15-mv beam than with 6-mv beam. since, the magnitude of the dose delivered to ptv was very high in this study, the slight differences at very low doses (less than 10%) are not reflected in this figure. but, from the data used to plot this graph, it is clear that greater volume received high dose with the 6-mv beam than with the 15-mv beam. as evident from figures 1e and 1f, the mean dvh - s of femoral heads favor 6 mv in comparison with 15 mv. for 42 % to 78% of doses, 4% to 5% more volume of femoral heads received high dose with the 15-mv beam. the different homogeneity indices for all the prostate patients and for both 6 and 15-mv energies are summarized in the table 1, table 2 and table 3. the relative efficacy values for h - index, modified h - index, hi - index, modified hi - index and sigma index are 1.015, 1.006, 1.031, 1.060 and 1.089, respectively. h and mod h - indices for eight patient intensity - modulated radiation therapy plans with 6 mv and 15 mv x - rays hi and mod hi - indices for eight patient intensity - modulated radiation therapy plans with 6 mv and 15 mv x - rays sigma indices for eight patient intensitymodulated radiation therapy plans with 6 mv and 15 mv x - rays conformity indices for eight patient intensity - modulated radiation therapy plans with 6 mv and 15 mv x - rays in figure 2, the cumulative dvh - s of 6-mv plan for patient 1 and patient 3 are shown in red and green solid colors, respectively. it is obvious that the dose homogeneity index of patient 1 is better than that of patient 3. but the h - indices are almost similar (1.128 and 1.126), as evident from table 1. the s - indices are 2.233 and 5.14 for patient 1 and patient 3, as evident from table 3, predicting good accuracy. the lower the value of the s - index, the better is the homogeneity. cumulative dose volume histogram of patient 1(red) and patient 3 (green) - 6 mv miften., have demonstrated the use of target conformity index (tci) and normal tissue - sparing index (ntsi) to assist in the process of judging the merit of a clinical treatment plan. but, in this work, the widely accepted conformity index was used to evaluate the conformity of the treatment plans. the mean conformity index was 1.172 and 1.194 for 6 mv and 15 mv, respectively. these small differences indicate that the plans are nearly identical in their conformity of dose to the target. in figure 1, 6-mv and 15-mv curves are shown in red and green color, respectively. figure 1a shows the mean dvh - s of ptv for both 6 mv and 15 mv. as mentioned earlier, the ptv dvh - s were normalized, to facilitate comparison, thus resulting in very similar curves for both 6 mv and 15 mv. in the mean dvh - s of rectum, shown in figure 1b, for doses greater than 80% (approximately > 64 gy), 2% to 4% more volume received higher dose in 6 mv in comparison to 15 mv. there was no significant difference between 6-mv and 15-mv for bladder, as clearly evident from figure 1c. a : mean dose volume histogram of planning target volume b : mean dose volume histogram of rectum c : mean dose volume histogram of bladder d : mean dose volume histogram of body e : mean dose volume histogram of left femoral head f : mean dose volume histogram of right femoral head in figure 1d, the mean dvh - s of the body are shown. for 20 % to 60 % of doses, 1% to 1.5% more volume was found to receive high dose with 6-mv beam than with 15-mv beam. for 15% to 20% of doses, approximately 0.5% more volume was found to receive high dose with 15-mv beam than with 6-mv beam. since, the magnitude of the dose delivered to ptv was very high in this study, the slight differences at very low doses (less than 10%) are not reflected in this figure. but, from the data used to plot this graph, it is clear that greater volume received high dose with the 6-mv beam than with the 15-mv beam. as evident from figures 1e and 1f, the mean dvh - s of femoral heads favor 6 mv in comparison with 15 mv. for 42 % to 78% of doses, 4% to 5% more volume of femoral heads received high dose with the 15-mv beam. the different homogeneity indices for all the prostate patients and for both 6 and 15-mv energies are summarized in the table 1, table 2 and table 3. the relative efficacy values for h - index, modified h - index, hi - index, modified hi - index and sigma index are 1.015, 1.006, 1.031, 1.060 and 1.089, respectively. h and mod h - indices for eight patient intensity - modulated radiation therapy plans with 6 mv and 15 mv x - rays hi and mod hi - indices for eight patient intensity - modulated radiation therapy plans with 6 mv and 15 mv x - rays sigma indices for eight patient intensitymodulated radiation therapy plans with 6 mv and 15 mv x - rays conformity indices for eight patient intensity - modulated radiation therapy plans with 6 mv and 15 mv x - rays in figure 2, the cumulative dvh - s of 6-mv plan for patient 1 and patient 3 are shown in red and green solid colors, respectively. it is obvious that the dose homogeneity index of patient 1 is better than that of patient 3. but the h - indices are almost similar (1.128 and 1.126), as evident from table 1. the s - indices are 2.233 and 5.14 for patient 1 and patient 3, as evident from table 3, predicting good accuracy. the lower the value of the s - index, the better is the homogeneity. cumulative dose volume histogram of patient 1(red) and patient 3 (green) - 6 mv miften., have demonstrated the use of target conformity index (tci) and normal tissue - sparing index (ntsi) to assist in the process of judging the merit of a clinical treatment plan. but, in this work, the widely accepted conformity index was used to evaluate the conformity of the treatment plans. the mean conformity index was 1.172 and 1.194 for 6 mv and 15 mv, respectively. these small differences indicate that the plans are nearly identical in their conformity of dose to the target. the mean dvh - s of ptv for both 6 mv and 15 mv show very little difference. greater than 64 gy, 2 % to 4 % more rectum volume received a higher dose with 6-mv beam because of high entry dose. femoral heads consistently received more dose with 15-mv beam for all patients as they received a higher depth dose. but, it is unlikely that such small magnitudes of doses can produce any complications. except for minor differences, this work also assessed widely used homogeneity indices like h, hi, modified h and modified hi., was successfully used to predict correct homogeneity, as it takes the entire dvh curve into consideration. s - index, was then, successfully used to evaluate the relative homogeneity between 6-mv and 15-mv groups. thus, the s - index value of 1.089, as seen in table 3, should be a good estimate of relative homogeneity between 6 mv and 15 mv energies. in addition, h - index, modified h - index, hi - index and modified hi - index were also used to evaluate the relative efficacy. evaluation of all these parameters clearly depicts less homogeneity for 6 mv than 15 mv group. the sample size was not a bad number, and h - index was also consistent with other parameters though they failed occasionally. a relative conformity index value of 0.976 between 6-mv and 15-mv energies shows that 6-mv group is more conformal than 15-mv group. in general, comparison of all above parameters showed that there was little difference between 6-mv and 15-mv groups. in practice, much tighter constraints are maintained in low - energy treatment plans such that the complication probability is kept within 5%. these tighter constraints might increase dose to other regions and other critical organs but are unlikely to increase their complication probabilities. bladder is a relatively large organ when compared to rectum and also exhibits a higher partial volume effect. usually, only the posterior part of bladder receives a high dose, and a small increase in dose is unlikely to increase its complication probability significantly. similarly, femoral heads are at a clinically insignificant distance from ptv (prostate and seminal vesicles) and usually experience small doses. hence the slight advantages of 15 mv in providing benefits of better normal - tissue sparing and better coverage can not be considered to outweigh its well - known risk of non - negligible neutron production. | the goal of the present study was to study the effects of low- and high - energy intensity - modulated photon beams on the planning of target volume and the critical organs in cases of localized prostate tumors in a cohort of 8 patients. to ensure that the difference between the plans is due to energy alone, all other parameters were kept constant. a mean dose volume histogram (dvh) for each value of energy and for each contoured structure was created and was considered as completely representative for all patients. to facilitate comparison between 6-mv and 15-mv beams, the dvh - s were normalized. the different parameters that were compared for 6-mv and 15-mv beams included mean dvh, different homogeneity indices, conformity index, etc. analysis of several indices depicts more homogeneous dose for 15-mv beam and more conformity for 6-mv beam. comparison of all these parameters showed that there was little difference between the 6-mv and 15-mv beams. for rectum, 2 to 4 % more volume received high dose with the 6-mv beam in comparison with the 15-mv beam, which was not clinically significant, since in practice much tighter constraints are maintained, such that normal tissue complication probability (ntcp) is kept within 5 %. such tighter constraints might increase the dose to other regions and other critical organs but are unlikely to increase their complication probabilities. hence the slight advantages of 15-mv beam in providing benefits of better normal - tissue sparing and better coverage can not be considered to outweigh its well - known risk of non - negligible neutron production. |
alzheimer s disease (ad) is a common neurodegenerative disorder, which accounts for 60%80% of all cases of dementia. as the population ages, the prevalence of ad will rise sharply in the next few decades.1 the gradual onset and slow progression of ad pose a challenge for early differentiation from other causes of cognitive decline, including healthy aging and mild cognitive impairment (mci). in recent years reliable and sensitive clinical biomarkers for early diagnosis of ad are particularly critical for ad identification. currently, some clinical biomarkers, such as pittsburgh compound b, amyloid beta 42, phosphorylated tau, and cerebrospinal fluid inflammatory factors,2,3 have not yet been widely used in large - scale clinical applications due to cost or lack of uniform clinical guidelines. hence, an inexpensive, simple, and practical diagnostic strategy for ad is urgently needed. olfactory dysfunction in ad has been reported as early as 1974.4 after 40 years of research, olfactory dysfunction in ad was better understood. some studies confirmed that olfactory dysfunction was possibly one of the earliest clinical symptoms of ad.5,6 in addition, typical ad pathology first involves the entorhinal cortex. the disease then gradually spreads to the whole brain and eventually affects the entire cerebral cortex.7 combining olfactory function tests with conventional diagnostic methods could help improve the sensitivity and specificity of ad diagnosis, thereby facilitating early recognition and diagnosis of ad.8 this review article summarizes and evaluates the research progress of olfactory dysfunction in ad to explore further its possible research directions in the future. recently, olfactory dysfunction has attracted the renewed interest of scientists, because olfactory dysfunction has the potential to be an early marker of neurodegenerative conditions, such as ad, parkinson s disease (pd), schizophrenia, and multiple sclerosis.9 but our understanding of olfactory dysfunction is still very limited. in addition, our knowledge of the prevalence of olfactory dysfunction in the population of normally aging individuals and in related diseases is very poor. doty assessed the sense of smell in 1,955 individuals aged from 5 years to 99 years using smell identification test and found that half of the population with ages ranging 6580 years had significant olfactory dysfunction. the prevalence of olfactory dysfunction at 80 years or older was > 75%.10 murphy conducted a cross - sectional population - based survey with 2,491 adults aged from 53 years to 97 years and found that the average prevalence of olfactory dysfunction of this population was 24.5%. patients with ages ranging 8097 years had a prevalence of olfactory dysfunction of 62.5%.11 smoking, stroke, epilepsy, nasal congestion, and upper respiratory tract infection were associated with an increased prevalence of olfactory dysfunction. in healthy adults, aging was the most relevant factor for a decline in the sense of smell and it was more significant than smoking.12 these data have been confirmed in cross - sectional and cohort studies.13 in general, age - related olfactory dysfunction was more severe in male than in female patients, although there were individual differences. this sex difference may be related to differences in the number of human olfactory bulb cells in individuals. a recent study confirmed sex differences in the total number of olfactory bulb cells in humans, indicating that females had 40%50% more olfactory bulb cells than males, which might affect olfactory function in different sexes.14 age - related olfactory dysfunction may be caused by age - related ossification and closure of the foramina of the cribriform plate, as well as accumulation of different types of olfactory receptor cell damage due to age - related brain degeneration throughout one s lifetime.12,13 olfactory dysfunction is an early symptom of dementia and has a relatively high prevalence in various types of dementia, reaching up to 100% in ad, 90% in parkinson s disease dementia, 96% in frontotemporal dementia (ftld), and 15% in vascular dementia.1517 olfactory dysfunction is often unnoticed. unlike auditory and visual changes therefore, clinicians and caregivers should be particularly alert to potential olfactory dysfunction in the elderly patients for early detection, diagnosis, and treatment of dementia. although different test methods for olfactory dysfunction and different demographic and sociological data result in heterogeneity in the epidemiology of olfactory dysfunction, the high prevalence of olfactory dysfunction among patients with dementia is an indisputable fact. data from existing studies are primarily from developed countries, with small survey sampling sizes and study designs mainly based on cross - sectional surveys but lacking incidence and cohort studies. epidemiological surveys of olfactory dysfunction in races and populations of developing countries have been rarely reported, primarily due to insufficiencies in health care coverage, awareness, and degree of attention to olfactory dysfunction in these countries. current research suggests that olfactory dysfunction in ad is associated with pathological changes of tau protein in the olfactory bulb and olfactory projection area.18,19 wilson performed olfactory function tests in 166 participants at baseline and performed brain autopsy in 77 ad patients who subsequently died, ad pathology and lewy bodies were quantified in multiple brain regions, including portions of the central olfactory system and found that the density of neurofibrillary tangles was the main pathological factor that affected olfactory function, especially in the entorhinal cortex, hippocampus ca1 region, and subiculum. no significant association was found between neurofibrillary tangles, senile plaque deposition, and olfactory function in other brain regions.20 bahar - fuchs conducted olfactory function tests and pittsburgh compound b - positron emission tomography (pet) on 19 healthy volunteers, 24 amnestic mci patients, and 20 ad patients and found that ad - related olfactory dysfunction was not directly related to amyloid beta burden. these studies also confirmed that ad - related olfactory dysfunction was induced by pathological changes in tau protein. pathological examination provides the most direct and powerful evidence of pathological changes within the entorhinal cortex for early stage ad. neurofibrillary tangles and neuritic plaques in stages i ii were mainly distributed in and throughout the transentorhinal cortex.7 olfactory system - related brain tissues in ad patients exhibited significant histopathological changes. christen - zaech analyzed autopsy information of 110 cases and found that the number of cases with olfactory impairment (degenerative changes such as senile plaques, neurofibrillary tangles, and curly fibers) was very high, > 84% of the cases with cortical ad - type lesions. degenerative olfactory changes were present in all 19 definite ad cases and only in two of the 19 controls. therefore, christen - zaech suggested that the olfactory bulb and olfactory tract were sites with some of the earliest pathological changes in ad patients. the olfactory nervous system has a variety of neurotransmitters, and the exact mechanisms through which neurotransmitters are involved in olfactory transduction remain unclear. under normal circumstances, the olfactory system is rich in acetylcholine, glutamate, -aminobutyric acid, and other neurotransmitters. a deficit in these neurotransmitters, especially acetylcholine, a previous study confirmed reduction of acetylcholine in ad patients, which might induce olfactory dysfunction.23 olfactory dysfunction in patients with idiopathic parkinson s disease could not be reversed or improved by treatment with dopaminergic agents.24 treatment efficacy of cholinesterase inhibitors in ad - related olfactory dysfunction has not been determined. velayudhan conducted an unblinded and uncontrolled study and demonstrated that the cholinesterase inhibitor, donepezil, could greatly improve olfactory function of ad patients. this result suggested that functional changes in olfactory recognition could be used to predict therapeutic effects in ad patients.25 hence, olfactory recognition may be used as an effective indicator in clinical tests to evaluate the treatment efficacy of ad therapy. nevertheless, the study of velayudhan was an unblinded and uncontrolled trial, and their findings only had preliminary significance. olfactory dysfunction in ad mainly presents as an impairment in olfactory recognition, which occurs during the early stage of the disease and worsens with the progression of ad.5 serby used the university of pennsylvania smell identification test (upsit) to conduct olfactory function tests in 55 ad patients. the study indicated that the early stages of ad showed impairment in olfactory recognition, and an increased olfactory threshold was only present during the late stage of the disease. the functional score of olfactory recognition was associated with the score of the mini - mental state examination, but olfactory threshold was not associated with mini - mental state examination scores.5 previous findings have suggested that changes in olfactory threshold did not occur in the early stage of ad.26,27 however, another study indicated that changes in olfactory threshold occurred in patients with early stage ad and even mci.28 inconsistent findings in the different studies might be due to different methods and/or stimulants used in olfactory threshold detection. reliability of some olfactory threshold detection methods was low, which could be due to small sample size. hence, further confirmation using a unified and effective test in a large - scale clinical trial with strictly selected samples will be necessary to study the relationship between changes in olfactory threshold and ad. rahayel conducted a meta - analysis on olfactory dysfunction in ad and pd (total inclusion of 81 studies) and showed that impairments of olfactory recognition and recognition tasks in ad and pd patients were more severe than the impairment of olfactory detection threshold. these results were more severe in ad patients than in pd patients, suggesting that olfactory recognition and olfactory identification are more likely to be impaired in ad. in addition, deficits in olfactory detection threshold of pd patients were more severe than those in ad patients, indicating that olfactory impairment in pd was primarily poor olfactory perception, whereas olfactory impairment in ad mostly involved advanced olfactory cognitive tasks. in summary, we believe that olfactory recognition and recognition tasks are among the most interesting research topics, which should be included in subclinical detection of ad. olfactory dysfunction occurs not only in ad but also in a variety of neurodegenerative diseases. the prevalence and severity of olfactory dysfunction in different neurodegenerative diseases vary drastically. olfactory recognition tests in ad, semantic dementia, ftld, and corticobasal degeneration showed that olfactory recognition was severely impaired in semantic dementia and ad patients, but only mildly impaired in ftld and corticobasal degeneration patients.30 severe olfactory dysfunction was found in patients with ad, pd, and the guam type of parkinson s disease dementia (with an upsit score < 20), while olfactory dysfunction in patients with huntington s disease, progressive supranuclear palsy, and amyotrophic lateral sclerosis was not severe.12 these data indicated that differential diagnosis of these neurodegenerative diseases using olfactory function tests helped in clinical practice. many neuroimaging studies have measured ad neuropathological changes in the regional processing center of the medial temporal lobe and other brain regions associated with ad. loss of left hippocampal volume was highly associated with the performance of oral and odor recognition tasks in ad patients.31 hippocampal volume of patients with olfactory recognition impairment associated with amnestic mci / ad was smaller than in normal healthy controls.32 using functional magnetic resonance imaging (fmri), wang showed that the blood oxygenation level - dependent signal in the primary olfactory cortex was weaker in patients with early stage ad than in healthy controls. furthermore, the intensity of the blood oxygen level dependent (bold) signal and the area of the brain in which this signal was, increased as the concentrations of the tested odorants increased in these ad patients, while no such change was found in the healthy controls. these findings confirmed that olfactory fmri was sensitive to ad - related degeneration in olfactory function and recognition in the early stage of the disease.33 murphy conducted another fmri study and showed that in the elderly patients, especially those with ad, the connection between the orbitofrontal cortex and the medial temporal lobe was interrupted, resulting in decreased activation in the entire cortex, particularly in the medial temporal lobe. functional network analysis has shown that interruptions in the connections between the orbitofrontal cortex and the medial temporal lobe might reflect age - related changes in the large - scale olfactory processing network.34 frster conducted resting - state fluorodeoxyglucose - pet to analyze different olfactory regions and assess olfactory performance in patients with early stage ad. the results showed that olfactory recognition was associated with peak values of normal fluorodeoxyglucose in the right superior parietal lobule, gyrus occipitotemporalis medialis, inferior frontal gyrus, and precuneus of patients with early stage ad, whereas odor discrimination scores correlated with a single cluster in the left postcentral cortex and odor threshold scores correlated with clusters in the right thalamus and cerebellum, supporting the theory of a parallel organized olfactory system.35 the study of imaging of olfactory dysfunction in ad still remains at the preclinical stage ; the existing research focuses on using fmri / pet to investigate the relation between the olfactory dysfunction and the olfactory cortex or neuronetwork. due to the complexity of the olfactory system, there is no specific clinical diagnostic value, and the clinical application of imaging in olfactory function still has a long way to go. a previous study showed that 18%30% of mci cases were at a risk of converting to ad 3 years after diagnosis.36 to improve the accuracy of ad diagnosis and to identify high - risk populations, improved prediction of conversion from mci to ad is needed. olfactory function tests have been used as markers to predict the risk of mci converting to ad. wilson conducted a cohort study in 471 healthy elderly individuals or mci patients and found a close relationship between the level of pathological changes in the cerebral cortex and the degree of risk in developing prodromal ad. this relationship still existed given the presence of other common behaviors and genetic markers of ad.37 other recent research monitored the predictive value of olfactory recognition and function tests in ad cases that had converted from mci. results of a 2-year follow - up interview showed that 47% of mci patients with olfactory impairment and 11% of mci patients with a normal sense of smell eventually developed ad.38 this study demonstrated that olfactory recognition and function testing was a very important tool for screening a population at high risk for ad. a cohort study of 148 mci outpatients in a 3-year follow - up showed that a combination of five out of eight potential predictors (olfactory function impairment, upsit, verbal memory, hippocampus volume, and entorhinal cortex volume) had a strong predictive value (90% specificity and 85.2% sensitivity) for ad converted from mci.39 lojkowska conducted a 24-month follow - up study in 49 mci patients and 33 controls. changes in olfactory functions, cognitive functions, and volume of medial temporal lobe structures (hippocampus, parahippocampal gyrus, and amygdala) were evaluated. in the mci group, a prediction of strong cognitive functions deterioration based on poor performance in olfactory identification tests shows sensitivity of 57% and specificity of 88%. the test based on cognitive functions only shows a sensitivity of 44% and specificity of 89%. combined tests having the criteria of poor olfactory identification performance and poor results of neuropsychological tests showed a sensitivity of 100% and specificity of 84%. the study reveals that the accuracy of predicting ad conversion from mci could be enhanced by using both olfactory and neuropsychological tests. a follow - up study of hippocampus volume reduction, olfactory identification performance, and cognitive functions deterioration will further increase prediction accuracy.40 devanand conducted a prospective observational study in a multiethnic community cohort in north manhattan, ny. a total of 1,037 participants without dementia were evaluated with the 40-item upsit. in 757 participants, follow - up occurred at 2 years and 4 years. in logistic regression analyses, lower baseline upsit scores were associated with cognitive decline (relative risk 1.067 per point interval ; 95% confidence interval [ci ] : 1.040, 1.095 ; p<0.0001) and remained significant (relative risk 1.065 per point interval ; 95% ci : 1.034, 1.095 ; p<0.0001) after including covariates. upsit, but not selective reminding test - total immediate recall, predicted cognitive decline in participants without baseline cognitive impairment. in discrete time survival analyses, lower baseline upsit scores were associated with transition to ad (hazard ratio 1.099 per point interval ; 95% ci : 1.067, 1.131 ; p<0.0001) and remained highly significant (hazard ratio 1.072 per point interval ; 95% ci : 1.036, 1.109 ; p<0.0001) after including demographic, cognitive, and functional covariates.41 the study by devanand confirmed that impairment in olfactory recognition in a population with normal cognitive function more accurately predicted decline of cognitive function than assessment of verbal episodic memory and supported the cross - cultural application of inexpensive olfactory recognition tests as bio - markers for predicting decline in cognitive function and early stage ad. in addition, in the future, olfactory recognition testing is expected to assist in patient selection and stratification in treatment trials for patients with cognitive impairment or prevention trials for healthy people with good cognition. control study to assess the effectiveness of a brief olfactory test for the diagnosis of ad. this study made the test subjects to close their eyes and assessed their ability to detect the odor of peanut butter through one nostril at a time and measuring the distance between the subject s nostril and the peanut butter container. the results showed that the distance to the left nostril for detecting the odor was significantly shorter in ad patients than in mci patients and normal controls. therefore, stamps proposed that left and right nostril odor detection was a sensitive and specific test for ad. however, a recent study by doty replicated and expanded the study by stamps but did not repeat the results or the significant asymmetry of odor detection in ad patients.42,43 the severity of olfactory dysfunction is correlated with certain clinical manifestations in ad patients. a previous study included 57 mild - to - moderate, late - onset ad patients and 24 age - matched healthy elderly individuals and showed that increasingly severe olfactory dysfunction heralded more clinical symptoms or severe illness of ad.44 another study that used olfactory recognition testing to evaluate the relationship between olfactory recognition and neuropsychiatric symptoms in 172 ad patients, 112 mic patients, and 132 neurologically and psychiatrically healthy controls showed that olfactory recognition was associated with the level of apathy but not with depression or other neuropsychiatric symptoms.45 since olfactory dysfunction is frequently overlooked by doctors and patients,46 it is not often the earliest clinical manifestation described in ad patients. patients with a low olfactory function score are considered more likely to develop ad, especially those with a low olfactory function score who are not aware of their problems in sense of smell. furthermore, only 6% of ad patients complained of decline in olfactory function during the early stage of the disease, but 90% of ad patients demonstrated a significant impairment of olfactory function in an olfactory test.6 patients with a low olfactory function score are considered more likely to develop ad, especially those who are not aware of their problems in sense of smell. stanciu conducted a population - based cohort study and showed that subjective olfactory dysfunction was an independent predictor for dementia. therefore, stanciu suggested that assessment of subjective olfactory dysfunction might play a complementary role in evaluating the risk of dementia.47 however, bahar - fuchs conducted a 12-month retrospective observational study and found no connection between decline of olfactory function and ad development from amnestic mci.48 further study to confirm the relationship between subjective olfactory dysfunction and risk of ad is necessary. in addition, no significant correlation was found between subjective and objective olfactory dysfunction.6,49 hence, the prediction accuracy of olfactory function scores using subjective olfactory dysfunction is extremely poor. olfactory function may be used as a clinical marker for severity and progression of ad. however, many questions must still be answered. for example, it is unclear how to identify and differentiate age - related olfactory changes and olfactory dysfunction caused by diseases. it is also unclear during which disease stage ad pathological changes are limited to the olfactory structures. moreover, the validity and clinical relevance in predicting ad using a combination of assessments including olfactory dysfunction and other biomarkers of ad remain unclear. since different olfactory tests have great variability, a brief, easy, sensitive, accurate, and convenient olfactory test is needed in daily clinical practice. although, there is sufficient evidence showing that olfactory tests can identify and differentiate ad cases from normal controls, further research to identify ad and other types of dementia using olfactory tests is needed. most ad - related olfactory studies had small sample sizes.8 many findings from existing reports must be confirmed by rigorously designed cohort studies.19 future studies should also investigate which combinations of biomarkers and olfactory assessments are most effective in predicting the conversion risk of dementia. based on currently available knowledge, we should recognize the importance of olfactory assessment in daily clinical practice. in addition, olfactory function tests should be incorporated in the assessment of populations at high risk for dementia to test methodologically and systematically for subclinical ad.50 | alzheimer s disease (ad) is a common neurodegenerative disorder with the earliest clinical symptom of olfactory dysfunction, which is a potential clinical marker for ad severity and progression. however, many questions remain unanswered. this article reviews relevant research on olfactory dysfunction in ad and evaluates the predictive value of olfactory dysfunction for the epidemiological, pathophysiological, and clinical features of ad, as well as for the conversion of cognitive impairment to ad. we summarize problems of existing studies and provide a useful reference for further studies in ad olfactory dysfunction and for clinical applications of olfactory testing. |
the e2f transcription factor is best known for its ability to regulate cell cycle progression by coordinating a large group of genes involved in g1-to - s phase transition. e2f regulated genes include cell cycle regulators, such as cyclin eand cdc25a, and genes that encode essential components of the dna replication machinery. in agreement with the idea that e2f is an important regulator of cell proliferation, studies in several experimental systems have shown that the ectopic expression of e2f is sufficient to drive quiescent cells into s phase, while the inhibition of e2f - dependent transcription blocks cell proliferation [24 ]. e2f cell cycle activity is controlled through the temporally regulated physical association of pocket proteins. the hypophosphorylated retinoblastoma protein prb represses e2f activity by shielding the e2f transactivation domain leading to restriction of cell cycle progression. during the course of cell cycle progression through the restriction point, prb becomes phosphorylated by the activity of cyclindependent kinases (cdks) resulting in the release of e2f, thereby causing its activation (fig. it is currently believed that most, and perhaps all, human cancers contain mutations that comprise prb function and that this contributes to their ability to proliferate in an environment that would cause normal cells to arrest. during transition of cells from g1 to s phase, the activity of e2f is controlled by prb, which binds e2f / dp, leading to transcriptional repression of e2f - responsive promoters in go / g1. in late g1 prb is phosphorylated by cyclin - dependent kinases (cdk), which results in the release of e2f. free e2f transactivates growth promoting genes such as cyclin e or cdc25a, resulting in s phase entry and cell cycle progression. e2f is a family composed of at least eight members that can be divided into distinct subgroups on the basis of their structural and functional similarities (reviewed in ref.). e2f1, e2f2 and e2f3a are transcriptional activators, whereas e2f3b, e2f4, e2f5, e2f6 and e2f7 act as repressors of transcription. the most recently identified e2f family member, e2f8, resembles the organization of e2f7 in the presence of two separate dna - binding domains. like e2f7 all e2f proteins except e2f7 and e2f8 function in heterodimeric complexes with dp family (dp1-dp4) proteins. from mutant mouse models it became clear that different e2f and dp subunits perform distinct, perhaps overlapping functions in the control of cell cycle progression, as well as unique roles during development, tissue homeostasis and apoptosis. for example, e2f3 is important for the control of cellular proliferation, while e2f4 and e2f5 are essential for g1 control and cell cycle exit as well as for normal mouse development. previous studies suggest a role for e2f7 in facilitating cell cycle arrest in both g1 and g2 phases. in contrast, e2f4 can associate with all pocket proteins, while e2f5 solely binds p130. e2f6 as well as the newest family members e2f7 and e2f8 lack a transactivation domain and do not interact with any pocket protein. recent genomic studies have found that the function of the e2f transcriptional program extends further than the g1/s transition. chromatin immunoprecipitation experiments [1013 ], expression profiling studies [1417 ] and a recently performed proteomic analysis showed that e2f proteins bind to and modulate expression of a large fraction of genes with diverse functions, suggesting a widespread role for e2f in the human genome. currently, the best - characterized activity, especially at the hands of e2f1, in addition to its traditional role in cell cycle progression is its ability to induce apoptosis [1922 ]. in some setting, another member of the e2f family, e2f3, also induces apoptosis. however, this e2f3-induced apoptosis was found to be e2f1-dependent and is largely attributed to the ability of e2f3 to transactivate the e2f1 gene, indicating that accumulation of crucial levels of e2f1 activity, and not total e2f activity, is essential for the induction of apoptosis. a large number of studies clearly support the role of e2f1 as a tumour suppressor rather than an onco - gene [2426 ]. under deregulated conditions the activity of e2f1 is linked to events that determine cell fate through the induction of apoptosis, thus protecting the organism against oncogenic transformation. mice, for example, lacking e2f1 (e2f1) exhibit defects in apoptosis together with an increased incidence of tumour development [24, 25 ], and the level of apoptosis seen in rbmice is suppressed by e2f1 deficiency. different mechanisms have been attributed to the ability of e2f1 to cause apoptosis, which can occur through both p53-dependent and independent path - ways (fig., p53 accumulates following e2f1 expression through activation of the cdkn2a transcript p14arf, which in turn interacts with mdm2, thereby preventing mdm2 from targeting p53 for ubiquitination and sub - sequent degradation. lately, additional arf - independent pathways have been described, and some reports even imply a negative feedback by arf since over - expression of arf inhibits e2f1-dependent apoptosis, and targets e2f1 for proteasomal degradation. e2f1-induced apoptosis in the absence of arf was shown to correlate with p53 phosphorylation at residues that are also phosphory - lated in response to dna damage [29, 31 ]. moreover, induction of both apoptosis and p53 phosphorylation by e2f1 are abolished by the atm and atr protein kinase inhibitor caffeine, supporting that e2f1 uses the atm signalling pathway to induce p53 and chk2 phosphorylation and thereby apoptosis [32, 33 ]. correspondingly, it was reported that the e2f1 transcription factor elevates atm promoter activity by direct binding, which is accompanied by enhanced p53 phosphorylation. in addition, e2f1 interacts directly with p53 via the cyclin a binding domain of e2f1, and this interaction enhances p53 apoptotic activity in response to dna damage e2f1 promotes apoptosis via the tumour suppressor p53 and independent of p53. in the p53-dependent pathway, e2f1 activates arf, which in turn stabilizes p53 by alleviating its proteosome degradation through mdm2. arf negatively regulates e2f1 in a feedback loop mechanism. in response to dna damage e2f1 is stabilized through phosphorylation by atm / atr and chk2 kinases (chk2 also stabilizes p53 by phosphorylation). in addition, e2f1 interacts directly with p53 via the cyclin a binding domain, thereby inducing p53-mediated apoptosis. alternatively, p53-independent apoptosis by e2f1 occurs via direct up - regulation of genes including p73 and apaf-1. the assembly of apaf1 with cytochrome c released from the mitochondria leads to formation of the apoptosome that catalyzes caspase-9, and successive initiation of proapoptotic effector caspases including caspase-3. e2f1-induced apoptosis occurs also independent of p53 in tissue culture and transgenic mice [3638 ], and prb has been shown to protect p53-null cells from apoptosis in an e2f1-binding dependent manner. apoptotic targets of e2f1 in the absence of p53 include the p53 homolog protein p73 [40, 41 ] and apoptosis protease - activating factor 1 (apaf1) both of which are transcriptionally regulated by e2f1. this initiates the assembly of apaf1 with cytochrome c followed by procaspase-9 activation and successive initiation of proapoptotic effector caspases including caspase-3, -6 and -7. mapping studies revealed that the apoptotic ability of e2f1 requires the dna - binding domain but not its transactivation function, since mutants of e2f1 that lack the transactivation domain are still able to induce cell death [36, 37, 43 ]. from these experiments, it was supposed that proapoptotic e2f1 target genes are activated by removal of e2f1/rb repression rather than direct transactivation. moreover, bell. recently reported dna - binding independent cell death from a minimal proapoptotic region of e2f1 that is consequently unable to transactivate, repress or derepress e2f target genes. however, since this activity is also present in e2f2 and e2f3 proteins, it might therefore define a distinct mechanism of death by e2f proteins. because normal cells proliferate without suffering e2f1-induced apoptosis, its proapoptotic potential integration of external signals appears to play an important role in determining the sensitivity to e2f1-induced apoptosis. for example, cell survival signals via the pi3/akt and the egfr / ras / raf pathway have been shown to promote e2f1-driven cell proliferation by suppressing e2f1-induced apoptosis [4547 ]. in addition, dna damage signals have been suggested to specifically activate e2f1-dependent transcription of proapoptotic genes [4850 ]. these changes stabilize e2f1, increase its transactivation potential and allow it to preferentially bind the promoters of some proapoptotic genes. overall, the final decision of whether e2f1 leads to cell proliferation or apoptosis might thus depend on the genetic status or molecular back - ground of the cell. this means that e2f1 would function strictly as a growth promoter if pathways that mediate e2f1-induced apoptosis are disabled. many of the drugs cause dna damage and the inflicted dna damage activates apoptotic programs that lead to cell death. forced expression of e2f1 has been shown to sensitize tumour cells to the proapoptotic signals generated by chemotherapeutic drugs or ionizing radiation [5156 ]. in addition, endogenous e2f1 is up - regulated after dna damage in a manner analogous to that of p53, suggesting its direct role in responsiveness to conventional genotoxic stress signals. in response to dna damage, the e2f1 protein is stabilized through distinct mechanisms, including direct phosphorylation by the ataxia - telangiectasia mutated (atm) kinase, the atm and rad3-related (atr) kinase and the chk2 kinase [32, 48, 58 ], and also by acetylation through p300/creb - binding protein - associated factor (p / caf). activation of the atm dna damage response pathway is commonly observed in a variety of early - stage tumours, suggesting that this checkpoint response functions to suppress the development of cancer. has demonstrated that e2f1 is a critical determinant of the cellular response in cancer cells to genotoxic stress. taking advantage of drosophila ddp and de2f1;de2f2 mutant animals, it was shown that the role of de2f1 within individual developing wing discs exposed to -irradiation is completely context - dependent. de2f1/ddp appears to protect non - proliferating cells and sensitizes proliferating cells to -irradiation - induced apoptosis. the loss of the prb - dependent cell cycle checkpoint might allow cancer cells to enter s phase and initiate apoptosis under conditions where normal cells would undergo a g1 arrest and initiate dna repair. in addition, e2f can increase the effectiveness of chemotherapeutic agents that are most active in s phase cells and/or that require the presence of a particular cell cycle dependent target for the induction of cell death [60, 61 ]. the frequent deregulation of e2f1 in human tumours, taken together with its apoptotic potential and its stabilization after damage suggest that e2f1 may play an important role in the enhanced sensitivity of tumour cells to dna damage - induced cell death. in agreement with this, increased levels of proapoptotic target genes of e2f1 such as adenoviral e1a and p73 have been invoked as a potential basis for selective killing of cancer cells, including p53-defective tumour cells, by dna - damaging agents relative to normal cells [6264 ]. in the past few years, a large number of novel e2f1-regulated target genes or gene networks functioning in the apoptotic program have been identified. from these studies it becomes evidently clear that e2f1 is a key regulator of the apoptotic machinery by being involved in many aspects of programmed cell death, both by activating proapoptotic genes and through the inhibition of antiapoptotic survival signals. e2f1 induces the expression of the proapoptotic bcl-2 homology 3 (bh3)-only proteins puma, noxa, bim, hrk / dp5 and bik in cancer cells through a direct transcriptional mechanism [65, 66 ]. in fact, e2f1 binds to the promoters of these genes and transactivates them by a p53-independent mechanism. bh3-only proteins are members of the bcl-2 protein family and are required for the execution of apoptotic cell death by integrating diverse apoptotic stimuli into a common death pathway governed by other multidomain bcl-2 family members. while some bh3-only proteins, such as bid, may chaperone the activation of bax and bak at the mitochondrial membrane, most others antagonize the functions of the anti - apoptotic bcl-2 family members. reversely, reducing the expression of noxa and puma by rna interference diminished apoptosis induced by e2f1. like e2f1 itself, some of these proteins, for example, bik and puma are also up - regulated in response to chemotherapeutic agents [65, 66 ]. consistent with their responsiveness to e2f1, it has been shown that the dna damage - induced elevation of puma and bik is abolished by suppression of e2f1 activity, indicating that both pathways contribute to the apoptotic response to damaging agents. increased expression of proapoptotic bcl-2 genes including bad, bak and bid after e2f1 activation in p53-deficient tumour cells was also reported in a gene array analysis by stanelle.. in the same study, kiaa0767, termed d(eath)-i(nducing)-p(rotein), was identified as a relevant apoptotic target of e2f1. the dip protein is localized in the mitochondria and mediates e2f1-induced apoptosis independently of p53 in a partially caspase - independent manner. in addition, transcriptional activation of the second mitochondrial activator of caspases or direct inhibitor - of - apoptosis - binding protein with low pi (smac / diablo) was reported as a mechanism by which e2f1 promotes p53-independent apoptosis via the mitochondria. these proteases are synthesized as inactive proenzymes and processed to an active state during apoptotic cell death. initiator caspases (caspase-2, -8 and -9) trigger a cascade that result in the activation of effector caspases (caspase-3 and -7), which in turn produce the characteristic morpho - logical changes associated with apoptosis. enforced e2f1 expression has been shown to result in the accumulation of caspase-2, -3, -7, -8 and -9 through a direct transcriptional mechanism. e2f1 can facilitate caspase activation through p53-dependent signals, resulting in mitochondrial release of cytochrome c, while simultaneously increasing caspase expression through direct caspase promoter binding that is independent of p53. by up - regulation of caspases, particularly caspase-8, e2f1 might sensitize cells to death - inducing ligands such as tumour necrosis factor (tnf). another novel death effector protein of e2f1 is siva, which was identified to be directly up - regulated by e2f1 and p53 via their corresponding consensus sites in the siva promoter. siva is a proapoptotic protein containing a death domain that is expressed on a broad scope of tissues, including cells of the immune system and the cns as well as tumour cells. as a potential mechanism for apoptosis induction by siva, its interaction with members of the tnf - receptor family and antiapoptotic bcl-2 family members has been considered. these studies suggest that siva may function through multiple mechanisms possibly dependent on the cell type and apoptotic stimulus. in addition to the findings by fortin. indicating that the siva gene exhibits a striking induction during p53-mediated neuronal apoptosis, up - regulation of siva was also observed in p53-deficient hepatoma cells in response to cisplatinum - induced apoptosis, suggesting that it is also regulated in a p53-independent manner, perhaps by e2f1. some clarity has been shed on the requirement of the ask1-p38 mapk pathway for e2f1-induced apoptosis. the ginsberg group showed that e2f1 modulates the activity of the p38 mapk path - way through up - regulation of p38 mapk phosphorylation. this involves transcriptional induction of the ask1 (apoptosis signal - regulating kinase 1) gene (also known as map3k5), a member of the mitogen - activated protein kinase family that phosphorylates p38 mkks. dna microarray analysis revealed map3k5 up - regulation following e2f1 over - expression in p53-positive u-2os osteosarcoma and in p53-deficient melanoma cells. for example, map3k5 has been shown to mediate rapamycin - induced apoptosis via phosphorylation of c - jun in p53-negative mouse embryonic fibroblasts, whereas suppression of kinase activity and ask1-jnk signalling through the interaction of ask1 with p21cip1 in p53 wild - type cells leads to apoptosis inhibition. the relevance of the ask1-p38 connection for e2f1-induced apoptosis is also evidently clear from data demonstrating that e2f1 regulates the activity of the p38 signalling inhibitor wip1. further insight into the mechanism by which e2f1 directs p53 activity towards apoptosis comes from studies showing that e2f1 up - regulates the expression of proapoptotic cofactors of p53, jmy, tp53inp1 and the apoptosis - stimulating proteins of p53 (aspp) family members aspp1 and aspp2 by a direct transcriptional mechanism [80, 81 ]. tp53inp1 was shown to mediate phosphorylation of p53 on serine 46. both aspp proteins modulate the cellular apoptotic threshold by direct interaction with p53, thereby enhancing the dna binding and trans - activation function of p53 on the promoters of proapoptotic genes in vivo. herein, aspp specifically renders p53-guided apoptosis by stimulation of the p53-regulated promoters bax and pig-3. while these studies provide additional pathways by which e2f1 cooperates with p53 to induce apoptosis, the aspp promoters can also be transactivated by e2f1 in a p53-deficient context as shown by chip and reporter gene assays [80, 83 ]. since aspp1 and aspp2 are specific activators of the apoptotic function of all p53 family members, e2f1 may use this pathway to increase the apoptotic function of p63 and p73 independently of p53. this ambivalence seems to be a common concept for death proteins since it has also been observed for other apoptosis factors such as siva and map3k5. siva seems to link the e2f / arf / p53- and the p53-independent apoptotic pathway. in a functional so - called technical knockout approach using the saos-2 er - e2f1 cell line, where e2f1 activity can be conditionally induced on 4-hydroxytamoxifen treatment, several other potential death targets of e2f1 were identified such as galectin-1 (or lgals1). e2f1-induced apoptosis was significantly abolished when galectin-1 anti - sense cdna was introduced into p53-negative cells. galectin-1 has been shown to play a key role in tumour - immune escape by killing antitumour effector t cells. it sensitizes, for example, human - resting t cells to fas (cd95)/caspase-8mediated cell death. e2f1 was earlier shown to be essential for t - cell apoptosis by a process called t - cell receptor (tcr) activation - induced cell death (aicd). lissy. demonstrated that t cells were protected from tcr - mediated apoptosis by disruption of the e2f1 gene or p73. together these findings support a function for e2f1 in balancing life and death of immune effector cells. beside direct activation of various apoptosis - related genes described earlier, a second major mechanism by which e2f1 sensitizes cells to apoptosis is via inhibition of antiapoptotic signalling. one example is the inhibition of nf-b that functions in most cases as a survival signal. activation of tnfr in response to tnf results in the stimulation of nf-b via traf2, which contributes to the inhibition of cell death. importantly, e2f1 expression sensitizes cells to apoptosis by down - regulation of traf2 protein levels, thereby inhibiting antiapoptotic nf-b signalling. the general principle of blocking survival factors by e2f1 to induce apoptosis is supported by the data from two other studies [89, 90 ]. they reported that over - expression of e2f1 suppresses the expression of the apoptotic antagonists bcl-2 and its family member mcl-1 which leads to apoptosis. in both cases, the e2f1-induced decrease in bcl-2 and mcl-1 levels occurs independently of the arf / p53 pathway. this scenario renders cells susceptible to apoptosis by altering the balance between anti- and proapoptotic bcl-2 family members in favour of those that induce cell death. together with the induction of bh3-only proteins and other proapoptotic bcl-2 family members by e2f1 there is increasing evidence that execution of the apoptotic program is achieved by an intense cross - talk between the mitochondria and the endoplasmic reticulum (er) (reviewed in ref.). a number of molecules involved in mitochondrial apoptosis are also involved in er stress - induced cell death, suggesting that the er might regulate apoptosis by sensitizing the mitochondria by a number of extrinsic and intrinsic stimuli, as well as by inducing apoptosis. bcl-2 family members, for example, localize to the er membrane, thereby disrupting er homeostasis by altering er membrane permeability as a result of mitochondrial dysfunction. unfolded protein response mediated cell survival or cell death is regulated by the balance between the er chaperones grp78 and gadd153. e2f1 can down - regulate the expression of grp78 (also known as bip), which normally protects cells from death induced by disturbance of er homeostasis [94, 95 ]. moreover, expression of another er chaperon, the 94 kd glucose - regulated protein (grp94), shown to be significantly up - regulated in human esophageal cancers, is also inhibited by e2f1. from these data it is evident that e2f1-mediated apoptosis involves both mitochondrial and er - related death pathways. e2f1 sensitizes cells to apoptosis by both direct activation of proapoptotic genes and through inhibition of antiapoptotic survival genes, thereby engaging different death signalling pathways via the mito - chondria (intrinsic), death receptors (extrinsic) and the endoplasmic reticulum. since apoptosis programs can be manipulated to produce massive changes in cell death, the genes and proteins controlling apoptosis are potential drug targets. as indicated earlier, acquired apoptotic defects during cancer progression involve three types of molecular changes : (i) inactivation of proapoptotic effectors, for example, p53 or apaf-1 ; (ii) activation of antiapoptotic factors, for example, bcl-2 and (iii) reinforcement of survival signals. thus, anti - cancer studies set out to either boost cell death or to impede antiapoptotic and proliferative pathways. two observations suggest that such strategies are feasible. first, most antiapoptotic mutations act relatively upstream in the program implying that tumour cells generally retain the machinery and latent potential for apoptosis. second, tumour - specific alterations in apoptotic programs provide opportunities to target cell death in a selective manner. restoration of the apoptotic pathway, for example, by re - introduction of p53 or activation of apaf-1 has been shown to increase the sensitivity of neoplastic cells to dna - damaging agents. the inef - fectiveness of a p53-based gene therapeutic strategy in certain conditions is, however, problematic (e.g. mutant p53 in tumour cells trans - dominantly impairs the function of wild - type p53). here, genes (such as proapoptotic e2f1 targets) are particularly useful that compensate or bypass cell death defects regardless of the p53 status. in the past few years, pre - clinical experiments mainly using e2f1 itself as anti - cancer therapeutic have been initiated. the effect of e2f1 over - expression on tumour growth has been evaluated in several types of human cancer in vitro and in vivo including glioma, melanoma, esophageal cancer, breast- and ovarian carcinoma, head and neck squamous cell cancer, gastric cancer ; pancreatic carcinoma and nonsmall - cell lung cancer [54, 100108 ]. these studies clearly indicate that apoptosis induction by adenoviral expressed e2f1 results in growth suppression and increased responsiveness of tumour cells to chemotherapy with relative sparing of normal tissues. this selectivity might be due to the fact that normal cells contain wild - type prb, which likely can bind to and antagonize the activity of exogenous e2f1. furthermore, transcription from e2f1-responsive promoters is higher in cancer cells than in normal cells. however, targeting e2f1 itself is a cumbersome mission possibly feasible for some but not suitable for all therapeutic purposes because of its dual role in cell cycle progression and apoptosis. regarding this issue, it has been shown that a transcriptionally inactive form of e2f1 is able to induce apoptosis without activating proliferation in human coronary vascular smooth muscle cells (vsmc). therefore, this strategy has widespread potential for preventing hyperproliferation in artherosclerosis, hypertension and restenosis after injury. in addition to e2f1, its downsteam target p73 was proven to be a valuable candidate for cancer therapy in tumour cells. infection with an adenoviral vector encoding the beta isoform of p73 resulted in potent cytotoxicity based on a combination of cell cycle arrest and apoptosis induction [64, 109, 110 ]. similar to e2f1, p73 can efficiently induce apoptosis and enhanced chemosensitivity of tumour cells primarily resistant to wild - type p53. although this strategy warrants further consideration, the antitumoural effect exhibited by over - expression of p73, in contrast to its homolog p53, is not restricted to tumour cells. although present data provide compelling evidence that effectors of e2f1-induced apoptosis can significantly kill cancer cells, their role as a target for cancer treatment is only beginning to emerge. so far, in clinical studies antiapoptotic e2f1 regulated genes were used to hamper tumour growth instead of using approaches for introducing proapoptotic molecules. antiapoptotic bcl-2, for example, has been targeted extensively by antisense oligonucleotide approaches which interfere with bcl-2 activity, resulting in less bcl-2 expression and elevated levels of apoptosis. in the phase i ii study, bcl-2 antisense oligonucleotide (g3139) treatment of chemoresistant melanoma patients led to minor to complete therapeutic response in 6 of 14 cases when the antisense therapy was combined with dacarbazine administration. in a phase ii study, bcl-2 antisense oligonucleotide treatment of patients with multiple myeloma led to therapeutic responses in 55% of cases when combined with chemotherapy. these data underline that inhibition of antiapoptotic bcl-2 is surely a promising treatment for otherwise resistant tumour entities. | abstractdefects in apoptotic programs contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy, and treatment failure. the genetic basis for apoptosis implies that cell death can be disrupted by mutations, raising the intriguing possibility that cell numbers can be regulated by factors that influence cell survival. it is well documented that the e2f1 transcription factor is a key regulator of apoptotic programs. e2f1-induced cell death occurs via multiple pathways, some of which involve the tumour suppressor p53, and autonomous of p53. this has led to the opinion that e2f1 functions as a tumour surveillance factor, detecting aberrant proliferation and engaging apoptotic pathways to protect the organism from developing tumours. frequently, novel players are discovered that expand the interpretation of apoptosis control by e2f1. this information will help to produce new strategies to exploit e2f1-induced apoptosis for therapeutic benefit. |
t - bet (t - box protein expressed in t cells, also called tbx21) was firstly described in 2000 in a report examining the effects of t - bet on the differentiation of t helper 1 (th1) cells. for the past 15 years, many studies have examined the functions of t - bet and have revealed multiple roles for this protein during th cell differentiation, with a focus on the molecular mechanisms involved, the novel functions of this transcription factor in innate immune cells, and t - bet - mediated modulation of inflammatory diseases [29 ]. it has been clarified that t - bet plays a critical role in the coordination of innate immunity and adaptive immunity and that it fulfills an important function in modulating chronic inflammatory diseases, including asthma and inflammatory bowel disease, by controlling a network of highly conserved genetic programs [1012 ]. thus, optimal regulation of t - bet expression and activity seems to be beneficial for preventing or treating chronic inflammation and autoimmune diseases. although attempts have been made at identifying the small molecules that control the expression and activity of t - bet that affect the t cell - mediated immune response, little progress has been made on this to date. given the importance of t - bet in the immune regulation, elucidating the functional mechanisms underlying the multiple roles of t - bet would facilitate the development of novel therapeutic interventions for treating chronic inflammatory and autoimmune diseases. this review summarizes the current state of knowledge about the molecular mechanisms underlying the multiple roles played by t - bet in th cell development. the t - bet contains an amino - terminus, a t - box domain, and a carboxyl - terminus, which show 82%, 100%, and 79% homology, respectively, between mice (530 amino - acid residue protein) and humans (535 amino - acid residue protein) (figure 1). the t - box domain, located between residues 135 and 326 in mouse t - bet, is highly conserved in 18 members of the t - box protein (tbx) family [13, 14 ]. common features shared by t - box proteins include a capacity for dna binding through the t - box domain and transcriptional regulatory activity, which plays a role in controlling the expression of developmental gene in all animal species. the t - box domain is made up of about 180 amino - acid residues and is both sufficient and necessary for binding to the consensus dna sequence tcacacct [1315 ]. brachyury (t) was the first t - box protein to be identified and, in dimeric form, interacts with the major and the minor grooves of dna through hydrophobic interactions and unusual main - chain carbonyl contact with a guanine as a dimer. tbx1 also binds to the dna sequence as a dimer, whereas tbx2 appears to bind to the same dna sequence as a monomer. although tbx1 and tbx2 share 61% identity in the t - box domain, the structure of the dna - t - box binding complex appears to be different, because of the low homology among the amino- and carboxyl - terminal regions. the t - box domain in t - bet shows 50% homology with the corresponding domain in brachyury (t), tbx1, and tbx2 ; however, the crystal structure of t - bet bound to the dna sequence remains to be characterized. t - bet directly binds to the consensus dna sequence within the ifng promoter and activates its transcription. the t - bet - induced expression of ifng derives th precursor cells to differentiate into th1 effector cells. while exogenous t - bet overexpression in nave th cells preferentially increases development of th1 cells, t - bet deficiency leads to a failure to produce sufficient ifn- and therefore reduces generation of th1 cells [1, 18 ]. t - bet expression is substantially increased by stimulation of the t cell receptor (tcr) and is augmented by cotreatment with ifn- and il-12. ifn- binds to its receptor and induces activation of signal transducer and activator of transcription (stat) 1 and transcription of t - bet gene (tbx21). subsequently, t - bet directly stimulates the transcription of ifng as well as il12rb2. expression of il-12 receptor (il-12r) 2 on the cell surface further enhances ifn- production through il-12 and the stat4 signaling pathway, thereby resulting in preferential th1 cell differentiation. interestingly, enforced t - bet expression can also convert the differentiated th2 cells into th1 cells. therefore, t - bet is positioned at the crux of the regulatory pathways that induce ifn- in th cells. this cytokine, an early t cell growth factor, is essential for activation, proliferation, and differentiation of th cells and is abundantly produced upon tcr stimulation. ectopically introduced t - bet significantly suppresses il-2 production through inhibition of nuclear factor b (nf-b) p65 activity, under conditions of both th1 and th2 differentiation. during th1 cell differentiation, il-2 transcription the t - bet - mediated il-2 inhibition may affect th cell expansion and exquisitely modulate the th1-mediated immune response upon exposure to a pathogenic antigen. furthermore, exogenous t - bet introduction into th cells suppresses the production of th2 cytokines, such as il-4, il-5, and il-13, via suppression of gata - binding protein-3 (gata-3). accordingly, a lack of t - bet induces spontaneous th2 cell development in vitro and in vivo [1, 18 ]. the th2-suppressive activity of t - bet was also confirmed in the absence of ifn-, indicating that t - bet has a discrete inhibitory function, independent of ifn- stimulation. recently, many studies have reported that t - modulates other th cell lineages, including th17, treg, and follicular th (tfh) cells, in coordination with many transcription factors, such as the retinoic acid - related orphan receptor-t (rort), runt - related transcription factor 3 (runx3), and b - cell lymphoma-6 (bcl6) [2025 ]. these findings suggest that t - bet is a transcription factor that is critical for fine - tuning th cell development. t - bet functions as a multitasking player in the regulation of th cell differentiation. however, the molecular mechanisms that underlie the stimulatory and inhibitory activity of t - bet in regulating target gene expression remain to be clarified. many multitasking proteins are known to undergo posttranslational protein modifications and to determine cell fates by exerting direct stimulatory and indirect inhibitory activity on target gene expression [26, 27 ]. antibody - based detection of t - bet proteins in western blots results in multiple bands, suggesting the posttranslational modification of t - bet in tcr - triggered th cells. tyrosine phosphorylation of t - bet protein occurs primarily during the early stages (days 2 to 3) of th cell development, upon tcr engagement, and declines afterwards. treating th cells with the tyrosine phosphatase inhibitor pervanadate enhances the tyrosine phosphorylation of t - bet. t - bet is mainly localized in the nucleus, and the nuclear tyrosine kinase il2-inducible tyrosine kinase (itk) was identified as the responsible upstream tyrosine kinase. itk deficiency prevents tyrosine phosphorylation of t - bet in th cells after stimulation with tcr and il-12. mutational research has revealed that tyrosine residue 525 (y525) is the relevant phosphorylation site and that phosphorylation at this site plays an important role in the interaction with gata-3. although t - box domain in t - bet is important for dna and protein - protein interaction, tyrosine phosphorylation of y525 is prerequisite for the suppression of gata-3-mediated th2 cell differentiation. blockade of y525 phosphorylation abrogates the interaction with and suppression of gata-3, resulting in impairment of th2 suppression. furthermore, another nuclear tyrosine kinase, c - abl, induces phosphorylation of t - bet at tyrosine residues 219, 265, and 304 in mouse t - bet. a deficiency in c - abl as well as mutation of t - bet at these residues (y219/265/304f mutants) leads to a failure to increase ifn- induction and to suppress th2 cytokine production, due to the loss of phosphorylation at these tyrosine residues. this, in turn, results in the aggravation of allergic lung inflammation in vivo. these findings suggest that itk- and c - abl - induced tyrosine phosphorylation of t - bet is essential for the modulation of th2 cell development and the allergic immune response. although t - bet - mediated suppression of th2 cell development is impaired by mutation of y525 and the absence of c - abl kinase, il-2 suppression is retained with a y525-mutant t - bet, suggesting the existence of an additional regulatory mechanism for il-2 modulation. interestingly, the appearance of multiple bands of t - bet protein on western blots could be eliminated by the addition of calf intestinal phosphatase, which predominantly eliminates phosphorylation at serine / threonine residues. mass spectrometric analysis then revealed serine 508 (s508) as another phosphorylation site in t - bet. mutation of s508 abolishes casein kinase- (ck-) and glycogen synthase kinase-3 (gsk-3-) mediated phosphorylation in t - bet, as well as the il-2suppressive activity of the protein. moreover, s508 phosphorylation is important for the interaction of t - bet with nf-b p65 and for prevention of binding of nf-b p65 to the il2 promoter. in accordance with the function of t - bet as an nf-b p65 inhibitor, t - bet - null th1 cells sustain nf-b p65 activity during th1 cell differentiation and thus produce more il-2. therefore, it has been suggested that t - bet is a physiological inhibitor of il-2 during th1 cell differentiation, through s508 phosphorylation - dependent suppression of nf-b p65. very recently, threonine 302 (t302) was characterized as a novel phosphorylation site in t - bet, although it remains unclear which kinase and phosphatase affect the phosphorylation of this residue. however, restoration of t - bet - null th cells with a t302-mutant t - bet stimulated ifn- production as much as did wild - type t - bet ; however, the mutant failed to suppress il-2 and other th2 cytokines. further analysis demonstrated that t302 phosphorylation is required for the interaction of t - bet with nuclear factor of activated t cells (nfat) and for downregulation of nfat - mediated il-2 and th2 cytokines, such as il-4, il-5, and il-13. nfat is not crucial for induction of ifn- production and t302-mutant t - bet is able to bind to the ifng promoter ; thus, ifn- production was comparable between wild - type and mutant t - bet. in other words, mutation of t302 abrogated the t - bet - mediated suppression of il-2 and th2 cytokine production but did not affect the dna - binding and ifn--stimulatory activity of t - bet. indeed, t302 is located in the dna - binding t - box, as is y304. the t - box domain consists of several repeats of -strands and -helices and is involved in both dimerization and dna binding. muller and herrmann predicted that the -helices h3 and h4 in brachyury (t) are important for the direct interaction of this protein with the minor and major grooves of dna. however, t302 may not be associated directly with the dna grooves, regardless of its phosphorylation status. it would be interesting to know which upstream kinase and phosphatase regulates t302 phosphorylation and whether t302 phosphorylation affects other protein modifications of t - bet. t - bet expression is critical for the transcriptional regulation of ifng and for the development of th1 cells, but the means of regulation of t - bet at the protein level is yet to be identified. jang. have recently reported that t - bet undergoes ubiquitination - mediated proteasomal degradation during the later stages of th1 cell differentiation. of the 16 lysine residues present in mouse t - bet protein, 11 are predominantly located within the t - box domain, and the remaining 5 are located at the carboxyl - terminus (residues 326 through 530), while no lysine residues are present in the amino - terminus (residues 1 through 134). interestingly, lysine residues within the t - box domain are preferentially ubiquitinated upon overexpression of ubiquitin. further analysis has identified that mutation of lysine 313 (k313) decreases ubiquitination - mediated t - bet degradation and enhances the expression level of t - bet in the nucleus and the cytoplasm. despite the increased levels of the k313 mutant, this mutation completely abrogated t - bet functions involving dna binding, transcriptional activation of ifng, and suppression of il-2 and th2 cytokine production. the crystal structure of the -helices of the t - box domain bound to dna strongly suggests that the amino group of k313 is associated with the phosphate of a dna base via hydrogen - bond interaction. in addition, mutation of k313 also leads to failure to suppress il-2 and th2 cytokine production ; however, the interaction with and suppression of gata-3 and nf-b p65 are not altered by mutation of k313. interestingly, nfat interaction is abolished in k313-mutant t - bet, which is also strongly associated with an absence of phosphorylation at t302. it is not clear yet whether and how k313 regulates t302 phosphorylation and vice versa. since mosmann. discovered th1 and th2 subsets that produce signature cytokines, ifn-, and il-4, il-5, and il-13, respectively, and that modulate inflammatory and allergic immune responses, further studies have identified novel subsets of th cells, such as th17, tfh, and treg cells [11, 21, 23, 24, 3134 ]. extensive studies have also characterized the cytokine signaling pathways and transcription factors involved in the regulation of immune responses to pathogens [1, 3554 ]. importantly, t - bet plays a fundamental role in controlling differentiation of several subsets of th cells and in modulating inflammatory and autoimmune diseases [1012 ]. a deficiency in t - bet spontaneously leads to the development of asthmatic symptoms, which is characterized by increased eosinophil infiltration into the airway, mucus - secreting goblet cell hyperplasia, and chronic airway remodeling with collagen accumulation and proliferative myofibroblasts ; these features are often also seen in asthmatic patients [5557 ]. restoration of t - bet expression shifts the immune balance to a th1 response and prevents and attenuates pathologic lung inflammation in vivo [58, 59 ]. abrogation of t - bet - induced ifn- production resulted in higher susceptibility to intracellular pathogens in vivo, including mycobacterium tuberculosis, leishmania major, and salmonella typhimurium [18, 60, 61 ], emphasizing the importance of ifn- production by t - bet - expressing th1 cells in the defense against bacterial infections. however, t - bet - deficient mice are resistant to infection by listeria monocytogenes, because inf- production by natural killer cells is both necessary and sufficient for the host defense against l. monocytogenes. furthermore, t - bet can aggravate the development of inflammatory and autoimmune diseases, including inflammatory bowel disease, experimental autoimmune encephalomyelitis, inflammatory arthritis, and type i diabetes, as these inflammatory diseases are attenuated in the absence of t - bet [6, 56, 63, 64 ]. these findings suggest that fine - tuning of the immune response by modulation of t - bet expression could have beneficial effects for patients with chronic asthma, inflammatory bowel disease, arthritis, multiple sclerosis, and diabetes. t - bet is a t - box domain - containing transcription factor that is typically involved in developmental regulation but exerts multiple functions in th cell differentiation ; transcriptional activation of ifn--expressing th1 cells, indirect suppression of th2, th17, and treg cell development, and fine - modulation of il-2 production in th1 cells. phosphorylation at y525 plays a role in gata-3 suppression during th2 regulation, phosphorylation at s508 causes nf-b p65 suppression in the context of il-2 regulation in th1 cells, phosphorylation at t302 plays a role in fine - tuning il-2 production, and ubiquitination at k313 plays a role in controlling t - bet protein stability. thus, posttranslational modification of t - bet facilitates its functional diversity and the complexity of its modulation of cytokine expression (figure 2). it is not known whether the various posttranslational modifications occur sequentially or simultaneously, whether one type of protein modification affects other modification, or whether changes in the posttranslational modification of t - bet are related to the development of infectious and chronic inflammatory diseases. further identification of novel protein modifications related to t - bet functions would provide valuable insights into the development of powerful therapeutic interventions for controlling chronic inflammatory and autoimmune diseases. | t - bet (t - box protein expressed in t cells, also called as tbx21) was originally cloned as a key transcription factor involved in the commitment of t helper (th) cells to the th1 lineage. t - bet directly activates ifn- gene transcription and enhances development of th1 cells. t - bet simultaneously modulates il-2 and th2 cytokines in an ifn--independent manner, resulting in an attenuation of th2 cell development. numerous studies have demonstrated that t - bet plays multiple roles in many subtypes of immune cells, including b cell, dendritic cells, natural killer (nk) cells, nk t cells, and innate lymphoid cells. therefore, t - bet is crucial for the development and coordination of both innate and adaptive immune responses. to fulfill these multiple roles, t - bet undergoes several posttranslational protein modifications, such as phosphorylation at tyrosine, serine, and threonine residues, and ubiquitination at lysine residues, which affect lineage commitment during th cell differentiation. this review presents a current overview of the progress made in understanding the roles of various types of t - bet protein modifications in the regulation of cytokine production during th cell differentiation. |
shock due to any reason carries a significant risk of mortality and morbidity, especially if the therapy is inappropriate. aggressive fluid and/or vasoactive therapy aimed at achieving the desired end - points of early goal directed therapy (egdt) is the cornerstone of management of both adults and children with septic shock, and also following cardiac surgery. specifically, achieving a central venous saturations (scvo2) > 70% is one of the key therapeutic goals that has been shown to improve outcomes, as this reflects tissue oxygenation and oxygen delivery. although the true value of mixed venous oxygen may be obtained by obtaining a sample from the pulmonary artery by swan ganz catherization, a scvo2 from an appropriately sited central venous catheter (cvc) is an acceptable surrogate. the gold standard to measure scvo2 is analysis of the venous blood gas sample by a co - oximeter (co - ox). this is because the saturation is directly measured in a co - ox, whereas this value is calculated (with many assumptions) in a standard blood gas machine. in sick patients, binding of hemoglobin (hb) to oxygen can not be assumed to be normal, and, furthermore, the position of the oxyhemoglobin curve may be shifted due to multiple reasons. however, due to the cost constraints, many intensive care units (icu) in india measure scvo2 by a standard abg (stand abg) machine, which is significantly less expensive. this prospective, observational study from a multidisciplinary (medical, cardiac, and surgical) pediatric icu (picu) of a tertiary level hospital (apollo hospitals, chennai) was conducted to assess the validity of scvo2 levels obtained by stand abg in comparison with co - ox. in particular, with reference to reaching egdt endpoints in septic shock, we aimed to elucidate the false positive rates (scvo2 > 70% by stand ab standard abg machine g but 7.42, respectively. temperature abnormality included patients with both hypothermia (97f) and hyperthermia (99f). patients were said to be in shock if they had at least three of the following clinical and laboratory evidence of shock : disproportionate tachycardia, low blood pressure for age, prolonged capillary refill time (> 3 sec), poor peripheral pulses, core to peripheral temperature difference of > 2c, low urine output, or elevated lactate (> 4 mmol / l). tests for correlation (pearson 's coefficient) and agreement (bland altman analysis) were further performed on scvo2 values obtained in these subgroups. sensitivity (true positive) and specificity (true negatives) for scvo2 values determined by stand abg versus the gold standard (co - ox) were calculated with particular reference to reaching egdt endpoints in septic shock. the baseline characteristics of the study population are summarized in table 1. from 82 patients, forty - four paired samples were collected in patients in shock, 54 in acidemic patients, 29 in alkalemic patients, 34 anemic patients, 32 in patients with either hypothermia or hyperthermia, and 31 in patients who did not fall in any of these categories (normal). baseline characteristics results obtained on blood gas analysis for the two groups are tabulated in table 2. despite a statistically significant difference in the ph and scvo2 values measured by both the methods, there was good linear correlation between these parameters [table 2, figures 1 and 2 ]. among the blood gas parameters, ph, paco2, and pao2 showed positive good correlation when obtained by either method [figures 1 and 2 ]. in the case of scvo2, a positive correlation was obtained when the scvo2 was analyzed by either machine (r = 0.69, p = 0.001) [figure 3 ]. limits of agreement between scvo2 measured by standard abg machine and co - ox by bland altman methodology gave 2.3% bias with 95% ci of 24.2% to 19.5% [figure 4 ]. comparison of blood gas parameters between standard blood gas machine (stand abg) and co - oximeter (co - ox) correlation between paco2 measured by stand abg and co - ox. scatter diagram with regression estimate shows the positive good correlation between paco2 by stand abg and co - ox (r = 0.84, p = 0.001) ; paco2 abg = stand abg ; paco2 c = co - ox scatter diagram correlation between ph by stand abg and co - ox (ph c). scatter diagram with regression estimate shows the positive good correlation between ph by stand abg and co - ox (r = 0.94, p = 0.001) ; ph abg = ph analyzed by stand abg ; ph c = ph analyzed by co - oximeter) correlation between scvo2 by stand abg and co - ox. scatter diagram with regression estimate shows the positive good correlation between scvo2 by stand abg and co - ox (r = 0.69, p = 0.001). (scvo2 a = central venous saturation measured by standard blood gas machine ; scvo2 c = central venous saturation measured by co - oximeter) agreement between scvo2 values obtained by the two machines. scvo2 a = central venous saturation measured by standard blood gas machine, scvo2 c = central venous saturation measured by co - oximeter the sensitivity of the standard abg machine machine in detecting scvo2 in patients with shock was 84.21%, while the specificity was 93.18%. in 4 (2.84%) samples, the standard abg machine gave false positive results, indicating that that shock endpoint had been reached (scvo2 > the scvo2 values obtained by the two methods in various subgroups are tabulated in table 3. all the subgroups, including patients with shock, had substantial correlation of the scvo2 values (0.6 - 0.8, p = 0.001) whether it was analyzed by standard abg machine or co - ox. in those with alkalosis, the scvo2 values had good substantial correlation (0.90, p = 0.001). however, presence of anemia showed only moderate correlation (0.49, p = 0.001). aggressive fluid and/or vasoactive therapy aimed at achieving the desired end - points of egdt, including scvo2 > 70%, is one of the key therapeutic goals that has been shown to improve outcomes. there have been no previous published studies comparing scvo2 values obtained by standard abg machine and co - ox. in our study, a positive good correlation (r = 0.69) was obtained in the scvo2 values measured by both the machines in a heterogeneous group of critically sick children, i.e., there is a strong, linear relationship unlikely to happen by chance (p = 0.001). 10 gm%, the correlation co - efficient showed only moderate positive correlation (r = 0.49, p = 0.001). the standard abg machine, which is widely used for blood gas analysis in many hospitals across india, measures the paco2, pao2, and ph of the sample. all other values are calculated by the algorithm inbuilt in the machine. as against this, most icus in the western world use the co - ox, which considered the gold standard. the advantage of a co - ox compared to a standard blood gas machine is its capability to measure concentrations of oxygenated hb, reduced hb, carboxyhemoglobin, methemoglobin, and bicarbonate. the co - ox is intuitively considered more reliable, especially in sick patients, where an algorithm - based calculation of scvo2 by a standard blood gas machine may be prone to fallacies. knowledge regarding the limits of agreement and correlation of scvo2 values analyzed by stand abg versus co - ox across a wide range of typical icu conditions is important for critical care practitioners. limits of agreement between scvo2 obtained by stand abg versus co - ox showed 2.3% bias with 95% ci of 24.2% to 19.5% by bland the same methodology was used in the subgroups with none, except the group with alkalosis showing agreement. thus, although there was good correlation, the agreement levels were wide, suggesting that the wide difference is possibly machine related. hemodynamic monitoring is a standard of care in the management of critically sick children to identify cardiovascular insufficiency, determine its cause, and evaluate the response to treatment. global tissue oxygen delivery is commonly monitored using mixed venous oxygen saturation (scvo2) level, which reflects the cardiac output, oxygen delivery (do2), peripheral extraction of oxygen, and consumption (vo2). its value is related to four determinants : vo2, cardiac output, hb, and oxygen saturation of hb. scvo2 trends measured with the central venous catheter tip in right atrium is more easily measurable than the mixed venous saturations and is an acceptable surrogate. inadequate do2 is presumed to be occurring if tissue oxygen extraction is markedly increased, as manifested by a decrease in scvo2 70%, the false positive rates were low and the degree of specificity and sensitivity was high, suggesting that the chance of missing a patient with uncorrected shock was low. two of the 4 patients who had scvo2 however, given the small sample size of false positives, we are underpowered at this stage to conclusively comment if fever tends to increase the rate of false positives. overall, scvo2 obtained by the stand abg machine showed good overall correlation with the scvo2 obtained by co - ox, except in anemic patients who had hb 70% as the end point of treatment. intensive care is making a foray in a big way in the developing world with several institutes offering tertiary level care. this machine is relatively cheaper (approximately rs 400,000) as compared to the much more expensive co - ox (approximately rs 10,00,000 - 14,00,000) and the cost per abg carried out by a stand abg is half of that performed by a co - ox. while it was encouraging that the less expensive equipment showed satisfactory correlation with the gold standard co - ox over a wide range of patient conditions, the wide range for limits of agreement was of concern. tertiary care icus in developing nations may prefer the stand abg as the co - ox is much more expensive and there was a general perception that the accuracy of the readings from a stand abg are in sync with the co - ox. our study indicates that the stand abg can be a satisfactory and cost - effective surrogate for the more expensive co - ox when assessing scvo2 for patients with shock. although the correlation was good over a range of conditions, the limits of agreement were wide and it performed particularly poorly in anemic patients | aims : aggressive therapy aimed at desired end - points of early goal directed therapy (egdt) is the cornerstone of septic shock management. a key endpoint that improves outcomes is achieving central venous saturation (scvo2) > 70%. the gold standard to measure scvo2 is by a co - oximeter (co - ox).settings and design : this prospective, observational study from a multidisciplinary pediatric intensive care unit (picu) was conducted to assess the validity of scvo2 levels by standard abg (stand abg) machine in comparison with co - ox in conditions that shifted the oxygen dissociation curve (odc) to the right or left in sick children and controls.materials and methods : data from paired samples was tested for correlation coefficient for ph, paco2, pao2, and scvo2. tests for correlation (pearson 's coefficient) and agreement (bland altman analysis) were performed on scvo2 values obtained in various subgroups. sensitivity and specificity for scvo2 values determined by standard abg machine versus co - ox were calculated in reference to egdt endpoints.results:a total of 141 paired samples were collected from 82 children. despite a statistically significant difference in the ph and scvo2, there was good linear correlation between these parameters. limits of agreement between scvo2 measured by standard abg machine and co - ox by bland altman gave 2.3% bias with 95% ci of -24.2% to 19.5%. sensitivity and specificity of standard abg machine in detecting low scvo2 in shock was 84.21% and 93.18% respectively, while it was false positive in 4 samples.conclusions:the less expensive standard abg machine showed satisfactory correlation with gold standard co - ox over a range of patient conditions ; however, the wide range for agreement was of concern and it performed particularly poorly in anemic patients. |
myeloid - derived, innate inflammatory cells provide early defense against invading pathogens by activating a diverse array of protective immune mechanisms. underlying these cells ' effector functions are the pattern recognition receptors (prrs), which detect repeating molecular motifs inherent to various pathogens (pathogen - associated molecular patterns (pamps)) and danger - associated molecular patterns (damps) generated as a result of tissue and cellular damage. a prominent prr known to elicit inflammation in response to gram - negative bacterial stimuli is the toll - like receptor 4 (tlr4). recognition of lipopolysaccharide (lps) by this receptor activates inflammatory signaling pathways to induce the expression of numerous antimicrobial and proinflammatory molecules. an important downstream target of tlr4 is the nod - like receptor pyrin domain - containing protein 3 (nlrp3) inflammasome. this multiprotein complex, responsible for cell pyroptosis and the caspase-1-dependent processing of pro - il-1 and pro - il-18 to their biologically active forms, has been shown to play a prominent role in regulating both chronic and acute inflammation as a responder to cell damage and stress. induction of the nlrp3 inflammasome is understood to proceed via a two - step process. ] serves as a priming stimulus by transcriptionally activating nlrp3 and pro - il-1 expression, thus inducing the production of essential inflammasome components. recent work, however, has also suggested this priming step to involve nontranscriptional regulation [3, 4 ]. signal 2 then activates the inflammasome by inducing cationic fluxes (k efflux and elevated intracellular ca), mitochondrial and lysosomal damage, and the subsequent generation of cathepsins and reactive oxygen species (ros) to elicit its effector functions [1, 2 ]. this activation step can be triggered by a large number of both endogenous [adenosine triphosphate (atp), alum, monosodium urate, and histones, etc. ] and exogenous (pore - forming toxins and bacterial rna, etc.) thus, it is the integration of multiple signals that activates the nlrp3 inflammasome to provide for a robust inflammatory response. the complement anaphylatoxin c5a is known to be a potent regulator of acute inflammatory responses and has been implicated to play a role in manifesting numerous inflammatory diseases, such as sepsis [5, 6 ]. c5a has two known receptors, c5ar1 (cd88) and c5ar2 (gpr77), which signal through phosphoinositide 3-kinase (pi3k), p38, and nuclear factor (nf)-b, amongst other pathways. it is well understood that c5a binds to and signals through c5ar1, a cell - surface g protein - coupled rhodopsin - like receptor, though the role of c5ar2 remains controversial [810 ]. however, even with c5ar2 's enigmatic function, c5a interactions with both receptors are important for eliciting the pathophysiology observed during sepsis. despite extensive efforts made to describe the crosstalk between c5a and tlr4 signaling [1215 ], no work to date has illuminated the ability of c5a to regulate tlr4-mediated nlrp3 inflammasome activation. furthermore, studies illustrating the interplay between complement and the inflammasome remain limited [1618 ]. in this study, we describe a mechanism by which c5a differentially modulates tlr4-induced nlrp3 inflammasome function in macrophages and monocytes. notably, c5a suppressed lps - induced nlrp3, il-1, and caspase-1 expression in mouse macrophages, leading to attenuated il-1 secretion following nlrp3 activation. conversely, acting through the mitogen - activated protein (map) kinase p38, c5a augmented the production of il-1 in lps - stimulated, ly6c monocytes. while this latter effect appeared to be entirely dependent upon c5ar1 signaling, our data surprisingly suggested that c5a 's suppression of the nlrp3 inflammasome in mouse macrophages might occur with limited dependency on c5ar1 engagement. together, these findings provide significant insight into the immunomodulatory role of c5a during acute inflammation in vivo, as wild - type mice produced significantly more il-1 during sublethal endotoxemia than their c5ar1 counterparts. procedures performed in this study were all in accordance with the us national institutes of health guidelines and were approved by the university of michigan committee on the use and care of animals. experiments were performed in male, age - matched c57bl/6 mice (1012 weeks old) purchased from jackson laboratories (bar harbor, me, usa) and c5ar1 mice bred and genotyped in facilities at the university of michigan (ann arbor, mi, usa). all mice were housed in pathogen - free conditions with free access to food and water. recombinant mouse c5a (r&d systems, minneapolis, mn, usa), lps (escherichia coli, o111:b4), adenosine triphosphate (atp, both from sigma - aldrich, st. louis, mo, usa), wortmannin (santa cruz biotechnology, santa cruz, ca, usa), and sb 203580 (invivogen, san diego, ca, usa) were all used at concentrations indicated in the figures. mice received a 10 mg / kg body weight intraperitoneal (i.p.) injection of lps. plasma was harvested after 8 hours by bleeding from the retroorbital venous plexus under isoflurane anesthesia. mouse peritoneal macrophages were harvested via the instillation and retraction of 8 ml sterile pbs (life technologies, carlsbad, ca, usa) 4 days following i.p. total bone marrow cells were harvested by flushing bilateral femurs with pbs, and erythrocytes were lysed in hypotonic buffer. all cells were cultured in rpmi 1640 media (life technologies) supplemented with 100 u / ml penicillin - streptomycin and 0.1% bsa. cell - free supernatants were stored at 80c until later use. to achieve nlrp3 inflammasome activation and il-1 secretion in vitro, 1 10 cells were incubated with lps in the copresence or absence of c5a for 4 hours followed by stimulation with atp for 45 minutes. in select experiments, cells were pretreated with signaling inhibitors 1 hour prior to the addition of lps and/or c5a. for mrna expression studies, total rna was harvested from cultured cells after lps treatment with or without c5a for 4 hours. cytokine concentrations were determined from log - transformed standard concentrations plotted on a standard curve. estimations of supernatant protein levels were determined by bicinchoninic acid assay (thermo fisher scientific, waltham, ma, usa), and 50 g total protein was loaded per well for page. supernatant protein was separated by sds - page and transferred onto nitrocellulose membranes (bio - rad, hercules, ca, usa). il-1 was detected using anti - mouse il-1 antibodies (r&d systems) followed by peroxidase - conjugated, rabbit anti - goat igg antibodies (jackson immunoresearch laboratories, west grove, pa, usa). total rna was isolated from cultured macrophages and bone marrow cells using trizol (sigma - aldrich). following purification, dnase (life technologies) was used to remove any contaminating genomic dna. cdna was generated using the reverse transcription kit provided by life technologies, and rt - pcr (sybr, life technologies) was performed on a 7500 real - time pcr system (applied biosystems, foster city, ca, usa). the following primers were used : gapdh, 5-cttcaacagcaactcccactcttcc-3 (forward), and 5-ggtggtccagggtttcttactcc-3 (reverse) ; nlrp3, 5-gagttcttcgctgctatgt-3 (forward) and 5-accttcacgtctcggttc-3 (reverse) ; il-1, 5-cctgctggtgtgtgacgttc-3 (forward) and 5-cagcacgaggcttttttgttgt-3 (reverse) ; asc, 5-gaagctgctgacagtgcaac-3 (forward), and 5-gccacagctccagactcttc-3 (reverse) ; caspase-1, 5-agatggcacatttccaggac-3 (forward), and 5-gatcctccagcagcaacttc-3 (reverse). cells were analyzed on a bd lsr - ii flow cytometer equipped with facsdiva software (both from bd biosciences, san jose, ca, usa). flowjo 7.6.4 software (tree star, ashland, or, usa) was used for data analysis. the following antibodies were used for cell - surface labeling : pe - anti - mouse cd45, pe - cy7-anti - mouse cd11b (both from ebioscience, san diego, ca, usa), apc - cy7-anti - mouse ly6c, and percp - cy5.5-anti - mouse ly6 g (both from bd biosciences). apc - anti - mouse il-1/il - f12 (r&d systems) was used for the intracellular labeling of il-1 in fixed and permeabilized (saponin) cells. data in this study (values expressed as means standard error of the mean) were analyzed using graphpad prism 6 graphing and statistical analysis software (graphpad inc. significance between multiple sample means was determined by one - way anova followed by tukey 's multiple comparisons test. where appropriate, significance between individual sample means was determined by two - tailed student 's t - test. given the importance of c5a - c5ar1 interactions in promoting the onset and development of sepsis, we sought to investigate the regulation of il-1 production by c5ar1 signaling in a tlr4-mediated, mouse endotoxemia model. injection of lps (10 mg / kg body weight), and plasma was harvested 8 hours later. as shown in figure 1, lps challenge in vivo strongly induced il-1 production in wild - type mice, which was significantly attenuated in the absence of c5ar1. attributable to a loss of c5ar1 signaling strongly suggested that c5a engages c5ar1 in vivo to amplify tlr4-mediated il-1 production in the endotoxemia model. assembly of the nlrp3 inflammasome is a major prerequisite to il-1 secretion in innate inflammatory cells that have been activated. given our observation that c5a modulated lps - induced il-1 production in vivo (figure 1), we sought to determine whether c5a functionally regulates nlrp3 inflammasome activation in macrophages. we harvested thioglycollate - elicited peritoneal macrophages and treated these cells with lps in the absence and copresence of various concentrations of c5a for 4 hours. using the inflammasome protocol, macrophages il-1 secretion was subsequently quantified by elisa in supernatant fluids of macrophages. in a dose - dependent manner, c5a significantly suppressed lps - induced macrophage il-1 release (figure 2(a) (top panel) and supplementary figure 1(a) in supplementary material available online at http://dx.doi.org/10.1155/2016/1340156). these data suggested that c5a suppresses tlr4-mediated inflammasome priming in macrophages, leading to attenuated il-1 secretion. to explore this hypothesis, we investigated the effects of c5a on the expression of pro - il-1, nlrp3, caspase-1, and asc (apoptosis - associated speck - like protein containing a carboxy - terminal card), an adapter protein that bridges the nlrs and caspase-1. for these mrna expression studies, we withheld atp treatment to specifically investigate c5a 's effects on the intracellular expression, rather than activation, of inflammasome machinery. in agreement with our il-1 protein data, the addition of c5a significantly dampened pro - il-1, nlrp3, and caspase-1 mrna expression levels in macrophages activated by lps (figure 2(a), middle and bottom panels). however, changes in asc mrna were refractory to lps and c5a cotreatment, and no effect was observed in the presence of c5a alone (figure 2(a), middle and bottom panels). these findings suggested that c5a reduces tlr4-mediated macrophage il-1 production via suppressed nlrp3 inflammasome priming. however, our data could not directly reconcile the observation that c5ar1 deficiency suppressed the in vivo il-1 response (figure 1). as the inflammatory response to endotoxin challenge involves several innate inflammatory cell types (e.g., macrophages, monocytes, and neutrophils), we considered the possibility that c5a may bidirectionally regulate nlrp3 inflammasome activation throughout the myeloid cell compartment to augment lps - induced il-1 production in vivo. to explore this, we harvested bone marrow cells from wild - type mice and induced inflammasome activation via cotreatment with lps followed by atp in the absence and copresence of various concentrations of c5a. as was the case for our macrophage studies, c5a was administered concomitantly with lps to investigate its potential regulation of inflammasome priming. interestingly, c5a robustly enhanced tlr4-mediated nlrp3 function in total bone marrow cells, and the magnitude of this response was largely sustained even upon treatment with a low dose (10 ng / ml) of c5a (figure 2(b), top panel). detection of the processed form (17 kda), but not unprocessed form (31 kda), of il-1 confirmed inflammasome activation under these cell culture conditions, albeit to a lesser extent than that observed for macrophages (supplementary figure 1). accordingly, qrt - pcr assays performed under atp - deficient conditions revealed that the addition of c5a significantly increased lps - activated pro - il-1 expression while also eliciting a nonsignificant, yet notable increase in nlrp3 mrna levels under these conditions (figure 2(b), middle panel). however, no changes in caspase-1 and asc expression were observed, and no effect was observed in the presence of c5a alone (figure 2(b), middle and bottom panels). taken together, our results indicated that c5a promotes il-1 production downstream of tlr4 activation in bone marrow cells, at least in part via transcriptional modulation of the nlrp3 inflammasome. as this effect was opposite the effect observed in macrophages (figure 2(a)), our data strongly suggested that c5a can bidirectionally regulate inflammasome responses throughout the myeloid cell compartment depending on the myeloid - derived cell type. since the bone marrow contains multiple cell types within the myeloid lineage, we next wanted to delineate which bone marrow cells were responsible for mediating c5a 's enhancement of nlrp3 function. monocytes and neutrophils are derived from a common myeloid progenitor in the bone marrow, and both contribute to pathogen clearance by activating inflammatory pathways. peripheral monocytes can be further subdivided into two major phenotypes based on the expression of ly6c [22, 23 ]. after being released from the bone marrow, ly6c inflammatory monocytes rapidly traffic to sites of inflammation where they respond to microbial stimuli and are thought to, over several days, differentiate into macrophages and dendritic cells [22, 23 ]. these cells are also capable of giving rise to ly6c monocytes, which are similarly recruited to sites of inflammation, but they appear to respond with different functions. thus, we decided to determine whether ly6c inflammatory monocytes or neutrophils in the bone marrow could demonstrate c5a - enhanced inflammasome responses. to investigate this, bone marrow cells were isolated from wild - type mice and treated with lps in the absence and copresence of c5a for 4 hours cells were then harvested and analyzed by flow cytometry to determine the relative contributions of cells to il-1 production by bone marrow monocytes and neutrophils. our gating strategy is outlined in figure 3(a) : within the cd45 leukocyte gate (gate 2), myeloid cells were first identified by cd11b expression (gate 3), and il-1 was detected intracellularly (gate 4). when bone marrow cells were cotreated with lps and c5a, a subset of myeloid cells demonstrated an il-1-high (il-1) phenotype, which was absent in the presence of either lps or c5a alone (figure 3(a)). monocytes and neutrophils were further defined by their expression of ly6c (cd11bly6c) and ly6 g (cd11bly6 g), respectively (gate 5). by determining the cell - surface phenotype of il-1 cells, our data revealed that most il-1-containing cells were cd11bly6c, whereas neutrophils represented only a small proportion of this il-1 population despite constituting the large majority of bone marrow cd11b cells (gate 5). furthermore, analyses of il-1 mean fluorescence intensity (mfi) demonstrated that monocytes had significantly upregulated lps - induced il-1 production when cotreated with c5a, whereas tlr4-mediated, neutrophil - specific il-1 production was not sensitive to c5a stimulation (figure 3(b)). a similar result was observed when we stimulated thioglycollate - elicited peritoneal neutrophils with lps and atp in in copresence of c5a. although c5a did elicit a significant increase in neutrophil il-1 secretion under these conditions (supplementary figure 2), its effects were modest when compared to the response exhibited by monocytes. taken together, our results suggested that ly6c inflammatory monocytes were the major cell type in the bone marrow to augment il-1 production when cotreated with lps and c5a, whereas the contribution to this response by neutrophils was marginal. therefore, these data suggest that ly6c monocytes, known to be released from the bone marrow during sepsis, are the target of c5a in vivo, which results in elevated il-1 production and release. however, our data do not exclude the possibility that c5a augments nlrp3 inflammasome function in these cells through an indirect, cell - extrinsic mechanism. as other myeloid subsets, including polymorphonuclear leukocytes (pmns), are known to activate in response to c5a, it is possible that the inflammatory milieu elicited by c5a, rather than c5a itself, is responsible for enhanced monocyte il-1 production under these conditions. exploration into a potential c5a - induced myeloid cell crosstalk mechanism will require follow - up experimentation. c5a stimulation of immune cells activates both the pi3k / akt and mapk / erk (extracellular signal - regulated kinase) signaling pathways [9, 1215 ]. as pi3k is known to mediate the anti - inflammatory effects of c5a on macrophage tlr4 signaling [1214, 25 ], we first investigated whether this pathway was also involved in c5a 's suppression of the nlrp3 inflammasome by using wortmannin, a selective pi3k inhibitor. as shown in figure 4(a) (upper panel) and supplementary figure 3(a), all concentrations of wortmannin tested (5,000, 500, and 50 nm) reduced the suppressive effects of c5a on lps- and atp - induced macrophage il-1 secretion. thus, these data suggested that pi3k is required for c5a 's inhibition of the il-1 response. however, when investigating c5a 's reliance on c5ar1 signaling for mediating its inflammasome - suppressing effects, we were very surprised to find that, similar to wild - type macrophages, c5ar1 macrophages demonstrated dampened il-1 production after treatment with lps and atp in the copresence of c5a (figure 4(a) (lower panel) and supplementary figure 3(b)). relative to positive control samples, c5a attenuated il-1 secretion by 38% in c5ar1 macrophages as compared to 55% in wild - type macrophages, thus representing only a modest loss of inhibition. this mechanism contrasts those elucidated in our previous studies, which have all implicated c5ar1 to be the critical c5a receptor that modulates lps - activated macrophage cytokine responses [1214 ], even when c5ar2 was shown to be functionally relevant. given this surprising result, we then wanted to explore the mechanism underlying c5a 's enhancement of lps - induced bone marrow il-1 production, which we largely attributed to augmented inflammasome activation in inflammatory monocytes (figure 3). as p38 signaling has been shown to potentiate il-1 and tnf- production in activated monocytes and to mediate c5a - enhanced monocyte il-6 secretion induced by lps, we investigated whether this pathway also assumed a functional role downstream of c5a to enhance the nlrp3 inflammasome response. to investigate this, we elicited il-1 secretion from bone marrow cells in the copresence of c5a and various concentrations of sb 203580 (a pharmacologic p38 inhibitor) and evaluated the effect of p38 blockade on inflammasome activation. as shown in figure 4(b) (upper panel) and supplementary figure 3(c), higher concentrations of sb 203580 largely negated the increase in lps - induced il-1 production evoked by c5a costimulation. thus, our results implicated p38 to act as a critical downstream target for c5a that elicits augmented inflammasome function in bone marrow cells. to then determine whether c5a depended on c5ar1 for potentiating the il-1 response, we performed our inflammasome activation protocol on bone marrow collected from c5ar1 mice and compared the subsequent il-1 response to that observed in wild - type bone marrow cells. in contrast to our macrophage studies, c5a 's enhancement of tlr4-mediated inflammasome activation in the bone marrow was entirely dependent on c5ar1 signaling (figure 4(b) (lower panel) and supplementary figure 3(d)). altogether, these data in conjunction with our flow cytometry results (figure 3)strongly suggested that, for inflammatory monocytes stimulated by lps, c5a engages c5ar1 to elicit downstream p38 signaling, which is critical for augmenting tlr4-mediated inflammasome activation and il-1 production. furthermore, as our in vivo data show (figure 1), this c5a - enhanced inflammasome function and il-1 release are likely important during experimental endotoxemia. in this study, we explored alterations in nlrp3 inflammasome function elicited by the complement anaphylatoxin c5a in vivo using a lps - induced endotoxemia model and in vitro by exploring innate inflammatory cell activation of nlrp3 in the absence and presence of c5a. interestingly, c5ar1 deficient mice had attenuated il-1 expression during experimental endotoxemia, thus suggesting that c5a - c5ar1 interactions in vivo augment the lps - activated il-1 response. this is in agreement with previous studies that have described a critical role for c5ar1 engagement in exacerbating sepsis immunopathology. given this result, we hypothesized that c5a modulates tlr4 signaling throughout the myeloid compartment to enhance nlrp3 inflammasome function. recent studies by an. have suggested that c5a was related to the generation of downstream monosodium urate / uric acid (msu) crystals during sterile inflammation, resulting in activation of monocyte inflammasome priming. although macrophage regulation was not explored under these conditions, their studies showed that c5a alone stimulated human monocyte pro - il-1 expression, which then required msu crystals for maximally activating nlrp3 function. in our hands, c5a similarly enhanced il-1 production in inflammatory monocytes, while suppressing this pathway in mouse macrophages through the attenuation of inflammasome transcriptional priming. however, in contrast to the mechanism proposed by an., we could not demonstrate that c5a by itself serves as a priming stimulus to modulate the transcription of nlrp3 machinery and/or its substrates. rather, our observations indicated that c5a depends on the copresence of a potent priming stimulus (e.g., lps) for regulating nlrp3 activity. one is that the mechanisms underlying c5a 's regulation of the inflammasome in human and mouse monocytes might be fundamentally different. another explanation stems from the concentration - dependency of c5a - regulated immune responses, which has been described in numerous studies. specifically, an. documented pro - il-1 expression occurring in human monocytes when c5a was administered at moderately low concentrations (50100 ng / ml), whereas c5a concentrations outside of this range did not elicit significant changes in inflammasome responsiveness. in our experiments, high c5a concentrations (1001,000 ng / ml) potently regulated lps - activated transcriptional priming, though this c5a treatment by itself did not enhance or suppress nlrp3 function. it is worth noting that low c5a concentrations were not as effective at modulating tlr4-mediated il-1 secretion as were high c5a concentrations. taken together, we conclude that the nature of c5a - nlrp3 crosstalk is likely dependent on both the copresence / absence of canonical nlrp3 priming stimuli along with the amount of c5a generated during an inflammatory response. bell - curve - like mechanism during sterile inflammation where, in the absence of pamps, inflammasome priming is regulated according to a small window of low c5a concentrations. however, our data show that during septic inflammation, c5a tunes strong tlr4-mediated inflammasome responses over a broader range of c5a levels with greater modulation occurring at higher concentrations. understanding how the complex crosstalk between complement, pamps, and damps regulates upon addressing the receptor dependency underlying c5a 's modulatory effects, we were very intrigued to find that while c5a required c5ar1 for enhancing inflammasome - mediated monocyte il-1 responses, its suppression of this pathway in macrophages was only moderately lost in the absence of c5ar1. this result suggested that c5ar2as opposed to c5ar1might play a more prominent role in regulating macrophage il-1 release, which contrasts its function in the context of other cytokine responses [1215 ]. additional studies using c5ar2 mice are needed to substantiate this hypothesis, though if c5ar2 is required for c5a 's suppression of nlrp3 inflammasome function, we are interested to investigate the il-1 phenotype of c5ar2 endotoxemic mice. it may be that macrophage c5ar2 engagement in vivo acts to prevent lps signaling from tipping the balance towards dysregulated inflammasome responses. as c5ar2 has been previously suggested to assume an anti - inflammatory function [28, 29 ], we would like to explore whether this receptor is critical for attenuating il-1-related immunopathology. however, given c5ar2 's enigmatic functions, it is unclear as to why c5a might favor this receptor over c5ar1 for suppressing the inflammasome. c5ar2 has been previously shown to signal downstream to pi3k, which is in agreement with our signaling data, though unraveling what happens proximal to pi3k activation to allow for noncanonical receptor specificity necessitates further investigation. in addition, while our data showed that c5a differentially regulated myeloid cell nlrp3 inflammasome function by activating divergent signaling pathways potentially due to disparate receptor dependencies we also can not rule out the possibility that c5ar1 expression differences amongst myeloid subsets might be important for regulating il-1 release. however, as comparable levels of c5ar1 have been previously quantified on murine monocytes and thioglycollate - elicited macrophages, we do not expect this to be the sole causal mechanism underlying our observations. while our in vitro studies suggested that c5a directly modulates tlr4 signaling to influence nlrp3 inflammasome transcriptional priming, it is likely that this regulation in vivo is multifaceted. specifically, our endpoints addressed mechanisms that would largely suggest cell - intrinsic nlrp3 regulation by c5a, but we can not rule out the possibility that c5a might modulate inflammasome activation via cell - extrinsic effects. a mechanism of interest to our group involves c5a - mediated neutrophil activation. specifically, we have shown that the c5a generated during acute lung injury and sepsis induces a robust release of nuclear histones from neutrophils, which has both lymphodepleting and pathophysiologic consequences [32, 33 ]. furthermore, recent studies have characterized extracellular histones as potent activators of the nlrp3 inflammasome, behaving as both signal 1 and signal 2 stimuli [3436 ]. thus, the augmented il-1 production elicited by c5a in our bone marrow stimulation experiments may have involved a neutrophil - driven histone release step. similarly, it is possible that proinflammatory cytokines and chemokines released by neutrophils following c5a stimulation might also play a prominent role in regulating the downstream monocyte il-1 response. these are intriguing hypotheses, as c5a - mediated inflammasome transcriptional modulation appeared to be less prominent in bone marrow cells, suggesting the potential involvement of cell - extrinsic monocyte regulatory mechanisms. furthermore, although our data demonstrated that c5a does not potently regulate neutrophil il-1 secretion, as mediators of c5a - induced histone release, these cells might play a critical role in eliciting the c5a - enhanced nlrp3 activation that we observed during physiologic endotoxemia. studies performed under neutrophil- and/or histone - depleting conditions are required to determine the functional relevance of this pathway. finally, while our experiments with bone marrow cells demonstrated that nlrp3 enhancement in inflammatory monocytes was uniquely sensitive to c5a stimulation, additional in vivo work is needed to understand the contribution of these cells to the inflammatory response observed during endotoxemia. ly6c monocytes in the bone marrow are mobilized in response to inflammatory stimuli and those coexpressing high levels of cc - chemokine receptor 2 (ccr2) traffic to sites of infection where ccr2 ligands are produced. once in tissues, these cells are capable of differentiating into a myriad of myeloid subsets with varying phenotypic and functional features, such as migratory dendritic cells (dcs), inflammatory macrophages, and/or tnf- and inos- (inducible nitric oxide synthase-) producing dcs. although our in vitro data suggested that inflammatory monocytes might be the target of c5a - augmented nlrp3 function during lps challenge in vivo, it remains to be determined whether they are indeed important for robust il-1 production. in addition, it would be beneficial to characterize these cells ' ultimate fate, and understand whether monocyte sensitivity to c5a - enhanced inflammasome function is maintained or lost upon achieving terminal differentiation. the results of our study suggest that c5a - c5ar1 interactions are critical for enhancing the innate il-1 response during lps - induced endotoxemia. in addition, our findings show for the first time that c5a bidirectionally regulates nlrp3 inflammasome activation throughout the myeloid cell compartment. specifically, c5a engages c5ar1 to augment tlr4-mediated il-1 production in inflammatory monocytes via the p38 mapk signaling pathway, while it suppresses macrophage il-1 secretion independent of c5ar1 engagement via the pi3k pathway. follow - up experiments will be required for understanding the relative contributions of these two mechanisms in eliciting the net il-1 response observed during sepsis. taken together, our data provide novel insight into the mechanisms underlying c5a 's immunomodulatory functions during acute inflammation while further characterizing nlrp3 regulation in vivo and in vitro. this crosstalk is likely to play a prominent role in regulating the il-1 produced during numerous inflammatory disease states in which complement, pamps, and damps are present. | c5a is an inflammatory mediator generated by complement activation that positively regulates various arms of immune defense, including toll - like receptor 4 (tlr4) signaling. the nod - like receptor pyrin domain - containing protein 3 (nlrp3) inflammasome is activated by pathogen products and cellular / tissue damage products and is a major contributor of il-1. in this study, we investigate whether c5a modulates lipopolysaccharide- (lps-) induced nlrp3 inflammasome activation in myeloid cells. appearance of plasma il-1 during endotoxemia was reduced in c5ar1/ mice when compared to wild - type mice. in vitro, c5a significantly enhanced lps - induced production of il-1 in bone marrow ly6c - high inflammatory monocytes, accompanied by augmented intracellular pro - il-1 expression. this effect was abolished during p38 blockade by sb 203580 and in the absence of c5ar1. conversely, c5a suppressed lps - induced macrophage production of il-1, which was accompanied by attenuated levels of pro - il-1, nlrp3, and caspase-1 expression. c5a 's suppressive effects were negated during phosphoinositide 3-kinase (pi3k) inhibition by wortmannin but were largely preserved in the absence of c5ar1. thus, c5a bidirectionally amplifies tlr4-mediated nlrp3 inflammasome activation in monocytes while suppressing this pathway in macrophages. however, as c5ar1 deficiency attenuates the il-1 response to lps challenge in vivo, our results suggest overall that c5a augments physiologic inflammasome responses. |
the blowflies (c. putoria) were reared at the laboratory for the study of diptera, department of microbiology and parasitology, universidade federal do estado do rio de janeiro. adults were obtained from a stock colony originated from insects collected at the rio de janeiro zoo, quinta da boa vista park, city of so cristvo, rj. three traps following the model of mello. adults and larvae of muscoid flies were taken to the laboratory, where they were sorted and identified following mello (2003). experimental flies were reared in plastic cages (40 by 30 by 20 cm) with an opening at the top for aeration and an anterior opening to allow access to the inside of the cage, which was covered with escaline - coated fabric. each cage received water, honey and water (50%) solution, and chicken gizzard or beef (which are protein sources and serve as substrate for oviposition and ovary maturation). first - instar larvae of the third laboratory generation were transferred with the help of a fine paintbrush to 100-ml beakers containing 60 g gizzard / agar 65% homogenate (ferraz. 2012). this diet was selected because it is practical and sterile (autoclaved), allowing the antibiotic to act fully and only on the larvae. each repetition received 1 ml of the antibiotic, resulting in the following three different diet concentrations : 4.44 m / ml, 13.33 mg / ml, and 66.66 mg / ml. the concentrations were chosen from the serum gentamicin concentration (established according to the dosage indicated in the prescription advisor), as follows : 23 daily intravenous or intramuscular doses ; maximum concentration 30 min/1 h after administration : 46 g / ml. avoid concentrations above 12 g / ml for extended periods (gentamicina 2011). the first concentration (t1) is close to the intravenous serum concentration ; the second (t2) is close to the maximum intravenous serum concentration ; the third (t3) is approximately five times the maximum serum intravenous concentration. each antibiotic concentration tested and the control were replicated four times using 40 larvae in each repetition. each beaker was put into a larger beaker containing sterilized sawdust, which was sealed with elastic escaline. the sawdust serves as a pupation substrate for the larvae after they abandon the diet. larvae were kept in a climatic chamber at 30c per day and 28c per night, 70 + 10% relative humidity (rh), and 14-h photoperiod. observations were made daily, always at the same time (12 h). as a precaution, all treatments in this study ensured that each individual larva had > 1 g of food available. we ensured this quantity because aguiar - coelho and milward - de - azevedo (1996) had previously suggested that this is the optimal amount of meat for rearing calliphoridae. ensuring that larvae have the optimal amount of food prevents exploitative competition and the stress associated with it, and with the chemical changes in the food substrate resulting from larval metabolism (khazaeli. the body mass after diet abandonment was recorded in batches of five larvae, with the help of an analytical scale. larvae were then stored in test tubes sealed with nylon and elastic fabric for further observation. the dates of pupation and emergence, as well as the sex ratio and morphological abnormalities in the adults, when present, were recorded. the program microsoft excel was used to analyze the raw data, and further analyses were conducted in the program past. variations in the mean body mass of larvae and the duration of larval and pupal stages and total development (from neolarvae to adult) were analyzed using the student s t - test (= 5%). the four treatments did not differ significantly in the following parameters : body mass of c. putoria larvae (table 1) ; mean duration of larval inoculation to abandonment ; and larval, pupal, and total development times (d) (table 2). table 1.body mass of larvae (g) of the blowfly c. putoria (wiedemann, 1830) (diptera, calliphoridae) after four treatments with different concentrations of gentamicin, reared in a climatic chamber (30c day and 28c night, 70 + 10% rh, and 14-h photoperiod)treatmentmean individual mass (g) standard deviationvariation intervalp = significance value (t - test)controlt1t2t3control0.049a 0.0070.0350.0590.5690.2390.483t10.053a 0.0120.0210.0680.5690.9090.974t20.054a 0.0030.0410.0620.2390.9090.826t30.053a 0.0080.0370.0640.4830.9740.826means followed by same letter in same column do not differ significantly by the t - test, 5% confidence. control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 g / ml ; t2 = gizzard homogenate agar + gentamicin 13.33 g / ml, t3 = gizzard homogenate agar + gentamicin 66.66 g / ml. table 2.average duration of the postembryonic development stages (d) of larvae of the blowfly c. putoria (wiedemann, 1830) subjected to four treatments with different concentrations of gentamicin and reared in a climatic chamber (30c day and 28c night, 70 + 10% rh, and 14-h photoperiod)daysvariation intervalstandard deviationp = significance value (t - test)controlt1t2t3duration until abandonmentcontrol4.478a450.5170.2140.4880.418t14.114a450.0890.2140.6450.150t24.225a450.4500.4880.6450.231t34.989a461.0560.4180.1500.231larval stagecontrol5.477a560.5160.5280.5730.494t15.270a560.3400.5280.9860.264t25.275a560.4410.5730.9860.287t35.845a570.8710.4940.2640.287pupal stagecontrol3.874a350.2140.4060.1810.652t13.610a360.5510.4060.1630.741t24.057a450.1140.1810.1630.278t33.741a250.5180.6520.7410.278total stagecontrol9.339a9100.3220.1240.9540.532t18.785a8110.5300.1240.1680.139t29.323a9100.4350.9540.1680.531t39.642a9110.8540.5320.1390.531means followed by same letter in same column do not differ significantly by the t - test, 5% confidence. control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 g / ml ; t2 = gizzard homogenate agar + gentamicin 13.33 g / ml, t3 = gizzard homogenate agar + gentamicin 66.66 g / ml. body mass of larvae (g) of the blowfly c. putoria (wiedemann, 1830) (diptera, calliphoridae) after four treatments with different concentrations of gentamicin, reared in a climatic chamber (30c day and 28c night, 70 + 10% rh, and 14-h photoperiod) means followed by same letter in same column do not differ significantly by the t - test, 5% confidence. control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 g / ml ; t2 = gizzard homogenate agar + gentamicin 13.33 g / ml, t3 = gizzard homogenate agar + gentamicin 66.66 g / ml. average duration of the postembryonic development stages (d) of larvae of the blowfly c. putoria (wiedemann, 1830) subjected to four treatments with different concentrations of gentamicin and reared in a climatic chamber (30c day and 28c night, 70 + 10% rh, and 14-h photoperiod) means followed by same letter in same column do not differ significantly by the t - test, 5% confidence. control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 g / ml ; t2 = gizzard homogenate agar + gentamicin 13.33 g / ml, t3 = gizzard homogenate agar + gentamicin 66.66 g / ml. the rates of diet abandonment, pupation, and emergence (%) obtained for c. putoria in the four different treatments (control = gizzard and agar homogenate ; t1 = gizzard and agar homogenate + gentamicin 3.33 mg / ml ; t2 = gizzard and agar homogenate + gentamicin + 13.33 mg / ml ; t3 = gizzard and agar homogenate + gentamicin 66.66 mg / ml) is shown in fig. diet abandonment peaked in the control group between 5 and 6 d, and predominantly on the fifth day in the three treatments. in t3, and only in this treatment, larval abandonment lasted until the seventh day. the pupation peak of control flies happened between the sixth and seventh days, whereas in the other treatments, it happened on day 6, extending up to day 8 in t3. in all treatments, the emergence peak occurred on day 10, lasting to day 12 only in t1 and t3. 1.rate of abandonment of the diet, pupation, and emergence (%) of c. putoria (wiedemann, 1830) subjected to four treatments : control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 mg / ml, t2 = gizzard homogenate agar + gentamicin 13.33 m / ml, t3 = gizzard homogenate agar + gentamicin 66.66 mg / ml. rate of abandonment of the diet, pupation, and emergence (%) of c. putoria (wiedemann, 1830) subjected to four treatments : control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 mg / ml, t2 = gizzard homogenate agar + gentamicin 13.33 m / ml, t3 = gizzard homogenate agar + gentamicin 66.66 mg / ml. the emergence rate of c. putoria males and females was similar in the control and t3 (fig. 2). in t1, males emerged first (beginning on day 9) and continued to do so up to day 12. females, in contrast, began to emerge a day later (day 10) and continued up to day 11. in t2, all female flies emerged on day 10, whereas males emerged from days 10 to 11. the sex ratio for each treatment was as follows : control, males = 47%, females = 53% ; t1, males = 48%, females = 52% ; t2 males = 50%, females = 50% ; and t3, males = 51%, females = 49%. the chi - square tests revealed that the sex ratios obtained for the four treatments did not differ from the expected (control : = 0.398, t1 : = 0.221, t2 : = 0.006, t3 : = 0.081 ; tabulated = 3.84, df = 1, = 5%). 2.rate of emergence, in days, of males and females c. putoria (wiedemann, 1830) subjected to four treatments : control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 mg / ml, t2 = gizzard homogenate agar + gentamicin 13.33 m / ml, t3 = gizzard homogenate agar + gentamicin 66.66 mg / ml. rate of emergence, in days, of males and females c. putoria (wiedemann, 1830) subjected to four treatments : control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 mg / ml, t2 = gizzard homogenate agar + gentamicin 13.33 m / ml, t3 = gizzard homogenate agar + gentamicin 66.66 mg / ml. all adults in the control and in the other treatments were normal. as for larval viability, only t2 differed significantly from the control (table 3). table 3.anova comparison of the viability of larvae, pupae, and total (neolarvae to adult) of c. putoria (wiedemann, 1830) subjected to four treatments : control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 mg / ml, t2 = gizzard homogenate agar + gentamicin 13.33 m / ml, t3 = gizzard homogenate agar + gentamicin 66.66 mg / mlviability (%) p = significance value (anova)controlt1t2t3larvalcontrol81.88a0.1960.0060.874t190.63ab0.1960.3260.419t296.88b0.0060.3260.185t380.00ab0.8740.4190.185pupalcontrol93.60a0.2630.7390.228t175.40a0.2630.2870.468t292.26a0.7390.2870.234t386.85a0.2280.4680.234totalcontrol76.88a0.7170.0730.517t170.63a0.7170.2840.948t289.38a0.0730.2840.093t369.38a0.5170.9480.093anova, analysis of variance. anova comparison of the viability of larvae, pupae, and total (neolarvae to adult) of c. putoria (wiedemann, 1830) subjected to four treatments : control = gizzard homogenate agar ; t1 = gizzard homogenate agar + gentamicin 3.33 mg / ml, t2 = gizzard homogenate agar + gentamicin 13.33 m / ml, t3 = gizzard homogenate agar + gentamicin 66.66 mg / ml anova, analysis of variance. means followed by the same letter do not differ at 5% significance. previous studies have detected toxins and controlled substances in samples collected from carrion in advanced stage of decomposition and the insects that visit them (goff and lord 2001). in some studies, decreased larval mass gain, and larval growth, as well as reduced larval activity, were correlated with the presence of certain drugs (soto 2008). (2012), who reared experimental larvae on meat, gizzard, and gizzard and agar homogenate, the mean larval mass obtained in our study was similar in all treatments. in the treatments, the total duration of development increased with increased antibiotic concentrations after all treatments in our study, the larval stage lasted longer than in the study of ferraz. this was expected, because meat is the natural diet of this species (leal. (2006) found that another blowfly, cochliomyia macellaria (f.) (diptera : calliphoridae), when reared under sterile conditions, developed faster and survived longer than flies reared in the presence of bacteria. in this study, no significant differences in the rate of diet abandonment, pupation, and adult emergence were found between the control and each of the treatments. according to our results (2009), who tested the effect of morphine on calliphora stygia (diptera : calliphoridae). however, in a study on the effects of cocaine on the development of c. putoria (reared on liver containing the substance), treatment larvae developed significantly faster, pupated, and emerged sooner than control larvae (carvalho 2004). other substances such as diazepam and anfepromona also accelerated the development of c. putoria larvae (carvalho. in contrast, the painkiller scopolamine slowed down larval development in treatments containing higher concentrations of the substance (grella. 2007), and buscopan accelerated the rate of growth of chrysomya megacephala (diptera : calliphoridae) (oliveira. 2009). additionally, a study on the effect of malathion on c. megacephala revealed that this insecticide can alter ipm estimates in up to 36 h (liu. 2009). in this study, the only statistically significant effect of gentamicin was on the viability of t2 larvae. larvae were more viable after this treatment than the others, although viability after all treatments was high. in diptera, viabilities above 60% are considered promising (loureiro. 2005). in a study using artificial diet plus scopolamine to rear c. putoria flies, mortality was higher with increased drug concentrations (grella. it is also possible that, in our study, the drug was not metabolized by the larvae. this is consistent with the results of grella and thyssen (2008), who reared chrysomya albiceps, c. megacephala, and c. putoria on artificial diet plus oxycodone hydrochloride and failed to find significant differences in development of the control and the experimental groups. larvae are able to efficiently eliminate various toxic substances during development (nuorteva and nuorteva 1982). also, at least in two other studies, treatments containing low drug concentrations paradoxically resulted in higher survival rates than the control (goff. this percentage is considered appropriate and is higher than the values obtained by soto (2008) for c. putoria larvae reared on artificial diet plus chicken heart, both for the control and the treatments with barbiturates. total survival was above 69% in all treatments, being higher than in the control and other treatments in the results of soto (2008). (1995) for several different larval densities of chrysomya ranged between 60 and 98%, also consistent with the findings of this study. the sex ratio found in our study, after chi - square analysis, was 1:1, indicating that the population is stable (fisher 1930). additionally, 100% of the control and treatment flies were normal, indicating that the flies metabolic adjustments buffered or neutralized the effects of the antibiotics (lomnaco and germanos 2001). this result is consistent with the effects of method on the sex ratio of lucilia sericata (diptera : calliphoridae). however, this substance delayed the development of those flies and altered their mortality rates (gosselin. entomotoxicology is the analysis of substances in necrophagous arthropods to identify drugs and toxins present in dead tissues, their effects on the development of arthropods, and how they interfere with ipm estimates (goff and lord 2001). such analyses are particularly relevant when blood, urine, and the internal organs of a body are no longer available. according to liu. (2009), understanding the effects of drugs and toxins on the development of flies is very important to make accurate ipm estimates. though laboratory studies to ascertain how certain drugs affect the development of flies can be made using rodents, rats, and rabbits (george. 2012), artificial diets containing these substances are a viable alternative. in the study of gosselin. (2011), 100 g of bovine heart plus 8% gelatin was given to every 50 larvae. / agar homogenate, this diet is low cost and is very rich in nutrients. because the final product is liquid, it can be easily mixed with different concentrations of gentamicin. two grams of diet in four repetitions were made available to the larvae, because this quantity is ideal for the development of this species and results in normal body mass, growth rate, viability, and sex ratio (ferraz. even though forensic entomologists have been conducting ipm calculations in different countries, a standard practice has not been adopted yet. united states statistics show that overdosing or drug incompatibility may lead to death, especially in older individuals. therefore, studies such as ours may foster the precision of pmi estimates using c. putoria, especially when antibiotics have been previously used and/or have been the cause of death. gentamicin did not alter the parameters tested for c. putoria in this work : body mass, larval, pupal, and total development. therefore, ipm calculations based on the biological parameters of this fly will not be affected when this antibiotic is present. | we evaluate the effects the antibiotic gentamicin on the development of chrysomya putoria (wiedemann, 1818). third - generation, first - instar larvae were reared in a climatic chamber on 60 g of homogenate + agar 65% and were treated with three concentrations of gentamicin : 4.44 mg / ml, 13.33 mg / ml, and 66.66 mg / ml. the control consisted of distilled water. the relationships between mean body mass of mature larvae (measured after diet abandonment, in batches of five individuals), duration of larval and pupal stages, and overall duration of development were analyzed. the actual sex ratio was compared against the expected using the chi square. none of the parameters measured differed significantly among the four treatments, with one exception : when gentamicin concentration was 13.33 mg / ml, larval viability differed significantly from the control. all larvae from all treatments were considered normal. we conclude that the antibiotic did not significantly alter the development of c. putoria (wiedemann) (diptera : calliphoridae). |
research by rodriguez (1) has asserted mcq items using three response options (one correct answer with two distractors) is comparable to, and possibly preferable over, traditional mcq item formats consisting of four response options (e.g., one correct answer with three distractors), or five response options (e.g., one correct answer with four distractors). some medical educators have also adopted the practice of using 3-option responses on mcq exams as a response to the difficulty experienced in generating additional plausible distractors. while it would seem solving the problem of creating a third or fourth plausible distractor could be fairly easily alleviated by tapping the expertise of one s colleagues, advanced students / residents, etc. (2 - 4), some remain committed to administering items with 3-option responses. while there is some research (and continually budding interest from faculty) supporting the use of items with 3-option responses, we must consider what effects this might have on guessing strategies and cut score determination decisions so as to avoid any unintended consequences (5). thus, the purpose of this work is to further explore these critically important considerations that have largely gone ignored in the medical education literature to this point. guessing on multiple - choice examinations (mcqs) has long been recognized as a serious threat to score validity. psychometricians generally have recognized three types of guessing : 1) informed guessing ; 2) cued guessing ; and 3) random guessing (6 - 7). while most educators recognize informed and cued types of guessing behaviors are common on medical school examinations, most educators have largely dismissed the notion that one could attain a desirable score result while relying purely on random guessing. after all, on the surface this appears to be a very poor strategy for completing an examination. however, let us look more closely at the impact of this particularly unassuming type of guessing behavior. with random guessing, an examinee s odds of correctly answering any given item increase with fewer response options. for example, for someone guessing purely at random, s / he would have a 1 in 5 (20%) chance of correctly answering an item with 5 response options ; a 1 in 4 (25%) chance of correctly answering an item with 4 response options ; and a 1 in 3 (33%) chance of answering an item with 3 response options. one may calculate the probability that an examinee will answer a given number of items correctly on any exam (of any length) by using the cumulative binomial distribution in excel. the formula for calculating these probabilities is presented below : p = (1 - binom.dist (a, b, c, true)) + binom.dist(a,b,c,false) where, p is the probability of an examinee getting the score of interest (or higher) by randomly guessing during a single administration, a is the score of interest (e.g., the number of correct responses), b is the length of the exam, and c is the probability of getting an item correct when randomly guessing (e.g., 1 divided by the number of response options). to illustrate why random guessing is a problem that medical educators should take more seriously, especially when cut scores are used, let us consider a hypothetical example. imagine a medical education scenario in which students complete an exam consisting of 10 items. the instructor opted to administer only 10 items because the exam is considered particularly rigorous due to challenging items assessing very complex material. further, the instructor opted to utilize a 3-option format for responses based on literature suggesting 3 options are largely comparable to 4 options when it is difficult to generate an effective third distractor. now, suppose the instructor conducted a standard setting exercise with colleagues and determined the following raw score performance ranges were appropriate and defensible : 0 - 4 = fail ; 5 - 7 = pass ; 8 - 10 = honors. in such a scenario, we used the cumulative binomial distribution formula presented previously to determine the probability that a student utilizing a random guessing strategy would attain a given number correct (see table 1). probability of attaining a given raw score with 3 response options on a 10-item test this example demonstrates that a student guessing completely at random would have a.2064 (about a 20.64%) probability of passing this exam. pass verdict is much too large, thus the current cut score between a pass / fail decision must be revised. with most education research it is customary to use a 95% confidence level, meaning education researchers are willing to accept a 5% chance of making an error. in this example, this would mean we must move the minimum cut score from 5 (50% correct in order to attain a pass verdict) to 7 (70% correct in order to attain a pass verdict) in order to ensure that no examinees would be likely to pass the exam by simply guessing at random. as stated previously, because this exam is particularly difficult a shift of the cut score from 5 to 7 would likely be indefensible. this creates quite the dilemma for a medical educator wishing to administer a rigorous assessment while maintaining fair and defensible performance standards. now, let us look at the same example using 4- and 5-option responses. in this scenario, let us assume the instructor was able to generate three and four plausible distractors, respectively, for each of the 10 items. the probability that a student utilizing a random guessing strategy would attain a given number correct is presented in table 2. probability of attaining a given raw score with 4- and 5- option responses in this scenario, a student guessing completely at random would have approximately a.07 (or about an 8%) probability of passing this exam when each item consisted of 4-response options, and about a.03 (about 3%) probability of passing when each item consisted of 5- response options. in both instances, the probability of passing the exam is much smaller than the exam consisting of 3-response options. interestingly, though, the exam consisting of 4-response options still produces a probability of passing that is greater than the typical 5% threshold we would prefer. if using 4-response options, the instructor would likely still need to revise the cut score in this scenario. the result would be a move from 5 (50% correct in order to attain a pass verdict) to 6 (60% correct in order to attain a pass verdict) in order to ensure that examinees would be unlikely to pass the exam by simply guessing at random. although this shift in the cut score seems less egregious than the shift given 3-response options, it still may be problematic and indefensible in such a scenario. so, what exactly does this mean for medical educators conducting routine classroom assessments ? while there is research and a continual budding of support from medical educators to support the use of mcq items with 3 response options, it is critical that one remains cognizant of what effect this will have on guessing strategies, and on cut score determination decisions. simply put, exams consisting of items with fewer response options will increase the likelihood that examinees utilizing a random guessing approach will pass that exam. the choice of using 3, 4 or 5 option responses also has significant implications with regard to setting cut scores and various performance standards. most medical school classroom assessments carry moderate - to - high stakes for students, thus it is imperative that the placement of a cut score is at a position in which it is highly unlikely an examinee could achieve by random guessing. of course, any decision about what constitutes highly unlikely is arbitrary, but given the conventions of education research a level of 5% seems reasonable for classroom assessments. table 3 provides recommendations for cut score thresholds for the minimum performance category (e.g., the threshold for a pass / fail decision) relative to exam length (from 10 to 100 items, by increments of 5) and the number of item response options. minimum recommended raw cut score placements for items with 3, 4, and 5 options a visual scan of table 3 illustrates the impact random guessing may have on minimum performance category threshold decisions. the table illustrates the number of items that would need to be answered correctly in order to be confident within a 5% error tolerance that the response pattern was not due to random guessing. specifically, the fewer response options provided, the greater the minimum (raw) cut score need be. for example, an exam consisting of 50 items would require a minimum (raw) cut score of 23 if the items consisted of 3 response options, and a minimum (raw) cut score of 16 if the items consisted of 5 response options to ensure examinees could not achieve the lowest meaningful performance category by random guessing given a 5% maximum error tolerance. the difference of requiring correct answers to 7 additional items when fewer options are presented offers a number of additional considerations for medical educators. specifically, what impact might the decision to use items with 3-response options relative to traditional item formats with 4- or 5-response options have on setting fair and defensible standards ? clearly, fewer response options equate to a higher minimum cut score, but what effects might this have on the substantive meaning of that minimum cut score ? what additional unintended consequences might result from using fewer response options ? practically speaking, mcqs with 3-options are typically easier to write because they require development of fewer plausible distractors than 4- and 5-option mcqs. educators should recognize that implausible distractors add to test error and therefore are not contributing meaningfully (in fact, they are doing quite the opposite). item distractors are often written to diagnose common misconceptions (e.g., order of operations) about a topic. it benefits medical educators when others, such as fellow content experts or residents, review items and their distractors for plausibility. in addition to distractor plausibility concerns, the amount of time per item can be problematic when trying to assess multiple content or competency domains. examinees sometimes run out of time and render random guesses in hopes of gaining additional points. when this occurs, scores are contaminated with error and the resulting measure of student performance will be inaccurate. proponents for 3-option responses contend examinees will require less time to complete each item, and may lead to more valid score results. alternatively, modifying exams in which a significant number of students have historically run out of time could give better estimates of difficulty and reliability than previous versions. however, in reducing the number of distractors that an item has, there is usually a small decrease in each item s difficulty and the exam s overall reliability (1). this translates to a need for more high - quality items per exam to compensate for diminished difficulty and reliability. it is well - documented that offering a mix of easy, moderate, and difficult items tends to increase reliability estimates (8). however, medical education classroom assessments are notorious for suffering from low reliability estimates, largely due to over - representation of easy items (p - values0.75). the extent to which items are easy due to their complexity, an artifact of student learning, instructional familiarity effects (9), or some other factor certainly must be considered as well. as royal and guskey (10) note, in the context of classroom assessment, when teaching is effective and learning is successful score results should resemble a negatively skewed distribution with few, if any, students receiving low marks. thus, the importance of reliability as an indicator of quality is often deceiving under these circumstances and becomes a less important indicator of quality. discrimination coefficients are often helpful for informing instructors of whether or not students performed as expected. to that end, instructors can have some, albeit subjective, estimate of the degree to which the exam functioned as intended when administered to the given sample. it should be noted that discrimination coefficients simply need to be positive (e.g., 0.01 or higher), as opposed to the guidelines proposed for norm - referenced examinations (e.g., greater than 0.2, 0.3, or 0.4) which have largely been misunderstood by most medical educators (11). that is, because classroom assessments often yield high scores for examinees and items difficulty estimates tend to indicate most items are easy, the discrimination coefficient becomes less important in this context than it would be in a norm - referenced assessment scenario (e.g., mcat exam, etc.) (10,12). exam length is another important consideration, as having too few items creates range issues and inflates error estimates. therefore, educators need to ensure there are enough items to adequately measure students abilities, while simultaneously ensuring someone can not pass an exam by utilizing a random guessing strategy. research by fisher (13) indicated exams should consist of a minimum of 25 items, when possible, because measures produced with this many items yield error estimates that generally are stable while reliability estimates can still reach levels exceeding 0.90. as a general rule, the more items administered the more statistically stable the scores will be ; however, at some point the cognitive load for examinees may become too great or examinees may have insufficient time, so these factors need to be appropriately considered and properly accounted for when determining an appropriate number of items to administer (14). of course, some individuals familiar with psychometric models might argue the solution for modeling guessing is simple : use a 3-parameter logistic (3-pl) item response theory (irt) measurement model to account for a pseudo - guessing parameter to adjust students scores. while this approach is certainly a possibility for individuals working in large - scale, medical licensure and certification testing environments, it is entirely inappropriate for classroom assessments as 3pl models require a minimum sample size of 1,000 examinees (15 - 17). therefore, corrections for guessing using 3-pl irt models is not a viable option for medical education classroom assessments. the use of rasch measurement models, however, is a possibility for classroom assessments (11) and may offer insights about guessing (18,19). guessing on multiple - choice examinations (mcqs) has long been recognized as a serious threat to score validity. psychometricians generally have recognized three types of guessing : 1) informed guessing ; 2) cued guessing ; and 3) random guessing (6 - 7). while most educators recognize informed and cued types of guessing behaviors are common on medical school examinations, most educators have largely dismissed the notion that one could attain a desirable score result while relying purely on random guessing. after all, on the surface this appears to be a very poor strategy for completing an examination. however, let us look more closely at the impact of this particularly unassuming type of guessing behavior. with random guessing, an examinee s odds of correctly answering any given item increase with fewer response options. for example, for someone guessing purely at random, s / he would have a 1 in 5 (20%) chance of correctly answering an item with 5 response options ; a 1 in 4 (25%) chance of correctly answering an item with 4 response options ; and a 1 in 3 (33%) chance of answering an item with 3 response options. one may calculate the probability that an examinee will answer a given number of items correctly on any exam (of any length) by using the cumulative binomial distribution in excel. the formula for calculating these probabilities is presented below : p = (1 - binom.dist (a, b, c, true)) + binom.dist(a,b,c,false) where, p is the probability of an examinee getting the score of interest (or higher) by randomly guessing during a single administration, a is the score of interest (e.g., the number of correct responses), b is the length of the exam, and c is the probability of getting an item correct when randomly guessing (e.g., 1 divided by the number of response options). to illustrate why random guessing is a problem that medical educators should take more seriously, especially when cut scores are used, let us consider a hypothetical example. imagine a medical education scenario in which students complete an exam consisting of 10 items. the instructor opted to administer only 10 items because the exam is considered particularly rigorous due to challenging items assessing very complex material. further, the instructor opted to utilize a 3-option format for responses based on literature suggesting 3 options are largely comparable to 4 options when it is difficult to generate an effective third distractor. now, suppose the instructor conducted a standard setting exercise with colleagues and determined the following raw score performance ranges were appropriate and defensible : 0 - 4 = fail ; 5 - 7 = pass ; 8 - 10 = honors. in such a scenario, we used the cumulative binomial distribution formula presented previously to determine the probability that a student utilizing a random guessing strategy would attain a given number correct (see table 1). probability of attaining a given raw score with 3 response options on a 10-item test this example demonstrates that a student guessing completely at random would have a.2064 (about a 20.64%) probability of passing this exam. pass verdict is much too large, thus the current cut score between a pass / fail decision must be revised. with most education research it is customary to use a 95% confidence level, meaning education researchers are willing to accept a 5% chance of making an error. in this example, this would mean we must move the minimum cut score from 5 (50% correct in order to attain a pass verdict) to 7 (70% correct in order to attain a pass verdict) in order to ensure that no examinees would be likely to pass the exam by simply guessing at random. as stated previously, because this exam is particularly difficult a shift of the cut score from 5 to 7 would likely be indefensible. this creates quite the dilemma for a medical educator wishing to administer a rigorous assessment while maintaining fair and defensible performance standards. now, let us look at the same example using 4- and 5-option responses. in this scenario, let us assume the instructor was able to generate three and four plausible distractors, respectively, for each of the 10 items. the probability that a student utilizing a random guessing strategy would attain a given number correct is presented in table 2. probability of attaining a given raw score with 4- and 5- option responses in this scenario, a student guessing completely at random would have approximately a.07 (or about an 8%) probability of passing this exam when each item consisted of 4-response options, and about a.03 (about 3%) probability of passing when each item consisted of 5- response options. in both instances, the probability of passing the exam is much smaller than the exam consisting of 3-response options. interestingly, though, the exam consisting of 4-response options still produces a probability of passing that is greater than the typical 5% threshold we would prefer. if using 4-response options, the instructor would likely still need to revise the cut score in this scenario. the result would be a move from 5 (50% correct in order to attain a pass verdict) to 6 (60% correct in order to attain a pass verdict) in order to ensure that examinees would be unlikely to pass the exam by simply guessing at random. although this shift in the cut score seems less egregious than the shift given 3-response options, it still may be problematic and indefensible in such a scenario. so, what exactly does this mean for medical educators conducting routine classroom assessments ? while there is research and a continual budding of support from medical educators to support the use of mcq items with 3 response options, it is critical that one remains cognizant of what effect this will have on guessing strategies, and on cut score determination decisions. simply put, exams consisting of items with fewer response options will increase the likelihood that examinees utilizing a random guessing approach will pass that exam. the choice of using 3, 4 or 5 option responses also has significant implications with regard to setting cut scores and various performance standards. most medical school classroom assessments carry moderate - to - high stakes for students, thus it is imperative that the placement of a cut score is at a position in which it is highly unlikely an examinee could achieve by random guessing. of course, any decision about what constitutes highly unlikely is arbitrary, but given the conventions of education research a level of 5% seems reasonable for classroom assessments. table 3 provides recommendations for cut score thresholds for the minimum performance category (e.g., the threshold for a pass / fail decision) relative to exam length (from 10 to 100 items, by increments of 5) and the number of item response options. minimum recommended raw cut score placements for items with 3, 4, and 5 options a visual scan of table 3 illustrates the impact random guessing may have on minimum performance category threshold decisions. the table illustrates the number of items that would need to be answered correctly in order to be confident within a 5% error tolerance that the response pattern was not due to random guessing. specifically, the fewer response options provided, the greater the minimum (raw) cut score need be. for example, an exam consisting of 50 items would require a minimum (raw) cut score of 23 if the items consisted of 3 response options, and a minimum (raw) cut score of 16 if the items consisted of 5 response options to ensure examinees could not achieve the lowest meaningful performance category by random guessing given a 5% maximum error tolerance. the difference of requiring correct answers to 7 additional items when fewer options are presented offers a number of additional considerations for medical educators. specifically, what impact might the decision to use items with 3-response options relative to traditional item formats with 4- or 5-response options have on setting fair and defensible standards ? clearly, fewer response options equate to a higher minimum cut score, but what effects might this have on the substantive meaning of that minimum cut score ? what additional unintended consequences might result from using fewer response options ? practically speaking, mcqs with 3-options are typically easier to write because they require development of fewer plausible distractors than 4- and 5-option mcqs. educators should recognize that implausible distractors add to test error and therefore are not contributing meaningfully (in fact, they are doing quite the opposite). item distractors are often written to diagnose common misconceptions (e.g., order of operations) about a topic. it benefits medical educators when others, such as fellow content experts or residents, review items and their distractors for plausibility. in addition to distractor plausibility concerns, the amount of time per item can be problematic when trying to assess multiple content or competency domains. examinees sometimes run out of time and render random guesses in hopes of gaining additional points. when this occurs, scores are contaminated with error and the resulting measure of student performance will be inaccurate. proponents for 3-option responses contend examinees will require less time to complete each item, and may lead to more valid score results. alternatively, modifying exams in which a significant number of students have historically run out of time could give better estimates of difficulty and reliability than previous versions. however, in reducing the number of distractors that an item has, there is usually a small decrease in each item s difficulty and the exam s overall reliability (1). this translates to a need for more high - quality items per exam to compensate for diminished difficulty and reliability. it is well - documented that offering a mix of easy, moderate, and difficult items tends to increase reliability estimates (8). however, medical education classroom assessments are notorious for suffering from low reliability estimates, largely due to over - representation of easy items (p - values0.75). the extent to which items are easy due to their complexity, an artifact of student learning, instructional familiarity effects (9), or some other factor certainly must be considered as well. as royal and guskey (10) note, in the context of classroom assessment, when teaching is effective and learning is successful score results should resemble a negatively skewed distribution with few, if any, students receiving low marks. thus, the importance of reliability as an indicator of quality is often deceiving under these circumstances and becomes a less important indicator of quality. discrimination coefficients are often helpful for informing instructors of whether or not students performed as expected. to that end, instructors can have some, albeit subjective, estimate of the degree to which the exam functioned as intended when administered to the given sample. it should be noted that discrimination coefficients simply need to be positive (e.g., 0.01 or higher), as opposed to the guidelines proposed for norm - referenced examinations (e.g., greater than 0.2, 0.3, or 0.4) which have largely been misunderstood by most medical educators (11). that is, because classroom assessments often yield high scores for examinees and items difficulty estimates tend to indicate most items are easy, the discrimination coefficient becomes less important in this context than it would be in a norm - referenced assessment scenario (e.g., mcat exam, etc.) exam length is another important consideration, as having too few items creates range issues and inflates error estimates. therefore, educators need to ensure there are enough items to adequately measure students abilities, while simultaneously ensuring someone can not pass an exam by utilizing a random guessing strategy. research by fisher (13) indicated exams should consist of a minimum of 25 items, when possible, because measures produced with this many items yield error estimates that generally are stable while reliability estimates can still reach levels exceeding 0.90. as a general rule, the more items administered the more statistically stable the scores will be ; however, at some point the cognitive load for examinees may become too great or examinees may have insufficient time, so these factors need to be appropriately considered and properly accounted for when determining an appropriate number of items to administer (14). of course, some individuals familiar with psychometric models might argue the solution for modeling guessing is simple : use a 3-parameter logistic (3-pl) item response theory (irt) measurement model to account for a pseudo - guessing parameter to adjust students scores. while this approach is certainly a possibility for individuals working in large - scale, medical licensure and certification testing environments, it is entirely inappropriate for classroom assessments as 3pl models require a minimum sample size of 1,000 examinees (15 - 17). therefore, corrections for guessing using 3-pl irt models is not a viable option for medical education classroom assessments. the use of rasch measurement models, however, is a possibility for classroom assessments (11) and may offer insights about guessing (18,19). practically speaking, mcqs with 3-options are typically easier to write because they require development of fewer plausible distractors than 4- and 5-option mcqs. educators should recognize that implausible distractors add to test error and therefore are not contributing meaningfully (in fact, they are doing quite the opposite). a good distractor targets learners insufficiency in the content or competency domain being assessed. item distractors are often written to diagnose common misconceptions (e.g., order of operations) about a topic. it benefits medical educators when others, such as fellow content experts or residents, review items and their distractors for plausibility. in addition to distractor plausibility concerns, the amount of time per item can be problematic when trying to assess multiple content or competency domains. examinees sometimes run out of time and render random guesses in hopes of gaining additional points. when this occurs, scores are contaminated with error and the resulting measure of student performance will be inaccurate. proponents for 3-option responses contend examinees will require less time to complete each item, and may lead to more valid score results. alternatively, modifying exams in which a significant number of students have historically run out of time could give better estimates of difficulty and reliability than previous versions. however, in reducing the number of distractors that an item has, there is usually a small decrease in each item s difficulty and the exam s overall reliability (1). this translates to a need for more high - quality items per exam to compensate for diminished difficulty and reliability. it is well - documented that offering a mix of easy, moderate, and difficult items tends to increase reliability estimates (8). however, medical education classroom assessments are notorious for suffering from low reliability estimates, largely due to over - representation of easy items (p - values0.75). the extent to which items are easy due to their complexity, an artifact of student learning, instructional familiarity effects (9), or some other factor certainly must be considered as well. as royal and guskey (10) note, in the context of classroom assessment, when teaching is effective and learning is successful score results should resemble a negatively skewed distribution with few, if any, students receiving low marks. thus, the importance of reliability as an indicator of quality is often deceiving under these circumstances and becomes a less important indicator of quality. discrimination coefficients are often helpful for informing instructors of whether or not students performed as expected. to that end, instructors can have some, albeit subjective, estimate of the degree to which the exam functioned as intended when administered to the given sample. it should be noted that discrimination coefficients simply need to be positive (e.g., 0.01 or higher), as opposed to the guidelines proposed for norm - referenced examinations (e.g., greater than 0.2, 0.3, or 0.4) which have largely been misunderstood by most medical educators (11). that is, because classroom assessments often yield high scores for examinees and items difficulty estimates tend to indicate most items are easy, the discrimination coefficient becomes less important in this context than it would be in a norm - referenced assessment scenario (e.g., mcat exam, etc.) exam length is another important consideration, as having too few items creates range issues and inflates error estimates. therefore, educators need to ensure there are enough items to adequately measure students abilities, while simultaneously ensuring someone can not pass an exam by utilizing a random guessing strategy. research by fisher (13) indicated exams should consist of a minimum of 25 items, when possible, because measures produced with this many items yield error estimates that generally are stable while reliability estimates can still reach levels exceeding 0.90. as a general rule, the more items administered the more statistically stable the scores will be ; however, at some point the cognitive load for examinees may become too great or examinees may have insufficient time, so these factors need to be appropriately considered and properly accounted for when determining an appropriate number of items to administer (14). of course, some individuals familiar with psychometric models might argue the solution for modeling guessing is simple : use a 3-parameter logistic (3-pl) item response theory (irt) measurement model to account for a pseudo - guessing parameter to adjust students scores. while this approach is certainly a possibility for individuals working in large - scale, medical licensure and certification testing environments, it is entirely inappropriate for classroom assessments as 3pl models require a minimum sample size of 1,000 examinees (15 - 17). therefore, corrections for guessing using 3-pl irt models is not a viable option for medical education classroom assessments. the use of rasch measurement models, however, is a possibility for classroom assessments (11) and may offer insights about guessing (18,19). while there is some research to support the use of items with 3-option responses, we must remain cognizant of what effect this will have on guessing strategies and cut score determinations. as demonstrated here, the probability of attaining an invalid score result tends to increase in scenarios involving a small number of items. much more research on items with 3-option responses are needed to better understand what value, if any, 3-option responses truly adds to assessments, and in what contexts potential benefits might be discernible. | introduction : research has asserted mcq items using three response options (one correct answer with two distractors) is comparable to, and possibly preferable over, traditional mcq item formats consisting of four response options (e.g., one correct answer with three distractors), or five response options (e.g., one correct answer with four distractors). some medical educators have also adopted the practice of using 3-option responses on mcq exams as a response to the difficulty experienced in generating additional plausible distractors. to date, however, little work has explored how 3-option responses might impact validity threats stemming from random guessing strategies, and what impact 3-option responses might have on cut - score determinations, particularly in the context of medical education classroom assessments. the purpose of this work is to further explore these critically important considerations that largely have gone ignored in the medical education literature to this point. methods : a cumulative binomial distribution formula was used to calculate the probability that an examinee will answer at random a given number of items correctly on any exam (of any length). by way of a demonstration, a variety of scenarios were presented to illustrate how examination length and the number of response options impact examinees chances of passing a given examination, and how subsequent cut - score decisions may be impacted by these factors. results : as a general rule, classroom assessments containing fewer items should utilize traditional 4-option or 5-option responses, whereas assessments of greater length are afforded greater flexibility in potentially utilizing 3-option responses. conclusions : more research on items with 3-option responses is needed to better understand what value, if any, 3-option responses truly add to classroom assessments, and in what contexts potential benefits might be discernible. |
on august 3, 2012, fever developed in a previously healthy 63-year - old woman who lived in chuncheon - si, gangwon province, south korea ; the same day, she noticed a lump on the left side of her neck. she visited a local clinic, and ciprofloxacin and ceftriaxone were started on the first day of illness. the patient reported that, 2 weeks before her fever started, she noticed an insect bite on her neck while she was working on a crop farm in hwacheon - gun, gangwon province (in the northernmost part of south korea). she did not recall having contact with any domestic animals on the farm and had no history of travel outside south korea in the month before illness onset. on the third day of her illness, she began having watery diarrhea, 6 times per day. on the fourth day of the illness, thrombocytopenia and leukopenia were recorded at the local clinic (table). a computed tomography scan of the neck showed an enlarged (1.6 cm), necrotic lymph node. multiple lymph nodes on the left cervical and left axillary areas were also swollen. on the sixth day, the patient was transferred to seoul national university hospital. day 1 was the onset day of the illness. platelets were transfused on day 7 and day 9, and fresh frozen plasma was transfused on day 9 and day 10. creatinine kinase - mb levels were 7, 5, 25.7, 117.5 and 186.2 ng / ml on days 6, 7, 8, 9, and 10, respectively (reference range 0.66.3 ng / ml). na, not available ; ast, aspartate aminotransferase ; alt, alanine aminotransferase ; ldh, lactate dehydrogenase ; aptt, activated partial prothromboplastin time ; inr, international normalized ratio. at admission to the hospital, the patient was febrile but alert. her temperature was 38.7c, blood pressure 126/70 mm hg, heart rate 86 beats per minute, and oxygen saturation 92% on room air. her face was puffy, with a sunburned appearance, and both conjunctivae were congested. the insect bite site on her posterior neck was swollen and erythematous, and the draining cervical lymph node was enlarged. laboratory test results showed pancytopenia and elevated serum aminotransferase levels ; prothrombin and activated partial thromboplastin times were normal, but fibrinogen level was decreased (table). a urine dipstick test showed albuminuria (+ + +), and microscopic examination of the urine revealed > 100 erythrocytes per high - power field. test results for antibodies against orientia tsutsugamushi, hantaan virus, and leptospira were negative. a chest radiograph showed bilateral increased vascular markings, and the plasma level of b - type natriuretic peptide increased to 134 pg / ml (reference range < 100 pg / ml) ; these findings suggested cardiac dysfunction. on the eighth day of her illness, a computed tomography scan of the brain showed no evidence of hemorrhage or infarction and no other abnormalities. on the tenth day of illness (august 12, 2012), the patient died of multiple organ failure. antiviral drugs, corticosteroids, immunosuppressive agents, or intravenous immunoglobulin were not given. because viral infection was suspected but no virus could be identified, an anticoagulated blood sample was obtained from the patient on the eighth day of illness and stored at 70c. when testing for sftsv became available 7 months later, we inoculated monolayers of vero cells with the patient s blood sample and cultured the cells at 37c in a 5% carbon dioxide atmosphere. the culture supernatant was also used to inoculate dh82 cells when the cell line became available ; 5 days after the inoculation, we observed a cytopathic effect of sftsv in dh82 cells. the sftsv - infected vero cell monolayer was fixed according to described methods (11) and cut on ultramicrotome (rmc mt - xl) at 65 nm. ultrathin sections were stained with saturated 4% uranyl acetate and 4% lead citrate before examination with a transmission electron microscope (hitachi-7100 ; hitachi high - technologies, ibaraki, japan) at 75 kv (figure 1). transmission electron microscopy image of vero cells infected with severe fever with thrombocytopenia syndrome virus (arrows). rna was extracted from the stored blood and from virus - infected vero cells by using a qiaamp viral rna mini kit (qiagen, hilden, germany). reverse transcription pcr (rt - pcr) was performed to amplify the partial large (l) segment of the viral rna from the stored blood to confirm sftsv, as described (12). rt - pcr results were positive, and direct sequencing was done. a blast search (http://blast.ncbi.nlm.nih.gov/blast.cgi) showed no sequences from organisms other than sftsv. using the culture supernatant, full lengths of all 3 genome segments (l, medium [m ], and small [s ]) were sequenced by rt - pcr and direct sequencing after polyadenylation of 3 ends of the genomic and complementary rnas, the sequences of the segment ends were obtained by rapid amplification of cdna ends. the complete sequences of the l, m, and s segments were deposited in genbank (accession nos. the l, m, and s segments of the isolate showed 95.8%99.8%, 94.1%99.9%, and 94.8%99.7% identity, respectively, to previously reported sftsv sequences. we also constructed a phylogenetic tree by the neighbor - joining method using rna - dependent rna polymerase gene nucleic acid sequences to compare the isolate we obtained to representative sftsv strains from china and japan ; the isolate and the other strains were closely related (95.9%99.9% sequence relatedness) but not identical (figure 2). phylogenetic tree for the rna - dependent rna polymerase (rdrp) gene sequences of the large segment of an isolate obtained from a patient in south korea who died of an illness retrospectively identified as severe fever with thrombocytopenia syndrome (sfts) (black dot) compared with representative sfts virus strains from china and japan. the tree was constructed on the basis of the nucleic acid sequences of the rdrp genes by using the neighbor - joining method. we confirmed a case of sfts in south korea in 2012 by isolation of sftsv from a stored blood sample collected shortly before the patient s death. the patient had a history of an insect bite while working on a crop farm in hwacheon - gun, gangwon province, the northernmost part of south korea. phylogenetic analysis of the rna - dependent rna polymerase gene showed that our virus isolate was closely related to sftsv strains reported from china and japan. as of july 5, 2013, the korea centers for disease control and prevention had confirmed 13 cases of sfts by rt - pcr ; of these patients, 8 were dead and 5 alive (13). except for our patient, who died in 2012, all cases occurred during 2013. | we report a retrospectively identified fatal case of severe fever with thrombocytopenia syndrome (sfts) in south korea from 2012. sfts virus was isolated from the stored blood of the patient. phylogenetic analysis revealed this isolate was closely related to sfts virus strains from china and japan. |
chronic pancreatitis is an inflammatory condition that may result in progressive parenchymal damage and fibrosis. the destruction of pancreatic tissue can lead to exocrine as well as endocrine dysfunction in addition to biliary, duodenal or gastric obstruction, pseudocysts, pancreatic ascites, pleural effusion, splenic or portal vein thromboses, splenic artery pseudoaneurysm, cancer and pancreatic fistulae. pancreatic fistulae have been reported to involve the pleura, peritoneum, pericardium, and other peripancreatic organs (i.e., esophagus, stomach, duodenum, and colon). this case report describes a pancreaticouretral fistula an exceedingly rare complication of pancreatitis, manifested as a fistula between the pancreatic duct and the ureter. a 69-year - old hispanic female with a history of recurrent episodes of pancreatitis and cholecystectomy presented with complaints of severe, sharp, and constant abdominal pain in the left upper quadrant (luq) and flank with radiation to the left lower quadrant (llq). the pain was associated with subjective fever, nausea, and nonbloody emesis and was exacerbated by oral intake. the patient denied additional gastrointestinal or genitourinary disturbances, sick contacts, travel, or consumption of unusual food. two months prior to presentation, she had an episode of pancreatitis complicated by pseudocyst formation along the greater curvature of the stomach, measuring 13 9.5 5.5 cm. she underwent ultrasound - guided drainage of approximately 100 ml of cloudy fluid with subsequent placement of a 12-french pigtail catheter. external bag drainage was continued for 2 weeks and removed once the drainage was minimal. one day after catheter removal, the patient developed progressive abdominal discomfort, which prompted repeat evaluation.. a computed tomography (ct) scan of the abdomen revealed a cystic structure with fistulous extension below the pancreas into the left para - aortic space. the reading further commented on the presence of mild left hydronephrosis due to the passage of the ureter near a cluster of lymph nodes and cystic structures from the fistula below the pancreatic tail measuring 2.2 3.9 4.5 cm [figure 1 ]. coronal ct scan demonstrating pancreatic pseudocyst (arrow) extending toward the left renal collecting system magnetic resonance cholangiopancreatography (mrcp) was obtained to further evaluate the pancreatobiliary anatomy. it demonstrated an abnormal pancreatic duct in the body and tail of the pancreas with a complex multiloculated pseudocyst extending anteriorly into the lesser sac, posteriorly and medially to the level of the left kidney with possible ureteral fistula formation, and mild left - sided hydronephrosis [figures 2 and 3 ]. an additional smaller fistula was noted to extend medially from the pseudocyst into a soft - tissue mass. axial t2-weighted sequence demonstrating the proximity of the pancreatic pseudocyst (arrow head) and the ureter (long arrow) 3d mrcp shows a fistula (long arrow) between the pancreatic pseudocyst (arrow head) and left renal collecting system (short arrow) in an effort to optimize pancreatic drainage, a sphincterotomy was performed with pancreatic stent placement during an endoscopic retrograde cholangiopancreatography (ercp). additionally, a left retrograde pyelogram was performed to evaluate the level / degree of ureteral obstruction and further assess for the presence of a fistulous tract. a fistulous tract was in fact visualized between the left renal pelvis (at the level of an upper pole calyx) and the pancreatic duct and a ureteral stent was placed to facilitate fistula closure [figure 4 ]. the patient was started on imipenem / cilastatin prophylaxis pending blood and pancreatic pseudocyst fluid cultures, which ultimately returned culture negative. left retrograde pyelogram demonstrating a fistulous tract between the left renal pelvis (at the level of an upper pole calyx) and the pancreatic duct. a ureteral stent was placed to facilitate fistula closure the patient remained intolerant of oral intake and experienced pain, even after the placement of pancreatic and ureteral stents. in an effort to relieve compression of the stomach by the pseudocyst and to facilitate eating, she underwent a successful pancreatic cyst gastrostomy. following the procedure, the patient attained symptomatic relief and oral intake was successfully resumed. a left retrograde pyelogram was repeated after 2 months demonstrating no evidence of a residual fistulous tract [figure 5 ]. left retrograde pyelogram, repeated 2 months after initial intervention for puf, demonstrating no evidence of a fistulous tract pancreaticoureteral fistulae (puf) are exceedingly rare. upon reviewing the literature only two case reports were identified. however, unlike our case, those fistulae evolved following traumatic injury to the ureter or pancreatic duct. no reports were found to describe puf as a complication of chronic pancreatitis. in trauma related cases, pancreatic fistula most likely form secondary to persistent leakage of pancreatic secretions from a disrupted pancreatic duct. many etiologies, including pancreatitis, trauma, biopsy, or surgery, can result in pancreatic duct disruption. one proposed etiology for the evolution of a puf in our patient may involve recurrent inflammatory infiltrate progressively eroding surrounding structures and intimately extending into the retroperitoneal space. ercp, mrcp, ct scan, and fistulography are often the main imaging studies implemented. ercp was found to be 100% sensitive and specific in the diagnosis of pancreatic ductal rupture in one prospectively study and was superior to ct scan and mrcp, which are reported to detect pancreatic duct abnormalities with similar accuracy. in addition to the common complications of pancreatic fistulae, complications specific for puf can include metabolic abnormalities (hyperchloremic metabolic acidosis, electrolytes imbalance), urological complications (chemical cystitis, urethritis, hematuria, urinary tract infection, and bladder stones), and reflux pancreatitis. metabolic complications are due to a loss of alkaline exocrine pancreatic secretions in the urine while pancreatic enzymes secreted in the urine may cause urological complications. up to 50% of internal pancreatic fistulas and 70% to 90% of external pancreatic fistulas for patients presenting with a main pancreatic duct dilatation, without ductal disruption or stricture, conservative therapy (broad spectrum antibiotics, enteral nutrition, and correction of fluid and electrolyte imbalances) should be pursued. of note, enteral nutrition is associated with significantly higher closure rates and shorter time to closure, than parenteral nutrition. additionally, somatostatin analog administration is reported to promote pancreatic fistula closure by decreasing the volume of fistulous tract output. if conservative measures fail or if the fistula becomes complicated by infection or bleeding, endoscopic or surgical interventions are warranted. ercp is a safe and effective modality and can be considered the first - line therapy in the management of pancreatic fistulae. early ercp and pancreatic stent insertion promote fistula resolution and may allow delay or avoidance of surgical measures. due to their significant complication profile, surgical interventions should be reserved for cases not responsive to conservative measures. a number of imaging modalities, such as ercp, mrcp, ct scan, or fistulography, can assist in the diagnosis of puf. nonoperative modalities, including medical and endoscopic measures, may initially be pursued for the management of puf. due to puf 's unclear etiology and possible variance of presentation, it is important for physicians to keep this rare complication of pancreatitis in mind while evaluating patients with recurrent pancreatitis, urinary symptoms, and/or imaging suggestive of abnormalities within the urinary collecting system and pancreas. | context : chronic pancreatitis is an inflammatory condition that may result in progressive parenchymal damage and fibrosis which can ultimately lead to destruction of pancreatic tissue. fistulas to the pleura, peritoneum, pericardium, and peripancreatic organs may form as a complications of pancreatitis. this case report describes an exceedingly rare complication, pancreaticoureteral fistula (puf). only two additional cases of puf have been reported. however, they evolved following traumatic injury to the ureter or pancreatic duct. no published reports describe puf as a complication of pancreatitis.case report : a 69-year - old hispanic female with a past medical history of cholecystectomy, pancreatic pseudocyst, and recurrent episodes of pancreatitis presented with severe, sharp, and constant abdominal pain. upon imaging, a fistulous tract was visualized between the left renal pelvis (at the level of an upper pole calyx) and the pancreatic duct and a ureteral stent was placed to facilitate fistula closure. following the procedure, the patient attained symptomatic relief and oral intake was resumed. a left retrograde pyelogram was repeated 2 months after the initial stent placement and demonstrating no evidence of a persistent fistulous tract.conclusion:due to puf 's unclear etiology and possible variance of presentation, it is important for physicians to keep this rare complication of pancreatitis in mind, especially, when evaluating a patient with recurrent pancreatitis, urinary symptoms and abnormal imaging within the urinary collecting system and pancreas. |
there are 2 major pathological types of lung cancer, which account for approximately 85% of lung cancers : small cell lung cancer (sclc) and non - small cell lung cancer (nsclc). lung cancer can be further divided into 3 histological types : squamous cell carcinoma, adenocarcinoma, and large cell lung cancer. currently, the primary treatment method for lung cancer is surgery, supplemented by comprehensive treatment with chemotherapy, radiation therapy, and targeted therapy. lung cancer does not usually cause any symptoms in the early stages of the disease, and thus is often diagnosed in late stages. therefore, the identification of early diagnostic markers is important for the diagnosis and treatment of lung cancer. chemokines are a family of small proteins of molecular weights ranging from 8 to 17 kda, with chemotactic activity. studies have shown that chemokines guide cell migration along a concentration gradient during inflammatory reactions. the chemokine superfamily can be divided into the following branches : c, cc, cxc, and cxxxc. chemokines play important roles in the occurrence and development of cancers due to their chemotactic activity in cancer cells xcl2 is a small protein of the xc chemokine family, and is closely associated with another chemokine, xcl1. it is mainly expressed in activated t cells, and is also expressed at low levels in inactivated t cells. in contrast to the typical structure of other chemokines, cx3cl1 has different spacing of the characteristic n - terminal cysteines. there are 3 amino acids separating the initial pair of cysteines in cx3cl1, with none in cc chemokines and only 1 intervening amino acid in cxc chemokines. soluble cx3cl1 has the ability to recruit t cells and monocytes, and thus enhances binding and adhesion capacity of cells. studies have shown that cx3cl1 is highly expressed in the hippocampus region during short - term memory, which may be regulated by glutamatergic neurotransmission. the interaction between cx3cl1 and glutamate led us to investigate the expression of xcl2 and cx3cl1 in lung cancer. a total of 38 tumor samples were collected from 18 male and 20 female patients with nsclc who underwent surgical resection in zhejiang provincial people s hospital between july 2014 and december 2014. based on the tnm classification for lung cancer staging (2009) by the union for international cancer control (uicc), the clinical staging of selected patients was as follows : 23 cases of squamous cell carcinoma, and 15 cases of adenocarcinoma ; tem staging : 21 cases of stage i ii, and 17 cases of stage iii iv ; lymph node metastasis for lung cancer staging : 24 cases of n0, 7 cases of n1, and 7 cases of n2. the study protocol was approved by the research ethics committee of zhejiang provincial people s hospital, and all patients gave their informed consent before study commencement. xcl2 and cx3cl1 antibodies were purchased from cell signaling technology ((beverly, ma, usa). total rna was extracted using trizol rna extraction kit (invitrogen ; carlsbad, ca, usa) according to the manufacture s instructions. aliquots of tissue or serum were mixed with equal amounts of trizol, and centrifuged at 12 000 rpm at 4c for 10 min. the supernatant was transferred into a new eppendorf (ep) tube, chilled on ice for 5 min, and mixed with 200 l of chloroform. the mixture was vortexed for 15 s and centrifuged at 12 000 rpm at 4c for 15 min. the top layer was transferred into a new ep tube and mixed with an equal volume of isopropanol. the mixture was allowed to precipitate at room temperature for 10 min, and centrifuged at 12 000 rpm / min at 4c for 10 min. the remnant was mixed with 75% ethanol and centrifuged at 7500 rpm at 4c for 5 min. rna was air - dried and dissolved in 20 l of depc - treated water. rnas were then transcribed into cdna, which was further amplified using a one - step rt - pcr kit. the reaction conditions were 94c for 45 s, followed by 35 cycles of refolding at 59c for 45 s and extension at 72c. briefly, sections were treated sequentially with xylene, absolute ethanol, and 95%, 90%, 85%, and 80% ethanol for 10 min each. the sections were then incubated with secondary antibodies, stained with hematoxylin solution, soaked in 1% hydrochloric acid alcohol, rinsed with running water anti - blue, dehydrated with an ethanol gradient, treated with xylene for transparency, and mounted with neutral gum. cytoplasm of xcl2- and cxc3cl1-positive cells were positively stained (colored yellow or brown). the positive rate was calculated as the ratio of the number of positive cells to the area of visual fields using image j software, and compared to determine the difference in the expression level of both proteins. after 90 min of reaction at 37c, 100 l of biotinylated antibody was added to each well and allowed to react at 37c for 60 min. after 30 min of reaction at 37c, the plate was rinsed 5 times with 0.01 m tbs. the plate was incubated with tmb at 37c in the dark and the reaction was terminated by adding tmb stop solution after 30 min. all tests were repeated at least 3 times and are presented as mean standard deviation. a total of 38 tumor samples were collected from 18 male and 20 female patients with nsclc who underwent surgical resection in zhejiang provincial people s hospital between july 2014 and december 2014. based on the tnm classification for lung cancer staging (2009) by the union for international cancer control (uicc), the clinical staging of selected patients was as follows : 23 cases of squamous cell carcinoma, and 15 cases of adenocarcinoma ; tem staging : 21 cases of stage i ii, and 17 cases of stage iii iv ; lymph node metastasis for lung cancer staging : 24 cases of n0, 7 cases of n1, and 7 cases of n2. the study protocol was approved by the research ethics committee of zhejiang provincial people s hospital, and all patients gave their informed consent before study commencement. xcl2 and cx3cl1 antibodies were purchased from cell signaling technology ((beverly, ma, usa). total rna was extracted using trizol rna extraction kit (invitrogen ; carlsbad, ca, usa) according to the manufacture s instructions. aliquots of tissue or serum were mixed with equal amounts of trizol, and centrifuged at 12 000 rpm at 4c for 10 min. the supernatant was transferred into a new eppendorf (ep) tube, chilled on ice for 5 min, and mixed with 200 l of chloroform. the mixture was vortexed for 15 s and centrifuged at 12 000 rpm at 4c for 15 min. the top layer was transferred into a new ep tube and mixed with an equal volume of isopropanol. the mixture was allowed to precipitate at room temperature for 10 min, and centrifuged at 12 000 rpm / min at 4c for 10 min. the remnant was mixed with 75% ethanol and centrifuged at 7500 rpm at 4c for 5 min. rna was air - dried and dissolved in 20 l of depc - treated water. rnas were then transcribed into cdna, which was further amplified using a one - step rt - pcr kit. the reaction conditions were 94c for 45 s, followed by 35 cycles of refolding at 59c for 45 s and extension at 72c. briefly, sections were treated sequentially with xylene, absolute ethanol, and 95%, 90%, 85%, and 80% ethanol for 10 min each. the sections were then incubated with secondary antibodies, stained with hematoxylin solution, soaked in 1% hydrochloric acid alcohol, rinsed with running water anti - blue, dehydrated with an ethanol gradient, treated with xylene for transparency, and mounted with neutral gum. cytoplasm of xcl2- and cxc3cl1-positive cells were positively stained (colored yellow or brown). the positive rate was calculated as the ratio of the number of positive cells to the area of visual fields using image j software, and compared to determine the difference in the expression level of both proteins. after 90 min of reaction at 37c, 100 l of biotinylated antibody was added to each well and allowed to react at 37c for 60 min. after 30 min of reaction at 37c, the plate was rinsed 5 times with 0.01 m tbs. the plate was incubated with tmb at 37c in the dark and the reaction was terminated by adding tmb stop solution after 30 min. all tests were repeated at least 3 times and are presented as mean standard deviation. as shown in figure 1a and 1b, qrt - pcr analyses demonstrated that the expression of xcl2 and cx3cl1 mrna in lung cancer was significantly higher than that in normal tissues (p<0.05). it was shown that the expression of both chemokines in lung cancer was also significantly increased compared with normal tissues (p<0.05, figure 1c, 1d). it was shown that the expression level of xcl2 was significantly increased with higher pathological stages (p<0.05 or p<0.001, figure 2a2f). xcl2 expression in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes (p<0.05 or p<0.001, figure 2 g). moreover, xcl2 expression in t4 lung cancer was significantly higher compared with t3 cancer (p<0.05, figure 2h). results of immunohistochemical analyses showed that cx3cl1 expression was significantly increased with increasing pathological stages (p<0.05, figure 3a3f). cx3cl1 expression in n1 lung cancer was also significantly higher compared with n0 cancer (p<0.001, figure 3 g). moreover, cx3cl1 expression in t4 lung cancer was significantly higher compared with t3 cancer (p<0.05, figure 3h). as shown in figure 1a and 1b, qrt - pcr analyses demonstrated that the expression of xcl2 and cx3cl1 mrna in lung cancer was significantly higher than that in normal tissues (p<0.05). it was shown that the expression of both chemokines in lung cancer was also significantly increased compared with normal tissues (p<0.05, figure 1c, 1d). it was shown that the expression level of xcl2 was significantly increased with higher pathological stages (p<0.05 or p<0.001, figure 2a2f). xcl2 expression in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes (p<0.05 or p<0.001, figure 2 g). moreover, xcl2 expression in t4 lung cancer was significantly higher compared with t3 cancer (p<0.05, figure 2h). results of immunohistochemical analyses showed that cx3cl1 expression was significantly increased with increasing pathological stages (p<0.05, figure 3a3f). cx3cl1 expression in n1 lung cancer was also significantly higher compared with n0 cancer (p<0.001, figure 3 g). moreover, cx3cl1 expression in t4 lung cancer was significantly higher compared with t3 cancer (p<0.05, figure 3h). the occurrence and development of lung cancer is a multi - step, multi - factor process involving the deactivation of tumor suppressor genes and activation of oncogenes. numerous studies have shown that chemokines play important regulatory roles in the pathogenesis of a variety of tumors, as well as in the proliferation, apoptosis, and signal transduction of tumor cells by acting as either tumor suppressors or promoters. wang. demonstrated that serum chemokine levels in patients with nsclc are higher compared with healthy controls. jia. reported that chemokine concentration in nsclc patients was lower than in patients with benign lung cancer. wu. found that chemokine expression in nsclc is associated with tnm staging, lymph node metastasis, survival rate, and overall response rate. it has also been shown that the expression of chemokines is closely related to tnm staging and distant metastasis of nsclc. these findings suggest that the changes in chemokine expression are strongly associated with the pathological stages of lung cancer. chemokines play important roles in the occurrence and development of cancers due to their chemotactic effect on cancer cells. studies have demonstrated that ccr1, the ccl5 receptor, and cx3cl1 synergistically induce liver metastases from colorectal cancer. high expression of xcl2 has been reported in a variety of cancers, including melanoma, nsclc, prostate cancer, and colorectal cancer. in our study, the expression of xcl2 mrna and protein in lung cancer was significantly higher compared with normal tissues. found that xcl2 is overexpressed in lung cancer and is associated with the prognosis of the disease. liu. demonstrated that cx3cl1 expression is gradually increased in benign lung tissue, primary lung tumors, and lymph node metastases, and that the overall survival rate of patients with stronger cx3cl1 expression is much lower compared with those with weaker cx3cl1 expression. in a study of cx3cl1 expression in nsclc, howard. demonstrated that cx3cl1 expression in cancer tissues is markedly higher than that in adjacent normal tissues it has also been shown that serum cx3cl1 expression is positively correlated with both serum vegf expression and microvessel density in tumors, suggesting that cx3cl1 overexpression is closely associated with the occurrence and development of lung cancer. in our study, in summary, the current study demonstrated that xcl2 and cx3cl1 expression in lung cancers were significantly higher compared to adjacent normal tissues. our findings indicate that both chemokines might be important targets in gene therapy for lung cancer, and their antagonists might have an anti - lung cancer effect. | backgroundchemokines are a family of small proteins secreted by cells with chemotactic activity, and they play important roles in cell adhesion. however, the expression of chemokine xcl2 and cx3cl1 in lung cancers in different pathological stages remains unclear.material/methodsxcl2 and cx3cl1 expression in lung cancers and adjacent non - cancerous tissues was detected by quantitative pcr and elisa. the relative expression of both chemokines in lung cancers in different pathological stages was compared by immunohistochemical assay.resultsthe relative expression level of xcl2 and cx3cl1 in lung cancer was significantly higher compared with adjacent normal tissues (p<0.001). the expression level of both chemokines was significantly increased with higher pathological stages, as indicated by immunohistochemical assay (p<0.05 or p < 0.001). their expression level in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes (p<0.05 or p<0.001).conclusionsthe expression of xcl2 and cx3cl1 increases with increasing degree of malignancy, indicating that both chemokines might be important targets in gene therapy for lung cancer. |
the crux of the problem of chronic periodontal diseases lies in the changes that occur in the bone. although, other tissues of the periodontium do get affected, yet it is the destruction of bone that is responsible for the loss of teeth. one objective of periodontal therapy is regeneration of the periodontal attachment including cementum, a functionally oriented periodontal ligament, and alveolar bone. bone grafting is used as part of surgical protocols aimed at regeneration of periodontal structures. autogenous bone grafts, bone derivatives like allografts, xenografts, and bone substitutes have been used for this purpose. among the biomaterials, autogenous bone has been adopted as the gold standard because of : autograft bone includes cells participating in osteogenesisa tissue reaction is induced without inducing immunological reactionsthere is a minimal inflammatory reactionthere is rapid revascularization around the graft particlesa potential release of growth and differentiation factors sequestered within the grafts (marx 1994). autograft bone includes cells participating in osteogenesis a tissue reaction is induced without inducing immunological reactions there is a minimal inflammatory reaction there is rapid revascularization around the graft particles a potential release of growth and differentiation factors sequestered within the grafts (marx 1994). in perspective, autograft bone has been considered to yield a high osteogenic potential and has thus been used with the intent to improve outcomes of periodontal regenerative procedures (dragoo and sullivan 1973, hiatt and schallhorn 1973, renvert.. however, the use of autograft bone presents potential disadvantages such as a need for a wider or second surgery region ; restricted donor sites make its use in cases with need for extensive grafting impractical. moreover, and perhaps most importantly, the use of autograft bone has been reported to be attributed to result in tooth debilitating ankylosis and root resorption in human (schallhorn and hiatt 1972, dragoo and sullivan 1973) and dog intrabony defects (levin 1975). allogenic bone obtained from a different person and commonly processed by tissue banks provides an alternative to autogenous bone if the problems associated with the autogenous grafts are considered. the initial use of demineralized allografts was based on compelling experimental data suggesting that it possesses osteoinductive potential. decalcified allogenic bone matrix (dabm) has its own limitations regarding the availability of the grafting material, for which the operator has to depend upon a hospital source. moreover, possibility of an immunological reaction and transmission of infective diseases are other disadvantages of dabm. a major concern with allografts in general is the potential for disease transfer, particularly viral transmission, and, even more particularly, hiv. treatment of cadaveric bone spiked with viral particles and cortical bone procured from a donor who had died of aids with a viricidal agent and demineralization in hcl has been found to inactivate hiv in both cases (mellonig.). both these materials have shown good results when tried individually,[58 ] but the literature is deficient in their comparative studies. in this study, therefore, an attempt has been made to evaluate and compare, radiographically, the relative efficacy of decalcified allogenic bone matrix and intra - oral free osseous autografts in the treatment of periodontal intrabony defects. thirty patients in the age group of 30 - 50 years were selected from amongst the patients visiting our department and were included in the study by taking written informed consent from each of them after explaining the study protocol. the criteria for the selection of the patients were as follows : presence of two almost identical intrabony defects, on either side of the mouth / upper and lower jaw, based upon the radiographic observations. only those patients were included who had an intrabony defect with a minimum of two walls, when viewed on surgical exposure. the other cases were excluded from the studyit was ensured that patients selected were not suffering from any systemic / debilitating diseaseit was ensured that patients selected were not smokersit was ensured that patients selected were not on any medication including antibiotics, corticosteroid, anti - hypertensive, immunosuppressant etc. presence of two almost identical intrabony defects, on either side of the mouth / upper and lower jaw, based upon the radiographic observations. only those patients were included who had an intrabony defect with a minimum of two walls, when viewed on surgical exposure. the other cases were excluded from the study it was ensured that patients selected were not suffering from any systemic / debilitating disease it was ensured that patients selected were not smokers it was ensured that patients selected were not on any medication including antibiotics, corticosteroid, anti - hypertensive, immunosuppressant etc. this bone graft was obtained intra - orally from the patient 's own mouth at the time of surgery from various sites viz., maxillary tuberosity region, edentulous regions of either maxilla or mandible, extraction sockets fresh or healed [figure 1 ]. the trephine method for procurement of cancellous bone from the edentulous region the bone was obtained after the reflection of the mucoperiosteal flap with the help of bur / chisel / bone rongeurs. the cortical bone was discarded, and the cancellous bone was utilized as the grafting material after being kept in sterile isotonic normal saline solution [figure 2 ]. the procured autogenous cancellous bone the decalcified allogenic bone matrix (dabm) used in our study was made from the fresh bone obtained from the corresponding dept of orthopedics, government medical college and hospital. the source of bone for preparing dabm was a government hospital recognized by the state government since decades. and, all the functioning of the hospital including biomedical waste disposal etc. the bone thus obtained was through proper channels with written records including patient 's details like name, age, sex, medical status etc. the following inclusionary / exclusionary criteria were followed while selecting the patients as donors : a)the patient should be in good general / systemic health, except for the orthopedic problem, for which he has visited the hospitalb)the patient should not be on heavy doses of anti - microbials for any reasonc)after the initial pre - operative investigations performed by the orthopedic dept. including screening for hiv, hbv etc., the patient should be free of any type of systemic disease, for example any type of microbial infection, hiv, hepatitis etcd)the bone thus obtained underwent a series of processes of demineralization and appropriate treatment with virucidal agents. the bone was sectioned into thin small pieces and then decalcified in 0.6 n hcl, for 6 to 7 days till the bone became resilient. the bone so formed was repeatedly washed in normal saline solution, and the acid was removed by repeated washings with 70% ethanol. decalcified allogenic bone matrix (dabm) so prepared was stored in 70% ethanol for 1 - 21 days before transplantation. on the day of the surgery, the graft material was first washed repeatedly in normal saline, and then boiled in distilled water for 30 minutes, and again kept in normal saline ready to be transplanted [figure 3 ]. the patient should be in good general / systemic health, except for the orthopedic problem, for which he has visited the hospital the patient should not be on heavy doses of anti - microbials for any reason after the initial pre - operative investigations performed by the orthopedic dept. including screening for hiv, hbv etc., the patient should be free of any type of systemic disease, for example any type of microbial infection, hiv, hepatitis etc the bone thus obtained underwent a series of processes of demineralization and appropriate treatment with virucidal agents. the bone was sectioned into thin small pieces and then decalcified in 0.6 n hcl, for 6 to 7 days till the bone became resilient. the bone so formed was repeatedly washed in normal saline solution, and the acid was removed by repeated washings with 70% ethanol. decalcified allogenic bone matrix (dabm) so prepared was stored in 70% ethanol for 1 - 21 days before transplantation. on the day of the surgery, the graft material was first washed repeatedly in normal saline, and then boiled in distilled water for 30 minutes, and again kept in normal saline ready to be transplanted [figure 3 ]. according to mellonig the use of exclusionary techniques in the procurement of bone allografts greatly reduced the chances of disease transmission. it was concluded by the authors that demineralization and the treatment with a virucidal agent inactivates hiv in spiked and infected bone. according to narang., the dabm grafts do not cause any immunologic response. routine investigations, prophylaxis, occlusal equilibration, and pre - operative intraoral peri - apical radiographs were taken using paralleling device, and the patients were instructed to adopt meticulous home care measures to control dental plaque. flap operations were carried out by a single operator each time to reduce the chances of biasing. the defects were randomly selected by the lottery method, and the intra - oral free osseous autograft (site a) and dabm graft (site d) were placed in the designated sites. we opted not to use gtr membranes to determine the sole role of the grafts used in producing radiographic bone fill and then have a comparison based truly on their individual abilities to form bone, as we know that gtr membranes in itself have the ability to form new bone if we provide enough space between the defect and the membrane. the patients were given post - operative medications including : amoxicillin 500 mg + lactic acid bacillus 6010 cells orally thrice - daily for 5 days post - operativelya 60-second rinse with 15 ml of 0.2% chlorhexidine digluconate was prescribed twice - daily for 3 weeksibuprofen 400 mg orally every 6 - 8 hours for 3 days and then as and when required by the patient was recommended for pain relief. amoxicillin 500 mg + lactic acid bacillus 6010 cells orally thrice - daily for 5 days post - operatively a 60-second rinse with 15 ml of 0.2% chlorhexidine digluconate was prescribed twice - daily for 3 weeks ibuprofen 400 mg orally every 6 - 8 hours for 3 days and then as and when required by the patient was recommended for pain relief. plaque levels were periodically checked in both groups using plaque index, and oral prophylaxis was done at each follow - up visit (approx. thirty patients in the age group of 30 - 50 years were selected from amongst the patients visiting our department and were included in the study by taking written informed consent from each of them after explaining the study protocol. the criteria for the selection of the patients were as follows : presence of two almost identical intrabony defects, on either side of the mouth / upper and lower jaw, based upon the radiographic observations. only those patients were included who had an intrabony defect with a minimum of two walls, when viewed on surgical exposure. the other cases were excluded from the studyit was ensured that patients selected were not suffering from any systemic / debilitating diseaseit was ensured that patients selected were not smokersit was ensured that patients selected were not on any medication including antibiotics, corticosteroid, anti - hypertensive, immunosuppressant etc. presence of two almost identical intrabony defects, on either side of the mouth / upper and lower jaw, based upon the radiographic observations. only those patients were included who had an intrabony defect with a minimum of two walls, when viewed on surgical exposure. the other cases were excluded from the study it was ensured that patients selected were not suffering from any systemic / debilitating disease it was ensured that patients selected were not smokers it was ensured that patients selected were not on any medication including antibiotics, corticosteroid, anti - hypertensive, immunosuppressant etc. this bone graft was obtained intra - orally from the patient 's own mouth at the time of surgery from various sites viz., maxillary tuberosity region, edentulous regions of either maxilla or mandible, extraction sockets fresh or healed [figure 1 ]. the trephine method for procurement of cancellous bone from the edentulous region the bone was obtained after the reflection of the mucoperiosteal flap with the help of bur / chisel / bone rongeurs. the cortical bone was discarded, and the cancellous bone was utilized as the grafting material after being kept in sterile isotonic normal saline solution [figure 2 ]. the procured autogenous cancellous bone the decalcified allogenic bone matrix (dabm) used in our study was made from the fresh bone obtained from the corresponding dept of orthopedics, government medical college and hospital. the source of bone for preparing dabm was a government hospital recognized by the state government since decades. and, all the functioning of the hospital including biomedical waste disposal etc. the bone thus obtained was through proper channels with written records including patient 's details like name, age, sex, medical status etc. the following inclusionary / exclusionary criteria were followed while selecting the patients as donors : a)the patient should be in good general / systemic health, except for the orthopedic problem, for which he has visited the hospitalb)the patient should not be on heavy doses of anti - microbials for any reasonc)after the initial pre - operative investigations performed by the orthopedic dept. including screening for hiv, hbv etc., the patient should be free of any type of systemic disease, for example any type of microbial infection, hiv, hepatitis etcd)the bone thus obtained underwent a series of processes of demineralization and appropriate treatment with virucidal agents. the bone so formed was repeatedly washed in normal saline solution, and the acid was removed by repeated washings with 70% ethanol. decalcified allogenic bone matrix (dabm) so prepared was stored in 70% ethanol for 1 - 21 days before transplantation. on the day of the surgery, the graft material was first washed repeatedly in normal saline, and then boiled in distilled water for 30 minutes, and again kept in normal saline ready to be transplanted [figure 3 ]. the patient should be in good general / systemic health, except for the orthopedic problem, for which he has visited the hospital the patient should not be on heavy doses of anti - microbials for any reason after the initial pre - operative investigations performed by the orthopedic dept. including screening for hiv, hbv etc., the patient should be free of any type of systemic disease, for example any type of microbial infection, hiv, hepatitis etc the bone thus obtained underwent a series of processes of demineralization and appropriate treatment with virucidal agents. the bone was sectioned into thin small pieces and then decalcified in 0.6 n hcl, for 6 to 7 days till the bone became resilient. the bone so formed was repeatedly washed in normal saline solution, and the acid was removed by repeated washings with 70% ethanol. decalcified allogenic bone matrix (dabm) so prepared was stored in 70% ethanol for 1 - 21 days before transplantation. on the day of the surgery, the graft material was first washed repeatedly in normal saline, and then boiled in distilled water for 30 minutes, and again kept in normal saline ready to be transplanted [figure 3 ]. according to mellonig., 1992, the use of exclusionary techniques in the procurement of bone allografts greatly reduced the chances of disease transmission. it was concluded by the authors that demineralization and the treatment with a virucidal agent inactivates hiv in spiked and infected bone. according to narang., the dabm grafts do not cause any immunologic response. routine investigations, prophylaxis, occlusal equilibration, and pre - operative intraoral peri - apical radiographs were taken using paralleling device, and the patients were instructed to adopt meticulous home care measures to control dental plaque. flap operations were carried out by a single operator each time to reduce the chances of biasing. the defects were randomly selected by the lottery method, and the intra - oral free osseous autograft (site a) and dabm graft (site d) were placed in the designated sites. we opted not to use gtr membranes to determine the sole role of the grafts used in producing radiographic bone fill and then have a comparison based truly on their individual abilities to form bone, as we know that gtr membranes in itself have the ability to form new bone if we provide enough space between the defect and the membrane. the patients were given post - operative medications including : amoxicillin 500 mg + lactic acid bacillus 6010 cells orally thrice - daily for 5 days post - operativelya 60-second rinse with 15 ml of 0.2% chlorhexidine digluconate was prescribed twice - daily for 3 weeksibuprofen 400 mg orally every 6 - 8 hours for 3 days and then as and when required by the patient was recommended for pain relief. amoxicillin 500 mg + lactic acid bacillus 6010 cells orally thrice - daily for 5 days post - operatively a 60-second rinse with 15 ml of 0.2% chlorhexidine digluconate was prescribed twice - daily for 3 weeks ibuprofen 400 mg orally every 6 - 8 hours for 3 days and then as and when required by the patient was recommended for pain relief. plaque levels were periodically checked in both groups using plaque index, and oral prophylaxis was done at each follow - up visit (approx. every month). this bone graft was obtained intra - orally from the patient 's own mouth at the time of surgery from various sites viz., maxillary tuberosity region, edentulous regions of either maxilla or mandible, extraction sockets fresh or healed [figure 1 ]. the trephine method for procurement of cancellous bone from the edentulous region the bone was obtained after the reflection of the mucoperiosteal flap with the help of bur / chisel / bone rongeurs. the cortical bone was discarded, and the cancellous bone was utilized as the grafting material after being kept in sterile isotonic normal saline solution [figure 2 ]. the procured autogenous cancellous bone the decalcified allogenic bone matrix (dabm) used in our study was made from the fresh bone obtained from the corresponding dept of orthopedics, government medical college and hospital. the source of bone for preparing dabm was a government hospital recognized by the state government since decades. and, all the functioning of the hospital including biomedical waste disposal etc. the bone thus obtained was through proper channels with written records including patient 's details like name, age, sex, medical status etc. the following inclusionary / exclusionary criteria were followed while selecting the patients as donors : a)the patient should be in good general / systemic health, except for the orthopedic problem, for which he has visited the hospitalb)the patient should not be on heavy doses of anti - microbials for any reasonc)after the initial pre - operative investigations performed by the orthopedic dept. including screening for hiv, hbv etc., the patient should be free of any type of systemic disease, for example any type of microbial infection, hiv, hepatitis etcd)the bone thus obtained underwent a series of processes of demineralization and appropriate treatment with virucidal agents. the bone was sectioned into thin small pieces and then decalcified in 0.6 n hcl, for 6 to 7 days till the bone became resilient. the bone so formed was repeatedly washed in normal saline solution, and the acid was removed by repeated washings with 70% ethanol. decalcified allogenic bone matrix (dabm) so prepared was stored in 70% ethanol for 1 - 21 days before transplantation. on the day of the surgery, the graft material was first washed repeatedly in normal saline, and then boiled in distilled water for 30 minutes, and again kept in normal saline ready to be transplanted [figure 3 ]. the patient should be in good general / systemic health, except for the orthopedic problem, for which he has visited the hospital the patient should not be on heavy doses of anti - microbials for any reason after the initial pre - operative investigations performed by the orthopedic dept. including screening for hiv, hbv etc., the patient should be free of any type of systemic disease, for example any type of microbial infection, hiv, hepatitis etc the bone thus obtained underwent a series of processes of demineralization and appropriate treatment with virucidal agents. the bone was sectioned into thin small pieces and then decalcified in 0.6 n hcl, for 6 to 7 days till the bone became resilient. the bone so formed was repeatedly washed in normal saline solution, and the acid was removed by repeated washings with 70% ethanol. decalcified allogenic bone matrix (dabm) so prepared was stored in 70% ethanol for 1 - 21 days before transplantation. on the day of the surgery, the graft material was first washed repeatedly in normal saline, and then boiled in distilled water for 30 minutes, and again kept in normal saline ready to be transplanted [figure 3 ]. according to mellonig the use of exclusionary techniques in the procurement of bone allografts greatly reduced the chances of disease transmission. it was concluded by the authors that demineralization and the treatment with a virucidal agent inactivates hiv in spiked and infected bone. according to narang., the dabm grafts do not cause any immunologic response. routine investigations, prophylaxis, occlusal equilibration, and pre - operative intraoral peri - apical radiographs were taken using paralleling device, and the patients were instructed to adopt meticulous home care measures to control dental plaque. flap operations were carried out by a single operator each time to reduce the chances of biasing. the defects were randomly selected by the lottery method, and the intra - oral free osseous autograft (site a) and dabm graft (site d) were placed in the designated sites. we opted not to use gtr membranes to determine the sole role of the grafts used in producing radiographic bone fill and then have a comparison based truly on their individual abilities to form bone, as we know that gtr membranes in itself have the ability to form new bone if we provide enough space between the defect and the membrane. the patients were given post - operative medications including : amoxicillin 500 mg + lactic acid bacillus 6010 cells orally thrice - daily for 5 days post - operativelya 60-second rinse with 15 ml of 0.2% chlorhexidine digluconate was prescribed twice - daily for 3 weeksibuprofen 400 mg orally every 6 - 8 hours for 3 days and then as and when required by the patient was recommended for pain relief. amoxicillin 500 mg + lactic acid bacillus 6010 cells orally thrice - daily for 5 days post - operatively a 60-second rinse with 15 ml of 0.2% chlorhexidine digluconate was prescribed twice - daily for 3 weeks ibuprofen 400 mg orally every 6 - 8 hours for 3 days and then as and when required by the patient was recommended for pain relief. plaque levels were periodically checked in both groups using plaque index, and oral prophylaxis was done at each follow - up visit (approx. out of 30 patients, 29 patients reported, and all the statistical analysis was done for 29 patients. it was observed that both the materials were well tolerated by the patients as far as post - operative healing was concerned and did not show any allergic manifestation. according to narang., the dabm 's replacement with new bone was seen within 8 weeks, and the areas were completely free of any remnant of dabm graft at the end of 12 weeks. therefore, we did the post - operative measurements at 12 weeks and 24 weeks post - operatively. post - operative assessment was done by taking intra - oral peri - apical radiographs 12 weeks and 24 weeks post - operatively. the x - rays were standardized using a paralleling device specially designed for this purpose and the following fixed angulations (from vertical plane to occlusal plane) [figure 4 ]. molar teeth 30 degreespremolar 40canine 50 molar teeth 30 degrees molar 0premolar -10canine -20 the mean bone fill was measured using an external metallic grid placed over the radiographic films. then, the measurements were made using a vernier caliper in 1/10 of a millimeter [figures 5 and 6 ]. radiographic assessment of bone fill with intra - oral free osseous autograft using grid 4ri- pre - operative, 4rii- at 12 weeks, 4riii- at 24 weeks radiographic assessment of bone fill with decalcified allogenic bone matrix using grid. 5ri- pre - operative, 5rii- at 12 weeks, 5riii- at 24 weeks the mean bone fill obtained for site a and site d after 12 weeks was 1.79 mm and 1.34 mm, respectively ; while after 24 weeks, it was 2.89 mm and 1.85 mm, respectively [figure 7 and table 1 ]. mean bone fill with two types of graft materials at various time intervals comparison of mean change in bone fill at various time intervals between both graft materials for statistical analysis, unpaired - t test was applied. statistical analysis showed that the difference between the two groups was non - significant ; p>0.05 from baseline to 12 weeks, significant with p=0.004 ; p0.05 from baseline to 12 weeks, significant with p=0.004 ; p<0.01 (significant at 1% significance level) from baseline to 24 weeks, and significant with p=0.005 ; p<0.01 (significant at 1% significance level) from 12 to 24 weeks [figure 8 and table 2 ]. standard deviations in bone fill at various time intervals within both sites standard deviations in bone fill at various time intervals within both sites the findings regarding the bone fill achieved with dabm are in conformity with the findings of narang., einhorn., gill. turek has reported that dabm itself gets resorbed and initiates host tissues to form new bone by its osteoinductive and osteoconductive property. the findings regarding the bone fill obtained with intra - oral free osseous autograft are in conformity with the findings of marvin, sindet - pederson s, oosterbeek. when the relative efficacy of intra - oral free osseous autograft and the dabm was tested, the bone fill for autograft was found to be highly significant as compared to the bone fill for dabm, both after 12 weeks and 24 weeks. the higher bone fill achieved with autograft as compared to dabm may be attributed to the fact that the graft material consisted of viable osteoblasts and was osteogenic. the difference of the bone fill achieved with both the grafts at 24 weeks post - operatively was much higher than at 12 weeks post - operatively meaning, thereby, that with the passage of time from 12 weeks to 24 weeks, the gain in the bone fill achieved with the autograft is much higher than that of dabm graft. given the limitations in this study, the result obtained establishes the superiority of the intra - oral free osseous autograft over that of dabm graft in correcting the intrabony defects. no doubt, an additional surgical site has to be created for the procurement of intra - oral free osseous autografts, yet, both the donor as well as the recipient site healed over the same period of time and showed no unusual finding as far as post - operative healing was concerned. | background and objectives : the primary goal of periodontal treatment is the maintenance of the natural dentition in health and comfortable function. the shift in therapeutic concepts from resection to regeneration has significantly impacted the practice of periodontology. the objective of the present study is to compare the relative efficacy of intra - oral autogenous graft and decalcified allogenic bone matrix (dabm) in the treatment of periodontal intrabony defects.materials and methods : in the present study, 30 patients in the age group of 30 - 50 years with two almost identical intrabony defects, on either side of the mouth / upper and lower jaw, based upon the radiographic observations were selected from amongst the patients visiting the department of periodontology and oral medicine, punjab government dental college and hospital, amritsar. one of the defect was selected randomly and filled with autogenous cancellous graft and the other with dabm. post - operative assessment was done by taking radiographs, 12 weeks and 24 weeks post-operatively.results:definite bone fill was achieved both with intra - oral free osseous autograft and with dabm at 12 weeks observation, which further increased significantly at 24 weeks observation. the bone fill obtained with intra - oral free osseous autograft was found to be significantly higher than that with dabm both at 12 weeks and 24 weeks post - operative observation.conclusion:within the limitations of this study, it establishes the superiority of the intra - oral free osseous autograft over that of dabm graft in correcting the intrabony defects. |
although any organ may be infiltrated by leukemic cells, breast infiltration is extremely rare. breast infiltration as the initial presentation or relapse of leukemia can be mistaken for a benign breast mass or primary breast cancer. to my knowledge, there has been no previous case report of leukemic infiltration of the breast by biphenotypic acute leukemia (bal). most case reports have described extramedullary leukemic infiltration by acute myeloid leukemia (aml) and acute lymphoblastic leukemia (all). here i present the case of a patient with extramedullary infiltration of the breast as a presentation of bal relapse and review the literature. in march 2014, a 28-year - old korean woman, at 24 weeks of gestation, presented with easy fatigue and an abnormal blood test. a complete blood count revealed a total white blood cell (wbc) count of 2.6910/l with 12% immature cells, a hemoglobin level of 6.8 mg / dl, and a platelet count of 8110/l. a bone marrow biopsy showed 78% blasts with various size nucleoli, fine chromatin, and basophilic cytoplasm. immunophenotyping of the bone marrow revealed that the patient was positive for the following : cd7, cd13, cd19, cd33, cd117, ccd79a, hla - dr, and terminal deoxynucleotidyl transferase (tdt) ; and negative for the following : cd2, cd3, cd5, cd20, ccd22, cd10, cd14, cd61, and myeloperoxidase. this result showed coexpression of markers for b lymphoid (cd19 and ccd79a) and myeloid lineage (cd117, cd13, and cd33), based on which a diagnosis of bal was confirmed. after a cesarean delivery at 28 weeks of gestation, the patient was treated with induction and consolidation therapy from may 2014 to september 2014. the induction regimen included daunorubicin 90 mg / m on days 1 to 3, vincristine 2 mg / m on days 1 and 8, prednisolone 60 mg / m on days 1 to 14, l - asparaginase 4,000 units / m on days 17 to 28, and intrathecal methotrexate 15 mg on days 2 and. for the first and third cycles, she was treated with daunorubicin 45 mg / m on days 1 and 2, vincristine 1.4 mg / m on days 1 and 8, prednisolone 60 mg / m on days 1 to 14, and l - asparaginase 4,000 units / m on days 1 to 7. for the second cycle, she received cytarabine 2,000 mg / m on days 1 to 4 and etoposide 150 mg / m on days 1 to 4. m on days 1 and 15 ; 720 mg / m on days 2 and 16). in november 2014 there was no evidence of relapse for 23 months after the stem cell transplantation. in september 2016, the patient presented with easy fatigue, a palpable right breast mass, and nipple discharge, which had developed 4 months prior. she had no history of bleeding, weight loss, or dizziness over the previous few months. on physical examination, a 6 cm6 cm firm, irregular, nontender mass was palpated in the upper inner area of her right breast. mammography showed extremely dense breast tissue but no well - defined mass (figure 1a). however, breast ultrasonography revealed 5.72.15.9 cm and 3.01.42.5 cm masses with partly indistinct margins and a heterogeneous echoic pattern in her right breast (figure 1b). a complete blood count showed a total wbc count of 7.4110/l, a hemoglobin level of 14.3 mg / dl, and a platelet count of 15110/l. a core needle biopsy (cnb) was performed on the 2 oclock direction mass in the right breast. microscopic examination revealed monomorphous infiltrates of blast cells with round nuclei and fine chromatin (figure 2a). the immunohistochemical (ihc) staining showed that the majority of blast cells were positive for cd34 and cd117 (figure 2b and c). a bone marrow karyotyping demonstrated 46, xy and no recipient xx cells, which suggested complete engraftment of transplanted donor cells. bone marrow differential counting showed 3.8% blasts and a 2.1:1 ratio of myeloid to erythroid precursors, which were within normal ranges. however, a bone marrow biopsy showed diffuse infiltration of immature cells with irregular nucleoli and a markedly decreased number of normal hematopoietic cells. infiltration of the breast by acute leukemia is rare, regardless of whether it is found at initial presentation or at relapse after treatment. of 235 patients with aml, only four had involvement of the breast (1.7%). although few cases have been described, breast infiltration seems more likely to occur in patients with aml rather than those with all. the expression of adhesion molecules such as cd56 was associated with extramedullary granulocytic sarcoma, which occurred in 3% to 7% of aml cases. patients with leukemic infiltration of the breast usually present with a palpable breast mass that mimics a benign breast lesion, such as a fibroadenoma. mammography and breast ultrasonography findings are variable and can be unremarkable with diffusely dense breast parenchyma. on ultrasonography, no definitive imaging pattern suggesting leukemic infiltration of the breast has been described because sonographic features of tumors vary from hypoechoic to hyperechoic masses. magnetic resonance imaging (mri) may be another diagnostic tool, especially in patients with dense glandular tissue, breast implants, or pregnancy. mri features include t2 hyperintensity and rapid ring - enhancement of the lesions. for a final diagnosis of leukemic infiltration of the breast a cnb under ultrasono - graphy guidance is an acceptable and safe method to establish the diagnosis and avoid unnecessary surgical biopsy. the cytologic features of breast infiltration of all include dispersed monomorphic blastic cells with variable size, high ratio of nuclear to cytoplasmic, round or convoluted nuclei with dispersed chromatin, and scanty cytoplasm. in the initial bone marrow biopsy of our case, cd7, cd13, cd19, cd33, cd117, ccd79a, hla - dr, and tdt were positive, indicating expression of both myeloid cell and lymphoid cell markers. in the biopsy of breast based on literature reviews, leukemic infiltration of the breast by acute leukemia has occurred in patients with aml and all. however, to the best of our knowledge, this is the first report of leukemic infiltration of the breast by bal. most experts believe that there is no role of surgery for breast leukemic infiltration because there is no evidence of a clinical benefit of surgery for breast lesions, and surgery may delay the initiation of systemic therapy. in conclusion, it should be considered in the differential diagnosis of breast masses, especially when there is a history of acute leukemia. mammography and breast ultrasonography may be useful to detect breast nodules. with cnb and ihc staining for leukemia markers, a definitive diagnosis can be established while avoiding unnecessary breast surgery, thus enabling early initiation of systemic therapy. | in acute leukemia, leukemic infiltration of the breast is extremely rare. we report a case of biphenotypic acute leukemia (bal) that presented as a breast mass. a 30-year - old woman presented with a 4-month history of a right breast mass with nipple discharge and easy fatigue. she had received chemotherapy and peripheral blood stem cell transplantation for bal and had been in complete remission for the last 2 years. core needle biopsy of the breast mass revealed monomorphous infiltrates of blast cells with round nuclei and fine chromatin, consistent with leukemic infiltration. subsequent bone marrow biopsy showed diffuse infiltration of immature cells. however, bone marrow karyotyping showed 46, xy, suggesting complete engraftment of transplanted donor cells. this is the report of bal recurring as a breast mass. in the differential diagnosis of a breast mass, extramedullary relapse should be considered when the patient has a history of leukemia. |
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