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Aniridia is a rare bilateral congenital malformation of iris due to neuro - ectodermal developmental defect secondary to a mutation in pax6 gene located on chromosome 11p13 . The incidence of aniridia is estimated to be 1 in 60,000 - 100,000 live births.1 it exists in both sporadic and familial form . It may occur in isolation or associated with number of syndromes such as wagr (wilm's tumor, bilateral aniridia, genitourinary abnormalities and mental retardation). The pax6 mutations cause a spectrum of ocular malformations, of which aniridia is the major sign . Other ocular defect that may be associated includes microcornea, corneal opacity, glaucoma, cataract, subluxated lens, foveal and optic nerve hypoplasia . We report two cases of familial total aniridia associated with microcornea, high myopia and aphakia . A 40-year - old male patient presented with marked dimness of vision for last 1 year . His elder brother (only sibling) had visual acuity of 6/6 with -1.5 dioptre sphere (dsph) without any other ocular abnormalities . Ocular examination revealed best corrected visual acuity in right eye (re) counting finger at 3 feet and 3/60 in left eye (le) with + 7.0 dsph . Slit lamp examination showed bilateral total aniridia with superiorly subluxated cataractous lens in the re [figure 1] and aphakia in the le . The radius of corneal curvature was 8.8 mm in the re and 8.6 mm in the le . The refractive power of cornea of the re in dioptre (d) was 39.0 in both vertical and horizontal meridian . The anterior chamber depth in the re was 1.54 mm and in the le was 1.48 . The axial length was 25.57 mm and 25.34 mm in the re and le respectively . Gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open . Aniridia with superiorly subluxated lens dislocated lens in vitreous a 7-year - old girl, the only child of case i, had poor vision since early childhood . Her best corrected visual acuity was 6/36 be with -7.0 dsph and -1.0 dioptre cylinder (dcyl) at 90. she had bilateral total aniridia, microcornea, microspherophekic lens [figure 3] and horizontal nystagmus . Average corneal diameter of be was 9.5 mm . Radius of corneal curvature was 8.6 mm in the re and 8.3 mm in the le . The refractive power of cornea of the re was 38.5d in vertical and 37.5d in horizontal meridian . The diameter of lens was 7.9 mm in be and iop in be with goldmann applanation tonometry was 14 mm hg . Gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open . Fundus examination of be revealed normal optic disc with foveal hypoplasia [figure 4]. A 40-year - old male patient presented with marked dimness of vision for last 1 year . His elder brother (only sibling) had visual acuity of 6/6 with -1.5 dioptre sphere (dsph) without any other ocular abnormalities . Ocular examination revealed best corrected visual acuity in right eye (re) counting finger at 3 feet and 3/60 in left eye (le) with + 7.0 dsph . Slit lamp examination showed bilateral total aniridia with superiorly subluxated cataractous lens in the re [figure 1] and aphakia in the le . The radius of corneal curvature was 8.8 mm in the re and 8.6 mm in the le . The refractive power of cornea of the re in dioptre (d) was 39.0 in both vertical and horizontal meridian . The anterior chamber depth in the re was 1.54 mm and in the le was 1.48 . The axial length was 25.57 mm and 25.34 mm in the re and le respectively . Gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open . A 7-year - old girl, the only child of case i, had poor vision since early childhood . Her best corrected visual acuity was 6/36 be with -7.0 dsph and -1.0 dioptre cylinder (dcyl) at 90. she had bilateral total aniridia, microcornea, microspherophekic lens [figure 3] and horizontal nystagmus . Average corneal diameter of be was 9.5 mm . Radius of corneal curvature was 8.6 mm in the re and 8.3 mm in the le . The refractive power of cornea of the re was 38.5d in vertical and 37.5d in horizontal meridian . The diameter of lens was 7.9 mm in be and iop in be with goldmann applanation tonometry was 14 mm hg . Gonioscopy of be revealed rudimentary frill of iris tissue in all quadrants and the angle was open . Fundus examination of be revealed normal optic disc with foveal hypoplasia [figure 4]. Aniridia is a rare congenital abnormality transmitted as an autosomal dominant trait with 85 - 90% penetrance.2 microcornea may rarely be associated with aniridia.34 high myopia is again a very rare association with aniridia and microcornea.5 both these abnormalities were present in our case . Aniridia and lenticular abnormalities like cataract and subluxated lens may co - exist.134] case i had subluxated cataractous lens in the re and aphakia in the le due to spontaneously dislocated lens in vitreous cavity in the le . To the best of our knowledge, in the literature of aniridia association of aphakia aniridia may be associated with defects in angle of anterior chamber and glaucoma, but the findings were not present in our cases . Foveal hypoplasia that may be associated with aniridia was present in our case.2 none of the patients was mentally retarded and thorough systemic examination was normal . Our cases highlight the rare association of congenital aniridia with microcornea, aphakia and high myopia.
Astigmatism treatment has always been a challenge for ophthalmologists and has followed an evolutionary pathway over the years, with experience in several surgical procedures . Since the approval of the excimer laser in 1995 for use in reshaping the cornea, significant developments in treating refractive diseases like myopia, hyperopia, and astigmatism have been achieved.1 photorefractive keratectomy (prk) in general is a surgical procedure using an excimer laser to reshape the central cornea to treat refractive errors . In prk, each method has special advantages and drawbacks; however, their outcomes are not as satisfactory as those of spherical ablation.2 wavefront sensors have been popular in astronomy for many decades, and appeared in the field of ophthalmological science quite recently, to identify and correct low- and high - order aberrations.3 wavefront - guided (wf) surgical procedures have shown to be promising in astigmatism treatment . 1 according to the literature, excellent clinical outcomes, safety, and efficacy of various wf and wavefront - optimized lasik treatments in low - to - moderate myopia are reported . However, data on high myopia are scarce.47 cross - cylinder prk is another technique of interest in astigmatism surgery . Cross - cylinder prk flattens the steepest meridian with central cylindrical ablation and steepens the flattened meridian with paracentral ablation . It may result in symmetrical corneal shape, better visual acuity, and less regression.8,9 although there are many studies of the efficacy and safety of wf and cross - cylinder prk, there are few studies on moderate - to - high astigmatism cases . The aim of this study was to compare the efficacy, safety, and predictability of wf and cross - cylinder prk in moderateto- high astigmatism . In a comparative cohort, the results of two before - and - after clinical trials conducted in nikoukari university hospital in tabriz, iran, between december 2009 and december 2010 were compared . Fifty eyes of 25 patients with stable refraction having moderate - to - high astigmatism were enrolled for a before - and - after clinical trial to undergo wf prk using the visx system (visx inc, santa clara, ca). The second clinical trial enrolled 48 eyes of 24 patients with stable refraction having moderate - to - high astigmatism to undergo prk by cross - cylinder method using a nidek ec-5000 (nidek co, ltd, gamagori, japan) excimer laser with repetition rate of 40 hz (v 1.26 w). All surgeries were performed by one surgeon (mrs) at the tabriz excimer laser center, tabriz, iran . Except for cohort timing, the inclusion criteria were stable refraction; astigmatism above 1.50 d; and age between 2050 years . Exclusion criteria were other ocular pathologies; pachymetry less than 470 m; connective tissue disease; asymmetric astigmatism; and tear film abnormality . The range of cylinders was from 1.5 d to 5.0 d. laser treatment parameters for cross - cylinder surgery were: hyperopic cylinder with 6.09.0 mm zone; myopic cylinder with 6.57.5 mm zone; and spherical equivalent treated at 6.07.0 mm (or 6.57.5 for large pupils). Under topical anesthesia (tetracaine 0.5%), the epithelium was removed by applying 20% alcohol for 15 seconds at the area of 8.0 or 9.0 mm optical zone marker and the laser was fired . Mitomycin c 0.02% was applied for 1545 seconds and a bandage contact lens was placed at the end of the procedure . The primary outcome of interest in this study was the amount of astigmatism corrected during a 6-month period after surgery . The endpoint measurement for this outcome was absolute change in refraction scores during the time period after surgery . The secondary outcomes were visual acuity measured using a snellen chart, and visual aberrations measured by aberrometry . Diclofenac and ciprofloxacin eye drops were discontinued after 2 days and following re - epithelialization, respectively . Betamethasone eye drops were replaced by fluorometholone 2 weeks postoperatively and continued for 3 months . Data were primarily analyzed using the stata statistical software package (v 11; stata, college station, tx). The surgically - induced astigmatism (sia) vector, target - induced astigmatism (tia) vector, astigmatic correction index (ci), index of success (i0s), angle of error, magnitude of error, flattening effect (fe), and flattening index were analyzed using methods described by alpins.10 sia is the vector of the astigmatic change actually induced by the surgery . Tia is the vector of the astigmatic change intended to be induced by the surgery . Ci> 1.0 and ci <1 indicate an overcorrection and undercorrection, respectively . Once the amount of astigmatism to be corrected had been determined, the nomogram was used by aligning the age and preoperative measures the difference vector (dv) is the magnitude and axis of astigmatic change that would enable the initial surgery to achieve its intended target . A ci of 1.00 and an ios of 0 indicate that the desired results have been obtained . The angle of error is the difference between the angles of the sia and the tia . Flattening effect (fe) is the amount of astigmatism reduction achieved by the effective proportion of the sia at the intended meridian (fe = sia cos2 * angle of error). The flattening index, which preferably equals 1, is obtained by dividing the flattening effect by the tia . Analysis of the mean magnitude sia was also performed after stratification of the groups based on the preoperative astigmatism (medium: 1.5 to 2.5 d and high: 2.75 d). Higher order aberrations including coma, trefoil, and spherical aberration were measured using the opd scan . Although visx custom has been recommended for astigmatism of <3.25 d, we included higher astigmatism up to 5 d to get the rate of correction in this subgroup too . The study was approved by the committee of ethics at tabriz university of medical sciences . The mean age of the patients was 30.4 6.7 years in the wf group (range 2348 years). The mean age of the patients was 28.4 7.4 years in the cross - cylinder group (range 1948 years). The percentages of patients with ucva 20/20, 20/25, and 20/40, or better were 80%, 100%, and 100%, respectively . In the cross - cylinder group the percentages of patients with ucva 20/20, 20/25, and 20/40, or better were 40%, 79%, and 98%, respectively . The change in visual acuity is compared between groups in figure 1 . Regarding safety, only one eye in the cross - cylinder group and no eyes in the wf group lost more than one line of bcva after 6 months of treatment . The percentage of eyes with no change in bcva was 54% and 58.3% in the wf and cross - cylinder groups, respectively . The attempted and achieved se refraction and astigmatic refraction for the two groups at the 6-month follow - up is shown in figures 2 and 3 . Total root - mean- square (rms) higher order aber rations were 0.05 0.22 m and 0.17 0.2 m for wf and cross - cylinder methods, respectively . For coma, the measurements were 0.03 0.2 m and 0.14 0.15 m, respectively, for wf and cross - cylinder methods . For trefoil, the measures were 0.02 0.25 m and 0.0 0.16 m, respectively, for wf and cross - cylinder methods . Spherical aberration measures were 0.0 0.25 m and 0.08 0.13 m for wf and cross - cylinder methods, respectively . Mean (sd) preoperative astigmatism measures were 2.91 1.3 and 2.51 0.98 d, respectively, in the wf and cross - cylinder groups . Mean change in refractive astigmatism after 6 months was 1.57 0.53 d in the wf group and 1.67 0.52 d in the cross - cylinder group . The vector analysis results based on the level of astigmatism are given in table 3 . Our findings support the hypothesis that both cross - cylinder and wf procedures are predictable, safe, and effective in treating high astigmatism . We found that 80% of patients achieved ucva 20/20 or better after the wf procedure . However this should be interpreted cautiously because the results come from a cohort of clinical trials instead of random assignment of procedures to control for known and unknown confounders . The advantage of the wf method may also not be generalizable to all types of astigmatism and may only apply to high astigmatism cases, even if the comparability of groups is fulfilled . This argument is based on previous research supporting better results for moderate versus high astigmatism.11,12 however this may not be the case for earlier prk methods used for astigmatism . Kremer et al, in a 1-year follow - up of prk for low, moderate, and high primary astigmatism found that the laser used in their study was an efficient tool to correct high and moderate astigmatism but less effective in low astigmatism.13 bababeygy et al found both better efficacy and higher safety for wf laser in situ keratomileusis in moderate astigmatism as opposed to high astigmatism.12 regarding safety and predictability, the two procedures appeared promising in a similar way, but with little superiority of the wf procedure . In this study, angle of error analysis demonstrated that both arithmetic means were slightly clockwise (0.71 and 1.26) and close to zero, which is consistent with the closeness of the vector mean tia and sia axes . Our study found both methods to be acceptable in efficacy, safety, and predictability . Regardless of the differences in percentages and means previously reported in the literature, which may be due to variation in settings and possible confounding factors, our results were in line with previous research . Mostly these studies were done separately for wf - guided astigmatism surgery4,6,12,1421 and cross - cylinder procedures.2225 several studies have used comparative designs, but we did nt find any studies that compared wf with cross - cylinder prk in moderate - to - high astigmatism . Our study did nt benefit from a randomized trial comparison, but had the advantage of substantial comparability considering common population, setting, and surgeon . In this study we found that the wf procedure provided better results regarding ucva, bcva, and refraction; although it did nt prove superiority in the final astigmatism assessment . Also it was found that the amount of hoas, coma, and spherical aberrations were lower in wf versus cross - cylinder procedure . It can be argued that the custom ablation of visx should be compared to its own platform of conventional toric ablation . Visx is a variable spot whereas nidek laser is a slit - beam ablation pattern so it would have been better to compare the nidek to its custom platform . Both methods of prk using the nidek ec-5000 and visx excimer laser systems are effective for correcting moderate - to - high astigmatism . Future randomized clinical trials, preferably on contralateral eyes, are recommended to provide detailed and more trustworthy comparison results . Future studies with larger sample size, stratification of cylinder study, and post - nomogram data gathering with longer follow - up are recommended.
Elastosonography is an ultrasound based diagnosis which measures the elasticity or stiffness of the tissue . Compressibility of the tissue is a parameter evaluated by an elastogram and it is expressed as the change in tissue displacement as a function of its distance from the probe which is recognised as strain . Among the 400450 lymph nodes (lns) of the human body, 6070 are located in the head and neck area . Lns display reactive enlargement in infection and also when they are primarily or secondarily involved in malignancies of head and neck . An important criterion is the hardness of the ln, because lns might demonstrate increased stiffness on the elastogram if affected by a malignant process [24]. The purpose of this study is to determine the reference sonoelastography values of cervical lns . After obtaining the approval of the ethical committee, patients referred to the radiology department for cervical vascular doppler ultrasonography examination from january to march 2013 were enrolled in this prospective study . 97 cervical lns of 89 consecutive patients (70 lns in 64 females, 27 lns in 25 males; mean age 54 years old; age range, 2184 years) who were referred to our clinic for the examination of cervical vasculature were analysed by elastosonography . One radiologist with 20 years of conventional sonography and 5 years of elastosonography experience performed the examination . The scanner used was esaote gold platform mylab 60 (italy) equipped with elasto software (elaxto). B - mode images were first obtained for each ln . Afterwards elastosonography was performed by applying light vertical pressure followed by decompression until a good quality image was obtained . Elaxto software also made the sonographer aware of the compression degree by the help of its unique optimal compression scale for measurement . Real time b - mode and elastosonographic images were demonstrated next to each other on the screen . A region of interest (roi) box was placed in the ln taking the adjacent subcutaneous fat tissue as the reference . Depth, length, width, length to width ratio, hilar thickness, and cortical thickness of the lns were measured . Lns with cortical thickness less than half of its hilar thickness were categorised as group a, and lns with cortical thickness equal to or more than half of the hilar thickness were categorised as group b. first strain ratio (sr) of the lns was measured . Afterwards, elastographic images were given 1 of 4 elasticity scores (ess) based on the percentage of stiff areas modified from choi et al . Which was introduced for the differential diagnosis of axillary lns in breast cancer . Es 1 (figure 1), an absent or a very small red area (i.e., stiff); es 2 (figure 2), small, scattered red areas, total red area <45%; and es 3 (figure 3), large red area, total red area 45%; and es 4 (figure 4), red area occupying the entire ln, were the classifications used in the study . 64 (66%) of the lns were found on the right side and 33 (34%) were found on the left side of the neck . The mean size of the lns was 17 mm with a range of 628 mm . The mean depth of the lns was 11 mm, with a range of 519 mm . 18 lns (18.6%) were categorised as group a. 79 lymph nodes (81.4%) were categorised as group b. 67 lns (69.1%) had sr less than 3 and were categorised as group 1 . 30 lns (30.9%) with sr equal to or more than 3 were categorised as group 2 . 32 lns (33%) displayed es 1; 30 lns (30.9%) demonstrated es 2; 22 lns (22.7%) showed es 3; 13 lns (13.4%) had es 4 . 62 lns (63.9%) with scores 1 and 2 were assigned as es grade 1; 35 lns (36.1%) having scores 3 and 4 were assigned as es grade 2 . There was a statistically significant correlation between thickness ratio groups and elasticity score grades (p: 0.011). A statistically significant correlation was also demonstrated between sr groups and elasticity score grades (p: 0.000). Elastosonography, a noninvasive imaging modality, can potentially help the differentiation of benign and malignant lns by reducing the number of unnecessary biopsies . In the literature, several studies have been published for the sonographic diagnosis of metastatic cervical lns by b - mode [610]. B - mode criteria for evaluating superficial lns are size, shape, the presence or the absence of the hilum, ratio of the cortex to hilum, borders, echogenicity, and homogeneity of internal structures . However no single ultrasonography criterion for malignant lns had high sensitivity and specificity [11, 12]. Specific criteria to differentiate reactive and metastatic cervical lns are still not well established . A simple, reproducible, noninvasive imaging technique for diagnosis of malignant lns is necessary . Sohn et al . Investigated the b - mode criteria to differentiate metastatic lns from benign lns taking fine needle aspiration biopsy as the gold standard . They evaluated the diagnostic performance of each ultrasound feature (loss of fatty hilum, presence of cystic change or calcification, hyperechogenicity, and round shape). They concluded that the most accurate ultrasound criterion to differentiate metastatic from benign lymph nodes was the presence of one of these malignant ultrasound findings, excluding the loss of fatty hilum . The 5-point elasticity scoring system was introduced by itoh et al . For breast lesions . Published their results in the diagnosis of cervical ln metastasis using gray - scale sonographic elastography . They utilized a 4-point scale including visibility, relative brightness, margin regularity, and margin definition of the ln . They measured strain index of the lns by comparing the absolute strain values of the lymph nodes with the absolute strain values values of the nearby muscles . Strain index greater than 1.5 was the most useful criteria in metastatic ln classification, having 98% specificity, 85% sensitivity, and 92% accuracy . Alam et al . Used a 5-pattern color scoring system based on distribution and percentage of area with high elasticity in the cervical lns . The cut - off line for reactive versus metastatic lns was set between patterns 2 and 3; patterns 35 were considered metastatic . They evaluated the sum of scores of five criteria, that is, short - axis diameter, shape, border (regular or irregular), echogenicity (homogeneous or inhomogeneous), and presence or absence of hilum for b - mode diagnosis . The cut - off line for reactive versus metastatic was set between scores 6 and 7 . Sensitivity, specificity, and accuracy of b - mode sonography were 98%, 59%, and 84%, respectively; 83%, 100%, and 89% for elastography; and 92%, 94%, and 93% for the combined evaluation . They concluded that the combination of elastography and b - mode sonography has the potential to improve the diagnosis of metastatic enlarged cervical lns . Concluded that elastosonography had 93.8% sensitivity and 89.5% specificity in the differentiation of benign and malignant cervical lymph nodes . In a meta - analysis of the real - time elastography for the differentiation of benign and malignant superficial lns ying et al . Concluded that even though es measurement had a good diagnostic accuracy, it had significant interobserver variability thus, a quantitative method such as strain ratio measurement was needed for analysis of elasticity of the ln . Although the accuracy of strain ratio measurement was similar to es measurement, it improved the diagnostic sensitivity value . Their meta - analysis suggested that real - time elastography could be used as a good identification tool for malignant lns . Nevertheless only a limited number of studies have been carried out to determine the elasticity of various tissues . Levinson et al . Evaluated the elasticity of skeletal muscles and arda et al . Conducted a study to determine the elasticity values of normal soft tissue including thyroid, parotid, and submandibular glands, gastrocnemius and masseter muscles, supraspinatus and achilles tendons, renal cortex, and pelvis, spleen, and pancreas of healthy volunteers . Estimation of tissue elasticity is useful for characterization of normal tissue . To our knowledge, the normal elasticity values of cervical lymph nodes have not been studied so far . In our study 36.1% of the evaluated cervical lns the aim of elastography is not necessarily to diagnose malignancies but to help reduce the number of unnecessary biopsies for benign processes by determining soft versus hard tissues due to its high specificity [2, 3, 19]. Elastosonography is a user dependent technique closely related to experience . Due to the shape of the neck, the pulsations from the neighboring great vessels may also make it difficult to obtain good quality elastograms . The primary aim of this research was to determine the reference values of cervical lns to guide elastographic studies not to establish the benign versus malignant nature of the lymph nodes.
Midfoot fractures and dislocations are uncommon because of the intrinsic stability of the tarsal structure . The arthrodesis of the talonavicular joint and calcaneal - cuboid joint is technically complex in the presence of bone defect and there is a high incidence of nonunion . Furthermore, in the presence of large skin exposures, the possibility of infection and the consequent negative effect on bone fusion associate the surgical procedure with a high risk of failure [1, 2]. In august 2012, a 49-year - old - man presented to our hospital with a traumatic forefoot subamputation after a work accident . The trauma caused a lacerated wound in correspondence of the dorsal surface of the right foot . Radiographs and computed tomography (ct) examination confirmed the diagnosis of the loss of talus head during the trauma (fig . 1). Figure 1:ap (a) and lateral (b) radiographs and ct images (c, d) confirm the diagnosis of the loss of talus head during the trauma . Ap (a) and lateral (b) radiographs and ct images (c, d) confirm the diagnosis of the loss of talus head during the trauma . Injuries of dorsalis pedis artery, tibialis anterior tendon, estensor digitorum longus tendons, peroneus tertium tendon were revealed, with the loss of talus head and with a complex lesion of the talonavicular ligament . Tendinous structures damaged were surgically sutured and subsequently an antibiotic - loaded cement spacer was positioned into the bone gap to re - establish the joint congruence . The foot was put in a cast and kept non - weight bearing for 8 weeks (fig . 2). Figure 2:an image of the foot before the first - time surgical repair reveals the injuries of dorsalis pedis artery, tibialis anterior tendon, estensor digitorum longus tendons, peroneus tertium tendon, with the loss of talus head and with a complex lesion of the talonavicular ligament . (a) tendinous structures damaged were surgically sutured and subsequently an antibiotic - loaded cement spacer was positioned into the bone gap to reestablish the joint congruence (b). Ap (c) and lateral (d) radiographs of the foot in the cast after the surgical time . An image of the foot before the first - time surgical repair reveals the injuries of dorsalis pedis artery, tibialis anterior tendon, estensor digitorum longus tendons, peroneus tertium tendon, with the loss of talus head and with a complex lesion of the talonavicular ligament . (a) tendinous structures damaged were surgically sutured and subsequently an antibiotic - loaded cement spacer was positioned into the bone gap to reestablish the joint congruence (b). Ap (c) and lateral (d) radiographs of the foot in the cast after the surgical time . Two months after the primary surgery, the patient underwent salvage arthrodesis of chopart joint via double dorsal incision . The patient was placed in a supine position with a support under the ipsilateral hip to allow easy access to the medial and lateral aspects of the foot . The skin incision was made along the lateral aspect of the foot, starting at the base of the fourth metatarsal, and extended proximally toward the tip of the fibula, stopping 1 cm short of the tip . The capsule of the extensor digitorum brevis was opened, its origin was released and the muscle was reflected distally 1 cm distal to the calcaneocuboid joint . The calcaneocuboid joint was identified and the soft tissue stripped plantarward and dorsally using a periosteal elevator . The articular cartilage was removed from the calcaneocuboid joint using a small, sharp osteotome . Placing a deep retractor into the wound along the dorsal aspect, the lateral aspect of the talonavicular joint opposite the calcaneocuboid joint was identified and articular cartilage was removed . The medial approach was through a longitudinal incision, starting at the tip of the medial malleolus and carried distally 1 cm past the naviculocuneiform joint . Figure 3:lateral approach at the base of the fourth metatarsal, and extended proximally toward the tip of the fibula, 1 cm short of the tip to identify the calcaneocuboid joint (a). Medial approach through a longitudinal incision, starting at the tip of the medial malleolus and carried distally 1 cm past the naviculocuneiform joint . Lateral approach at the base of the fourth metatarsal, and extended proximally toward the tip of the fibula, 1 cm short of the tip to identify the calcaneocuboid joint (a). Medial approach through a longitudinal incision, starting at the tip of the medial malleolus and carried distally 1 cm past the naviculocuneiform joint . An elevator was passed over the dorsal aspect of the talonavicular joint, completely freeing the joint . It was removed the antibiotic - loaded cement spacer . Using a towel clip embedded into the proximal portion of the navicular, the talonavicular joint was distracted by pulling the foot in an adducted position and longitudinally . An autologous graft, harvested from the omolateral iliac crest, was placed into the talonavicular joint . It was evaluated that there was no gap at the calcaneocuboid joint when the foot was brought into a plantigrade position . Before placing the internal fixation, the bone ends were heavily scaled using a 4 mm osteotome . The talonavicular joint was fused in situ using two 4.0-mm cannulated screws across the talonavicular joint . The foot was then manipulated into proper alignment; the guide pin for the 4.0-mm cannulated screw was placed across the talonavicular joint starting at the distal end of the navicular at the naviculocuneiform joint . The navicular was overdrilled with a 4.0-mm drill bit, after which 4.0-mm long threaded screws were inserted . The screws were brought from proximal to distal, starting in the anterior process area and brought obliquely across into the cuboid (fig . Figure 4:the placement of an autologous graft into the talonavicular joint harvested from the omolateral iliac crest (a). The fixation of the calcaneocuboid joint was carried out using two 4.0-mm cannulated screws (b) and the talonavicular joint was fused in situ using two 4.0-mm cannulated screws across the talonavicular joint (c). The placement of an autologous graft into the talonavicular joint harvested from the omolateral iliac crest (a). The fixation of the calcaneocuboid joint was carried out using two 4.0-mm cannulated screws (b) and the talonavicular joint was fused in situ using two 4.0-mm cannulated screws across the talonavicular joint (c). The foot was put in a cast and kept non - weight bearing for 10 weeks, with range - of - movement exercises of the knee and hip allowed . The patient was reevaluated during a clinical and radiographic follow - up at 1, 3, 6 months after surgery . There were no infectious problems and the patient has resumed normal work activities . At the sixth month follow - up, the patient had returned to work and remained pain free while walking, with good fusion of both joints (fig . Figure 5:ap (a) and lateral (b) radiographs of the foot in the cast after the surgical time . Ap (c) and lateral (d) radiographs of the foot at the sixth month follow - up . Ap (a) and lateral (b) radiographs of the foot in the cast after the surgical time . Ap (c) and lateral (d) radiographs of the foot at the sixth month follow - up . Dislocations of the midfoot are uncommon because of the constrained configuration of multiple articular surfaces augmented by capsular attachments, strong ligaments and tendons [5, 6]. Foot trauma of this severity can result in articular incongruities, complex derangement of the arc geometry, ligamentous instability, and, eventually, long - term disability secondary to joint subluxation and posttraumatic arthritis . In our patient, the surgical management was based on the principles for treatment of chopart joint fracture dislocations: realignment of both medial and lateral columns of the foot, restoration of joint congruity, alignment of axes, temporary fixation and ligament reconstruction to ensure proper ligament healing [7, 8]. Early anatomic reduction, stable fixation and ligament reconstruction could have achieved a better outcome . Primary arthrodesis is a viable option for severe midfoot fracture dislocations, because it facilitates rehabilitation and functional recovery and obviates the need for a secondary arthrodesis should arthritis arise . Furthermore, in the presence of large skin exposures, the possibility of infection and the consequent negative effect on bone fusion make the surgical procedure at high risk of failure . The manuscript is original, it is not under consideration for publication elsewhere, and has not been previously published . The manuscript has been read and approved by all the authors.
Biological evolution is a complex process allowing species and organisms to try to resist the second law of thermodynamics . Over time, this has led to the evolution of increasingly sophisticated brains allowing organisms to adapt and survive longer by learning from experience and predicting future events . Yet, this increase in adaptability also leads to an increasingly difficult resource allocation problem of how to best select between competing wants and likes (kringelbach and berridge, 2009). Human consciousness can be thought of as one particular successful solution to this taxing problem, primarily facilitated by the processes of attention and awareness . The scientific study of human consciousness is growing beyond its infancy and many interesting properties of its underlying neuronal substrates have been established (changeux and lou, 2011). Yet countless fundamental discoveries remain, however, and in particular understanding the precise relationship between attention and awareness has remained elusive (although this special issue is beginning to address the issue). In addition to the main tenet of the present discussion, namely of a bidirectional and intimate relationship between attention and self - awareness networks, it should be noted that in particular for the attentional network, there are asymmetries between hemispheric involvement . For instance, mesulam (1999) drew attention to the prime role of the right hemisphere in generating spatial neglect, and more recently it has been demonstrated that the functional connectivity of the insula to the paralimbic network is asymmetrical as well (cauda et al ., 2011). One way to conceptualize the relationship between attention and awareness is to think of attention as an executive function allowing the focusing of the mind, while awareness is its closely associated counterpart at the highest level of sensory processing . Yet, this conceptualization leaves out the importance of reward and emotion in guiding the crucial decision making involved in optimizing resource allocation of brain resources, which we discuss in this review . Some progress has been made in identifying how self - awareness can help in shaping the decision making . Interestingly, part of this progress has been made possible by studying the introspective states involved in meditation . Studies of highly experienced yoga - nidra practitioners have shown that there are significantly different brain networks involved in the states related to relaxation meditation (yoga - nidra) and normal resting state (i.e., at rest, without intended motor, or mental function), and that these networks vary greatly with the changing contents of consciousness during the two conditions (lou et al ., 1999). In contrast, common to both states and traits is sustained activity in a paralimbic network consisting of midline frontal regions (anterior cingulate and medial prefrontal cortices), parietal regions (primarily precuneus), and striatum (kjaer and lou, 2000; see figures 1 and 2). Principal component analysis was used to identify brain regions common to conscious experiences during yoga - nidra meditation and the normal resting conscious state . The paralimbic network consists of a set of three regions which contributed to both clusters: striatum, medial prefrontal, and medial parietal cortices (precuneus). Being active in a wide variety of conscious experiences, we hypothesize that this paralimbic network represented self - reference, as a common denominator for conscious experiences to account for a sense of unity of consciousness . A medial paralimbic network common to conscious experiences . The figure shows brain maps of activity related to self and other (midline, right, and left hemisphere). (a) emergence of self - representation . Differential activity is noted in medial paralimbic regions, together with bilateral occipital and parietal regions (p <0.05, corrected for multiple comparisons). (b) emergence of representation of other (danish queen). Differential activity is noted in medial paralimbic regions, together with bilateral occipital and parietal regions and a confluent left inferior prefrontal and temporal region . Activity in the paralimbic network is similar across the two conditions, an indication of the pivotal role of this network in higher - order consciousness with self - reference . This discovery led to the hypothesis that the paralimbic network contributes to the regulation of conscious experience by acting as a common reference of self - perspective for changing conscious states and traits (kjaer and lou, 2000). Such a role for the paralimbic network would be consistent with ontogenetic considerations of brain development (feinberg and keenan, 2005). Accordingly, animals with a paleomammalian brain, a brain with developed limbic system, but scarcely developed neocortex, may display quite sophisticated social behaviors like maternal nurturing, separation distress calls, and play, while the adjacent neocortex is essential for perception of the self in space and time, a prerequisite for abstract thinking . The neuroanatomical evidence also supports the hypothesis that the paralimbic network plays a special role in guiding the allocation of brain resources by mediating between attention, awareness, and emotion . The paralimbic regions are anatomically located at the interface between the limbic and the neocortical brain, the former providing information on the bodily state and emotions, while the heteromodal neocortical association regions provide extrapersonal sensory and mnemotic information on the highest level of integration available for attention and awareness . Relying on this broad outline and on anatomical, physiological, and behavioral experiments in the monkey, mesulam (1998) proposed that the cerebral cortex is organized into five hierarchically arranged subtypes: limbic, paralimbic, heteromodal association, unimodal association, and primary sensory motor regions . In the present review we synthesize the evidence from the literature to show that the paralimbic network is active and efficient in self - processing, a prime organizer of conscious experience . We show how the paralimbic network uses information from both the external world and introspection to balance the resource allocation, and how the access and balance in this system is regulated by emotion and by neurotransmitters such as dopamine . Finally, we show how disturbances in the paralimbic network may be involved in developmental disorders such as autism . Before proceeding, however, a brief note of caution regarding the limits of neuroimaging . As such it is important to realize that neuroimaging findings resulting from a comparison of active and control conditions usually are correlative only and will not imply causality . Instead, what neuroimaging may reveal are the changes in activity in the important nodes and hubs of the human brain which can bring about changes in the balance of resource allocation between different brain networks, the associated behavior, and the resulting experience . One example is the fact that microsaccades may elicit gamma synchrony in the frontal cortex, while the parietal lobe is unaffected (carl et al ., 2012). The parietal lobe is a key constituent of the paralimbic network discussed in this review . The conscious sense of self serves as a common reference for making decisions about the allocation of resources ensuring survival, whether related to any of the fundamental pleasures of food, sex, or other people or even higher - order pleasures such as music or monetary reward . Establishing the functional neuroanatomy of the self is thus potentially a fruitful strategy for identifying the hubs and key nodes in the brain networks important for maintaining the balance between the competing processes of attention, awareness, and emotion . One essential role for the self is to be a common reference point in space and time for conscious experiences (gallagher, 2000). Together, they are all experienced in unity, coherent in space and time . Even when someone enters the room, or the phone rings, and the scene and our focus of attention shifts completely, we usually experience the changed scene as continuous with the preceding (tononi and edelman, 1998). To account for this coherence of widely different conscious percepts, the self offers a possibility: per definition, we cannot have free - floating sensations with no self to experience them, and we cannot have a self completely devoid of sensory experiences, memories, or feelings . In other words: non - self sensations and memories and the self are two sides of the same coin . If each of the different, ever - changing sensations and memories are attached to the same coherent self - structure across space and time, these types of information will be bound to each other via the self . A coherent self across time requires a system to retrieve memories of personal experiences, i.e., episodic memory . Correlational evidence suggests that this system is involved not only in retrieval of past personal memories, but also in conceptualizing the future (ingvar, 1985; andreasen et al ., 1995). Consequently, episodic memory retrieval appears as an indispensable component of the more complex forms of self - awareness and consciousness, which are described in various terms such as extended self, meta - consciousness, autonoetic consciousness, or narrative self (gallagher, 2000). In a particular amnesic syndrome, transient global amnesia, a rudimentary sense of self this state is characterized by loss of episodic memory, while working memory, and semantic memory are relatively spared (quinette et al ., 2003). The neural organization of episodic memory retrieval involves hemodynamically synchronized activity in medial paralimbic regions including the anterior cingulate and posterior cingulate cortices and adjacent neocortical structures, as well as in the thalamus (henson et al ., 1999;, 1999; wiggs et al ., 1999; cabeza and nyberg, 2000; gardiner, 2001; tulving, 2002). Further hemodynamic studies have confirmed that the paralimbic network is active in self - awareness (kjaer et al ., 2002b), and single pulse transcranial magnetic stimulation confirmed the specificity and causality of the network in extended self - awareness (lou et al ., 2004 the figure demonstrates the effects of applying transcranial magnetic stimulation (tms) in targeting the precuneus / posterior cingulate region in a task probing self - reference . The causality of this region in self - reference is seen by the fact that retrieval of self - judgment was significantly less efficient with tms at a latency of 160 ms than with a latency of 0 ms (p = 0.003), suggesting that neural activity at that time interval after stimulus presentation is particularly important for self - representation . Specificity of this region in self - reference is provided by the fact that the difference between self and other (best friend) is significant at most latencies (p <0.05), except at the specific causal latency . In contrast to extended self - awareness, the minimal self is pre - reflexive, immediate, normally infallible, and involves the sense of ownership of experiences . For the minimal self, data on the neural organization are scarcer, but indicate that the same paralimbic structures are active here as well (vogeley et al ., 2004), being a common denominator for the self, independent of its complexity, and consisting mainly of a network of paralimbic regions . Functional magnetic resonance imaging (fmri) have shown a consistent pattern of activity in self - processing for a paralimbic network including anterior cingulate / medial prefrontal cortices, posterior cingulate / medial parietal cortices (with precuneus), and pulvinar thalamus (lou et al ., 2004). This network of paralimbic regions not only shows correlated activity with self - reference, but has also been shown to be causally related to self - reference . Transcranial magnetic pulses (tms) can transiently disturb the neural function of key regions in the paralimbic network with decreased efficiency specifically of self - reference as a result (lou et al ., 2004). Anatomically, the medial prefrontal / anterior cingulate and medial parietal / posterior cingulate regions are connected directly via the cingulum bundle, and indirectly, through their rich connections via the limbic and intralaminar thalamic nuclei, located centrally at the base of the forebrain (lou et al ., 2004). Each structure in the paralimbic circuitry contributes fundamental properties to extended self - awareness (northoff and bermpohl, 2004). The evidence shows that many of the regions on the midline of the prefrontal cortex are involved in self - reference . The medial orbitofrontal cortex at the base of the frontal lobes has been shown to be a related to evaluating the valence of the convergence of intero- and exteroceptive stimuli, including the remarkable valence of human infants (nauta, 1971; kringelbach and rolls, 2004; kringelbach et al ., 2008). This region has been called the entrance door to self - awareness based on eeg and meg studies showing comparatively early engagement after onset of emotional stimuli (northoff and bermpohl, 2004). Self - referential stimuli are then monitored in the dorsal anterior cingulate cortex in the sense that stimuli are selected here among competing stimuli for access to consciousness . The supplementary motor area is closely functionally related to the anterior cingulate cortex in preparation for action, in particular when there are no external cues to tell the subject what to do . Self - referential stimuli are thought to be evaluated in the dorso - medial prefrontal cortex as judged to be pertaining to one - self or other persons (northoff and bermpohl, 2004). Theory of the mind, or attributing mental states to others, is also associated with activity in the region, which therefore may constitute a link between introspection and understanding others (frith and frith, 1999). These frontal monitoring and evaluative functions of self - awareness are complemented by functions in the posterior midline establishing spatial and diachronic unity of self and of consciousness: spatial organization in a first person framework involves posterior cingulate / medial parietal cortex (northoff and bermpohl, 2004) which is also active in linking new information with prior knowledge on the subject matter (maguire et al ., 1999). In autonoetic consciousness, the region is active in retrieval of episodic memory for autobiographical self - consciousness (cavanna and trimble, 2006). This has been shown by tms targeting precuneus to transiently disrupt the normal function of this region (lou et al ., 2004). The central role of the intralaminar nuclei for consciousness is illustrated by the fact that even minute lesions here may cause loss of consciousness (schiff, 2008). Neuroimaging experiments support the proposed role of the paralimbic network as a central integrator and arbitrator of resource allocation in the human brain . In particular, in addition to evidence from indirect techniques such as fmri and pet, more direct and faster methods such as magnetoencephalography (meg) have recently provided very useful information regarding the dynamics of the underlying physiology . When sampling data from the most important hubs of the paralimbic network including the anterior cingulate / medial prefrontal and posterior cingulate / medial parietal cortices as well as thalamic regions, it has been demonstrated that gamma synchrony is a common neural event in both minimal self - reference and extended self - reference, where the degree of synchrony is clearly related to the degree of self - reference (lou et al ., 2010). As noted above, this mechanism of gamma synchrony is not likely to be affected by the recent discovery that microsaccades may give rise to gamma oscillations . These gamma oscillations are induced by extra cerebral sources primarily in the frontal lobe, and have not been found in the parietal cortices (carl et al ., 2012). In addition we used beamforming for source localization which allows a clear distinction between sources . The degree of gamma synchrony in the paralimbic network is a potential mechanism between conscious experiences and widely different degrees of self - reference . This mechanism of gamma synchrony in the paralimbic network thus meets one of the main requirements needed to bind conscious experience in the so - called neuronal global workspace theory (dehaene et al . Yet, theories of consciousness abound and while the global workspace theory has been highly influential, other potential candidate theories exist . An equally important concept developed over the last decade is the default mode theory (gusnard and raichle, 2001; raichle et al ., 2001). According to this theory, sensory information arriving from the outside world will lead to a decrease in perfusion and energy metabolism of the medial default mode network, in analogy with the re - direction of brain activity with shifting modalities of sensory stimulation (lam et al ., 1999). At first glance these two theories would seem contradictory, with the global workspace theory referring to a single global brain network processing consciousness, while the default mode theory posits one brain network for processing the outer world, and another network for the inner world . The hypothesized paralimbic network offers a solution to this apparent paradox . The evidence from meg, which is a direct measure of brain activity, shows that paralimbic network becomes synchronized to varying degrees during experiences of both the outer world with only minimal self - reference, and more so in introspection during extended self - reference (lou et al ., 2010). In contrast, we reanalyzed some of our data and found that the paralimbic network showed a significant reduction in gamma power only during a minimally self - related task (syllable counting, see figure 4), while there were no significant change in gamma power during extended self - reference (retrieval of own judgment on one - self). Changes in gamma power in the paralimbic network may serve to guide allocation of brain resources . The figure shows the changes in gamma power within the paralimbic network in the pre - stimulus baseline in a task involving autobiographic memory retrieval of previous personal judgments of visually presented words . A significant decrease in gamma power (p <0.05) was found in minimal self - awareness (syllable counting: syl), compared to the pre - stimulus control state . This was not the case in extended self - awareness (retrieval of own previous judgment of one - self). The figure is generated from new analysis based on data from the previous experiment shown in figure 3 (lou et al ., compare this with the evidence from indirect measures of brain activity such as fmri which shows that regions involved in the default mode network have reduced blood oxygenation level dependent (bold) signal during goal directed (non - self - referential) tasks relative to rest (raichle et al ., 2001). The reduction in gamma power as measured with meg during an active task (syllable counting) could reflect the deactivation in the default mode network during active tasks . Some of the evidence suggests that changes in gamma power are indeed correlated with bold which is an indirect measure of the synaptic activity (logothetis and wandell, 2004). In sum, the evidence from meg studies show that the paralimbic network can provide the gamma synchrony needed for the global coherent workspace of consciousness, while also fulfilling the criteria of reduced activity (i.e., changes in gamma power in minimal self - reference) in the midline paralimbic regions of self - reference elicited by minimal self - reference . Yet, general gamma synchrony on its own is not sufficient for consciousness but would also seem to require amplification of activity within recurrent networks . This is suggested by some intriguing data gathered over 30 years ago, when (libet, 1982) discovered that conscious sensations emerge as a function of both intensity and duration of electric stimulation of the human somatosensory cortices . They reported a delay of conscious experience of approximately 400 ms from onset of a sustained stimulus at threshold intensity and assumed that this latency reflected the need for maintaining the initial frequency and amplitude for an extended period of time . This suggests that amplification of perceptual processing is required to elicit conscious experience, a process which in the neuronal global workspace theory is termed ignition . In addition, evidence also indicates that high signal intensity in cortico - thalamic interaction is essential for emergence of consciousness . Several researchers have speculated that extensive recursive activity to bootstrap neuronal processing is a general mechanism for stimuli to become available for consciousness (tononi and edelman, 1998). In spite of this long - standing interest in recurrent neuronal activity, beyond some demonstrations in primary visual cortices, such activity was only recently demonstrated in higher - order, modality non - specific regions using meg and the so - called granger causality analysis, a mathematical method initially developed to analyze econometric data but now also used to identify the directionality of flow of information between brain regions (granger, 1969). Autobiographic memory retrieval of previous personal judgments of visually presented words was used to probe the temporal flow of activity within the paralimbic network (lou et al ., 2011a). It was demonstrated that the pre - stimulus condition is characterized by causal, recurrent oscillations which are maximal in the lower gamma range . When retrieving previous judgments of visually presented adjectives, this activity is dramatically increased during the stimulus task as ascertained by granger causality analysis, demonstrating not only recurrent gamma activity in higher - order, modality non - specific regions but the paralimbic network described here does not only include midline structures in medial anterior and posterior regions which are continuously active in different states and traits of consciousness, but the evidence has also implicated the striatum . Therefore interest has recently focused on the possible role of dopaminergic neurotransmission in the regulation of conscious experience . At the system level we now know that conscious experience is linked to interacting regions of parietal and prefrontal cortices, which are not only active, but also effective in self - reference (lou et al ., 2004). However, on the molecular level our knowledge has until recently merely been suggestive . Functional brain imaging has established that abnormal conscious experiences in schizophrenia, like hallucinations and delusions, are associated with abnormal dopaminergic neurotransmission (changeux and lou, 2011). For instance, striatal dopamine transporter availability is inversely correlated with hallucinations (schmitt et al ., 2006). One way to measure conscious experience is to approach the problem as one of probability of signal detection (and subsequent subjective interpretation). Following this approach, conscious experience can be determined by the setting of a criterion for when the sensory signal - to - noise ratio warrants sufficient subjective confidence that a stimulus is present . In schizophrenia, psychotic symptoms could be explained as a result of setting too liberal a signal - to - noise criterion . This is thought to be due to abnormally upregulated dopaminergic neurotransmission, an effect of dopamine on the cellular level being to influence the signal - to - noise ratio (lou et al ., 2011b). Furthermore, in the previously mentioned study of yoga - nidra meditation it was shown that such meditation is accompanied by a strong increase in sensory awareness, and that this phenomenon is linked to dopamine release in the nucleus accumbens in the ventral striatum, which has been implicated in the paralimbic network consistently active in different conscious states and traits (kjaer et al ., 2002a). We directly examined the effect of increasing dopamine activation and showed that dopaminergic stimulation with the d1 and d2 receptor agonist pergolide is effective in increasing confidence in seeing words, a valid measure of awareness (lou et al ., 2011b). These results demonstrate how dopamine can influence activity across the various nodes and hubs of the paralimbic network, giving rise to changes in both conscious states and content . The evidence presented above supports the hypothesis that the paralimbic network subserves self - processing in the human brain, and that specific changes in synchrony between the nodes and hubs of this network can regulate the dynamics of widespread connected brain networks . In addition, the evidence also suggests that dopamine has a special role in modulating activity in this network . The evidence thus supports the overall notion that the paralimbic network plays a key role in the conscious brain resource allocation associated with predicting and selecting behaviors that ensures survival . The fundamental rewards afforded by evolution to ensure survival are food, sex, and conspecifics; and the timecourses of the associated behaviors are cyclical (kringelbach and berridge, 2009). There are distinct wanting, liking, and learning phases in the reward cycle, which have been shown to have partly dissociable neural substrates (kringelbach, 2005). Over time the human brain has to use attentional mechanisms to detect potential rewards which, depending on the current state of the organism, may (or may not) initiate a specific reward cycle . For example, after becoming aware of hunger signals, the food reward cycle is initiated and our attention invariably turns to finding potential food rewards . We propose that the paralimbic network plays a key role in integrating awareness, attention, and emotion processing to optimize the brain resource allocation . The inputs from the senses such as vision, auditory, taste, smell, and touch (linked to survival - related rewards such as food and sex) and their subsequent evaluation have the potential to temporarily shift the focus of the brain networks to allow for efficient processing and control over behaviors (berridge and kringelbach, 2008; kringelbach et al ., 2008). The paralimbic network helps to ensure that the processing remains balanced over longer time periods . Mounting evidence has demonstrated that frontal regions such as the medial orbitofrontal and the anterior cingulate cortices are situated anatomically at the crossroad between interacting networks of attention, awareness, and emotion as necessary nodes linking the networks (tsuchiya and adolphs, 2007; kringelbach et al ., 2011). It should, however, be noted that these regions are heterogenous regions with many sub - regions which have been implicated in different networks (kringelbach and rolls, 2004; beckmann et al ., 2009). Direct causal evidence for the interaction has been given by studies showing that direct electrical stimulation of the anterior cingulate cortex can help to alleviate severe chronic pain in patients (kringelbach et al ., 2010). Recently, we further demonstrated that reward can directly impact conscious experience and this integration is directly reflected in the neural activity in the anterior cingulate cortex as measured by local field potentials rmer thomsen et al ., 2011). In general, emerging evidence shows that perturbations to the paralimbic network may manifest in an unbalancing of widespread brain network as seen for example in the anhedonia associated with mental illness (kringelbach et al ., 2011). Development perturbations to the paralimbic network are not uncommon (lou, 2011). Here we will briefly focus on an example of developmental dysfunction found in the asperger syndrome (see box 1). Autism is a neurodevelopmental disorder of genetic origin, characterized by impaired social interaction and communication as well as repetitive behavior and restricted interests . The symptomatology being highly variable, the condition is often referred to as autism spectrum disorder . Asperger syndrome is a subgroup where the affected individuals have normal language development and normal or above normal iq, together with the typical social communication impairments, obsessions, and narrow interests, suggesting impaired binding of attention, awareness, and emotion . After a normal pregnancy and birth he was delayed in motor development and slightly delayed in development of language . He did not babble until he suddenly spoke perfectly correct . As a toddler he was always playing with construction toys and puzzles, but not with other children . At school he behaves oddly in social interaction where he is rigid and clumsy, with a very formal, monotonous language . He speaks without restraint about his special interests, in an exalted, gesturing, and noisy way . The psychological investigation shows that he is of normal intelligence, with high scores in memory of facts, especially meaningless facts, and he is eminent at puzzles and labyrinths . In contrast, he scores below normal in reasoning and social comprehension, in particular sequential understanding of social activities . Individuals with asperger syndrome would appear to have problems with introspection and narrative self - awareness . They also have peculiar concrete thought patterns and a tendency to focus on external events rather than inner experiences (hill et al ., 2004). Given the hypothesis of the paralimbic network presented here, this failure of subjectivity may be predicted to be linked to dysfunction in the paralimbic loop and medial neocortical structures . While still scarce, some of the experimental data would seem to support this link . One neuroimaging study involved higher - order consciousness based on linguistic information, sentence comprehension (just et al ., 2004). In autists compared to normal controls, higher activity was seen in the wernicke region, while lower activity was found in broca s region and in the medial paralimbic regions . This finding supports the hypothesis that the medial paralimbic circuitry could be defective in autism . Yet, some caution is needed given that the autists and the normal control participants were not compared directly, but only indirectly via a common reference to a fixation point . Deficiency of the paralimbic loop of self - reference could potentially be explained by the evidence for volume reduction and reduction in glucose metabolism in the entire cingulate cortex in autism (haznedar et al . In fact, related deficiencies may be seen in another disorder of self - awareness and higher - order consciousness: schizophrenia (haznedar et al ., 2004). There is a clear lesson for consciousness research initially emerging from studying the brain activity in experienced yoga - nidra teachers during meditation but subsequently supported by a wealth of other experiments: a common paralimbic network serves to regulate and balance the dynamic resource allocation needed to ensure survival . This process is guided by processes linked to awareness, attention, and emotion in order to support memory - dependent self - reference, which in narrative self - consciousness is extended into adjacent neocortical regions . Dire clinical consequences are linked to its impediment in pathological conditions . Yet, as a common reference of conscious experiences, it provides us with an adaptive tool for building a unified self and personality over the years by successfully balancing the allocation of brain resources for survival and procreation . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
A rare, potentially fatal and usually self - limiting adverse effect of therapy with enalapril, the angioedema, is always a challenging encounter for an intensive care specialist . The unpredictability of clinical course and the risk of airway compromise with not so high end airway management gears at hand make it more challenging in a rural health setup . Incidence of enalapril - induced angioedema is three times more common in population of african origin . Botswana provides universal health care to its citizens, and health sector is almost completely under government management . We came across two cases in a very short window of about a month at a rural health center . A 74-year - old female was admitted to the emergency department of sekghoma memorial hospital, serowe village, botswana, past midnight with progressive swelling of her face, tongue and breathing difficulty for about 8 h. the family gave a history of change of antihypertensive medication recently . She has been started on tablet enalapril, 20 mg, once daily, orally, 2 days back . On examination, she was conscious, coherent but very anxious . On auscultation of chest, air entry was good bilaterally and there were some conducted sounds from pharynx due to excessive secretions . The tongue was grossly swollen, hard in consistency, it was wedged between the teeth and she was not able to close her mouth [figure 1]. It was not possible to transfer this case to a tertiary care hospital by helicopter as there were landing issues in darkness . The patient was started on oxygen support at the rate of 5 l / min by facemask for building up her oxygen reserves in case of airway emergency . Lateral view x - ray of neck was done which showed airway patency as fine [figure 3]. Elective blind nasal intubation as a proactive airway management was risky due to less chances of success and more risk of further airway compromise . There were no ear, nose, and throat (ent) specialists available for either evaluation or surgical airway management . She was injected with 100 mg of hydrocortisone, intravenously and 0.5 ml of injection adrenaline (1:1000) subcutaneously . She was observed for about 30 min in which she gradually became worse and swelling of face and tongue increased . We decided to go ahead with fresh - frozen plasma infusion under intravenous beta - blocker antihypertensive coverage . Her blood group was o - positive and hospital being a peripheral center; we had only blood storage facility where o - positive fresh - frozen plasma was not available . We decided to go ahead with transfusion of the only pint of o - negative fresh - frozen plasma available at our storage facility . Injection metoprolol, 5 mg, intravenously was instituted and then we started with o - negative fresh - frozen plasma infusion intravenously, 220 ml over next 30 min . On post - fresh - frozen plasma infusion,, she recovered grossly and after 6 h she was able to close her mouth completely . The patient on arrival to intensive care unit the patient after fresh - frozen plasma transfusion lateral x - ray of neck taken to decide the airway management ace inhibitors are one from the most widely used antihypertensive worldwide, particularly for the diabetics to prevent nephropathy and in cases of left ventricular dysfunction or heart failure . Soon after the development of ace inhibitors, wilkin et al . Reported angioedema and proposed enhanced kinin effects from inhibition of kininase ii as the underlying mechanism . Later, reports of ace inhibitor - related angioedema estimated an incidence between 1/1000 and 3/1000 . Angioedema was about three times more likely to occur among black patients (1.62% vs. 0.55% for white patients) and slightly more likely in women (0.84% vs. 0.54% for men). The incidence of angioedema was lower in diabetic patients (0.43%) and higher in patients with renal disease (1.63%) or a history of rash (2.48%). The most common sites of angioedema were the lips (49%) and the face (52%). Swelling of the tongue, neck, or eyelids occurred in approximately 1 of 5 patients with angioedema . The incidence of angioedema was not affected by prior or current use of ace inhibitors at randomization . They identified black race, history of drug rash, age> 65 years, and seasonal allergies as independent risk factors for angioedema related to enalapril . Slater et al . Reported a 14-fold higher incidence of angioedema in the 1 week compared with later time intervals . Hedner et al . Reported that about half of the cases occurred in the 1 week of therapy ., these patients should be carefully examined for any evidence of respiratory compromise such as stridor, a markedly enlarged tongue, symptoms of dyspnea, acutely presenting dysphagia, and drooling of saliva . Lateral view x - ray of neck may be an important tool to decide about intubation . The decision of when to intubate should be made early based on standard physiologic criteria, recognizing that intubation may be difficult secondary to edema of the airway . Cricothyrotomy or emergency tracheostomy is required for maintenance of the airway if oropharyngeal and nasopharyngeal intubation fails . Fluid resuscitation and therapy with vasopressors may be required if the patient is hemodynamically unstable . Hypotension can be seen in severe cases secondary to the extravasation of large amounts of fluid into the extravascular space . Patients presenting with respiratory symptoms should immediately receive 0.5 ml epinephrine (1:1000) subcutaneously . Therapy with antihistamines and steroids should be started initially in all patients, as a true allergic reaction cannot be excluded . Although the role of epinephrine, antihistamines, and corticosteroids is controversial in nonmast cell - mediated angioedema, i.e. Ace inhibitor - related angioedema, in our case, we used all the three above - mentioned agents but there was no clinical improvement . Fresh - frozen plasma has been used successfully for the treatment of resistant, life - threatening angioedema induced by ace inhibitor . The benefit of fresh - frozen plasma in this condition may be due to the effect of kininase ii in breaking down accumulated bradykinin . In this case, we used fresh - frozen plasma, and the clinical improvement was observed . We recommend that in case of life - threatening enalapril - induced angioedema in rural setup, where advanced airway management procedures may be difficult, we must give an early thought about starting fresh - frozen plasma . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
The dosimetric accuracy of brachytherapy delivery is fundamental to the achievement of clinical treatment aims, tumour control and minimised normal tissue toxicity . As early as 1993 van dyk et al . Defined a requirement for brachytherapy treatment delivery of 3% accuracy in dose at distances of 0.5 cm or more at any point for any radiation source . Control of dose delivery is particularly difficult to achieve in brachytherapy due to small treatment distances, very high dose gradients and a multitude of aspects that affect accuracy . A quality assurance system in radiotherapy is essential to ensure treatment delivery is consistent and as intended . This will include a multitude of quality control (qc) tests designed to evaluate actual operating performance in comparison to goal values and to enable rectification / reconciliation of any differences . Brachytherapy is currently undergoing a period of significant innovation and rapid modernisation, including a shift from 2d to 3d basis, the enhanced use of imaging, patient - specific treatment plan optimisation, fully volume - based prescribing, inverse - planning, advanced planning algorithms, use of advanced treatment applicators [9, 10], and in - vivo dosimetry verification systems . It is essential that qc test schedules keep pace with the changing technology and clinical practice . This includes re - assessing the use of historic qc tests that are no longer fit for purpose and replacing with more relevant qc, or where system performance is verified by other means avoiding unnecessary redundancy . Quality assurance of all brachytherapy techniques has recently received increased attention following low dose rate brachytherapy incident in 2009 at the philadelphia va medical centre in which a number of patients received poor quality prostate seed brachytherapy treatment . There have also been errors in brachytherapy due to confusion of source strength units, including incorrect entry into treatment planning systems . In addition, there are numerous publications on more subtle equipment - related quality issues in high dose rate (hdr) delivery, such as unexpected, irregular spacing of source dwell positions in ring applicators . It is important that qc testing is robust and comprehensive and meets the needs of modern equipment and treatment techniques . It is over twenty years since a comprehensive assessment has been undertaken of brachytherapy qc practice in the united kingdom (uk): reproduced in the institute of physics and engineering in medicine (ipem) report 81 . The ipem guidance on qc for radiotherapy is currently being revised, and it is therefore timely to undertake a repeat benchmark exercise of current qc practice for brachytherapy . A similar survey was conducted in 2002 in the netherlands and belgium, which reported large variations in test frequencies and methods, and differences in qc - philosophy and available equipment . The publication of a comprehensive assessment of current qc practice has several potential benefits for individual radiotherapy departments: centres may be reassured that their qc systems are in - line with accepted practice; alternatively, centres may identify discrepancies against standards of practice . Following investigation, this may lead to either reduction of tests or frequencies and hence efficiency savings, or resolution of deficiencies and potential improvements in safety and quality . However, the details of qc tests presented here should not be interpreted as guidelines or recommendations, but as a snapshot of current uk practice . It is important to be aware that specific qc testing is a local decision, based on many local factors, and should ideally be based on risk - assessment approaches . All 64 radiotherapy centres in the uk were contacted by email in june 2012 to request their contribution to the study, with collation of responses taking place during june to august 2012 . Centres were asked to complete a detailed questionnaire on their routine qc practices and other aspects of hdr and pdr service provision . To enable a contextual review of qc practice, initial questions were asked included equipment type, average patient workload, sites treated, source strength calibration methods, level of image guidance, and prescribing practice . A spreadsheet containing a comprehensive list of possible quality control tests for treatment and planning equipment centres were asked to document whether they routinely perform each test, at what frequency, by which staff group, and the acceptable tolerance values used . They were also asked to comment whether the target test frequencies were actually achieved in practice . Forty - seven (73%) of the 64 uk radiotherapy centres that were invited to take part in the survey of hdr and pdr qc responded . Thirty - one centres had appropriate brachytherapy equipment and provided fully completed questionnaires on their qc practice . Thirteen centres reported no hdr or pdr brachytherapy facilities, and further 3 intended to commence hdr brachytherapy in the near future . The majority (29) of radiotherapy centres had hdr units, with only three having access to pdr treatments, two having exclusively pdr . The equipment profile of the responding centres included 20 nucletron / elekta microselectron, 7 varian gammamed, 4 nucletron / elekta / isodose control flexitron, 1 eckert & ziegler bebig hdr multisource, and 1 varian varisource . One centre had 3 treatment units, 1 hdr and 2 pdr, all others had 1 unit . One centre had a co-60 source (hdr), the others were all using ir-192 . The latter isotope being exchanged at 3-monthly intervals in all but two centres: one at 4-monthly intervals and another pdr centre at between 3 and 6 months . The mean number of hdr fractions delivered per year at each centre was 281, with interquartile range 173 to 359 (minimum 80 and maximum 730). There was a lack of agreement as to whether the locally measured value or the manufacturer's supplied source certificate should be used for the source strength value in treatment planning calculations; 18 centres (58%) preferring to use their own measurement . The current uk code of practice for hdr brachytherapy dosimetry recommends a well chamber for the primary source strength measurement, but allows some flexibility in the method used to obtain the second independent verification value . Table 1 lists methods used for source calibration and their relative popularity within uk centres . Methods employed and their popularity, for source strength measurement of hdr & pdr sources at centres in uk the quoted origin of the tg-43 source model data used in the treatment planning systems also varied between centres . Fourteen (45%) used the supplied manufacturer data, 8 (26%) used journal published data, and 8 (26%) used manufacturer data and verified this against publications (with 1 (3%) not answering the question). There was also a variety of methods quoted as an independent check of the output of the treatment planning system . Methods employed and their popularity, to independently verify treatment planning system (tps) calculations at centres in uk treatment plan optimisation in some form was used in 23 centres (73%), including for cervix (majority), prostate (next most common), skin / limb moulds, interstitial anus, vaginal vault, lung, head & neck, multilumen mammosite breast, intraluminal, and keloid scars . Twenty - seven centres (87%) optimised treatment plans for individual patients; 19 (61%) employing manual methods and the others inverse planning optimisation, often with final manual adjustment . 6 centres (19%) stated they used pre - optimised standard plan libraries . The level of image - guidance varied significantly between centres . In cervix treatments, 16 (52%) used ct alone for treatment planning, 12 (39%) mri with ct, 2 (6%) mri alone, and 1 (3%) c - arm 2d imaging alone . When mri was available this was often used for the first fraction, with ct used in subsequent treatments . For vaginal vault treatments, 13 (42%) did not image, 10 (32%) used orthogonal 2d x - ray, and 8 (26%) used ct . Some centres responded they would only image vault treatments for complex cases or if individualised plans were required . There were an insufficient number of responses on imaging used for other treatment sites for statistical significance . Gynaecology cancers were the most commonly treated . In cervix, 22 centres (71%) still prescribe treatment doses to manchester point a. for those prescribing instead to high - risk clinical target volume, hr - ctv, all centres additionally record the point a dose . Only 2 centres (6%) exclusively recorded icru organ at risk (oar) point doses, likely when only orthogonal imaging is used, the others recorded either just gec - estro dose - volume histogram (dvh) data (48%), or both icru point dose and dvh data (46%). Centres were asked to list the primary sources of guidance used in establishing their hdr or pdr quality control schedules . Table 3 provides a list of the documents that were indicated and their popularity, quoted as the percentage of centres citing the document . All centres stated they had reviewed the content of their hdr / pdr qc schedule within the last two years, except two which did not answer the question . Primary sources of guidance for establishing qc schedules and their popularity at centres in uk table 4 provides detail from the hdr and pdr qc survey . The table shows the percentage of centres that include each of the specific tests in their planned qc schedules . A centre is deemed to have included the test in their regular qc if it is performed within the department whether it is in the specific physics qc documentation or other standard operating procedures, for plan checking for example . A test is deemed not to be in regular qc if it was only intended to be performed once at initial equipment commissioning . The mean and range of frequencies of testing and acceptable tolerance levels are provided in the table . Inclusion of tests in qc schedules varies from 31 centres (100%) for source strength measurements to just 2 centres (6%) for mri tests, the latter of course being due to limitations of access to mri for brachytherapy - specific clinical use and qc testing (it was not recorded how many centres routinely used mri for brachytherapy planning). The consistency of frequency of testing and acceptable tolerance levels also varies markedly between the individual qc tests, between complete or lack of agreement for specific tests . Of the 45 tests included in the survey, 21 were performed at greater than 75% of centres, and 4 were performed at less than 25% of centres . There were no significant differences in the qc techniques employed for hdr and pdr equipment, and certainly within the range of practice between centres . Only 2 centres (6%) reported achieved measurement frequencies were below planned measurement frequencies, and then only for up to two tests each . Hdr & pdr qc survey: response to questionnaire on test popularity, measurement frequency and tolerance values d day, w week, m month, y year, tps treatment planning system table 5 documents additional qc tests suggested by responding centres, which were not included in the original list of tests in the distributed survey . These are generally proposals made by single centres and there is no information of the popularity of these tests across uk, however they are included for interest . Hdr & pdr qc survey: additional tests identified by responding centres, not included in original survey questionnaire quality control testing of dosimetry equipment associated with hdr and pdr use has not been included in the results tables; secondary standard calibrations and consistency testing of well chambers and farmer - type ionisation chambers, which are covered elsewhere and in published codes of practice . Forty - seven (73%) of the 64 uk radiotherapy centres that were invited to take part in the survey of hdr and pdr qc responded . Thirty - one centres had appropriate brachytherapy equipment and provided fully completed questionnaires on their qc practice . Thirteen centres reported no hdr or pdr brachytherapy facilities, and further 3 intended to commence hdr brachytherapy in the near future . The majority (29) of radiotherapy centres had hdr units, with only three having access to pdr treatments, two having exclusively pdr . The equipment profile of the responding centres included 20 nucletron / elekta microselectron, 7 varian gammamed, 4 nucletron / elekta / isodose control flexitron, 1 eckert & ziegler bebig hdr multisource, and 1 varian varisource . One centre had 3 treatment units, 1 hdr and 2 pdr, all others had 1 unit . One centre had a co-60 source (hdr), the others were all using ir-192 . The latter isotope being exchanged at 3-monthly intervals in all but two centres: one at 4-monthly intervals and another pdr centre at between 3 and 6 months . The mean number of hdr fractions delivered per year at each centre was 281, with interquartile range 173 to 359 (minimum 80 and maximum 730). There was a lack of agreement as to whether the locally measured value or the manufacturer's supplied source certificate should be used for the source strength value in treatment planning calculations; 18 centres (58%) preferring to use their own measurement . The current uk code of practice for hdr brachytherapy dosimetry recommends a well chamber for the primary source strength measurement, but allows some flexibility in the method used to obtain the second independent verification value . Table 1 lists methods used for source calibration and their relative popularity within uk centres . Methods employed and their popularity, for source strength measurement of hdr & pdr sources at centres in uk the quoted origin of the tg-43 source model data used in the treatment planning systems also varied between centres . Fourteen (45%) used the supplied manufacturer data, 8 (26%) used journal published data, and 8 (26%) used manufacturer data and verified this against publications (with 1 (3%) not answering the question). There was also a variety of methods quoted as an independent check of the output of the treatment planning system . Methods employed and their popularity, to independently verify treatment planning system (tps) calculations at centres in uk treatment plan optimisation in some form was used in 23 centres (73%), including for cervix (majority), prostate (next most common), skin / limb moulds, interstitial anus, vaginal vault, lung, head & neck, multilumen mammosite breast, intraluminal, and keloid scars . Twenty - seven centres (87%) optimised treatment plans for individual patients; 19 (61%) employing manual methods and the others inverse planning optimisation, often with final manual adjustment . 6 centres (19%) stated they used pre - optimised standard plan libraries . The level of image - guidance varied significantly between centres . In cervix treatments, 16 (52%) used ct alone for treatment planning, 12 (39%) mri with ct, 2 (6%) mri alone, and 1 (3%) c - arm 2d imaging alone . When mri was available this was often used for the first fraction, with ct used in subsequent treatments . For vaginal vault treatments, 13 (42%) did not image, 10 (32%) used orthogonal 2d x - ray, and 8 (26%) used ct . Some centres responded they would only image vault treatments for complex cases or if individualised plans were required . There were an insufficient number of responses on imaging used for other treatment sites for statistical significance . Gynaecology cancers were the most commonly treated . In cervix, 22 centres (71%) still prescribe treatment doses to manchester point a. for those prescribing instead to high - risk clinical target volume, hr - ctv, all centres additionally record the point a dose . Only 2 centres (6%) exclusively recorded icru organ at risk (oar) point doses, likely when only orthogonal imaging is used, the others recorded either just gec - estro dose - volume histogram (dvh) data (48%), or both icru point dose and dvh data (46%). Centres were asked to list the primary sources of guidance used in establishing their hdr or pdr quality control schedules . Table 3 provides a list of the documents that were indicated and their popularity, quoted as the percentage of centres citing the document . All centres stated they had reviewed the content of their hdr / pdr qc schedule within the last two years, except two which did not answer the question . The table shows the percentage of centres that include each of the specific tests in their planned qc schedules . A centre is deemed to have included the test in their regular qc if it is performed within the department whether it is in the specific physics qc documentation or other a test is deemed not to be in regular qc if it was only intended to be performed once at initial equipment commissioning . The mean and range of frequencies of testing and acceptable tolerance levels are provided in the table . Inclusion of tests in qc schedules varies from 31 centres (100%) for source strength measurements to just 2 centres (6%) for mri tests, the latter of course being due to limitations of access to mri for brachytherapy - specific clinical use and qc testing (it was not recorded how many centres routinely used mri for brachytherapy planning). The consistency of frequency of testing and acceptable tolerance levels also varies markedly between the individual qc tests, between complete or lack of agreement for specific tests . Of the 45 tests included in the survey, 21 were performed at greater than 75% of centres, and 4 were performed at less than 25% of centres . There were no significant differences in the qc techniques employed for hdr and pdr equipment, and certainly within the range of practice between centres . Only 2 centres (6%) reported achieved measurement frequencies were below planned measurement frequencies, and then only for up to two tests each . Hdr & pdr qc survey: response to questionnaire on test popularity, measurement frequency and tolerance values d day, w week, m month, y year, tps treatment planning system table 5 documents additional qc tests suggested by responding centres, which were not included in the original list of tests in the distributed survey . These are generally proposals made by single centres and there is no information of the popularity of these tests across uk, however they are included for interest . Hdr & pdr qc survey: additional tests identified by responding centres, not included in original survey questionnaire quality control testing of dosimetry equipment associated with hdr and pdr use has not been included in the results tables; secondary standard calibrations and consistency testing of well chambers and farmer - type ionisation chambers, which are covered elsewhere and in published codes of practice . The survey data presented in this report represents the current practice in the majority of brachytherapy centres within the uk . Whilst there is a high level of consistency in inclusion, frequency and tolerance values for some tests, such as source strength measurement, this is likely due to differences in local planning and treatment procedures in clinical use, availability of equipment, and differing functionality or performance of equipment . Local assessment of qc needs is essential in determining schedules, rather than simple reliance on the majority view. However, benchmarking against accepted practice is a good starting point for local review . A risk assessment approach including local known factors is advocated for final decisions on qc testing . All schedules must include measurement of source strength, source position and dwell time, but the specific details require knowledge of local clinical practice and equipment in use . While there was some variation in staff groups involved in qc testing between centres, physics staff most commonly performed all of the qc tests except facilities testing (table 4) which was almost exclusively performed by radiographers . Within each centre, a specific qc test may be performed in multiple ways, including staff group involved, equipment used, frequency of measurement, and tolerance value . For example, decay correction accuracy at treatment unit may be performed prior to each patient treatment by radiographers, and separately by radiotherapy physics after each source change, but to a tighter investigation tolerance level . The achievable tolerance value for this test is also dependent on the equipment design, whether the software makes hourly corrections for source decay or 12-hourly for example . The standard operating procedures of individual departments also have a significant affect on the qc testing that is performed . This includes all aspects such as whether optimised or standard / tabulated planning is used, whether 2d or 3d imaging is utilised, and whether electronic transfer of data is available . An independent method for the verification of the accuracy of treatment plans is required for individually - optimised treatments, but may not be required for each patient if a standard plan is used that has previously been verified and is checked for consistency . Some tests are adopted by all centres such as source strength measurement and source position in a straight catheter. However, others such as x - ray imaging of applicators is undertaken by only 32% of centres . The difference may be attributed to whether the process is already being assessed by alternative means, and there is some evidence from the survey to support this . For applicator dimensions and angles, a specific measurement may not be necessary if the consistency of shape is evaluated through agreement to planning system library applicators used for each individually - planned treatment (provided both physical applicator and library applicator have already been tested at commissioning). The number of centres including a routine measurement of actual source dwell positions in clinical applicators was surprisingly low, at 17 centres (55%). Such testing should be performed at commissioning and at regular intervals, particularly if ring applicators are in use, in which the actual and tps planned dwell positions should be compared . Ipem report 81 was the most frequently cited document used for guidance on required hdr or pdr qc tests, but it is surprising that only 61% of uk centres cited this document, being the uk professional body's recommendations for qc . More recent, but again surprisingly cited by only 48% of centres is the estro booklet no . There is a large range in the documents identified by individual centres as their primary sources of guidance for qc testing, supporting the need for an update of ipem report 81 in uk . A benchmark data set of brachytherapy hdr and pdr qc testing has been presented which is representative of practice across the uk . This updates a previous survey conducted over twenty years ago . A modern approach to qc is required to ensure continued safety and highest quality brachytherapy into the future as technology and procedures continue to increase in complexity, alongside increasing workforce pressures . Qc testing schedules must be designed intelligently, including risk - based assessments of need rather than simply maintaining historic tests . The contents of this report should not be interpreted as professional advice as to the requirements of a brachytherapy qc schedule and are presented as a benchmark data set . Local decisions on qc testing must be made based on full risk - assessment and local factors.
In order to better respect the wishes of terminally ill patients about their medical treatment and to best protect their rights, the taiwan government passed a piece of legislation titled the hospice palliative care act in 2000 . In the same year, the taiwan national health insurance (nhi) program, which was created in 1995 and covers 99.6% of the national population, announced a new reimbursement regulation that provides coverage for inpatient palliative care (ipc). According to these regulations, terminal cancer patients have the autonomy to choose either hospice care or curative care after hospitalization . Currently, the taiwan nhi reimburses three kinds of hospice palliative care programs for terminal patients . In the first program, which began in 1996, hospice home care is provided for patients living in the community or in institutes for the terminally ill . The second program, which began in 2000, provides ipc for patients who need to be hospitalized and who are willing to be admitted to a hospice ward . The third program, also called hospice shared care, which began in 2005, provides care for patients who are admitted to a typical hospital ward and who are in need of hospice care . In taiwan, before the launch of hospice shared care in 2005, ipc is the main type of hospice care, utilized by up to 80% of patients who had received hospice care . In recent years, about 25% of terminal cancer patients who ever received hospice care is ipc . A previous study by enguidanos et al found that seriously ill older patients who received a consultation from an ipc team and then received hospice or home - based palliative care postdischarge experienced a significantly lower odds of hospital readmission . In a recent study by oconnor et al, patients who received an inpatient palliative consultation had a lower 30-day readmission rate - adjusted odds ratio, and when the palliative team involved patients in discussions regarding their goals for care, patients experienced a lower readmission rate . Terminal cancer patients are admitted to ipc units for pain as well as physical, psychosocial, and spiritual problems . An important issue in the continuity of care revolves around where these patients are moved after their initial symptoms improve . Although a majority of these patients wish to return home, their vulnerable health status and fluctuating conditions might prevent these patients from returning home and puts them at risk for frequent readmissions at different hospitals . In addition, readmission is an indicator of quality of care and is related to healthcare costs . Therefore, the aim of this study was to investigate the characteristics surrounding readmission for cancer patients after their first ipc admission in taiwan from 2002 to 2010 with the goal of informing future policy discussions . This study was a secondary data analysis using information from the national health insurance database (nhird), which comprised anonymized secondary data derived from patient registries and claims data from the taiwan nhi program . The taiwan nhi program, which began in 1995, covers more than 99% of the national population . The taiwan national health research institute (nhri) collects and publishes the registry and claims data released by the nhi on an annual basis . We identified all cancer patients who were added to the registry of catastrophic illness dataset from 1996 to 2010 . We included subjects 20 years old with diagnoses of malignant neoplasm (icd-9-cm code 140 - 209). Patients diagnosed with cancer before january 1, 2002 or who had ever been admitted to an inpatient hospice palliative care unit before the study period began were excluded . Terminally ill cancer patients who received ipc for the first time between 2002 and 2010 were included for analysis . Readmission was defined as hospital readmission at least once after discharge from first ipc admission until mortality or the end of this study . The outcomes in this study included characteristics of patients during their first ipc admission and all subsequent readmissions . Covariates included subject income levels, urbanization of patient residence, comorbidities, and hospital level . Subject income levels were grouped into three categories according to the premium paid (ntd$40,000, 20,00039,999, 119,999, and dependent). We designated levels 1 and 2 as urban areas, levels 3 and 4 as suburban areas, and levels 4 and 7 as rural areas . Comorbidities were identified and classified by severity according to the charlson comorbidity index as described in previous studies . Hospitals were classified by level, such as tertiary hospital, regional hospital, and local hospital . Sex distributions, age at cancer diagnosis, age at first ipc admission, length of stay of first ipc admission, and survival days from first ipc discharge were also calculated . Data linkage was performed with microsoft sql server 2012 (microsoft corporation, redmond, wa). Statistical analyses were performed with ibm spss statistics 20.0 (ibm corporation, armonk, ny). Continuous variables were presented as the mean standard deviation (sd) or as a median and range, and these were compared using the student t test or the manny whitney u test . Categorical variables were presented with a number and percentage, and these were compared using the chi - squared test . Hazard ratios (hrs) were presented as crude ratios and adjusted ratios according to the covariates of sex, age at first ipc admission, insurance premium level, residence urbanization, age at diagnosis, charlson comorbidity index, teaching status of admitting hospital, hospital level, and survival days from first ipc discharge until mortality . A 2-tailed p - value <0.05 was considered to be statistically significant . This study was a secondary data analysis using information from the national health insurance database (nhird), which comprised anonymized secondary data derived from patient registries and claims data from the taiwan nhi program . The taiwan nhi program, which began in 1995, covers more than 99% of the national population . The taiwan national health research institute (nhri) collects and publishes the registry and claims data released by the nhi on an annual basis . We identified all cancer patients who were added to the registry of catastrophic illness dataset from 1996 to 2010 . We included subjects 20 years old with diagnoses of malignant neoplasm (icd-9-cm code 140 - 209). Patients diagnosed with cancer before january 1, 2002 or who had ever been admitted to an inpatient hospice palliative care unit before the study period began were excluded . Terminally ill cancer patients who received ipc for the first time between 2002 and 2010 were included for analysis . Readmission was defined as hospital readmission at least once after discharge from first ipc admission until mortality or the end of this study . The outcomes in this study included characteristics of patients during their first ipc admission and all subsequent readmissions . Covariates included subject income levels, urbanization of patient residence, comorbidities, and hospital level . Subject income levels were grouped into three categories according to the premium paid (ntd$40,000, 20,00039,999, 119,999, and dependent). We designated levels 1 and 2 as urban areas, levels 3 and 4 as suburban areas, and levels 4 and 7 as rural areas . Comorbidities were identified and classified by severity according to the charlson comorbidity index as described in previous studies . Hospitals were classified by level, such as tertiary hospital, regional hospital, and local hospital . Sex distributions, age at cancer diagnosis, age at first ipc admission, length of stay of first ipc admission, and survival days from first ipc discharge were also calculated . Data linkage was performed with microsoft sql server 2012 (microsoft corporation, redmond, wa). Statistical analyses were performed with ibm spss statistics 20.0 (ibm corporation, armonk, ny). Continuous variables were presented as the mean standard deviation (sd) or as a median and range, and these were compared using the student t test or the manny whitney u test . Categorical variables were presented with a number and percentage, and these were compared using the chi - squared test . Hazard ratios (hrs) were presented as crude ratios and adjusted ratios according to the covariates of sex, age at first ipc admission, insurance premium level, residence urbanization, age at diagnosis, charlson comorbidity index, teaching status of admitting hospital, hospital level, and survival days from first ipc discharge until mortality . A 2-tailed p - value <0.05 was considered to be statistically significant . There were 637,272 patients identified from the catastrophic illness registry who were older than age 20 and who were diagnosed with cancer between january 1, 2002 and december 31, 2010 . Among them, we identified 42,022 patients who were first admitted for ipc after being added to the catastrophic cancer registry . The majority of these patients were male (n = 25,359, 60.4%). The mean age at cancer diagnosis was 64.0 14.4 years for males and 64.5 14.7 years for females . The mean age at first hospice ward admission was 65.2 14.2 years for males and 65.9 14.9 years for females . The majority (48.6%) of patients who received ipc had a middle insurance premium level between 20,000 and 39,999 ntd and lived in an urban area (58.8%). The top 5 diagnoses were lung cancer (18.4%), liver cancer (18.0%), colorectal cancer (11.8%), oral cancer (7.5%), and stomach cancer (6.8%). Most patients (n = 38,012, 90.7%) had a charlson comorbidity score of 5 . The median length of stay for first ipc admission was 8.0 days with an interquartile range (iqr) of 12 days, and 24,876 (59.2%) patients died during their first ipc admission (table 1). The majority of patients were first admitted for ipc at a tertiary hospital (n = 22,855, 54.4%) and teaching hospital (n = 37,875, 90.1%). Demographics and clinical characteristics of patients received first inpatient palliative care during 2002 and 2010 (n = 42,022) table 2 shows the characteristics of patients according to 30-day readmission and nonreadmission status . Ages at cancer diagnosis and at first ipc admission were significantly younger in the readmission patients (both p <0.001). According to distribution by insurance premium level, readmission patients had a significantly higher premium level when compared to nonreadmission patients (p <0.001). In regards to cancer diagnosis, more readmission patients had been diagnosed with lung, colorectal, oral, esophageal, prostate, cervical, nasopharyngeal, and ovarian cancer . Readmission patients had a significantly higher charlson comorbidity index score than nonreadmission patients (p <0.001). Characteristics of 30-day readmission and nonreadmission patients in table 3, we further analyzed the characteristics of patients who were readmitted after their first ipc discharge until mortality . Two thousand one hundred fifty - six (19.4%) patients were readmitted on the same day of their first ipc discharge, 5005 (45.0%) patients were readmitted between days 1 and 14 of their first ipc discharge, and 1932 (17.4%) patients were readmitted between days 15 and 30 of their first ipc discharge . In terms of readmission frequency, most patients were readmitted once (n = 6093, 94.8%), 2654 (23.9%) patients were readmitted 2 times, 1105 (9.9%) were readmitted 3 times, and 1277 (11.5%) were readmitted 4 times . Characteristics of readmissions from first inpatient palliative care discharge to mortality (n = 11,129) table 4 shows risk factors for 30-day readmission after first discharge from an ipc stay . After adjusting for covariates, statistically significant risk factors for 30-day readmission included being male (adjusted hazard ratio [adjusted hr] = 1.087; 95% confidence interval [ci], 1.0361.140, p = 0.001), higher insurance premium level (adjusted hr = 1.107, 95% ci, 1.0151.208, p = 0.022), length of stay during first ipc admission (adjusted hr = 1.006, 95% ci, 1.0051.008, p <0.001), being admitted to a teaching hospital (adjusted hr = 1.105, 95% ci, 1.0031.218, p = 0.044), and being admitted to a tertiary hospital (adjusted hr = 1.113, 95% ci, 1.0061.232, p = 0.038). Patients who were older at first ipc admission were less likely to be readmitted (adjusted hr = 0.996, 95% ci, 0.9940.997, p <0.001). There are very few studies focused on readmissions of cancer patients after discharge from ipc, but it is an important issue when considering continuity of care, healthcare utilization, and healthcare resource allocation . In this study, we found that lung cancer, liver cancer, and colorectal cancer were the most common diagnoses . The majority of these patients died during their first admission for ipc, and the median length of stay for first ipc admission was 8 days . Our results showed that 19.4% were readmitted on the same day of discharge, 45.0% were readmitted within 14 days, and 81.7% were readmitted within 30 days . Males, patients with a higher insurance premium level, patients with a longer length of stay during first ipc admission, admission to a teaching hospital, and admission to a tertiary hospital all increased the adjusted hr for readmission . The target population in this study included terminal cancer patients with limited life expectancy . In addition, in clinical practice we found that the majority of patients who received ipc were very vulnerable and in a relatively unexpected condition . Patients with a longer stay during their first ipc admission, admitted to a teaching or tertiary hospital might indicate a relatively unstable or severe condition of these patients that may attribute to increased risk for readmission . A previous study in hong kong reported that progression of previous condition is one of the factors related to readmission . A previous study on rehospitalization of gynecological oncology patients examined the possible factors related to readmissions, including planned treatments, symptom management, and end - of - life care . A previous study by jencks et al reported a 19.6% readmission rate within 30 days of initial hospitalization among medicare beneficiaries; they also found that rehospitalization was costly . The authors reported that poor communication with patients, insufficient outpatient follow - up, and a lack of care coordination may be related to this phenomenon . Ranganathan et al reported that palliative home care may help patients to remain at home and avoid 30-day rehospitalizations . In taiwan, hospice palliative home care has been provided for terminal patients since 1996, but in a study by hu et al regarding the beliefs of healthcare professionals about why taiwanese hospice patients prefer staying in the hospital, the main concerns included an inability to manage emergent medical conditions, better quality of care in hospitals, and an insufficient number of caregivers . Furthermore, our results revealed that 2156 (19.4%) patients were readmitted on the same day of discharge . In our clinical practice experience, some patients whose family cannot take them back home will ask for transferring to another hospital for continual hospitalization . This might be one of the reasons for this high rate of readmission on the same day of hospital discharge; however, further study is needed . Besides, although studies in taiwan have reported that the preferred place of death for terminal cancer patients is at home, there were discrepancies regarding this preference between patients, their caregivers, and even physicians . Chiu et al reported that the location of care was one of the prevailing ethical dilemmas surrounding the provision of terminal care for cancer patients . In regards to the readmission phenomenon found in our study, it may indicate a need for improved continuity of care and increased support for caregivers, and may be associated with the unstable nature of the patient's underlying diseases and comorbidities . A previous study evaluated the effects of ipc consultation on subsequent hospice use and place of death in patients with advanced gastrointestinal cancers and found that receiving a palliative care consult during admission increased the odds of home death and decreased the odds of hospital death, but had no effect on hospital readmission . The live discharge rates were 95% and 89% for curative care and palliative care, respectively, and the hospital readmission rates 6 months after discharge were 68% and 67% for curative care and palliative care, respectively . In our study, 59.2% patients died during their first ipc admission, with a median length of stay of 8 days . The results of our study reflect a problem with late referrals to hospice palliative care, as other studies in taiwan have also found . The hospice palliative care act of 2000 and its recent amendments in 2013 are intended to better protect terminal patients right to choose hospice palliative care . However, further studies are needed to better understand the effects of the law and its amendment on hospice palliative care utilization . This study is a secondary data analysis and, as such, it has several limitations . First, the claim database did not contain information about cancer stage; therefore, we could not know how terminal these patients were . To mitigate this issue, we used the charlson comorbidity index to measure the severity of comorbid conditions among these patients . Second, the claim database did not contain information about education level, marital status, and actual income . To address this problem, we assessed insurance premium level, which has been widely used in many studies as a surrogate for income level . Despite the aforementioned limitations, this study was a nationwide population - based cohort study that included all terminal cancer patients admitted for ipc with limited selection bias and good representativeness . In conclusion, our study found that 59.2% of patients died during their first ipc admission and that the median stay of first ipc was 8 days . Among those discharged alive after first ipc admission, 64.9% were readmitted and 19.4% of them were readmitted on the same day of discharge . From first ipc discharge to mortality, 54.8% were readmitted once, 23.9% were readmitted twice, 9.9% were readmitted 3 times, and 11.5% were readmitted 4 or more times . The results may help inform policy discussions to improve the relatively late referral rate to ipc and to reduce the high readmission rate among terminal cancer patients in taiwan.
Reverse shoulder prosthesis is an excellent surgical option for patients with certain shoulder pathologies, of which rotator cuff arthropathy is one . One area where we are gaining a greater understanding is that of managing acromial pathology . Recent studies are showing that not all acromial pathology is a contraindication to reverse shoulder arthroplasty; in both pre- and post - operative patients, small lateral fractures of the acromion can be treated non - operatively or fixed surgically with a tension band technique . Overall, results comparable to that of reverse arthroplasty can be achieved with either of these treatment options . However, fractures at the base of the acromion or of the scapular spine present a different problem . In these types of fractures, a larger length of deltoid muscle is detached, and therefore defunctioned . In reverse shoulder arthroplasty, in order to achieve a stable shoulder, the deltoid must be tensioned a great deal . This in turn causes a deforming force and distraction at the fracture site, making for a poor healing potential . Also, as the reverse shoulder prosthesis relies on the deltoid muscle to move the arm, if the deltoid is detensioned or improperly tensioned, this will lead to poor functional capacity . We present a case of treatment of a fracture at the base of the acromion using a 90/90 plating construct that healed in a good position . A 71-year - old right - handed woman presented with a 2-year history of right shoulder pain and dysfunction, which began abruptly when lifting a heavy load . At the initial evaluation, her range of motion was limited, particularly in active abduction as she achieved only 45 degrees in this plane . A cuff arthropathy (hamada grade 3) was diagnosed and the decision was taken to proceed with reverse shoulder arthroplasty . To complete the pre - operative assessment, a ct scan was performed which confirmed there was no acromial pathology involved . A reverse shoulder prosthesis and a biceps tenodesis was carried out (delta xtend, depuy, warsaw, in, usa) through a deltopecoral approach [figure 1]. The patient's right arm was immobilized in a sling and the range of motion exercises were started 6 weeks after arthroplasty [figure 1a]. Five months after surgery, she sustained a blow to the right shoulder but sought medical attention 1 month later, at her scheduled follow - up visit . Radiographs showed a displaced fracture at the base of the acromion [figure 1]. This showed that both the humeral and glenoid components were well fixed and the only pathology was the fracture . Antero - posterior radiographs showing the patient's right shoulder immediately after reverse shoulder arthroplasty (a) and after fracture, 5 months later (b). The post - operative image shows no pre - existing acromial pathology she was taken to the operating room and her previous incision was extended into a sabre incision to better expose the acromion . Two small fragment (3.5 mm) locking plates (synthes, west chester, pa, usa) were used in a 90/90 configuration; one was a fragment specific clavicular plate and the other a reconstruction plate . The fragment specific clavicular plate was applied on the superior edge of the scapular spine in a compression mode . A second reconsrtuction plate was applied from the posterior acromion to the posterior cortex of the scapular spine, in the infraspinatus fossa . Fixation was solid, but in order to protect the construct, the patient was placed in an abduction brace for 6 weeks . Eighteen months after fracture fixation, the patient was satisfied with the clinical result [figure 2]. She was capable of 125 degrees of abduction, 160 degrees of forward flexion, 85 degrees of external rotation in adduction, and 60 degrees of internal rotation . Her quickdash score was 29.5 (compared to 82.5 pre - op) and constant score was 69 on the affected side compared to 85 on the left side, for a good functional outcome . A medline search using the key words [acromion fracture], [scapula fracture], [reverse arthroplasty] was performed . We then sought to classify and separate the cases by location; there were 30 cases of fractures at the base of the acromion, and scapular spine . When our case is added, we have a total of 31 cases . Results are summarized in table 1 . In brief, of the 30 more proximal cases, 21 were treated non - operatively in a sling, 7 were treated with open reduction and open fixation and one with revision of the prosthesis . Of those treated non - operatively, and for whom results are published, fourteen had a non - union, four had a malunion and in two cases, it was unclear whether union was achieved . For patients receiving fixation of the fracture, there was one non - union, two repeat fixations, and one patient required removal of the fixation . The patient who had a revision of the prosthesis had a malunion of the acromion . Results of the literature review . In the fracture location column, the fracture description provided by the authors is presented . Although recent studies have shown that good outcomes may be achieved with non - surgical management of lateral acromial fractures, the same does not hold true for basal acromial fractures . The likely reason for this is that with fractures at the base of the acromion, a large length of the deltoid is defunctioned and the deltoid muscle is essential to the functioning of the reverse shoulder prosthesis . Previously described techniques, such as tension band fixation achieved poor functional results for fractures at the base of the acromion . We postulate that this is because the fixation method is insufficient to withstand the forces generated by the deltoid muscle a tension band can neutralize forces parallel to the axis of the band but in the case of the deltoid, force vectors are generated in different directions . It also allows for compression along the fracture site and resists motion in all directions . Good screw purchase can be achieved by angling the screws either toward the scapular spine or the coracoid . The locking option enables good fixation and improves cut - out strength in osteopenic or osteoporotic bone . In addition to the stable fixation achieved with this technique, we further recommend immobilization of the affected shoulder using a sling and an abduction pillow to detension the deltoid, thus at least partially removing the distractive forces at the fracture site . Also essential to achieving a good functional result we thus also recommend a program of range of motion exercises followed by strengthening exercises to maximize functional capabilities . Based on results from previous studies, fractures of the tip were those of the most lateral or anterior portion of the acromion . Fractures of the body of the acromion are those medial to the tip of the acromion and lateral to the beginning of the scapular base . The scapular base is the lateral border of the scapular spine, which is smooth and round . In our nomenclature, fractures at the scapular base are termed fractures at the base of the acromion as functionally, this zone connects the acromion to the rest of the scapula and this term avoids confusion with any more medially occurring fractures . Coronal (a) and axial (b) representations of the proposed classification scheme for acromial fractures associated with revere arthroplasty we propose the above mentioned classification system as it provides a nomenclature for acromial fractures that is descriptive and is based on the anatomy and functionality of the scapula . As the results from the review of the literature indicate, the more medial the fracture, the worse the prognosis with non - operative treatment . Thus, this classification system may be used to predict outcomes and determine treatment offered . It differs from other classification systems (crosby) in that it further subclassifies more medial fractures, which are the more ominous fractures . As more research is carried out in this field
As the extra - cellular domain of fz, mainly the wnt binding crd, is necessary and sufficient for the non - autonomous function of fz, we revisited these issues and asked systematically, whether and how wnts might function in drosophila pcp generation . Wg, dwnt4 and wnt6 are all expressed in identical patterns during drosophila imaginal disc development . Their expression domain at the wing margin is near perpendicular to the initial pcp axes, radial towards the wing margin (fig . S1a; also), and also in the eye pcp - specification occurs at the stage when r3/r4 are aligned perpendicular to the wnt expression domains at the poles of the eye field (fig . S1a', ref), suggesting that they could provide directional cues . In adult wings, pcp orientation is still pointing towards wing margin in areas near the margin (fig . Planar polarization in the wing progressively strengthens during early pupal stages, suggesting that pcp requires also later input for its strengthening, refinement and maintenance . The wing is an ideal model for these analyses, being a simple epithelial tissue and, importantly, canonical wg - fz/-catenin signaling requirements are much reduced at pupal stages and (at least partially) temporally separable from fz / pcp - establishment . To bypass global patterning roles of wnts (especially wg) via canonical arm/-catenin signaling, which precludes pcp analyses at pupal stages, we examined the role of wnts in pcp signaling during early pupal development, when most other wing patterning events are completed . At early pupal stages 17h apf), cellular pcp orientation is radial and polarity directed towards the margin (fig . 1d - d), where wg and other wnts are expressed throughout wing development (fig . 1e), consistent with a hypothesis that wnts could provide directional long - range control to fz / pcp - signaling . To circumvent potential redundancy associated with overlapping expression domains of several wnts (see above), we first used gain - of - function (gof) approaches by misexpressing each drosophila wnt in developing wings (using the gal4/uas system with either clonal or regional [dppgal4] expression). Whereas most wnts did not display pcp effects (all wnt - genes in the genome were tested), expression of dwnt4 and wg caused striking and classical pcp defects, affecting cellular (wing hair) orientation of neighboring wild - type cells (fig . 3). These data are consistent with earlier reports, suggesting that misexpression of wnt4 can cause pcp phenotypes in the wing, and also with suggestions that wg overexpression can affect pcp . Strikingly, the phenotypes of wg and dwnt4 misexpression were similar to non - autonomous polarity defects associated with fz clones (surrounding wild - type cells orienting towards mutant area) and opposite to fz gof defects (wild - type cells orienting away from fz - overexpression regions). To establish that these gof phenotypes are mediated by fz / pcp signaling, and not by potential effects of the canonical wnt/-catenin pathway, we induced wnt4-expressing clones in fz null mutant wings (the entire animal lacks fz, but can signal canonically via the dfz2 receptor). Consistent with a direct effect of wnt4 via fz / pcp signaling, the cellular reorientation of cells neighboring wnt4-expressing clones was eliminated in fz backgrounds (fig . 2d) and cellular orientation was basically identical to the patterns in fz mutant wings alone (figure 2d e; 8/8 wnt4-expressing clones caused no change to fz mutant wing cell orientations; also suppl . S1e - e'). Further confirming that the pcp effects were not mediated through canonical wg - wnt/-catenin signaling, wnt4-expressing clones did not affect expression of any canonical wnt / wg - targets in larval or pupal wings (e.g. Dll and fj - lacz, fig . D). Taken together, the genetic epistasis data (fig . 2d e and suppl . Fig . D) indicate that the wnt gof pcp phenotypes are mediated through fz / pcp - signaling and that fz acts downstream of wnts in this context . If wnts regulate fz / pcp - activity, their misexpression should not only affect wing hair orientation, but also alter core pcp factor polarization and hence pcp axis orientation at critical early stages of pcp establishment . To examine this functionally, we used wnt4 clonal expression (which has no effect on canonical wnt - signaling, see above) and analyzed pupal wings throughout pcp patterning . At early stages (prepupae-16h apf), when pcp axes are radial, wnt4 clones strikingly reorient core pcp factor polarity (determined via fmi staining) to a near 90 angle relative to clonal borders (fig . 4a - a, c, d - d, compare to wild type, fig . Similarly, at later stages (~3032h apf) when pcp orientation has been realigned to the proximo - distal axis (and actin prehairs have started to form) wnt4-expressing clones continue to reorient surrounding cells towards clonal borders (fig . 2c - c' and 3b - b, d; see legend for statistical evaluations). Of note, the near 90 reorientation of cells (relative to clonal borders, fig . 4a, c, d) is mirrored by border cells on the inside of clonal margins, an effect likely mediated by intercellular feedback loops . Also, wnt4-expressing cells inside clones are less polarized compared to surrounding non - expressing cells as measured by their nematic order (length of polarity lines in fig . 3b, and 4a, d; see figure legend for statistics), the exception again being border cells (marked by orange dots in fig . 4a), which are polarized relative to their outside neighbors (blue dots in fig . 4a; see legend for details). Overall, the frequency (72%) and effects seen with the wnt4 misexpression clones are very comparable to fz- null clones [around 80% frequency in our hands], consistent with the notion that the wnt effect and fz / pcp mediated orientation are linked . Taken together, our data indicate that (1) wnt4 and wg can reorient cell polarity towards their expression domain (similar to fz- clones), and (2) that this polarity depends on a wnt expression border (and possibly graded wnt distribution), rather than high uniform wnt levels (as demonstrated by the reduction of nematic order / polarity strength inside wnt - expressing clones; fig . Both observations indicate that wnts serve an instructive role in establishing a pcp orientation axis . Taken together, we hypothesize that dwnt4 and wg instruct the direction of fz / pcp function and based on the opposing phenotypes of fz and wg / dwnt4-expressing clones, possibly by inhibition of the non - autonomous fz function in pcp establishment . To determine whether wnt4 and/or wg are also necessary for fz / pcp axis orientation, we examined loss - of - function (lof) phenotypes . As wg and dwnt4 are expressed in an identical pattern and both display comparable gof pcp effects (see above), the lack of a lof phenotype in dwnt4 genetic null mutants (fig . 5d; dwnt4 mutants also do not show pcp phenotypes in the eye) could be due to redundancy with wg . Lof wg studies reveal strong canonical wnt/-catenin signaling defects throughout development, precluding simple pcp analyses . To limit the early wg - requirements, we used a temperature sensitive allele, wg, and removed wg function only later . Wg is a hypomorphic allele with reduction in wg expression at the margin (suppl . S2), accompanied by genetic wing margin defects . In wg/ wings that were shifted to the non - permissive temperature in early prepupae (see methods), most wings displayed wild - type pcp features (fig . 5a - a). To further reduce wnt function and to address potential redundancy between wg and dwnt4, we examined double mutant wings for wnt4, wg/ (completely removing wnt4 and partially wg, shifted as above, see methods; importantly, the stronger wnt4wg double homozygous animals were lethal prior to 3 instar stages even at the permissive temperature, and hence the need for the hypomorphic wg/ combination). Strikingly, wings from such animals displayed robust pcp defects, both in pupal wings (fig . C'; note that such animals did not eclose and thus were analyzed at pupal stages with molecular markers) as well as at early stages (prepupa -16h apf; fig . Pcp defects were not only evidenced by misoriented cellular hairs (actin staining in fig . 5b, green; f for orientation defects in rare adult escapers), but importantly also by defects in the early pupal polarization axes of core pcp factors (detected via fmi staining, fig . Importantly, these results suggest a requirement of wg, dwnt4 at the critical, early stages of pcp axis definition . Whereas wild - type cells polarize towards the wing margin and display strong polarization (measured by nematic order, length of yellow lines in individual cells in fig . 6a,b,c', longer lines correspond to stronger polarization), the wg, dwnt4 double mutant wings lacked coordinated cellular orientation, mutant cells showed a wide distribution of polarity angles (fig.6b c') and displayed reduced overall polarization (evident in nematic order levels reduced to 60% [p=10] in the double mutant; see fig . Interestingly, cells near the wing margin were oriented parallel to the margin in the double mutant background (e.g. Pink nematic order lines in fig . 6b), which is consistent with the proposal that cells in / at the margin respond to cues from the antero - posterior organizer (in 3 instar stages) and are less polarized in wild - type at early pupal stages (pink nematic order lines in cells in fig . 6ac and ref). Together with the gof data, these results indicate that wnts are not only instructive but also necessary for drosophila pcp axis establishment . Fz and vang / stbm interact physically with each other through their extracellular domains between cell membranes of neighboring cells in culture and in vitro and thus presumably also in vivo . This interaction is thought to serve as a sensing mechanism of fz levels / activity, and thus a determinant of pcp direction . Since wnts bind fz crds and affect pcp orientation (figs . 26), we hypothesized that a wnt - fz association could modulate the fz - vang interaction, thus creating the sought - after fz - pcp activity gradient; it would be oriented along a wnt - gradient emanating from the source of wg / wnt4, as suggested by our gof and lof studies . We first tested whether wg and/or wnt4 can interfere with the fz - vang interaction in a cell - based recruitment assay . This assay is well suited, as it directly detects an intercellular fz - vang interaction across cell membranes of neighboring cells, similar to the context of pcp axis specification . Fz - expressing s2+r cells recruit vang in neighboring cells to the membrane via cell - cell contact (fig . As s2+r cells are normally not adhesive and only a very small fraction of fz and vang expressing cells touch each other, we aided adhesion by co - transfecting with drosophila e - cadherin (e - cad; to promote cell - cell contacts via homophilic e - cad interactions) with either fz or vang . Fz / e - cad expressing cells recruit vang to the membrane at cell contacts with vang / e - cad expressing cells (fig . 7a - a). To test whether wg or wnt4 can affect this fz - vang recruitment, we co - transfected the fz / e - cad cells with wg . Strikingly, co - transfection of wg, reduced vang recruitment in a specific and dosage dependent manner (fig . S4a; quantification reflects vang membrane localization in individual cells in contact with fz expressing cells; control transfections included non - related secreted ligands, e.g. The jak / stat - signaling ligand upd; fig . 7c - c'). To confirm that this is a non - autonomous (extracellular) effect, we tested conditioned media (cm) containing secreted wg or wnt4 (and again upd as control) in the same assay . The fz - vang recruitment was similarly decreased in wg - cm and wnt4-cm conditions (fig . 7f; also suppl . D), indicating that extracellular wg and wnt4 are interfering with intercellular fz - vang interactions . These data confirm that wg / wnt4 can act as dosage dependent modulators of the fz - vang interaction during pcp establishment . To confirm these effects on fz activity in vivo, we tested whether the fz gof pcp phenotype can be modified by wnt4 co - expression . Strikingly, co - expression of wnt4 with fz (in the dpp - gal4 domain) markedly reduced the non - autonomous effects of fz overexpression on cells outside the dpp domain (fig . S5). These data are consistent with a previous report showing that wnt4 can antagonize fz overexpressing phenotypes (although a different fz phenotype was assessed there). In summary, wnt4/wg can modulate the fz - vang interaction, possibly by antagonizing fz signaling activity to neighboring cells . Our data indicate that wg / dwnt4 regulate the establishment of fz / pcp axes by modulating the fz - vang intercellular interactions in a graded, dosage dependent manner . Consequently they might generate different levels of fz - vang interactions across a wg / dwnt4 gradient experienced by cells (see model in fig . This process is reiterated across the tissue and the directionality of fz - vang binding is subsequently reinforced by intracellular core pcp factor interactions . Our data are consistent with a model in which wg / dwnt4 generate a fz-activity gradient by modulating its capacity to bind to vang, consistent with proposals of fz-activity gradient models . Accordingly, pcp axes are orientated towards the wg / dwnt4 source, which is evident in (at least) the wing and eye . The early wing pcp axis (late larval to early pupal stages) correlates well with wg / dwnt4 margin expression and, similarly, in the eye polarity is oriented in the dorsal - ventral axis towards the poles where wg / wnt4 are expressed (illustrated in suppl . This model relying on a fz - vang interaction is also compatible with the addition of fmi to this scenario, with intercellular (homophillic) fmi - fmi interactions also being required for pcp specification . As fmi forms complexes with both fz and vang, the full complement of intercellular interactions includes fz / fmi - fmi / vang complexes, and these interactions would also be modulated by wnt binding to fz, either directly as proposed in our model or possibly by modulating the fmi - fmi interactions by having fmi associated with fz that is bound to different levels of wg / wnt4 . In vivo, fmi helps to enrich both fz and vang to the sub - apical junctional region, and fmi - fmi interactions bring fz and vang to close molecular proximity . Intercellular fmi - fmi interactions are strong, as fmi expressing s2 cells form cell - aggregates through homophilic fmi interactions . The interaction between fz and vang is weaker and cell - cell contacts between the two cell groups are infrequent . It was suggested that pcp signal sensing complexes include both fmi and fz on one cell interacting with fmi / vang at the surface of a neighboring cell . Within these complexes, fz is required for sending a polarity signal, whereas fmi and vang are involved in its reception, consistent with our data and model . Although it has been suggested that fmi is capable of sensing fz / fmi signals in the absence of vang, the fz - sensing capability of cells with fmi alone (lacking vang) is much weaker than that of cells with vang . It will be interesting to determine if there are additional pcp regulators directly involved in modifying fmi - fmi interactions . How does our data relate to previous models and why was the wg / wnt4 requirement not observed before? Previous work attempted to address the role of the wing margin on pcp by examining either mutants affecting wing margin cells without eliminating wg / wnt expression or in clones . Although cellular hairs near the site of wing margin loss pointed towards remaining wing margin areas, the effect was considered weak . We also examined potential effects of wnt lof clones of df(2l)nl, lacking wnt4, wg, wnt6 and wnt10 . In contrast to the global reduction of wg / wnt4 through the temperature sensitive wg - allele, such clones do cause only mild pcp perturbations (figure s3a there are several reasons why clonal loss of wnt expression in the margin only mildly affect pcp orientation: (1) cells can respond to wnts from several sources / cells from remaining wnt - expressing wing margin regions (illustrated in fig . S3a'); (2) polarization strengths (measured by nematic order) in the first few rows of cells near the margin are much weaker than cells further way (at 1417h apf ref) and weak pcp reorientation in cells neighboring wing margin clones could thus reflect the initial weak polarization in these cells; and (3) pcp orientation changes from its initial radial polarity towards the proximo - distal polarity during hinge contraction morphogenesis and associated cell flow, likely leading to significant corrections of subtle defects near the margin . Similarly, pcp orientation in cells near the margin is only very weak early (at 1416h apf), likely because cells close to the wnt - producing cells are exposed to saturated wnt - levels (and not a wnt gradient) or the presence of other organizers (directing polarity parallel to the margin) weakens the effect of wnts . Pcp in these cells gets established / corrected through more local interactions during the feedback loops among neighboring cells . To determine the direct role of wg / wnts on fz / pcp signaling, we examined it at pupal stages as the patterning role of canonical wg - signaling is much reduced then and pcp still correlates well with wg / wnt4 expression (ref and this study). Importantly, wnt4 does not affect expression of patterning genes via canonical signaling at larval or pupal stages (fig . S1), yet wnt4 alters pcp orientation, consistent with the model that wnt4/wg act directly on fz / pcp - interactions . It is likely that besides the wg / wnt4 input and mechanism identified here, both early and late pcp axes depend on additional cues, provided for instance by the parallel ft / ds - pcp system or other morphogenetic organizers . Strikingly, such a scenario would suggest that wg regulates pcp directionality through both pcp systems, affecting fz / pcp interactions directly (this study) and through canonical wg - signaling transcriptionally regulating graded fj and ds expression in eyes and wings . In summary, our data provide insight into wnt - mediated mechanisms to directly regulate long - range fz / pcp orientation by modulating fz - vang / pcp interactions during tissue morphogenesis.
Parkinson's disease (pd) affects neurological, physiological and psychological functions and quality of life (qol). Physical contributors such as dyskinesias, decreased mobility, fatigue and motor complications worsen qol . Psychological variables like depression correlate with reduced qol . Although treatments target motor symptoms, proper identification and subsequent treatment for psychological measures may be overlooked . Compromised pulmonary function may also lead to impairment in activities of daily living (adls) and physical function and affect exercise tolerance, causing fatigue and thereby reducing qol . Several forms of exercise show improvement in adls, perceived health status, fall risk, qol and motor performance . Yoga also improves respiratory pressure, while yogic breathing techniques have an immediate effect on lowering blood pressure in healthy people . The purpose of this study was to discern if physiological and qol - related benefits of yoga exist in individuals with pd . Because overall function measured by the unified parkinson's disease rating scale (updrs) was investigated, we wanted to discern whether yoga affects the non - motor aspects of pd . This pilot study presents additional findings . Medical information and self - reported symptoms included time of pd diagnosis, current medications, pd - related symptoms, physical and social activity levels, and fall incidents . If there was a major medication change, participants were allowed to complete yoga training but data were not used after the medication change . Health - related qol measures included the modified falls efficacy scale (fes), the geriatric depression scale (gds) and the sf-36 . Physiological measures included weight, resting heart rate, respiratory rate, blood pressure and standard pulmonary function tests calculated by a puritan - bennett renaissance ii spirometer (covidien - puritan bennett, boulder, co, usa). Data were analyzed using the sigma plot 11.0 (systat software, san jose, ca, usa). The primary analysis used independent t - tests to assess the baseline group differences and one - way repeated measures anova to measure change over time, with p = 0.05 considered statistically significant . Gender recruitment was fairly even, but random allocation into groups divided males predominantly into the control group and more females into the yoga group . The only baseline group differences existed in bodily pain (p = 0.006) and role limitations due to emotional problems (p = 0.038) sub - scales of the sf-36 . Participant characteristics the updrs showed a significant improvement in the yoga group over time, with the largest gain in the first 6 weeks of intervention [figure 1a]. Although updrs mentation, behavior and mood subsection scores improved with yoga, they were not significantly different in either group [figure 1b]. At baseline, one control and two yoga participants had reached the ceiling on the modified fes . At the final assessment,, no control participants displayed depression measured by the gds . Only three yoga participants displayed mild depression . At the final evaluation, the baseline group differences in the two domains of the sf-36 were no longer significant at the final assessment (p = 0.435 and p = 0.724). There was a significant reduction in diastolic blood pressure in the yoga group only (p = 0.036). All participants had mild pulmonary obstruction as interpreted from flow volume graphs and volume time graphs . The only pulmonary function test, average forced vital capacity (fvc), showed significant improvement in the yoga group (p = 0.03). There was also a trend of improved forced expiratory volume in 1 s over time in the yoga group (p = 0.059) but not in the control group (p = 0.962). (a) updrs score improved significantly with the yoga group (*) over time (p = 0.006, f = 7.593, df = 2) but not the control group (p = 0.876). Both groups showed minimal symptoms in this subsection, with 0 being no symptoms and 12 being severe symptoms medical information and self - reported symptoms are summarized in table 2 . At baseline, the yoga group reported swallowing difficulties and disinterest or loss of initiative in activities, while the control group reported none . The yoga group more commonly reported hand shaking, stiffness, clumsiness, falling, sensory disturbances or pain, lack of energy or exhaustion and forgetfulness than the control group . Between 20 and 40% of the control group reported worsening of seven symptoms over time (). Sleep disturbances, lack of energy, shaking hands, stiffness and clumsiness showed mixed results in improvement in the yoga group . Self - report of symptoms and activity in the medical questionnaire yoga participants reported more energy and visible tremor reduction lasting several hours immediately following yoga plus more relaxation with less fatigue, positive social relationship building, improved overall mood and paying more attention to body signals . The purpose of this study was to determine the potential effects of yoga on non - motor symptoms, specifically depression, qol and physiological measures . Certain qol and physiological measures resulted in improvement or maintenance in activity and exercise tolerance, suggesting that positive effects of yoga may be possible . Because of the progressive nature of pd, lack of deterioration of yoga participants' self - reported symptoms and improvement in clinical outcome measures suggest that yoga is potentially an effective intervention to maintain the current activity level . Updrs scores were significantly improved, especially during the initial 6 weeks of yoga training . The 95% confidence intervals indicate that clinical benefit is likely to result from the intervention . Improvement in updrs scores could be explained by improved motor symptoms, but it is possible that non - motor symptoms could cumulatively contribute to the score . It is possible that with more participants, improvements would have been noted in other sf-36 domains or the group gender discrepancy could alter these perceptions . Our results resemble aerobic and strengthening exercise interventions where emotional reactions, social interactions, physical activity and qol scores improve in pd participants . Depression has a major impact on the symptoms of pd and is often overlooked and undertreated . Yoga practice shows improvements in memory, stress, depression and anxiety in a healthy population, possibly due to improved cortisol levels . People with chronic pain who participated in yoga demonstrate improvements in depression, anxiety, fatigue and pain, while yoga in those with multiple sclerosis has a significant impact on qol, fatigue and cognition . Our results of improved fvc after 6 weeks is consistent with another study showing that as little as 8 weeks of yoga in inactive older adults improves respiratory function, blood pressure and aspects of mental wellbeing . Improvement in cardiorespiratory fitness, specifically pulmonary function parameters, rate of perceived exertion and exercise tolerance after aerobic exercise training has been reported in pd . It is known that different types of yoga breathing techniques have different effects on blood pressure . Healthy participants increase absolute and relative maximal oxygen uptake in addition to improved strength and flexibility, showing health - related improvement in physical fitness . Our results support these findings and those that found improvements in blood pressure, but changes in blood pressure may be confounded by the existence of multiple comorbidities and medications . Yoga may contribute to efficacy via improvement in physiological and psychological variables associated with pd . A proposed mechanism of mental and physical health benefits has been postulated through the down - regulation of the hypothalamic - pituitary - adrenal axis . Differences in stress responses between novice and expert yoga practitioners allude to this pathway as having health benefits . Improved mood with decreased anxiety was correlated with acute changes in thalamic gaba levels in a healthy yoga group, which was different than a metabolically matched walking group . Studies are needed to confirm the psycho - neuro immunological hypotheses in neurological disease . Given the pilot nature of this study, there are a number of limitations: small sample size, coin toss randomization method, no control for social interaction, confounding variables like comorbidities, no correction for multiple comparisons between outcome measures and the measures selected may not have captured important changes in other relevant non - motor symptoms that might be positively impacted by yoga . Yoga appears to improve physiological and non - motor factors that can affect qol over a relatively short period of time for pd participants . It is important to determine conservative intervention to treat physiological functions and clinical outcomes contributing to qol in pd for which yoga appears to be a viable intervention.
Early preterm birth (defined as delivery before 32 weeks) is a major cause of infant morbidity and mortality, and the determinant for these is known to be gestational age at birth (12). Among very preterm infants staying in the intensive care unit, the occurrence of bronchopulmonary dysplasia (bpd), necrotizing enterocolitis (nec), severe intraventricular hemorrhage (ivh), and periventricular leukomalacia (pvl) is a major concern because these morbidities are at increased risk for later death or neurosensory impairments (345). However, to what extent the risks of these morbidities are directly related to premature birth or to biological mechanisms of preterm birth remains uncertain . Evidence has shown that intra - amniotic infection and/or inflammation, defined as elevated pro - inflammatory cytokine levels in amniotic fluid (af), is strongly associated with preterm birth in women with preterm labor or preterm premature rupture of membranes (pprom) (678). Thus, a large proportion of very preterm infants born from these women are already exposed to af inflammation in utero . In fact, several studies have shown a systemic inflammatory response, with elevated af cortisol levels, in the fetus exposed to infected af (91011). In particular, it has been reported that af inflammation may also contribute to the pathogenesis of adverse short- and long - term neonatal pulmonary and neurologic sequelae, especially in very preterm infants (12131415). However, most of these studies have been relatively small (131416), and recruited patients with non - infection - mediated preterm birth (e.g., preeclampsia) (1416) or premature infants with advanced gestational age as a study cohort (1214151617), adjusted for gestational age at enrolment but not at birth (17), and did not consider whether af inflammation can modify the effect of gestational age on infant morbidities (1213141516). As a result, studies that examined the risk of af inflammation for infant morbidities may have concluded that af inflammation poses a high risk for these morbidities . The purposes of this study were to examine the effect of exposure to af infection and elevated pro - inflammatory cytokine levels on the mortality and pulmonary, intestinal, and neurologic outcomes of preterm infants, and to determine whether these associations persist after adjustments are made for gestational age at birth . In this retrospective cohort study, we included all consecutive women who underwent amniocentesis and their infants who were admitted to a neonatal intensive care unit (nicu) at the seoul national university bundang hospital (seongnam, korea) between august 2004 and august 2013 . The inclusion criteria were: 1) singleton gestation; 2) preterm labor or pprom; and 3) delivery with a gestational age between 23 + 0 and 32 + 0 weeks . Exclusion criteria included major congenital anomalies, twin and higher order multiple births, and transfer to another hospital after amniocentesis . Gestational age was calculated from the first day of the last menstrual period, and confirmed by first or second trimester ultrasound . The primary outcome measure was adverse perinatal outcome defined as the presence of one or more of the following: mortality (stillbirth or neonatal death), bpd, nec, ivh, and pvl . Additionally, we investigated the associations of 4 individual morbidity variables with af infection and elevated cytokine levels in infants who survived for at least 30 days after birth . Af was obtained aseptically by transabdominal amniocentesis, and was cultured for aerobic and anaerobic bacteria and genital mycoplasma according to previously described methods (18). The remaining af was centrifuged at 1,500 g at 4c for 10 minutes, and the supernatant was aliquoted and immediately stored at 70c until assayed . Interleukin-6 (il-6) and il-8 in stored af were measured by an enzyme - linked immunosorbent assay human duoset kit (r & d systems, minneapolis, mn, usa). The range of the il-6 and il-8 standard curves was 7.8600 and 31.22,000 pg / ml, respectively . The intra- and inter - assay coefficients of variation for 2 different proteins were <10% and <15%, respectively . Culture proven af infection was defined as an infection with positive cultures of af, regardless of the inflammatory state of the af . Our primary explanatory variables for outcome parameters were af culture and af il-6 and il-8 . The other explanatory variables investigated were maternal and infantile demographic characteristics (maternal age, parity, gestational age at birth, birth weight, and gender), cause of preterm delivery, antenatal use of medications, mode of delivery, clinical diagnosis of chorioamnionitis, apgar scores at 1 and 5 minutes, mechanical ventilation, and surfactant application . Diagnostic criteria and management of preterm labor and pprom have been described in detail elsewhere (1819). The following clinical definitions have also been described in detail elsewhere: clinical chorioamnionitis, bpd, nec, ivh, and pvl (61920212223). The severity of nec was graded according to the modified bell's staging criteria; and stage iia or higher was defined as the presence of nec (21). Bivariate analysis of the association of adverse perinatal outcome with risk factors was conducted using student's t - test, the mann - whitney u test, fisher's exact test, or test, as appropriate . The normality for continuous variables in groups was determined using the shapiro - wilk test . Odds ratio (or) with 95% confidence interval (ci) was calculated for categorical variables, whereas continuous variables were summarized by the mean and standard deviation (sd) or by the median and range if not normally distributed . Multivariate logistic regression was then performed to determine the independent relationships of adverse perinatal outcome with af infection, as well as with elevated cytokine levels, after adjusting for baseline variables showing a significant correlation or tendency towards an association with this outcome in bivariate analysis (p <0.1). We checked for multicollinearity among the variables by using a -test, the pearson's or spearman's rank correlation test and variance inflation factor (vif). Variables with a high correlation were summarized in the analysis; gestational age at birth alone was included instead of both gestational age and birth weight, and apgar score at 5 minutes alone was included instead of apgar score at both 1 minute and 5 minutes . Although the af il-6 and il-8 levels were highly correlated, as main explanatory variables of interest, they were analyzed in separate models . The receiver operating characteristic (roc) curve was used to identify the best cut - off values for independent risk factors in predicting adverse perinatal outcome . The optimal cut - off values were obtained from the youden index maximum ([sensitivity + specificity] 1). The study was approved by the institutional review board at seoul national university bundang hospital (irb no . B-1105/128 - 102). All women provided written informed consent for the amniocentesis procedure and use of af samples . The study was approved by the institutional review board at seoul national university bundang hospital (irb no . B-1105/128 - 102). All women provided written informed consent for the amniocentesis procedure and use of af samples . During the study period, 152 women with preterm labor and intact membranes (n = 81) or pprom (n = 71) and their babies met eligibility criteria and were included in the final analysis . Infants who were transferred to another hospital prior to evaluation (n = 1) were excluded . The mean gestational age at birth was 29.1 weeks (sd 2.2 weeks; range 23.432.0 weeks) and the mean birth weight was 1,283 g (sd, 376 g; range, 5002,275 g). The overall perinatal mortality was 5.2% with 1 stillbirth and 7 neonatal deaths occurring in the first 30 days of life . Seventy - four of 152 infants (48.7%) had an adverse perinatal outcome . Among 144 infants who survived for at least 30 days after birth, any stage bpd, nec (stage ii), ivh (grade ii) or pvl developed in 56 (39%), 13 (9%), 10 (7%), and 5 (4%), respectively . The microorganisms isolated from the amniotic cavity included ureaplasma urealyticum (n = 55), mycoplasma hominis (n = 43), escherichia coli (n = 1), streptococcus spp . (n = 4), staphylococcus aureus (n = 4), candida albicans (n = 1), bacillus spp . (n = 1), gram - positive bacteria (n = 5), and gram - negative bacteria (n = 2). The levels of af il-6 and il-8 were significantly correlated with each other (r = 0.799; p <0.001). Gestational age at birth was also significantly correlated with af il-6 (r = 0.270; p = 0.001), af il-8 levels (r = 0.306; p <0.001), or gestational age at amniocentesis (r = 0.726; p <0.001), whereas gestational age at the time of amniocentesis was correlated with neither af il-6 (r = 0.091; p = 0.263) nor af il-8 levels (r = 0.057; p = 0.484). Based on the bivariate analyses (tables 1 and 2), elevated af il-6 and il-8 levels were significantly associated with adverse perinatal outcome, as well as with bpd and ivh . Interactions between gestational age at birth and af il-6 and il-8 levels were not found for the presence of adverse perinatal outcome, as well as of bpd and ivh (fig . The mortality, nec and pvl were not associated with elevated il-6 and il-8 levels in af . Intra - amniotic infection was not associated with adverse perinatal outcome, mortality and the 4 individual morbidity variables . Data are presented as number (percentage), median (range) or mean sd . Af = amniotic fluid, bpd = bronchopulmonary dysplasia, il = interleukin, pprom = preterm premature rupture of membranes, nec = necrotizing enterocolitis, ivh = intraventricular hemorrhage, pvl = periventricular leukomalacia, sd = standard deviation . Based on 144 subjects who survived for at least 30 days after birth . Median in infants with or without the indicated outcome variable is given for all numerical variables, or is given for all categorical variables . Only predictors with p <0.1 are presented, except for the primary explanatory variables (i.e., af culture and af il-6 and il-8). Af = amniotic fluid, ci = confidence interval, il = interleukin, or = odds ratio, nec = necrotizing enterocolitis, ivh = intraventricular hemorrhage, pvl = periventricular leukomalacia . Based on 144 subjects who survived for at least 30 days after birth . The presence of composite adverse perinatal outcome, bpd, and ivh according to af il-6 and il-8 levels and ga at birth . (): cut - offs for il-6 and il-8 levels; (): cut - off for ga at birth . Af = amniotic fluid, bpd = bronchopulmonary dysplasia, ga = gestational age, ivh = intraventricular hemorrhage, il = interleukin . In bivariate analyses (tables 1 and 2), the demographic and perinatal variables significantly associated with adverse perinatal outcome were gestational age at birth and amniocentesis, birth weight, low apgar scores (<7) at 1 and 5 minutes, mechanical ventilation, and surfactant application . The results for bpd and nec were largely the same as those for adverse perinatal outcome . For ivh, an association was limited to gestational age at birth, birth weight, and surfactant application, and the association for pvl was further limited to amniocentesis - to - delivery interval . The associations of adverse perinatal outcome, mortality and individual morbidity variables with af infection as well as with elevated cytokine levels after adjusting for baseline parameters were compared using multivariate logistic regression analyses (table 3). Because af il-6 and il-8 levels were highly correlated, 2 separate multivariate analyses were carried out, whereby each excluded one of these terms . For adverse perinatal outcome, bpd and ivh, elevated af il-6 and il-8 levels that were significantly associated in bivariate analyses remained significant risk factors for the aforementioned 3 outcomes, when we adjusted for low apgar scores at 5 minutes, antenatal corticosteroids, mechanical ventilation, and surfactant application (af il-6 [ng / ml] for adverse perinatal outcome: adjusted or [aor], 1.022; 95% ci, 1.0051.040; p = 0.010; af il-8 [ng / ml] for adverse perinatal outcome: aor, 1.025; 95% ci, 1.0051.046; p = 0.016, data on bpd and ivh not shown). However, the independent effect of elevated il-6 and il-8 levels in af disappeared when additionally adjusted for low gestational age at birth; as a result, low gestational age at birth remained, in regression analyses, strongly associated with the risk of an adverse perinatal outcome and bpd (table 3). The range of vif in our models was from 1.012 to 1.821 which indicated absence of multicollinearity between our explanatory variables . The area under the curve (auc) value for gestational age at birth predicting adverse perinatal outcome was 0.879 (95% ci, 0.8220.936), and a cutoff value of <29.0 weeks was identified as the optimal threshold, with a sensitivity of 81.1% and specificity of 82.1% . Af = amniotic fluid, bpd = bronchopulmonary dysplasia, ci = confidence interval, ga = gestational age, il = interleukin, ivh = intraventricular hemorrhage, nec = necrotizing enterocolitis, or = odds ratio . Af il-6 and il-8 levels were highly correlated with each other (r = 0.804, p <0.001), and thus 2 separate regression models were used in multivariate analyses, in which each excluded one of these terms; interaction term between gestational age at birth and af il-6 or af il-8 levels; adjusted for gestational age at birth, low apgar scores (<7) at 5 minutes, mechanical ventilation, and surfactant application; based on 144 subjects who survived for at least 30 days after birth; adjusted for gestational age at birth, antenatal corticosteroids, and surfactant application . The principal findings of this study are as follows: 1) in bivariate analysis, elevated af il-6 and il-8 levels were significantly associated with the risk of adverse perinatal outcome, but this relationship disappeared after adjustment for the gestational age at birth; and 2) culture - proven af infection was not associated with the development of adverse perinatal outcomes . (17) showing that af il-6 level was stronger than microbial invasion of the amniotic cavity as a predictor of composite perinatal morbidity and death, but il-6 level was no longer predictive after adjustment for gestational age at delivery . Collectively, these findings suggest that the role of prenatal exposure to pro - inflammatory cytokines in the development of adverse neonatal outcomes may be overestimated, and underscored the importance of gestational age at preterm delivery to the risk of adverse neonatal outcomes in infants born at 32 weeks . In the bivariate analyses, the risk of adverse perinatal outcome for infants exposed to elevated inflammatory cytokine levels in af was increased, compared to those not exposed in utero . However, when low gestational age at birth was included in the multivariate model, the risk of elevated pro - inflammatory cytokine levels in af disappeared, indicating the increased risk of elevated levels for adverse perinatal outcome may be mainly due to the large proportion of infants exposed to elevated levels who were born at lower gestational age (fig . 1), given the tight inverse correlation between af il-6 and il-8 levels and gestational age at birth . These findings suggest that delaying delivery in women with preterm labor or pprom might reduce adverse outcomes in infants, especially in very preterm infants born at less than 29 weeks' gestation, despite prolonged exposure of the fetus to af infection / inflammation in utero . Several case reports should be noted, in which antibiotic therapy eradicated microorganisms in af in some patients with af infection / inflammation, with subsequent continuation of pregnancy near or at term (242526). Further studies are needed to confirm whether or not prolongation of pregnancy would be beneficial for the subgroup of women with both af infection / inflammation and threatened birth of an infant of extremely low gestational age (e.g., 2228 weeks of gestation), if antibiotic therapy can be targeted appropriately . Previous studies have reported an association between elevated af pro - inflammatory cytokines and the development of bpd, independent of gestational age at birth (1316). However, these studies have the limitation of a small sample size (1316) and the inclusion of a heterogeneous group of patients with regard to disease entity (16), cases with advanced gestational age as a study cohort (16), and the use of diagnostic criteria that differ from new criteria (20) for diagnosis and severity of bpd proposed by the national institutes of health in 2001 (1316). In contrast to the results of these previous studies, we found that elevated af il-6 and il-8 levels were significantly associated with the subsequent development of bpd in the bivariate analyses, but the association disappeared after adjustment for the gestational age at birth; traditional risk factors for adverse neonatal outcomes, such as gestational age at birth, rather than af inflammation, remained significantly independently associated with bpd . Similarly, our observation of a lack of association between af inflammation and grade ii iv ivh in gestational age - adjusted analysis, was different from the finding of only one study in which this association was investigated (15). This discrepancy may be related to which factor was adjusted for in the analyses (birth weight vs. gestational age at birth), which cytokines were used to define af inflammation (tumor necrosis factor- vs. il-6 and il-8), and different gestational age at enrolment (34 weeks vs. 32 weeks). In terms of association of af inflammation with nec, similar explanations can be applied to the discrepancy between our study and that of hitti et al . The finding that positive af cultures were not associated with adverse perinatal outcomes is consistent with previous studies (131416). This observation is not surprising given 1) a recent report indicating that microbial colonization without inflammatory response appears relatively benign (17); and 2) the limitation of the standard microbiological culture technique used in the current study, which depends on many factors, including inoculum size, whether samples were obtained from an infected site, and the properties of the strains . With regard to microbial footprints in af, a recent study in which culture and/or polymerase chain reaction (pcr) technique were used to detect ureaplasma spp . Showed that the presence of ureaplasma spp . At the time of preterm cesarean delivery was strongly associated with bpd and ivh in preterm infants, even after adjustment for multiple risk factors (27). A recent study suggested a synergistic detrimental effect of gestational age at birth and histologic and clinical chorioamnionitis as surrogate markers for prenatal inflammation on adverse neonatal outcomes (28). However, we did not find an interaction between gestational age at birth and elevated af pro - inflammatory cytokines in association with adverse perinatal outcomes . The reason for this discrepancy may be related to the time difference at which the measured markers were taken to assess whether adverse neonatal outcomes were present . The measured markers in a previous study, which were late findings of antenatal inflammation, were assessed in samples taken at or near the time of delivery, and thus directly reflected adverse neonatal outcomes, whereas the measured markers in the current study, being early findings of antenatal inflammation, were assessed in samples taken remote from delivery . In fact, we found that gestational age at amniocentesis modified the association with elevated af il-8 levels on adverse perinatal outcome (data not shown). First, we did not use molecular techniques, such as pcr, to diagnose cases with actual af infection that were falsely negative by standard microbiological technique for af culture . Second, there were a relatively small number of patients in this study, which resulted in a low prevalence of certain individual adverse perinatal outcomes, such as pvl and mortality, with diminished statistical power for comparison with other groups . Third, the results of af culture and white blood cell (wbc) counts were routinely reported to caregivers, which might affect the timing of delivery and initiation of antibiotic and tocolytic therapy . However, this bias is unlikely to change our main findings, because af culture results take several days to become available, the results of af il-6 and il-8 were unavailable to the caregivers for clinical use, and caregivers were not likely to deliver a pregnant woman solely based on the results of af culture and wbc counts . In conclusion, elevated levels of pro - inflammatory cytokines in af are associated with increased risk of adverse perinatal outcomes, but this risk is not independent of low gestational age at birth . Low gestational age at birth is a major contributor to the risk of adverse perinatal outcomes . Culture - proven af infection is not associated with the development of adverse perinatal outcomes.
Radical cystectomy (rc) is the standard surgical treatment for clinically localized muscle - invasive bladder cancer (mibc). However, nearly 50% of patients with mibc develop metastases and die of bc . Such a high failure rate there are several factors to consider while offering perioperative chemotherapy to a patient undergoing rc . The advantages and disadvantages of neoadjuvant chemotherapy (nac) and adjuvant chemotherapy (ac) are discussed in table 1 . Neoadjuvant versus adjuvant chemotherapy for bladder cancer: advantages and disadvantages after a decision is made to administer chemotherapy, to achieve maximum benefit the planned regimen should be initiated and the planned number of cycles must be completed at an optimal dosage . However, due to various reasons chemotherapy is often not initiated or it is initiated but not completed . The aim of our study is to evaluate and compare the likelihood of initiating and completing neoadjuvant and adjuvant chemotherapy in patients with mibc . Upon obtaining an institutional review board approval, we performed a retrospective analysis of patients who underwent rc and urinary diversion (ud) by a single surgical team between 1992 and 2011 . Data on whether nac or ac was recommended, initiated, discontinued, or completed was obtained and analyzed . Prior to 2004, nac was recommended for patients with clinical stage t3 or higher stage, hydronephrosis, extensive lymphovascular invasion (lvi), or prostatic stromal invasion . Since 2004, the typical chemotherapy regimens were gemcitabine plus cisplatin or methotrexate, vinblastine, doxorubicin, and cisplatin (mvac). On occasions, carboplatin was substituted for cisplatin at the discretion of the oncologist . At any given instance, both ac and nac were not recommended to patients who were unlikely to tolerate chemotherapy due to renal impairment or other debilitating morbidities . A total of 363 patients underwent rc and ud and were considered for perioperative chemotherapy141 for nac and 222 for ac . The mean age was 66 years in nac group and 67.5 years in ac group (p = 0.13). Of these patients, 79% were men and 21% were women . Completion of chemotherapy was defined as receiving the planned number of cycles predetermined by the medical oncologist . Among further, 98 patients (78.4%) completed at least 3 cycles, 20 (16%) completed 2 cycles as planned, and 7 (5.6%) did not complete the planned number of cycles . From the 222 patients recommended to receive ac, 151 (68.0%) initiated the treatment . Of patients who initiated ac, 79 (52.7%) completed at least 4 cycles and 72 (47.3%) overall, only 79 (35.5%) of patients who are candidates for ac received the prescribed number of cycles, while 118 (83.5%) of nac candidates received the planned number of cycles . Patients who were offered nac were more likely to initiate (p <0.001) and complete (p <0.001) chemotherapy [figure 1]. The reason for not initiating nac was patient preference, while for ac the reasons were either patient preference or presence of medical condition or surgical complication interfering with chemotherapy initiation . The reasons for discontinuing nac or ac were patient preference, chemotherapy intolerability, or development of complications [table 2]. Initiation and completion rates for nac vs ac reasons for not initiating / completing the chemotherapy there are several difficulties in administering chemotherapy in the adjuvant setting . The typical patient undergoing rc is in his / her sixth or seventh decade of life and has significant associated comorbidities . Moreover, rc and ud are associated with complications in approximately 30% of patients . In a study by donat et al ., 83% of patients who experienced complications following rc and ud had significant complications (grade 2 - 5, modified clavien system), which prevented or delayed the use of adjuvant chemotherapy . The psychological impact of a major surgery may further impede adjuvant therapy, and thus fewer patients receive ac . Our study shows that 32% of the patients who were recommended to receive ac rejected the treatment and elected to be monitored instead . Patients who undergo a curative treatment for mibc in the form of rc might be more reluctant to receive additional treatment . The associated morbidity brought by such a major surgery alone would interfere with the initiation of ac . On the other hand, patients who were offered nac were more willing to initiate treatment to improve their chance for a cure as well as survival (88.6%). Furthermore, among patients who initiated perioperative chemotherapy, nac patients were more likely to complete the number of planned cycles (94.4%), while only 52.7% completed the ac treatment . Currently, nac followed by rc is an established approach for mibc . Supported by several randomized trials, nac is associated with a survival benefit of 6.5% and a lower risk of bc - specific mortality . On the other hand, our study shows that patients are more likely to accept and complete nac, and this further supports the administration of nac for mibc . Chemotherapy protocols were not the same throughout the study period as novel chemotherapy regimens with less toxicity became available . However, the protocols offered were the same in the nac and ac setting at a specific time . This study is limited to the likelihood of initiation and completion of nac and ac . Patients with mibc are more likely to initiate and complete the planned number of cycles of neoadjuvant chemotherapy as compared to adjuvant chemotherapy . Therefore, we advocate administration of chemotherapy in the neoadjuvant setting in patients who are likely to benefit from chemotherapy.
Acute lymphoblastic leukemia (all), while frequently diagnosed in children, can also be seen in adulthood . Patients with t - cell all and the m4 and m5 subtypes of acute myeloblastic leukemia are at a higher risk for extramedullary disease, including renal parenchymal involvement, which is the most frequent extramedullary metastatic site.1 consequences of leukemic infiltration of the kidneys are asymptomatic bilateral renal enlargement, acute renal failure, and/or secondary hypertension, as reported previously . We report a patient with end - stage renal disease (esrd), receiving hemodialysis, and uncontrolled hypertension due to leukemic cell infiltration of the kidneys . A 34-year - old man who had esrd was admitted to the emergency room with complaints of productive coughing, shortness of breath, and hypertension not controlled by his previous antihypertensive medication . He had a history of generalized seizures and had used carbamazepine for 15 years . Despite iron supplementation and erythropoetin - stimulating agent therapy, he had normochrome - normocytic anemia . His blood pressure (bp) was under control with ramipril 5 mg once daily and amlodipin 5 mg once daily . Before admission, he also had had peripheral facial nerve palsy (bell s palsy) and was treated with corticosteroid therapy for 3 weeks . On admission, he seemed pale, dyspneic, and agitated . His heart rate was regular at 98 bpm, he was afebrile and had a 2/6 systolic ejection murmur on auscultation . He had crackles at the bases of the lungs and + /+ pedal edema bilaterally . The initial biochemistry and complete blood counts of the patient are shown in table 1 . Urinalysis revealed trace protein, and no red blood cells per high - power field . 24-hour urine protein was 1 g. anti - nuclear antibody, anti - ena, anca, and cryoglobulins were negative, and c3 and c4 levels were normal . Hepatitis b surface antigen, anti - hbs, anti - hav, and anti - hcv were also negative . His previous ultrasonography (usg) revealed bilaterally diminished kidney sizes (the right kidney measured 90 40 mm, and the left kidney measured 85 45 mm). On a blood smear, atypical lymphocytes were seen, and bone marrow biopsy showed hypercellularity, with cells containing minimal cytoplasm and abnormal nuclear - cytoplasmic ratio . Flow cytometric studies of bone marrow tissues showed a population of t - cells that expressed cd2, cd3, cd4, cd5, cd7, and cytoplasmic cd3 . These findings were consistent with precursor t - cell all . Despite the combination of 5 different classes of antihypertensive therapy (ramipril 10 mg / day, losartan 100 mg / day, amlodipin 10 mg / day, doxazosin 4 mg / day, and intravenous nitroglycerin), and aquate hemodialysis, his blood pressure did not drop under 180/110 mmhg . To establish the diagnosis, we performed abdominal computed tomography (ct), which showed bilaterally enlarged kidneys (figure 1). We consulted over the patient with a hematologist and initiated peripheral vascular disease (pvd) chemotherapy (daunorubicine 45 mg / m per day for 7 days, vincristine 2 mg / m per day for 7 days, and prednisolone 64 mg / day for 28 days). To treat hyperuricemia, after the first cycle of pvd, his blood pressure dropped under 130/80 mmhg, and the renal usg revealed bilaterally atrophic kidneys . In this case kidney biopsy was considered before chemotherapy, but this procedure could not be performed because of uncontrolled hypertension and severe thrombocytopenia . After the first chemotherapy, his neutrophil counts dropped under 500/mm, and febrile neutropenia developed . On the following days, despite the appropriate antibiotherapy, he had severe dyspnea and tachypnea . To establish the diagnosis, we performed thoracic ct . We initiated antifungal therapy and the patient was entubated in an intensive care unit . Despite the intensive therapy, he died secondary to respiratory failure . All is a malignant disorder that orginates from a single b- or t - lymphocyte progenitor . All represents about 12% of all leukemias diagnosed in united states, and 60% of all cases occur in patients younger than 20 years.2 leukemic patients who are at a higher risk for extramedullary disease, including renal parencymal involvement, include those with t - cell all, as well as those with the m4 and m5 subtypes of acute myelogenous leukemia.1 although all and its extramedullary infiltrations can frequently be seen in children, these manifestations have also been reported in adults previously.3 the kidneys are the most frequent extramedullary site of leukemic infiltration, which was identified in 63% of autopsies performed on patients who had died with either lymphoid or myeloid leukemia.4 kidney involvement of leukemic cells might be related to the embryological orgin of hematopoietic organ developments.5 we present a hemodialysis patient with new onset of uncontrolled hypertension, esa - resistant anemia, thrombocytopenia, and bell s palsy, secondary to all . The case presented here is unusual because the patient did not have a previous history of all . Leukemic infiltration of the kidney is usually silent, and the hospital admission of patients with renal dysfunctions such as renal enlargement, hematuria, proteinuria, treatment - resistant - hypertension, or acute kidney injury is uncommon . In a german multicenter trial of 938 adult patients with all, 10% of patients had organ infiltration, and only 0.4% had clinical evidence of renal involvement.6 patients with enlarged kidneys may suffer abdominal fullness or pain . Physical examination reveals a palpable abdominal mass in 3%5% of patients.7 multiple reports suggest that enlarged kidneys due to leukemic infiltration, detected by usg, ct, or excretory urography, is the most common imaging manifestation observed in renal involvement of all.1 olgar et al showed that renal leukemic infiltration is a risk - factor for developing hypertension, and they also determined that hypertension might be a risk factor for renal parenchymal disease . In that study, therefore, several factors affecting hypertension, such as age, elevated uric acid levels, blood transfusions, and treatment - induced encephalopathy should be considered.8 in the present case, he had well - controlled hypertension following treatment with an angiotensin - converting enzyme- (ace) inhibitor and a calcium channel blocker (ccb). However, after the onset of all, the blood pressure of the patient did not drop under 180/100 mmhg with 5 different groups of antihypertensive drugs, including ace - inhibitor, angiotensin - receptor blocker, ccb, -blocker, and intravenous nitroglycerin . We also hypothesized that the renin - angiotensin - aldosterone system may be responsible for his therapy - resistant hypertension, due to compression of tubules by leukemic cells . Tumor lysis syndrome (tls) is another important complication in all patients, especially those treated with chemotherapy . This syndrome may lead to both acute uric acid and phosphate nephropathy, which can also progress to acute renal failure . The patient presented had tls, with increased uric acid and lactate dehyrogenase levels after chemotherapy that also contributed to resistant hypertension due to hyperuricemia . Interestingly, the patient had bell s palsy before admission, which also supports leukemic cell infiltration . Prednisolone was given to treat the situation before admission, which could also have contributed to tls and hypertension in this patient . The patient presented here also had skin rashes, such as petechiae and dry purpura, when he was first admitted to our medical center . Since bleeding into the skin is one of the most common findings in thrombocytopenia, this part of the examination should be the most detailed . The differential diagnoses of skin rashes in patients with thrombocytopenia vary according to concomitant symptoms and physical examination findings . Sites of bleeding should be noted, especially in the dependent parts of the body . These are normally the feet and ankles in ambulatory patients, but may be the presacral area in bedridden patients . It is generally felt that the presence of wet purpura is the more serious, and is a prognostic sign for potentially life - threatening hemorrhage . Although rare, malignancy - associated vasculitis occurs more often with hematologic rather than solid malignancies . The classic presentation is that of a necrotizing leukocytoclastic vasculitis involving the skin, with palpable purpura, typically in dependent areas.9 it is hypothesized that tumor - related antigens, or cryoglobulins, form immune complexes, which are then deposited in the skin and result in a vasculitis . It may be especially common in acute myelomonocytic leukemia and precede the clinical onset of the leukemia . Systemic vasculitis in patients with lymphoproliferative disorders (lymphocytic lymphoma, waldenstrom s macroglobulinemia, or chronic lymphocytic leukemia) is most often caused by cryoglobulinemia.9 however, our patient did not have other clinical symptoms, such as fever, headache, arthralgia and myalgia, or laboratory findings of vasculitis, such as increased erythrocyte sedimentation rate, ana, anca, cryoglobulins, or hepatitis b and c. in conclusion, pathological findings such as uncontrolled hypertension, flank pain, skin rashes, and abnormal blood count could be a part of a systemic disease or malignany in patients with esrd who are receiving renal replacement therapy . Although all and its extramedullary infiltrations can frequently be seen in children, these manifestations are also reported in adults . Leukemic cell infiltration of the kidneys can cause either renal failure, hypertension, or asymptomatic kidney enlargement . Treatments to be considered for hypertension associated with leukemic cell infiltration should include multiple drug combination, such as antihypertensive agents, chemotherapeutics, and antihyperuricemic agents . Despite the appropriate therapy, management of patients with all and esrd
A 72-year - old man presented with a two - week history of a red painful right eye . He was a soft contact lens wearer and wore his lenses for one month at a time, taking them out at the end of each day and disposing of the lenses at the end of the month . He had no past ocular, medical, or drug history of note . On examination he had an inflamed eye with a central corneal epithelial defect 3.0 mm 3.5 mm in size, with surrounding superficial and midstromal infiltration . A corneal scrape of the right eye was performed for gram staining and cultures were obtained on blood, chocolate, and sabouraud s agar, as well as non - nutrient agar overlaid with escherichia coli . He was started on empirical antimicrobial treatment with topical ofloxacin hourly by day and night . There was no growth of any other organisms, and the topical ofloxacin was discontinued . His treatment was switched to topical, ie, hourly polyhexamethylene biguanide 0.02%, hourly propamidine isethionate 0.1% (brolene), prednisolone 0.5% four times daily, and atropine 1% three times daily . Over the following three months, his treatment was slowly tapered to topical, ie, polyhexamethylene biguanide four times daily, brolene four times daily, and prednisolone 0.1% twice daily as his clinical picture improved . At three months after initial diagnosis he complained of the right eye being red and sore again for several days . On examination the gram stain showed gram - positive cocci in chains and he was empirically commenced on topical penicillin hourly, which was added to his prophylactic treatment for acanthamoeba . After 48 hours, the blood agar grew streptococcus viridans which was shown to be sensitive to penicillin . Topical penicillin was tapered down to twice daily as his condition improved over the following month . Four months after initial diagnosis, he again complained of a red painful right eye for one week . On examination fine debris and hairs were noted in the base of the abscess and were removed by scraping (figure 1). The penicillin drops were stopped and he was treated with topical ofloxacin initially hourly and then tapered off over one month . Five months after initial diagnosis he complained of a one - day history of a painful right eye . Fine strands were noted on the surface of a central corneal abscess and were removed during the corneal scrape . He was treated with topical ofloxacin and gentamicin, to which the organism was found to be sensitive . The organism was resistant to ciprofloxacin, chloramphenicol, penicillin, cefuroxime, and fusidic acid . As he improved, fine fibrils were again seen in the healing cornea and were removed and sent for microscopy . This confirmed that the fibrils were synthetic and were not observed in previous / present cultures, and presumed to be derived from tissues which the patient was constantly using to wipe his eye . The cornea continued to heal and topical treatment for acanthamoeba (including steroids and ofloxacin) were continued and tailed off over a further three months . In all episodes of infection, antibiotic sensitivities were determined using disc diffusion susceptibility testing . In all episodes of reinfection, scraping for acanthamoeba was performed to exclude reactivation of the initial infection . This patient had acanthamoeba keratitis secondary to contact lens wear and then developed three episodes of bacterial keratitis . Microbial keratitis results from the interaction of a broad spectrum of pathogens and a diverse range of host responses . Recurrence is rare in the absence of predisposing factors, such as contact lens wear, ocular surface and corneal disease, corneal anesthesia, exposure, trauma, or previous corneal surgery.1 when suspected, corneal scraping is mandatory in order to provide material for a microbiological diagnosis, debride necrotic tissue, and enhance antibiotic penetration . Corneal biopsy may be necessary in some cases of recurrence . The initial bacterial infection in this patient presumably was facilitated by the underlying acanthamoeba keratitis and the use of corticosteroids.2,3 the presence of synthetic fibrils on the ulcer base presumably contributed to the infection recurrences by acting as a nidus for organisms and by interfering with corneal healing . It is noteworthy that the cornea finally healed with no further infections once the patient was instructed not to wipe his eye with a paper tissue . Unfortunately it was not possible to obtain cultures directly from the tissue fibrils to confirm their direct association with the infective organisms . The first two bacterial infections were due to virulent organisms, such as pseudomonas and streptococcus . The culture of stenotrophomonas maltophilia from the third corneal abscess was feasible because this is an opportunistic organism and infections typically occur in patients with compromised ocular surface and trauma.4,5 it has been reported previously in patients following penetrating keratoplasty.3,6,7 this is the first case report of acanthamoeba keratitis occurring in a contact lens wearer with preceding protozoal and bacterial keratitis . The characteristically resistant antibiogram of s. maltophilia may limit the therapeutic options . Fortunately in this patient treatment
The uptake of large amounts of lipids from plasma lipoproteins via endocytosis by macrophages in the artery wall to form lipid - engorged foam cells is an important initiating event in the development of atherosclerosis, a major cause of heart disease . The main lipoprotein implicated is low - density lipoprotein (ldl), but oxidative modification of the particles, a process which can occur within the vessel wall, is required before foam cell formation is induced . The myosin superfamily is a group of actin - associated motor proteins that use atp hydrolysis to generate force or directional movement along actin filaments [2, 3]. Many new classes of myosin have been identified since the discovery, over seventy years ago, of myosin ii (conventional myosin), which produces contractile force in muscle cells; to date, 35 classes of myosin proteins have been characterised in mammalian cells . It is now known that these various classes of myosin motor are involved in a diverse range of intracellular processes, ranging from cell migration and division to cell anchorage and transport of cargo proteins along actin filaments . Myosin vi (myo6), a member of the unconventional myosin classes, is an unusual member of the myosin superfamily in that its movement along actin filaments is towards the pointed () end of an actin filament, the opposite direction to myosins of other classes . For this reason, myo6 is ideally suited for a role in endocytosis, which involves vesicle formation and/or transport away from the plasma membrane . Evidence suggests that it may play a part in organization and/or anchorage of endocytic machinery, in the provision of force required for invagination and/or vesicle fission, and during transport of vesicles through the cell cortex [68]. Attachment to vesicles is achieved through direct interaction with adaptor proteins (aps), including disabled 2 (dab2) (which binds directly to receptors or indirectly via adaptor protein-2 (ap2)) and gaip interacting protein c terminus 1 (gipc1) [79]. Myo6 can be alternatively spliced in two regions of its cargo binding domain, giving rise to either a small insert (9aa) (si) and/or a large insert (up to 32aa) (li). Thus, four variants are possible: myo6(+si, + li), myo6(si, + li), myo6(+si, li), and myo6 without inserts (myo6(i)). These variants are differentially expressed and appear to have distinct cellular targets and functions [6, 7]. Although the inserts contain no functional motifs or binding sites, they may affect the structure and therefore the affinity of myo6 for its binding partners [9, 11]. Endocytosis may occur by phagocytosis, the uptake of large particles, or by pinocytosis, the internalization of fluid and small particles [10, 12]. Phagocytosis is a regulated and selective process requiring receptor - mediated target recognition, but pinocytosis can be nonselective (macropinocytosis) or can be receptor - mediated . Clathrin - mediated endocytosis (cme) is the most studied form of receptor - mediated pinocytosis, but other forms include caveolin - mediated endocytosis, as well as clathrin - and - caveolin - independent endocytosis [10, 12]. Most evidence supporting a role for myo6 in endocytosis relates to cme: it has been shown to colocalise with clathrin, dab2, and ap-2 in polarised cells [1315] and with uncoated vesicles and gipc1 in unpolarised cells [13, 16]. In addition to cme, myo6 has been found localized to sites of macropinocytosis in fibroblasts, and holt et al . Have reported that loss of myo6 function in bone marrow - derived murine dendritic cells causes enhancement of macropinocytotic uptake, but its exact function in this process remains unclear [7, 18]. Given the importance of endocytosis in the uptake of lipoproteins by macrophages, it is possible that myo6 may have a role in foam cell formation and thus play a part in atherogenesis . Little is known, however, about the expression and function of myo6 and its binding partners in macrophages . Expression of myo6, dab2, and ap-2 has been detected in murine macrophages and expression of gipc in human peripheral blood monocytes, but, except for one report which found myo6 mrna in macrophages derived from the human monocyte cell line, thp-1, no information is available on expression of these proteins in human macrophages . Moreover, as far as we are aware, splice variant expression, subcellular localisation, and protein - protein interactions of myo6 have not been studied previously in any macrophage type . The aims of this study were to investigate the expression and function of myo6 and related proteins in human macrophages and to test the hypothesis that myo6 plays a role in the endocytosis of ldl and/or oxldl by these cells . Expression of myo6 (including splice variants), dab2, ap-22 (one of the large subunits of the ap-2 heterotetrameric complex), and gipc1 was demonstrated in both thp-1 macrophages and primary human monocyte - derived macrophages (hmdm), and their subcellular location and interactions within the cells were investigated . To study the possible role of these proteins in macrophage endocytosis, thp-1 macrophages were used to evaluate the effects of ldl and oxldl on the expression of mrna and protein for myo6, dab2, ap-2, and gipc1, as well as the effects of inhibition of myo6 and dab2 expression by small interfering rna (sirna) on the uptake of the lipoproteins by the cells . The thp-1 cell line was established 30 years ago, and since then it has been used very extensively to study the role of monocyte / macrophages in cardiovascular disease . A recent comprehensive review of the value of this model concluded that, in defined conditions, thp-1 cells resemble hmdm and mimic the changes seen in atherosclerosis and that, provided results are interpreted cautiously, they are suitable for the study of the mechanisms by which these cells affect vascular function . The present work shows that the expression and subcellular location of myo6 and related proteins are generally similar in hmdm and thp-1 macrophages, but because of the practical difficulties in obtaining large numbers of hmdm, it was not possible to use primary cells in all of our experimental approaches . Fetal bovine serum and penicillin / streptomycin were obtained from gibco (paisley, uk), and dmem and l - glutamine were from paa uk (yeovil, uk). Rpmi 1640 medium, -mercaptoethanol, 4-phorbol 12 myristate 13-acetate (pma), oil red o, ethidium bromide, and fluorescein - isothiocyanate- (fitc-) conjugated goat anti - mouse igg were supplied by sigma aldrich (poole, uk). 4,6-diamidino-2-phenylindole (dapi) 1,1-dioctadecyl-3,3,33-tetramethylindo - carbocyanine perchlorate (dii), rhodamine conjugated phalloidin, alexa488 conjugated goat anti - rabbit-, and alexa555 donkey anti - rabbit igg were purchased from invitrogen molecular probes (paisley, uk). Myo6 (h-215) and dab2 (h-110) rabbit polyclonal igg and normal donkey serum were from santa cruz biotechnology inc . Other antibodies including mouse polyclonal anti - ap-22 and rabbit polyclonal anti--microglobulin igg (abcam, cambridge, uk), mouse polyclonal anti - gipc1 igg (abnova, heidelberg, germany), rabbit polyclonal anti--actin (cell signaling technology, hitchin, uk), and horse radish peroxidase - conjugated goat anti - mouse and goat anti - rabbit igg (thermo fisher scientific, cramlington, uk) were from various suppliers as indicated, and normal goat serum was from dako uk ltd (ely, uk). Thp-1 monocytes were cultured in suspension in rpmi-1640 media, supplemented with 10% heat inactivated (56c, 30 min) fetal bovine serum (fbs), 1% penicillin / streptomycin, and 0.1% -mercaptoethanol (culture medium). For differentiation into macrophages, monocytes were incubated with 200 ng / ml phorbol 12-myristate 13-acetate (pma) (200 ng / ml) for 72 h. prior to experimental treatment, the medium containing pma and nonadherent or dead cells was removed, the cells were washed twice, and fresh culture medium was added . Cells were incubated with or without treatment for up to 5 days, with media changes every 48 h. for hmdm preparation, primary human monocytes were isolated from the blood of healthy adult volunteers with ethical approval from the east london research ethics committee . Blood was collected into tubes containing 15% edta (v: v) and processed immediately by the addition of pbs (1: 1, v: v), layering over 15 ml lymphoprep (axis healthcare ltd, borehamwood, herts, uk) and centrifugation at 800 g (30 min, 20c). The mononuclear cell layer was collected, mixed with an equal volume of ice - cold pbs containing 0.4% (v / w) trisodium citrate, and centrifuged at 800 g (5 min, 4c). After removal of the supernatant, the cell pellet was re - suspended in 0.2% (w: v) nacl for 30 sec at 4c to lyse any remaining red blood cells and 1.6% (w: v), nacl was added, and the tubes were centrifuged as previously mentioned . Resuspension in pbs containing 0.4% (w: v) trisodium citrate and centrifugation were then repeated 6 to remove contaminating platelets . The final pellet was resuspended in rpmi-1640 medium containing 5% fbs and 1% penicillin / streptomycin, and the cells were incubated at 37c in 5% co2 for 7 days to allow differentiation into macrophages . Examination by light microscopy indicated that the cells showed a macrophage phenotype after 6 days; cells were used on the 7th day after washing with pbs (4) to remove any remaining nonadherent cells . Cos-7 adherent cells were cultured in dmem maintenance media supplemented with 10% fbs, 1% penicillin / streptomycin, and 1% l - glutamine . Total rna was extracted from hmdm, thp-1 macrophages, and cos-7 cells using an extraction kit (sigma - aldrich, poole, uk) and rnase - free dnase (qiagen, crawley, uk) as recommended by the manufacturers . For reverse transcription, rna (1 g) was used to generate cdna using omniscript reverse transcriptase (qiagen) and oligo-(dt) primers according to the manufacturers' instructions . For expression studies in untreated cells, pcr was used to amplify gene products for myo6, the small and large insert regions of myo6, dab2, ap-22, gipc1, and in geneamp pcr system 9700 (applied biosystems) using the primers shown in table 1 and the following protocol: denaturing at 95c (5 min), followed by 35 cycles consisting of 30 sec at 94c, 90 sec at the appropriate specific annealing temperatures and extension at 72c for 60 sec, with a final extension at 72c for 10 min . Pcr products were visualised on 1.2% or 3% agarose gels containing 0.01% ethidium bromide (v: v). Gels were viewed and photographed under uv light using the chemidoc xrs scanner (bio - rad, hemel hempstead, uk). To determine the effects of ldl or oxldl on mrna expression, thp-1 macrophages were incubated with the lipoproteins (50 g protein / ml) for 8 or 24 h, and total rna was then extracted and reverse transcribed as described previously . The abundance of mrna transcripts for myo6, dab2, ap-22, gipc1, -actin, 2-macroglobulin, and ribosomal protein l13a was assessed by quantitative pcr (qpcr) in an opticon 2 lightcycler system (mj research, waltham, massachusetts, usa) using the primers shown in table 1 employing the following protocol: denaturing at 94c (2 min), followed by 37 cycles of amplification consisting of denaturation at 94c (15 sec), incubation at an appropriate, specific annealing temperature (1 min), followed by extension at 72c (1 min). Threshold cycle values were determined using opticon monitor 3 software and quantified using the standard curve for each gene . Normalisation factors (nf) were calculated from the geometric means of the three most stably expressed reference genes (-actin, 2-macroglobulin, and ribosomal l13a protein) as determined using genorm software or, for sirna studies, to the reference gene 2-microglobulin run on the same qpcr plate with its own standard curve . In the latter case, annealing temperatures (table 1) were adjusted to a temperature suitable for both the genes of interest and 2-microglobulin . For analysis of protein expression by western blotting, cells were washed twice with pbs and treated with ripa lysis buffer (150 mm nacl, 50 mm tris (ph 8.0), 1% triton x-100, 0.5% deoxycholate (doc), 0.1% sds) containing protease inhibitor cocktail (10 l / ml) (5 min, 4c). Lysates were agitated for 30 min at 4c, centrifuged at 93 g (10 min at 4c) to remove cell debris, and then denatured and reduced in nupage lds sample buffer (2: 1, v: v) containing 10% -mercaptoethanol solution at 70c for 10 min . Proteins (1030 g) were separated by sds - polyacrylamide electrophoresis (8% or 10% resolving gel with 5% stacking gel or precast mini - protean tgx biorad graded gels (415%) (biorad)) and transferred to polyvinylidene fluoride (pdvf) membranes . Blocking was carried out in 5% (w: v) nonfat milk powder at room temperature for 1 h, and membranes were then incubated with polyclonal rabbit anti - myo6 (1: 200), anti - dab2 (1: 400), anti--actin (1: 1,000) or anti--microglobulin (1: 700), or mouse anti - gipc1 (1: 500) igg overnight at 4c with agitation . After washing in tbst (4 15 min), horseradish peroxidase - conjugated goat anti - rabbit or anti - mouse secondary antibodies (2-microglobulin, dab2, myo6, 1: 8,000; -actin, gipc1, 1: 10,000) were added and incubations were continued for 1 h at room temperature with agitation . Finally membranes were washed in tbst (4 15 min) and bands were visualized using enhanced chemiluminescence (ge healthcare, little chalfont, uk). Band densities were digitized from film to 12 bit images using a chemidoc xrs (bio - rad) and quantified as mean grey values measured over identical areas using quantity one software (bio - rad). Cls fluorescence microscopy was conducted using either a leica sp5 or a zeiss lsm510 confocal microscope . For all experiments, thp - macrophages or hmdm were incubated in 24-well plates containing coverslips, which were removed for mounting at the end of the procedure . Coverslips were washed twice in 1 pbs, fixed with 4% formalin (v: v) for 15 min, and followed by cell permeabilisation in 0.25% triton x-100 for 5 min prior to blocking (1 h, 3% (w: v) bsa) with normal goat (ngs) or donkey (nds) serum (1% w: v). Igg polyclonal rabbit anti - myo6, -gipc1 (1: 100, v: v), -dab2 (1: 200, v: v), or polyclonal mouse anti - ap-22 (1: 100, v: v) (diluted in 0.1% (w: v) bsa and 1% ngs / nds in pbs) was then added . After 1 h, cells were washed (0.5% bsa in pbs, 5 min 4 and 15 min 1) and incubated with igg polyclonal secondary antibodies conjugated to a specific fluorochrome as follows: goat anti - rabbit antibody conjugated to alexa fluor 488 (1: 1,000, v: v), donkey anti - rabbit antibody conjugated to alexa fluor 555 (1: 1,000, v: v), or goat anti - mouse antibody conjugated to fluorescein isothiocyanate (fitc) (1: 25, v: v) dilutions in 0.1% bsa and 1% ngs in pbs (w: v). Where double immunofluorescence staining was used, compatible pairs of primary or secondary antibodies were incubated with cells simultaneously . Cells were then washed in pbs (5 min 3, then 15 min 1). To visualise cell nuclei, cells were incubated (3 min) with 300 nm 4,6-diamidino-2-phenylindole (dapi), a nucleic acid stain . The structure of f - actin was preserved prior to visualisation, with minimal loss of free myosin, by incubating cells (45 sec) with buffer containing nacl, 137 mm; kci, 5 mm; na2hpo4, 1.1 mm; kh2po4, 0.4 mm; nahco3, 4 mm; glucose, 5.5 mm; mgcl2, 2 mm; egta, 2 mm; pipes, 5 mm; ph 6.0 - 6.1 [25, 26] supplemented with 0.32 m sucrose, 0.1% triton x-100, and 1 g / ml phalloidin . Cells were immediately incubated with 165 nm phalloidin conjugated to rhodamine, as a fluorescent marker (20 min). Areas of fluorescence signal were detected by application of a threshold and the mean fluorescence intensity quantified using volocity (5.4) 3d image analysis software (perkinelmer, waltham, ma, usa). G / ml) was isolated from human plasma obtained from the national blood service (north london, uk), using sequential density gradient ultracentrifugation . Final preparations of native ldl (nldl) were dialysed against 0.9% nacl (w / v) containing 10 m edta (5l 4). For ldl used in oxidation studies, human plasma was used within 1 month of purchase and ldl within 2 weeks of isolation . Ldl was oxidised by incubation with cuso4 (5 m) for 6 h, oxidation was terminated by the addition of edta (1%, 50 l / ml v / v), and the extent of oxidation was assessed by measuring the concentration of malondialdehyde (mda) (nmol / mg ldl protein) using the thiobarbituric acid reactive substances (tbars) assay . Cuso4 was removed by dialysis at 4c against 0.9% nacl (w / v) containing 10 m edta (5l 4). For labelling of nldl and oxldl with the fluorescent probe 1,1-dioctadecyl-3,3,3,3-tetramethylindo - carbocyanine perchlorate (dii), lipoprotein deficient serum (lpds), prepared from human plasma by sequential density gradient ultracentrifugation, was added to the ldl preparations (1 mg ldl protein/2 ml lpds) followed by dii (300 g dii / mg ldl protein). To prevent oxidation, edta was added to the preparations at a final concentration of 10m, and the mixture was incubated for 8 h at 37c with shaking . The dii labelled lipoproteins (dii - nldl / oxldl) were then isolated by ultracentrifugation at d 1.063 g / ml (96,919 g, 15 h, 4c) and dialysed as described previously before use . Small interfering rna (sirna) sequences designed to target myo6 (combination of two sequences), dab2, ap-22 (table 2), or gipc1 (sequence not released by the manufacturer) were used to inhibit the expression of their cognate genes . Thp-1 macrophages (6 10 well) were transfected with sirna using hiperfect transfection reagent (qiagen, crawley, uk). 5 nm (ap-22, gipc1, dab2) or 40 nm (myo6) sirna was added to 100 l ripa-1640 medium, then 20 l hiperfect was added, and the mixture was incubated for 10 min at room temperature to allow sirna - hiperfect complexes to form prior to their addition (100 l) to cells . In all experiments, a nonsilencing scrambled sirna (allstars negative control, qiagen), which has no known homology to any mammalian genes, was used as a control . To assess the effects of inhibition of gene expression on the uptake of nldl and oxldl by thp-1 macrophages, lipoproteins were labelled with the fluorescent probe dii and uptake was assessed using fluorescence wide - field microscopy . Cells were seeded in 24-well plates (1.6 10 cells / well) and incubated with sirna for 4872 h. dii - labelled ldl or oxldl (50 g protein / ml) was then added, and the incubations, continued for periods up to 24 h. cells were fixed and imaged using a leica inverted dmrib wide - field microscope . Protein concentrations of cell lysates were determined by the method of lowry and those of nldl and oxldl were obtained using peterson's modification of lowry's method . The total cholesterol content of lipoproteins was determined by enzymatic analyses using commercially available kits (thermo fisher scientific, cramlington, uk). Statistical analysis was performed using student's t test or one- or two - way anova (followed by bonferroni's multiple comparison test), as indicated in the text . To determine whether myo6 and its binding partners are expressed in macrophages, total rna was isolated from untreated thp-1 macrophages and hmdm and then used to generate cdna for amplification of gene products by conventional pcr . Bands at the expected product size (table 1) were detected for myo6 (figure 1(a)) and also for dab2 (figure 1(b)), the large subunit of ap-2, ap-22, which was used to detect ap-2 expression (figure 1(c)) and gipc1 (figure 1(d)). In all cases, no bands were found for any of the genes when pcr was carried out in the absence of either primers (data not shown) or cdna . These results demonstrate that both thp-1 macrophages and hmdm express mrna encoding myo6, dab2, ap-22, and gipc1 . Although previous studies in mammalian cells have established that myo6 can be expressed as four variants due to alternative splicing of the li and si regions of the tail domain [6, 11], it is not known which splice variants are expressed in macrophages . Dance et al . Demonstrated the presence of myo6 both with and without the li and si in cos-7 cells, and we have used the same primer sequences to investigate their expression in thp-1 macrophages and hmdm . We found bands identical to those reported by dance et al . For myo6 variants in cos-7 cells (figure 2), and these were used as markers of band identity . Figure 2 shows that bands corresponding to myo6(li) (figure 2(a)) and myo6(si) (figure 2(b)) in cos-7 cells were also found in thp-1 macrophages (figures 2(a) and 2(b), white arrows). A band of product size equivalent to myo6(+si) mrna (figure 2(b), green arrows) was also detected in thp-1 cells, although at a lower level relative to myo6(si) in comparison to cos-7 cells . Expression of myo6(+li) was low in cos-7 cells, but a band corresponding to this variant was clearly present in thp-1 macrophages, and these cells also expressed number of other transcripts of larger size (figure 2(a), blue arrows). Hmdm showed similar patterns of expression of myo6 splice variants to those observed in thp-1 cells, with myo6(li) and multiple bands of myo6(+li) (figure 2(c)) and myo6(si) (figure 2(d)) present; however, myo6(+si) was not detected in these cells (figure 2(d)). Immunoblotting indicated the presence of a protein of the molecular weight of dab2 (96/67 kda) in 3 lysates from thp-1 macrophages and in cells from 3 individual donors for hmdm . In addition, a band corresponding to the molecular weight of myo6 was clearly present in 3 lysates of thp-1 cells (one example is shown in figure 3(b)). For hmdm, a weak band corresponding to myo6 was visible in cells from 2 donors when 45 g protein was loaded (one example is shown in figure 3(b)) but not when a lower amount of protein (35 g) was used (data not shown). The expression of myo6 in hmdm, however, was clear in the immunofluorescence experiments described later . In addition, ap-22 protein could not be detected by immunoblotting, although its presence in thp-1 macrophages was demonstrated by immunofluorescence (see later). Immunofluorescence was used to explore the distribution and interactions of myo6 and associated proteins in thp-1 macrophages and hmdm . The proteins were detected using alexa488- (myo6, dab2) or fitc-(ap-22) conjugated secondary antibodies (green) and where appropriate, dapi (blue) and rhodamine - conjugated phalloidin (red) were employed as counterstains for the nucleus and f - actin, respectively . No green fluorescence was observed in negative controls when cells were incubated with fitc- or alexa488-conjugated secondary ab in the absence of the primary antibodies . Ap-22 and dab2 were located mainly around the cell periphery in a punctate staining pattern in both thp-1 macrophages (figures 4(a) and 4(c)) and hmdm (figures 4(b) and 4(d)). By contrast, myo6 was found to exhibit a more diffuse staining in both cell types (figure 5) and was found within membrane protrusions and membrane ruffles along the leading edge of the cells (thp-1 cells figures 5(a), and 5(b), hmdm, figures 5(c) and 5(d)). Association between myo6 and f - actin was studied using fluorescence labelling techniques in both thp-1 macrophages and hmdm (figure 6). Confocal images showed that, as expected, myo6 appeared to be associated with f - actin, with similar distributions mainly found around the cell periphery in both types of macrophages (figures 6(a)(ii) and 6(b)), including possible podosome attachment sites in thp-1 cells (figure 6(a)(i)). To assess the extent of interaction between myo6 and ap-22, double immunofluorescence experiments were carried out in thp-1 macrophages in which myo6 was labelled with alexa555-linked secondary antibody to give red fluorescence (figure 7(a)). Ap-22 immunofluorescence was observed mainly at the cell periphery, while that for myo6 was also found intracellularly (figure 6(a)). Regions of overlap were rare and imperfect (figure 7(a)(ii)), suggesting that the two proteins are not substantially associated within the cells . Experiments using an alexa555-linked secondary antibody for dab2, however, indicated that there was substantial colocalisation between ap-22 and dab2 (figure 7(b)). The compositions (protein, total cholesterol, and tbars content) of the nldl and oxldl preparations used are shown in table 3 . No significant differences were found in the protein or total cholesterol content of nldl as compared to oxldl . The concentration of tbars, however, was approximately 19-fold higher in oxldl, indicating a significantly increased oxidative state . Thp-1 macrophages were incubated with or without nldl or oxldl (50 g protein / ml) for 8 h or 24 h, and lysates were then used to determine the abundance of transcripts for myo6, dab2, ap-22, and gipc1 by a combination of rt - qpcr and immunoblotting (the latter for myo6 and dab2). Incubation with nldl caused no significant change in myo6 or dab2 mrna levels at either time point (figures 8(a) and 8(b)). Mrna abundance for ap-22, however, was decreased by about 35% after 24 h (p <0.001) and that for gipc1 by 31% after 8 h (p <0.05) or 39% after 24 h (p <0.001) (figures 8(c) and 8(d)). Oxldl, however, caused a marked decrease in mrna levels for myo6 at both time points (8 h, 47%, p <0.01; 24 h, 61%, p <0.05) and also significantly reduced mrna levels for ap-22 (39%, p <0.001) and gipc1 (22%, p <0.05) compared to control values . Myo6 and dab2 protein concentrations showed little change when macrophages were incubated with either nldl or oxldl, with a small decrease in dab2 (19%) in the presence of nldl being the only difference from control values (figures 8(e)8(h)). Myo6 proteins levels, however, were significantly lower after treatment with oxldl as compared to nldl after 8 h (+ 47%, p <0.01, figure 8(f)), while dab2 levels were significantly higher (+ 24%, p <0.01, figure 8(h)). The effects of inhibition of the expression of myo6, dab2, ap-22, and gipc1 on the uptake of nldl and oxldl by thp-1 macrophages were studied using sirna sequences designed to target their cognate genes . Mrna abundance for all test proteins was maximally decreased in thp-1 macrophages 2448 h after transfection with antisense oligonucleotides as compared to scrambled controls (reduction in abundance was 7585%, myo6, dab2, and ap-22, p <0.0001; 50% gipc, p <0.001) (figures 9(a)9(d)). After 72 h (ap-22) or 96 h (myo6 and dab2), however, the inhibitory effect was reduced for all genes except gipc1 . In addition, protein concentrations of myo6 were reduced by 7583% between 48 and 96 h and those for dab2 by> 75% between 24 and 96 h (p <0.01) (figures 9(e) and 9(f)). Uptake of nldl and oxldl by thp-1 macrophages was measured using dii - labelled lipoproteins (50 g / ml) and fluorescence microscopy . In non - sirna - treated cells, the area of fluorescence signal increased between 2 h and 24 h, and, as expected, the rate of increase was faster with oxldl as compared to nldl (figures 10(a) and 10(b)). Comparison of the areas of fluorescence signal in macrophages transfected with scrambled sirna, or sirna targeting myo6 showed no significant differences (figures 10(c) and 10(d)); similar results were obtained when sirna targeting dab2, ap-22 or gipc1 were used (data not shown). Myo6 is an intracellular motor protein found to be associated with f - actin in the cytoskeleton [7, 31]; in cells where it functions in endocytosis, it is also found in association with other proteins that have assigned roles in endocytosis, namely, ap-2, dab2, and/or gipc1 . Myo6 and the interactive adaptor proteins dab2, ap-2, and gipc1 are widely expressed and play diverse, often essential, roles in cellular functioning and signalling . Furthermore, these proteins have been shown to function and interact during cme, and myo6 is thought to provide a driving force for vesicle formation and trafficking . However, although the endocytosis of lipoproteins by macrophages to form foam cells is crucial to atherosclerotic development, the potential roles for myo6 and binding partners in this process have not been explored till now . Indeed, little information is available about the expression of these proteins or their roles in human macrophages, and nothing is known about their subcellular location or mutual interactions in these cells . We have demonstrated the mrna expression for myo6, dab2, ap-2, and gipc1 in primary human macrophages (hmdm) as well as in macrophages derived from the human monocyte cell line, thp-1 (figure 1). Furthermore, the presence of myo6, dab2, and ap-2 protein was demonstrated in these two cell types using both immunoblotting and immunofluorescence (figures 35). Previous studies have detected dab2 mrna in mouse bone marrow macrophages and myo6, dab2, and ap-2 protein in various murine macrophage cell lines [1921, 32]. In addition, myo6 mrna and low levels of gipc1 mrna or protein have been found in human peripheral blood leukocytes . Nonetheless, this is the first report of the expression of these proteins in human macrophages, except for one study showing myo6 mrna expression in thp-1 cells . As positive controls, we demonstrated splice variant expression of myo6 in cos-7 cells, previously shown to express myo6 with and without the li and si insert sequences . Thp-1 macrophages and hmdm were found to express myo6(li), but while only one weak band corresponding to myo6(+li) was observed in cos-7 cells, numerous bands in this region were seen within the two macrophage cell lines (figures 2(a) and 2(c)). Dance et al . Found a similar pattern of multiple bands for myo6(+li) in the epithelial cell lines arpe-19 and llc - pk1 and determined that these resulted from alternative splicing within the li region . Expression of myo6(si) was demonstrated in thp-1 macrophages and hmdm in our experiments (figures 2(b) and 2(d)), but although myo6(+si) was clearly present in thp-1 cells, its expression was not detected in hmdm . Alternative splice variants of myo6 are differentially expressed and have distinct subcellular locations and functions in various cell types . In polarised epithelial cells, for example, myo6(li) has been shown to associate with uncoated endocytotic vesicles via gipc1, while myo6(+li) is recruited to clathrin coated pits / vesicles via dab2 . Thus, the expression of various myo6 splice forms in human macrophages further indicates that myo6 is likely to have multiple functions, including cme, within these cells . In the current study, myo6 was found to be distributed throughout the cytoplasmic pool in both thp-1 macrophages and hmdm, with particular localisation to actin - generated protrusions of the plasma membrane, and possibly membrane ruffles (figure 5), often expressing a punctuate pattern in the cell periphery indicative of association with vesicular structures, as noted previously [6, 14, 15]. As expected, myo6 appeared to be associated with f - actin, particularly at the apical membrane and at putative podosome attachment sites (figure 6). Similar associations of myo6 to plasma membrane protrusions and membrane ruffles have been reported in a number of migratory cell types other than macrophages, including epithelial, fibroblast, and ovarian border cells [13, 35], suggesting that it plays an important part in the coordination of the various processes required for cell migration, including cell adhesion and directed cell progression . Chibalina and colleagues have demonstrated that myo6 functions in endocytosis and vesicle recycling in such cells, and in conjunction with gipc, it has also been shown to participate in the endocytosis of activated 51 integrin, which links it directly to focal adhesion turnover . Have also suggested that the association of myo6 with membrane ruffles in fibroblasts may be indicative of a role in macropinocytosis . During cme, ap-2 is known to act in the formation of clathrin - coated pits / vesicles, while dab2 can either associate with ap-2 as an accessory protein or act as an adaptor protein in its own right . Dab2 also has numerous functions outside of cme, and so the extent of its association with clathrin - coated vesicles varies between cell types [40, 41]. In this study cls fluorescence microscopy was used to show that ap-22 and dab2 (figure 4) are located adjacent to the periphery of both thp-1 macrophages and hmdm in a punctate staining pattern, indicative of vesicle association . To assess the extent of interaction between myo6, dab2, and ap-22 in thp-1 macrophages, double immunofluorescence labeling experiments were used . Dab2 and ap-22 were shown to be partially colocalised (figure 7(b)) in a punctate pattern, indicative that dab2, the direct binding partner of myo6, is associated with clathrin - coated pits / vesicles in thp-1 macrophages . This finding is consistent with previous work showing that dab2 is associated with ap-22 in cos-7 cells . However, although association between myo6 and ap-1 has been reported in other cell types, the present experiments suggest that the two proteins do not have substantially overlapping distributions in macrophages (figure 7(a)). Having shown that myo6, dab2, ap-2, and gipc1 are expressed in human macrophages and that there are some interactions between them, we explored the possibility that they might play a part in macrophage foam cell formation . Thp-1 macrophages were used for these studies since there are serious technical difficulties in obtaining sufficient hmdm for the types of experiment needed . These cells have some advantages over hmdm in that, because they are genetically homogeneous, there is less variability in their phenotype, and they can be stored indefinitely at 80c . Another factor which was important for our experiments is that transfection efficiency of thp-1 macrophages with sirna is much greater than that with hmdm . The uptake of nldl and oxldl by macrophages is known to be mediated by different receptors: nldl undergoes endocytosis via the ldl receptor (ldlr) which is down - regulated when intracellular cholesterol levels increase, while oxldl is recognised by unregulated scavenger receptors, thus its uptake is the main driver of foam cell formation . Since numerous studies have demonstrated that nldl may also directly contribute to foam cell formation [4345], both nldl and oxldl were used in our studies . The expression of the scavenger receptors cd36, cd68 and scavenger receptor - a (sr - a) has been shown to be upregulated in the presence of oxldl and to a lesser extent nldl [4649], while ldlr expression is downregulated in response to cell exposure by nldl [50, 51]. In the present work, the effects of nldl and oxldl on the expression of mrna and protein for myo6, dab2, ap-2, and gipc1 in thp-1 macrophages were investigated . Compared to control levels, nldl significantly decreased mrna transcript abundance for ap-22 and gipc1 (figures 8(c) and 8(d)), while incubation with oxldl resulted in reduced levels of myo6 and ap-22 mrna (figures 8(a) and 8(c)). Little change was seen at the protein level, however, except for a small decrease in dab2 with nldl (figures 8(e)8(h)). Both myo6 protein and mrna levels were significantly lower after incubation with oxldl compared to nldl (figures 8(a) and 8(f)), suggesting that nldl and oxldl have different effects on the endocytic machinery, possibly reflecting their uptake via different receptor - mediated endocytic routes [52, 53]. We also examined the effects of knockingdown the expression of myo6 and associated proteins on the uptake of nldl and oxldl . Although substantial knockdown of gene expression was achieved (figure 9), we did not detect any significant effect on the uptake of lipoprotein type as assessed by fluorescence microscopy (figure 10). These results, together with our findings of relatively small effects of the lipoproteins on mrna and protein expression (figure 8), suggest that myo6 and its binding partners do not play a major role in the uptake of nldl or oxldl by macrophages during induction of foam cell formation . The results presented here show that myo6 and its associated proteins dab2, ap-2, and gipc are expressed in human macrophages, displaying similar patterns of expression and subcellular location in both the thp-1 cell line and in primary cells (hmdm). Myo6 was found to be particularly prominent in protrusions of the plasma membrane and membrane ruffles, where it was associated with f - actin . Ap-2 and dab2 appeared to be associated with cell vesicles, and ap-2, but not myo6, was colocalised with dab2 . Although nldl and oxldl had some effects on the expression of the genes for the proteins investigated in macrophages, attenuation of their expression through the action of cognate sirna molecules did not affect their uptake by the cells . Because the nature of these experiments requires large number of cells, however, hmdm could not be used, thp-1 cells being used instead; for this reason we remain cautious in extrapolating our findings to primary cells, as would be the case with any study involving immortalized cell lines . Nevertheless, the findings presented here on expression and subcellular localisation suggest that, in macrophages, myo6 functions in cell adhesion and progression as well as in macropinocytosis . Data from the experiments on the uptake of ldl do not support the idea that it plays a major role in foam cell formation.
Metabolic syndrome (ms) is a group of cardiovascular (cv) risk factors including abdominal obesity, hypertriglyceridemia, low high - density lipoprotein cholesterol (hdl - c), dysglycemia, and high blood pressure (bp). Numerous studies have shown that a significant increase in systemic inflammatory markers, such as high sensitivity c - reactive protein (hscrp), interleukin-6, and tumor necrosis factor- (tnf-), among others, is observed in subjects with ms . High sensitivity c - reactive protein is a good predictor of ms and is strongly associated with abdominal obesity . Moreover, this biomarker predicts cv events in healthy people as well as in those with atherosclerotic disease [5, 6]. It has been reported that hscrp is elevated in an urban population of santiago, chile, with ms, and there is a clear correlation between ms, hscrp, and subclinical atherosclerosis in this population . Thus, the clustering of cv risk factors and inflammation observed in ms increase the risk of atherosclerosis . Lipoprotein - associated phospholipase a2 (lp - pla2) is a recently described inflammatory marker . It is an enzyme produced by macrophages that hydrolyzes phospholipids of oxidized low - density lipoprotein (ldl), releasing oxidized fatty acids and lysophosphatidylcholine, which are potent proinflammatory and prooxidative molecules . Since oxidized ldl is mostly located in the intima of the artery, the products of lp - pla2 activity are generated mainly in the vascular wall . Given that atherosclerosis is an inflammatory disease which begins in the vascular wall, lp - pla2 may have a prominent role in its pathophysiology . Both lp - pla2 and hscrp are associated with coronary and cerebrovascular atherosclerotic disease in populations with and without a history of cv events [912]. Furthermore, lp - pla2 is related to cv risk factors, with a strong correlation with ldl, as well as various components of ms (e.g., abdominal obesity). The vast majority of published studies do not show any association between lp - pla2 and hscrp; hence, these inflammatory markers have been considered to be independent of each other . The objective of this study was to analyze and compare the levels of lp - pla2 and hscrp as predictors of ms in subjects without atherosclerotic disease . This was a descriptive cross - sectional study in 152 subjects (69 women), without history of atherosclerotic disease, who were recruited in two preventive cardiology centers of santiago between october 2011 and june 2012: hospital clnico de la pontificia universidad catlica de chile and hospital de la previsin de carabineros de chile . Exclusion criteria were (a) history of coronary heart disease and/or carotid or peripheral vascular disease; (b) lipid - lowering therapy (statins, ezetimibe, fibrates, nicotinic acid, and omega 3); (c) use of oral contraceptives or hormone replacement therapy; (d) chronic intake of anti - inflammatory medicines (steroidal and nonsteroidal); (e) acute and/or chronic inflammatory diseases; and (f) pregnancy . Subjects were contacted by telephone and invited to participate by nurses or doctors in charge of the study . After signing the written, informed consent, patients were subjected to clinical data collection and anthropometric and laboratory measurements, all detailed below . During the visit to the centers, subjects were interviewed about demographics, medical history, education, physical activity, family medical history, and intake of medications . Weight, height, body mass index (bmi), and hip and waist circumferences were measured . The latter was measured in the midpoint between the last rib and the iliac crest . Blood pressure was measured following the recommendations of the seventh joint national committee, with the subject sitting and after 5 minutes of rest . Venous blood samples were taken after 12-hour fasting for determining the following biochemical parameters.lp-pla2: samples for analysis of lp - pla2 activity were frozen and stored at 70c . Levels of lp - pla2 activity were determined by enzymatic method (diadexus, usa), cobas 8000 analyzer, c702 module . Prior to the analysis, precision and trueness of the enzymatic test were assessed locally, and a calibration curve was constructed, which was sent to the international center for approval . Following the approval, the analysis of the subjects' samples was performed.total cholesterol (total - c): colorimetric enzymatic method, roche diagnostics cobas analyzer cobas 8000, c702 module.hdl-c: colorimetric homogeneous enzymatic method, roche diagnostics cobas, cobas 8000 analyzer, c702 module.triglycerides: white colorimetric enzymatic method with glycerol, roche diagnostics cobas, cobas 8000 analyzer, c702 module.ldl cholesterol (ldl - c): calculated by friedewald formula.blood glucose: enzymatic method (hexokinase), roche diagnostics cobas, cobas 8000 analyzer, c702 module.creatinine: jaff method, roche diagnostics cobas, cobas 8000 analyzer, c70 module.hscrp: nephelometric method on bn prospec analyzer, siemens (detection limit 0.16 mg / l).fibrinogen: clauss method in acl top 500 analyzer, instrumentation laboratory . Lp - pla2: samples for analysis of lp - pla2 activity were frozen and stored at 70c . Levels of lp - pla2 activity were determined by enzymatic method (diadexus, usa), cobas 8000 analyzer, c702 module . Prior to the analysis, precision and trueness of the enzymatic test were assessed locally, and a calibration curve was constructed, which was sent to the international center for approval . Following the approval, the analysis of the subjects' samples was performed . Total cholesterol (total - c): colorimetric enzymatic method, roche diagnostics cobas analyzer cobas 8000, c702 module . Hdl - c: colorimetric homogeneous enzymatic method, roche diagnostics cobas, cobas 8000 analyzer, c702 module . Triglycerides: white colorimetric enzymatic method with glycerol, roche diagnostics cobas, cobas 8000 analyzer, c702 module . Ldl cholesterol (ldl - c): calculated by friedewald formula . Blood glucose: enzymatic method (hexokinase), roche diagnostics cobas, cobas 8000 analyzer, c702 module . Creatinine: jaff method, roche diagnostics cobas, cobas 8000 analyzer, c70 module . Hypertensives were subjects who had a prior diagnosis according to the jnc 7, with or without pharmacologic therapy, and/or those with an average blood pressure 140/90 mm hg . Dyslipidemics were subjects who had ldl - c level 130 mg / dl, hdl - c level <40 mg / dl in men, <50 mg / dl in women, or non - hdl - c level 160 mg / dl in the laboratory assessment . Subjects were considered diabetics if they had a prior diagnosis, with or without drug treatment, and/or if they had a fasting blood glucose 126 mg / dl during the study, according to the american diabetes association criteria . Current smokers were those subjects who smoked daily during the last month, and ex - smokers were those subjects who had at least six consecutive months without smoking . The recent harmonized criteria were used for the diagnosis of ms, which includes a waist circumference 90 cm in men and 80 cm in women for latin american populations . A logistic regression analysis was performed with each biomarker, adjusted for age and gender . Smooth receiver operating characteristic (roc) curves were constructed with cubic splines for ms (area under the curve c value = 0.50 implies a predictive value equivalent to chance). Comparisons of means (expressed as mean standard deviation [sd]) are based on the analysis of variance and linear regression models . This study included 152 subjects (45% women) with a mean age of 46 11 years . Prevalence rate for hypertension, diabetes, dyslipidemia, and current smoking was 30%, 5%, 62%, and 31%, respectively, with no significant differences observed between genders . Prevalence of ms was 38% (57 subjects) in the total sample with no significant differences between genders: 42% and 32% in men and women, respectively . Men had significantly higher waist circumference (p <0.01), systolic and diastolic bp (p <0.0001), and creatinine levels (p <0.0001) than women . Mean level of hscrp was 2.1 2.2 mg / l, and there were no significant differences between men and women (table 2). Likewise, there were no significant differences in the prevalence of high levels of hscrp (defined as> 2 mg / l) between genders (39% in men and 36% in women). Of note, hscrp was significantly and directly correlated with bmi, waist circumference, blood glucose, systolic bp, non - hdl - c, and fibrinogen, and it was inversely and significantly correlated with hdl - c (table 3). Mean level of lp - pla2 activity was 185 48 nmol / ml / min in the total sample with significant differences between men and women: 201 nmol / ml / min and 166 nmol / ml / min (p <0.0001), respectively (table 2). Lp - pla2 was significantly and directly correlated with bmi, waist circumference, diastolic bp, ldl - c, non - hdl - c, and plasma creatinine; hdl - c was inversely and significantly correlated with lp - pla2 . No significant correlations were found between lp - pla2 and blood glucose, systolic bp, hscrp, and fibrinogen (table 3). No differences were found between lp - pla2 activity of active smokers and nonsmokers . As shown in table 4, both lp - pla2 and hscrp were significantly higher in subjects with ms than in those without ms . In subjects with ms, lp - pla2 was 198 45 nmol / ml / min versus 180 48 nmol / ml / min in subjects without ms (p = 0.03). Mg / l in subjects with ms versus 2.2 3.2 mg / dl in those without ms (p = 0.0001). Both biomarkers significantly increased with the number of ms components: lp - pla2 levels increased from 163 nmol / ml / min to 198 nmol / ml / min (p <0.01) and hscrp increased from 1.5 mg / l to 4.1 mg / l (p = 0.04) when subjects with 0 and 3 components of ms were compared (figures 1 and 2). The results of the logistic regression analysis for determining ms, which included both biomarkers adjusted for age and gender, were as follows: odds ratio (or) for lppla2: 1.02 (confidence interval [ci]: 1.001.02, p = 0.03) and or for hscrp: 2.5 (ci: 1.653.80, p <0.0001). Figure 3 shows the roc curves for both biomarkers adjusted for age and gender: lp - pla2, c value = 0.66 [0.570.74] and hscrp, c value = 0.73 [0.650.81]. According to this analysis, although hscrp shows a better area under the curve, from a statistical point of view, it cannot be shown that it is better than lp - pla2, as the cis of both biomarkers overlap . In this study, we have demonstrated in a population without atherosclerotic disease that levels of lp - pla2 activity and hscrp are elevated in subjects with ms . Both biomarkers significantly increased with the number of metabolic risk factors, and both were shown to be independent and statistically significant predictors of ms . The ms corresponds to a clustering of cv and metabolic risk factors and inflammation . In our country, ms prevalence is approximately 30%, and it is occurring at younger ages, probably due to the overweight, obesity, and insulin resistance epidemic . Although, the pathophysiologic mechanism involved in ms is not entirely clear, it has been shown that it confers an increased risk for diabetes and atherosclerotic cv disease [9, 15]. Recent studies have reported that the presence of ms and inflammation (determined by hscrp) confers more cv risk than the presence of ms alone [16, 17]. High sensitivity c - reactive protein is also a predictor for the development of ms and diabetes . In our country, we have previously reported that subjects with ms and elevated hscrp have more subclinical atherosclerosis and carotid atherosclerotic plaques . Therefore, many inflammatory markers related to ms have been investigated in the search of a possible explanation for the elevated cv risk associated with it . The most studied biomarker is hscrp; however, there are new biomarkers associated with atherosclerotic disease that may be more specific to vascular inflammation . Among them is lp - pla2, an enzyme that acts on oxidized ldl and produces strong inflammatory and oxidative mediators of the intima . Epidemiologic and clinical studies have shown an association between atherosclerotic disease and lp - pla2 concentration, in a similar way as reported with hscrp [911, 13]. In this study, we have demonstrated that both biomarkers, lp - pla2 and hscrp, predicted ms with a similar power . Regarding the association of hscrp with ms, this study confirms the available evidence showing that levels of hscrp are increased in subjects with ms [16, 17, 20]. Our results also support the association between hscrp and overall / visceral obesity, hdl - c, non - hdl - c, and glycemia . Similar to the findings of ballantyne and colleagues, we demonstrated that hscrp is not associated with lp - pla2, suggesting that their inflammation pathways are different from a physiologic point of view . Conversely, hscrp was related to fibrinogen, also an acute phase protein that has a similar mechanism of production by the liver . Hscrp is produced by the liver, primarily by the action of interleukin-6, a cytokine that increases in the conditions of visceral obesity and insulin resistance . Based on this evidence, although hscrp is an important inflammatory marker, it is unspecific . Activity levels of lp - pla2 were also significantly higher in subjects with ms in our study . These findings are similar to those from the bruneck study in 2009 . In that study, tsimikas et al . Found significant associations between lp - pla2 and ldl - c, non - hdl - c, hdl - c, and homeostasis model assessment insulin resistance (homa - ir), whereas correlations with bmi and waist circumference were weaker . Our results are identical to those reported by these authors regarding lipid factors and obesity . However, we did not find any direct correlation with blood glucose, and we did not measure insulin resistance . Furthermore, lp - pla2 was not correlated to bp and smoking in our study, which was seen in the bruneck study . Lastly, we found no association between lp - pla2 and fibrinogen, which confirms that lp - pla2 inflammatory pathway is not through acute phase reaction . It is important to analyze the relationship between lp - pla2 and ldl - c and non - hdl - c (atherogenic cholesterol). After adjusting for gender and age, our population had an average ldl - c level of 128 mg / dl, which suggests that, even at moderate levels of ldl - c, subjects with ms had elevated activity of this enzyme . This result could be because many subjects with ms have primarily small and dense ldl molecules, which are prone to oxidation and therefore they are substrate for lp - pla2 . The inflammation produced by lp - pla2 is primarily in the vascular level, because lysophosphatidylcholine and oxidized fatty acids, generated after lp - pla2 action over oxidized ldl, induce adhesion molecules and other cytokines in the same vascular wall . Thus, it can be hypothesized that, unlike hscrp (which occurs as a result of inflammation caused by other cytokines from the visceral fat), lp - pla2 is directly involved in the inflammatory process, and, subsequently, the atherogenic process of the plaque . Thus, lp - pla2 could be a more specific marker of atherosclerotic events in patients with ms . Confirmation of this hypothesis must be demonstrated by studies of cv events in patients with ms, who have these biomarkers measured, and with findings adjusted for all the risk factors associated with hscrp and lp - pla2 . In this regard, it should be noted that a meta - analysis evaluating the clinical use of hscrp has shown that this biomarker adds little in the prognosis of cv risk, when adjusting by the risk factors involved in ms . Conversely, the fact that lp - pla2 was weakly related to these metabolic factors should preserve its power as a biomarker . The reasons for not having completely cleared lp - pla2 prognosis utility are based primarily on the different techniques used for its measurement, which makes it impossible to compare the results of different studies . In this study, we did not expect to find the same power for hscrp and lp - pla2 as predictors of ms . We expected hscrp to be a better predictor, given its close association with metabolic risk factors included in ms . However, from a statistical point of view (as demonstrated by the roc curves), both had a similar level of prediction . These results suggest that lp - pla2 is related to ms through other pathways that are not fully known and most likely not associated with the metabolic risk factors . We know that lp - pla2 is linked through oxidized ldl, which is elevated in subjects with ms, although it cannot be perceived due to the moderate levels of ldl - c observed in the subjects of our study . Oxidized ldl, and, for measuring oxidized ldl in the clinic, it required more expensive and sophisticated methods not frequently used in clinical practice . Since ms is highly prevalent in our population, our results could provide knowledge about nontraditional risk factors that could help to identify which patients with ms must be treated earlier and more aggressively . This is important, because currently there is a pharmacologic inhibitor of lp - pla2 activity, darapladib, which is being investigated in subjects with atherosclerotic disease . In addition, another study is currently investigating the use of a monoclonal antibody against interleukin-1 for the intervention against high hscrp . If these studies have positive results, the measurement and targeting of these biomarkers might become important components of clinical management of patients . Finally, it must be noted that we found no studies in literature that compare lp - pla2 and hscrp as predictors of ms in the general population . Our study has some limitations: (1) it is a cross - sectional study, and hence causality inferences cannot be made; (2) it was done in a small, nonrandom sample of subjects; (3) we did not include homa - ir, which could explain the relationship of insulin resistance with ms and inflammatory markers; (4) finally, we only determined lp - pla2 activity instead of mass . This decision was subject to the availability of the laboratory determination in our country . However, currently, it is the most used technique, and it has shown the best correlation with ms . The strengths of the study include the following: (1) the recruitment of subjects was performed prospectively; (2) subjects with lipid - lowering therapy and women with hormone replacement therapy were excluded, among others; (3) this study has validated the measurement of lp - pla2 in our country . In conclusion, prospective studies must confirm which of the markers or if both markers are causally related to the atherosclerotic consequences observed in subjects with ms.
There is a weakness in the spigelian fascia that gives rise to a spigelian hernia, and this may be either congenital or acquired . Spigelian hernias were named after the flemish anatomist, adrian van den spiegel, who was the first to describe the semilunar line . However, it was josef klinkosch who was credited for recognizing a clinical entity associated with the spigelian area in 1764 . Spigelian hernias are rare, representing only 12% of abdominal wall hernias, with a slightly higher incidence in women . Here we discuss a case of a spigelian hernia discovered through diagnostic laparoscopy, in the absence of pertinent physical exam or imaging findings . We present the case of a 75-year old, obese female who presented to our facility for evaluation of left lower quadrant abdominal pain that radiated to the lower back, daily, after a transvaginal bladder suspension surgery performed last year . The pain was described as an intense, intermittent pain, exacerbated by abdominal flexion and partially alleviated by evacuations and sometimes spontaneously . In review of systems the patient denied changes in bowel functions, hematuria or vaginal discharge . Patient s past medical history reveals hypertension, dyslipidemia, gerd, diabetes mellitus, osteoporosis, arthritis, recurrent utis, and thyroid disease . Her surgical history revealed a prior lumbar laminectomy, appendectomy, abdominoplasty, hysterectomy and lysis of adhesions . She had been seen multiple times by multiple specialists for this pain without any success in reaching a proper diagnosis . The abdominal computerized tomography (ct) showed no evidence of acute abdominopelvic disease but reported findings consistent with constipation . Due to the persistence and severity of the patient s symptoms, and the lack of definitive evidence from ct and ultrasound, the decision was made to take the patient in for a diagnostic laparoscopy . After adequate preparation, a 5 mm camera was inserted at the level of the umbilicus . Upon entry into the abdominal cavity no evidence of bleeding or injury to internal organs was noted, multiple adhesions were visible in the area of the left lower quadrant and right lower quadrant, and were subsequently lysed using the harmonic scalpel . Upon removal of these adhesions, a spigelian hernia was visible in the left lower quadrant, containing a segment of omentum (fig . 1). There was no relationship between the previous laparotomy incision site and the location of the spigelian hernia found . Two additional 5 mm ports were placed, one at the left midclavicular line in the left upper quadrant and another at the right midclavicular line in the right upper quadrant . The segment of omentum trapped inside the spigelian hernia was reduced and resected using a harmonic scalpel, which permitted visualization of the defect in the spigelian fascia (fig . 3a) and then secondarily reinforced using a 4 6 inch proceed mesh secured with sutures at the 3 and 9 oclock position and then anchored to the abdominal wall using the securestrap fixation device (fig . Postoperatively, the patient made an uneventful recovery and was discharged on the first postoperative day . Spigelian hernias are ventral hernias that occur at the linea semilunaris; most often where it crosses douglas line spigelian hernias occur through the spigelian fascia which is composed of the aponeurotic layer between the rectus muscles medially, and the semilunar line laterally . . The spigelian fascia is the space between the lateral border of the rectus abdominis and the semilunar line, also called the spigelian line . Spigelian hernias are also referred to as hernias of the semilunar line, hernias through the conjoint tendon, or as spontaneous lateral ventral hernias . As previously mentioned, they are a rare form of hernia, representing less than 2% of all abdominal hernias . Spigelian hernias may occur secondary to increased intraabdominal pressure due to obesity, pregnancy, chronic cough and prostate disease . Additionally, a history of previous abdominal surgeries may predispose patients to the development of spigelian hernias due to the resultant weakening of the semilunar line . They tend to develop during the fourth to seventh decade of life, with a higher prevalence in females . It is believed that they develop as a result of weakness in the fascia created by perforating vessels . These hernias have a very narrow neck, increasing the risk of incarceration and strangulation, . Studies suggest that the risk of incarceration could be as high as 20% at the time of diagnosis . Therefore, the presence of a spigelian hernia is an indication for its surgical repair . Once repaired, reoccurrence is uncommon, . Spigelian hernias can be repaired using an open conventional approach, or laparoscopic repair . Despite the increased popularity of the laparoscopic approach an open approach is more feasible in emergent presentations associated with viscus incarceration, as this prevents undue delays and allows for rapid reduction with possible revival of ischemic tissues, as well as reduction of the rate of iatrogenic bowel injury during trocars insertion for the laparoscopic approach . Although direct approximation may be performed, studies have shown a distinct advantage in recurrence rates with the usage of prosthetic mesh . A prospective randomized trial comparing outcomes in patients who underwent open repair vs. those who underwent laparoscopic repair showed an advantage regarding morbidity (p <0.05) and hospital stay (p <0.001), for those who underwent laparoscopic repair . The study took 11 patients who underwent open repair and compared their outcomes with 11 patients who underwent laparoscopic repair . They reported an average hospital stay of five days for the open repair group vs. one day for the laparoscopic group . This study demonstrated a clear advantage to the laparoscopic approach in terms of hospital stay and morbidity . When done laparoscopically, the trans - abdominal approach is preferred . The advantage of tep repair is that it significantly decreases the risk of complications related to penetrating the peritoneal layer, therefore decreasing adhesions and additional complications, . In our case, the tep approach was not employed because the diagnosis was made only after inserting a laparoscope into the peritoneal cavity . Another consideration besides the surgical approach is the method used for fixation of the mesh . Huber and colleagues described a novel approach using a fibrin sealant in place of staples to secure the mesh . This is believed to reduce the risk of complications such as nerve entrapment, hematomas, and post - operative chronic pain . Spigelian hernias present with localized pain at the hernia site which may or may not be palpable during the physical exam . During the physical exam, a tender spot is often palpable over the hernia defect when the abdominal muscles are tensed . Palpation of the hernia is facilitated by having the patients alternately tense and relax their abdominal muscle . The diagnosis is unequivocal if the patient presents with a palpable mass and hernia defect along the spigelian aponeurosis . However, palpable masses are often not well localized during physical exam, as the hernia may dissect through the layers of the abdominal wall and localize away from the semilunaris line . This may lead to confusion during an open surgical approach since the incision is often made over the palpable mass, but the actual defect may exist away from the mass . Additionally, patients often have localized pain in the area, but no bulge, because the hernia lies beneath the external oblique aponeurosis, . Although ct and ultrasound are often useful to make the diagnosis, spigelian hernias are often not visible with either study . When the mass is not palpable during physical exam and not visible through ultrasound or ct scan, diagnostic laparoscopy may be indicated we experienced an unusual case where the diagnosis of a spigelian hernia was especially challenging due to vague symptomatology and the lack of a palpable defect; likely as a consequence of the patient s obesity . We encountered a patient with chronic left lower quadrant pain whose physical examination was only positive for pain on palpation and during abdominal flexion . The laparoscopic approach was central to this case since it permitted both diagnosis and surgical repair . Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Raul mederos: attending surgeon, performed the procedure . Supervised every step of the case report production and data collection . Javier alvarado: assisted in surgery, contributed to study design, data collection, and writing . Moises matos: corresponding author, contributed to study design, data collection, and writing.
In total, we collected 70 horse samples including two european - origin breeds (thoroughbred; n=4, selle franais; n=1), one south american - origin breed (criollo; n=5), three native japanese breeds, and a native korean breed . The japanese samples were from hokkaido (n=31), taishu (tsushima; n=20), and yonaguni (n=1). We excluded offspring or full - siblings from our samples when estimating the allele frequency of each breed . We extracted genomic dna from blood, buccal swabs, or hair using a qiaamp blood and tissue kit (qiagen, valencia, ca, usa). For some hair samples, an instagene matrix (bio - rad laboratories, hercules, ca, usa) was used . We amplified 504 base pairs of the exon3 region in equine drd4 (genbank: ab080626.1) by polymerase chain reaction (pcr). Pcr products were purified using a high pure pcr product purification kit (roche, basel, switzerland), and their nucleotide sequences were determined by cycle sequencing using a big dye terminator v3 . 1 cycle sequencing kit and an applied biosystems 3130xl genetic analyzer (applied biosystems, foster city, ca, usa). Polymorphisms in the sequences (snps and vntr) were detected by the alignment using mega software, version 5 . For snps found in more than one breed, the allele frequencies of each breed were statistically compared using fisher s exact test . In equine drd4 exon3 region we found two types of polymorphisms, vntr and snps . The sequence of the exon3 region and locations of polymorphisms we found are shown in fig . 1.sequences of the drd4 exon3 region in four breeds (criollo, hokkaido, taishu and thoroughbred). Vntr regions are marked by lines and bold numbers indicate the number of repeats . Novel sequences determined in this study (criollo and taishu) were deposited in the ddbj database (accession nos . Ab817803 and ab817804).. vntr was found only in criollo horses; these horses had a nine - repeat allele and a ten - repeat allele (fig . The ten - repeat allele was longer than previously reported alleles (nine and eight repeats), and its frequency was approximately 0.30 . No length polymorphisms were found in the other breeds (all of them had the nine - repeat allele). Sequences of the drd4 exon3 region in four breeds (criollo, hokkaido, taishu and thoroughbred). Locations of snps are marked by squares . Vntr regions are marked by lines and bold numbers indicate the number of repeats . Novel sequences determined in this study (criollo and taishu) were deposited in the ddbj database (accession nos . We also found six snps, three of which (c199 t, c415 t and g445a) have not previously been reported . The allele frequencies of each snp in the five breeds (criollo, hokkaido, korean, taishu and thoroughbred) are shown in table 2table 2 . The allele frequencies of snps in five breedscriollohokkaidokoreantaishuthoroughbredn5318204vntr90.701.001.001.001.00100.300.000.000.000.00he0.470.000.000.000.00c147tc1.000.971.001.000.88t0.000.030.000.000.12he0.000.060.000.000.25c199tc0.701.001.001.001.00t0.300.000.000.000.00he0.470.000.000.000.00g292ag0.700.970.620.920.38a0.300.030.380.080.62he0.470.060.500.140.54c318ac1.001.001.000.721.00a0.000.000.000.280.00he0.000.000.000.410.00c415tc1.001.001.000.651.00t0.000.000.000.350.00he0.000.000.000.470.00g445ag0.900.810.880.901.00a0.100.190.120.100.00he0.200.320.230.180.00he: expected heterozygosity .. he: expected heterozygosity . Among the snps we identified, c147 t and c318a are synonymous snps, which cause no amino acid changes . We found the c147 t polymorphism only in hokkaido and thoroughbred horses, and the c318a polymorphism only in taishu horses . The nucleotide substitution reported at the c318a location in a previous study was from cytosine to thymine, but the substitution we found in taishu horses was from cytosine to adenine . In both cases, these substitutions cause no amino acid change . The other four snps (c199 t, g292a, c415 t and g445a) are non - synonymous snps, which cause amino acid changes . C199 t was unique to criollo horses and c415 t was unique to taishu horses . G292a is a non - synonymous snp that causes an amino acid change from asparagine to aspartic acid, and it was linked to horse personality in a previous study . The a allele of g292a is associated with low curiosity and high vigilance scores . We compared the allele frequency of g292a among five breeds (hokkaido, taishu, korean, criollo and thoroughbred) and found a significant difference among breeds (fisher s exact test, p=4.4 10). In native japanese breeds (hokkaido and taishu) the frequency of the a allele was very low (approximately 0.03 for hokkaido, 0.08 for taishu). In contrast, the frequency of the a allele was considerably higher (approximately 0.62) in thoroughbreds . One selle franais horse and one yonaguni horse were heterozygous (their genotypes were a / g). G445a is a novel snp that causes an amino acid change from alanine to threonine . This polymorphism was found in four breeds (hokkaido, taishu, korean and criollo), but the allele frequencies did not differ significantly among these breeds (fisher s exact test, p=0.65). The novel sequence data determined in this study was registered in the ddbj nucleotide database with the accession numbers ab817803 (criollo) and ab817804 (taishu). In this study we found several novel polymorphisms in equine drd4 exon3 region . Some of these seem to be breed - specific . Criollo horses had a unique non - synonymous snp (c199 t) and longer alleles (ten - repeat) in the vntr region than other breeds . Taishu horses also had a unique non - synonymous snp (c415 t). These polymorphisms may change the amino acid sequence of the protein and modify its function . We also compared the allele frequencies of polymorphisms among different breeds . Although samples from unrelated individuals are needed to estimate the allele frequency more accurately, it is difficult to collect completely unrelated samples in the case of rare native breeds . So, we excluded offspring or full - siblings from our samples when estimating the allele frequency . We found that the allele frequency of g292a, which has been reported to be associated with horse personality, was different among breeds . In particular, native japanese horses had quite different allele frequencies of g292a from thoroughbred horses . Although our sample size of thoroughbreds was relatively small, the a allele seems to be a major allele in these horses, because the frequency of this allele was also high in a previous study (approximately 0.54). This suggests that the a allele is a minor allele of horses in general, except for thoroughbreds . It may be a byproduct of the special breeding history of thoroughbred horses for racing performance . The low frequency of the a allele, linked with low curiosity and high vigilance, suggests that japanese horses should be more curious and less vigilant than thoroughbreds . In fact, a questionnaire - based behavioral evaluation of kiso horses revealed these animals are curious and friendly with humans . The differences of the a allele frequency of g292a may be used as a predictor of behavioral traits of horse breed . Also polymorphisms within each breed may be useful for management or training suited to each individual trait . However, the association between g292a and behavioral traits requires further confirmation using more objective behavioral tests . Behavioral tests used in canine studies showing the association between drd4 and behavioral traits [4, 6] may be useful, following modification, for use with horses . In conclusion, this study showed that there are differences in drd4 polymorphisms among breeds of horses . These differences may be a result of different breeding histories or of geographical variation . Further studies of both functional aspects of genes and behavioral variations are needed to validate the effect of these polymorphisms.
Pilomatricoma usually appears in the first two decadesusually, an asymptomatic lesion with an occasional inflammation . Pilomatricoma usually appears in the first two decades usually, an asymptomatic lesion with an occasional inflammation . Usually, they present as a solitary nodule / cystic lesion distributed on head, neck, and upper trunk area . Sometimes, there is associated inflammation . A 36-year - old obese, hypothyroid female patient presented in surgery outpatient department of a tertiary care medical college of eastern bihar with a bluish - red, nodular lesion over the upper back [figure 1]. She gave a history of 68 months duration and complained of some sticky material coming out from the lesion intermittently whenever there was pain and redness . She was treated outside with intermittent antibiotic and anti - inflammatory agents with an apparent resolution of the symptoms followed by recurrence in a few weeks . On examination, we found a violaceous nodulo - cystic lesion on the upper back with a size of 2 cm 1.5 cm; the center of the lesion was eroded, and there was a visible yellow - colored cheesy discharge from that area [figure 2]. Depending on the site, the nodulo - cystic nature of the lesion, the recurrent history of inflammation and discharge, a provisional diagnosis of the ruptured epidermal cyst was placed . The patient was managed conservatively till discharge subsided, and the epithelialization of central area was complete . Nodule covered with yellow cheesy discharge and crust in scanner view, the epidermis was acanthotic . In the dermis, there was a nodular basaloid cell proliferation extending from papillary to reticular dermis [figure 3]. There was also the proliferation of squamoid cells with the disappearance of nucleus leaving behind a shadow (ghost cells / shadow cells) [figures 4 and 5]. The dermis was covered by mononuclear inflammatory infiltrate along with the formation of foreign body type giant cells [figures 6 and 7]. In view of these findings h and e (scanner view): basaloid cells h and e (scanner view): ghost cells ghost cells with nuclear shadow, (h and e, 40) stromal inflammation, (h and e, 10) foreign body giant cells, (h and e, 40) in scanner view, the epidermis was acanthotic . In the dermis, there was a nodular basaloid cell proliferation extending from papillary to reticular dermis [figure 3]. There was also the proliferation of squamoid cells with the disappearance of nucleus leaving behind a shadow (ghost cells / shadow cells) [figures 4 and 5]. The dermis was covered by mononuclear inflammatory infiltrate along with the formation of foreign body type giant cells [figures 6 and 7]. In view of these findings h and e (scanner view): basaloid cells h and e (scanner view): ghost cells ghost cells with nuclear shadow, (h and e, 40) stromal inflammation, (h and e, 10) foreign body giant cells, (h and e, 40) in scanner view, the epidermis was acanthotic . In the dermis, there was a nodular basaloid cell proliferation extending from papillary to reticular dermis [figure 3]. There was also the proliferation of squamoid cells with the disappearance of nucleus leaving behind a shadow (ghost cells / shadow cells) [figures 4 and 5]. The dermis was covered by mononuclear inflammatory infiltrate along with the formation of foreign body type giant cells [figures 6 and 7]. In view of these findings h and e (scanner view): basaloid cells h and e (scanner view): ghost cells ghost cells with nuclear shadow, (h and e, 40) stromal inflammation, (h and e, 10) foreign body giant cells, (h and e, 40) usually, they appear in early age, the average age of onset being childhood to adolescence . They usually present as a solitary firm nodule or cyst distributed on any nonglabrous area, but mostly restricted to head - neck and upper trunk . Sometimes, the lesion shows an inflammatory sign . In histology, usually there is a cyst formation with matrical keratinization . There are two kinds of cells; outer basaloid matrical cells and central squamatized cells with the remnant of nuclear outline (shadow or ghost cells). In the early stage, particularly, in the elderly population, the basaloid cells predominate, and these tumors are called proliferating pilomatricoma . The treatment is basically surgical excision; recurrence has been reported following the surgical excision . In our case, the pilomatricoma appeared at 36 years of age, and clinically mimicked ruptured epidermal cyst . The goal of this presentation is to highlight the fact that pilomatricoma may appear late in life and matrical cyst of pilomatricoma can rupture and give rise to a clinical picture mimicking ruptured epidermal cyst.
The potential use of the affected upper extremity of children with hemiplegia often fails due to learned non - use phenomenon . Constraint - induced movement therapy (cimt) is one of the treatment strategies which utilizes the principles of neural plasticity to help acquire motor skills of the affected upper extremity. [27] this therapeutic approach involves constraining of the unaffected upper extremity using sling, plaster cast, mitt or splints and intensive training of the affected upper extremity with task - specific, goal - oriented activities by reinforcement (shaping technique). A five - year - old female child presented with right hemiplegia with the history of delayed motor milestones and limited motor skills of the right upper extremity since birth . The objective details of the cause of hemiplegia could not be established as there were no medical records available . Presently, she has achieved the highest level of functional independence (able to walk and run independently). She displayed no voluntary effort to initiate any motor skills of the right upper extremity unless verbally prompted, even otherwise initiating only minimal response suggesting learned non - use phenomenon . The following criteria were considered for use of cimt in this child (adapted from cochrane review study). Observed learned non - use of affected upper extremity.a possible movement of at least 10 extension at metacarpophalangeal and inter - phalangeal joints and 20 extension at wrist of the affected upper extremity.no cognitive impairment and shall cooperate with treatment . Observed learned non - use of affected upper extremity . A possible movement of at least 10 extension at metacarpophalangeal and inter - phalangeal joints and 20 extension at wrist of the affected upper extremity . No cognitive impairment and shall cooperate with treatment . The quality of upper extremity skills test (quest) is a criterion - referenced measure that evaluates the quality of upper extremity function in four domains: dissociated movements (19 items with one level of response for each item), grasps (six items with three to five levels of response for each item), weight - bearing (five items with six levels of response for each item) and protective extension (three items with six levels of response for each item). It is designed to be used with children who exhibit neuromotor dysfunction with spasticity and has been validated with children 18 months to eight years of age . The data collected during the neuro developmental therapy / casting study by law et al ., were used to analyze the validity and responsiveness of the quest . The parents of the child were counseled for the treatment approach that could improve the motor skills of the affected right upper extremity . The parents were interested and were keen in subjecting their child to this treatment approach and gave informed consent . The pre - intervention assessments of the right upper extremity motor skills were measured using quest . The unaffected left upper extremity was constrained with posterior slab plaster cast extending above the elbow to the interphalangeal joints of fingers and supported with a sling . The affected right upper extremity was then subjected to task - specific goal - oriented activities that aimed to improve reaching, grasps, manipulation and release of the object using the arm and hand . The activities were encouraged using play way method and reinforcements using visual (postural mirror) and verbal feedback . The therapy session usually lasted for one hour a day for five days a week . The parents were instructed to constantly encourage the same activities that were carried out during the treatment session in daily activities . At the end of the two - week period, post - intervention assessment of the right upper extremity was done using quest . As there was an incremental response in the outcome measure, the investigators convinced the parent to continue the treatment for another week . At the end of three weeks, once again post - intervention assessment was done . The quality of upper extremity skills test (quest) is a criterion - referenced measure that evaluates the quality of upper extremity function in four domains: dissociated movements (19 items with one level of response for each item), grasps (six items with three to five levels of response for each item), weight - bearing (five items with six levels of response for each item) and protective extension (three items with six levels of response for each item). It is designed to be used with children who exhibit neuromotor dysfunction with spasticity and has been validated with children 18 months to eight years of age . The data collected during the neuro developmental therapy / casting study by law et al ., were used to analyze the validity and responsiveness of the quest . The parents of the child were counseled for the treatment approach that could improve the motor skills of the affected right upper extremity . The parents were interested and were keen in subjecting their child to this treatment approach and gave informed consent . The pre - intervention assessments of the right upper extremity motor skills were measured using quest . The unaffected left upper extremity was constrained with posterior slab plaster cast extending above the elbow to the interphalangeal joints of fingers and supported with a sling . The affected right upper extremity was then subjected to task - specific goal - oriented activities that aimed to improve reaching, grasps, manipulation and release of the object using the arm and hand . The activities were encouraged using play way method and reinforcements using visual (postural mirror) and verbal feedback . The therapy session usually lasted for one hour a day for five days a week . The parents were instructed to constantly encourage the same activities that were carried out during the treatment session in daily activities . At the end of the two - week period, post - intervention assessment of the right upper extremity was done using quest . As there was an incremental response in the outcome measure, the investigators convinced the parent to continue the treatment for another week . At the end of three weeks as the quest measure analyzes the quality of motor skills of both the right and left extremity, it may be noted that the pre - intervention percentage score was 53.04% indicating full percentage score of unaffected left upper extremity and marginal percentage score of affected right upper extremity [table 1]. Following intervention, increments in percentage score by 26.79% and 07.51% were observed at the end of two weeks and three weeks respectively which should be attributed to the improvements in the quality of motor skills of the affected right upper extremity . It may also be noted that the grasp percentage score was greater than other domains indicating better improvement in fine motor skills than gross motor skills . The main concern for the parents in the use of cimt for improving the motor skills of the affected upper extremity in infantile hemiplegia was the fact that it restricts the use of the unaffected extremity . The success of the use of cimt in infantile hemiplegia depends on the parents, their proper understanding of the concept of the approach and their deep motivation in carrying out home exercises . The increments in percentage scores observed in this case report are attributed to the increased demands for the use of the affected upper extremity while constraining the unaffected upper extremity through task - specific goal - oriented activities that were reinforced with visual and verbal feedback . The observation of this case report indicates that the use of cimt could reverse the learned non - use phenomenon of the affected upper extremity in infantile hemiplegia and thus reduce disability to greater extent.
The phosphoinositide 3-kinase (pi3k)/akt survival pathway functions upstream to positively regulate the activity of the mtor axis . Recent studies show that extracellular signal - regulated kinases 1/2 (erk1/2) and 90 kda ribosomal protein s6 kinase 2 (rsk2) also positively regulate mtorc1 through respective phosphorylation of tsc2 and raptor, linking ras to positive regulation of mtorc1 signaling, . Thus, mtor serves as a convergence point of the pi3k / akt and mitogen - activated protein kinase (mapk)/erk signaling pathways, which are often hyperactivated in cancer . In contrast, the tumor suppressors liver kinase b1 (lkb1) and p53 both negatively regulate the mtor axis . Lkb1 inhibits mtorc1 signaling through activation of amp - activated protein kinase (ampk) and tsc2 . Similarly, p53 inhibits mtor1 signaling through sestrin - mediated activation of ampk and tsc2, . Hence, alteration (e.g., activation and inactivating mutations) of genes (e.g., pten, kras, and lkb1) whose products regulate the mtor axis will result in hyperactivation of the axis in certain types of cancers (figure 1). This category of cancer is expected to be addicted to the mtor axis for survival and growth, hence increasing susceptibility to mtor - targeted therapy . Compared with mtorc1 signaling, little is known about the upstream regulators of the mtorc2 axis . Various oncogenes (e.g., ras and pik3ca; in blue) or tumor suppressors (e.g., pten, lkb1 and p53; in red) positively or negatively regulate the mtorc axis . Mtor, mammalian target of rapamycin; pik3ca, phosphoinositide-3-kinase, catalytic, alpha polypeptide; pten, phosphatase and tensin homolog; lkb1, liver kinase b1; mtorc, mammalian target of rapamycin complex . The tumor suppressor pten is a lipid phosphatase that negatively regulates the pi3k signaling pathway . Sawyers's group revealed that pten - deficient mouse cells or human cancer cells showed enhanced sensitivity to cci-779, both in vitro and in vivo . Soon after that study, shi et al . Reported similar findings in multiple myeloma cells . They found that 3 of 4 pten - deficient cell lines with constitutively active akt were remarkably sensitive to growth inhibition and g1 arrest induced by cci-779, with id50 concentrations of <1 nmol / l . In contrast, myeloma cells expressing wild - type pten were> 1,000-fold more resistant . Acute expression of a constitutively active akt gene in cci-779resistant myeloma cells containing wild - type pten and quiescent akt did not convert them to the cci-779sensitive phenotype . Conversely, expression of wild - type pten in cci-779-sensitive, pten - deficient myeloma cells did not induce resistance . Thus, the level of pten and akt activity does not regulate sensitivity per se . In a recent study, suppressing pten function by expressing a pten mutant lacking lipid (g129e) or lipid and protein (c124s) phosphatase activity in mcf-7 cells conferred sensitivity to rapamycin . Pi3ks are heterodimeric lipid kinases composed of p110 catalytic and p85 regulatory subunit variants encoded by separate genes and alternative splicing . Mutations in the p110 catalytic subunit (pi3kca) occur in certain types of cancer, such as breast cancer, and lead to the activation of pi3k / akt signaling . In a study with 31 breast cancer cell lines, weigelt et al . Reported that breast cancer cells harboring pik3ca mutations, but not pten loss, were selectively sensitive to rad001 and the mtor kinase inhibitor pp242 . However, in another study with 31 human cancer cell lines, meric - bernstam et al . Reported that cell lines with pik3ca and/or pten mutations were more likely to be rapamycin - sensitive . Interestingly, di nicolantonio et al . Recently reported that human cancer cells carrying alterations in the pi3k pathway were responsive to rad001, both in vitro and in vivo, except when kras mutations occurred concomitantly or were exogenously introduced . In cancer cells with mutations in both pik3ca and kras, genetic ablation of mutant kras reinstated response to the drug . Mahoney et al . Reported that lkb1/kras mutant nsclcs constitute a genetic subset of nsclc with increased sensitivity to mapk (ci-1040) and mtor (rapamycin) signaling inhibition, whereas lkb1 and kras mutations alone do not confer similar sensitivity . Contreras et al . Reported that lkb1 inactivation - driven endometrial cancer is highly responsive to rapamycin monotherapy . In their study, they found that rapamycin monotherapy not only greatly slowed disease progression, but also led to striking regression of pre - existing tumors . To date, few clinical studies have investigated the impact of genetic alterations on mtor - targeted cancer therapy . In a cohort of metastatic cancer patients who had received single - agent rad001, di nicolantoni et al . Found that the presence of kras mutations was significantly associated with lack of benefit (partial response and stable disease) after rad001 therapy . In this study, 11 of the 12 patients with kras mutant tumors had disease progression, whereas 15 of 31 of wild - type cases benefited from treatment (p = 0.0171). Unfortunately, the impact of pik3ca mutations on patient response to treatment was not analyzed because of limited sample size . Recently investigated the genetic basis of remission of a patient with metastatic bladder cancer treated with rad001 using whole - genome sequencing . They found that the mutation of tsc, which occurs in about 8% of bladder cancer cases, correlated with rad001 sensitivity . After identification of tsc1 mutation from this responsive bladder cancer case, the authors further analyzed samples from 13 additional bladder cancer patients treated with rad001 in the same trial . This analysis revealed 3 patients with tumors harboring nonsense mutations in tsc1, including 2 patients who had minor responses to rad001 (17% and 24% tumor regression). A fourth patient with 7% tumor regression had a somatic missense tsc1 variant of unknown functional consequence . In contrast, tumors from 8 of the 9 patients showing disease progression were tsc7-wild type . Benefit from rad001 lasted longer in patients with tsc1-mutant tumors than in those with wild - type tumors (7.7 months vs. 2.0 months, p = 0.004), with a significant improvement in time to recurrence (4.1 months vs. 1.8 months; p = 0.001). Sega is a benign, slow - growing tumor that usually forms in the walls of fluid - filled spaces in the brain . It is common in patients with tsc, which is caused by the mutation of tsc1 or tsc2 gene . In an early single - arm trial, rad001 reduced sega tumor volume 50% in 9 of 28 patients aged 3 to 34 years . In the recently completed double - blind, placebo - controlled phase iii trial involving 117 patients with tsc in 24 centers across 10 countries worldwide, 24 weeks of oral rad001 treatment caused 50% reduction in sega tumor volume in 35% (27/78) of patients, whereas placebo did not have this effect in any patient (0/39). These results prompted fda approval of rad001 for the treatment of pediatric and adult sega . In a prospective clinical trial, janku et al . Sequenced pik3ca in tumor samples from patients with advanced breast, cervical, endometrial, and ovarian cancers that were refractory to standard therapies . Of the 140 patients analyzed, 25 (18%) had phoca mutations, and 23 were then enrolled in a clinical trial that included a pi3k / akt / mtor pathway inhibitor (e.g., cci-779 alone; cci-779 plus bevacizumab; rapamycin plus docetaxel; or cci-779, bevacizumab plus liposomal doxorubicin). Partial response was observed in 30% (7/23) of the patients harboring a pik3ca mutation . In contrast, only 10% (7/70) of patients with wild - type pik3ca who were treated on the same protocols and had the same disease types responded to treatment (p = 0.04). Thus, this study suggests that screening for pik3ca mutations may reveal a subset of patients who are sensitive to treatment regimens that include a pi3k / akt / mtor inhibitor . Several preclinical studies have consistently suggested that tumors with pik3ca mutations are likely to be sensitive to rapalog monotherapy . However, the clinical data to confirm these preclinical findings are largely lacking, although a clinical study has shown that patients with pik3ca mutations responded better than those without the mutation to treatment regimens with a pi3k / akt / mtor inhibitor . Moreover, preclinical data regarding the impact of pten mutation or loss on cancer cell response to mtor inhibition are not consistent and need further clarification or validation, particularly in the clinic . The finding of an association between kras mutations and cell resistance to rapalogs is intriguing . Another important observation is that the concomitant presence of kras and pik3ca mutations may confer resistance to rad001, though mutations in pik3ca alone predict cell sensitivity to rad001 . It is crucial to fully understand the biological bases or molecular mechanisms for these findings . In this way, we may eventually develop more efficacious, mechanism - driven therapeutic strategies to overcome resistance . Although mutations in pik3ca and/or pten seem to impact cancer cell sensitivity to mtor - targeted therapy, this may not be helpful for predicting the sensitivity of cancers in which these genes have low mutation rates, such as nsclc (<10% for combination of pik3ca and pten), . This also applies to the findings that lkb1 mutation or inactivation confers cell sensitivity to rapalogs,, since lkb1 is primarily mutated in nsclc (up to 30%) but is rarely mutated in other cancers . Nonetheless, we have made considerable progress towards identifying subsets of cancer patients who may benefit from mtor - targeted therapy, though some studies are preliminary so far . These efforts represent the first step toward personalized mtor - directed cancer medicine . Considering the limited single - agent activity of rapalogs in the majority of cancers, development of rapalog - based combination regimens one successful example is the combination of rad001 and exemestane for the treatment of advanced hormone receptor positive, her2-negative breast cancer in postmenopausal women . However, the impact of genetic alterations on tumor response to rapalog - based combination treatments is largely unknown . Demonstration of effective and mechanism - driven rapalog - based combination regimens in patients should be a key effort in this direction.
The extracellular ph in all tissues is generally controlled within a narrow range (7.40.05) to maintain normal physiological processes . However, the extracellular ph can fall as low as ph 5.5 in several pathological conditions, including inflammation, ischemia / hypoxia, injury, and malignant tumors, and under normal physiological conditions, including the generation of action potentials and synaptic transmission . This tissue acidosis has a broad impact on several tissues that have electrically excitable membranes, such as nerves, heart, and muscle . In the case of peripheral tissues, light has been shed on the role of tissue acidosis on nociceptive transmission, since acidic ph induces pain and exacerbates painful conditions by activating acid - sensitive ion channels (asics) to increase the excitability of sensory neurons . However, since all proteins are affected by ph, the functional properties of ion channels and receptors expressed in the nervous system can be altered by the ph level . For example, various voltage - gated ion channels, transient receptor potential vanilloid 1, and gamma - aminobutyric acid a (gabaa) receptors are modulated by an acidic ph, suggesting that local and systemic changes in the extracellular ph may affect basic functions, including neuronal excitability . The somata of sensory neurons innervating orofacial tissues are located in the trigeminal ganglia and trigeminal mesencephalic nucleus (vmes). While sensory neurons within the trigeminal ganglia process somatosensory information from orofacial tissues, including pain and innocuous tactile sensation, those within the vmes process proprioception and mechanosensation from the muscle spindle of the masseter muscle and periodontal ligaments . Unlike other sensory neurons, the somata of vmes neurons are located in the central nervous system (cns); thus, their excitability can be affected by peripheral as well as central environments . For example, local and/or systemic acidosis may affect the proprioception and mechanosensation mediated by vmes neurons by activating asics . However, given that peripheral information is conveyed to the cns through action potentials, the role of an acidic extracellular ph in the generation and conduction of action potentials should also be examined . In this regard, we have recently shown that an acidic ph had a minor effect on voltage - gated na channels in acutely isolated vmes neurons . However, the generation and conduction of action potentials, and synaptic transmission at presynaptic nerve terminals also require other voltage - gated ion channels, such as voltage - gated k and ca channels and hyperpolarization - gated and cyclic nucleotide - activated cation (hcn) channels . Therefore, in the present study we investigated whether acidic ph modulates the function of voltage - gated k and ca channels, and hcn channels in acutely isolated rat vmes neurons . All experiments complied with the guiding principles for the care and use of animals approved by the council of the physiological society of korea and the national institutes of health guide for the care and use of laboratory animals, and every effort was made to minimize both the number of animals used and their suffering . Sprague dawley rats (12~16 days old, either sex) were decapitated under ketamine - induced anesthesia (100 mg / kg, intraperitoneally [i.p . ]). The midbrain was dissected and transversely sliced at a thickness of 400 m using a microslicer (vt1000s; leica, nussloch, germany). Slices containing the vmes were kept in an incubation medium (124 mm nacl, 3 mm kcl, 1.5 mm kh2po4, 24 mm nahco3, 2 mm cacl2, 1.3 mm mgso4 and 10 mm glucose) saturated with 95% o2 and 5% co2 at room temperature (22~24) for at least 1 h before the mechanical dissociation . For dissociation, slices were transferred into a 35 mm culture dish (primaria 3801; becton dickinson, rutherford, nj, usa) containing a standard external solution (150 mm nacl, 3 mm kcl, 2 mm cacl2, 1 mm mgcl2, 10 mm glucose, and 10 mm hepes; ph of 7.4 with tris - base, 320~330 mosm), and the vmes region was identified under a binocular microscope (smz-1; nikon, tokyo, japan). Briefly, mechanical dissociation was accomplished using a custom - built vibration device and a fire - polished glass pipette oscillating at 50~60 hz (0.3~0.5 mm) on the surface of the vmes region . The slices were removed and the mechanically dissociated neurons were left for 15 min to allow the cells to adhere to the bottom of the culture dish . All electrophysiological measurements were performed using a conventional whole - cell patch clamp method in a voltage - clamp mode (axopatch 200b; molecular devices, union city, ca, usa). Patch pipettes were made from borosilicate capillary glass (g-1.5; narishige, tokyo, japan) using a pipette puller (p-97; sutter instrument co., novato, ca, usa). The resistance of the recording pipettes filled with an internal solution was 1.0~2.0 m. the liquid junction potential (9 to 11 mv; measured by exchanging the bath solution from the internal solution to the standard external solution) and pipette capacitance were compensated for . Neurons were viewed under phase contrast on an inverted microscope (te2000; nikon). Membrane currents were filtered at 2~5 khz, digitized at 10~20 khz, and stored on a computer equipped with pclamp 10.2 (molecular devices). All experiments were performed at room temperature (22~25). To record voltage - dependent k currents, the pipette solution containing 140 mm kmehso3, 10 mm kcl, 2 mm egta, 2 mm mg - atp, and 10 mm hepes (ph 7.2 with tris - base, 300~310 mosm) was used, and neurons were held at a holding potential (vh) of 120 mv . The standard external solution contained 10 m sr95531, 3 mm kynurenic acid, 300 nm tetrodotoxin (ttx), and 100 m cd to block ionotropic gabaa and glutamate receptors, and voltage - gated na and ca channels, respectively . To record hcn channel - mediated currents (also called ih), the pipette solution containing 140 mm csmehso3, 5 mm tea - cl, 5 mm cscl, 2 mm egta, 2 mm mg - atp, and 10 mm hepes (ph 7.2 with tris - base, 300~310 mosm) was used and neurons were held at a vh of 50 mv . The standard external solution contained 10 m sr95531, 3 mm kynurenic acid, 300 nm ttx, and 100 m cd to block ionotropic gabaa and glutamate receptors, and voltage - gated na and ca channels, respectively . To record voltage - dependent ca currents, the pipette solution containing 140 mm csmehso3, 5 mm cscl, 1 mm cacl2, 4 mm egta, 2 mm mg - atp, and 10 mm hepes (ph 7.2 with tris - base, 300~310 mosm) was used and neurons were held at a vh of 100 mv . The standard external solution contained 10 m sr95531, 3 mm kynurenic acid, and 300 nm ttx to block ionotropic gabaa and glutamate receptors, and voltage - gated na channels, respectively . Each extracellular solution was adjusted to a ph of 7.4, ph 6.5, or ph 6.0 with tris - base, and was applied using the y tube system for rapid solution exchange . The amplitudes of voltage - gated ion currents were measured by subtracting the baseline from the peak currents using the clampfit program (molecular devices). The effects of acidic ph were quantified as the percentage change in current amplitudes compared to the control values . The amplitude of the voltage - gated ion current was transformed into conductance (g) using the following equation: g = i/(v eion), where eion represents the equilibrium potential of the relevant ion channels (k channels: 98.5 mv of ek, ca channels: + 75.5 mv of eca, hcn channels: + 0.5 mv of ecation, respectively) calculated by the nernst equation . The averaged steady - state activation curves were fitted to the boltzmann equation: g / gmax=1/{1+exp[(v50,act v)/k]}, where gmax is the maximum conductance, v50,act is a half - maximum potential for activation, and k is the slope factor . Numerical values are provided as the meanstandard error of the mean (sem), except where indicated, using values normalized to the control . Significant differences induced in the current amplitude by acidic ph levels were tested using a two - tailed student's t - test, using absolute values rather than normalized values . Differences with a p<0.05 the effects of acidic ph on voltage - gated k channels were examined in acutely isolated vmes neurons using a whole - cell patch - clamp technique . Vmes neurons were easily distinguished from other central neurons based on their morphological properties, such as round or oval somata without any dendritic processes (fig . As shown in our previous study, the application of an acidic solution induced an inwardly directed membrane current, which is mediated by asics, in most vmes neurons . However, the acid - induced currents were desensitized within 10 s, and the input resistance was not changed by the prolonged application of a solution with ph 6.0 (150.331.5 m for the control and 134.928.4 m for 30 s after the application of a ph 6.0 solution, respectively, n=6, p=0.61; fig . Therefore, in all subsequent experiments the effects of an acidic solution on voltage - gated ion channels were examined at least 30 s after the application of the acid . The voltage - gated k currents were induced by depolarizing step pulses (120 mv to + 30 mv, 500 ms duration every 10 s), and the stable voltage - gated k currents were recorded (fig . While the ph 6.5 solution had no effect on voltage - gated k channels (99.52.1% of the control, n=8, p=0.85), the ph 6.0 solution slightly but significantly decreased the peak amplitude of voltage - gated k currents (93.63.5%, n=9, p<0.05; fig . Next, the effects of acidic ph on the voltage - activation relationship of voltage - gated k channels were examined . The voltage - gated k currents were induced by 500 ms depolarizing pulses from 120 mv to + 30 mv in 10 mv increments at ph 7.4 and acidic ph (fig . While the ph 6.5 solution did not affect the current - voltage (i~v) and conductance - voltage (g~v) relationships (fig . 2b), the ph 6.0 solution shifted both the i~v and g~v relationships slightly to the right (fig . 2c). In addition, the conductance at ph 6.5 or ph 6.0 was normalized to the maximal conductance at ph 7.4, and the data were individually fitted to the boltzmann function . The ph 6.5 solution had no effect on the midpoint voltage for activation (v50,act) (20.21.0 mv for the control and 20.31.5 mv for the ph 6.5 condition, n=8, p=0.49; fig . However, the ph 6.0 solution significantly shifted the v50,act towards the depolarization range (22.71.3 mv for the control and 16.81.0 mv for the ph 6.0 condition, n=10, p<0.01, fig . The slope factor k was not affected by acidic ph (15.11.1 mv and 15.31.3 mv for the control and ph 6.5 conditions, respectively, n=8, p=0.74; 15.21.0 mv and 15.40.8 mv for the control and ph 6.0 conditions, respectively, n=10, p=0.82; fig . The effects of acidic ph on hcn channels were examined in acutely isolated vmes neurons . Vmes neurons were held at a vh of 50 mv and the stable hyperpolarization - induced inward currents were recorded by voltage step pulses (50 mv to 130 mv, 1,000 ms duration every 10 s). The application of 1 mm cs, a general hcn channel inhibitor, greatly reduced the hyperpolarization - induced membrane currents (12.61.1% of the control, n=6, p<0.01; fig . 3a and b), suggesting that these hyperpolarization - induced currents are mediated by hcn channels . Under these conditions, while the ph 6.5 solution had no effect on hyperpolarization - induced currents (97.01.3% of the control, n=9, p=0.33), the ph 6.0 solution significantly decreased the peak amplitude of hyperpolarization - induced currents (90.12.1% of the control, n=9, p<0.05) (fig . Next, the effects of acidic ph on the voltage - activation relationship in hcn channels were examined . The hyperpolarization - induced currents were stimulated by 1000 ms hyperpolarizing pulses from 50 mv to 160 mv in 10 mv decrements at ph 7.4 and acidic ph (fig . 4b), or the v50,act of the hyperpolarization - induced currents (111.35.6 mv for the control and 111.44.8 mv for the ph 6.5 condition, n=9, p=0.68; fig . The slope factor k was not affected by the ph 6.5 solution (10.20.3 mv for the control and 10.50.4 mv for the ph 6.5 condition, n=9, p=0.93; fig ., the ph 6.0 solution induced a unique pattern in the i~v and g~v relationships in hcn channels (fig . When the hyperpolarization - induced currents were stimulated by strong hyperpolarization (130 mv~160 mv), the ph 6.0 solution decreased the amplitude of these currents . However, the ph 6.0 solution increased the amplitude of the hyperpolarization - induced currents stimulated by weak hyperpolarization (80 mv~100 mv). The v50,act of the hyperpolarization - induced currents was not affected by the ph 6.0 solution (113.75.2 mv for the control and 109.55.9 mv for the ph 6.0 condition, n=10, p=0.47; fig . However, the slope factor k was significantly increased (10.30.3 mv for the control and 11.70.5 mv for the ph 6.0 condition, n=10, p<0.05; fig . The effects of acidic ph on voltage - gated ca channels were examined using acutely isolated vmes neurons . Vmes neurons were held at a vh of 100 mv and the stable voltage - gated ca currents were recorded by voltage step pulses (100 mv to 0 mv, 50 ms duration every 20 s). Application of 100 m of cd, a general voltage - gated ca channel inhibitor, completely blocked these currents (1.50.1% of the control, n=8, p<0.01; fig . 5a and b). Unlike the voltage - gated k channels or hcn channels, the acidic extracellular solution profoundly decreased the peak amplitude of voltage - gated ca currents (for ph 6.5: 72.32.3% of the control, n=9, p<0.01; for ph 6.0: 48.22.8% of the control, n=9, p<0.01; fig . Next, the effects of acidic ph on the voltage - activation relationship of voltage - gated ca channels were examined . The voltage - gated ca currents were induced by 50 ms depolarizing pulses from 100 mv to + 20 mv in 15 mv increments at ph 7.4 and acidic ph (fig . In particular, the ph 6.5 solution shifted the v50,act towards the depolarization range (24.41.8 mv for the control and 17.82.1 mv for the ph 6.5 condition, n=8, p<0.01; fig . 6b and da). The slope factor k was increased by acidic ph 6.5 (6.10.4 mv for the control and 7.20.4 mv for the ph 6.5 condition, n=8, p<0.05; fig . The v50,act was shifted towards the depolarization range (25.62.2 mv for the control and 22.71.9 mv for the ph 6.0 condition, n=10, p<0.05; fig . 6c and da). The slope factor k was increased (6.10.4 mv for the control and 7.00.4 mv for the ph 6.0 condition, n=8, p<0.05; fig . The effects of acidic ph on voltage - gated k channels were examined in acutely isolated vmes neurons using a whole - cell patch - clamp technique . Vmes neurons were easily distinguished from other central neurons based on their morphological properties, such as round or oval somata without any dendritic processes (fig . As shown in our previous study, the application of an acidic solution induced an inwardly directed membrane current, which is mediated by asics, in most vmes neurons . However, the acid - induced currents were desensitized within 10 s, and the input resistance was not changed by the prolonged application of a solution with ph 6.0 (150.331.5 m for the control and 134.928.4 m for 30 s after the application of a ph 6.0 solution, respectively, n=6, p=0.61; fig . Therefore, in all subsequent experiments the effects of an acidic solution on voltage - gated ion channels were examined at least 30 s after the application of the acid . The voltage - gated k currents were induced by depolarizing step pulses (120 mv to + 30 mv, 500 ms duration every 10 s), and the stable voltage - gated k currents were recorded (fig . While the ph 6.5 solution had no effect on voltage - gated k channels (99.52.1% of the control, n=8, p=0.85), the ph 6.0 solution slightly but significantly decreased the peak amplitude of voltage - gated k currents (93.63.5%, n=9, p<0.05; fig . Next, the effects of acidic ph on the voltage - activation relationship of voltage - gated k channels were examined . The voltage - gated k currents were induced by 500 ms depolarizing pulses from 120 mv to + 30 mv in 10 mv increments at ph 7.4 and acidic ph (fig . While the ph 6.5 solution did not affect the current - voltage (i~v) and conductance - voltage (g~v) relationships (fig . 2b), the ph 6.0 solution shifted both the i~v and g~v relationships slightly to the right (fig . 2c). In addition, the conductance at ph 6.5 or ph 6.0 was normalized to the maximal conductance at ph 7.4, and the data were individually fitted to the boltzmann function . The ph 6.5 solution had no effect on the midpoint voltage for activation (v50,act) (20.21.0 mv for the control and 20.31.5 mv for the ph 6.5 condition, n=8, p=0.49; fig . However, the ph 6.0 solution significantly shifted the v50,act towards the depolarization range (22.71.3 mv for the control and 16.81.0 mv for the ph 6.0 condition, n=10, p<0.01, fig . The slope factor k was not affected by acidic ph (15.11.1 mv and 15.31.3 mv for the control and ph 6.5 conditions, respectively, n=8, p=0.74; 15.21.0 mv and 15.40.8 mv for the control and ph 6.0 conditions, respectively, n=10, p=0.82; fig . The effects of acidic ph on hcn channels were examined in acutely isolated vmes neurons . Vmes neurons were held at a vh of 50 mv and the stable hyperpolarization - induced inward currents were recorded by voltage step pulses (50 mv to 130 mv, 1,000 ms duration every 10 s). The application of 1 mm cs, a general hcn channel inhibitor, greatly reduced the hyperpolarization - induced membrane currents (12.61.1% of the control, n=6, p<0.01; fig . 3a and b), suggesting that these hyperpolarization - induced currents are mediated by hcn channels . Under these conditions, while the ph 6.5 solution had no effect on hyperpolarization - induced currents (97.01.3% of the control, n=9, p=0.33), the ph 6.0 solution significantly decreased the peak amplitude of hyperpolarization - induced currents (90.12.1% of the control, n=9, next, the effects of acidic ph on the voltage - activation relationship in hcn channels were examined . The hyperpolarization - induced currents were stimulated by 1000 ms hyperpolarizing pulses from 50 mv to 160 mv in 10 mv decrements at ph 7.4 and acidic ph (fig . 4b), or the v50,act of the hyperpolarization - induced currents (111.35.6 mv for the control and 111.44.8 mv for the ph 6.5 condition, n=9, p=0.68; fig . The slope factor k was not affected by the ph 6.5 solution (10.20.3 mv for the control and 10.50.4 mv for the ph 6.5 condition, n=9, p=0.93; fig ., the ph 6.0 solution induced a unique pattern in the i~v and g~v relationships in hcn channels (fig . When the hyperpolarization - induced currents were stimulated by strong hyperpolarization (130 mv~160 mv), the ph 6.0 solution decreased the amplitude of these currents . However, the ph 6.0 solution increased the amplitude of the hyperpolarization - induced currents stimulated by weak hyperpolarization (80 mv~100 mv). The v50,act of the hyperpolarization - induced currents was not affected by the ph 6.0 solution (113.75.2 mv for the control and 109.55.9 mv for the ph 6.0 condition, n=10, p=0.47; fig . However, the slope factor k was significantly increased (10.30.3 mv for the control and 11.70.5 mv for the ph 6.0 condition, n=10, p<0.05; fig . The effects of acidic ph on voltage - gated ca channels were examined using acutely isolated vmes neurons . Vmes neurons were held at a vh of 100 mv and the stable voltage - gated ca currents were recorded by voltage step pulses (100 mv to 0 mv, 50 ms duration every 20 s). Application of 100 m of cd, a general voltage - gated ca channel inhibitor, completely blocked these currents (1.50.1% of the control, n=8, p<0.01; fig . 5a and b). Unlike the voltage - gated k channels or hcn channels, the acidic extracellular solution profoundly decreased the peak amplitude of voltage - gated ca currents (for ph 6.5: 72.32.3% of the control, n=9, p<0.01; for ph 6.0: 48.22.8% of the control, n=9, p<0.01; fig . Next, the effects of acidic ph on the voltage - activation relationship of voltage - gated ca channels were examined . The voltage - gated ca currents were induced by 50 ms depolarizing pulses from 100 mv to + 20 mv in 15 mv increments at ph 7.4 and acidic ph (fig . In particular, the ph 6.5 solution shifted the v50,act towards the depolarization range (24.41.8 mv for the control and 17.82.1 mv for the ph 6.5 condition, n=8, p<0.01; fig . 6b and da). The slope factor k was increased by acidic ph 6.5 (6.10.4 mv for the control and 7.20.4 mv for the ph 6.5 condition, n=8, p<0.05; fig . The v50,act was shifted towards the depolarization range (25.62.2 mv for the control and 22.71.9 mv for the ph 6.0 condition, n=10, p<0.05; fig . 6c and da). The slope factor k was increased (6.10.4 mv for the control and 7.00.4 mv for the ph 6.0 condition, n=8, p<0.05; fig . Potassium channels play a pivotal role in neuronal excitability by shaping action potentials and regulating resting membrane potentials . Potassium channels are largely divided into three families based on their transmembrane domains (tm), i.e., 2tm, 4tm, and 6tm k channels . The 2tm k channel family includes inward rectifier k channels (kir2.x), g - protein - activated inward rectifier k channels (kir3.x), and atp - sensitive k channels (kir6.x). The 4tm channel family includes a number of two - pore domain k channels (k2p), such as trek and tresk . This family is responsible for the leak k currents and act as background k channels determining resting membrane potentials in electrically excitable cells . Voltage - gated k channels belong to the 6tm family, and various subtypes, such as kv1.x (shaker - related), kv2.x (shab - related), kv3.x (shalr - related), kv4.x (shaw - related), kv7.x (kcnq), kv10 . X (erg), kvv11.x (herg), play roles in the excitability of several tissues . With regard to function, kv3.3, kv3.4, kv1.4, and kv4.x subtypes mediate the a - type k currents (ia), which show faster inactivation kinetics . In contrast, kv7.x mediates the slowly inactivating k - type k currents (ik), and kv11.x mediates the hyperpolarization of cardiac action potentials . While a number of factors regulate the physiological functions of voltage - gated k channels, the modulation of voltage - gated k currents by the extracellular ph is largely undetermined . A previous study performed in hippocampal ca1 pyramidal neurons has shown that an acidic extracellular ph (ph 6.4) does not change the peak amplitude or the voltage - activation relationship of ia and ik but shifts the inactivation relationship of voltage - gated k channels to the depolarization range . In the present study, we recorded the voltage - gated k currents from vmes neurons, which are mediated by a variety of kv subtypes . Although we could not determine which kv subtypes are responsible for the recorded k currents owing to the absence of subtype - selective antagonists, both the peak and steady currents might be responsible for the components of ia and ik evoked by depolarization, respectively . We found that a weakly acidic ph (ph 6.5) had no effect on the peak amplitude or the voltage - activation relationship of k currents, which is consistent with a previous study . However, we found that the ph 6.0 solution slightly but significantly inhibited the peak amplitude of k currents, and shifted the voltage - activation relationship of k currents to the depolarization range . These results suggest that the voltage - gated k channels expressed in vmes neurons are difficult to activate at an acidic ph below ph 6.0 . Hcn channels are non - selective cation channels that are activated by membrane hyperpolarization . To date, four subtypes of hcn channels, i.e., hcn1, hcn2, hcn3, and hcn4, have been identified . By providing additional depolarization at the end of action potentials, hcn channels play a crucial role in the generation of repetitive and rhythmic action potentials in electrically excitable cells . Hcn channels are widely expressed in the central and peripheral nervous system, including in nociceptive neurons, and therefore dysfunction of these channels is closely related to hyperexcitable neurological disorders, such as epilepsy and pain . Although functional hcn channels are found in vmes neurons, it is still unknown which hcn channel subtypes are expressed in these cells . To our knowledge, no previous study has been conducted to determine whether the extracellular ph regulates the function of hcn channels . In the present study, we found that a weakly acidic ph (ph 6.5) had no effect on the peak amplitude or the voltage - activation relationship of hcn channels . In the present study, the hcn currents induced by weak hyperpolarization (80 ~100 mv) were increased at ph 6.0; however, those induced by strong hyperpolarization (140~160 mv) were increased at ph 6.0 . Given that the resting membrane potential of vmes neurons is about 65 mv, and that the membrane potential is unlikely to fall below 100 mv during the repolarization period, the hcn currents elicited by physiological hyperpolarizing stimuli might be increased by acidic ph (ph 6.0). This would suggest that an acidic ph contributes to the increased excitability of vmes neurons . Similarly, the cyclic amp - mediated potentiation of hcn channels at near resting membrane potentials results in the activation of a larger hcn current following an action potential in a variety of excitable tissues . Voltage - gated ca channels are divided into six subtypes, i.e., l, n, p, q, r, and t - types, based on the pore - forming -subunits . Of these, t - type ca channels have a low activation threshold; however, other ca channel subtypes need higher voltage stimuli to be activated . The voltage - gated ca currents recorded from vmes neurons might be mediated by the various ca channel subtypes described above . While voltage - gated ca channels are not directly related to the generation and conduction of action potentials, they play pivotal roles in synaptic transmission at presynaptic terminals, and in ca - dependent cellular signal transduction pathways in somata . Previous studies have shown that voltage - gated ca channels are highly sensitive to the extracellular ph . The proton inhibition of voltage - gated ca channels seems to be mediated by a direct interaction with the channel pore, as well as changes in the voltage - dependence for activation of ca channels . In the present study, we found that an acidic ph inhibited the membrane currents mediated by voltage - gated ca channels more strongly than those of the other voltage - gated ion channels tested . In addition, an acidic ph shifted the v50,act value of voltage - gated ca channels to the depolarization range, indicating that these channels need further depolarization to become activated under acidic extracellular ph conditions . The increase in the slope factor induced by an acidic ph suggests that the steepness of the voltage - dependence is decreased under acidic ph conditions . The acidic ph - mediated inhibition of voltage - gated ca channels may suppress action potential - dependent neurotransmitter release from presynaptic terminals in vmes neurons . In the present study we have shown the effects of acidic ph on several voltage - gated ion channels in acutely isolated vmes neurons . The mechanisms underlying the acidic modulation of voltage - gated ion channels are still unknown; however, an acidic ph may affect the various ion channels tested in this study either directly or indirectly . For example, an acidic ph indirectly affects these ion channels by activating asics, since vmes neurons express multiple asic subtypes . However, the acidic ph - induced modulation of voltage - gated ion channels might not be due to the activation of asics, as the membrane currents mediated by asics disappeared during the prolonged application of an acidic solution . The direct acidic modulation of voltage - gated ion channels, such as the neutralization of negative charges near ion channels and/or the membrane surface, and protonation of extracellular parts of ion channels, may result in shifts in both voltage dependence and channel conductance . Similarly, we found that an acidic ph shifted the voltage dependence of voltage - gated k and ca channels . Alternatively, the increased number of protons at acidic ph levels might compete with cations for the pore region of voltage - gated ion channels, like voltage - gated na+ channels . Further studies are required to elucidate the detailed mechanisms underlying the acidic modulation of voltage - gated ion channels . In general, the generation and conduction of action potentials in single neurons in addition to voltage - gated na channels, which are responsible for the upstroke of action potentials, other voltage - gated ion channels tested in the present study play important roles in the shape and frequency of action potentials in response to generator potentials . In particular, voltage - gated k channels and hcn channels play important roles in the generation of repetitive action potentials . Voltage - gated ca channels are not related to the generation and conduction of action potentials; however, these channels are involved in neurotransmitter release at the central terminals of vmes neurons . Therefore, acidic modulation of voltage - gated ion channels related to action potentials and synaptic transmission would have a broad impact on neuronal activity . In this regard, since the somata of vmes neurons are located in the central region, the excitability of vmes neurons can be regulated by changes in extracellular ph in peripheral tissues, as well as in the cns . Further study is needed to evaluate whether the acidic modulation of voltage - gated ion channels shown here affects the unique functions mediated by vmes neurons, including the regulation of mastication in pathological conditions.
Metabolic syndrome (mets) comprises a cluster of abnormalities, with insulin resistance and adiposity as central features [13]. Five diagnostic criteria for mets have been identified by the national cholesterol education program adult treatment panel iii (ncep - atp iii), and the presence of any of these three features central obesity, dyslipidemia (high triglycerides, low high - density lipoprotein [hdl] cholesterol), hypertension, and impaired fasting glucose is considered sufficient to diagnose the syndrome . About 24% of us adults have mets, and the prevalence increases with age (44% at age 60 years). Individuals with mets have an increased burden of cardiovascular disease (cvd) [68]. In the kuopio ischemic heart disease study, lakka et al . Convincingly showed that men with mets, even in the absence of baseline coronary artery disease (cad) or diabetes, had a significantly increased mortality from cad . In the botnia study, mets was defined as the presence of at least two of the following risk factors: obesity, hypertension, dyslipidemia, or microalbuminuria . Cardiovascular mortality was assessed in 3,606 individuals, with a median follow - up of 6.9 years . In women and men, respectively, mets was seen in 10% and 15% of participants with normal glucose tolerance, 42% and 64% of those with impaired fasting glucose / impaired glucose tolerance, and 78% and 84% of those with type 2 diabetes mellitus . In individuals with mets, the risk for coronary heart disease (chd) and stroke was increased threefold (p <0.001) and the risk for cardiovascular mortality was increased sixfold (12.0% vs 2.2%; p <0.001) [68]. Using data from the third national health and nutrition examination survey (nhanes iii), alexander et al . Reported that mets is very common, with 44% of the us population over 50 years of age meeting the ncep - atp iii criteria . Individuals with mets without diabetes had higher chd prevalence (13.9%), and those with both mets and diabetes had the highest prevalence of chd (19.2%) compared with those with neither . The hoorn study examined 615 men and 749 women aged 50 to 75 years without diabetes or a history of cvd at baseline and reported that the ncep - atp iii definition of mets was associated with about a twofold increase in age - adjusted risk of fatal cvd in men and nonfatal cvd in women [68]. The lower but significant risks were also obtained using the world health organization, american college of endocrinology, and european group on insulin resistance definitions of mets . Ford [5,] using the modified ncep - atp iii criteria on the nhanes cohort, also reported significantly increased prevalence of mets in the us population . Besides the effect on cardiovascular morbidity and mortality, the components of mets have been associated with diabetes . Hanson et al . Used factor analysis to identify the components of mets on 1918 native americans of the pima tribe . Insulin resistance factor was strongly associated with diabetes in a 4-year follow - up . Also, the body size and the lipid factor predicted diabetes whereas the blood pressure factor did not . In the west of scotland coronary prevention study (woscops), mets increased the risk for chd events and diabetes . Participants with four or five features of the syndrome had a 3.7-fold increase in risk for chd and a 24.5-fold increase in risk for diabetes compared with those with no features of the syndrome . The prospective cardiovascular mnster (procam) study also reported a 2.3-fold increased incidence of cvd in participants with mets, and these effects persisted after adjustment for conventional risk factors . Although individual components of mets independently contribute to increased cardiovascular risk, in concert they do not explain the increased propensity of vascular disease in individuals with mets, and the precise mechanisms for this increased propensity remain to be elucidated . Inflammation is pivotal in all phases of atherosclerosis, from foam cell formation to culmination in acute coronary syndromes . It appears that low - grade chronic inflammation is a central feature of mets and could contribute to increased risks of both cvd and diabetes in mets . Evidence supporting the hypothesis that elevated c - reactive protein (crp) levels contribute to increased cardiovascular risk is now available from at least six major prospective studies . These are the physician s health study (phs), women s healthy study (whs), atherosclerosis risk in communities (aric) study, and air force / texas coronary atherosclerosis prevention study (afcaps / texcaps) in the united states and the monitoring trends and determinants on cardiovascular diseases / cooperative health research in the region of augsburg (monica / kora augsburg) and the age gene / environment susceptibility (ages)-reykjavik studies in europe . The largest of the american cohorts is the whs, a prospective evaluation of 27,939 initially healthy american women who underwent high - sensitivity crp (hscrp) evaluation along with a full lipid panel and framingham risk assessment and were monitored over a period of 8.3 years for the occurrence of first - ever cardiovascular events . Overall, baseline hscrp levels in the whs were a strong predictor of future vascular events; the relative risks for those with lowest to highest quintiles of hscrp at baseline were 1.0, 1.8, 2.3, 3.2, and 4.5 (p <0.001). After adjustment for age, smoking, diabetes, blood pressure, and hormone replacement therapy, the risk in the top quintile of hscrp was 2.3 (95% ci, 1.63.4). A combined approach, using both a lipid panel and hscrp, provided improved prediction of cardiovascular event free survival . Most importantly, hscrp levels remained a highly significant predictor of risk in the whs after adjustment for the framingham risk score . Data on hscrp from the phs and whs have been corroborated by similar analyses from other large cohorts from the united states and europe . In a case - cohort analysis of 12,819 apparently healthy middle - aged men and women participating in the aric study over a 6-year follow - up period, the relative risks of incident chd for those with baseline hscrp levels less than 1.0, 1.0 to 3.0, and greater than 3.0 mg / l were 1.0, 1.6, and 2.5, respectively, after adjusting for age, gender, and ethnicity . Almost identical data were derived from a prospective evaluation of 3435 german men participating in the monica / kora augsburg cohort study, in which 191 incident coronary events occurred during 6.6 years of follow - up . In this study of men, as in the whs study of women, hscrp levels at baseline were independently associated with incident coronary events . These effects remained significant (p <0.001) after adjustment for framingham risk score . Although this prospective study used an hscrp cut - point for relative risk of 2.0 rather than 3.0, and thus would tend to underestimate effects compared with other cohorts, a highly significant fully adjusted odds ratio of 1.5 was observed . Within the afcaps / texcaps analysis of 5,742 apparently healthy individuals enrolled in a randomized primary prevention trial of lovastatin versus placebo, each quartile increase in baseline hscrp was associated with a 21% increase in the risk of a first cardiovascular event (95% ci, 441%), an effect that again persisted after control for all individual components of the framingham risk score . Similarly, in an analysis of 1,666 individuals free of cvd enrolled in the pravastatin inflammation / crp evaluation (prince) study, hscrp levels correlated modestly with 10-year framingham risk scores yet showed minimal relation to any individual component of the score itself . Furthermore, it has been proposed that hscrp be added as a clinical criterion for mets and for creation of an hscrp - modified chd risk score . With regard to mets, yudkin et al ., conducted z - score analyses in a study 107 nondiabetic individuals and found a very significant correlation between inflammatory markers and several features of the mets . Crp levels were shown to be strongly associated with insulin resistance calculated from the homeostatic model assessment, blood pressure, low hdl, and triglycerides, and also to levels of the proinflammatory cytokines interleukin-6 (il-6) and tumor necrosis factor (tnf). There is a linear relationship between the number of metabolic features and increasing levels of hscrp . Furthermore, festa et al ., in the insulin resistance and atherosclerosis study (iras), showed that hscrp was positively correlated with body mass index, waist circumference, blood pressure, triglycerides, cholesterol, low - density lipoprotein (ldl) cholesterol, plasma glucose, and fasting insulin, and that it was inversely correlated with hdl cholesterol and the insulin sensitivity index . The strongest associations were observed between crp levels, central adiposity, and insulin resistance . The largest study to date that examined the association between inflammation and mets was nhanes iii . In a representative sample of the us population (8570 participants> 20 years of age), individuals with mets, defined using ncep - atp iii criteria, were more likely than those without the syndrome to have elevated levels of markers of inflammation such as crp, fibrinogen, and leukocyte count . [12, 14] evaluated inter - relationships between crp, mets, and incident cardiovascular events among 14,719 apparently healthy women who were followed for an 8-year period for myocardial infarction, stroke, coronary revascularization, or cardiovascular death; 24% of the cohort had mets at study entry . At baseline, median crp levels for those with zero, one, two, three, four, or five characteristics of the metabolic syndrome were 0.68, 1.09, 1.93, 3.01, 3.88, and 5.75 mg / l, respectively (p for trend <0.0001). Over the 8-year follow - up, cardiovascular event - free survival rates based on crp levels above or below 3.0 mg / l were similar to survival rates based on having three or more characteristics of mets . At all levels of severity of mets, crp added prognostic information on subsequent risk . Additive effects for crp were also observed for those with four or five characteristics of mets . Thus, in this study, those who had hscrp levels of less than 3 mg / l without mets had the best cardiovascular survival, whereas those who had hscrp levels greater than 3 mg / l with mets had the worst cardiovascular survival . An almost identical additive interaction between hscrp, mets, and subsequent vascular risk was observed in woscops, a randomized intervention trial of pravastatin that monitored 6447 middle - aged men over a 5-year period . In woscops, mg / l at baseline was highly predictive of incident vascular events after stratification by the presence or absence of mets . Specifically, the observed relative risks of future coronary events in the low - crp / mets - absent, high - crp / mets - absent, low - crp / mets - present, and high - crp / mets - present subgroups within woscops were 1.0 (referent), 1.6, 1.6, and 2.8, respectively (p <0.05). Studied 4,017 men and women 65 years of age without baseline congestive heart failure (chf) or diabetes participating in the cardiovascular health study, an observational study with 12.2 years follow - up and 966 cases of incident chf . Baseline crp / mets or il-6/mets were defined as presence of three of six components, with elevated crp (3 mg / l) or il-6 (2.21 pg / ml) as a sixth component added to ncep - atp iii criteria . Mets and elevated inflammation markers were independently associated with chf risk (hazard ratio = 1.32 [95% ci, 1.161.51] for mets; 1.53 [95% ci, 1.341.75] for crp; and 1.37 [95% ci, 1.191.55] for il-6). There was a 20% relative excess risk attributed to the combination of mets and crp (95% ci, 44% to 88%). Crp / mets and il-6/mets definitions reclassified 18% and 13%, respectively, of participants as having mets . Both crp / mets and il-6/mets increased risk of chf by 60% compared with those without mets . In this study, mets and inflammation markers provided additive information on chf risk in this elderly cohort . In a cross - sectional study of 3,873 individuals (weighted to 156 million) aged 18 years participating in the nhanes 19992000, participants were classified as having diabetes, mets according to modified ncep - atp iii criteria, or neither condition by low (<1 mg / l), intermediate (13 mg / l), or high (> 3 reported that after adjusting for age, gender, smoking, and total cholesterol, compared with those with neither mets nor diabetes and low crp levels, the odd ratios of cvd were 1.99 (95% ci, 1.103.59) for those with no disease and high crp levels and 2.67 (95% ci, 1.305.48) for those with mets and intermediate crp . Persons with mets but high crp had an or of 3.33 (95% ci, 1.806.16), similar to those with diabetes and low crp (3.21; 95% ci, 1.278.09). The likelihood of cvd was highest in those with diabetes who had intermediate crp levels (6.01; 95% ci, 2.5414.20) and in those with diabetes and high crp (7.73; 95% ci, 3.9914.95). . Showed in the nurses health study and health professionals follow - up study that whereas mets was a strong predictor of chd in both men and women, crp was additive in men only . It should be emphasized that in this study, a modified definition of mets was used because waist circumference, blood pressure, and glucose were not available at baseline . In a smaller japanese study by takeno et al . Of 461 patients with acute myocardial infarction, crp levels were additive to mets in predicting future major adverse cardiac events . Furthermore, recent investigation relating increased crp levels and mets in 1,044 older (65 years) individuals has also led to the conclusion that mets is associated with low - grade systemic inflammation and that the association is mainly supported by a strong independent correlation between waist circumference and high hscrp levels . Collectively, all these studies support the hypothesis that an increased crp level in the setting of mets confers an increased risk of future cardiovascular events . Additionally, a genome - wide association study has been performed among 6,345 apparently healthy women in whom 336,108 single nucleotide proteins were evaluated as potential determinants of plasma crp concentration . Overall, seven loci that associate with plasma crp at levels achieving genome - wide statistical significance were found . Two of these loci (gckr and hnf1a) are suspected or known to be associated with maturity - onset diabetes of the young, one is a gene - desert region on 12q23.2, and the remaining four loci are in or near the leptin receptor protein gene, the apolipoprotein e gene, the il-6 receptor protein gene, or the crp gene itself . The protein products of six of these seven loci are directly involved in mets, insulin resistance, -cell function, weight homeostasis, and/or premature atherothrombosis . Thus, it was concluded that common variations in several genes involved in metabolic and inflammatory regulation have significant effects on crp levels, consistent with the identification of crp as a useful biomarker of risk for incident vascular disease and diabetes . These findings have sparked increased discussion about the formal addition of hscrp to the criteria of mets . In addition to the prognostic information that hscrp evaluation might add to the current definition of mets, there are several other practical benefits of hscrp measurement . First, hscrp is strongly associated with components of mets that are difficult to measure in routine clinical practice, such as impaired fibrinolysis and insulin resistance [19, 20]. Also, the widespread availability of commercial assays for hscrp has made its measurement simple and inexpensive . In addition, as hscrp does not display diurnal variation and demonstrates long - term stability comparable with cholesterol, it can be reliably evaluated with a single nonfasting measurement [29, 30]. The addition of hscrp measurement to our present diagnosis of the mets may significantly improve the early detection of risk for future diabetes and cardiovascular events in individuals . Overall, it appears that in individuals with mets, an elevated crp confers a greater risk for cardiovascular events by activation of monocytes and induction of endothelial cell dysfunction . Endothelial dysfunction (ed) is now recognized to play a critical role in the initiation and progression of atherosclerotic vascular disease [3335]. Furthermore, endothelial function assessment by brachial flow - mediated dilatation is a surrogate marker of cardiovascular risk [36, 37] and has been shown to be decreased in mets [3843]. Previous studies have shown that obesity, low hdl cholesterol, impaired glucose tolerance, hypertriglyceridemia, and hypertension are associated with decreased endothelium - dependent vasodilatation [44, 45]. An impressive amount of data now implicates crp in inducing endothelial cell activation and dysfunction in vitro as well as in vivo [4852]. Several observations demonstrated that crp levels correlated inversely with endothelial vasoreactivity in vivo [53, 54].the most compelling data implicating crp as a determinant of ed were studies demonstrating that human crp reduced basal and stimulated nitric oxide release from arterial and venous endothelial cells [55, 56]. Our group has explored various mechanistic events involved in crp - mediated endothelial nitric oxide synthase (enos) inhibition and documented that increased nadph oxidase activation and gtpch1 downregulation are associated with crp - mediated enos uncoupling in human airway epithelial cells in vitro [57]. In vivo studies have shown that crp impairs endothelial vasoreactivity and decreases enos activity [48, 49, 5154]. Guan et al . [58] showed that a single intravenous injection of adeno - associated virus (aav) vector with hscrp to male rats resulted in efficient and sustained expression of crp in the liver and other tissues and an increase in serum crp to 15 g / ml at 2 and 4 months . The authors went on to show impaired endothelium - dependent vasoreactivity in the aav - hscrp rats versus the control rats administered aav - green fluorescent protein . Previously, we have shown that crp inhibits prostacyclin synthase, resulting in decreased prostacyclin, which is a potent vasodilator . Thus, crp, by inducing ed, could put individuals with mets at further risk for hypertension and cvd . Individuals with mets are in a procoagulant state, as evidenced by increased circulating plasminogen activator inhibitor-1 (pai-1). We have shown that crp induces pai-1 and decreases tissue plasminogen activator in endothelial cells [59, 60]. Crp appears to induce pai-1 antigen and activity via upregulation of nuclear factor-b (nf-b) and rho kinase activities . Individuals with mets have increased circulating levels of cell adhesion molecules (cams). In 943 70-old participants (50% women) of the prospective investigation of the vasculature in uppsala seniors (pivus) study, ingelsson et al . Reported that vascular cell adhesion molecule-1 (vcam-1), e - selectin, and crp demonstrated the strongest associations with mets and insulin resistance . Also, in the women s health study, women with mets had significantly higher crp and soluble intercellular adhesion molecule-1 (icam-1) levels that correlated significantly with increased vascular risk . Furthermore, kressel et al . Showed that individuals with mets had significantly higher values of crp, soluble icam, and soluble vcam compared with those without mets . Rosso et al . Demonstrated increased vcam in individuals with mets, and this correlated significantly with the quantitative insulin - sensitivity check index (quicki). We have previously shown in shear stress and static conditions that crp upregulates icam, vcam, nf-b, and monocyte - endothelial cell adhesion in a dose - dependent manner . The proinflammatory effects of crp that have been documented in monocyte - macrophages include induction of tissue factor, proinflammatory cytokines, reactive oxygen species, chemokine receptor ccr2, release of matrix metalloproteinase (mmp), cd11b expression, and oxidized ldl uptake as well as inhibition of cholesterol efflux and lipopolysaccharide (lps)-induced il-10 release [6773, 74]. Another important in vivo demonstration made recently is from our group on the induction of myeloperoxidase activity in macrophages by crp administration . Several lines of experimental evidence support the role of monocyte chemotactic protein-1 (mcp-1) in atherogenesis, insulin resistance, and adipose tissue - mediated inflammation . Also, crp has been reported to induce mcp-1 in endothelial cells and its receptor ccr2 on monocytes . Furthermore, esposito et al . Have reported that in both obese and non - obese women, il-10 levels were significantly lower in women with mets . In this regard, we have reported that crp inhibits lps - induced il-10 release from human monocyte - derived macrophages . Recently, bisoendial et al . Examined whole - blood expression for 95 inflammatory markers before and after infusion of 1.25 mg / kg of recombinant human crp in five male volunteers using quantitative real - time polymerase chain reaction analysis . Relevant transcript levels were measured at baseline and 4 and 8 h after recombinant human crp infusion . Crp caused significant upregulation of mmp-9, mcp-1, plasminogen activator urokinase, macrophage inflammatory protein 1, and nf-b mrnas in peripheral leukocytes . The corresponding increase in plasma protein levels of mmp-9 (78 32 ng / ml to 109 41 ng / ml; p = 0.014) and mcp-1 (312 92 pg / ml to 2,590 898 pg / ml; p = 0.007) closely mirrored mrna findings . Thus, mets individuals, who have persistently high crp levels, may demonstrate increased leukocyte activation compared with those with low crp levels, and this may contribute to increased cardiovascular risk in these individuals . Oxidative stress, mainly superoxide, plays a critical role in the pathogenesis of mets parameters . They also demonstrate increased superoxide, nitrotyrosine, and oxidized ldl in mets patients compared with controls, although mets patients studied in this report were on various medications, including statins (39%) and oral hyperglycemics (21%). Thus, there is an emergent need to study monocyte biology in drug - nave mets patients with low and high crp levels, a subject that has not yet been explored . Furthermore, crp has been shown by numerous investigators, including our group, to result in increased superoxide production as a result of enhanced nadph oxidase activity in endothelial cells as well as in human peripheral blood monocytes [55, 57, 68, 73, 81, 82]. Also, in situ hybridization revealed the presence of crp mrna that co - localized with p22phox, an essential component of nadph oxidase . We also demonstrated in wistar rats that crp stimulates superoxide production in macrophages via upregulation of nadph oxidase . Increased oxidized ldl in the vessel wall and circulation has been shown in patients with acute coronary syndrome and is associated with ed and predicted cardiovascular events . Furthermore, patients with mets exhibit increased plasma oxidized ldl levels . In addition, we recently showed that crp promotes oxidized ldl uptake and cholesterol ester accumulation in wistar rats [74]. Tissue factor is increased in morbidly obese persons with abnormal glucose tolerance compared with those with normal glucose tolerance . Diamant et al . Also demonstrated increased tissue factor containing microparticles in type 2 diabetes mellitus that correlated significantly with features of mets in this population . In vivo, we have shown that crp promotes tissue factor activity in vitro and in vivo in the rat model and shown that it occurs via activation of reactive oxygen species and nf-b . Mmps and their tissue inhibitors of metalloproteinase (timp) may play a role in cardiovascular complications associated with mets . Studies have shown increased levels of pro - mmp-9, mmp-8, and timp-1 in mets patients compared with controls . Furthermore, we have confirmed in vitro and in vivo that crp augments mmp-9 release and activity when injected into wistar rats . Studies in animal models have demonstrated the importance of ikk in the pathogenesis of insulin resistance in obese and diabetic rodents . Recently, it has also been shown in humans that obesity is associated with an increase in nf-b binding in the nucleus and a decrease in the inhibitory b in the mononuclear cells, with increased mrna for tnf, il-6, macrophage inhibitory factor, and mmp-9, consistent with the proinflammatory state . Recently, we have shown that crp induces nf-b activity in rat macrophages in vivo [68, 74]. Also, we have previously shown that in mets patients, simvastatin therapy decreased hscrp levels and mononuclear nf-b activity . Emerging laboratory and clinical evidence has shown a strong relationship between crp and various features of mets . The addition of crp to the present definition of the mets may help identify patients at high risk for future diabetes and cvd . Further investigation is clearly needed not only to clarify the molecular role of crp in the pathogenesis of mets, but also to shed new light on the role of crp, specifically in mediating vascular effects and conferring increased risk of cardiovascular events in mets patients with high crp levels . In this regard, it should be emphasized that the justification for the use of statins in prevention: an intervention trial evaluating rosuvastatin (jupiter) [90] study showed a clear benefit of statin therapy in patients with crp greater than 2 mg / l, and the published evidence base supports a role for statin therapy in mets.
Standard care for chronic hepatitis c (chc) has been a combination of pegylated interferon - alpha (peg - inf) and ribavirin, although this treatment has suboptimal antiviral efficacy and significant adverse events . Even in the latest treatment recommendation using novel direct acting antiviral agents, interferon (ifn)-based treatments are still optional . The main drawbacks of interferon - based treatment are unsatisfactory response rate and various adverse effects, which often lead to premature termination of treatment followed by treatment failure . Myasthenia gravis (mg) is an autoimmune neuromuscular junction disorder characterized by muscular weakness and fatigability . Mg is occasionally associated with invasive thymoma that needs surgical resection and may progress to severe respiratory failure . Development of mg in patients with chc has been rarely reported before or during antiviral treatment with interferon . A total of 12 cases of mg were reported in association with interferon treatment for chc and 6 of them went through myasthenia crisis that require respiratory support . We experienced a rare case of myasthenia crisis during antiviral therapy for chc, in whom mediastinal thymoma was discovered and successfully managed with surgical thymectomy and meticulous medical care . We reviewed the clinical course of this life - threatening complication of ifn treatment along with the already reported myasthenia crisis cases in chc patients . A 47-year - old male patient presented with a complaining of sudden diplopia that developed one week after 11-week of antiviral combination treatment for chronic hepatitis c (figure 1a). He had been on antiviral combination treatment with peg - ifn-2a (180 g / wk) (pegasys; roche, basel, switzerland) and ribavirin (1000 mg / d) (lg ribavirin; lg life sciences, seoul, korea) for the genotype 1b hepatitis c virus (hcv) infection . The pretreatment serum hcv rna level was 2.32 10 iu / ml and ast / alt levels were 255/323 u / l . At 6-week of treatment, the dosage of peg - ifn-2a was reduced to 90 g / wk, but wbc count continued decreasing to 2000/mm . Five weeks later, the absolute neutrophil count was 500/mm and the serum hcv rna level was below the limit of detection (<15 iu / ml). Consequently, the antiviral treatment was promptly discontinued and the neutropenia improved on serial blood tests . One week after discontinuation of antiviral treatment, the patient complained of sudden diplopia and right eye ptosis was observed . One week later, right eye ptosis and upper extremities weakness (grade iii) were noted . He was immediately hospitalized and initial physical examination was normal except facial palsy with the right ptosis, mild dyspnea, and upper extremities weakness . Upon admission, he denied any other oral medications and had no history of vascular or thyroid diseases . Ptosis and restricted movement on the right upper lid was improved after treatment (b). On further evaluation, the low rate repetitive nerve stimulation test (jolly test) and neostigmine test were positive findings for myasthenia gravis (table 1) and serum antiacetylcholine receptor (anti - achr) antibody level of 14.95 nmol / l (normal range: below 0.2 initial symptoms of ocular mg progressed to difficulties in chewing and swallowing, followed by respiratory failure in 3 days . Administration of pyridostigmine (mestinon) 180 mg / day was started and chest ct scan was performed which revealed anterior mediastinal mass measuring 7 cm in its greatest dimension, whereas brain imaging study was normal (figure 2). The patient received a surgical thymectomy and a high - grade thymoma invading pericardium and pleura (figure 3) was observed on the pathological examination of the surgical specimen, which confirmed invasive thymoma (type b3, who classification). The patient's respiratory failure aggravated after surgery, leading to an increased ventilator dependency . In response to the worsening respiratory condition, pyridostigmine dose was increased up to 600 mg / d and intravenous immunoglobulin and high - dose steroid pulse therapy were started . After 40 days of intensive care, the patient has his ocular symptoms back to normal (figure 1b) and was successfully weaned from the mechanical ventilator . Results of low rate repetitive nerve stimulation test (rnst) and stigmine test chest ct findings . There was 7-cm sized lobulated solid mass with internal low density and calcified portion at anterior mediastinum . (a) h&e stain (40) shows a multilobular growth pattern and infiltration into lung parenchyma . (b) h&e stain (100) shows thymoma b3 findings: sheet - like growth of medium - size round or polygonal cells with slight atypia (sheet - like growth pattern); epithelial cells are mixed with a minor component of intraepithelial lymphocytes . Treatment for chc with combination of peg - inf and ribavirin eradicates hcv rna in 40% to 50% of treatment - naive patients infected with hcv genotype 1 . In early clinical trials, 10% to 14% of treated patients discontinue combination treatment prematurely due to various adverse events and ifn was the major concern in most of them . Unsatisfactory response rate and high rate of unbearable side effects have been the major issues of conventional combination therapy . Recently, direct acting antiviral agents (daa) against hcv were developed and showed markedly improved clinical efficacy than the conventional combination treatment in terms of both potency and safety aspects . New standards of treatments using direct acting antivirals are being established and rapidly replacing the conventional combination therapy . However, the conventional combination treatment is still widely prescribed for chc and ifn is not completely excluded from some of the daa - based new regimens yet . Therefore, for the treatment of chc it is still major concern of clinicians to cope with the various side effects of ifn . Mg in patients with chc were mostly associated with ifn treatment, but it may develop without ifn administration . Two case reports described development of mg in chc patients who did not receive antiviral treatment . A 35-year - old male patients with established chc for several years without any treatment developed mg and a 59-year - old male with liver cirrhosis as a result of long - standing hcv infection developed typical mg symptoms and died due to myasthenia crisis . The authors suggested the cross - reactivity between hcv epitopes and the acetylcholine receptor as the underlying mechanism . The etiologic roles of various viral infections including hcv were suggested for the development of mg, but the exact significance of hcv infection per se is not clear . Though mg was not reported in large - scale analyses of chc patients who received ifn treatment, literatures suggesting the association between mg and ifn administration have been published in the form of case reports . There were 12 cases of mg by ifn for chc including 6 myasthenia crisis cases (table 2). In case reports of myasthenia crisis that developed during ifn - based antiviral treatment for chc, male patients> 44 were most commonly affected and the mean age of patients was 57 years and mean interval between starting ifn administration and diagnosis of mg was 4.57 months (6 weeks to 15 months). The mean duration of ifn treatment was 4.96 months (6 weeks to 15 months). The mean level of anti - achr antibody was high as 16.3 nmol / l . In most patients, respiratory distress followed advanced progression of mg symptoms and a case of severe respiratory failure was reported at 6 months after ifn discontinuation . The clinical courses of mg cases by ifn for chc were either mild or severe enough to suffer myasthenia crisis, but most of the patients were recovered eventually with medical care after discontinuing ifn treatment . List of cases of myasthenia crisis by interferon treatment for chronic hepatitis c to our knowledge, this is the second case of thymoma - associated mg during ifn treatment for chc . A case of mg that progressed to myasthenic crisis was reported at 1996 in japanese patient who had thymomectomy for known thymoma - associated mg, 19 years before starting antiviral treatment . Thymomas are responsible for 21% of mg cases and 30% of patients with thymoma have symptoms of mg . Myasthenic crisis is the most serious complication of mg and thymoma is one of the risk factors attributed to it . The mechanism of mg development by ifn therapy is not completely understood, but some of the complex immunological actions of ifn such as enhanced lymphocyte cytotoxicity, production of pro - inflammatory cytokines, inhibition of t suppressor cell function, activation of t helper lymphocytes by autoantigens, and differentiation of antigen - presenting cell might contribute to the development of ifn - induced autoimmune diseases . The thymus plays a primary role in early - onset mg mediated by anti - achr antibodies . Ifn - beta could play a central role in thymic events leading to mg by triggering the overexpression of a - achr probably leading to thymic dendritic cells autosensitization, the abnormal recruitment of peripheral cells, and germinal center formation . The epithelial neoplastic cells of thymoma are capable of presenting epitopes, which cross - reacts with different neuromuscular antigens . In addition to achr antibodies, antibodies against striated muscle titin and ryr antigens are found in most of mg thymoma patients . On the other, it has also been suggested that some infectious viruses and hcv itself may lead to mg via mechanism of cross - reactivity between viral epitopes and the acetylcholine receptor . It is not known whether the patients who experienced mg during ifn treatment for chc had subclinical mg before initiating treatment because pretreatment screening for mg is not routinely recommended . In some patients, pre - existing subclinical mg that might be associated with hcv infection itself, progress to overt mg during ifn treatment, whereas, in others, de novo mg might be induced by ifn administration . In our patient, relatively large thymoma so it seems that thymoma - associated subclinical mg were unrecognized before treatment, which was provoked by ifn administration to progress to life - threatening myasthenic crisis . For the better understanding of clinical implication of ifn - induced mg in chc treatment, screening for mg in chc patient might be considered before starting treatment or at least, during treatment when the patient complained of fatigue, muscle weakness . Screening for mg with serum anti - achr antibody test is simple and helpful in many patients, but it needs further investigation in this clinical condition because some of mg patients have serum anti - achr antibody levels within normal range . In patients with chc who is going to receive ifn - based antiviral treatment, whether screening for thymoma or subclinical mg should be included in basal evaluation is not clear but physicians need to keep in mind the potential life - threatening manifestations of mg before and during antiviral treatment especially when patients complain of muscular weakness and easy fatigability.
The most recent estimates indicate that the prevalence of autism spectrum disorders (asd) in the united states has raised to 1 in 54 boys and 1 in 252 girls . Although increased awareness and changes in diagnostic criteria have been proposed as the major contributors to this increased prevalence, as of today, the etiopathology of disorders like asd and schizophrenia (sz) remains largely unknown . Several studies have suggested that impaired mitochondrial function and altered energy metabolism in individuals with asd may contribute to their social and cognitive deficits [35], and recent reports indicate the presence of mitochondrial dysfunction (md) in brain, skeletal muscle, and peripheral blood mononuclear cells (pbmc) from children with asd . The md in asd is generally characterized by lower complex i activity accompanied, in a subset of cases, by deficits in other complexes [68]. Beside their critical role in a number of pathways, spanning from atp production (via oxidative phosphorylation), one - carbon metabolism regulation, heme biosynthesis, fatty acid catabolism, and branched chain amino acid metabolism for example, human neutrophil mitochondria are involved in several functions such as chemotaxis, respiratory burst activity, maintenance of cell shape, and apoptosis [1317]. Furthermore, neutrophil phagocytosis may involve the incorporation of some mitochondrial proteins into the phagosome . In addition, mitochondria can be involved in the immune response by providing part of the metabolic pathway for gln, in a process named glutaminolysis [19, 20]. Interestingly, gln is implicated in the expression of nadph oxidase components, cytokine production in lymphocytes, and macrophage, and as a provider for substrates required for nucleic acid synthesis [2125]. Taken together, these lines of evidence unveil a link between mitochondria and immune response [1012]. Indeed, deficits in bioenergetics have been reported in lymphocytes from children with asd enrolled in the case - control population - based childhood autism risk genetics and environment (charge) study [26, 27]. Children with asd in this study display a number of immune dysfunctions including abnormalities in monocytes, t cells and nk cell responses . These observations suggest the presence of a genetic background that results in a distinct immune profile in responses to a variety of triggers, among them psychological stressors, exposure to chemical triggers, and infectious agents [29, 30]. Considering that (i) mitochondria are inherited maternally via oocyte, (ii) maternal diet or immune activation during pregnancy has an impact on fetal metabolic and immune programming [3133], and (iii) offspring born to pregnant mice injected with poly(inosinic: polycytidylic acid) (poly(i: c)), a synthetic double - stranded rna that mimics viral infection via activation of toll - like receptor-3 (tlr3), at embryonic day 12.5 (e12.5), display core behavioral symptoms of asd [34, 35] and sz, it is hypothesized that prenatal exposure of mothers to an immunogenic response, that is, poly(i: c) elicits changes in mitochondrial function in splenocytes from progeny lasting into adulthood . Exposure to tlr ligands can lead to maternal hypertension, vascular dysfunction, and proteinuria in pregnant animals but not in nonpregnant animals [3638] suggesting the occurrence of a differential immune response / pathway during pregnancy . Differences are also evident between pregnant individuals with human placentas and patients with preeclampsia showing greater expression of tlr3, along with tlr2, tlr4, and tlr9, compared to nonpreeclampsia mothers [39, 40]. These data suggest that tlr signaling may be involved in placental deficiencies / abnormalities that may provide a framework for altered fetal programming . Of note, trophoblastic inclusions, which are also observed in preeclampsia and other placental defects, were reported to be increased in placenta from mothers of children with asd compared to controls . Furthermore, maternal exposure to various pathogens, including viruses, significantly increases the risk for asd and sz [4248]. Considering that maternal exposure to various pathogens is associated with asd and sz, the critical link between prenatal maternal infection and postnatal brain and behavioral pathology seem to be the maternal immune response, including cytokine production [47, 4954], which may contribute to the fetal imprinting of the neuroimmune response and, possibly, mitochondria - mediated metabolic responses . Although it is already known that upon poly(i: c) injection, the induction of maternal cytokines alters the expression of several cytokines in the fetal brain (with only il-1 remaining elevated at 24 h with only a few changes during adulthood), it is unknown if maternal immune activation (mia) also causes chronic changes in the bioenergetics of immune cells (such as splenocytes) of adult offspring . In this study, we sought to determine whether mia in pregnant dams alters mitochondrial function in splenocytes from affected offspring . To test this, dams were exposed to poly(i: c) on gestational day 12.5 to induce mia . This stage of gestation correlates with the late first trimester in humans, coincidental with the time that infections are most closely linked to increased incidence of asd and sz [47, 48]. The present study reports for the first time that mia activation by poly(i: c) at early gestation, which can lead to impairments in multiple psychological domains, is associated with mitochondrial changes in immune the cells of adult offspring . Male and female c57bl/6j (jackson laboratory, sacramento, ca, usa) mice were bred and maintained by the center for laboratory animal research, at university of california, davis, and maintained at ambient room temperature on a 12 h light / dark cycle (lights on at 06:00 h). All procedures were performed with approval by the institutional animal care and use committee, university of california, davis, and in accordance with the guidelines provided by the national institutes of health guide for the care and use of laboratory animals . Mice were mated overnight and females were checked daily for the presence of seminal plugs, noted on gestational day 0.5 (g0.5). On g12.5, pregnant female mice were weighed and injected with a single dose (20 mg / kg; i.p .) Of poly(i: c) (sigma aldrich, st louis, mo, usa) or saline vehicle (sham) as previously described . Each dam was returned to its cage and left undisturbed until the birth of its litter . All mice pups remained with the mother until weaning on postnatal day 21, at which time mice were group - housed 3 - 4 per cage with same - sex littermates . Mice born from poly(i: c)-treated dams exhibited autism - like behavioral deficits including reduced social approach, increased ultrasonic vocalizations, and repetitive marble burying behaviors . One week following behavioral testing, 12 wk old mice were sacrificed by cervical dislocation and spleens were collected for tissue processing . While spleen is constituted by a variety of cells relevant to the immune response (including t- and b - lymphocytes, dendritic cells, and macrophages), it has recently been shown that spleens from the offspring of mia mice elicited by poly(i: c) provide a more homogeneous preparation enriched in granulocytes compared to the preparation obtained from whole blood . Briefly, spleens were homogenized into single cell suspensions by gently pushing them through a 100 m nylon mesh filter (fisher sci) into pbs at 4c . Cells were then pelleted, and rbcs were lysed using ack lysis buffer according to the manufacturer's instructions (gibco). Cell viability was determined by trypan blue staining and found to be about 90% . The oxygen uptake of intact cell suspensions (10 cells / ml) obtained as described above was measured by using a clark - type o2 electrode from hansatech (king's lynn, uk) at 22c . Cells were incubated in the presence of 5 mm glucose in calcium and magnesium - supplemented hbss buffer without phenol red at 2022c . Nadh, succinate, and cytochrome oxidase activities were evaluated under phosphorylating conditions as described before [6, 30]. To this end, cells were permeabilized with a controlled treatment with digitonin by adding 60 g / ml 2x recrystallized digitonin for 2 min . Oxygen consumption rates were evaluated in the presence of 1 mm adp plus 1 mm malate-10 mm glutamate followed by the addition of 5 m rotenone; 10 mm succinate followed by the addition of 3.6 m antimycin a, and 10 mm ascorbate and 0.2 mm n, n, n,n-tetramethyl - p - phenylenediamine followed by the addition of 1 mm kcn . Student's two - tailed t - test was used to evaluate the differences between offspring of poly(i: c)-treated and sham - treated dams . Splenocytes from adult mice born to either sham- or poly(i: c)-treated dams were isolated for mitochondrial function testing . Given that most of the oxygen uptake by cells is linked to atp production via oxidative phosphorylation, this parameter was evaluated in intact cells in the presence of glucose (figure 1(a)). The rate of oxygen uptake by intact cells from sham - treated dams was 0.22 0.03 nmol oxygen (min 10 cells). Under the same conditions, this rate was decreased by 36% in poly(i: c)-treated dams (figure 1(a)). Addition of oligomycin, an inhibitor of atpase, was used to stop the fraction of oxygen utilized to synthesize atp via mitochondria . In both groups, more than 90% of the total oxygen uptake was inhibited by oligomycin, supporting the previous assumption that most if not all oxygen uptake by these cells was derived from oxidative phosphorylation . The oxygen uptake resistant to oligomycin, considered somewhat equivalent to state 4 (nonphosphorylating mitochondria), was not different between groups . This result indicated that the proton leak across the inner mitochondrial membrane was similar in both groups, suggesting no major mitochondrial membrane damage by either treatment . Addition of fccp, an uncoupler of electron transport and atp synthesis, increased significantly the basal oxygen uptake to a similar extent in both groups (2.5- to 3-fold) with no changes between treatments, suggesting that the maximum respiratory capacity was similar in both groups . Coupling between oxygen uptake and atp production was evaluated by the respiratory control ratio in intact cells (rcr). Mitochondria from either treatment showed a significant coupling with glucose as a substrate (with malate - glutamate, rcr = 3.5 0.4 and 2.7 0.3; with succinate, rcr = 4 1 and 6.2 0.5, for saline and poly(i: c), resp .) With no statistical differences between treatment groups . This result indicated that mitochondria were highly coupled and provided a means of support for their integrity during the testing process . Phosphorylating mitochondria from splenocytes of sham animals in the presence of an nad - linked substrate (such as malate) consumed oxygen at a rate of 0.31 0.04 nmol oxygen (min 10 cells). Phosphorylating mitochondria from offspring of poly(i: c)-treated dams showed a significant decrease in oxygen consumption (by 55%; p <0.01; figure 1(b)). By adding rotenone, an inhibitor of complex i, and succinate, a substrate for complex ii, the segment comprising from complex ii to complex v was evaluated . No differences in terms of oxygen uptake were observed between controls and poly(i: c) suggesting that the deficit in offspring of poly(i: c)-treated dams was located at the level of complex i. confirming this result, complex iv activity was not different between treatments (figure 1(b)) suggesting that mitochondrial mass was equivalent between groups . However, attempts to directly evaluate complex i activity were unsuccessful due to the limited amount of biological material . The ratios among complexes need to be preserved to provide suitable oxidation of substrates . To this end, the ratios of electron transport chain activities indicated that both treatments allow oxidizing fad - linked substrates (such as fatty acids) similarly, whereas a significantly lower oxidation of nad - linked substrates (such as glucose) was evident in the poly(i: c)-treated condition compared to controls (figure 1(c)). This imbalance in the complexes' ratios suggests that splenocytes from offspring of dams exposed to poly(i: c) use preferentially fatty acids over glucose as their main substrate for mitochondrial oxidative phosphorylation . These results are consistent with the md observed in lymphocytes from asd children characterized by lower complex i activity and accompanied, in some cases, by deficits in other complexes and/or pyruvate dehydrogenase [68]. The cytokine production (il-1, il-6, il-10, il-17, and tnf-) from cona - activated splenocytes obtained from adult offspring of poly(i: c)-treated animals was not different from that of sham - treated animals (see supplementary material available online on http://dx.doi.org/10.1155/2013/609602). This is consistent with the findings of others utilizing a similar mia model in which only a handful of cytokines was still increased in early adulthood (frontal corteces il-1, il-6, il-10, and il-9; cingulate corteces il-10 and ifn-; none in hippocampus or serum). The aim of this study was to evaluate mitochondrial function in splenocytes from offspring gestationally exposed to an acute viral mimetic, that is, poly(i: c), to induce mia . Our results indicate that the exposure of dams to a single dose of poly(i: c) at gestational day 12.5 likely triggers a tlr3-mediated response in the mother that is transmitted transplacentally to the offspring . In particular, the proinflammatory cytokine il-6 has been proved to be a key intermediary in the behavioral changes observed in the offspring of dams treated with poly(i: c). Moreover, blocking il-6 with antibodies prevents behavioral changes in the offspring, and poly(i: c)-induced mia in il-6 ko does not result in behavioral changes in the offspring . These data seem to suggest a role for il-6 in mia - induced behavioral changes, although we cannot exclude other inflammatory agents such as type 1 interferons which have also been shown to take part in the response to poly(i: c). This mia imprints a fetal programming that can still be detected during adulthood characterized by abnormal behaviors resembling those of asd [34, 35] and sz and, biochemically, by a lower oxidative phosphorylation capacity in mitochondria within intact cells and isolated mitochondria . This suggests that prenatal immune changes ensuing the maternal poly(i: c) administration are likely to imprint the long - lasting changes in the bioenergetics of the adult offspring splenocytes . While, at the normal murine fetomaternal interface, immune cells such as neutrophils, macrophages, and nk cells are assumed to be excluded from the placenta and localized only in the decidua, treatment with poly(i: c) disrupts this normal distribution and induces a significant increase in the levels of proinflammatory cytokines in the placenta and a large migration of immune cells, primarily nk cells from the decidua towards the placenta, invading the spongiotrophoblast and then the labyrinth . Trophoblasts, which express tlr3, play a role in coordinating the maternal innate immune response to infection at the fetomaternal interface [6264] and, especially in this case, in response to viral infection . These results beget the question, what is the link between lower complex i activity (or lower oxidative phosphorylation) in the offspring and an acute maternal immune response? A growing body of evidence is placing mitochondria at the center of bioenergetics and immune response / inflammation . Immunity to infection is also dependent on mitochondria function by regulating the synthesis of both pro- and anti - inflammatory cytokines [6569]. More recently, the view that mitochondria act as a platform facilitating innate immune responses adds to our understanding of the molecular complexity of sensor and adaptor interactions that promote effective host defense [11, 70]. Therefore, an emerging concept is that innate immune signaling is regulated by basic host metabolic functions . For instance, toll - like receptor signaling activates mitochondrial biogenesis during critical illness [7173], perhaps in response to increased oxidative damage in host cells [71, 74, 75]. Acute inflammation is accompanied by increases in inflammatory cytokines sustained by glycolysis, whereas chronic inflammation is sustained by less inflammatory cytokines, with more reparative features fueled mainly by mitochondria - derived atp . Thus, in this study, mia seems to imprint the immune cells of adult offspring with this more glycolytic stage resembling the influence of an acute inflammation, without switching back to the less inflammatory response . Without pointing at cause or consequence, it is interesting to note that the changes in bioenergetics in the immune cells (and not their immune response or the immune response in brain or serum) segregate with the abnormal behaviors observed in this mia model . However, a number of studies have shown differential mia induction and behavioral responses depending on the gestational exposure stage [7779]. This would suggest the existence of a window of vulnerability to infection during gestation for the onset of different behavioral defects, which may be reflected also on the mitochondrial function of the offspring . The above effects can be explained by (i) the transfer of immune cells and/or cytokines from mother to the fetus at the maternal - fetal interface and (ii) a genetic predisposition / susceptibility of the offspring that, in association with maternal viral or bacterial infections, might increase the risk of long - lasting behavioral and immune changes . Furthermore, we cannot exclude the possibility that the relatively high doses of poly(i: c) used in this study could have affected the well - being of the mother and therefore that of the fetus . For instance, poly(i: c) inhibits the development of diabetes in the nod mouse whereas the development of diabetes in diabetes - prone bb rats is poly(i: c)-dose dependent [8184]. Indeed, a recent report indicated that at least some nongenetic risk factors are shared between asd and sz, in particular, diabetes, exposure to drugs, nutritional deficiencies, and infectious agents among others . Considering the mechanisms described above, studies are now needed to clearly identify the key players affected in this acute viral response in order to evaluate the increase in risk of either asd or sz that is associated with these (and other [30, 86, 87]) modifiable environmental factors to elicit public health interventions.
Bovine babesiosis, caused by babesia bigemina, is an important tick - borne disease in the tropical and subtropical counties, transmitted by rhipicephalus (boophilus) microplus . India suffers losses of about 57.2 million us dollars annually due to babesiosis and anaplasmosis in livestock (mcleod and kristjanson 1999). Microscopic techniques like giemsa stained blood smear is the most appropriate for the diagnosis of acute babesiosis but the low sensitivity of these method do not permit to identify the carrier animals (juyal et al . Serological tests, though employed for epidemiological survey of babesiosis, lack discrimination between previous and current exposure to infection (passos et al . 1998). Molecular tests, such as pcr assays, are highly specific and sensitive method over the existing diagnostic techniques for the diagnosis of subclinical infections (figueroa et al . The molecular tests can depict the status of active infect in exposed animals and further their correlation with haemato - biochemical parameters will provide an insight into the pathogenicity of subclinical infection primarily diagnosed by pcr assay . The present study was therefore undertaken to diagnose subclinical cases of babesiosis by pcr based molecular diagnosis in relation to the haemato - biochemical parameters to study the pathogenicity induced by latent infection of b. bigemina . The province of punjab covers a total area of 50,362 square kilometers between 2930n to 3232n latitude and 7355e to 7650e longitudes . The study area was divided into three regions: western punjab (western and western plain zone), central punjab (central plain zone) and north - eastern punjab (submountain and undulating zone) (fig . 1). To study the status of molecular prevalence of the babesiosis caused by b. bigemina, the expected prevalence to be 50% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples was considered . The number of samples thus calculated was adjusted for finite population (thrusfield 2005) and correlated with 542 samples (cattle 466 and buffaloes 76) collected from month of may to october, 2011 from jugular vein of dairy animals having history of tick infestation, fever, hemoglobinurea or anemia from punjab state (india) also including the samples from the outbreaks of b. bigemina in central and north - eastern regions . About 5ml of blood was collected aseptically from the jugular vein of each animal in edta coated vials for dna isolation and serum separation, respectively . Geographic distribution clinical and subclinical babesiosis of bovine babesiosis in punjab animals diagnosed positive by giemsa stained thin blood smears technique were considered clinically positive cases . For sub - clinical diagnosis, dna was extracted using dna isolation kit (hipura blood genomic dna miniprep purification spin kit) as per the protocol of the manufacturer . The bg3/bg4 set of oligonucleotide primer was used to amplify 18s ribosomal rna gene (ellis et al . The nucleotide sequence of the primer was bg3 5 tagttgtatttcagc ctcgcg 3 and bg4 - 5 aacatccaa gcagctahtttag 3. the pcr reaction mixture (25l) mixture constituted of 12.5l of kapa 2 g fast hot start ready mix (2x containing kapa2 g fast hot start dna polymerase, kapa 2 g fast hot start pcr buffer, 0.2 mm dntp each, 1.5 mm mgcl2),3 l of 25 mm mgcl2, 1.5 l of 10 pmol bg3/bg4 primers, 1.5 l depc - treated water and 5 l of dna template . The reaction was carried on in automated thermocycler (eppendrof, master cycler personal) on the following programme: initial denaturation at 95 c (5min), 30 cycles of denaturation at 95 c (30 sec), annealing at 57 c (1min), and extension at 72 c (1.5 min) with final extension at 72 c for 10 min . The amplified pcr products were separated by electrophoresis on 1% agarose gel and visualized under uv transilluminator for detection of 689 bp amplified product . The hematology was done on advia 2120 hematology system (siemens health care diagnostic inc . Deerfield, il, usa) and serum biochemical profile was drawn on vtros dt 6011 system chemistry using ortho - clinical diagnostics kit (johnson and johnson company). The 689 bp product obtained by the bg3/bg4 primers specific for b. bigemina pcr were custom sequenced from xcelris genomics, ahmedabad, india . The nucleotide sequences were subjected to blastn analysis (altschul et al . 1990) for determining the similarity with the sequences present in the nucleotide database . Chi - square test was applied to evaluate association of disease prevalence with various districts under study . One - way analysis of variance (anova) was applied to various haematological and biochemical parameters to determine the variance in these parameters using spss software . Agreement between the results of the two techniques was analyzed through cohen s kappa coefficient . The province of punjab covers a total area of 50,362 square kilometers between 2930n to 3232n latitude and 7355e to 7650e longitudes . The study area was divided into three regions: western punjab (western and western plain zone), central punjab (central plain zone) and north - eastern punjab (submountain and undulating zone) (fig . 1). To study the status of molecular prevalence of the babesiosis caused by b. bigemina, the expected prevalence to be 50% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples was considered . The number of samples thus calculated was adjusted for finite population (thrusfield 2005) and correlated with 542 samples (cattle 466 and buffaloes 76) collected from month of may to october, 2011 from jugular vein of dairy animals having history of tick infestation, fever, hemoglobinurea or anemia from punjab state (india) also including the samples from the outbreaks of b. bigemina in central and north - eastern regions . About 5ml of blood was collected aseptically from the jugular vein of each animal in edta coated vials for dna isolation and serum separation, respectively . The extracted dna and collected sera were stored at 20 c for further analysis . Geographic distribution clinical and subclinical babesiosis of bovine babesiosis in punjab animals diagnosed positive by giemsa stained thin blood smears technique were considered clinically positive cases . For sub - clinical diagnosis, dna was extracted using dna isolation kit (hipura blood genomic dna miniprep purification spin kit) as per the protocol of the manufacturer . The bg3/bg4 set of oligonucleotide primer was used to amplify 18s ribosomal rna gene (ellis et al . The nucleotide sequence of the primer was bg3 5 tagttgtatttcagc ctcgcg 3 and bg4 - 5 aacatccaa gcagctahtttag 3. the pcr reaction mixture (25l) mixture constituted of 12.5l of kapa 2 g fast hot start ready mix (2x containing kapa2 g fast hot start dna polymerase, kapa 2 g fast hot start pcr buffer, 0.2 mm dntp each, 1.5 mm mgcl2),3 l of 25 mm mgcl2, 1.5 l of 10 pmol bg3/bg4 primers, 1.5 l depc - treated water and 5 l of dna template . The reaction was carried on in automated thermocycler (eppendrof, master cycler personal) on the following programme: initial denaturation at 95 c (5min), 30 cycles of denaturation at 95 c (30 sec), annealing at 57 c (1min), and extension at 72 c (1.5 min) with final extension at 72 c for 10 min . The amplified pcr products were separated by electrophoresis on 1% agarose gel and visualized under uv transilluminator for detection of 689 bp amplified product . The hematology was done on advia 2120 hematology system (siemens health care diagnostic inc . Deerfield, il, usa) and serum biochemical profile was drawn on vtros dt 6011 system chemistry using ortho - clinical diagnostics kit (johnson and johnson company). The 689 bp product obtained by the bg3/bg4 primers specific for b. bigemina pcr were custom sequenced from xcelris genomics, ahmedabad, india . The nucleotide sequences were subjected to blastn analysis (altschul et al . 1990) for determining the similarity with the sequences present in the nucleotide database . Chi - square test was applied to evaluate association of disease prevalence with various districts under study . One - way analysis of variance (anova) was applied to various haematological and biochemical parameters to determine the variance in these parameters using spss software . Agreement between the results of the two techniques was analyzed through cohen s kappa coefficient . Out of total 542 blood samples (466 cattle and 76 buffaloes), pcr based (fig . 2) diagnosis revealed 89 animals (16.42%, 95% ci= 13.7319.1) positive for babesia that included 83 cattle (17.81%, 95% ci= 14.8120.81) and 6 buffaloes (7.89%, 95% ci= 2.6613.12). The relative prevalence of b. bigemina by pcr amplifying 689 bp fragment for 18s rrna gene showed highest prevalence of the disease in north - eastern region (64.28%, 95% ci= 51.7976.78) (fig . 1) and lowest in western region (3.89%, 95% ci= 1.456.32) of punjab . Overall overall percentage of animals positive for b. bigemina b. bigemina by giemsa - stained thin blood smears was 1.66% (9/542), the higher in cattle 1.73% (8/463) than in buffaloes 1.31% (1/76). Chi - square test showed significant difference in the prevalence of the infection as revealed by blood smear examination and pcr . Kappa contingency test indicated a slight agreement between blood smear examination and pcr assay with latter being highly sensitive for the detection of latent infections . Agarose gel (1.5%) electrophoresis showing amplified dna (689bp) from babesia bigemina targeting ssu rrna gene using primer bg3/bg4 the 689bp sequence showed homology (99%) with b. bigemina isolate brco2 18s ribosomal rna gene (genbank no . 2) showed significant (p <0.05) decrease in the tec, hb, pcv and mcv, while significant increase (p <0.05) in mch and mchc in clinically and subclinically infected animals as compared to non - infected healthy control group (fig . Hematological alterations indication anemia in clinically and subclinically infected animals (rbc (red blood cells): 10cells/l, hb (hemoglobin level): g / dl, pcv (packed cell volume):%, mcv (mean corpuscular volume: fl, mch (mean corpuscular hemoglobin): pg, mchc (mean corpuscular hemoglobin concentration): g / dl). Bars with different alphabets a, b and c differ significantly for the parameter a significant increase (p <0.05) was seen in alkp and ast of clinically infected group while the increase as non - significant in subclinically infected animals as compared to non - infected control group . The increase in alt levels was non - significant (p <0.05) in both the infected groups (fig . Liver function biochemical alterations in clinically and subclinically infected animals (alkp (alkaline phosphatase), ast (aspartate aminotransferase), alt (alanine aminotransferase) there was no significant difference in the level of creatinine and bun in the three groups under study, however the level of total bilirubin had increased significantly in the infected groups as compared to non - infected group (p <0.05) (fig . Kidney function biochemical alterations in clinically and subclinically infected animals (crsc (cretinine), tbil (total bilirubin), bun (blood urea nitrogen) significant response in terms of decrease in wbc, tp, glo and plt was seen only in clinically infected animals (p <0.05), while the alterations in albumin level was non - significant in both the infected groups as compared to non - infected control group (fig . Blood cellular and biochemical response in clinically and subclinically infected animals (wbc (white blood cells), tp (total protein), glo (globulin), alb (albumin), plt (platelets) lane m, molecular size marker 100 bp plus, lane p positive control and n negative controls, lane a d showing amplified b. bigemina genomic dna from the blood of animals positive for infection, lane e showing no amplification of b. bigemina genomic dna from the blood of animal negative for infection . Out of total 542 blood samples (466 cattle and 76 buffaloes), pcr based (fig . 2) diagnosis revealed 89 animals (16.42%, 95% ci= 13.7319.1) positive for babesia that included 83 cattle (17.81%, 95% ci= 14.8120.81) and 6 buffaloes (7.89%, 95% ci= 2.6613.12). The relative prevalence of b. bigemina by pcr amplifying 689 bp fragment for 18s rrna gene showed highest prevalence of the disease in north - eastern region (64.28%, 95% ci= 51.7976.78) (fig . 1) and lowest in western region (3.89%, 95% ci= 1.456.32) of punjab . Overall overall percentage of animals positive for b. bigemina b. bigemina by giemsa - stained thin blood smears was 1.66% (9/542), the higher in cattle 1.73% (8/463) than in buffaloes 1.31% (1/76). Chi - square test showed significant difference in the prevalence of the infection as revealed by blood smear examination and pcr . Kappa contingency test indicated a slight agreement between blood smear examination and pcr assay with latter being highly sensitive for the detection of latent infections . Agarose gel (1.5%) electrophoresis showing amplified dna (689bp) from babesia bigemina targeting ssu rrna gene using primer bg3/bg4 the 689bp sequence showed homology (99%) with b. bigemina isolate brco2 18s ribosomal rna gene (genbank no . 2) showed significant (p <0.05) decrease in the tec, hb, pcv and mcv, while significant increase (p <0.05) in mch and mchc in clinically and subclinically infected animals as compared to non - infected healthy control group (fig . Hematological alterations indication anemia in clinically and subclinically infected animals (rbc (red blood cells): 10cells/l, hb (hemoglobin level): g / dl, pcv (packed cell volume):%, mcv (mean corpuscular volume: fl, mch (mean corpuscular hemoglobin): pg, mchc (mean corpuscular hemoglobin concentration): g / dl). A significant increase (p <0.05) was seen in alkp and ast of clinically infected group while the increase as non - significant in subclinically infected animals as compared to non - infected control group . The increase in alt levels was non - significant (p <0.05) in both the infected groups (fig . Liver function biochemical alterations in clinically and subclinically infected animals (alkp (alkaline phosphatase), ast (aspartate aminotransferase), alt (alanine aminotransferase) there was no significant difference in the level of creatinine and bun in the three groups under study, however the level of total bilirubin had increased significantly in the infected groups as compared to non - infected group (p <0.05) (fig . Kidney function biochemical alterations in clinically and subclinically infected animals (crsc (cretinine), tbil (total bilirubin), bun (blood urea nitrogen) significant response in terms of decrease in wbc, tp, glo and plt was seen only in clinically infected animals (p <0.05), while the alterations in albumin level was non - significant in both the infected groups as compared to non - infected control group (fig . Blood cellular and biochemical response in clinically and subclinically infected animals (wbc (white blood cells), tp (total protein), glo (globulin), alb (albumin), plt (platelets) lane m, molecular size marker 100 bp plus, lane p positive control and n negative controls, lane a d showing amplified b. bigemina genomic dna from the blood of animals positive for infection, lane e showing no amplification of b. bigemina genomic dna from the blood of animal negative for infection . In the clinically positive (9) cases the marked clinical symptoms of high fever (39.440.5c), history of hemoglobinurea, jaundice, icterus, tachycardia, weight loss and decrease in milk yield were recorded . The blood smear of these animals revealed typical pyriform bodies of b. bigemina at an acute angle inside the erythrocytes . Previously, parasitological prevalence of babesiosis due to b. bigemina in cattle is reported to be 5.94% in punjab (aulakh et al . 2005). However, the status of subclinical infection is essential to be drawn as this kind of infection can be the source of infection to other animals of the herd as well as may flair in the condition of stress . Thus, it has been observed that the molecular approaches based on nucleic acids such as polymerase chain reaction (pcr) assays offer greater sensitivity and specificity over the existing diagnostic tests (fahrimal et al . The trend of geographic distribution of babesiosis can be corroborated with the availability of favorable conditions of high humidity and soil moisture content (ghai et al . 2008) and higher population of tick vector, r. microplus (singh et al . 2000), in northeastern parts are punjab as it covers the sub mountain and undulating zones . Ticks were observed on 78.6% of the affected cattle and 9.4% of the affected buffaloes . Population of the ticks was mainly constituted by r. (boophilus) microplus followed by hyalomma annatolicum anatolicum, correlating the fact that incidence of b. bigemina is a tick borne pathogenesis . Above all the major fraction of sampling was done from cattle and they primarily considered susceptibly to the infection (vohara et al . Findings of hematobiochemical profiling indicate that the subclinical infection of babesiosis in bovines is also responsible for causing anemia . Marked anemia has been documented in clinical cases of bovine babesiosis in previous studies (sharma et al . A number of factors contribute to anemia in babesiosis including lyses of erythrocytes by emerging parasites (dwivedi et al . 1976), indiscriminate phagocytosis of infected and non - infected erythrocytes by activated macrophages system (ruprah 1985), suppression of erythopoitietic activity of bone marrow (pandey and misra 1987) and antigen antibody mechanism (ferris et al . 1967). Non - significant difference was seen in glucose level among the three groups, when compared with the normal range (4275 g / dl) (latimer et al . The apparent increase in the levels of ast, alt, alkp, bun and creatinine signifies harmful effect of toxic metabolites of babesia sp . On liver cells 2007 reported the significant increase in aspartate aminotransferase, alanine aminotransferase (alt) and gamma glutamyltransferase (ggt) in babesiosis . A significant increase in total bilirubin in subclinically infected group hemolytic crisis of babesiosis (panday and misra 1987) and hepatic damage (yeruham et al . Overall, the degenerative changes in the internal organs as indicated by biochemical response may be due to anemic hypoxia . The comparison of prevalence of bovine babesiosis by pcr and giemsa stained thin blood smear in dairy cattle of punjab, revealed highest prevalence in north - eastern region of punjab by both the techniques . As the transmission of b. bigemina is transovarian, the presence of rather few infected ticks on even a subclinically infected animal may acts as nidus of infection for the whole herd, posing severe economic losses and pathognomic effects on the animal as indicated by altered vital parameters contemplated though significant decrease in the level of rbc, hb, pcv, and mcv with significant increase in tbil, mch and mchc levels.
In modern society, more people spend long periods working at computers, and experience increased work - related neck and back discomfort1 . Remaining seated for long periods can cause problems for the spine, circulation, muscles, and joints2 . Prolonged sitting is a risk factor for low back pain (lbp)2 . Osullivan et al.2 showed that decreased trunk muscle endurance is associated with habitually adopting a passive sitting posture and reduced activity levels . According to recent findings, the form of resting and the work configuration are important to muscle activation and recovery of the circulation4, 5 . The purpose of this study was to examine the effects of a relaxation chair and resting with simple range of motion (rom) exercises during computer work, and low back muscle strengthening exercises after computer work on pain and the flexion - relaxation (fr) ratio of one computer worker with lbp . The subject of this study was a 37-year - old male who complained of severe lbp pain at the l4 level . Ethics approval was obtained from the yonsei university faculty of health science human ethics committee . He did not have back pain in the morning but did have it in the evening after working on a computer all day . When he performed forward flexion in the standing position with his knees fully extended, he experienced pain in his lower back with a visual analogue scale (vas) score of 7 ., he rested three times for approximately 10 min each at irregular intervals . The rest of the time, he was performing internet searches or shopping on a computer . In the study, he worked on a computer for 5 h each day for 3 days and followed a different program each day . The first day (session 1), he spent 10 min in a relaxation chair resting after each hour of computer work . The second day (session 2), he spent 10 min performing simple trunk rom exercises (trunk flexion, extension, lateral bending, and rotation) while standing after each hour of computer work . The third day (session 3), he performed low back muscle strengthening exercises for 20 min after 5 h of using the computer . The exercises consisted of a bridging exercise in the supine position (three sets of ten repetitions) and hip extension exercises in the quadruped position with knee extension (left and right leg, five sets each of ten repetitions). This study measured the fr ratio and vas score when he performed forward flexion in the standing position with his knees fully extended, before and after each 5-h work session . Electromyography (emg) signals were preamplified by a preamplifier placed close to the electrodes, and sent to the data acquisition unit of an mp150 system (biopac systems, santa barbara, ca, usa), which amplified and sampled the emg input at 1000 hz . The emg signals were band - stop filtered at 60 hz, and the root mean square (rms) values were calculated . The subject was required to stand comfortably, bend forward slowly with his arms dangling freely (bending period), and then hold this position for 3 s (hanging period). The fr ratio (%) was calculated as the ratio of the rms activity in the hanging period to that in the bending period . In session 1, the fr ratios before and after work were 19% and 38% (+ 19%), respectively . The respective vas scores before and after work were 5 and 8 (+ 3). In session 2, the fr ratios before and after work were 18% and 21% (+ 3%), respectively . The respective vas scores were 5 and 6 (+ 1). In session 3, the fr ratios before and after work were 22% and 29% (+ 7%), respectively, and the vas scores were 5 and 6 (+ 1). The fr response is reported to occur in the lumbar region of more than 90% of healthy people who do not have lbp8 . In individuals with lbp, the trunk muscle activation pattern is altered, and the lower back muscles are strongly activated over a long period8 . It is necessary to evaluate the fr response for lbp . Mathieu and forin9 suggested that if the erector spinae (es) muscle activity during the hanging period is less than 10% of the muscle activity during the bending period, it indicates the presence of fr . In session 1, the fr ratio (+ 19%) and vas score (+ 3) increased to the greatest degree . Therefore, the relaxation chair was not effective for muscle activation and recovery of the circulation . In session 2, the fr ratio increased by 3% and the vas score increased by + 1 . In session 3, the fr ratio was increased by 7% and the vas score increased by + 1 . Therefore, after computer work, the back muscles require exercises to prevent lbp . There was no difference in pain between sessions 2 and 3 . However, difference in fr ratio was the lower in session 2 than session 3 . The fr ratio is known to be the more sensitive indicator for prediction of lbp8, 9 . Also, session 3 required additional time after work, and it also required exercise with a high load when compared with session 2 . So, this study suggests that session 2 (regular trunk rom exercises after each hour of computer work) was more effective than session 3 (strengthening exercises after work). However, this report has a limitation . The carryover effects could have an influence on the results . This study suggests that it is more effective to perform regular, passive exercises to prevent lbp in computer users . It also suggests that poor resting is a risk factor for a change in the muscle pattern.
Hyperpolarization - activated and cyclic nucleotide - gated (hcn) channels, first identified in 1976 in the heart by noma and irisawa and characterized by brown and difrancesco and weiss and his colleague, are cation channels that open when the membrane potential is hyperpolarized . The general structure of hcn channels resembles that of the voltage - gated k channels . Hcn channels consist of four subunits that have six transmembrane segments, the canonical gyg sequence in the pore forming region, and the positively charged s4 segment [4, 5]. But the k permeability of hcn channels is not so selective as typical k channels and is permeable to na . Thus a typical current reversal potential of hcn channels is around 30 mv . For voltage - dependent gating, inward movement of s4 segment in response to hyperpolarization is reported [79], but molecular aspects of channel opening are still unknown . For example, hcn channels require extracellular cl and extracellular k to open . Cyclic - amp-(camp-) binding site locates near the c terminus, and camp affects the voltage dependence of activation in some hcn channel isoforms . Phosphatidylinositol 4,5-bisphosphate (pip2) is also known as a modulator of hcn channels; it shifts the voltage dependence through a different mechanism from that of camp [11, 12]. These multifaceted features endow hcn channels to work in many functions described below . In this paper, we try to understand the physiological significance of these functions by using simple numerical simulations . Previous literatures reported that hcn channels are expressed in heart, brain, taste buds, and pancreatic cells . This paper focuses on the electrophysiological function of hcn channels in the central nervous system . In the brain, hcn1 isoform is expressed strongly in cerebral cortex, hippocampus, and superior colliculus, and hcn2 is expressed ubiquitously in the brain . Expression of hcn3 and hcn4 is found in localized regions in the brain . In the neuron expression in axon terminals is reported at the calyx of held and the globus pallidus neurons . Previous reports showed that expression of hcn channels on the cell surface is uneven in some neurons . Hcn channels are expressed more strongly in distal dendrites than in somas in thalamic reticular neurons, whereas they distribute nearly evenly in thalamocortical relay neurons . For example, hcn1 is expressed strongly in dendrites in layer 5 pyramidal neurons of matured rats, whereas hcn2 is mainly expressed in the soma . Expression and kinetics of hcn channels are affected by unusual neuronal activity like epilepsy and environmental stimuli . Recent studies showed that channel current and surface expression of hcn channels decrease in one hour after pilocarpine - induced status epilepticus, but decreases in total channel protein and mrna occur in the subsequent week . Involvement of activation of calcineurin and inactivation of p38 mitogen - activated protein kinase (p38 mapk) in the hyperpolarizing shift of voltage - dependence is reported . On the other hand, some reports showed that sensory inputs are necessary to increase or keep the expression level of hcn channels . Deprivation of whiskers decreases the expression of dendritic hcn channels in the rat somatosensory cortex . Reported increased hcn2 in ca3 and decreased hcn1 and hcn2 in ca1 in the same epilepsy model . Based on the multifaceted features, hcn channels are involved in many kinds of functions that range from cellular to behavior levels (working memory task, initiation of neuropathic pain). In this paper, we focus on neuron - level functions, which include regulation of resting membrane potential, normalization of synaptic inputs, selective filtering for coincident inputs, modulation of intrinsic cellular frequency characteristics, and regulation of membrane resistance . We summarize these functions and show typical examples of stimulations obtained by using neuron simulator (figure 1(a)). Conditions for the simulation are summarized at the end of the text . Because a typical current reversal potential of hcn channels is about 30 mv that is higher than the normal threshold for the generation of action potential, hcn channel currents depolarize and control the membrane potential near the resting membrane potential . When hcn channel conductance is removed, a simulated neuron shows a hyperpolarized membrane potential (figure 1(b)). Although hcn channel conductance decreases the epsp amplitude (see below and figure 2(a)), this inhibitory effect is masked by depolarization of the membrane potential (figure 1(c)). In addition, the effect of inhibitory synaptic inputs is suppressed by hcn channels, because hyperpolarizing conductance of inhibitory synaptic input opens hcn channels to evoke depolarizing conductance . When voltage dependence of hcn channels is shifted to the depolarizing direction by camp or pip2, hcn channels can be active at around the resting membrane potential and can work as an excitatory factor . Furthermore, in an interesting case, dendritic hcn channels are necessary to generate dendritic spikes at a short and fixed latency following optic fiber stimulation in the rat superior colliculus . As hcn channels activate more, the amplitude and kinetics of postsynaptic potentials become smaller and shorter (figures 2(a), 2(b), 2(c), and 2(d)). Current density of hcn channels increases along with the distance from the soma in hippocampal and neocortical pyramidal neurons . Such dendritic hcn channels scale the epsp amplitude measured at the soma and suppress the location dependency of synaptic inputs in hippocampal and neocortical pyramidal neurons . We use two models with the gradient and even hcn channel distributions (figure 3(a)), and computer simulation can reproduce a similar effect (figure 3(b)). Interestingly note that, despite of different hcn channel distributions, both models show similar current amplitudes and overlapping voltage dependence of hcn channels in this condition (figures 3(c) and 3(d)). Activated hcn channels are involved in the detection of coincidental synaptic inputs . Because hcn channel conductance shortens the rise time and decay time of the epsps through the decrease in membrane resistance (figure 2), summation of repetitive epsps that occur at a low frequency is suppressed . This suppression filters out low - frequency inputs, and thus improves the selectivity for synchronous synaptic inputs (figure 4(a)). Active conductance of ion channels enables neurons to produce intrinsic membrane potential oscillation and resonance . Such rhythmic activity can filter inputs at certain frequencies, and can influence the precision of spike timing [39, 40]. Our simulation successfully shows that hcn channel conductance suppresses intrinsic activities particularly around 10 hz (figures 4(b) and 4(c)). Because hcn channels can work as both excitatory and inhibitory factors, it is difficult to predict which is the major role of hcn channels in controlling neural activity without conducting actual experiments . For example, k channels and cav3 (t - type) ca channels can indirectly affect the activation of hcn channels through modulation of membrane potential and resistance, and vice versa [43, 44]. Especially, delayed - rectifier m - type k channels (km), also called kv7 or kcnq channels, have a large impact on the role of the hcn channels, because km channels open slowly at relatively polarized potentials from about 60 mv and do not inactivate . (2009) showed that km channels determine whether hcn channels function as an excitatory or an inhibitory factor, on the basis of recordings from hippocampal pyramidal neurons and computational simulation of model neurons . We further advanced the simulation study and examined the role of km channel conductance on the threshold potential for spike generation by using a model neuron with conductances for hcn, km, delayed rectifier k, na, and ca channels . Our simulation reproduces a similar effect of km channels on the hcn channel function (figures 5(a) and 5(b)). For a weak synaptic input, the membrane potential at the peak epsps (vpeak) is more depolarized with hcn channel conductance than without hcn channel conductance (figure 5(b)). But as the synaptic input becomes stronger, the relation between vpeak with and without hcn channel conductance reverses . The crossing point of the relationship shifts in a km conductance - dependent manner (figure 5(b)). Because our model contains na conductances, we can also analyze the effect of hcn channel conductance on the threshold potential to generate action potential in various km conductance conditions . Our simulation suggests that when km conductance is very small, hcn channel conductance reduces the minimum intensity of synaptic input to generate action potential . But when km conductance becomes large, hcn channel conductance increases the minimum intensity of synaptic input to generate action potential (figure 5(c)). The relationships between required intensity of synaptic input and km conductance with and without hcn channel conductance crosses (figure 5(d)). Conversely, when t - type ca channel conductance was altered to shift the action potential threshold, the relation of the threshold does not reverse in our simulation condition (figures 5(e) and 5(f)). A previous study showed that presynaptic hcn1 channels regulate cav3.2 channel activity and neurotransmission at specific cortical synapses . In order to uncover how hcn channels contribute to the neural activity, it will be necessary to compare the effects of k channels and ca channels by actual experiments . To elucidate the role of hcn channels in the neuron, modulators of hcn channels are indispensable . A number of modulators are reported for hcn channels (summarized by lewis et al . ). Previous studies showed that camp, pip2, and ethanol shift the voltage dependence to the depolarizing direction and accelerate the gating kinetics [48, 49]. In contrast, trip8b shifts the voltage dependence to the hyperpolarizing direction and slows the kinetics . These modulators can shift the voltage dependence by as large as 10 mv in either direction . We examined the effect of such shifts of voltage dependence on neural activity as well as effects of km and t - type channel conductance . Briefly, hyperpolarizing and depolarizing shifts of the voltage dependence, respectively, result in decrease and increase in channel conductance (figures 5(g) and 5(h)). Importance of modulated voltage dependence of hcn channels in the nervous system has already reported . For example, hcn channels are inhibited via suppression of camp by the activation of 2a - adrenoceptors that are colocalized with hcn channels in dendritic spines in the prefrontal cortex . This inhibition results in improved working memory in monkey and rat . Increased neural activity caused by enhanced hcn channel activity with camp previous reports showed the involvement of hcn channels in brain disorders, including absence seizures, febrile seizures, and parkinson's disease . Reduced hcn channel activity following the establishment of status epilepticus is reported in many epilepsy models [2326]. Decreased hcn channel activity is also reported in the external segment of the globus pallidus in rodent models of parkinson's disease . It is not known well, however, whether such reductions precede or follow the onset of the diseases . Genetic mutations of hcn channels are found in human epilepsy ([51, 52], and references therein). Null mice show absence epilepsy, and hcn1 null mice are more susceptible to kainic acid - induced seizures . These reports imply, at least, dysfunction of hcn channel can promote the onset of seizures . Because hcn channels can work both excitatory and inhibitory, and these effects can be altered by pharmacological modulation, hcn channels are one of the promising pharmacological targets in treating diseases of the brain . Further studies about complex relations between hcn channels and other ion channels and about mechanisms underlying modulation of voltage dependence will provide us a concrete step for clinical usage of hcn channel modulation . For computer simulation, a simplified neuron model is used, because the complicated dendritic structure can be compactified according to the rall's rule (figure 1(a)). Membrane capacitance (1.4 f / cm) and axial resistance (150 cm) are even throughout the neuronal structure . The model neuron contains conductances for two kinds of na channels, delayed rectifier k channel, muscarinic k (km) channel, and hcn channel . In addition, nonspecific leak conductance is set in all models (conductance, 40 s / cm; reversal potential, 70 mv). Conductance for the t - type ca channel is included in some of the simulations, where addition is explicitly stated . Most conductances, except for delayed rectifier k and km channels, are taken from a model of the rat subthalamic nucleus . In order to change the voltage dependence, synaptic input is always applied to the soma using the exp2syn function of the neuron simulator (tau1, 1.5 ms; tau2, 4 ms). Reversal potentials of excitatory and inhibitory inputs are 0 and 85 mv, respectively.
Recurrent epithelial ovarian carcinoma has a poor prognosis and is almost always fatal [1, 2]. The most frequent relapse site involves the peritoneal cavity and the infradiaphragmatic lymph nodes . Outside the abdomen, the most frequent metastatic sites are the pleura and the mediastinal lymph nodes [3, 4]. Although nodal metastases are well known in the course of epithelial ovarian carcinoma, solitary lymph node relapses are rarely described . The cervical, supraclavicular, groin and axillary areas are unusual sites of isolated lymph node involvement . In a retrospective study evaluating 640 ovarian cancer patients, prognosis of isolated lymph node relapse seems better than that of metastatic recurrence at other sites and in the event of an asymptomatic relapse, immediate or delayed therapy should be assessed . Recurrence of ovarian carcinoma is commonly suspected when there is a progressive increase of the serum ca-125 level, but it does not allow differentiation between localized and diffuse tumor spread . Ct scan is the imaging technique of choice, but its capability to detect residual tumor is limited in case of small metastases, such as peritoneal, mesenteric and omental recurrences . F - fdg - pet can detect residual tumor with a higher accuracy than ct and even with a higher sensitivity than the tumor markers ca-125 [7, 8]. Recently, we have observed internal mammary lymph node metastases diagnosed by f - fdg - pet / ct as a solitary recurrence site in 3 ovarian cancer patients . A 65-year - old woman had been treated by total abdominal hysterectomy, bilateral salpingooophorectomy, pelvic lymphadenectomy and omentectomy followed by chemotherapy (carboplatin and cyclophosphamide) for ovarian papillary serous adenocarcinoma stage ic in 1997 . At her annual followup, 9 years later (may 2006), the serum ca-125 level was increased (83 u / ml, normal value <35 u / ml). A pet / ct scan showed increased f - fdg uptakes in the bilateral internal mammary lymph nodes (fig . 1a, b), with suspected infiltration of the right side of the sternum (fig . A ct scan confirmed an abnormal left internal mammary lymph node of 1.7 cm in size and a thickening of the right parasternal tissue . She was treated with 7 courses of chemotherapy (carboplatin plus gemcitabine). At the end of the treatment, the serum ca-125 level was stable (75 u / ml) and a ct scan confirmed stable disease . External radiation therapy was administered to the bilateral internal mammary lymph node chains at a total dose of 4,680 cgy . Pet / tc was performed 4 months after the end of the radiation therapy and showed a reduction of the f - fdg uptake in the right internal mammary lymph node (suv max . 6 vs. 11) and a complete normalization in the contralateral lymph node . In november 2007, she was retreated with chemotherapy (6 courses of carboplatin) for disease progression in the internal mammary lymph nodes and the sternum . In july 2008, she developed 2 subcentimetric brain metastases that were treated with stereotaxic radiosurgery, obtaining a complete remission . In september 2008, an increase of the serum ca-125 level occurred (265 u / ml) and the ffdg - pet / ct scan showed multiple pathological uptakes in the supradiaphragmatic lymph nodes (bilateral supraclavicular, internal mammary chains and mediastinal). She was treated with 6 courses of carboplatin + paclitaxel and the serum ca-125 level was normalized . During 2010, disease progression was further documented and monochemotherapy with pegylated doxorubicin was started . On december 31, 2010, she was still on treatment . A 47-year - old woman had been treated by total abdominal hysterectomy, bilateral salpingooophorectomy and lombo - aortic lymphadenectomy followed by chemotherapy (cisplatin and cyclophosphamide) for ovarian papillary serous carcinoma stage iii in 1991 . Subsequently, she first received anterior sigmoid resection for pelvic relapse and adjuvant carboplatin (1996), and secondly, chemotherapy with cisplatin + paclitaxel, followed by liver dissections for liver metastases and carboplatin + topotecan chemotherapy (1998). During these treatments, there was no evidence of disease . In 2001, at a follow - up examination, a right supraclavicular lymph node was palpable . In absence of other signs of localizations, the patient experienced partial remission and subsequently, she was submitted for dissection of the right supraclavicular lymph node . Radiation therapy was delivered postoperatively . In december 2002, a pet / ct carried out during a follow - up examination, showed an increased f - fdg uptake in a left internal mammary lymph node (fig . She was operated on and a lymph node metastasis was removed and histologically confirmed (poorly differentiated adenocarcinoma). No postoperative treatment was administered . In january 2006, a pet / ct showed an increased f - fdg uptake in 3 supradiaphragmatic nodules located in the right anterior costophrenic sinus . She was treated with supradiaphragmatic lymphadenectomy and partial diaphragm resection; 3 out of 14 lymph nodes were massively infiltrated by a poorly differentiated adenocarcinoma of ovarian origin . At the last follow - up examination (december 2010), almost 5 years later, the patient was well and showed no evidence of disease . A 51-year - old woman had been treated by total abdominal hysterectomy, bilateral salpingooophorectomy and omentectomy, followed by chemotherapy (carboplatin and paclitaxel) for ovarian papillary serous carcinoma stage iiib in 2002 . At her annual follow - up, 6 years later, the serum ca-125 level showed an increase above the normal level (> 35 u / ml). A pet / ct scan revealed an increased ffdg uptake in the right internal mammary lymph nodes and the cardiophrenic nodule . Thus, 1 month later, she was submitted for right internal mammarian lymphoadenectomy; the histological examination confirmed a papillary adenocarcinoma . From april to july 2009, she was treated with 6 courses of carboplatin and paclitaxel . The serum ca-125 level was within the normal range . At the last follow - up visit, in september 2010, she was well and showed no signs of recurrent disease . A 65-year - old woman had been treated by total abdominal hysterectomy, bilateral salpingooophorectomy, pelvic lymphadenectomy and omentectomy followed by chemotherapy (carboplatin and cyclophosphamide) for ovarian papillary serous adenocarcinoma stage ic in 1997 . At her annual followup, 9 years later (may 2006), the serum ca-125 level was increased (83 u / ml, normal value <35 u / ml). A pet / ct scan showed increased f - fdg uptakes in the bilateral internal mammary lymph nodes (fig . 1a, b), with suspected infiltration of the right side of the sternum (fig . A ct scan confirmed an abnormal left internal mammary lymph node of 1.7 cm in size and a thickening of the right parasternal tissue . She was treated with 7 courses of chemotherapy (carboplatin plus gemcitabine). At the end of the treatment, the serum ca-125 level was stable (75 u / ml) and a ct scan confirmed stable disease . External radiation therapy was administered to the bilateral internal mammary lymph node chains at a total dose of 4,680 cgy . Pet / tc was performed 4 months after the end of the radiation therapy and showed a reduction of the f - fdg uptake in the right internal mammary lymph node (suv max . 6 vs. 11) and a complete normalization in the contralateral lymph node . In november 2007, she was retreated with chemotherapy (6 courses of carboplatin) for disease progression in the internal mammary lymph nodes and the sternum . In july 2008, she developed 2 subcentimetric brain metastases that were treated with stereotaxic radiosurgery, obtaining a complete remission . In september 2008, an increase of the serum ca-125 level occurred (265 u / ml) and the ffdg - pet / ct scan showed multiple pathological uptakes in the supradiaphragmatic lymph nodes (bilateral supraclavicular, internal mammary chains and mediastinal). She was treated with 6 courses of carboplatin + paclitaxel and the serum ca-125 level was normalized . During 2010, disease progression was further documented and monochemotherapy with pegylated doxorubicin was started . On december 31, 2010, she was still on treatment . A 47-year - old woman had been treated by total abdominal hysterectomy, bilateral salpingooophorectomy and lombo - aortic lymphadenectomy followed by chemotherapy (cisplatin and cyclophosphamide) for ovarian papillary serous carcinoma stage iii in 1991 . Subsequently, she first received anterior sigmoid resection for pelvic relapse and adjuvant carboplatin (1996), and secondly, chemotherapy with cisplatin + paclitaxel, followed by liver dissections for liver metastases and carboplatin + topotecan chemotherapy (1998). During these treatments, there was no evidence of disease . In 2001, at a follow - up examination, a right supraclavicular lymph node was palpable . In absence of other signs of localizations, she was treated with gemcitabine + vinorelbine over a 4-month period . The patient experienced partial remission and subsequently, she was submitted for dissection of the right supraclavicular lymph node . Radiation therapy was delivered postoperatively . In december 2002, a pet / ct carried out during a follow - up examination, showed an increased f - fdg uptake in a left internal mammary lymph node (fig . She was operated on and a lymph node metastasis was removed and histologically confirmed (poorly differentiated adenocarcinoma). No postoperative treatment was administered . In january 2006, a pet / ct showed an increased f - fdg uptake in 3 supradiaphragmatic nodules located in the right anterior costophrenic sinus . She was treated with supradiaphragmatic lymphadenectomy and partial diaphragm resection; 3 out of 14 lymph nodes were massively infiltrated by a poorly differentiated adenocarcinoma of ovarian origin . At the last follow - up examination (december 2010), almost 5 years later, the patient was well and showed no evidence of disease . A 51-year - old woman had been treated by total abdominal hysterectomy, bilateral salpingooophorectomy and omentectomy, followed by chemotherapy (carboplatin and paclitaxel) for ovarian papillary serous carcinoma stage iiib in 2002 . At her annual follow - up, 6 years later, the serum ca-125 level showed an increase above the normal level (> 35 u / ml). A pet / ct scan revealed an increased ffdg uptake in the right internal mammary lymph nodes and the cardiophrenic nodule . Thus, 1 month later, she was submitted for right internal mammarian lymphoadenectomy; the histological examination confirmed a papillary adenocarcinoma . From april to july 2009, she was treated with 6 courses of carboplatin and paclitaxel . The serum ca-125 level was within the normal range . At the last follow - up visit, in september 2010, she was well and showed no signs of recurrent disease . We have been unsuccessful in finding reports on internal mammary lymph nodes metastases from ovarian cancer in the recent literature . In this case report, we have presented 3 cases of recurrent ovarian cancer in which pet / ct detected internal mammary lymph node metastases as unique site of disease, 6, 9 and 11 years after the primary surgical operation, respectively . In the first and third case, pet / ct confirmed the recurrence suspected by the increase of the serum ca-125 level; in the second case, pet / ct was carried out as a follow - up examination in absence of symptoms or increased serum ca-125 levels . In these 3 patients, the pet / ct scan has enabled a local therapeutic approach: a surgical treatment in two patients and radiation therapy in 1 patient, permitting a local control of the disease . At, respectively, 4.5, 8 and 2 years after the diagnosis of internal mammary lymph node metastases our patients are still alive . To our knowledge, this is the first report on internal mammary lymph nodes as a site of late and very late solitary ovarian cancer recurrence and on the determining role of pet / ct scan for the diagnosis of isolated tumor involvement and therapeutic decisionmaking . Surgical dissection or radical radiotherapy
Stress is broadly defined as an actual or anticipated threat of well - being or disruption of organism homeostasis . Although the sensing and reaction to stress evolved to promote adaptation, modern workstyles and lifestyles represent challenges that render individuals susceptible to physical and mental disorders [25]. Multiple factors influence an individual's ability to cope with stress, for example, early life experiences, gender, or personality traits . Both vulnerability and resilience may be determined by genetic and epigenetic (gene environmental interactions) background [59]. Since the discovery of the communication between hypothalamus and pituitary in early 70s that opens a new window in our understanding of the brain - body communication, there are plethora of studies describing the high biological significance of stress and its responses which enables various adaptive processes to changing conditions . The most easily measureable and critical physiological response to stress involves the release of glucocorticoids (glucocorticoids, gcs). These hormones are synthesized and secreted into systemic circulation from the adrenal glands following stimulation by the anterior pituitary hormone adrenocorticotropic hormone (acth). The release of acth itself is increased in response to the secretion of corticotropin - releasing hormone (crh) and arginine vasopressin (avp) from neurons in the hypothalamic paraventricular nucleus (pvn). Together, the hypothalamus, pituitary, and adrenal glands constitute the so - called hypothalamo - pituitary - adrenal (hpa) axis, which plays an essential role in the adaptive response to psychogenic (e.g., fear) and physical (e.g., cellular lesion or pathogen invasion) stressors . The adaptive responses that are initiated by gcs occur in multiple tissues and involve alterations in numerous physiological (e.g., metabolic, cardiovascular, and immune) as well as behavioral (e.g., emotion, cognition, and motor) processes [1, 1012]. Normally, gc - driven negative feedback mechanisms at the different levels of the hpa axis serve to normalize gc secretion and restore homeostasis; however, and depending on the type, duration, and intensity of the stressful stimulus, gc hypersecretion may persist and become a potential threat for health . There is now abundant evidence that gcs can exert profound modulatory effects on a variety of brain functions from early development through to late life . Their actions are mediated by two receptors: the mineralocorticoid receptor (mr) and glucocorticoid receptor (gr), which belong to the superfamily of nuclear receptors that act as transcription modulators [13, 14]. In the brain, gr is ubiquitously expressed, whereas mr expression is more restricted to just a few structures (hippocampus, locus coeruleus, amygdala, prefrontal cortex, and nucleus of the solitary tract, as well as pvn neurons). Mr is also present in nonneuronal cells, namely, in glia and epithelial cells of the choroid plexus and ependyma . Binding assays using [h] corticosterone have shown the mr has a 10-fold higher affinity (kd = 0.5 nm) for gc compared to gr (kd = 5 nm), which means that, at basal gc levels, mr is occupied and activated whereas gr is only activated when gc levels reach a certain level, for example, during the circadian peak of gc secretion and during stress . Importantly, brain mr and gr both respond to the same endogenous ligand (cortisol in humans and larger mammals, corticosterone in rodents); further, mr and gr were reported to colocalize in the same pyramidal and granular neurons of the hippocampus . Given the gr and mr colocalization and relatively small difference in their affinity for endogenous gcs, the question arises as to whether they regulate distinct genes and/or coregulate transcription by heterodimerization . Heterodimerization of gr and mr was shown with high concentration of gc (stress level) in the nuclei of cultured hippocampal neurons . Moreover, evidence suggests that their cellular responses through regulation of distinct gene expression (as homodimers) depend strongly upon specific recruitment of coregulators [18, 19]. Synthetic gcs (e.g., dexamethasone, methylprednisolone) are routinely used in clinical situations due to their powerful anti - inflammatory and immunosuppressive actions . However, a growing body of evidence suggests that high gc exposure in early life can adversely program the hpa axis and increase the susceptibility to develop metabolic, neuropsychiatric, and neurodegenerative disorders [5, 20, 21]. In addition, there is now ample experimental evidence where elevated gc levels and prolonged exposure to stressful conditions induce structural remodeling of neurons with synaptic loss as well as alterations in glial functions, which are frequently maladaptive; see also figure 1 . In this brief review we discuss some of current knowledge about cellular targets and mechanisms through which stress and altered gc levels trigger changes in the brain that may lead towards the development and progression of neurodegenerative pathologies such as alzheimer's (ad) and parkinson (pd) disease . In addition to nongenomic mechanisms that are still incompletely identified, chronic stress and gc levels most likely influence neuronal function and connectivity by activating gr - mediated transcription . Grs are normally located in the cytoplasm in association with chaperone proteins such as the heat shock proteins hsp90 and 70 and the immunophilins fkbp51 and fkbp52 . Upon gc binding, conformational change of the gr - chaperone complex results in nuclear translocation of the gr [24, 25]. In the nucleus, gr binds to specific regions of dna, which possess glucocorticoid response elements (gre) within the promoters of target genes, leading to cell - type and context - dependent gene expression [2628]. Transcriptional regulation by gr may occur by (a) direct binding of gr homodimers to gre within dna sequences to stimulate transcription, for example, mitogen - activated protein kinase phosphatase-1 gene; (b) direct binding to negative gre elements to repress transcription; the gene encoding the prohormone from which acth is derived (proopiomelanocortin, pomc), crh, and the crh receptor genes are examples of negatively regulated genes; and (c) trans - repression or tethering, that is, association with other transcriptional factors that inhibit the transcriptional activity of gr . In the brain, identification of gr - modulated genes is difficult due to the anatomical complexity and cellular heterogeneity . Nevertheless, transcriptomic studies in the hippocampus have identified functional classes of gr target genes which include genes coding for neurotransmitter catabolism, neurotrophic factors and their receptors, signal transduction, energy metabolism, and cell adhesion . In addition to altering gene expression, growing evidence suggests that epigenetic mechanisms represent a means through which stress and gcs can leave long - lasting memories of past experiences which, in turn, contributes to shaping the organism's physical and mental health trajectory [21, 30, 31]; see figure 1 . Broadly, epigenetics refers to stable changes in the regulation and/or function of dna, rna, and/or proteins that do not involve alterations of their primary sequences . Two well - known examples of epigenetic marks induced by environmental stimuli (e.g., stress) are dna methylation and histone modification . The first evidence of epigenetic programing in the brain by early life adversity showed that poor maternal care in rats leads to methylation of exon 17 in the gr promoter, being accompanied by aberrant behaviors and altered hpa axis responses during adulthood [32, 33]. Subsequently, similar mechanisms were reported in humans who had experienced childhood adversity and in infants born to depressed mothers . The earlier studies in rats were replicated in mice in paradigms of prenatal gc exposure and early postnatal stress; we showed that these pre- and postnatal manipulations resulted in epigenetic modifications of the promoters of neurotransmitter (dopamine receptor 2), gr, and various gr target genes [37, 38] with long - lasting maladaptive behavioral consequences . Recent studies also suggest that early life events (e.g., intrauterine infections, maternal stress, and poor maternal and perinatal nutrition) may play a role in the onset of alzheimer's disease (ad), an age - related neurodegenerative disorder characterized progressive memory and cognitive deficits . From this perspective, ad is probably not determined by a single etiologic factor but results from the interplay between genetic and environmental factors throughout life, possibly explaining why monozygous twins can be discordant for ad . Albeit this is still controversial and the literature is sparse, it has been suggested that adverse events in early life, for example, maternal stress and poor maternal and perinatal nutrition, can potentially predispose eventually to ad through epigenetic programing of specific genes / pathways related to ad neurodegeneration . For example, maternal separation for the first 3 weeks of rodent life is shown to result in increase of ad cellular pathways (e.g., app misprocessing and tau hyperphosphorylation; see below) followed by synaptic and neuronal damage as well as cognitive deficits in adulthood suggesting the potential impact of early - life stress exposure to the precipitation of ad neurodegeneration later in life . While most current research on epigenetic mechanisms focuses on dna methylation, one recent study demonstrated that gc, acting via gr, increase the levels of histone deacetylase 2 (hdac2), an enzyme regulating dna expression, in the ck - p25 mouse . In general, how early life stressors reprogram the fetal brain and contribute to late - life development of neurodegenerative disorders (e.g., ad) is emerging as an exciting, new research field . Experimental evidence in animal studies indicates that stressful events in early life can impact the etiopathogenesis of another neurodegenerative disorder, parkinson's disease (pd), which is characterized by both motor and nonmotor symptoms . Depression, anxiety, apathy and interestingly fatigue are common nonmotor features occurring in around 30 to 58% of patients before the onset of motor symptoms in pd patients . In addition, the prevalence of cognitive impairment in pd ranges from 19 to 36% . The cellular mechanisms underlying these nonmotor symptoms in pd may share similarities to ad, particularly with respect to the molecular pathways activated by stress . Maternal separation was reported to exacerbate motor deficits and nigrostriatal lesion in an experimental model of pd . In an interesting study, pups of female animals, exposed to the bacterial endotoxin lipopolysaccharide (lps) during pregnancy, showed loss of dopaminergic (da) neurons . Since loss of dopaminergic neurons as well as related motor deficits is a characteristic feature of pd pathology, the above findings suggest that high lps levels in mothers might interfere with the development of da neurons in the fetus, thus enhancing susceptibility to pd . Accordingly, developmental stress may represent the first imprint in the brain and accumulatively with later stressful stimuli to affect nigrostriatal neurochemical reserve and precipitate the pd phenotype . Ad is a multifactorial neurodegenerative disorder with complex etiopathology . Besides early life stress (see above), accumulating clinical evidence strongly suggests that chronic stress in adulthood as well as elevated gc levels may have a role in the development of ad pathology and related dementia [47, 48]. In fact, high levels of cortisol are commonly found in ad patients' plasma, saliva, and/or csf [4953]; ad patients also show higher total daily secretion of cortisol . The potential link between stress / gc and ad described above is strengthened by emerging evidence that stress may advance the age of onset of the familial form of ad [47, 48, 55] and that cortisol levels in ad patients correlate with their memory deficits [56, 57] suggesting a role for gc on ad . Nevertheless, in the absence of longitudinal studies it is not clear from the available evidence as to whether elevated gc secretion is a cause or a consequence of ad disease . An important brain area in unraveling the interrelationship between stress, elevated gc, and ad pathology is the hippocampus, which is among the first areas affected in ad patients . Hippocampal lesions in ad brain are not only associated with the deficits in declarative, spatial, and contextual memory but could also be responsible for the suggested hpa axis dysregulation and the subsequent overproduction of gc found in ad patients due to the inhibitory role that hippocampus exhibits on hpa axis . Indeed, previous studies from our laboratories (and others) have shown that hippocampal neurons are particularly vulnerable to the adverse effects of stress and gc, their effects being manifested as dendritic atrophy and apoptotic cell death [22, 58]. Moreover, a large number of studies have shown that stress and elevated gc levels affect neurogenesis in adult brain with subsequent impairments of mood and cognitive behavior [59, 60]. More specifically, both acute and chronic exposure stress reduces adult neurogenesis, affecting hippocampal cell proliferation and, in certain studies, survival of newborns [61, 62]. In addition, administration of corticosterone showed the ability of glucocorticoids to damage neurogenesis in adult brain by inhibiting cell proliferation, differentiation and survival while the deleterious effect of stress and/or corticosterone on neurogenesis is gc - dependent . In a vicious cycle, alteration in neurogenesis of adult brain is recently shown to impact on gc negative feedback on the central elements regulating hpa axis activity [65, 66]. Moreover, perturbations in adult neurogenesis may also be related to the cognitive deficits associated with ad whereas contradictory findings support both increases and decreases of neurogenesis in brain of ad patients and tg animal models . Here, it is also worthwhile noting that stress and gc interfere with hippocampal - prefrontal cortex (pfc) connectivity and dendritic and synaptic plasticity in the pfc, thus disrupting executive functions . These pfc structural deficits are also likely to have consequences for central regulation of the hpa axis providing another neuroanatomical link between hpa axis dysregulation and subsequent gc hypersecretion and ad pathology . At the molecular level, ad pathology is characterized by amyloid beta (a) that forms deposits (senile plaques) and hyperphosphorylated forms of the cytoskeletal protein tau that aggregate into neurofibrillary tangles (nft) [6971]. A is the proteolytic product of a large transmembrane protein called amyloid precursor protein (app), which is sequentially cleaved by -secretase (bace-1) and -secretase (a complex of enzymes) to generate the production of a; this cellular pathway is often called app misprocessing . Many studies have demonstrated that the products of app misprocessing trigger neuropathological processes associated with ad such as synaptic malfunction (including impairment of long - term potentiation), neuronal atrophy and synaptic disintegration and loss as well as mitochondrial dysfunction, oxidative stress, and glial activation . Although still a subject of debate, several studies suggest that a also triggers the abnormal hyperphosphorylation of tau, nft formation, and neuronal loss . Moreover, cumulative evidence suggests that the detrimental effects of a are abolished in tau - ko mice, highlighting the essential mediatory role of tau protein in the neuro- and synaptotoxic effects of a [7377]. Further support for an essential role of tau in the establishment of ad pathology derives from clinical findings that have consistently shown that the cognitive deficits in ad patients correlate better with nft rather with a deposition per se . Moreover, gmez - isla et al . Demonstrated a strong correlation between neuronal loss in the cerebral cortex and increased nft burden with disease progression; no such correlation was found with a. in addition, the reduction of hippocampal volume in ad patients correlates better with csf levels of phosphorylated tau than with those of a . The evidence of a causal relationship between stress / gc and ad includes that from studies showing that either high gc levels and/or stress increase the production of a and exacerbate memory deficits in transgenic mouse models of ad [80, 81]. Specifically, chronic immobilization stress in transgenic mice expressing the amyloid precursor protein (app) v717ict-100 (a mutation which results in aggressive early onset ad) accelerates the appearance of extracellular a deposits and worsens memory deficits . Similar findings were obtained in vivo when young (prodromal) 3xtg - ad mice were treated with the synthetic gc, dexamethasone; the same authors also reported dexamethasone - induced app misprocessing in the n2a cell line, a finding matched by our own observations in pc12 cells . Further, green et al . Demonstrated that gcs upregulate the transcription of app and -secretase, whose promoters contain a glucocorticoid response element (gre). Consistent with the above, our studies in middle aged rats showed that stress and chronic gc drive app processing towards the generation of a and its precursor molecule (c99), both of which have neurotoxic and cognition - impairing properties (see also figure 1). The latter changes were accompanied by increases in the levels of -secretase (bace-1) and nicastrin, a protein found in the -secretase complex . Further experiments that attempted to mimic intermittent stressful events that may exert cumulative effects over the lifetime indicated that gc potentiate the app misprocessing pathway in previously stressed rats receiving a-infusions (see figure 2). In addition to triggering the amyloidogenic pathway, high levels of gc and stress can also instigate the aberrant hyperphosphorylation of tau protein that also characterized ad brain . Among the first reports suggesting a potential connection between gcs and tau was that from stein - behrens et al . Who demonstrated that gc exacerbate kainic acid - induced hippocampal neuronal loss with a contemporaneous increase in tau immunoreactivity . A later study showed that chronic treatment of 3xtg ad mice with dexamethasone leads to the somatodendritic accumulation of tau in the hippocampus, amygdala and cortex . Supporting those earlier studies, we showed that chronic stress or gc increase the levels of aberrantly hyperphosphorylated tau in the rat hippocampus and pfc (see figure 2). Importantly, the hyperphosphorylation occurred at certain tau epitopes that are strongly implicated in cytoskeletal dysfunction and synaptic loss (e.g., pser262) [86, 87] and hippocampal atrophy (e.g., pthr231) in ad patients . Here, it is pertinent to note that the extent of phosphorylation at thr231- and ser262-tau correlates strongly with severity of memory impairment, speed of mental processing, and executive functioning in ad patients [8991]. Although chronic stress and gc treatment exert similar, but not identical, effects on individual tau phosphoepitopes in vivo and in vitro, the overall evidence points to gc as the key mediator of the ad - like pathology induced by stress . On the other hand, some studies have suggested a role for at least one other stress - related molecule, namely, corticotrophin - releasing hormone (crh), as deletion of the crh receptor 1 gene in mice was found to block the detrimental effects of stress on tau phosphorylation [92, 93]. As shown at figure 2, information on the mechanisms underlying stress / gc - induced hyperphosphorylation of tau is only just beginning to emerge . For example, in vitro experiments indicate that the effects of stress / gc are mediated by glycogen synthase kinase 3 (gsk3) and cyclin - dependent kinase 5 (cdk5), both of which have well - established roles in tau hyperphosphorylation and the subsequent disruption of microtubules, features seen in the ad brain . We now also know that gc exposure increases tau accumulation by affecting turnover of the protein by reducing its degradation; the latter appears to result from dysregulation of molecular chaperones (e.g., hsp90 and hsp70) that are responsible for tau proteostasis (see figure 2). Interestingly, both these heat shock proteins also serve to maintain gr in a high affinity state, suggesting that these proteins may be the point at which gc / gr signaling intersects with the cellular machinery that regulates tau degradation . Using a transgenic mouse that expresses human p301l - tau (the most common tau mutation), we recently showed that chronic stress triggers different aspects of tau pathology in addition to inducing, its aberrant hyperphosphorylation and aggregation of tau into insoluble forms . Adding to the mechanistic understanding of stress - driven aggregation of tau, we also showed that chronic stress enhances caspase 3-mediated truncation of tau at its c - terminal, leading to an abnormal conformation of tau in the hippocampus (figure 2). This truncation - dependent misfolding of tau into an abnormal conformation is known to facilitate nucleation and recruitment of other tau molecules into neurotoxic aggregates [95, 96] before nft are formed [95, 97, 98]. It is interesting to note that chronically elevated gc secretion, usually in response to stress, is a major cause of major depressive illness . In light of the increasing volume of data implicating high gc levels in ad, it is important to consider that epidemiological studies implicate depression as a risk factor for the development of ad; this is supported by the observation that previously depressed subjects have increased amyloid plaque and nft loads . Different studies have in fact sought to discriminate between subjects undergoing normal aging from those suffering from depression or ad through the measurement of the various app cleavage products [101104]. While much remains to be discovered about the potentially important role of depression in ad pathology, it is interesting to note that antidepressant drugs, whose actions often involve reductions in gc secretion, inhibit the proteolytic cleavage of app into amyloidogenic products [104, 105]. Lastly, it deserves mentioning that a recent epidemiological study found that the prevalence and incidence of dementia in war veterans suffering from posttraumatic depression (ptsd) is twice as high as that in age - matched ptsd - free subjects . While ptsd is a condition quite distinct from major depression, these findings hint at the important influence lifetime stressful experiences can have on mental health, possibly through epigenetic mechanisms . The findings are also interesting since ptsd patients usually show hypoactivity of the hpa axis (versus hyperactivity in depression), suggesting that just a single but major stressful event involving transient gc hypersecretion can have long - lasting neuropathological consequences . Chronic inflammation is one of the central pathological features of ad with reactive microglia and astrocytes surrounding senile -amyloid plaques observed in both postmortem ad brain and animal models [107, 108]. Evidence from human studies suggests that glial activation is an early event; thus inflammatory markers are present in mild cognitive impairment cases that eventually progress to ad . Thus proinflammatory cytokines produced by activated glia in response to amyloid fibrils would be expected to activate hpa axis and increase gc levels . In vitro studies clearly show that a can be taken up through phagocytosis in microglia and thereafter degraded [110, 111]; thus, in ad setting, microglial likely have a beneficial role early in pathology . However, elevation of proinflammatory cytokines such as il-1 may also participate in mood disorders such as depression in ad . The importance of immune - related responses in the emergence of a burden, tau pathology, and dementia is gaining momentum as molecular comprehension of their actions is increasingly unraveled by human genetic and animal studies . Recent genome - wide association studies have identified variants in at least 16 genes involved in microglia / macrophage functions as risks for developing ad . Among them, 4 allele of apoe gene is a known strong risk factor, accelerating the age of onset of ad . Apoe is produced by both microglia and astrocytes; it regulates not only lipid and a metabolism but also microglial chemotaxis and proinflammatory cytokine expression . Trem2 is specifically expressed in myeloid cells where it promotes phagocytosis whilst inhibiting cytokine production . These and most other gwas genes identified are involved in aberrant microglial / macrophage responses with regard to a clearance and spread of tau pathology . Thus, crucial microglial functions such as motility and phagocytosis were impaired in app / ps1 mice; also in these mice the levels of a receptors (sra, cd36, rage) and a degrading enzymes (neprilysin, mmp9) were decreased with concomitant increase in proinflammatory cytokines tnf- and il-1 . Age, a primary risk factor for ad, is also an important contributor to dysfunction of innate immune responses . Microglial dystrophy and fragmentation observed in aging brain occur before the appearance of abnormal tau suggesting dysfunctional microglia could contribute to appearance of tau pathology . Chronic stress through gcs is known to prime and augment neuroinflammatory processes in the cortex and hippocampus upon subsequent proinflammatory challenges such as lps [119, 120]. Peripheral infections and stress are both known to affect the activation state of microglia and in ad pathology both could have detrimental effects on the functions of microglia . There is little known on how glucocorticoids influence glial functions during prodromal to emergence and progression of ad pathology . It would be important to understand whether gc through gr has any role in a degradation in astrocytes or myeloid cells . Parkinson's disease (pd) is a complex systemic and progressive neurodegenerative disease associated with both motor and nonmotor symptoms . The cardinal motor symptoms such as akinesia, resting tremor and rigidity mostly arise from preferential and substantial loss of dopaminergic neurons (5060%) in the substantia nigra pars compacta (snpc) with significant dopamine depletion in the sensorimotor striatum . The nonmotor symptoms include olfactory dysfunction and sleep behavior disorder as well as mood changes and cognitive impairment as discussed above . One principle histopathological feature is the presence of lewy bodies (lbs), which are proteinaceous inclusions containing mainly structurally altered presynaptic protein, alpha - synuclein, which, as recent evidence shows, plays a central role in pd pathology . Alpha - synuclein lb deposition was used by braak et al . As a principle pathological marker to monitor the progression and severity of pd . Pd is believed to originate from olfactory nucleus and autonomic nervous system progressing in an ascending manner to many brain regions such as substantia nigra, striatum, raphe, locus coeruleus, hypothalamic nuclei, hippocampus, amygdala, and cerebral cortex accounting for both motor and nonmotor symptoms [121123]. Thus, for example, pd patients with cortical lbs also suffer from dementia and visual hallucinations . While several gene mutations have been identified in familial forms of pd, the majority of pd cases are sporadic and of unknown etiology . Nevertheless, significant advances in the last decade on pd genetics, particularly genome - wide association, as well as pathophysiological mechanisms in various pd model systems, have contributed much to our comprehension of pd . Cellular processes such as oxidative and nitrative stress, mitochondrial dysfunction, and deregulated intracellular calcium levels as well as damaged proteostasis related to alpha - synuclein aggregation are the most studied and relate to dopamine neurodegeneration . As in ad patients, specifically, previous studies [54, 126128] including our own work show that plasma cortisol levels are significantly higher in idiopathic pd patients compared to control subjects; however, these high levels do not correlate to disease duration or to l-3,4-dihydroxyphenylalanine (l - dopa) treatment . Interestingly, the diurnal pattern of cortisol secretion in pd patients, in particular the normally quiescent nocturnal cortisol secretory pattern, is affected . Chronically elevated gc levels in pd patients suggest that hpa regulated - stress responses may impact pd pathology . Indeed, the role of stress was proposed as one of the underlying causes of pd as clinical reports show that stress triggers the appearance of pd symptoms or exacerbates the motor symptoms [130132]. The role of stress in pd is supported by few experimental studies such as food deprivation and tail - shock and maternal separation aggravate motor deficits in the 6-hydroxydopamine (6-ohda) pd model (6-hydroxydopamine local injections lesions the nigrostriatal pathway). In combined chronic stress exposure with 6-ohda lesion, stress was shown to worsen the 6-ohda - driven motor deficits, aggravate the neurodegeneration of nigrostriatal system, and completely block compensatory recovery of motor tasks [131, 134]. The precise actions of high gc levels in motor control following nigrostriatal lesions are yet not known . Analysis of gr expression in pd brains revealed that gr levels were reduced in the snpc and augmented in the putamen, compared to age - matched control subjects; similar results were found in mptp- (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine-) treated mice . The role of gc on the limbic arm of the dopaminergic circuitry related to reward and motivation as well as neuropsychiatric diseases has been extensively investigated (see below). Thus, from its known roles in mesolimbic circuitry, it has been postulated that gr also likely affect motor automated or habitual skills of the sensorimotor circuitry in the striatum by influencing nmda / ampa receptor functions in d1 and d2 receptor - medium spiny neurons (figure 3). Indeed, it has been shown that chronic stress leads to opposing structural changes in the limbic / associative and sensorimotor striatal circuitry with atrophy in the former and hypertrophy of sensorimotor striatum, leading to habit behavior . In addition, the roles of both glucocorticoids and noradrenaline were recently reported in habit memory . It is possible that gr - mediated changes in the putamen during the prodromal stage of pd play a role in preventing the appearance of motor symptoms, culminating in dopamine depletion and death of dopaminergic neurons in the substantia nigra . Altered stress responses most likely play an important role in nonmotor pd symptoms, particularly anxiety, depression, and mild cognitive impairment, which often precede motor symptoms . Interestingly, there is also evidence in pd for lower novelty - seeking and high harm avoidance personality traits with anxiety - associated symptoms [43, 137]. These observations suggest that, in the initial disease stage, stress - related alterations in gc - gr activity could impact both the motivation / cognitive - associated dopaminergic as well as nondopaminergic (serotonergic and noradrenergic) neuronal circuitry . This would also implicate dopaminergic neurons in the ventral tegmentum area (vta), which although relatively spared in pd are well - known to regulate reward and aversion by stress and have been implicated not only in addiction but also depression involving the transcriptional factor creb and bdnf [138141]. On the other hand, dorsolateral dopamine neurons in the sn (vulnerable in pd) were shown to respond to tasks involving working memory; thus, their demise could explain, in part, the cognitive deficits observed in pd . In fact, firing patterns of dopamine neurons in vta correlated with depressive - like behaviors in mice, although the effect appears to depend on the stress paradigm used to induce the depressive - like behavior [139, 143]. Electrophysiological evidence implicates changes in both d1r and d2r - medium spiny neurons (msns) in the ventral striatum, but the depressive - behaviors seems to preferentially affect d1r msns (figure 3). Glutamatergic receptors, nmda and ampa receptor functions were shown to be also altered in the d1r msns, notably nmdar - dependent ltd, reduced ampa / nmda receptor ratio and increased endocytosis of ampa receptors . Accumulating evidence points to inflammation resulting from chronic activation of innate and adaptive immune cells as playing an important role in both neurodegenerative processes and in nonmotor symptoms of pd . Using radiolabeled ligand c - pk-11195 for translocator protein, positron emission tomography (pet) studies in pd patients revealed an early activation of microglia in many brain regions including basal ganglia and midbrain [147, 148]. Furthermore, postmortem studies as well as analyses of serum and cerebrospinal fluid from pd showed high levels of proinflammatory mediators such as tnf-, il-1, inos, ifn-, and cox-2 . In line with observations in pd patients, presence of inflammatory mediators and glial reactivity in striatum and substantia evidence from recent genome - wide studies points to involvement of the immune system in the etiology of idiopathic pd . A number of susceptibility loci identified relate to genes expressed in immune cells such as hla - dqb1, lrrk2 or bst-1 [151, 152]. In addition, identified pd risk factors [such as age, environmental toxins (e.g., heavy metals or pesticides,) traumatic brain injury, and bacterial or viral infections] activate immune responses in periphery and brain . Activated microglia functioning as innate - immune competent cells are likely involved in releasing the above inflammatory molecules, thereby inducing dopamine neurodegeneration . Indeed, the important role of these proinflammatory mediators in promoting degeneration of dopaminergic neurons of substantia nigra was demonstrated using mice with specific knockout of these genes [153156]. The synthetic analogue of gcs, dexamethasone, was shown to attenuate dopamine neuronal loss by precluding activated microglia from releasing toxic inflammatory molecules [157, 158]. In adrenalectomized mice (lacking endogenous production of gcs), dopamine neuronal loss was augmented following mptp intoxication indicating that endogenous gcs do play a role in protecting dopamine neurons . Examination of gr in microglia revealed an increase in nuclear localization of gr following mptp treatment in mice, which coincided with a rise in systemic corticosterone levels, indicating that gr is activated in microglia during the degeneration of dopamine neurons . The unequivocal evidence that gr in microglia normally protects dopamine neurons was provided by experiments with mice in which the gr gene was selectively deleted in microglia / macrophages . Mptp treatment in these mice resulted in increased dopamine neuronal loss as well as increased microglial activation and expression of proinflammatory mediators . Indeed, the absence of gr in microglia resulted in sustained activation of nf-b as was shown in these microglial gr mutants . The above findings have a significant relevance for pd pathogenesis as nuclear expression of p65 subunit of nf-b, indicative of transcriptional activity, was found in the substantia nigra microglia of pd postmortem . Inflammatory reaction mediated by immune - competent cells such as microglia is normally a very tightly regulated process of limited duration . It is very likely that the processes involved in the regulation of glial immune responses including the expression and secretion of inflammatory mediators are compromised in pd and also ad resulting in a chronic inflammatory state with sustained activation of glia spanning many years . Immune - regulatory processes are compromised in aging (immunosenescence) and also during chronic stress where there is an increased susceptibility to infections as well as proinflammatory cytokine production . In aging, microglia show enhanced sensitivity to inflammatory stimuli, a process called priming which could be also induced by chronic stress and a dysregulated hpa axis . In this regard, there are several studies showing that chronically elevated gcs levels in response to different stressors cause proinflammatory cytokine production and sensitization or priming of microglia . Importantly, subsequent inflammatory or toxic stimuli result in aggravation of neuronal injury [119, 120, 163]. Moreover high and sustained gcs can exacerbate inflammation because of gc resistance whereby gr activity is affected . Thus it is plausible that gr transcriptional activity regulating inflammatory response of microglia is compromised in ad and pd patients who display persistently high gc levels . Recent experimental evidence shows that glia and peripheral immune cells are activated upon chronic psychogenic stress and that their actions are important in mood and behavior [164167]. Glial production of potent proinflammatory cytokines such as tnf-, il-6, and inf- are implicated in depression through stimulation of the kynurenine pathway (shift of serotonin synthesis from tryptophan to kyneurin) in activated astroglia, microglia, and infiltrating peripheral immune cells . Kynurenine, produced from tryptophan by activation of indoleamine 2,3-dioxygenase (ido), can be further converted to kynurenic acid or quinolinic acid, the latter affecting the function of both monoaminergic and glutamatergic neurons . Quinolinic acid toxicity with increased glutamate release results in lipid peroxidation and nitrative stress [168, 169] evidence shows that the kynurenic acid / tryptophan ratio is altered in csf and serum in pd patients . Another means by which glial activation and proinflammatory cytokines promote mood anomalies in pd is through reducing neurogenesis in hippocampal subgranular zone, thus affecting hippocampus - mediated regulation of mood and cognition . Clinical and preclinical studies suggest that chronic stress / elevated gc levels may be an etiological factor in the development and progression of both ad and pd pathologies . Growing evidence indicates that the pathological manifestations of chronic stress include neuronal and synaptic atrophy / malfunction as well as immunosuppression, but our understanding of the underpinning mechanisms is still poor and calls for more research not only to identify therapeutic inroads but, also, preventative measures or ways to delay onset of disease.
The chemistry of metallocenes rapidly expanded in the 1970s with organozircocene emerging as one of the most useful classes of transition metal derivatives for use in organic synthesis . One important reagent belonging to this class of organometallic compounds is the chlorobis(cyclopentadienyl)hydrido - zirconium 1, also called zirconocene hydrochloride but better known as schwartz's reagent . Wailes and weigold were the first to prepare 1 and, subsequently, schwartz and many other scientists exploited its potential in the functionalization of organic compounds . Selected examples of the utility of this reagent in organic synthesis are depicted in figure 1 . Schwartz's reagent is most often used in the hydrozirconation of triple and double bonds (figure 1, reaction a). However, its use in this reaction is sometimes hampered by the functional group compatibility of the process that is limited by the oxophilic, hard lewis - acid character of the schwartz reagent . This characteristic has been therefore exploited in other useful synthetic organic transformations such as the chemoselective reduction of tertiary amides to aldehydes (figure 1, reaction b), and the reduction of secondary amides to n - substituted imines (figure 1, reaction c). The latter transformation, when performed with most other metal hydride reagents, results either in over - reduction of the imine to give the corresponding amine or in the reductive cleavage of the amide . Interestingly, 1 has also been employed as a reducing agent for phosphine oxides to the corresponding phosphines (figure 1, reaction d). Recently, bhat and co - workers reported a very mild and useful deacetylation procedure of acetamides involving reagent 1 . The methodology proved to be very efficient for aromatic, heteroaromatic, and aliphatic amides and moreover no epimerization was observed during the n - deacetylation of chiral acetamides . The amine functionality is present in many compounds of biological interest, for instance natural products and pharmaceuticals . Nucleoside and nucleotide chemistry frequently involves protecting procedures at the amine and hydroxyl groups and benefit of selective deprotection methods, in order to obtain the target compound efficiently without any side reactions . N - acetyl protection is one of the most widely used in nucleoside and nucleotide chemistry . However, the requirement of strong base or acid and/or high reaction temperatures during the deprotection step (n - deacetylation) is often not compatible with some nucleosidic substrates and especially their prodrugs . Harsh conditions are also likely to yield fully deprotected products, an outcome often undesired . Zinc bromide and hydrazine hydrate were initially reported as selective and mild n - deacetylating agents for nucleosides, although their compatibility with other protecting groups was not explored broadly . Our goal was the identification of a selective n - deacetylation methodology involving mild reaction conditions that could be applied to a broad spectrum of differently protected nucleosides . Therefore, we became immediately interested in bhat's methodology and in its possible application in nucleoside chemistry . To the best of our knowledge, no attempt to use schwartz's reagent in the n - deacetylation of nucleosides and their prodrugs has been previously reported . Herein, we would like to report our efforts directed at deacetylation of n - acetyl purine and pyrimidine nucleoside analogues bearing a variety of protecting groups (pgs) at the hydroxyl moiety, commonly used in nucleoside chemistry, such as o - acetyl, o - tert - butyloxycarbonyl, o - tert - butyldimethylsilyl, o - tetrahydropyranyl, o - benzoyl, and o - isopropylidene groups, with the aim of evaluating the ability of reagent 1 to selectively remove the acetyl group from the amino moiety, leaving the other pgs untouched . Our investigation began with the preparation, according to literature procedures, of differently substituted n - acetyl-2-deoxycytidine (2a - c) and n - acetylcytidine (2d - g), as indicated in table 1 . With all the o - protected nucleosides in hand, we proceeded to test the deacetylation reaction . First, compound 2a was subjected to the action of three equivalents of reagent 1 in a thf solution, under a nitrogen atmosphere, for 30 minutes (table 1, entry 1). Pleasingly, we were able to isolate after aqueous work - up and column chromatography compound 3a in 57% yield, where only the n - acetyl group was removed . Application of these conditions to 2b, once again, led to the selective removal of the acetyl group from the amino moiety without affecting the silicon protected hydroxyl groups and yielding 3b in 49% yield (table 1, entry 2). Table 1 deacetylation reaction of differentially protected n - acetyl-2-deoxycytidines and n - acetylcytidines, promoted by schwartz reagententrycpdr1r2r31 (eq)temp (c)time (h)prod.yield (%) recovered sm (%) 12ahacac3rt0.53a574022bhtbdmstbdms3rt0.53b493332chthpthp3rt33c682042dohbocboc3rt0.53d395552eoacacac3rt33e702362fobzbzbz3rt33f534172gcme2 h3rt33g553882ahacac6rt33a554292ahacac370123a4839 similar results were obtained testing the compatibility of schwartz's reagent with other protecting groups such as tetrahydropyranyl, tertbutoxycarbonyl, benzoyl, and isopropylidene (table 1, entries 37). In all examples, the o - protecting groups were unaffected by this procedure, and only the acetyl group on the nucleobase was removed, affording the desired compounds in moderate to good yields (3970%), along with the recovery of unreacted starting material . Attempts to increase the yield of the desired n - deacetylated nucleoside either by increasing the equivalents of 1 (up to six) or by adding it portionwise over a period of three hours, proved to be unsuccessful and some amount of starting material was always recovered (table 1, entry 8). Incrementing the temperature up to 70c did not enhance the yield (table 1, entry 9). Subsequently, we decided to broaden the scope of the methodology by testing it on examples of other pyrimidine and purine nucleoside derivatives bearing different pgs (table 2). Lamivudine was acetylated on both oxygen and amine groups and the resulting n, o - diacetylated compound 2h was then submitted to the deacetylating protocol (table 2, entry 1). As expected, the acetyl group was removed only from the nitrogen on the nucleobase, leaving the o - acetyl group untouched . Table 2 n - deacetylation of protected pyrimidine and purine nucleosides, promoted by schwartz's reagententrycompoundproductyield (%) 1 56 5 53 2 25 3 47 4 76 n - acetyl - o - trityl - lamivudine 2i was found to be a poor substrate for this protocol (table 2, entry 2). The desired product 3i was obtained only in 25% isolated yield, most probably due to the partial trityl group instability to the reaction conditions . No starting material was isolated in this case . When the protocol was tested on purine nucleoside analogues, reagent 1 proved efficient in removing the acetyl group selectively from the amine group on both the guanine ring of peracetylated acyclovir 2j and the adenine ring of n - acetyl-5-o - acetyl-2,3-o - isopropylidene adenosine 2k, affording 3j and 3k respectively in 47% and 76% isolated yields (table 2, entries 3,4). Finally, n, n - diacetyl-2,3-isopropylidene adenosine 2l was submitted to the optimized protocol . In this case schwartz's reagent was able to remove both n - acetyl groups, affording compound 3k in 53% yield, with only traces of mono - n - acetylated derivative 2k . . Reported a practical method to generate schwartz's reagent in situ using li(ot - bu)3alh, demonstrating the advantage of this procedure both in reducing tertiary amides to aldehydes and in hydrozirconation reactions, in comparison to the use of the commercial schwartz's reagent . We, therefore, decided to assess this efficient procedure in the deacetylation of nucleoside 2 g in order to be able to compare the in situ methods with the use of the preformed 1 (scheme 1). Therefore, to a thf solution of 2 g and 1.5 equivalents of cp2zrcl2 in thf, solid li(ot - bu)3alh was added in one portion and the mixture was stirred for 40 minutes . Aqueous workup, followed by column chromatography, afforded compound 3 g in 80% isolated yield, demonstrating a yield improvement from the previous 55%, possibly due to the partial air, light, and moisture sensitivity of 1, which are reported to be responsible for the reduction of its effectiveness with time . Figure 1 n - deacetylation of n - acetyl-2,3-o - isopropylidene cytidine 2 g with in situ generated schwartz's reagent 1 . Nucleoside analogues (nas) are an important class of molecules accounting for half of all antiviral drugs currently on the market and a number of anticancer agents that are widely used . Unfortunately, many nucleoside analogues are not phosphorylated effectively in vivo, and thus their therapeutic potential is often quite limited . Using various approaches, the ionizable phosphate group can be masked by derivatization, generating prodrugs with increased biological activity . Among different nucleoside monophosphate prodrug strategies is the phosphoroamidate prodrug approach (protide), consisting of an amino acid ester promoiety linked via p gilead has just launched on the market its anti - hcv protide, sofosbuvir (psi-7977), whereas nucana biomed has taken to trial a gemcitabine protide (nuc-1031), for patients with advanced solid tumours . Considering the importance that the protide technology is assuming in the antiviral and anticancer scenarios, we were interested in checking if schwartz's reagent is compatible with the phosphoramidate motif and whether this protocol can be used at later stage in the synthesis of this class of prodrugs . Figure 2 synthesis and deacetylation of n - acetyl-2,3-o - isopropylidene- cytidine-5-o-[napthyl (cyclohexyl - l - alanyl)] phosphate (5) promoted by schwartz reagent . Phosphoroamidate 5 was synthesized from 2 g and (2s)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate 4 in 40% yield (scheme 2), according to a previously described method . Phosphoroamidate 5 was then treated with three equivalents of the schwartz's reagent, yielding the corresponding deacetylated prodrug 6 in 65% isolated yield . At the same time we were conducting our investigations, snieckus et al . Reported a practical method to generate schwartz's reagent in situ using li(ot - bu)3alh, demonstrating the advantage of this procedure both in reducing tertiary amides to aldehydes and in hydrozirconation reactions, in comparison to the use of the commercial schwartz's reagent . We, therefore, decided to assess this efficient procedure in the deacetylation of nucleoside 2 g in order to be able to compare the in situ methods with the use of the preformed 1 (scheme 1). Therefore, to a thf solution of 2 g and 1.5 equivalents of cp2zrcl2 in thf, solid li(ot - bu)3alh was added in one portion and the mixture was stirred for 40 minutes . Aqueous workup, followed by column chromatography, afforded compound 3 g in 80% isolated yield, demonstrating a yield improvement from the previous 55%, possibly due to the partial air, light, and moisture sensitivity of 1, which are reported to be responsible for the reduction of its effectiveness with time . Figure 1 n - deacetylation of n - acetyl-2,3-o - isopropylidene cytidine 2 g with in situ generated schwartz's reagent 1 . Nucleoside analogues (nas) are an important class of molecules accounting for half of all antiviral drugs currently on the market and a number of anticancer agents that are widely used . Nas mostly require phosphorylation to be active . Unfortunately, many nucleoside analogues are not phosphorylated effectively in vivo, and thus their therapeutic potential is often quite limited . Using various approaches, the ionizable phosphate group can be masked by derivatization, generating prodrugs with increased biological activity . Among different nucleoside monophosphate prodrug strategies is the phosphoroamidate prodrug approach (protide), consisting of an amino acid ester promoiety linked via p gilead has just launched on the market its anti - hcv protide, sofosbuvir (psi-7977), whereas nucana biomed has taken to trial a gemcitabine protide (nuc-1031), for patients with advanced solid tumours . Considering the importance that the protide technology is assuming in the antiviral and anticancer scenarios, we were interested in checking if schwartz's reagent is compatible with the phosphoramidate motif and whether this protocol can be used at later stage in the synthesis of this class of prodrugs . Figure 2 synthesis and deacetylation of n - acetyl-2,3-o - isopropylidene- cytidine-5-o-[napthyl (cyclohexyl - l - alanyl)] phosphate (5) promoted by schwartz reagent . Phosphoroamidate 5 was synthesized from 2 g and (2s)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate 4 in 40% yield (scheme 2), according to a previously described method . Phosphoroamidate 5 was then treated with three equivalents of the schwartz's reagent, yielding the corresponding deacetylated prodrug 6 in 65% isolated yield . In conclusion, we have demonstrated that the general and mild method for the deacetylation of n - acetyl group developed by bhat et al . Can be successfully applied to purine and pyrimidine nucleosides, with diverse o - protection . Moreover, we have shown that schwartz's reagent is compatible with the phosphoramidate moiety and can be used for n - deacetylation reaction in the later stages of the synthesis of this class of nucleoside monophosphate prodrugs . Lastly, we have proved that modification of the reported procedure involving in situ generation of schwartz's reagent leads to yield improvement and is promising of similar results on other nucleoside substrates . Schwartz reagent was purchased from sigma - aldrich or prepared in situ as described and all nucleosides used as starting materials were purchased from carbosynth . H nmr (500 mhz), c nmr (125 mhz), and p nmr (202 mhz) spectra were recorded on a bruker avance 500 mhz spectrometer at 25c . Chemical shifts () are quoted in parts per million (ppm) relative to internal cd3od (3.34 h nmr, 49.86 c nmr) and cdcl3 (7.26 h nmr, 77.36 c nmr) or external 85% h3po4 (0.00 p nmr). The following abbreviations are used in the assignment of nmr signals: singlet (s), doublet (d), triplet (t), quartet (q), quintet (qn), multiplet (m), broad singlet (bs), doublet of doublet (dd), and doublet of triplet (dt). High and low - resolution mass spectrometry analyses were performed on a bruker microtof - lc system . To a stirred solution of n - acetyl nucleoside (100 mg) in anhydrous thf (2 ml), schwartz's reagent (36 eq .) Is added at room temperature and the reaction mixture is stirred for 0.53 hours . The combined organic layer is washed with brine solution and dried over anhydrous na2so4, filtered and evaporated to afford the crude product, which is purified by silica gel column chromatography to finally afford pure deacetylated nucleoside . N - deacetylation reaction via in situ generation of schwartz's . To a solution of n - acetyl nucleoside (100 mg) and cp2zrcl2 (1.5 equiv) in thf (2 ml) at rt, solid lialh(otbu)3 (1.5 equiv) was rapidly added . The resulting solution was stirred at rt for 40 minutes and the reaction was monitored by tlc analysis . The combined organic layer is washed with brine solution and dried over anhydrous na2so4, filtered and evaporated to afford the crude product, which is purified by silica gel column chromatography to finally afford pure deacetylated nucleoside . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3a as a white solid (0.050 g, 57% yield). H nmr (500 mhz) cdcl3 h 2.08 (3h, s, ococh3), 2.09 (3h, s, ococh3), 2.192.12 (1h, m, h-2a), 2.58 (1h, dd, j = 4.0, 14.0 hz, h-2b), 4.244.26 (1h, m, h-4), 4.32 (2h, d, j = 12.5 hz, h-5), 5.185.19 (1h, m, h-3), 6.11 (1h, d, j = 6.5 hz, h-5), 6.22 (1h, t j = 6.5 hz, h-1), 7.57 (1h, d, j = 6.5 hz, h-6). C nmr (125 mhz) cdcl3 c 20.90, 20.98 (ch3), 38.24 (ch2 - 2), 63.82 (ch2 - 5), 74.12 (c-3), 82.63 (c-4), 88.18 (c-1), 95.87 (c-5), 140.21 (c-6), 170.32 (c - o), 155.15 (c-2), 164.90 (c-4); ms (es+) m / z (312) [m+1]; hrms (es) m / z found 312.1192 [calcd for c13h18n3o6 (m+h) 312.1190]. The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3b as a white solid (0.044 g, 49%). H nmr (500 mhz) cdcl3 h 0.12 (6h, s, 2 ch3), 0.15 (6h, s, 2 ch3), 0.91 (9h, s, c(ch3)3), 0.98 (9h, s, c(ch3)3), 2.092.13 (1h, m, h-2a), 2.352.40 (1h, m, h-2b), 3.83 (1h, dd, j = 2.5, 11.5 hz, h-5a), 3.92 (1h, dd, j = 3.0, 11.0 hz, h-5b), 3.933.96 (1h, m, h-4), 4.474.49 (1h, m, h-3), 5.89 (1h, d, j = 7.5 hz, h-5), 6.24 (1h, t j = 6.0 hz, h-1), 7.97 (1h, d, j = 7.5 hz, h-6); c nmr (125 mhz) cdcl3 c 4.92, 4.58 (sich3), 17.93, 18.94 (c(ch3)3) 26.04, 26.22 (c(ch3)3), 42.13 (c-2), 62.04 (c-5), 70.44 (c-3), 85.80 (c-1), 87.25 (c-4), 94.26 (c-5), 141 (c-6), 155.94 (c-2), 165.87 (c-4); ms (es+) m / z 456 [m+1]; hrms (es) m / z found 456.2710 [calcd for c21h42n3o4si2 (m+h) 456.2708]. 3,5-o - bis(tetrahydropyranyl)-2-deoxycytidine (3c). Prepared from 2c according to standard procedure for the n - deacetylation reaction . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3c as a white solid (0.061 mg, 68%). H nmr (500 mhz) cd3od h 1.441.48 (8h, m, ch2 py), 1.611.76 (4h, m, ch2 py), 2.342.50 (1h, m, h-2b), 1.952.12 (1h, m, h-2a), 3.433.47 (2h, m, ch2o py), 3.51 (0.25h, dd, j = 3.0, 11.0 hz, h-5b), 3.53 (0.25h, dd, j = 3.0, 11.5 hz, h-5b), 3.543.61 (0.5h, dt j = 3.9, 16.5 hz, h-5b), 3.713.82 (2h, m, ch2o py), 3.803.86 (0.5, m, h-5a), 3.903.95 (0.5h, m, h-5a), 4.074.11 (0.5h, m, h-4), 4.134.15 (0.25h, m, h-4), 4.174.19 (0.25h, m, h-4), 4.294.31(0.25h, m, h-3), 4.344.36 (0.5h, m h-3), 4.404.41 (0.25h, m, h-3), 4.554.67 (2h, m, cho py), 5.775.80 (1h, m, h-5), 6.116.16 (1h, m, h-1), 7.88 (0.25h, d, j = 7.5 hz, h-6), 7.89 (0.25h, d, j = 7.5 hz, h-6), 7.94 (0.25h, d, j = 7.5 hz, h-6), 7.97 (0.25h, d, j = 7.0 hz, h-6); c nmr (125 mhz) cd3od c 20.47, 20.50, 20.68, 20.77 (ch2 py), 26.51, 26.55 (ch2 py), 31.69, 31.75, 31.94, 32.01 (ch2 py), 39.54, 39.71, 40.82, 40.98 (c-2), 63.69, 63.73, 63.75, 63.92 (ch2o), 68.30, 68.42, 68.49, 68.80 (c-5), 77.39, 77.82, 78.25 (c-3), 85.41, 85.47, 85.97, 85.18 (c-4), 87.65, 87.83, 87.87, 87.97 (c-1), 95.80, 95.90 (c-5),99.29, 99.36, 99.79, 99.88, 100.14, 100.33, 100.97,101.03 (ch py), 142.38, 142.81 (c-6), 158.17 (c-2), 167.63 (c-4); ms (es+) m / z = 396 [m+1]. Hrms (es) m / z found 396.2127 [calcd for c19h30n3o6 (m+h) 396.2129]. The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3d as a white solid (0.035 g, 39% yield). H nmr (500 mhz) cd3od h 1.47 (9h, s, c(ch3)3), 1.50 (9h, s, c(ch3)3), 3.77 (1h, dd, j = 3.0, 12.5 hz, h-5), 3.88 (1h, dd, j = 2.5, 12.5 hz, h-5), 4.224.20 (1h, m, h-4), 5.27 (1h, dd, j = 5.5 hz, j = 5.5 hz, h-3), 5.32 (1h, dd, j = 4.0, 4.0 hz, h-2), 5.90 (1h, d, j = 7.5 hz, h-5), 6.10 (1h, d, j = 4.5 hz, h-1), 8.0 (1h, d, j = 7.5 hz, h-6); c nmr (125 mhz) cd3od c 27.96, 28.02 (c(ch3)3), 31.70 (c(ch3)3), 61.96 (c-5), 74.17 (c-3), 77.25 (c-2), 83.93 (c-4), 89.94 (c-1), 96.64 (c-5), 143.07 (c-6), 153.61, 153.89 (coc(ch3)3), 158.01(c-2), 167.56 (c-4); ms (es+) m / z = 444 [m+1]. Hrms (es) m / z found 444.1973 [calcd for c19h30n3o9 (m+h) 444.1977]. The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3e as a white solid (0.063 g, 70% yield). H nmr (500 mhz) cd3od h 2.10 (3h, s, coch3), 2.11 (3h, s, coch3), 2.12 (3h, s, coch3), 4.344.40 (3h, m, h4, h5), 5.41 (dd, j = 5.0, 5.9 hz, 1h, h3), 5.48 (dd, j = 4.6, 6.0 hz, 1h, h3), 5.96 (1h, d, j = 7.5 hz, h5), 5.98 (1h, d, j = 4.6 hz, h1), 7.69 (1h, d, j = 7.5 hz, h6); c nmr (125 mhz) cd3od c 20.40 (coch3), 20.45 (coch3), 20.69 (coch3), 64.32 (c5), 71.68 (c3), 74.69 (c2), 80.99 (c4), 91.09 (c1), 96.76 (c5), 143.23 (c6), 157.91 (c4), 167.82 (c2), 171.35 (coch3), 171.39 (coch3), 172.19 (coch3); ms (es+). Hrms (es) m / z found 370.1243 [calcd for c15h20n3o8 (m+h) 370.1245]. Cytidine 2, 3, 5-tribenzoate (3f). Prepared from 2f according to standard procedure for the n - deacetylation reaction . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 96/4 to give 3f as a white solid (0.049 g, 53% yield). H nmr (500 mhz) cd3cl h 2.12 (3h, s, coch3), 4.63 (1h, dd, j = 3.9, 12.2 hz, h5), 4.724.68 (1h, m, h4), 4.76 (1h, dd, j = 2.8, 12.2 hz, h5), 5.745.78 (1h, m, h2), 5.795.84 (1h, m, h3), 6.31 (1h, d, j = 4.2 hz, h1), 7.317.24 (5h, m, ph, h5), 7.377.42 (2h, m, ph), 7.437.49 (2h, m, ph), 7.507.55 (1h, m, ph), 7.817.89 (5h, m, ph, h6), 7.988.08 (2h, m, ph), 9.58 (1h, br s, nhcoch3). C nmr (125 mhz) cd3cl c 63.92 (c5), 71.20 (c3), 74.45 (c2), 80.15 (c4), 89.25 (c1), 95.45 (c5), 128.46 (ch ar), 128.49 (ch - ar), 128.68 (ch - ar), 128.75 (c - ar), 129.40 (c - ar), 129.71 (ch - ar), 129.85 (ch - ar), 129.97 (ch - ar), 133.49 (ch - ar), 133.61 (ch - ar), 133.62 (ch - ar), 141.36 (c6), 155.28 (c5), 165.33 (coph), 165.37 (coph), 165.39 (c4), 166.14 (coph); ms (es+) m / z (556) [m+1]; hrms (es) m / z found 556.1711 [calcd for c30h26n3o8 (m+h) 556.1714]. 2, 3-o - isopropylidene - cytidine (3 g). Prepared from 2 g according to standard procedure for the n - deacetylation reaction . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3 g as a white solid (0.043 g, 49% yield). Prepared according to the in situ schwartz's reagent generation procedure for the deacetylation reaction . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3 g as a white solid (0.070 g, 80% yield). H nmr (500 mhz) cd3od h 1.25 (3h, s, ch3), 1.45 (3h, s, ch3), 3.62 (1h, dd, j = 4.5, 12.0 hz, h-5a), 3.69 (1h, dd, j = 3.5, 12.0 hz, h-5b), 4.114.13 (1h, m, h-4), 4.724.74 (1h, m, h-3), 4.784.80 (1h, m, h-2), 5.75 (1h, s, h-1), 5.81 (1h, d, j = 7.5 hz, h-5), 7.73 (1h, d, j = 7.5hz, h-6); c nmr (125 mhz) cd3od c 25.55 (cch3)2), 27.55 (cch3)2), 63.19 (c5), 82.25 (c3), 86.40 (c2), 88.69 (c4), 95.45 (c1), 96.20 (c5), 115.03 (cch3)2), 144.42 (c6), 154.90 (c2), 167.82 (c4).ms (esi+) m / z = (284) [m+1].hrms (es) m / z found 284.1243 [calcd for c12h18n3o5 (m+h) 284.1241]. The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3h as a white solid (0.048 g, 56% yield). H nmr (500 mhz) cd3od h 1.90 (3h, s, coch3), 2.95 (1h, dd, j = 3.0, 12.5 hz, h-4), 3.35 (1h, dd, j = 5.5, 12.5 hz, h-4), 4.20 (1h, dd, j = 3.0, 12.5 hz, ch2oh), 4.35 (1h, dd, j = 5.0, 12.5 hz, ch2oh), 5.20 (1h, dd, j = 3.5, 5.0 hz, h-2), 5.70 (1h, d, j = 7.5 hz, h-5), 6.10 (1h, dd, j = 3.5, 5.5 hz, h-5), 7.68 (1h, d, j = 7.5 hz, h-6); c nmr (125 mhz) cd3od c 20.62 (ch3), 38.15 (c-4), 65.64 (ch2oh), 84.44 (c-2), 89.11 (c-5), 96.03 (c-6), 142.49 (c-5), 157.83 (c-2), 167.72 (c-4), 172.08 (coch3); ms (esi+) m / z = 272 [m+1]. Hrms (es) m / z found 272.0701 [calcd for c10h14n3o4s (m+h) 272.0700]. The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3i as a white solid (0.023 g, 25% yield). H nmr (500 mhz) cdcl3 h 3.09 (1h, dd, j = 2.5, 12.5 hz, h-4), 3.44 (1h, dd, j = 5.5, 12.5 hz, h-4), 3.49 (2h, d, j = 3.5 hz, ch2oh), 5.19 (1h, t, j = 3.5 hz, h-2), 5.38 (1h, d, j = 7.0 hz, h-5), 6.27 (1h, dd, j = 3.0, 5.5 hz, h-5), 7.267.15 (10h, m, ph), 7.397.37 (5h, m, ph), 7.93 (1h, d, j = 7.0 hz, h-6); c nmr (125 mhz) cdcl3 c 36.72 (c-4), 61.31 (ch2oh), 83.90 (c-2), 84.75 (c-5), 84.88 (c - ph), 91.12 (c-5), 124.39, 125.59, 126.23 (ch - ph), 139.06 (c-6), 140.08 (c - ph), 153.01 (c-2), 163.16 (c-4); ms (esi+) m / z = 472 [m+1]. Hrms (es+) m / z found 472.1685 [calcd for c27h26n3o3s (m+h) 472.1689]. The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3j as a white solid (0.041 g, 47%). H nmr (500 mhz) (cd3)2so h 1.95 (3h, s, coch3), 3.65 (2h, dd, j = 7.0, 7.5 hz, ch2o), 4.10 (2h, dd, j = 5.0, 6.0 hz, ch2o), 6.55 (2h, s, nch2o), 7.80 (1h, s, h-8), 10.65 (1h, brs, nh); c nmr (125 mhz) (cd3)2so c 20.53 (ch3), 62.70 (nch2), 66.48 (ch2o), 71.80 (nch2o), 116.45 (c-5), 137.62 (c-8), 151.39 (c-4), 153.92 (c-2), 156.71(c-6), 170.22 (coch3); ms (esi+) m / z = 268 [m+1]. Hrms (es) m / z found 268.1044 [calcd for c10h14n5o4 (m+h) 268.1040]. Prepared from 2k or 2l according to standard procedure for the n - deacetylation reaction . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 3k as a white solid (0.068 g, 76% from 2k; 0.034 g, 53% from 2l). H nmr (500 mhz) cd3cl h 1.33 (3h, s, ch3).1.55 (3h, s, ch3), 1.92 (3h, s, coch3), 4.15 (1h, dd, j = 6.2 hz, 11.7 hz, h5), 4.29 (1h, dd, j = 4.4 hz, 11.7 hz, h5), 4.434.38 (1h, m, h4), 4.99 (1h, dd, j = 3.4 hz, 6.3 hz, h3), 5.41 (1h, dd, j = 2.0 hz, 6.3 hz, h2), 5.94 (2h, br s, nh2), 6.04 (1h, d, j = 2.0 hz, h1), 7.82 (1h, s, h8), 8.28 (1h, s, h2); c nmr (125 mhz) cdcl3 c 20.68 (coch3), 25.41 (cch3), 27.14 (cch3), 64.09 (c5), 81.73 (c4), 84.23 (c3), 85.04 (c2), 91.06 (c1), 114.59 (c5), 120.35 (cch3), 139.71 (c8), 149.27 (c4), 153.22 (c2), 155.71 (c6), 170.41 (coch3). Ms (es+) m / z (350) [m+1]; hrms (es) m / z found 350.1451 [calcd for c15h20n5o5 (m+h) 350.1459]. N - acetyl-2,3-o - isopropylidene-5-o-[napthyl (cyclohexyl - l - alanyl)] pho - sphate cytidine (5). To a solution of 2 g (0.5 g, 1.53 mmol) and (2s)-cyclohexyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propa - noate (4, 1.02 g, 3.073 mmol) in anhydrous thf (10ml), 1 m bumgcl (3.08 ml, 3.073 mmol) is added dropwise and the reaction mixture is stirred at room temperature overnight . After this period the crude is purified by column chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 . The compound 5 is recovered as a white solid (0.420, 40% yield). H nmr (500 mhz) cd3od h 1.281.40 (11h, m, chch3, ch3, 2 ch2-chex), 1.521.58 (4h, m, ch3, ch2-chex), 1.691.78 (4h, m, 2 ch2-chex), 2.15, 2.18 (3h, s, coch3), 3.994.02 (1h, m, chch3), 4.404.52 (3.5h, m, ch-2, h-4, h-5), 4.684.72 (2h, m, h-2, h-3), 4.90 (0.5h, m, h-3), 5.80 (1h, d, j = 2.5 hz, h1), 7.25 (0.5h, d, j = 7.5 hz, h-5), 7.35 (0.5h, d, j = 7.5 hz, h-6), 7.397.42 (1h, m, naph), 7.477.49 (1h, m, naph), 7.527.58 (2h, m, naph), 7.707.72 (1h, m, naph), 7.877.89 (1.5h, m, naph, h-5), 7,96 (0.5h, d, j = 7.5 hz, h-5), 8.088.10 (0.5h, m, naph), 8.138.15 (0.5h, m, naph); c nmr (125 mhz) cd3od c 20.45 (d, jcp = 7.5 hz, chch3), 20.62 (d, jcp = 7.5 hz, chch3), 24.57 (coch3), 24.60, 24.62 (ch2-chex), 25.42, 25.52, (ch3), 26.38 (ch2-chex), 27.37, 27.43 (ch3), 32.35, 32.45 (ch2-chex), 51.89, 51.97 (chch3), 68.01, 68.02 (d, jcp = 7.5 hz, c-5), 75.00 (cho), 82.22, 82.24 (ch3), 86.57 (ch2), 87.51 (d, jcp = 2.5 hz, c-4), 87.57 (d, jcp = 2.5 hz, c-4), 96.58, 96.65 (c-1), 98.02, 98.12 (c-6), 115.06, 115.21 (c - naph), 116.22 (d, jcp = 3.7 hz, ch - naph), 116 . 49 (d, jcp = 3.7 hz, ch - naph), 122.60, 122.68, 126.15, 126.53 (ch - naph), 126.59, 127.60, 127.70, 127.80, 127.85, 127.93, 128.97 (ch - naph), 136.28 (c - naph), 147.20, 147.35 (c5), 147.85 (d, jcp = 7.5 hz, ipsoc - naph), 148.00 (d, jcp = 7.5 hz, ipsoc - naph), 157.55, 157.59 (c-2), 164.57 (c-4), 172.85, 172.91 (coch3), 174.32 (d, jcp = 3.7 hz, co2), 174.63 (d, jcp = 3.7 hz, co2); p nmr (500 mhz) meod p 3.92, 4.15 . Ms (esi+) m / z = 685 [m+1]. Hrms (es+) m / z found 685.2631 [calcd for c33h42n4o10p (m+h) 685.2633]. 2,3-o - isopropylidene-5-o-[napthyl (cyclohexyl - l - alanyl)] phosphate cytidine (6). Prepared from 5 according to standard procedure for the n - deacetylation reaction . The crude compound is purified by flash chromatography on silica gel gradient elution of ch2cl2/meoh from 98/2 to 95/5 to give 6 as a white solid (0.061 g, 65% yield). H nmr (500 mhz) cd3od h 1.321.41 (12h, m, chch 3, ch3, 3 ch2- chex), 1.54 (3h, s, ch3), 1.551.84 (4h, m, ch2-chexyl), 4.004.05 (1h, m, chch3), 4.344.44 (3h, m, h-4, h-5), 4.644.77 (2h, m, h-2, h-3), 4.814.84 (m, 1h, cho), 5.74 (0.5h, d, j = 7.5 hz, h-6), 5.785.81 (1.5h, m, h-1, h-6), 7.437.48 (1.5h, m, ch - naph . ), 7.507.53 (1.5h, m, ch - naph), 7.557.59 (2h, m, ch - naph, h-5), 7.73 (1h, d, j = 5.0 hz, ch - naph), 7.907.92 (1h, m, ch - naph), 8.168.20 (1h, m, naph); c nmr (125 mhz) cd3od c 20.45 (d, jcp = 7.5 hz, chch3), 20.60 (d, jcp = 7.5 hz, ch3), 24.61 (ch2-chex), 25.50, 25.56, (ch3), 26.39 (ch2-chex), 27.45, 27.47 (ch3), 32.36, 32.45 (ch2-chex), 51.91, 51.96 (chch3), 68.11, 68.13 (d, jcp = 5.0 hz, c-5), 74.98 (cho), 82.23, 82.27 (c-3), 86.04, 86.09 (c-2), 86.63 (d, jcp = 2.5 hz, c-4), 86.70 (d, jcp = 2.5 hz, c-4), 95.65 (c-1), 96.24 (c-6), 115.24, 115.31 (c - naph), 116.31 (d, jcp = 3.7 hz, ch - naph), 116 . 48 (d, jcp = 3.7 hz, ch - naph), 122.74, 126.08, 126.54, 126.58, 127.55, 127.60, 127.84 127.87, 128.93 (ch - naph), 136.33 (c - naph), 144.19 (c-5), 147.85 (d, jcp = 7.5 hz, ipsoc - naph), 148.00 (d, jcp = 7.5 hz, ipsoc - naph), 157.84 (c-2), 167.84 (c-4), 174.32 (d, jcp = 3.7 hz, co2), 174.63 (d, jcp = 3.7 hz, co2); p nmr (500 mhz) cd3od p 3.96, 4.11; ms (es+) m / z = 643 [m+1]. Hrms (es+) m / z found 643.2521 [calcd for c31h40n4o9p (m+h) 643.2527]. Supplementary file includes experimental procedures for the synthesis of protected nucleosides and analytical data for all new compounds . Go to the publisher's online edition of nucleosides, nucleotides and nucleic acids to view the free supplementary files.
Systemic chemotherapy can lead to a variety of ocular complications, such as cicatricial ectropion, nasolacrimal duct stenosis, conjunctivitis, keratitis, cataract, macular edema, retinopathy, and optic neuropathy (1, 2). Although bulbar perforation with orbital cellulitis has been reported in an immunocompromised patient, corneal perforation has not been documented in patients undergoing systemic chemotherapy (3). We report a case of corneal perforation with preseptal cellulitis in a patient treated with systemic chemotherapy for acute lymphocytic leukemia (all). A 17-yr - old female patient undergoing systemic chemotherapy for all was referred to our hospital due to swelling and pain of the right upper lid for two days . Laboratory examination showed leukocytes 40 cells/l, erythrocytes 3.610 cells/l, hemoglobin 10.8 g / dl, hematocrit 30.6%, and thrombocytes 3310 cells/l . The patients received induction chemotherapy (vincristine, prednisolone, daunorubicin, and l - asparaginase). There were no abnormal findings, other than diffuse swelling of the right upper eyelid . Artificial tear eye drops were used for the treatment of superficial punctate erosions . On the 12th day, spontaneous bloody and purulent discharge from the upper palpebral conjunctiva occurred in the right eye . Systemic antibiotics (meropenem) were maintained per the antibiotic sensitivity test . On the 16th day, periorbital swelling decreased, however, corneal melting and perforation with iris prolapse was noted in the right eye (fig . Seven months after surgery, visual acuity in the right eye was 20/300, and intraocular pressure was 14 mmhg . Ocular complications associated with systemic chemotherapy can be divided into complications in the adnexa, anterior segment, and posterior segment . Previously reported complications in the anterior segment include conjunctival injection, conjunctivitis, corneal edema, keratitis, and corneal opacity (1, 2). Anticancer agents such as cytosine arabinoside, 5-flurouracil, carmustine, deoxycoformycin, and tamoxifen have been known to cause corneal toxicity (1, 2, 4, 5). In our case, vincristine, prednisolone, daunorubicin, and l - asparaginase were used for combination chemotherapy . To the best of our knowledge, corneal toxicity associated with these anticancer agents has not been reported . Serratia marcescens, a motile, gram - negative coccobacillus, is an emerging opportunistic pathogen noted for causing urinary, respiratory, blood stream, and ocular infections (6). Preseptal cellulitis in the present case was caused by s. marcescens because the patient was immunocompromised . Extracellular protease produced by s. marcescens is considered a major corneal destructive factor . In an experimental study of rabbit cornea, serratia protease preparations have caused rapid and extensive liquefactive corneal necrosis, descemetocele formation, and corneal perforation (7). It is possible that s. marcescens in the purulent discharge directly invaded the cornea with superficial punctate erosions and caused corneal melting and perforation in the immunocompromised patient . This hypothesis is supported by two facts: first, s. marcescens is a virulent organism . Second, corneal melting and perforation occurred in the area in direct contact with the infected upper palpebral conjunctiva . In conclusion, physicians should consider the possibility of serious ocular complications, such as severe bacterial keratitis or corneal perforation in cases of preseptal cellulitis caused by a virulent organism, particularly in immunocompromised patients.
Microleakage around resin composite restorations results from the formation of gaps where the restoration and the cavosurface margin of the natural tooth structure are joined . Gap formation may be related to shrinkage of the resin during polymerization and/or poor adhesion of dentin bonding agents between the dentin and composite material which leads to consequences, such as discoloration of the restoration, marginal break down, recurrent caries, pulpal inflammation and post operative sensitivity which may affect the longevity of restoration and ultimately the vitality of the dental pulp . When we cut the tooth structure with rotary instruments such as burs, an amorphous layer of organic and non - organic debris named the smear layer covers the cavity surface . Now it is clear that the quality and quantity of the smear layer severely depends on how it is created and in different conditions it has various properties [35]. Many variations in the smear layer of the prepared teeth with different dental instruments have been reported that can affect microleakage of composite resin restorations . The use of different burs to increase the surface roughness of preparations was evaluated as early as 1987 . Mowery, parker and davis used four different grits of sandpaper to prepare the dentin surface . They found that increasing the surface roughness may increase bond strength primarily because of the increase in the total surface area . In addition, surface irregularities were created; subsequently, increasing the mechanical locking of resin into these irregularities . While most of these studies concentrated on improving the bond strength of resin composites to the tooth structure, microleakage does not always decrease in direct proportion to increased bond strength . Many studies indicate that the quality and quantity of the smear layer is different when we use high speed or low speed cavity preparation devices or when we use different cutting instruments [35] formation of the smear layer by various diamond burs with different grits was evaluated by tani et al . He understood that the smear layer which covered the dentin when preparing with 50150 grit diamond bur is thicker than the smear layer formed with 1530 grit diamond bur . He also revealed that using different adhesive systems have affected the microleakage of composite restorations . Studies have evaluated different dentin bonding agents designed to improve the bond between the tooth structure and restorative materials . Bonded enamel is generally reliable in decreasing microleakage; however, bonded dentin is not as predictable at reducing microleakeage of the gingival margins at or near the cement - enamel junction . Dentin bonding agents have substantially reduced microleakage in the gingival margin but have not eliminated it completely . Deliperi and others compared the degree of microleakage in self etch and total etch adhesive systems . They reported that i - bond, the one step self etch adhesive system, has more dye penetration in both gingival and occlusal margins and there was no signifcant difference between occlusal and gingival margins in xenoiii (one step self etch adhesive), nt (total etch adhesive) and i - bond (one step self etch adhesive). On the other hand clearfil se bond had more dye penetration in the occlusal margin than the gingival margin . In different studies, it has been revealed that the cutting efficacy of cutting instruments is reduced while applying and it may affect the quality and quantity of the underlying smear layer . Therefore, this study evaluated the effects of cutting efficacy of different diamond burs on the microleakage of resin composite restorations using total etch and self etch adhesive systems, and it also determined whether or not bur cutting efficacy had an impact on the results . Ninety non - carious extracted human third molars, stored in 0/2% thymol solution after extraction, were cleaned of calculus, soft tissue and debris with hand instrumentation . In order to omit the inter - operator bias, each sample was assigned randomly in equal numbers (n=15) to one of six groups . Conservative class v composite preparations were made using one of three different burs; a coarse new diamond bur, a coarse used diamond bur and a fine diamond bur in an air / water cooled high speed headpiece (ch-4t5nsk b2/b3, japan a1101800). The cavity preparations were standardized with a width of 3 mm and a height of 2.5 mm and a depth of 1.5 mm . The occlusal wall of the cavity was limited in the enamel wall and the gingival wall of the cavity was extended beyond the cej onto the cementum . First group: teeth were cut with used coarse diamond bur (tizkavan - iran) and were conditioned with clearfil se bond (kurary medical inc . Japan pef 1975-wd batch no: 1-primer: lot 00670a2-bond: 00957a) adhesive system . Second group: teeth were cut with new coarse diamond bur (tizkavan - iran) and were conditioned with se bond adhesive system . Third group: teeth were cut with fine diamond bur (komet brasseler germany) and were conditioned with se bond adhesive system . Fourth group: teeth were cut with fine diamond bur and were conditioned with single bond (3 m dental product st . Fifth group: teeth were cut with new coarse diamond bur and were conditioned with single bond adhesive system . Sixth group: teeth were cut with used coarse diamond bur and were conditioned with single bond adhesive system . All prepared cavities were washed for 15 seconds with an air / water spray and the excessive water was removed with a gentle air spray, leaving the preparation slightly moist . Se bond adhesive system was applied for the cavities of group 1, 2 and 3, according to the manufacturer s instruction the primer was applied in the cavity, 10 seconds air dried gently, then applied a single layer bond in the cavity, the bonding agent was thinned with intermittent one - to - two second air blasts, which was followed by 20-second light polymerization by a led light curing unit (led turbo light cure- taiwan) with 600 mw / cm light intensity . Single bond adhesive system was applied for the cavities of group 4,5 and 6, according to the manufacturer s instruction, 20 seconds total etch time of the enamel, dentin and cementum with ultra etch 37% phosphoric acid (ultraetch 505 west 10200 south south jordan, utah 84095 ultradent, usa), 15 seconds rinse, then a light one - to - two seconds stream of air leaving the surface slightly moist . This was followed by applying single bond into the preparation and rubbing the bonding resin into the dentin enamel and cementum with the applicator brush tip . Single bond was thinned with intermittent one - to - two second air blasts to the point of not losing its glossy appearance . This was followed by 20 second light polymerization using an led light curing unit (led turbo light cure - taiwan) with 600 mw / cm light intensity . In all groups, the composite restorative material (z100 - 3 m, shade a2 usa) was placed and condensed incrementally until the preparations were completely filled . Each increment of restorative material attempted to involve only two walls of the preparation to reduce shrinkage and direct stress strain away from the internal walls . Each increment was light polymerized for 20 seconds prior to placement of the subsequent increment . All specimens were then subjected to 500 thermocycles at 5c, 55c with a 20 second dwell time . All specimens were then subjected to 500 thermocycles at 5c and 55c with a 20- second dwell time (vafaei - iran). After 24 hours all the restorations were finished, although the required finishing was minimal . After cycling, the apices of all root surfaces were sealed with adhesive wax and two coats of finger nail were applied to within approximately 1 mm of the tooth - composite interface . After sealing, the teeth were immersed in a 5% solution of methylene blue dye for 12 hours . Upon retrieval from the dye, the teeth were washed under running water and left to dry for dye fixation . The embedded samples were then sectioned once vertically approximately midway through the facial surface using a diamond coated cutting disk and nonstop section machine (bego nonstop - germany). Dye penetration was evaluated using a 10x stereomicroscope (m6c- 10- germany) at the occlusal and gingival margins . Microleakage scores were based on the degree of dye penetration according to the criteria described in table 1 . Microleakage scores were recorded for both the occlusal and gingival margins as shown in table 2 . The kruskal - wallis for non parametric data was used to analyze inter group comparisons of microleakage, while mann - whitney u and wilcoxon w tests with bonferoni s correction were used to test for differences in microleakage between pairs of groups in dentin and enamel margins . Microleakage raw data scores by examiners are presented in table 2 . Mean rank and means of dye penetration for the experimental groups at occlusal and gingival margins are presented in table 3 . Kruskal - wallis test showed that new / se group has the most and used / sb group has the least microleakage in the occlusal margin (p - value=0.05). In the gingival margin new / sb group has the least and used / sb group has the most microleakage as shown in table 3 . The kruskal - wallis test revealed that using the same adhesive system in gingival margins, significant difference was seen between bur types . The se bond adhesive system had the most leakage in the used diamond bur and the least leakage in new diamond bur (p - value=0.05). The single bond adhesive system had the most leakage in the new diamond bur and the least leakage in the used diamond bur (p - value=0.001). The kruskal - wallis test revealed that using the single bond adhesive system in occlusal margins, significant difference was seen between bur types (p - value=0.029). The results showed that using the se bond adhesive system in occlusal margins there was no significant difference between bur types (p - value=0.127). The mann - whitney test was used to test for differences in microleakage between pairs of groups in gingival and occlusal margins . The results have been shown in table 4 . The mann - whitney test with bonferoni s correction showed that using the same bur type in occlusal margins, there was a significant difference (p - value<0.0055) between used se and used sb groups . There was no significant difference using the same bur type in gingival margins between two types of adhesive system . The mann - whitney test with bonferoni s correction was used to compare microleakage at the occlusal and gingival margins of the samples for each group . The results showed that the gingival margins leaked significantly more than the occlusal margins in the used sb group (p - value=0.000). Reviewing the previous literature makes it clear that the efficiency of the instrument used for cavity preparation has great effect on quantity and quality of the smear layer and the amount of leakage in cavities prepared with burs having different efficiency is not the same . In 2005, von fraunhofer evaluated the effect of re - using of disposable diamond burs on restoration leakage . He showed that leakage of the first and third uses of the bur were similar to each other, but was much greater for the fifth use . He said that disposable diamond burs can cut preparations in up to three teeth before adversely affecting leakage behavior . Oliveria reported that the roughness of the surface varied strongly with the degree of coarseness . The surface roughness and also the thickness of the smear layer increased significantly with the coarseness of the abrasive but did not differ significantly with the abrasive type . Evaluating the smear layer modification; they found a significant inverse association between the degree of coarseness and the tubule openness . The sem study carried out by sanitini et al suggested that the smear layer produced by rough diamond burs were consistently thicker than those produced by tungsten carbide fissure burs, which in turn were thicker than those produced by fine diamond burs . Using the inverted cone at speeds of 6000 rpm without water spray, the research says that an understanding and recognition of appropriate treatment of smear layers is crucial to the development of improved dentin bonding systems . The research results showed that gingival margins leaked more than occlusal margins for all bur types . As well as organic in its nature, dentin is a complex structure of collagen and dentinal tubules . Depending on the location of the preparation, the size, number and direction of dentinal tubules are different . Dentin tubules may be absent if the preparation ends at the cej or below; this will affect the bond and also microleakage . In the se bond group, we found that the used diamond bur had more leakage than the new diamond bur, which may be the result of the quality of the smear layer, which is unlike in different bur types and high ph of self etch primer in this system . Vonfraunhofer related the higher restoration leakage in multiple uses of the disposable bur to the effect of greater smearing of the surface of cavity preparation together with some redeposition of cutting debris on the surface from the repeatedly used bur . We also found that when the same adhesive systems were used, there was significant difference between bur types in dentin margins, so when the single bond adhesive system was used, the used diamond bur had the most and a new diamond bur had the least microleakage and it had the same results when we used the se bond adhesive system . This might suggest that the smear layer which is produced by used diamond bur is denser and stickier, because of the low rate of efficiency, in order to prepare the cavity, more pressure of the hand is used by the dentist unintentionally . But when the bur types were the same in dentin margins, there was no significant difference between different adhesive systems . The results of this study revealed that using the same adhesive system in enamel margins caused no significant difference between bur types . But when the same bur type was used, there was significant difference between the types of adhesive systems . This might show that the phosphoric acid etchant in single bond system can totally remove the smear layer and resin can easily infiltrate into the demineralized space . But as the ph value of self - etching / priming solution in se bond system is generally low enough to demineralize the smear layer and the underlying dentinal surface, we have more leakage in se bond system than the single bond system . Pashley and carvalho suggested that the smear layer interferes with the self - etching primer adhesion . In conclusion, the cutting efficiency of bur had a great increasing effect on microleakage of composite resin restorations . So long term use of burs may result in an increased microleakage of composite resin restorations . On the other hand,
Cervical spine spondylolysis is the most commonly caused by high energy trauma to the upper cervical spine in the form of a " hangman's " fracture of c2 vertebrae7). However, congenital cervical spine spondylolysis is discovered by incidental radiographic finding, neck pain after minor trauma, a clunking or clicking neck and rarely neurologic compromise1 - 3,6,8,10). This rare condition potentially signifies an unstable cervical spine . We will report a case of congenital isthmic defect of cervical spine and describe a cause of cervical isthmic defect, diagnostic radiographic finding and appropriate treatment of cervical spondylolysis or spondylolithesis a 23-year - old female suffered from a hyperextension injury to cervical spine after a traffic accident . Following this injury, she experienced mild to moderate degree of nuchal and back pain . Plain radiograph of the cervical spine showed bilateral pars interarticularis defect at the c6 level with cleft - bow - tie configuration, hypoplastic pedicle, grade i spondylolisthesis of c6 on c7 and spinal bifida at c6 (fig . 1). Computed tomography of the c6 vertebrae showed bilateral corticated defects of the c6 articular mass, bilateral hypoplastic pedicles, lamina and spinal bifida of c6 and apparent neural foraminal enlargement at c6 - 7 (fig . The magnetic resonance image of spine showed grade i spondylolisthesis c6 - 7, degenerative change of c6 - 7 disc and acute compressed fracture at 4 thoracic vertebrae . She was subjected to controlled flexion - extension views of the cervical spine, which confirmed stability of the segment with less than 10 degrees of anterior angulations (fig . She was treated with the semihard - orthosis brace immobilization for 8 weeks followed by repeated lateral cervical spine radiography . After 10 months to injury, she was symptom free with full range of motion of cervical spine . Reported cervical spondylolysis are incidentally discovered except the " hangman's " fracture1 - 3,6,8,9,10). Cervical spondylolysis is less prevalent than lumbar spondylolysis, only about 100 cases have been reported in the literature worldwide, mainly in adults1). There is often no clear uniform mechanism of the injury, or the force causing the injury is often not strong enough to explain all of observed cervical anomaly . Incidentally discovered cervical spondylolysis could combine with another anomaly like spinal bifida, dysplastic lamina or transverse process (table 1). The etiology of cervical spondylolysis is unknown, but congenital basis is asserted because no history of trauma is elicited in the majority of cases2,3,5). Another basis is nonunion of vertebra after birth, infantile fracture or repetitive microtrauma1,6,9). The vertebral body will articulate with the vertebral arch at the neurocentral joints at birth, with fusion occurring between the ages of 5 and 8 years5,7). If congenital defect is invalid, problem of ossification or chondrification might occur during vulnerable period from birth to fusion at neurocentral joint . Proposed that this condition was caused by repetitive microtrauma resulting in stress fractures to the pars region similar to the process proposed in lumbar spondylolysis5). They further proposed c6 to be the commonest site to be affected as, this being a transitional vertebra, it was subjected to stress more often . Some of these theories can be put together and it may be suggested that a dysplastic spine is more susceptible to trauma or stress and leads to spondylolysis6). We could clarify that our case was associated with congenital basis because of well corticated margin in defected area combined with another congenital lesion and no trauma history . Congenital cervical spondylolysis, an embryologic developmental defect, occurs the most commonly at c61,2,6,9,10). The overall frequency is unknown, and most cases are detected incidentally (table 1) conventional radiolographic findings of cervical spondylolysis included a deformed articular mass, hypoplastic or dysplastic pedicle, spinal bifida and anterolisthesis1 - 3,6,8 - 10). The computed tomography is the most useful image modality, because it shows the exact bony structures and three dimensional images . The magnetic resonance image can aid identification of combined soft tissue damage, cord injury and bony contusion at trauma lesion . However, magnetic resonance image is not useful for diagnosis of cervical spondylolysis . In our case, cervical magnetic resonance image showed acute compressive fracture at 4 thoracic vertebrae and degenerative disc change . Misdiagnosis occurred for traumatic unilateral interfacet dislocation, a chronic nonunited articular mass fracture or congenitally absent pedicle4,10). For example, unilateral interfacet dislocation is characterized by spondylolisthesis greater than 3 mm, rotation without a superimposed articular mass, and misalignment of the spinous process4). The cervical spondylolysis can cause variable symptoms like neck pain, muscle spasm or neurologic deficit . Symptomatic cervical spondylolysis need adequate treatment because previously existing cervical spondylolysis is vulnerable to trauma . The treatment of cervical spondylolysis depends on instability . If the cervical spine is stable, conservative approach is desirable for treating symptomatic cervical spondylolysis . Our case was stable but needed hard orthosis because of the fracture of 4 thoracic vertebrae.
A major factor leading to cardiovascular disease in hemodialysis patients is vascular calcification, which causes vascular stiffness, higher pulse wave velocities, and elevated pulse pressures . It is thought that vascular calcification is caused by disrupted mineral metabolism and abnormal regulation of phosphorus and calcium . Fibroblast growth factor 23 (fgf-23) is an osteocyte - derived hormone that participates in phosphate and vitamin d metabolism . Elevated fgf-23 levels are observed in phase 2 of chronic kidney disease and are inversely correlated with glomerular filtration rate . Patients with esrd may reach fgf-23 blood levels around 101000 times higher than normal . Even within the normal range, a strong association was observed between high serum fgf-23 levels and atherosclerotic burden, endothelial dysfunction, and arterial stiffness . Elevated fgf-23 serum levels are particularly implicated in the calcification of peripheral vasculature and the coronary artery . High - flux hemodialysis (hfhd) is a dialysis procedure designed to eliminate blood toxins that accumulate in esrd . Whether hfhd is better than low - flux hemodialysis (lfhd) is still under debate . In this study, we compared the efficacy of hfhd and lfhd in reducing vascular calcification and serum fgf-23 levels in patients receiving maintenance hemodialysis . The study was conducted according to the declaration of helsinki and was approved by the ethics committee of xiangya hospital, zhongnan university . This study was registered as a clinical trial (chinese clinical trial registry, www.chictr.org.cn, chictr - opc-15006249). From june 2013 to june 2014, fifty patients receiving maintenance hemodialysis were recruited to participate . Inclusion criteria was as follows: 1880 years of age, previously treated with hemodialysis for at least 3 months (3 per week; 4 h per session), dry weight was relatively stable, arteriovenous fistula was created for hemodialysis, and blood flow rate was 200 ml / min . Exclusion criteria were as follows: patients with severe infections in the past three months, hepatic disease, or previous hemodiafiltration or peritoneal dialysis treatments . Patients were randomly allocated into either the hfhd group or the lfhd group (25 per group). Patients received respective treatments for 12 months, which was performed 4 h per session three times per week . The hemodialysis machines utilized in this study were a fresenius 4008s (bad homburg, germany) and a gambro ak96 (lund, sweden). The dialysate flow rate was set at 500 ml / min, blood flow was set at 250300 ml / min, and the urea clearance index (kt / v) was 1.2 . A ca - hp170 dialyzer (baxter, deerfield, usa) was used for lfhd . The surface area of the tri - cellulose acetate (tca) membrane was 1.7 m and the ultrafiltration coefficient was 10.0 ml / hr / mmhg . A ct-190 g dialyzer (baxter, deerfield, usa) was utilized for hfhd . The surface area of the tca membrane was 1.9 m and the ultrafiltration coefficient was 36.0 ml / hr / mmhg . Fasting venous blood was collected at baseline, six months, and twelve months after treatment . Fgf-23 levels were measured by commercial elisa kits (millipore inc, usa) according to the manufacturer s instructions . Calcium, phosphorus, akp, albumin, creatinine, and lipid levels were measured by standard methods . Aorta abdominalis calcification scores (aacs, figure 1) were calculated according to the length of calcified deposits on the anterior and posterior walls of the aorta abdominalis, which corresponds to lumbar vertebrae 14 . Anterior and posterior calcification scores were recorded separately for each vertebra (i.e. Eight separate scores were recorded). Scoring was performed blindly by two independent doctors as follows: 0 point, no calcification; 1 point, calcification of <1/3 of the vascular wall; 2 points, calcification of 1/32/3 of the vascular wall; 3 points, calcification of> 2/3 of the vascular wall [2123]. Final aacs were calculated by summing the eight separate scores, ranging from 0 to 24 points . T - tests were performed for comparing between different groups of the patients characteristics data . Repeated measures analysis of variance was used to examine changes in serum calcium, phosphorus, pth, akp, fgf-23, and aacs from baseline to 12 months after treatment . Multiple regression analyses were also carried out to screen potential factors influencing the development of vascular calcification . These factors included age, duration of hemodialysis, blood pressure, fgf-23 levels, therapy alternatives, blood calcium levels, and blood phosphorus levels . Seventy - three patients were initially screened in the study . However, 7 patients declined to participate and 16 patients were excluded (2 cases for severe infection within 3 months, 11 cases for hepatic disease, and 3 cases for previous hemodiafiltration or peritoneal dialysis treatments). The remaining 50 patients were randomly allocated into either the hfhd or lfhd group (25 in each). The hfhd group included 23 chronic glomerulonephritis patients, one diabetic nephropathy patient, and one obstructive nephropathy patient . The lfhd group was comprised of 19 chronic glomerulonephritis patients, four diabetic nephropathy patients, and two hypertensive nephropathy patients . Over the one - year follow - up period, one hfhd participant was excluded for receiving a kidney transplant . In the lfhd group, one patient was excluded for kidney transplants, one patient died, and three others dropped out of the study . No significant differences in basic patient characteristics (e.g. Age, sex, co - morbidity diseases, blood pressure, lipid levels, and kt / v) were detected between the hfhd and lfhd groups (p>0.05; table 1). Six months after treatment, we found no significant difference in calcium, phosphorus, parathyroid hormone (pth), alkaline phosphatase (akp), fgf-23, or aacs between the two groups (p>0.05). After 12 months of treatment, fgf-23 levels and aacs significantly decreased in the hfhd group, but not the lfhd group (p=0.049 and p=0.002, respectively; table 2). Fgf-23 levels and aacs in the hfhd group decreased by 41.423.4% and 30.040.3% respectively, while they increased by 0.326.4% and 76.45.6% in the lfhd group, respectively . Pearson correlation analysis revealed that aacs were positively correlated with fgf-23 levels in all patients (p=0.004; figure 2a), the hfhd group alone (p=0.040; figure 2b), and the lfhd group alone (p=0.037, figure 2c). Multiple regression analysis indicated that older patients (p=0.048), those with higher blood phosphorus levels (p=0.003), and higher fgf-23 levels (p=0.001) had significantly increased risks of aorta abdominalis calcification . It also showed that hfhd reduced the risk of aorta abdominalis calcification, compared to lfhd (p=0.003; table 3). Our study supports findings that serum fgf-23 levels are positively correlated with aorta abdominalis calcification . Further, it shows that hfhd is more effective than lfhd in clearing serum fgf-23 and reducing aorta abdominalis calcification . Moreover, multiple - regression analysis shows that advanced age, serum fgf-23, and blood phosphorus are risk factors for the development of vascular calcification . Fgf-23 is a mineral regulatory factor that regulates phosphate homeostasis in chronic kidney disease patients . Fgf-23 decreases renal phosphate reabsorption and increases urinary phosphate waste by decreasing the expression of sodium - phosphate transporter in proximal tubular epithelial cells . Compromised renal function may result in the accumulation of phosphorus and decreased clearance of blood fgf-23 levels . Previous studies show that increased fgf-23 levels are correlated with increased mortality and vascular calcification in hemodialysis patient, which is supported by the results presented here . Molecular weight cut - offs of traditional hemodialysis membranes range from 20 to 30 kda . Thus, fgf-23 is not removed since it has a molecular weight of 32 kda . Though we detected a small fluctuation of fgf-23 levels in the lfhd group, significant increases were not observed in our study . Torres et al . (2008) found that lfhd treatment did not decrease serum fgf-23 concentration . In contrast, it has been shown that alpha1-microglobulin, which has a molecular weight of 33 kda, is reduced by 21% using a high flux polyether - sulfone membrane . Another study found that the fgf-23 clearance rate by hfhd reaches to 36.228.6%, which is substantially lower than on - line hemodiafiltration, which has a fgf-23 clearance rate of 55.725.2%). In the present study, the clearance rate of fgf-23 in the hfhd group was 41.423.4% after 12 months of treatment, while fgf-23 increased 0.326.4% in the lfhd group . Interestingly, we found that aorta abdominalis calcification is better controlled by hfhd than lfhd . Further, we find that advanced age, blood phosphorus levels, and serum fgf-23 levels are all independent risk factors for the development of vascular calcification, which agrees with a previous study . Another study, including 1130 healthy males, also suggests that circulating fgf-23 is associated with mineral metabolism, including bone metabolism - regulating cytokines, and with severe aacs independent of traditional risk factors . (2009) report that higher serum fgf-23 levels, even within normal range, are independently correlated with increased arterial stiffness and impaired vasoreactivity . These elevated fgf-23 concentrations may then stimulate vascular calcification by acting directly on the vascular wall to induce a local reduction of the enzyme klotho . Interestingly, a recent study indicates that hemodiafiltration appears to offer no extra benefit over hfhd in terms of controlling vascular stiffness . This may due to the limited follow - up period or that the efficacies of hemodiafiltration and hfhd are similar in controlling vascular calcification . Here, we show that the improvement of aacs in hfhd compared to lfhd is not significant at 6 months, but becomes significant at 12 months (p=0.002). First, the reliability of the study may be impaired by the small cohort size and short follow - up time . Second, in 2009, kdigo (kidney disease: improving global outcomes) guidelines suggested that an echocardiogram should also be used to detect the valvular calcification in esrd patients . Hfhd is superior to lfhd and benefits hemodialysis patients by reducing serum fgf-23 levels and reducing vascular calcification.
Three villages (manduo, luoping, and tuofeng; figure) in tengchong county, located in the westernmost part of yunnan province, were selected for study because of their 20112013 malaria incidence, ecologic features related to malaria transmission (i.e., altitude and proportion of land used for rice cultivation), and housing and economic status . The villages range in altitude from 1,276 to 1,893 meters and have 1,115 households and a population of 4,904 (detailed methods in technical appendix). Location of 3 villages in tengchong county, yunnan province, china, in which study of transmission risk from imported plasmodium vivax malaria was conducted . During 20112013, a total of 24 p. vivax malaria cases were reported from the study villages to the chinese information system for disease control and prevention; all were classified as imported (technical appendix), as determined by patients travel history (3). All patients were adult men 1849 years of age, and most worked in business or mining . Patients were radically cured with a regimen of chloroquine and primaquine, as recommended by national treatment guidelines . Malaria vulnerability or importation risk (i.e., incidence of imported malaria cases per 1,000 population per year) averaged 1.6 cases in the study area during 20112013 (technical appendix table 1). The average interval between symptom development and diagnosis of malaria was 2.4 days; average interval between diagnosis and treatment was 1.5 days (table 1). All cases were reported within 1 day and investigated within 3 days (table 1). Health workers performing active fever surveillance during the 2013 transmission season (may september) visited each house at 2-week intervals, conducted a total of 7,680 household interviews and 38,960 interviews with village residents, and collected 399 blood samples from persons with history of fever during the previous 2 weeks . A total of 268 (67.2%) samples were from local residents who reported no travel outside tengchong county within the past 2 weeks; 131 were from mobile populations reporting travel . Vivax isolates were detected by microscopy and pcr in 10 (7.6%) persons in the mobile population; no malaria infection was found in local persons (technical appendix table 2). To estimate human biting rates, mosquitoes were collected in each study village every 2 weeks by using volunteer outdoor human - landing catches during may a total of 5,576 mosquitoes were caught; most (95%) were anopheles sinensis mosquitoes . The average number of mosquitoes landing on a single person per night (human landing rate) was 2.7 (table 2; technical appendix table 3). Sinensis mosquitoes tested for the villages of manduo, luoping, and tuofeng was 0.52, 0.54, and 0.61, respectively (technical appendix table 4). The average human blood index (i.e., proportion of tested mosquitoes with ingested human blood) was 3.8% in the surveillance areas (table 2; technical appendix table 5). Average vectorial capacity (i.e., expected number of new human infections from 1 infected person within 1 day, assuming all mosquitoes with sporozoites are potentially infective) of an . Sinensis mosquitoes was 0.02 (table 2), indicating that 50 cumulative days would be needed for transmission of malaria from an infected person to another person, assuming that all female mosquitoes biting malaria - infected persons become infected and transmit . Sinensis mosquitoes found to have ingested human blood (i.e., blood feeding rate) was 15.6% (31/199). The proportion of infected mosquitoes was 16.1% (5/31), the rate of susceptibility of local an . * vectorial capacity, expected number of new human infections from 1 infected person within 1 day, assuming all mosquitoes with sporozoites are potentially infective . Hbi, human blood index (proportion of tested mosquitoes having ingested human blood . Average number of mosquitoes landing on a single person per night (ma). Human blood index divided by days needed to complete gonotrophic cycle (cycle of taking a blood meal and laying eggs). Probability (p) of a mosquito surviving 1 whole day . #time (n) needed for parasites to complete development from ingested gametocytes during blood meal to sporozoites in salivary glands, when parasites are transmissible to humans . * * fraction of infected mosquitoes after duration of sporogony . Duration of vector's life, in days, after surviving the extrinsic incubation period, calculated as the negative logarithmic reciprocal of the daily survival rate: 1/ln(p). An . Sinensis mosquitoes were the main local vector for p. vivax and were widely distributed throughout this study area . The vector s susceptibility to imported p. vivax infection indicates potential for p. vivax malaria to be sustained in this region . Although a recent field survey showed secondary transmission from patients with imported p. vivax malaria in other villages in tengchong county (2), we identified no secondary infections in the study villages . Sinensis mosquitoes are the most widely distributed malaria vector in china but have relatively low susceptibility to parasites compared with other malaria vectors (4). Sinensis mosquitoes were susceptible to imported p. vivax infection; however, vectorial capacity of an . Sinensis mosquitoes was lower than it was during the 1990s (0.05) in the same region (5) and much lower than reported in central china (0.3) in the 1980s (6). The reduced vectorial capacity in the study region since the 1990s is likely attributable to the change in the dominant malaria vector species . Historically, an . Kunmingensis mosquitoes were the main malaria vector in tengchong county and accounted for 77% of total malaria vector density; its vectorial capacity (0.3) was 10 times higher than that of an . Kunmingensis mosquitoes (<5% of total malaria vectors) were captured in our study, likely because of extensive residual insecticide use and improved housing, which reduce contact with this mosquito and vectorial capacity . High coverage (> 90%) of long - lasting insecticidal nets and indoor residual spraying has been achieved in southern china along the myanmar border since implementation of the national malaria elimination program in july 2010 . In early 2012, a real - time surveillance system and response strategy (1 - 3 - 7) was rolled out nationally (8) and substantially improved timeliness of malaria surveillance and response activities . During 20112013, all malaria cases in the study area were reported within 1 day and investigated within 3 days, and screening of persons with fever was conducted within 7 days . Other countries reporting few days (range 3.08.2 days) before testing and treatment of imported malaria reported similar results and no subsequent secondary infections (911). In our study, additional testing by pcr for those reporting fever found no subpatent infections (i.e., slight infections with low parasitemia), a finding consistent with a recent review that showed no difference between pcr and microscopy for detecting parasites in symptomatic persons . However, a large proportion of p. vivax infections were subpatent in a cross - sectional survey of the general population in china (12). Rigorous evaluation of malaria elimination programs is essential for continuously improving the programs, targeting limited resources, and maintaining financial and political support . We used robust entomologic and epidemiologic metrics to assess malaria elimination in a border region . Many national malaria control programs lack capacity to conduct entomologic surveillance to assess vectorial capacity . Although our study shows evidence of successful malaria elimination, additional validated metrics to ascertain success are needed . Recent efforts to compare the basic reproductive rate (total number of malaria cases derived from 1 infective case and distributed by mosquitoes in the absence of immunity) for imported versus local malaria cases provide a more nuanced and stable metric for measuring malaria elimination (13). Because this region has a unique ecology and distinct mosquito species composition, our findings need further validation to determine whether they can be extrapolated to other areas of china s border region (14). Technical appendix . Detailed study methods and additional tables summarizing data on imported malaria cases, active fever surveillance, and entomologic investigations for study of transmission risk from imported plasmodium vivax malaria along the china
Diabetes represents a challenging health issue in the 21st century with a growing incidence estimated to be 381.8 million patients globally in 2013 and high morbidity and mortality rates . Though population data are often scarce, the african continent is expected to bear the most important burden of diabetes during the next coming decades [13]. However, a few countries have developed national strategies to contain this coming epidemic . In many cases, lack of data is the major barrier for setting up efficient programs for prevention and management of diabetes in disadvantaged populations . In senegal, there is not yet available data on diabetes burden at national level but one recent survey in saint - louis city (northern region) reported a prevalence of 10.4% with two - thirds of patients uncontrolled . Moreover, marked disparities had been reported between urban and rural regions where lifestyle habits and access to care are different [24]. The objective of this study was to compare prevalence and risk factors of diabetes in adult populations living in rural and urban areas in senegal . We performed a community - based cross - sectional survey in saint - louis (northern region of senegal). All individuals aged 18 years and living in saint - louis for 3 months were eligible to participate in the study . A two - stage cluster sampling method was used to select a representative sample of adults living in urban and rural areas of saint - louis . We firstly selected 17 localities as clusters (9 urban areas and 8 rural areas). Then, we randomly took a number of households proportional to population size of each locality (data available from the national agency of statistics and demography). From each household a maximum of two participants considering -error of 0.05 and a power of 80%, the required sample size was 855 individuals and we added a 20% attrition rate to get a sample of 1026 participants . Finally, a total of 1056 persons were sampled to enter the study . Data were collected on site during house - to - house visits that were conducted between 7 a.m. and 12 p.m. or at the nearest health centre when patients did not live far away from this facility . A modified version of the who stepwise questionnaire (http://www.who.int/chp/steps/) was pretested and validated before its use to collect data . Researchers assisted by medical students, trained nurse practitioners, and community health workers had to fill the data collection form, to document the sociodemographic status (age, sex, marital status, education, profession, and education), personal and family health history (regarding, particularly, hypertension, diabetes, stroke, and heart and kidney disease), and lifestyle (nutritional habits, physical activity, smoking, and alcohol consumption) of each participant . Anthropometric measurements (weight, height, waist, and hip circumference) were performed using standard methods and calibrated devices . Blood pressure was measured twice at five minutes intervals by a semiautomatic sphygmomanometer and the mean of the two readings was calculated . If the difference between the readings was greater than 10 mm hg, a third measurement was performed . Hypertension was defined as a systolic blood pressure of 140 mm hg or more, diastolic blood pressure of 90 mm hg, any prescription of antihypertensive medication in the past two weeks, or any self - reported history of hypertension . Serum total cholesterol, ldl cholesterol, hdl cholesterol, and triglycerides were measured with colorimetric method . Fasting blood glucose (fbg) was measured with a glucose oxidase method, and serum total cholesterol, hdl cholesterol, and total triglycerides were measured by an enzymatic calorimetric method . Diabetes was defined as fbg 126 mg / dl or by prescription of hypoglycemic agents despite fasting plasma glucose or any self - reported history of diabetes . Physical inactivity was defined as less than 30 minutes of moderate activity per week or less than 20 minutes of vigorous activity three times per week, or the equivalent . Statistical analyses were performed with stata 12.0 (stata corp, tx, usa). Comparison of proportions and means were done using pearson's chi - square test or student's t - test as appropriated . The age - standardized prevalence rates were calculated with the direct method, using the results of senegalese general population census as the standard (http://www.ansd.sn/). Clinical and biochemical parameters associated with diabetes were assessed, in bivariate analysis, comparing the group with diabetes and the group without diabetes using student's t - test or a chi - square test . Variables significantly associated with diabetes were then included in a multivariate logistic regression model with age, gender, and urbanization as forced variable . Odds ratio (ors) with 95% ci and p values of the final model are presented . A total of 1056 participants were involved in the study with a response rate of 99.1% . But 21 of them were excluded from the analysis because of incomplete collected data and 1026 individuals were finally analyzed . The crude prevalence of diabetes in our sample was 10.8% (95% ci: 6.9%14.2%). The age - adjusted prevalence of diabetes was 7.6% (95% ci: 5.4%10.5%) with a difference between men (6.0%) and women (9.0%). The mean age of diabetic patients was 46.8 13.5 years (1876 years). The crude prevalence of diabetes in urban and rural settings was, respectively, 12.7% and 6.8% . Adjusted prevalence of diabetes was higher in urban areas (8.1%) compared to rural areas (4.6%) (figure 1). Also, in both settings, there was an increase in diabetes prevalence with age as shown in figure 2 . Comparing cardiovascular risk of people living in urban and in rural settings, we found similarly high prevalence of traditional cardiovascular risk factors such as hypertension, obesity, and physical inactivity contrasting with low proportion of smokers . Regarding the disease awareness, 43% of patients were diagnosed during the survey, 36% were previously declared diabetic but without any medical follow - up, and 21% were regularly treated by a physician . The presence of diabetes was associated with common risk factors like age, gender, obesity, and familial history of diabetes (see table 2). Univariate analysis showed that age 35 years (10% of diabetics versus 5.5% in people aged <35 years), female gender (9.0% of diabetics versus 6.0% in males), family history of diabetes (diabetes prevalence of 8.4% versus 5.1%), and obesity (diabetes prevalence of 13.9% versus 8.3%) were significantly associated with a higher chance to get diabetes . Conversely, physical activity and daily consumption of 3 fruits / vegetables were associated with lower risk of diabetes in this population (prevalence differences of 19.5% and 4.4%, resp . ). After adjustment for age, gender, smoking, familial history, and obesity, the risk of diabetes was similar in individuals living in urban area compared to those living in rural areas (they presented a diabetes risk excess of 27%). Only age> 35 years (or = 1.63, 95% ci = [1.482.06]), existence of family history of diabetes (or = 1.42, 95% ci = [1.123.77]), and abdominal obesity (or = 1.17, 95% ci = [1.001.78]) remained significantly associated with diabetes (see table 3). Prevalence of diabetes in sub - saharan africa is variable across countries, ethnic groups, and settings considered . Reported data from community - based studies range from 2.8% in rural populations in angola to 28.2% found in south african mixed ancestry populations living in urban areas [8, 9]. Epidemiological studies in rural populations are scarcer but they generally show a lower prevalence [1012] compared to surveys in urban settings [1315]. In this study, the prevalence of diabetes in urban areas is quite twice the one in rural areas . Comparable prevalence ratios (urban / rural) were found in kenya and in democratic republic of congo . As already reported in the literature, we found an increasing prevalence with age which is a major risk factor for type 2 diabetes . Some studies found higher prevalence in men [10, 11] and others reported the contrary [9, 16]. In the present study, female gender, absence of school education, and living in urban setting were associated with diabetes at univariate analysis . However, these associations were no longer significant after adjustment for true risk factors that were abdominal obesity, age, and familial history of diabetes . As underlined in many studies, we found a higher prevalence of diabetes in women compared to men and this difference was more striking among rural populations . However, these rates are better than what was reported in rural tanzanians (8.3 to 13.2%) [17, 18] or south africans (15.3%) or in a previous survey in dakar (capital city) where 90% of newly diagnosed diabetics were not aware of their disease . Recent forecast suggests an alarming increase of diabetes incidence in africa during the next decade in addition to other noncommunicable and infectious diseases . In the us population between 1980 and 2011, the crude prevalence of diagnosed diabetes increased from 2.5% to 6.9% while age - adjusted prevalence rose in the same proportions indicating that changes in the population age structure do not explain the epidemic transition . The true explanation of this rising burden of diabetes in both urban and rural africa is probably multifactorial . With life expectancy increase, the most important part might be driven by lifestyle modifications (fast urbanization, physical inactivity, and dietary transition) which promote obesity and insulin resistance but also environmental and genetic factors have not been well explored . Data on the changes in the -cell function and insulin resistance in the early stages of the disease process in african populations are scarce . A few genetic studies performed in small groups from northern and western africa had identified some polymorphism associated with diabetes but epigenetic factors which should play an important role in the disease onset are still unknown [2224]. Other conditions like hiv and sickle cell diseases are also incriminated in the current epidemics of diabetes in africa [25, 26]. Despite its epidemiological importance of describing diabetes face in senegalese populations firstly, the cross - sectional design is not suitable for inferential analysis about causality or direction of association between diabetes and other cardiovascular risk factors . Secondly, incidence of diabetes could not be calculated to estimate the disease potential progression in the population . Age, familial history of diabetes, and abdominal obesity are the main risk factors identified . Prevention program targeting both urban and rural populations are urgently needed in african countries in order to reduce the morbidity and mortality due to diabetes.
The visual system is a well - developed sensory organ that involves effortless complex interaction between the visual organ and the brain . It involves physiological and a series of hierarchical neural processes from the photoreceptors, retinal ganglion cells, the lateral geniculate nuclei (lgn) of the thalamus, and the visual cortices . The american optometric association identified that prolonged use of the visual system can result in inefficient visual processing functions generally called visual fatigue . In line with that, the american association of ophthalmologist (aao) diagnosed visual fatigue as an ophthalmological disease called asthenopia with the medical diagnostic code of icd-9, 368.13 with the subclassifications shown in table 1 [3, 4]. The last two digits of the medical code indicate the seriousness of the ophthalmological disease . The larger the last two digits, the more severe the symptoms of the disease . Visual fatigue commonly arises when people view stereoscopic motion images . In a stereoscopic image, the viewer watches two images corresponding to the right and left eyes with convergence eye movement . Based on individual characteristics and instrumentation issues, conflicts can occur between convergence or divergence eye movements and the accommodation function thus causing visual fatigue . One of the major problems with stereoscopic displays is that a stereoscopic display produces a mismatch between the focus (accommodation distance) and the fixation (convergence distance) of human eyes . Thus, stereoscopic images may present different depth information to the accommodation and convergence functions . Stereoscopic displays require that the viewer converges and accommodates at different depth planes, thus creating a conflict between the two visual mechanisms . This problem is common in 3d display systems such as head mounted displays (hmds) and displays that make use of a lenticular lens . Imbalanced visual information may be one of the reasons for the visual load and eventually can cause the visual fatigue associated with 3d images . Further, inoue and ohzu noted that the common cause of visual fatigue in a stereoscopic display is the fact that 3d spaces generated by stereoscopic images are artificial spaces which are different from real spaces . As a result, the perceived images through 3d glasses do not really exist . . Showed that improvements of image quality, when a stereoscopic system is used, increase the discrepancy of accommodation and convergence and may cause increased visual fatigue . For example, in one of their experiments, kuze and ukai had participants playing a video game using a stereoscopic hmd or monoscopic tv display and found significantly greater increases in subjective ratings of general discomfort, nausea, and headache after using the hmd compared to the increases observed with a conventional tv display . Vertical alignment error is an upward or downward tilt in the optical axis of one image resulting in differences in vertical position of an image . Rotational alignment error is a tilt in one of the images with the degree of alignment error increasing from the center . Lastly, magnification difference is an error due to the size difference between the left and right stereo pairs from the field of view . Firstly, these are errors in optics due to a shift in the image placement of the left and right stereo pairs . These optical errors are due to either a shift in rotation, a difference in magnification, or a difference in reduction . Second, filtration errors in photometry caused by the difference in luminance, color, contrast, chromatic aberration, or crosstalk (images switching back and forth between the eyes). The existing past studies have not explicitly addressed the issues of image alignment errors on the cerebral cortical responses and visual fatigue severity ratings . This study investigates the effect of prolonged use of stereoscopic display on visual fatigue and dorsolateral prefrontal cortex hemodynamic responses during visual display tasks . The intellectual contribution of this paper can be realized in ophthalmological disease etiology, optometry, vision science, and 3d devices and systems . Specifically, the neuroimaging of the brain in relation to visual fatigue can yield rich neurophysiologic data to objectively and clinically classify and diagnose onsets of visual fatigue . This study included 21 males and 3 females recruited from north carolina agricultural and technical state university community . Participants' visual acuity and color blindness score were limited to 20/40 and 16 out of 22 points, respectively, and they were compensated for their participation . Nec lt silver screen, nec lt 245 dlp projectors, nec lt 245 polaroid glasses, a 22 1.58 ghz 0.99 gb ram gateway dcdi desktop, a snellen chart, and a simulator sickness questionnaire (ssq), a neurophysiological assessments, and an ophthalmological questionnaire . Several software systems were used for the study . These were air traffic control interface model (atcim), cognitive optical brain imaging studio (cobi) provided by fnirs devices llc (2010) for the fnirs . Raw materials used included 70% isopropyl alcohol swabs, cotton squares, and astromed bandages . Is comprised of four parts: nausea rating, oculomotor fatigue rating, disorientation rating, and a total score rating . It consists of thirty - two symptoms and for each symptom, a higher rating indicates that a participant experienced more symptoms . The symptoms are rated as none = 0; slight = 1; moderate = 2; and severe = 3 . Fnirs is a brain imaging device for exploring brain responses to a variety of stimulated cognitive and physical tasks in the field of cognitive neuroscience . This device consists of an infrared light source with a wavelength range of 650850 nm into the head and a detector that receives light after it has interacted with the tissue . The emitted photons after interacting with head tissues undergo two types of interaction, namely, absorption (loss of energy to the medium) and scattering [1618]. Researchers have shown that by placing the probes on a participant's forehead, fnirs provides accurate measures of activities within the frontal lobe of the brain which are responsible for many high order cognitive functions, such as memory and problem solving . The light passing through the frontal tissues is sufficiently low to allow for tissue imaging at depths up to 2030 mm . Deoxygenated (hbb) and oxygenated (hbo2) hemoglobin are the main absorbers of near infrared light in tissues, and they provide relevant markers of hemodynamic and metabolic changes associated with neural activity in the brain . It is not possible to arrive at absolute values of concentration using fnirs imaging on living samples . Once the hbb and hbo2 levels are computed, they are used to calculate the levels of oxygenation (in m) and values that may be approximately treated as percent changes in blood volume . The task stimulus is an air traffic control interface model (atcim) developed by wiyor and designed with labview 8.1 . Provided by national instrument . The simulated testbed provides a situation awareness (sa) environment similar to an atc task domain . The use of atcim as an atc simulator is specifically designed to allow for close experimenter control over display features and task performance, while providing a moderate degree of realism . In the atcim domain, an operator perceives and responds to aircraft conflict separation tasks which occur when two or more aircrafts are in self - separation violation . The separation violation is set to a loss of separation of 5 nm laterally or 500 ft . Vertically or both conditions can occur simultaneously . The atcim is shown in figure 1 . The atcim system consists of two main interfaces, namely, the control setting interface and the dashboard interface . These are (a) two closest aircraft color identifications; (b) a color status bar in which two mutually exclusive red and green colors appear with green for safe mode and red for crash mode; (c) an alert distance control that allows the user to preset the minimum safe distance between two planes; (d) a display time control which allows for the user to input the maximum time allowed for a participant to perceive and respond to a collision alert; (e) a number of aircraft to monitor which allows the user to preselect the number of aircraft for the experiment; (f) a collision alert used by the participants to respond before collision time; and (g) an aircraft information - radii mode in which the plane path, latitude, longitude, speed, and bearing are displayed on the radii and move with the aircraft . This consists of a 5 nm (nautical miles) radius sector in which the number of aircrafts is selected to be monitored by the participants . Based on the predetermined mode selected from the setting section, the required output will be shown in the dashboard section at each simulation run . It should be noted that a particular treatment replication is completed before moving to the next treatment . For example, if the randomization order is vertical shift, magnification difference, and rotational error, the vertical shift treatment will be replicated twice and each replication will last for 10 minutes, hence 20 minutes for vertical shift as atc1, followed by magnification difference; two replications as atc2; and lastly, the rotational error with two replications as atc3 . Table 2 contains the information on the independent variable, levels, and measurement units for the experiment . The number of aircraft ran from low being 3 aircraft and high being 9 aircraft . Task difficulty was characterized by separation conflicts labeled as vertical, horizontal, combined horizontal and vertical, and none, respectively . A high maneuver (atc3) had a maximum number of nine aircraft (high cognitive load) with either combined horizontal and vertical or none for separation conflicts, and a low maneuver (atc1) had 2 - 3 aircraft conflicts in the airspace . The independent variable was the stereoscopic alignment errors observed at three levels, namely, vertical shift, rotational error, and magnification difference . For the vertical shift, the focused images on the left and right retina were displaced by 0.5; the rotational error had the images displaced from each other in the left and right retina by 0.25; and for magnification difference, an error was caused by 6.25% enlargement in one image . The two response measures in the study were cerebral hemodynamic response and visual fatigue ratings . The cerebral hemodynamic responses were continuous variables measured with fnirs and visual fatigue symptoms were subjective ratings measured by ssq . The ophthalmological survey was used to ensure that the participants under 40 years had no optometric and/or ophthalmological symptoms . Available studies have shown that after age 40, most people experience the onset of ocular - motor problems, such as accommodations and convergence [22, 23]. The national eye institute estimates that, currently, more than 38 million americans age 40 and older experience blindness, low vision, or an age - related eye disease, such as age - related macular degeneration (amd), glaucoma, diabetic retinopathy, or cataracts . In addition, a subjective neurological questionnaire was administered to participants for life styles and/or accidental damage that might affect the brain electrophysiological data . Each participant was assigned an alphanumerical identification number instead of using their real names; the same identification number was also assigned to a particular randomized experimental treatment in accordance with the irb protocol . The participants' foreheads were cleaned with 70% isopropyl alcohol swab and air - dried for two minutes . The fnirs sensor pad was placed on the participant's forehead and held in place by a head band . The participants were then seated about 120 inches in front of the 8 ft 6 ft silver screen as shown in figure 2 . They were then given ssq consisting of 32 symptoms to rate their perception of visual fatigue as none (0), slight (1), moderate (2), and severe (3). The participants proceeded to perform the atc task presented in alignment error labeled as atc1 . Subsequently, they proceeded to perform the same atc task but with a different alignment error labeled as atc2 . Lastly, the participants performed the atc task (labeled atc3) under alignment errors, followed by ssq ratings . It should be noted that throughout the experiment participants were continuously asked if they were willing to continue the experiment . The tasks involve continuous monitoring where the participants visually scanned the display to detect any two or more self - separation violations . . However, if they detected no self - separation violations, no response was issued and after 20 seconds, the display refreshed itself to a new display . The self - separation violation was set to a loss of separation of 5 nmi laterally or 500 ft . Vertically . After the completion of each session, participants were asked to rate their perception of visual fatigue . The participants were required to perform the experimental tasks as quickly and as accurately as possible . 45 mins, inclusive of irb briefing, visual acuity test, conducting the ophthalmological and neurological survey questionnaires, performing the experiments, and cleaning the participants and debriefing them . Four ssq instruments were administered per participant after each experimental treatment labeled as batc, atc1, atc2, and atc3 . For analyses, the files were generated by conducting frequency tallying each participant's responses for each experimental treatment . After obtaining the total number of responses per symptom per treatment, the total adjusted rating for each symptom was calculated as (1)adjusted ratings for symptoms=n=03wnxnn=03xn, where n = 0, 1, 2, and 3 are the index counts for none, slight, moderate, and severe ssq ratings of visual fatigue for a particular symptom, wn is the total rating for xn, and xn has the values (2)xn={x0=0;nonex1=1;slightx2=2;moderatex3=3;severe . The total weighted rating across all 32 symptoms per treatment was calculated as shown in: (3)({(n=03wnxnn=03xn)}s1 + {(n=03wnxnn=03xn)}s32)(n=24)1, where n is total number of participants; s1, s2,, s32 represent the types of symptoms rated . An fnirs sensor pad was placed on the participant's forehead specifically at the dorsolateral prefrontal cortex and held in place by a head band . The hemodynamic data was recorded using fnirs (model: 1000, fnir devices llc, md, usa) at a sampling rate of 100 khz, a frame rate of 500 ms, and samples per voxels of 25 . The data acquisition settings included an led drive current of 10 ma, an analog - to - digital (a / d) gain of 1 ma, and an initial gain drift of 1 with a balance of 4000 . These responses were changes in oxyhemoglobin concentration chbo2 (t) and deoxyhemoglobin concentration chbb (t) from the 16 voxels . The dlpfc roughly corresponds to broadman areas 9 and 46, and it covers portions of the middle and inferior frontal gyri . These regions of interest for task execution in the prefrontal area had been identified based on a previous fnirs study by izzetoglu et al . And based on a meta - analysis of other neuroimaging studies performed by cabeza and nyberg . To reduce the data dimensionality, participant averages were computed using microsoft excel 2009 for both oxyhemoglobin and deoxyhemoglobin concentration for the 16 voxels as hbo2 (mean) and hbb(mean), respectively . As indicated by fnir devices llc, the voxels 1 to 8 correspond to the left (l) dlpfc while voxels 9 to 16 correspond to right (r) dlpfc . For each of the two traditional hemodynamic variables, the mean value (hmean), the maximum value (hmax), and standard deviation value (hstdev) were computed . First, each of the dependent variables was preprocessed for outliers, missing values, or nonresponses . For the missing values, the second approach involved applying the appropriate software and technique to each dependent variable to make it suitable for statistical analysis . A model adequacy check was performed to test for the three anova assumptions of normality, independence, and homogeneity of variance . If the original data violated any of the assumptions, an appropriate transformation was applied to the data until all the assumptions were met . The cronbach was evaluated based on participants overall ssq scores for each atc task session (alignment errors). Thus, the dataset consisted of twenty - four observations and four attributes representing batc, atc1, atc2, and atc3 . This shows stable responses considering 0.7 as the cutoff for acceptable response [3134]. A paired t - test statistical technique (= 0.05) was used and the data was analyzed by sas . At 5% significance level, there was enough evidence to conclude that a prolonged use of stereoscopic display was likely to induce visual fatigue as there was a significant difference between the ssq responses between before air traffic control task (batc) and after air traffic control task (atc 3), t (23) = 15.27, p <0.05 . An exploratory analysis revealed that twenty - one of the thirty - two symptoms were pronounced after atc3 . These observed significant symptoms include ache, blurred vision, boredom, difficult concentrating, difficult focusing, dizziness eye open, dizziness eye closed, double vision, drowsiness, eyestrain, faintness, fatigue, fullness of the head, general discomfort, headache, loss of appetite, increased appetite, mental depression, salivation increase, salivation decrease, and tearing . Further, one - way anova analysis (= 0.05) was conducted to determine the effect stereoscopic alignment errors on ssq ratings . At 5% level of significance, there was enough evidence to conclude that at least one of the stereoscopic alignment errors was different, the one - way anova results, f (2, 69) = 35.38, p <0.05 . A tukey post ad hoc analysis revealed that there was a significant difference between magnification difference and vertical shift (p <0.05) and magnification difference and rotational error (p <0.05). There was no significant difference between vertical shift and rotational error . A one - way manova statistical technique (= 0.05) was used to analyze the effect of display alignment errors on cerebral tissue hemodynamic responses with sas . For any significance, a tukey post ad hoc analysis was conducted to reveal the significant difference . For the hemodynamic responses, the hbb variable, w = 0.406, p <0.05, and hbo2 variable w = 0.476, p <0.05 . The normality tests were violated . As a result of significant violations of the model adequacy checks by the hemodynamic data various transformation approaches were applied to the hemodynamic response data and further analyzed for model adequacy check . After the transformation, shapiro - wilks test showed that w = 0.04, p = 0.65; for hbo2 variable, w = 0.015, p = 0.548 . Levene's test for homogeneity of variance for hbb and hbo2 variables, analyzed with anova of squared deviations from group mean, had significant results for hbb, f (5, 272) = 1.23, p = 0.541, and hbo2, f (5, 272) = 0.74, p = 0.75 . All the variables were positively correlated with the exception of (l) dlpfc - hbb and (r) dlpfc - hbo2, which had negative correlation between them as shown in table 4 . This means that as an activity increases in the left dorsolateral prefrontal cortex, the less oxygen is demanded in the right dorsolateral prefrontal cortex . From table 4, this means that an increase in activity in the right dorsolateral prefrontal cortex requires an increase in oxygen demand . We noted that r dlpfc - hbo2 was also positively correlated with l dlpfc - hbo2 . Thus, any oxygen requirement in the right dorsolateral prefrontal cortex results in equivalent increase in demand in the left dorsolateral prefrontal cortex (a positive correlation of 0.44). Further, r dlpfc - hbb has a positive correlation of 0.55 with l dlpfc - hbb, meaning that performance of visual activities is concurrent in both right and left dorsolateral prefrontal cortex . The one - way manova results showed wilk's lambda = 0.924, f (8, 132) = 0.66, p <0.05 . Thus, at 5% level of significance, there was enough evidence to conclude that the transformed hemodynamic response as a composite score was significant . Further, at 5% level of significance, there was enough evidence to conclude that at least one of the stereoscopic alignment errors was different: for l dlpfc - hbb transformed dataset, the anova result, f (2, 69) = 0.10, p <0.05, for l dlpfc - hbo2 transformed dataset, the anova result, f (2, 69) = 0.15, p <0.05, and r dlpfc - hbb transformed dataset, the anova result, f (2, 69) = 1.09, p <0.05 . Tukey analyses for l dlpfc - hbb, l dlpfc - hbo2, and r dlpfc - hbb results are shown in table 5 . It was possible to elicit tissue hemodynamic response and simulator sickness questionnaire (ssq) ratings based on visual task in stereoscopic alignment errors . From the second to the third experimental sessions, the participants' perceptual ratings of visual fatigue increased from slight to moderate or moderate to severe . The left dorsolateral prefrontal cortex was affected more than the right dorsolateral prefrontal cortex . The oxygenated hemoglobin and deoxygenated hemoglobin in the left dorsolateral prefrontal cortex were significantly affected by the stereoscopic display alignment error . While in the right dorsolateral prefrontal cortex, the stereoscopic display alignment error had an impact on only the deoxygenated hemoglobin ., responses to stimuli changes result in increase or decrease of regional cerebral blood flow (rcbf) to this localized brain region . The cortical tissue oxygenation requirement in the left hemisphere indicates that the effect of visual fatigue is more pronounced in the left dorsolateral prefrontal cortex . Using the oxygenated hemoglobin, deoxygenated hemoglobin, blood volume, and oxygenation levels, a 3d scatter plot for neuroimaging data comprising deoxygenated hemoglobin, oxygenated hemoglobin, and oxygenation level was plotted as shown in figure 4 . This was depicted by the upward movements toward the vertex of the oxygenation level and oxygenated hemoglobin variables . The same pattern was depicted in the right dorsolateral prefrontal cortex . However, for the right dorsolateral prefrontal cortex, the distribution was narrower than the left dorsolateral prefrontal cortex . The atc tasks induced more cognitive processes in the left dorsolateral prefrontal cortex than the right dorsolateral prefrontal cortex . As a result of cognitive load, more energy was required by the brain with a corresponding oxygen requirement . According to hansen, the concentration of oxygen in the brain is about 0.1 mol g, of which 90% is in oxy - hbb in brain capillaries . This concentration can support the normal oxygen consumption of about 3.5 mol g min for two seconds . For this reason, any increase in neural activity in the brain is followed by the rise in regional cerebral blood flow (cbf). Oxygen is transported to neural tissue via oxygenated hemoglobin (oxy - hbb) in the blood . The demand for glucose and oxygen by neuronal tissues in a particular brain region may vary due to particular processing requirements at a particular time . Increases in hemoglobin and oxygenation levels in left dorsolateral prefrontal cortex mean that the left side of the brain is more engaged with visiomotor cognitive processes than the right side of the brain . The post ad hoc analyses revealed that there were significant differences between magnification error and rotational error and magnification error and vertical shift error . For the right hemisphere of the dorsolateral prefrontal cortex, oxygenated hemoglobin, blood volume level, and oxygenation levels were significant . Since magnification difference errors were significant, we conducted a further analysis to identify likely points of visual fatigue . The hemodynamic responses revealed that significant differences exist between left and right dorsolateral prefrontal in the alignment errors in visual attention tasks, f (1, 47) = 0.034, p <0.05 . Generally, oxygenation levels were increased in both left and right dorsolateral prefrontal; however, it was more pronounced in the left dorsolateral prefrontal . In the left dorsolateral prefrontal, the increased oxygenation levels resulted from the corresponding increased oxygenated hemoglobin and blood volume . Contrasting to the left dorsolateral prefrontal, oxygenated levels increased without any blood flow to that region . More interestingly, the pronouncements in left dorsolateral prefrontal can be traced to the fact that all participants were right - handed . Regrettably, it is well documented that the introduction of three - dimensional displays such 3d tv, high - definition multimedia interface (hdmi) desktop, head - mounted display, helmet - mounted display, and 3d glass into consumer markets has resulted in the increase of oculist visits for visual complaints [38, 39]. From the empirical findings, stereoscopic display alignment errors can induce visual fatigue with accompanying underpinning physiological effects associated with visual task performance . Visual fatigue is likely to increase the hemodynamic response in the left dorsolateral prefrontal cortex of the brain and delta band waves can be used to predict cognitive fatigue across all the occipital cerebral cortex . To improve 3d or stereoscopic display systems for minimum visual fatigue to the users, the optical axes system in display devices should be designed with minimum magnification difference and rotational error, as these errors were observed to be most prevalent . 3d display designs with minimum magnification difference and rotational error are more likely not only to reduce visual fatigue but also to increase performances and satisfaction of the users such as aviators and flight pilots.
Acute myocarditis is associated with various electrocardiogram (ecg) alterations, including st - t changes, q - waves, atrioventricular- and intraventricular - conduction delays, atrial and ventricular tachyarrhythmia, and low voltage . Fulminant myocarditis with ventricular arrhythmia or atrioventricular block is associated with a high mortality rate . Extracorporeal membrane oxygenation (ecmo) is instituted for the management of life threatening pulmonary or cardiac failure, when no other forms of treatment are likely to be successful . We describe life salvage of ecmo in a patient with cardiogenic shock and malignant arrhythmia caused by acute fulminant myocarditis . A 53-year - old korean - chinese woman was brought into the emergency department of our hospital by an ambulance, with complaints of progressive chest pain and dyspnea . She had a high fever, cough, and general fatigue over the three days . Upon arrival, she was fully conscious and had signs of cold sweating, cyanosis and general pallor . The initial creatine kinase (ck), ck - mb and troponin - i levels were 626 u / l, 47.27 ng / ml,> 50 ng / ml, respecitvely . Emergent coronary angiogram was performed because of low blood pressure (88/56 mm hg), st - segment depression in the precordial leads of ecg (fig . Coronary angiographic findings were normal, and an intra - aortic balloon pump (iabp) was immediately inserted for hemodynamic support (fig . 2). On the basis of prodromal symptoms, elevated cardiac enzymes and normal coronary angiographic findings with ventricular wall motion abnormality, we contemplated acute decompensated heart failure due to clinical suspicion acute myocarditis . She was admitted to the coronary care unit, and mechanical ventilation and inotropic agents were started . We decided to support the patient with ecmo, which was inserted in a veno - arterial configuration by cannulation on both femoral vessels . We were able to reduce the amount of inotropic agents using mechanical circulatory support . However, ecg showed wide qrs tachycardia with left bundle branch block (fig . 3a). The serum creatinine level was elevated, and the amount of urination was decreased . Because of low blood pressure and the lack of diuretic effect, continuous renal replacement therapy was started using the ecmo circuit on day 1 . Ventricular wall movement was gradually decreased, and ecg showed little or no electrical activity (figs . 1d and 3b). On day 2, iabp had to be removed because of its malfunction . Ecg showed little or no electrical activity . On day 5, ecg revealed weak electrical activities (fig . Mechanical ventilator was weaned off on day 18, and ecmo was weaned off on day 20 . She underwent cardiac magnetic resonance imaging after making sure that she was medically stable, which showed diffuse wall thinning, decreased wall motion and delayed enhancement of diffuse ventricular wall sparing of the infero - septal wall (fig ., she was complicated with ischemic insult in the lower extremities, hepatitis with jaundice, and acute tubular necrosis . On day 76, she was discharged from the hospital . Fulminant myocarditis is an inflammatory process that occurs in the myocardium and has a fatal course due to the rapid development into acute heart failure or cardiogenic shock.1) fulminant myocarditis is associated with various ecg alterations, including st - t changes, q - waves, atrioventricular- and intraventricular - conduction delays, atrial and ventricular tachyarrhythmia, and low voltage.2) our case demonstrated various alterations of ecg, which are some of the characteristics that are observed in the fulminant myocarditis . At first, ecg showed st segmental changes associated with myocardial ischemia, then ecg showed various manifestations, such as ventricular tachycardia, atrial fibrillation with left bundle branch block, and complete atrioventricular block . It also displayed very little electrical activity on the whole leads, resembling an ecg after arrest . Low voltage on ecg can be observed in obesity, lung emphysema, pericardial effusion, pulmonary edema, hypothyroidism and amyloidosis, however low voltage on precordial leads, which is less common than on limb leads, implies cardiac tamponade or post - defibrillation state.3) low voltage ecg in our case was probably due to pulmonary edema, pericardial effusion and edema of the ventricular wall . Patients presented with syncope, new ventricular arrhythmia or atrioventricular block, ejection fraction of less than 40 percent, and lack of response to usual care within 1 to 2 weeks are associated with a high mortality rate in acute myocarditis.4)5) extracorporeal membrane oxygenation is instituted for providing emergent circulatory support to patients with cardiogenic shock . Extracorporeal membrane oxygenation is not only useful for acute decompensated heart failure with low cardiac output, but also for life - threatening arrhythmia.6) it provides a bridge - to - recovery or decision regarding either heart transplantation or ventricular assist device . Its adaptation can physiologically reduce wall stress, decrease cytokine activation, and improve myocardial contractility . However, its use is limited by complications such as thromboembolic events.7) it is reported that patients with ischemic insult in the lower extremities or multi - organ failure due to thromboembolic events have poorer prognosis.6) long term mechanical circulatory support such as implantable left ventricular assist devices is better treatment modality for advanced heart failure patients with nyha functional class iii b or iv, intermacs level 1 to 5 as a bridge to recovery or bridge to transplantation.8) unfortunately the device is not yet commercially abailable in korea . In summary, this patient had multiple factors associated with high mortality, such as low cardiac output and malignant arrhythmia . In addition, ischemic insults in the lower extremities and multi - organ failure (lung, kidney and liver) occurred during intensive therapy . However, early adaptation of mechanical circulatory support, such as iabp and ecmo, combined with intensive medical therapy could save our patient with high - risk fulminant myocarditis.
Brachytherapy can be considered the ultimate conformal therapy in the armamentarium of radiation therapy techniques . It implements sophisticated tools for applicator placement, dose optimization and delivery, but its inherent physical charateristics (internal sources, rapid fall - off of the dose and gradient generation at the edge of target volumes) causes brachytherapy to become self - optimized by nature . No other conformal therapy (except maybe proton therapy) can achieve the degree of conformation and low integral doses to the rest of the anatomy . Since many years brachytherapy has played a major role in the treatment of cancer . It has been used, combined with external beam radiotherapy in the treatment of gynecologic malignancies with good results . Prostate brachytherapy has open a new era in organ and function conservation and became the most prominent example of highly conformal therapies . The use of brachytherapy in breast cancer has contributed to the change of paradigm in breast conserving therapies for early stage low risk breast cancer . Finally, the use of intraoperative brachytherapy approaches will contribute in the following years to more radical surgical results . In the last forty years a considerable effort was made to understand the relationships between delivered dose, dose rate and irradiated volume . By the late seventies it became clear that low dose rate brachytherapy was an optimal treatment in a variety of tumours, including head and neck and breast . This created the basis of the clinical radiobiology, applied to optimize treatments with radiotherapy . Brachytherapy can exploit the properties of the tissues it interacts with to improve the therapeutic ratio, creating special conditions depending on the technological solution used . Every modality (low - dose - rate, high - dose - rate, pulsed - dose - rate, permanent implantation) creates a different dose - rate condition, and the mechanisms involved at the molecular level are probably different . The knowledge of the tissue kinetics parameters can lead to optimized brachytherapy treatments with more antitumoral effect without excess of normal tissue toxicity . Modern brachytherapy relies on the paradigm built around the triad dose - volume - fraction . With the advent of ct - based dose planning a more detailed knowledge of however this knowledge was only partial due to the poor resolution of ct for target volume delineation when applicators are in place and the lack of temporal information of organ motion, very important in brachytherapy due the marked gradients involved in dose delivery . On the other hand, clinical results with different dose rates (or doses per fraction) and modeling studies set the basis for the knowledge of the basic rules for tissue response to ionizing radiation . Moreover, in the last five years, technological advances in radiology and nuclear medicine gave us more understanding of the topography and metabolism of tumors, and a new dimension to optimize radiation therapy, including brachytherapy . Genomics, on the other side, opens the possibility to know individual tumor characteristics, and to personalize radiation therapy to the patient . All those developments are leading to a change in the paradigm, forcing us to revisit our concepts and to adapt to the new circumstances . Intraoperative dynamic dose calculation (iddc) represents a paradigm shift in dose prescription and specification and source delivery for brachytherapy . It mirrors the igrt paradigm in ebrt in that an intended prescription dose is adaptively matched to a changing 3d target volume, or selectively sculpted to paint the different functional volumes . This process of matching may result in alteration of a previously accepted isodose distribution at any time, until the end of the procedure when a satisfactory dose distribution is achieved . Several workflows have been outlined for intraoperative dynamic dose calculation in the field of permanent seed implantation [3, 4]. The general scheme performing iddc consists of three steps: first, at some point during the implant, coordinates of implanted vectors are identified . Second, vector images are projected onto the reference frame of the intraoperative images for planning; and finally the plan is reoptimized . To accomplish this objective, precise imaging, dose planning and in - vivo dosimetry will be needed . . It also increases the precision requirement for target volume localization and for securing geometrical precision before and during irradiation . Different technologies are available for clinical use, including pure intraoperative trus based tracking, trus - fluoroscopy fusion, intraoperative mr and intraoperative cone - beam ct . Trus provides adequate imaging of the soft tissue anatomy but it does not allow for robust localization of the implanted vectors . Various researchers have tried to use trus to segment the seeds in permanent prostate brachytherapy [57]. Fluoroscopy can be used in combination with trus to create a reliable method for intraoperative applicator capture and dosimetry optimization . Combining their images by spatial co - registration (fusion) trus offers the ability to identify the soft tissues (prostate, gynecological relapses), and fluoroscopy can provide the data needed to perform three - dimensional (3d) applicator reconstruction . Flat - panel based cone - beam computed tomography (cbct) is a strong candidate technology for intraoperative imaging in image - guided procedures such as brachytherapy [8, 9]. Cbct uses a two - dimensional detector to produce a ct reconstruction from a single rotation of a point source . The soft - tissue imaging performance and potential navigational utility have been investigated by several authors both in pre - clinical and in clinical situation . In image - guided permanent seed implantation, soft - tissue imaging performance and seed detection could satisfy the imaging and navigation requirements . The demonstrated soft - tissue visibility, excellent spatial resolution, low imaging dose, and convenient form factor make c - arm based cone - beam ct a powerful new technology for image - guidance applications, including brachytherapy, vertebroplasty and neurosurgery . Magnetic resonance imaging (mri) can overcome some of the limitations inherent to trus and cbct . Mri offers a three - dimensional dataset, arbitrary multiplanar reconstruction and better soft - tissue resolution with good correlation with trus - based evaluations and pathologic findings, making it an attractive image modality for brachytherapy dosimetry [1017]. Different experiences using mri have been reported, including prostate seed implantation and gynecologic malignancies [1820]. Over the last few years, the use of molecular imaging, particularly the use of 18f - fdg based pet, has become increasingly popular in oncology, opening a new dimension to management for patients with cancer . The potential role of functional imaging in radiation oncology can be broadly divided into four main areas . First, functional imaging is emerging as a powerful technique in radiation treatment planning assisting in target delineation . Second, functional imaging would help in the modulation of the dose to the target volume (dose painting by numbers). Finally, functional imaging can be utilized for in vivo predictive testing and in the assessment of response to radiation therapy . Recent advances now allow highly specific and sensitive detection of cellular and molecular events non - invasively . The diagnostic imaging for radiation oncology aims to map in three dimensions the distribution of a tumor, tissue, or functional feature, and to provide information about the clinical response of tumors or healthy tissues to radiotherapy . In solid tumors, the aim is to provide images of phenotypic or microenvironmental characteristics known to affect the clinical response . Most research has been done to detect tumor burden and clonogen density, tumor hypoxia or proliferation . In the new paradigm (dose - guided brachytherapy), imaging is used to know the coordinates of the tumor cells, and to guide applicator insertion to the correct position . To map cancer cells, a number of new image modalities have been developed in the last years: pet, mri - mrs and power doppler us imaging are among them . All those image modalities give twofold information: morphological in one side and metabolical in the other . Combining the two different aspects it is possible to define areas where it is likely that tumor burden is present, or certain hypoxic areas, or areas of repopulation or intrinsic radiosensitivity load . Those areas are supposed to be liable to be boosted by high - precision modalities . In this the rapid fall - off of the dose would serve to precisely sculpt dose around these sub - volumes . This process is known as dose - painting, as we can paint the different dose levels we want to achieve within the target volume . Imaging is also required for precise deposition of the prescribed dose . Beyond ct - based 3d planning and us needle guidance for prostate implantation there is a brand new field of dose guidance in which the brachytherapist could see in real time the relationship between the planned dose, the applicator and the anatomical volumes of interest . Different tools can be used (ct, mri, us), every one adapted to different clinical situations . It can be intraoperative, it is fast, no radiation exposure to the staff, it is cheap and would allow direct visualization of the applicator and the intended dose overlaying together with anatomical and functional information . The new paradigm in brachytherapy relies on the new image modalities for tumor mapping and dose guidance, and brachytherapy will obtain a clear advantage from this modalities that could translate in better treatments, more conformal to the target volume, more dose - intense, and less toxic to the surrounding tissues . Magnetic resonance imaging (mri) provides numerous techniques for image - based surrogates of different functional pathways: angiogenesis (perfusion mri), metabolism (mr spectroscopy), tissue at risk and tumor cellularity (diffusion weighted imaging), and motion (4d mri). Contrast - enhanced dynamic mri provides information about tissue perfusion, visualising differences between tissues in the behaviour of a gadolinium - based contrast medium . It has been applied to many different tumors, such as cancers of the breast, prostate, cervix, rectum and liver, as well as gliomas, pulmonary nodules and multiple myeloma . From signal intensity time curves descriptive parameters such as lag time, amplitude, slope and area under the curve (wash - out) can be calculated . Blood oxygen level - dependent (bold) mri can depict clinically significant prostate tumor hypoxia . Co - registration studies shown a significant correlation between pimonidazole immunostaining of coregistered histological sections and the value of mr relaxivity parameter r(2) * . Mr spectroscopy (mrs) can provide metabolic information about tumour cells and the surrounding tissue . Shifts in the distribution of certain metabolites provide important hints towards both differential diagnosis and the tumor biological behaviour . Mrs can well distinguish cancer dominant regions from the normal prostate tissue within the prostate gland based on the depletion of citrate relative to choline and creatine . In addition, cancer - positive voxels detected by 3d mrs study can be precisely overlaid on the corresponding mr images, which provides morphological imaging of the lesions . This information can be loaded in the planning of transperineal seed implantation [27, 28]. Fdg - pet (18f - fluoro - deoxyglucose positron emission tomography) is an established imaging technique, developed 30 years ago . Fdg is an analogue of glucose, which is taken up into cells by glut-1 receptors, which are over - expressed in tumor cells . Intracellularly, the fdg is phosphorylated by hexokinase with the metabolite retained in the cell . The radiolabel can be detected and usually demonstrates a high uptake in tumour relative to normal tissue . Although fdg uptake is not tumor specific, fdg - pet is a very useful tool in oncology, with its use is spreading in the last few years . Positron emission tomography has been explored by some authors for planning brachytherapy in cervix cancer [3033]. Feasibility of using pet images for treatment planning was demonstrated with a phantom study showing acceptable reconstruction accuracy . Despite the limited number of patients included in these studies, the authors conclude that pet - based treatment planning allowed for improved coverage to the planning target volume in cervix cancer endocavitary brachytherapy . Future developments using functional ultrasound (power doppler imaging, elastography) could bring functional imaging into the operating room . Those imaging techniques should have far - reaching therapeutically implications in some tumor localizations, like prostate and cervix cancer . A progressive increase in the microvessel density (mvd) has been shown by immunohistochemical staining as prostate cancer progresses through various pathological stages . It has been reported that mvd is an important prognostic marker, and is an independent predictor of pathological stage and of the malignant potential of prostatic carcinoma . The use of vascular imaging for detecting neo - vascularity is a rational choice, since it has been demonstrated that tumors larger than 1 mm in area must recruit new blood vessels to grow larger . This neovascularity is expected to give rise to detectable flow using the power doppler principle . Tumor vascularity can be quantified in power doppler images by computing the color pixel density (cpd), which is equal to the percentage of image voxels within a region of interest that exhibit detectable flow . Nakanouchi et al . Reported a clinical study revealing correlation between the degree of blood flow signals on pdi and the microvascular density as determined in radical prostatectomy specimens . With the advent and the fast evolution of the ultrasound contrast agents a new horizon for doppler imaging sonocontrast agents increase the echoes obtained from the arterial blood with a factor of 3 - 20, and so the efficacy of doppler examination . Contrast enhanced ultrasound examinations are reported to improve the detection of malignancies within the prostate, suggesting that perfusion based imaging techniques have the potential to improve the detection of the intraprostatic dominant cancer lesions . Three - dimensional contrast enhanced power doppler techniques appear to improve the detection of prostate cancer and have the potential to visualize lesions with increased microvessel density . This semiquantitative evaluation of blood flow signals in prostate cancer lesions, as evaluated by pdi might be of clinical use when planning prostate brachytherapy . However, up to date, no clinical results have been presented using this imaging approach . Taking the classic concepts of dose - rate and volume effect, modern brachytherapy moves to personalized treatments, using predictive assays and detailed functional information of the tumor to model response of the individual patient to the given treatment . As we already mentioned above, functional imaging gives a picture of the tumor biology, allowing dose delivery to be much more adapted to the actual tumor . Dose prescription will be individualized, with different dose levels to the whole target volume and the different sub - volumes, including the dominant lesion or the more radio - resistant hypoxic regions . Predictive assays are very useful tools to model the behaviour of different tumors based on their individual genetic profiling . Microarray technology has been exploited for its predictive ability in disease development, clinical outcome and prognosis - based treatment . Microarrays have been used in to discriminate which patients treated with brachytherapy for head and neck tumors would need prophylactic neck dissection as part of their treatment . However, predictive assays for local relapse after surgery or radiation therapy are lacking, but needed to personalize local treatments (giving different dose levels to good and poor responders, adding bio - modulators or other local strategies). Targeted brachytherapy is a new integrative paradigm, where the goal is to improve therapeutic ratio through the integration of detailed information of the tumor coordinates and genetic profiling and precise delivery of the prescribed dose using image guidance . Technology for intraoperative functional imaging is already available (us power doppler, and elastrography). The work in progress is promising and the whole paradigm could be a reality in a few years . In conclusion, the combination of functional imaging with the new tools for intraoperative dose calculation and optimization opens new and exciting times in brachytherapy . New optimized protocols are needed and should be tested in controlled trials, to demonstrate an advantage of such a new paradigm.
Foot - and - mouth disease (fmd) is a highly infectious disease of cloven - hoofed animals . The causative agent, foot - and - mouth disease virus (fmdv), is a member of the genus aphthovirus within the family picornaviridae and has seven serotypes: o, a, c, asia1, and southern african territories (sat) 1, sat2, and sat3 [1, 2]. The fmdv genome includes a single open reading frame (orf) flanked by 5 and 3 untranslated regions, 5-utr and 3-utr . Upon infection, the viral rna is translated into a single polyprotein that is concurrently processed by three virus - encoded proteinases, leader (l), 2a, and 3c into precursors and consequent mature structural (vp1, vp2, vp3, and vp4) and nonstructural (l, 2a, 2b, 2c, 3a, 3b, 3c, and 3d) proteins [4, 5]. In the late 1950s, a fmd - like disease occurred in baoshan county, yunnan province, china . A virus related to the outbreak was isolated and named as the fmdv zb (zhongguo baoshan) strain . Subsequently, a live attenuated vaccine was developed by serial passaging wild - type virulent zb strain in suckling rabbits for more than 100 passages, in order to prevent fmd asia1 outbreaks on the border between china and myanmar from 1960s to 1990s . A review of the passaging history of the virulent zb strain attenuation process revealed that the virus after the 60th passage still caused clinical fmd but did not cause visible clinical symptoms in cattle after 109th passage . The virus passaged to the 114th passage was therefore used as a live vaccine after its safety and efficacy were confirmed . However, the criteria for the selection of the attenuated strain were highly empirical, and little is known about the molecular mechanisms causing attenuation . The complete genome sequence of the attenuated strain and its virulence determinants must be clear for quality control of the vaccine . Although the entire genomes of a cell - culture rabbit - attenuated zb / cha/58(att) strain and an inactivated vaccine ynbs/58 strain deriving from the same origin of zb strain had been previously compared, the virulence determinants of the zb strain have not been elucidated . A total of 25 amino acid substitutions were observed between strains zb / cah/58(att) and ynbs/58 . Therefore, we compared the entire genomes of three different rabbit - passaged attenuated zb strains (zb / cha/58(att), zbrf168, and zbrf188) and their virulent parental viruses (zbcf22 and ynbs/58), in order to identify genomic changes that occurred during the attenuation process of the zb strain . Virulent fmdv asia1 zbcf22 strain was passaged 22 times in cattle via needle inoculation and then passaged on tongue epithelium in baoshan county, china . It was maintained as a challenge virus strain at yunnan provincial research center for veterinary biological products . The attenuated zbrf168 and zbrf188 strains were derived from virulent parental zbcf22 strain via consecutive passage for 168 or 188 times in suckling rabbits following the established protocol with modifications . Briefly, a 5-day - old suckling rabbit was subcutaneously inoculated with the viral agent . The rabbits showed clinical signs of short breath and leg paralysis 1620 hrs after inoculation and died between 18 and 28 hrs . The carcasses of dead rabbits were harvested and homogenized in pbs buffer at 1/10 (w / v) followed by clarification by centrifugation at 1000 g for 30 min . The supernatant containing the viral agent was then used as inoculums for the next passage . Zb / cha/58(att) strain is a cell - adapted rabbit - attenuated strain that was passaged 187 times in suckling rabbits and adapted to bhk-21 cells . Synthesis of cdna was carried out using 6 random primers and superscript ii reverse transcriptase (invitrogen, usa). Six cdna fragments covering the entire fmdv genome were amplified by pcr using six primer sets (table 1). Pcr amplifications with pyrobest pfu polymerase (takara biotechnology co. ltd ., dalian, china) were performed according to the manufacturer's protocol . For pcr amplification, pcr reaction conditions were as follows: 1 cycle predenaturation for 5 min at 95c, 30 cycles for amplification at 94c for 30 sec, 58c (for f1 fragment) or 52c (for f2, f3, f4, f5, and f6 fragment) for 30 sec, 72c for 1 min (for f1) or 3 min (for f2, f3, f4, f5, and f6), and 1 cycle for final extension at 72c for 10 min . The pcr products were purified from agarose gel electrophoresis and sequenced directly using a abi - pris mtm 377xl dna sequencer at two companies, sangon biotech (shanghai, china) and taihe biotech (beijing, china). Fmdv reference sequences were acquired from the genbank database of the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/). The sequence data were assembled using the program assemble (vector nti 8.0 suite, informax, north bethesda, md, usa). Multiple sequence alignments were performed using a clustalx multiple sequence alignment program, version 1.83 . A phylogenetic tree was constructed by the neighbor - joining method with the kimura 2-parameter nucleotide substitution method using 1000 bootstrap replicates in the mega version 3.1 . The genome sequences of the virulent zbcf22 and the rabbit - attenuated strains (zbrf168 and zbrf188) were determined to be 8,164 nucleotides (nt) in length (excluding the poly - c and poly - a tracts) and contain an orf of 6,990 nt that encodes a polyprotein of 2,329 amino acids, which is consistent with the previously reported zb / cha/58(att) genome . The orfs of these strains were shown to be flanked by a 5-utr of 1,081 nt and a 3-utr of 93 nt . A neighboring - joining (nj) tree construct was based on the sequence alignment of 21 selected genomes, which were distinctly divided into seven serotypes of fmdv and the zb strains were tightly clustered in the asia1 serotype (figure 1). The results demonstrate that zb strains belong to the fmdv serotype asia1 from the perspective of comparative genomics . The nucleotide sequences were compared for the three attenuated zb strains, the two virulent strains, and other fmdv asia1 reference strains . Observed differences in the 5-utr between the virulent and attenuated zb strains were limited, with only one common nucleotide mutated in the internal ribosome entry site (ires) region (c573-g) (table 2). The critical role of the ires element in mediating efficient translation of the viral rna suggests that this mutation may be involved in the attenuated phenotypes of the zb strains, presumably through a disruption of rna secondary structure of ires . It has been reported that fmd virus after 100 passages in bhk-21 cells carried two point mutations in the ires and showed increased virulence in cells . In contrast, previous studies have shown that nucleotides changes in the utr between virulent and attenuated fmdv strains (i.e., strains o1 campos and c3 resende) may not be the key determinants of egg - adapted attenuated fmdvs . The fmdv 3-utr was predicted to fold into two well - defined stem - loop (sl) structures, highly conserved among isolates and essential for viral infection and ires activity . Of the two, the first stem - loop (sl1) is to be dispensable for infectivity acting as a replication enhancer . In the genomes of zbcf22 and zb / cha/58(att) strains, guanine was located at the 24 position of sl1 in 3-utr (figure 2), but guanine was replaced by adenine (table 2) in the genomes of the zbrf168 and zbrf188 strains . We conclude that this noncommon mutation in the 3-utr is unlikely to be involved in the process of the attenuation . Comparison of the protein coding regions between the virulent and attenuated zb strains revealed no deletion / insertion mutations . A total of 33 amino acid substitutions were observed scattered across nine proteins (l, vp2, vp1, 2a, 2b, 2c, 3a, 3c, and 3d). Five of them (vp2: d133-g, vp1: p146-f, g155-r, 2a: k8-e, and 3a: a51-g) only existed in the rabbit - attenuated zb / cha/58(att) strain, whereas 28 common amino acid changes were found during the process of attenuation of the zb strains . No amino acid changes in the vp4, vp3, and 3b protein had been found after attenuation (table 3). In addition, a total of 21 characteristic nucleotide substitutions, all of which produce silent mutations, were found scattered across ten proteins (l, vp4, vp2, vp3, vp1, 2b, 2c, 3a, 3b, and 3d) (table 4). In protease 3c of the zb strains, a common substitution v74-i was observed during the attenuation process, which was identical to the other fmdv asia1 reference strains (table 3). Residue v74-i substitution might not be essential for attenuation since both amino acids belong to the same hydrophobic group, although fmdv 3c is critical for viral pathogenesis and plays vital roles in both the processing of the polyprotein precursor and rna replication [15, 16]. In fact, proteases play an essential role in viral polyprotein processing and have been shown to be important virulence determinants in many pathogens [1719]. In protease l of zb strains, three common amino acid changes (n2-d, m143-l, and e147-g) have been found . Two of them (n2-d and e147-g) changed the charges of the amino acids and thus may likely contribute to the attenuated phenotype of zb strains . Residue n2-d substitution was located at the inter - aug region of fmdv, which has been associated in the attenuation of fmdv a24 strain . Fmdv l was shown to be a virulence determinant based on experiments with l lacking virus, which is highly attenuated in cattle and pig [21, 22] and not required for viral replication . L plays a central role in pathogenesis through regulation affecting the host innate immune response [2325]. Residue l143 of the fmdv l is a determinant of cleavage specificity, but a hydrophobic residue (m / l) substitution in the l may not be the key virulence determinant for the zb strain attenuation . In the conserved rna - dependent rna polymerase 3d, three common amino acid changes (r84-h, v158-a, and h378-q) were found after attenuation by passaging in vivo . However, four picornaviral polymerase peptide motifs kdelr, psg, yggd, and flkr [27, 28] were conserved among the virulent and attenuated zb strains, and three hypervariable and hydrophobic antigenic regions (aa 1 to 12, 64 to 76, and 143 to 145) were also stable in all zb strains [29, 30]. Furthermore, it has been recently reported that the r84-h mutant is a high fidelity variant and does not correlate with virus attenuation . We suspect that residues r84-h and v158-a substitutions are not the key determinants of attenuation, but that the h378-q mutation (a basic amino acid changed to an amide amino acid) may be related to the attenuation of zb strains in cattle . In the structural protein p1 - 2a region of the zb strains, no substitution was observed in the vp4 or vp3 protein after the attenuation process . It was previously reported that residue r56-h substitution in the vp3 of o1campos strain could lead to thermostability and induced typical clinical signs of fmd . We found that a common substitution (v184-l) in the vp2 protein of the zb strains located at the previously identified t - cell epitopes (aa 179 to 187). Fmdv vp2 associates mainly with virion structural stability and maturation . Therefore, considering that valine and leucine residue share the same amino acid properties, we assume that substitution v184-l may not play a critical role in the attenuation of the zb strains . The highest mutation rate was found in the vp1 protein with eight amino acid substitutions during the attenuation process of the zb strains (table 3). Four of them (a4-t, p45-l, a81-v, and s147-a) included amino acids with similar nature, while two others (a21-e and s142-r) resulted in a change of amino acids charge . It was previously proposed that changes of viral surface may play an important role in attenuation process . Indeed, a recent study demonstrated that the capsid proteins of the o1kaubeuren b64 strain were responsible for its attenuation in cattle . Moreover, the adaptive replacement of l147-p in vp1 of the guinea pig - adapted c - s8c1 strain abolished growth of the virus in different established cell lines and modified its antigenicity . Additionally, substitution of d143-a in the g - h loop antigenic site (vp1 residues 138 to 150) abolished infectivity of virus in suckling mice . After zb strain attenuation, the influence of the s142-r change in the 1 position and s147-a change in the + 2 position of rgd is not clear (figure 3(a)). The amino acid substitutions around the rgd motif suggest advantages of these substitutions in host cell recognition and binding during the attenuation process of the zb strains . It is conceivable that these amino acid substitutions may modify the surface properties of the virion in a way that may reduce its virulence in cattle . The amino acids immediately following the rgd motif have a major influence on the ability of the different integrin species, and rgd + 1 position is important in the receptor recognition process . Residue phenylalanine at rgd + 1 position of the cell - adapted rabbit - attenuated zb / cha/58(att) was different in other zb strains, suggesting that this substitution may be associated with the cell adaptation in bhk-21 cells . In the fmdv genomic p2 region, during the virulent zb strain attenuation process, three common residues with a similar nature (n19-s, a74-t, and v136-i) of substitutions were found in 2b (table 3), making their influence in attenuation difficult to assess . Fmdv 2b protein is an integral membrane protein and localizes to endoplasmic reticulum - derived outer surface vesicles which are sites of genome replication . In the 2c protein, four common amino acid substitutions were observed after attenuation (table 3), and we suspect that amino acid substitutions k64-e and y313-h play a role in viral virulence by interactions with several host factors during infection . Fmdv 2c, with atpase activity, localizes to membrane - associated virus - replicating complexes . The 2c protein in the hepatitis a virus is one of the virulence genes in this virus . In the case of the fmdv c - s8cl strain, residue t248-i mutation in 2c was not required for virulence of virus adaptation to infect the guinea pig, and residue r55-w mutation in 2c had regained competence in plaque development on bhk21 . In the 3a protein, sequence analysis showed that no deletion or insertion mutations existed between the attenuated zb strains and their parental viruses, while five common amino acid mutations were observed (table 3). A residue substitution (a51-g) was only found in zb / cha/58(att), and another substitution (d107-n) was found in zbrf188 and zb / cha/58(att) (figure 3(b)). We propose that these amino acid substitutions are likely to be the major determinants of attenuation, especially residues e78-g, h80-c, k84-n, and r127-i, which will result in a less charged and more hydrophobic 3a protein than the one in virulent zbcf22 . Residue e78-g substitution was also observed in the fmdv r strain and its chick - attenuated r304 strain . It was previously demonstrated that the q44-r amino acid substitution in 3a of fmdv strain c - s8cl can mediate adaptation of fmdv to the guinea pig . The 3a protein has been found to be associated with bovine attenuation of egg - adapted fmdv, and deletions in 3a have been shown to be associated with fmdv attenuation in cattle and high virulence in pigs [45, 46]. It was recently shown that the partial deletion in 3a can attenuate fmdv in cattle . It would be interesting to examine by reverse genetics if these amino acid substitutions in the 3a protein of the zb strain may contribute to an attenuation phenotype in cattle . In conclusion, we identified genomic changes between the virulent and attenuated fmdv zb strains in the untranslated regions, as well as in the protein coding regions, and discussed those changes correlated with the virus attenuation in context of the current knowledge of the fmdv's molecular biology . Study data indicated that candidate amino acid substitutions might play roles in the attenuation phenotype of the zb strains . However, determining which and how those amino acid substitutions are directly involved in the attenuation of the virulent viruses will require animal experiments and cell infections to determine the pathogenesis of different mutated viruses . We are now using reverse genetic approaches to construct a series of mutants to confirm the virulent determinants during the attenuation process.
L.c.c . Owns equity in, receives compensation from, and serves on the board of directors and scientific advisory board of agios pharmaceut - icals . Agios pharmaceuticals is identifying metabolic pathways of cancer 135 cells and developing drugs to inhibit such enzymes in order to disrupt tumor cell growth and survival . Was partially supported by a pancan - aacr pathway to leadership award and a dale f. frey award for breakthrough scientists from the damon runyon cancer research foundation (dfs-09 - 14).
This study was a case - control that was conducted in isfahan; groups included 33 transurethral resection of the prostate (turp) patients (group a) and 33 patients who underwent open prostatectomy (group b); the cases were interviewed from 12 weeks to 12 months after undergoing surgery (a total of 66 patients in case groups). A three - month lag to recruit the patients was set because primary symptoms of surgery might be recovered after 3 months and ibs symptoms would occur during these months after surgery . Control groups were formed by enrollment of 33 turp candidate patients (group c) and 33 cases that were candidate for open prostatectomy (group d). Both of these groups were interviewed before their operation . Patients in both case and control groups were divided into two subgroups of middle aged (age 64) and old (age> 65). Patients in both case and control groups were selected using convenience time - based sequential sampling method from a single academic center where they were under treatment by a unique urology surgeon . Those with history of prostatic malignancy, chemotherapy or radiotherapy, operation on abdominal or pelvic region as well as those with any positive findings indicating malignant prostatic disease, even during the current illness before and after prostatectomy, were excluded . Major psychiatric problems were ruled out by direct interview as well as reviewing medical history . The project was approved by the organizational research governance council as well as the regional bioethics committee . All cases were informed about the objectives of the study and enrolled after getting consent . The patients were interviewed in outpatient setting along with their routine follow - up consultation visit . A structured interview was designed for data gathering and performed by one of the research teams using field coding . The questions covered irritable bowel syndrome criteria according to rome guidelines including diarrheal symptoms, constipation, pain and discomfort initiation or aggravation . To categorize the data of questionnaires, responses of instances were divided into negative where the answers were never or seldom and positive in the case of other answers . Quantitative variables were compared and analyzed between groups using t - test; in the case of need for non - parametric tests due to small sample size and similar problems, mann - whitney - u test was substituted . Chi - square and fisher's exact tests were used to study qualitative variables . For ordinal factors spearman mean age in case groups was 68.7 years (range: 51 - 84) and in control groups was 70.9 years (sd = 8.64, range: 52 - 79). Ten patients in case groups (group a and group b) and 5 patients (8%) in control groups (group c and group d) had ibs . Data were analyzed using chi - square test and there was no significant difference in ibs between control and case groups (p = 0.117, odd's ratio = 2.18). Seven out of ten patients in case groups were from group a and the other 3 was from group b. using the fisher's exact test, no significant difference was revealed in relative frequency of ibs between control groups and group a (p = 0.98, odd's ratio ci 95% = 0.954 - 11.306); also there was no significant difference in definite ibs between control groups and group b (p = 1.00, odd's ratio ci 95% = 0.273 - 5.449). Table 1 shows answers of control and case groups to questions regarding presence of functional bowel symptoms . On the basis of collected data, spearman test was used and relative frequency of diarrhea in case groups were found to be significantly higher than control groups (p = 0.012, odd's ratio = 1.75). Control groups data were compared separately with group a and group b. relative frequency of post - operative diarrhea in the patients who underwent open prostatectomy was higher than the control groups (p = 0.034, odd's ratio = 3.06). No significant difference in relative frequency of diarrhea was found between control groups and group b (patients underwent turp) (p = 0.87, odd's ratio = 0.92). Within the case groups, the relative frequency of post - operative diarrhea was higher in group a (patients underwent open prostatectomy) than the group b (patients underwent turp) (p = 0.026, odd's ratio = 0.30). Frequency distribution of bowel symptoms in patients with bph before and after surgery no significant differences in relative frequency of constipation were found between case groups and control groups (p = 0.61, odd's ratio = 0.74). Constipation was reported to be seen in similar extend by both open prostatectomy and turp group (p = 0.632, odd's ratio = 0.72). One part of the study referred to the onset of abdominal pain related with defecation in patients; no significant difference was seen between subgroup a and subgroup b in this regard (p = 0.87, odd's ratio = 0.92). Three patients in open prostatectomy group reported feeling better regarding these symptoms following surgery whereas in turp group no one felt better . Aggravation of functional bowel symptoms, including diarrhea, abdominal pain and bowel habit alternation was evaluated in the case groups between open prostatectomy subgroup and turp subgroup after surgery and no significant difference in symptoms alteration was found (p = 0.427, odd's ratio = 2.77), except for occurrence of bowel habit change after surgery that were higher in open surgery group . Twenty patients were middle aged and forty six patients were old in case groups; twenty two patients in control groups were middle aged and forty four were old . There was no significant difference between groups regarding patients ages (p = 0.70, odd's ratio = 0.87). Then association of age with bowel symptoms aggravation was evaluated by chi - square test . Aggravation of functional bowel symptoms after surgery was reported by three middle aged patients while four old patients reported this change too . No statistically significant difference in ibs symptoms aggravation between middle aged and old patients was found (p = 0.425, odd's ratio ci 95% = 0.109 - 2.672). The main idea of this study was to determine the association between prostate surgery and change in functional bowel symptoms . In our knowledge, although the study sample was small, this is the first forward study regarding bowel symptom changes following prostatectomy; it is also notable that the present study compared minimally invasive versus routine surgery . Also to cover possible limitations, the control groups were selected from the same population that the case groups came from . The instances in case groups were recruited 3 months after the surgery15; this time range was chosen since the initial stressful period should be passed before evaluating the patient for chronic pain and symptoms following operation . Also according to most of routine criteria of functional bowel disorders 12 it was previously reported in other cross - sectional studies that functional bowel symptoms were significantly correlated with history of abdominopelvic surgery; within these operations cholecystectomy was the most prevalent and those with irritable bowel symptoms had four fold history of gallbladder operation.14 these type of studies had a temporal ambiguity of causality relationships . The main positive finding of this study is that open prostatectomy was followed by significant increase in diarrhea and bowel habit alternation associated with onset of abdominal pain . Specifically the change was found after open operation but not after turp . As it could be seen in table 1, other bowel functional symptoms may also be altered after open prostatectomy but the main limitation of the present study was the limited sample size which would limit the power of the statistical tests to show the real differences . This is the problem of forward or prospective post - surgical functional bowel disorders studies and is related to difficulty of case finding and follow - up.16 it is also interesting that the majority of patients faced new functional problems after surgery especially after open operation although the symptoms may be minor or short term in most of them and will not extend for a long time as a chronic disorder . One assumption for development of post - surgical bowel functional changes is an inflammatory process in the pelvic region that involves sensory nerves of this region with activation of mast cells and consequently visceral hypersensitivity.17 other explanation of these functional bowel changes is triad of psychological stress, early post - operative pain and psychological background of the patient . It was considered that the higher is the psychological catastrophe related pain following surgery, the more would functional bowel be changed.18 surely one of the main preventive means would be reducing pain and anxiety due to surgery that predispose the patients to consequent complications . It is more important in those with possible psychological backgrounds that are more prone to develop symptoms.19 prostatectomy is similar to hysterectomy in some extend, since it is a pelvic surgery, relates with genital organs and possibly with personal gender - related body - image and identity and is done after youth period . Of course it is different in some aspects: extension of the operation and possible nerve manipulation, patients gender and its related psychological backgrounds, and age of the patients; but it is assumed that due to lack of data regarding postprostatectomy bowel function changes, hysterectomy - related studies may be considered as a similar model . In the patients underwent open prostatectomy, surgery led to increase in relative frequency of diarrhea and bowel habit alternation associated with onset of abdominal pain . The present data are in agreement with van dam study on bowel habit alteration, in which, hysterectomy was followed by a significant increase in the incidence of disturbed bowel function.20 but the report by heaton emphasized on higher prevalence of constipation and defecation problems in hysterectomized patients.21 previous reports showed that those with ibs who underwent operation may develop more pain and will have worse symptoms.22 although for some ibs patients, surgery may have a placebo effect on their pain and discomfort symptoms and even recovery from ibs may occur.23 previously, in an interesting study, it was reported that in 60% of cases, patients would relief from pain and discomfort after hysterectomy . To explain the etiology of changing symptoms in these patients, the author raised the idea of non - ibs related symptoms and real somatic effect of surgery on relief; also placebo effect was mentioned in the causality chain of recovery.24 similar changes in bowel symptoms were reported in minor proportion of the current open surgery group (3 out of 33); if 10 percent would be considered as the acceptable error of the tests, abdominal surgery aggravate symptoms in patients with functional bowel discomfort . On the other hand the present study revealed no increase or decrease in irritable bowel syndrome incidence or severity after surgery whereas hysterectomy was found to be a possible risk factor for definite ibs . Although, if referring to data presented in table 1 regarding initiation of new pain or discomfort, it might be understood that in both groups of post - prostatectomy patients, pain or discomfort presents in large extend, although due to possible methodological limitations and small sample size, demonstration of definite ibs increment was impossible in this study . In conclusion, it seems that prostatectomy, in both forms of open and transurethral, may cause onset of abdominal discomfort and bowel habit change that may resolve in time but significantly may induce bowel habit change in form of diarrheal symptoms . Pa and hm carried out the design, coordinated the study, and participated in most of the experiments . Ad provided assistance in the design of the study, coordinated and carried out all the experiments and participated in manuscript preparation . At
Acute disseminated encephalomyelitis (adem) is an autoimmune inflammatory disorder of the central nervous system (cns). Etiopathogenesis is thought to be immune mediated, because in up to three - fourths of the cases; it follows an antecedent infection or immunization . Currently for adem, magnetic resonance imaging (mri) is the imaging modality of choice to demonstrate lesions in white matter of brain . There are no specific biomarkers available currently to diagnose adem; hence, diagnosis is made after excluding clinical and laboratory findings and suggestive neuroradiological features of other disease . A 4-year - old boy was brought to our hospital with complaints of slurring of speech and difficulty in swallowing and fever for 2 days . He was evaluated at emergency and his gcs was 10/15 (e4v2m4). On examination, he was in altered sensorium; tone was increased in upper and lower limb with brisk deep tendon reflexes . He was started on with iv antibiotic (ceftriaxone), acyclovir and supportive measures . Prior to this he had fever for 5 days, 8 days back along with mild cough and cold . On day 4 of fever child had one episode of seizure . Mri of brain was done which was unremarkable except for mildly dilated lateral and third ventricle . He was managed with iv ceftriaxione and iv acyclovir iv phenytoin and other supportive measures . Two days later patient developed slurring of speech and difficulty in swallowing with fever for which he was admitted in our hospital . Initial investigations revealed hb - 11.8 gm / dl, tlc -15200/mcl, platelet - 3.13 lac . / l, typhi dot, widal test, blood c / s and malarial antigen test was negative . Mri of brain was done which showed inhomogeneous area of increased signal in t2 weighted images in basal ganglia, pons, midbrain, medulla, thalami, and frontal cortex [figures 13]. Lumbar puncture revealed csf cell count was 3 cells (100% lymphocytes), protein - 32 mg / dl and glucose - 56 mg / dl (capillary blood glucose was 86 mg / dl) ldh - 12 u / l . Csf bacterial antigen test causing encephalitis (streptococcus group b, h. influenzae b, s. pneumoniae, n. meningitidis, e. coli k1), herpes igm test and herpes pcr were negative . Child was started on iv methylprednisolone (500 mg) pulse therapy for 3 days, following which he was started on oral steroid therapy . Gradually his sensorium became better . By day 5, he started responding to commands . On day 6 he was discharged and at the time of discharge he was conscious oriented with gcs of e4 v5 m6 . He was seen on follow up after 7 days and neurological examination revealed alert child with mild slurring of speech . Mri t2 weighted image showing areas of hyper intensity involving brainstem mri t2 weighted image showing areas of hyper intensity involving caudate nucleus and thalamus mri t2 weighted image showing areas of hyper intensity involving white matter the annual incidence of adem is reported to be 0.40.8 per 100,000 and the disease more commonly affects children and young adults in winter / spring . Most of the case are reported post - exanthematous infection or vaccination . It may have abrupt, acute, or may evolve over a period of few days . Adem typically presents as a monophasic illness but sometimes may have a biphasic or multiphasic course depending on the neuraxis affected . Characteristic clinical features include sudden onset multifocal neurologic disturbances such as visual field defects, aphasia, motor and sensory deficits, ataxia, movement disorders, a depressed level of consciousness, focal or generalized seizures, and psychosis . As in our case, child presented with slurring of speech with difficulty in swallowing, altered sensorium and seizure . Csf is usually normal, but sometimes mild elevation of protein with lymphocytic pleocytosis can be found . Markers such as oligoclonal immunological bands, igg or myelin basic protein (mbp) are sometimes detectable, but not diagnostic . The electroencephalogram (eeg) often shows non - specific features of an encephalopathic process, and visual evoked potential (vep) responses may be delayed . In the absence of specific biologic markers, the diagnosis of adem is based on the clinical and radiologic features . In our case csf and vep studies were normal . With the wider use of mri, adem mri t2 enhancing images shows disseminated multifocal lesions in the white matter, basal ganglia, thalamus, and brainstem consistent with edema, inflammation, and demyelination . Sometimes during initial course of disease we may find a normal mri brain . Initial mri scan in our case had no evidence of adem but later mri revealed finding suggestive of adem . However, the recovery is incomplete in patients with adem not receiving any form of immunomodulatory treatment . Most of the literature is in consensus with the use of high - dose intravenous methyl prednisolone, intravenous immunoglobulin (ivig), and plasmapharesis as various modality of treatment . Intravenous methyl prednisolone is the first - line drug (1030 mg / kg / day, up to a maximum of 1 g / day) for 35 days followed by oral corticosteroid treatment continued with gradual tapering over 6 weeks to reduce the risk of relapses . Intravenous immunoglobulin (ivig) (0.4 gm / kg / day for 5 days) is another option . Either plasma exchange or ivig, could be the second - line treatment, when corticosteroids fail . Due to lack of any pathognomonic clinical feature or specific biomarker few differential diagnoses first priority should be to rule out infective causes of meningoencephalitis after ruling out infective causes demyelinating inflammatory process should be looked for . The outcome of adem is generally good, with 5789% of children making a full recovery . Adem is considered to be monophasic illness but relapse may occur and if it represents same acute monophasic immune process, the term multiphasic disseminated encephalomyelitis (mdem) is used . But it must be differentiated from second attack of multiple sclerosis which may take months to years and more commone in older age groups . Patient presenting with optic neuritis, ocular lesions, oligoclonal bands in csf examination, disseminated in space and time and periventricular lesion in mri goes in favor of multiple sclerosis (ms). It is very necessary to differentiate adem / mdem from ms as early institution of therapy may alter course of ms.
Colonic necrosis has been described as a rare complication after the administration of kayexalate [1, 2]. In this case study, we present a case of calcium polystyrene sulfonate - induced colonic necrosis and perforation to remind clinicians of this rare, but dangerous, toxicity associated with this commonly used medication . A 78-year - old woman with stage 4 chronic kidney disease (ckd) due to chronic pyelonephritis, and a right hypoplastic kidney was presented to our emergency department with a 2-day history of abdominal pain . Her medical history included epilepsy that was treated with carbamazepine, hypertension and hyperlipidaemia, for which she received carvedilol and atorvastatin . Because of the persistent hyperkalaemia, she was treated with calcium polystyrene sulfonate at 30 g / day (anti - potassium granule; assos drug, istanbul, turkey). As a result of the ckd, she was prescribed calcitriol for a mineral and bone disorder, darbepoetin alpha for anaemia and sodium hydrogen carbonate for acidosis . Upon initial physical examination, her abdomen was non - distended with normoactive bowel sounds, but there was diffuse tenderness in her abdomen . Significant laboratory values at the time of admission included sodium 138 meq / l, potassium 4.6 meq / l, ph 7.20, bicarbonate 13 meq / l, blood urea nitrogen 71 mg / dl, creatinine 2.6 mg / dl, leukocytes 15 500/mm, eosinophils 110/mm, haematocrit 32.7%, platelets 221 000/mm and c - reactive protein 10.9 mg / dl . On the second day of hospitalization, the abdominal pain worsened and free air under the diaphragm was found on abdominal radiography . Her sigmoid colon was found to be necrotic and perforated . A biopsy was performed and the perforated segment was repaired by primary closure . The surface of the deep ulcer contained necroinflammatory debris and various sized fragments of basophilic crystalloid material with angulated margins on microscopic examination (figure 1a and b). Also, there were no features of chronic colitis, including inflammatory bowel disease or chronic ischaemic colitis . (a) the basophilic crystalloid material in the necroinflammatory background of the ulcer surface (haematoxylin and eosin, original magnification, 100). (b) the basophilic crystalloid material in the necroinflammatory background of the ulcer surface (haematoxylin and eosin, original magnification, 400). Sodium polystyrene sulfonate can also bind intraluminal calcium, leading to constipation, fecal impaction and subsequent bowel obstruction or perforation . Gerstman et al . Reported a 0.27% overall incidence, with a higher incidence (1.8%) during the postoperative period . Sorbitol enemas, along with experimental evidence suggesting that the necrosis was due to sorbitol rather than the kayexalate in presence of uraemia . Extensive transmural necrosis was noted in rats receiving enemas of sorbitol or kayexalate in sorbitol in both the uraemic and non - uraemic groups . As in this case report, renal failure may be an important facilitating factor in the pathogenesis of the necrosis . In contrast to the experimental data, our case was treated with calcium polystyrene sulfonate orally, not rectally . Rashid et al . Noted that kayexalate in sorbitol given as an enema or orally to treat hyperkalaemia has been reported to induce intestinal necrosis in uraemic patients . They studied clinical and pathologic features of 15 patients and observed kayexalate crystals in tissue specimens from surgical resections and endoscopic biopsies . One possibility is elevated renin levels, commonly seen in renal insufficiency, that predispose the patient to non - occlusive mesenteric ischaemia via angiotensin - mediated vasoconstriction . One gram of kayexalate possesses a theoretical in vitro exchange capacity of 23.1 meq of potassium and in vivo capacity of 1 meq . Emmett et al . Reported that in vivo potassium - binding capacity may be lower than previously estimated, more on the order of 0.40.8 meq / g of kayexalate resin . In contrast to other minor digestive complications associated with kayexalate treatment, colonic perforation results in significant morbidity and mortality . As a result, potassium exchange resins may, although rarely, induce a colonic perforation, and this diagnosis should be considered in a patient treated as such in case of acute abdomen . The clinicians must be aware of the possible rare and serious complications of potassium exchange resins.
Intra - articular injections of corticosteroids have been used for several decades in the management of inflammatory and degenerative joint conditions when first - line conservative therapies such as rest, ice, and anti - inflammatory medications fail to provide adequate symptom relief . Based in part on this long history of successful utilization coupled with the findings of several randomized controlled trials, consensus statements and meta - analyses have concluded that intra - articular corticosteroid injections provide short - term patient benefit and clinical efficacy for chronic knee pain.13 more recently, various injectable hyaluronic acid agents have become commercially available and have enjoyed widespread clinical acceptance as an effective treatment for knee osteoarthritis . These agents are indicated for the treatment of the pain associated with osteoarthritis of the knee in patients who have failed to respond adequately to conservative nonpharmacologic therapy and simple analgesics, eg, acetaminophen . Traditionally, intra - articular injections have been performed using anatomical landmarks to identify the correct trajectory for needle placement . However, different anatomical - guided injection techniques have yielded inconsistent intra - articular needle positioning due, in large part, to the fact that the physician cannot directly visualize the area of interest, and variations in anatomy are common . Incorrect needle placement has been partially attributed to variable clinical outcomes.410 furthermore, inaccurate corticosteroid injections in the knee, for example, may result in post - injection pain, crystal synovitis, hemarthrosis, joint sepsis, and steroid articular cartilage atrophy, as well as systemic effects, such as fluid retention or exacerbation of hypertension or diabetes mellitus.1 therefore, identification of methods and proper training to aid in correct needle placement during these procedures is warranted . Various imaging modalities can be used to improve the accuracy of intra - articular injections, including fluoroscopy, computed tomography, and magnetic resonance imaging . However, musculoskeletal ultrasound is one of the most practical because it is rapid, safe, relatively inexpensive, emits no ionizing radiation, and can be performed in the outpatient clinical setting.11,12 ultrasound utilizes high - frequency sound waves to visualize soft tissues and bony structures and is a frequently used imaging modality to diagnose musculoskeletal pathology or to aid with needle guidance during interventional procedures . Importantly, unlike fluoroscopy, ultrasound allows identification of vascular and nervous structures as well as demonstrating needle movement in real time to aid in needle positioning without the use of contrast medium.13 the purpose of this review was to determine the effect of ultrasound guidance on the accuracy of needle placement, clinical outcomes, and cost - effectiveness in comparison with anatomical landmark - guided intra - articular large joint injections, with particular emphasis on the knee . The peer - reviewed literature was searched using medline and relevant bibliographies published in english language journals through december 31, 2011 that compared the accuracy of intra - articular large joint injections with imaging guidance versus conventional anatomical guidance . After screening titles and abstracts and excluding irrelevant articles, 47 articles were considered for inclusion into this review . Full - text manuscripts of these studies were retrieved and reviewed . Of the 47 articles screened, 13 studies met the criteria for inclusion in this review . All studies of large joints (ie, knee, hip, and shoulder) utilizing any imaging modality to aid with needle placement were analyzed . The imaging - guided group was further sub - divided by ultrasound or other imaging modalities . Injection accuracy rates between groups were analyzed with fisher s exact test and odds ratios with 95% confidence intervals were calculated . Clinical outcome and cost - effectiveness data were summarized in a narrative review due to inconsistent reporting that made objective data analysis impractical . A total of 13 studies were identified that met the entry criteria;1426 five studied the knee,15,17,20,21,26 seven studied the shoulder,14,16,18,2225 and one studied both the knee and shoulder.19 no comparative studies of the hip were identified . Ultrasound was used in seven studies,15,1921,23,25,26 and the remaining studies utilized air arthrography,17 fluoroscopy,14,16,24 magnetic resonance arthrography,18 or magnetic resonance imaging22 (table 1). Across all studies (using all imaging modalities and all joints), needle placement accuracy ranged from 63% to 100% with ultrasound and from 39% to 100% with conventional anatomical guidance (table 2). Imaging guidance improved the accuracy of intra - articular injections of the knee (96.7% versus 81.0%, p <0.001) and shoulder (97.3% versus 65.4%, p <0.001, table 3). In particular, ultrasound guidance of knee injections resulted in better accuracy than did anatomical guidance (95.8% versus 77.8%, p <0.001). The five studies comparing the accuracy of ultrasound guidance with anatomical guidance of intra - articular knee injections demonstrated superior accuracy with ultrasound, with an odds ratio of 6.4 (95% confidence interval 2.914, figure 1). Despite the evidence for improved accuracy of intra - articular injections using ultrasound guidance, some researchers have questioned whether improved injection accuracy translates directly into better clinical outcomes.19,27 jones et al4 reported that clinical improvement was reported in 52% (28 of 54) of subjects with correctly placed intra - articular corticosteroid injections, but in only 23% (seven of 30) with incorrectly placed extra - articular injections . This finding was corroborated by cunnington et al who demonstrated that accurate intra - articular injections improve joint function significantly at 6 weeks compared with inaccurate injections.19 furthermore, injections guided by ultrasound were associated with better self - reported health - related quality of life at 6 weeks.19 in a randomized controlled trial of 148 painful joints (62 of 148, 41.9% knee) comparing ultrasound - guided and anatomical - guided corticosteroid injections in rheumatoid arthritis (n = 100) and osteoarthritis (n = 48), sibbitt et al28 reported that ultrasound guidance resulted in a 43% reduction in procedural pain (p <0.001), a 59% reduction in absolute pain scores at the 2-week outcome (p <0.001), a 75% reduction in significant pain (defined as visual analog score 5 cm; p <0001), a 26% increase in the responder rate (defined as reduction in visual analog score 50% from baseline; p <0.01), and a 62% reduction in the nonresponder rate (defined as reduction in visual analog score <50% from baseline; p <0.01). Ultrasound also increased detection of effusion by 200% and volume of aspirated fluid by 337% . A previously unappreciated finding was the marked difference in procedural pain favoring ultrasound guidance . Although the causes of this significant beneficial reduction in procedural pain are uncertain, the authors speculated that better control and direction of the needle away from pain - sensitive structures during introduction of the needle into the intra - articular space are the most likely explanations.29 indeed, a randomized controlled trial demonstrated that procedural pain during needle procedures is a surrogate measure for direct needle trauma to patient tissues, with better needle control being significantly associated with less intra - articular bleeding, less tissue trauma, reduced pain, and less bruising.30 sibbitt et al also conducted a randomized controlled trial of intra - articular corticosteroid injections for inflammatory arthritis.31 two hundred and forty - four joints (85 of 244, 34.8% knee) were randomized to injection with conventional anatomical guidance (n = 120) or ultrasound guidance (n = 124). Compared with anatomical guidance, ultrasound guidance reduced injection pain by 81% (p <0.001), lowered 6-month pain scores by 35% (p <0.02), and increased therapeutic duration by 32% (p = 0.01). The same research group recently reported the findings of a controlled trial of 94 noneffusive knees with osteoarthritis randomly allocated to intra - articular corticosteroid treatment with either ultrasound guidance or anatomical guidance.32 ultrasound guidance yielded 48% less procedural pain (p <0.001), a 36% increase in therapeutic duration (p = 0.01), and 42% less knee pain at 2 months (p <0.03) but no difference at 6 months . Finally, sibbitt et al conducted a randomized controlled trial comparing the clinical outcome of ultrasound - guided (n = 42) or anatomical - guided (n = 22) arthrocentesis and intra - articular corticosteroid injection.33 patients who received ultrasound - guided injections reported 48% less procedural pain (p = 0.001) and 46% less pain at 2 weeks (p = 0.03). The limited evidence suggests that improved injection accuracy and correspondingly better clinical outcomes achieved with ultrasound guidance are also cost - effective . In their large, randomized, controlled trial in inflammatory arthritis, sibbitt et al31 found that ultrasound injection guidance modestly reduced the cost per patient per year by 8% ($7) relative to anatomical guidance . More importantly, ultrasound guidance significantly reduced the cost per responder per year by 33% ($64; p <0.001). In their follow - up study in osteoarthritis, sibbitt et al32 reported that ultrasound guidance reduces the costs of treating a hospital outpatient by 13% ($17), particularly in responders (reduction of 58% or $224). A total of 13 studies were identified that met the entry criteria;1426 five studied the knee,15,17,20,21,26 seven studied the shoulder,14,16,18,2225 and one studied both the knee and shoulder.19 no comparative studies of the hip were identified . Ultrasound was used in seven studies,15,1921,23,25,26 and the remaining studies utilized air arthrography,17 fluoroscopy,14,16,24 magnetic resonance arthrography,18 or magnetic resonance imaging22 (table 1). Across all studies (using all imaging modalities and all joints), needle placement accuracy ranged from 63% to 100% with ultrasound and from 39% to 100% with conventional anatomical guidance (table 2). Imaging guidance improved the accuracy of intra - articular injections of the knee (96.7% versus 81.0%, p <0.001) and shoulder (97.3% versus 65.4%, p <0.001, table 3). In particular, ultrasound guidance of knee injections resulted in better accuracy than did anatomical guidance (95.8% versus 77.8%, p <0.001). The five studies comparing the accuracy of ultrasound guidance with anatomical guidance of intra - articular knee injections demonstrated superior accuracy with ultrasound, with an odds ratio of 6.4 (95% confidence interval 2.914, figure 1). Despite the evidence for improved accuracy of intra - articular injections using ultrasound guidance, some researchers have questioned whether improved injection accuracy translates directly into better clinical outcomes.19,27 jones et al4 reported that clinical improvement was reported in 52% (28 of 54) of subjects with correctly placed intra - articular corticosteroid injections, but in only 23% (seven of 30) with incorrectly placed extra - articular injections . This finding was corroborated by cunnington et al who demonstrated that accurate intra - articular injections improve joint function significantly at 6 weeks compared with inaccurate injections.19 furthermore, injections guided by ultrasound were associated with better self - reported health - related quality of life at 6 weeks.19 in a randomized controlled trial of 148 painful joints (62 of 148, 41.9% knee) comparing ultrasound - guided and anatomical - guided corticosteroid injections in rheumatoid arthritis (n = 100) and osteoarthritis (n = 48), sibbitt et al28 reported that ultrasound guidance resulted in a 43% reduction in procedural pain (p <0.001), a 59% reduction in absolute pain scores at the 2-week outcome (p <0.001), a 75% reduction in significant pain (defined as visual analog score 5 cm; p <0001), a 26% increase in the responder rate (defined as reduction in visual analog score 50% from baseline; p <0.01), and a 62% reduction in the nonresponder rate (defined as reduction in visual analog score <50% from baseline; p <0.01). Ultrasound also increased detection of effusion by 200% and volume of aspirated fluid by 337% . Although the causes of this significant beneficial reduction in procedural pain are uncertain, the authors speculated that better control and direction of the needle away from pain - sensitive structures during introduction of the needle into the intra - articular space are the most likely explanations.29 indeed, a randomized controlled trial demonstrated that procedural pain during needle procedures is a surrogate measure for direct needle trauma to patient tissues, with better needle control being significantly associated with less intra - articular bleeding, less tissue trauma, reduced pain, and less bruising.30 sibbitt et al also conducted a randomized controlled trial of intra - articular corticosteroid injections for inflammatory arthritis.31 two hundred and forty - four joints (85 of 244, 34.8% knee) were randomized to injection with conventional anatomical guidance (n = 120) or ultrasound guidance (n = 124). Compared with anatomical guidance, ultrasound guidance reduced injection pain by 81% (p <0.001), lowered 6-month pain scores by 35% (p <0.02), and increased therapeutic duration by 32% (p = 0.01). The same research group recently reported the findings of a controlled trial of 94 noneffusive knees with osteoarthritis randomly allocated to intra - articular corticosteroid treatment with either ultrasound guidance or anatomical guidance.32 ultrasound guidance yielded 48% less procedural pain (p <0.001), a 36% increase in therapeutic duration (p = 0.01), and 42% less knee pain at 2 months (p <0.03) but no difference at 6 months . Finally, sibbitt et al conducted a randomized controlled trial comparing the clinical outcome of ultrasound - guided (n = 42) or anatomical - guided (n = 22) arthrocentesis and intra - articular corticosteroid injection.33 patients who received ultrasound - guided injections reported 48% less procedural pain (p = 0.001) and 46% less pain at 2 weeks (p = 0.03). The limited evidence suggests that improved injection accuracy and correspondingly better clinical outcomes achieved with ultrasound guidance are also cost - effective . In their large, randomized, controlled trial in inflammatory arthritis, sibbitt et al31 found that ultrasound injection guidance modestly reduced the cost per patient per year by 8% ($7) relative to anatomical guidance . More importantly, ultrasound guidance significantly reduced the cost per responder per year by 33% ($64; p <0.001). In their follow - up study in osteoarthritis, sibbitt et al32 reported that ultrasound guidance reduces the costs of treating a hospital outpatient by 13% ($17), particularly in responders (reduction of 58% or $224). Intra - articular knee injections are commonly performed by orthopedic surgeons and rheumatologists, and as the role of general practitioners in chronic disease management expands, joint injections are now frequently being performed in the primary care setting . This trend underscores the necessity to standardize the procedure to assure patient comfort and safety by employing the most accurate injection techniques possible . Numerous imaging modalities may be used to aid the clinician in identifying the correct trajectory for intra - articular diagnostic and/or therapeutic injections including ultrasound, fluoroscopy, computed tomography, and magnetic resonance imaging . However, ultrasound represents one of the most practical options because it is safe, quick, comparatively inexpensive, and emits no radiation . Although numerous studies have reported the accuracy of intra - articular joint injections using imaging or anatomical guidance, few controlled studies comparing the accuracy of these methods have been performed . The results of the current analysis demonstrate that use of imaging guidance improves the accuracy of intra - articular injection in large joints including the knee . Furthermore, the use of ultrasound guidance specifically at the knee greatly increases the likelihood of correct needle placement . These findings confirm and extend similar conclusions reached by daley et al34 in their systematic review of injection accuracy . Relative to corticosteroids that act to quell the inflammatory reaction in intra - articular and peri - articular structures, injection accuracy may be particularly important for hyaluronic acid because this therapeutic agent directly confers a number of protective properties to joint fluid, including shock absorption, traumatic energy dissipation, protective coating of the articular surface, and lubrication . A large meta - analysis by the cochrane collaboration found that hyaluronic acid viscosupplementation provides beneficial effects on knee pain, function, and patient global assessment comparable with systemic forms of active intervention, and that hyaluronic acid products have more prolonged effects than intra - articular corticosteroids.35 notably, bannuru et al36 demonstrated the time - varying effects of both agents for knee pain . Specifically, from baseline to 4 weeks, intra - articular corticosteroids are relatively more effective for pain relief than intra - articular hyaluronic acid . However, by week 4, both interventions show similar efficacy, with hyaluronic acid administration reaching peak effectiveness at 8 weeks and exerting a residual detectable effect at 24 weeks . The use of ultrasound guidance with hyaluronic acid injection has potential to improve clinical outcomes further, although prospective trials are required to confirm this theory . We also noted that accurate intra - articular injections with ultrasound guidance result in improved clinical outcomes, and preliminary evidence suggests that these patient benefits result in long - term health care savings . On balance, the assessment of cost - effectiveness of ultrasound - guided injections in these studies is likely to be somewhat overestimated by failing to account for the initial equipment costs, equipment maintenance costs, costs associated with staff training, and the additional procedural time required for ultrasound guidance, all of which are factors that may partially explain why ultrasound remains infrequently utilized . In the us, only one in five rheumatologists regularly utilizes musculoskeletal ultrasound in their practice, although three out of four agree that it should be a standard clinical tool for diagnosis, injection guidance, and gauging treatment response.37 limitations of this review include heterogeneity of the evaluation methods, lack of consistent outcome blinding methods, and a relatively small number of relevant studies identified . Of the five comparative studies of ultrasound versus anatomical injection guidance at the knee, two utilized patients with osteoarthritis, one used inflammatory arthritis, one used various forms of arthritis, and one used cadavers . The procedural methods used in these comparative studies also varied, with two studies using hyaluronic acid injection while fluid aspiration, corticosteroid injection, and dye injection in cadavers were used in one study each . Lastly, the results of this systematic review lack robustness and, with the inclusion of future trials, the reported accuracy rates may be quite sensitive to change . Overall, the use of imaging guidance, in particular ultrasound, improves the accuracy of intra - articular injection in large joints, including the knee . Furthermore, accurate ultrasound - guided intra - articular knee injections improve clinical outcomes and lower health care costs.
Development of spontaneous pneumopericardium is a very rare complication of tuberculosis with coexisting human immunodeficiency virus (hiv) infection . To the best of our knowledge only three cases of pneumopericardium with pulmonary tuberculosis concomitant with hiv infection[13] have been reported so far . A 30-year - old hiv - positive male smoker on antiretroviral therapy (art) presented with complaints of cough with expectoration and fever for two and a half months . Cough was moderate in intensity and was present throughout the day with worsening at night . Patient had intermittent fever with an evening rise . There were associated complaints of decreased appetite and loss of weight . Bp was 110/70 mm of hg and temperature was 99.2f with a respiratory rate of 20/min . A thin hyper - lucent line was present lining the lateral cardiac borders suggesting possibility of pneumopericardium [figure 1]. The air or gas in x - ray chest did not rise above the upper level of pericardium in standing erect position differentiating it from pneumothorax and pneumomediastinum . Computed tomography (ct) scan of the chest confirmed the presence of pneumopericardium [figure 2]. It also showed a well - defined thick walled cavity with necrotic areas in apicoposterior segment of left upper lobe and bilateral infiltration with minimal bronchiectatic changes [figure 3]. X - ray chest pa view showing patchy infiltration in both the upper zones with pneumopericardium ct scan showing bilateral infiltration with pneumopericardium ct scan showing cavitary lesion on the left side with pneumopericardium barium swallow fluoroscopy ruled out any esophagopericardial fistula . The patient did not have any symptoms related to pneumopericardium and its detection was purely incidental . Patient was started on anti - tubercular treatment (att) as per the guidelines of revised national tuberculosis control program . Pneumopericardium was first described by bricheteau in 1844 who named bruit de moulin (water wheel sound) associated with pneumopericardium . Pneumopericardium occurs typically because of the breech of the pericardium by traumatic or nontraumatic causes . In adults, 60% of pneumopericardium results from trauma either sharp or blunt . Iatrogenic factors include thoracocentesis, thoracic surgery, endotracheal intubation, sternal bone marrow puncture, or positive pressure mechanical ventilation . Noniatrogenic causes can be underlying disease processes like infected fluid or gas producing organisms in the pericardial sac, fistulous communication between pericardium and other air containing structures such as bronchus, esophagus, or stomach and amebic liver abscess . Other causes also include foreign body aspiration, physical exertion, parturition, severe cough, acute asthma, cocaine inhalation, chlorine gas exposure, and forceful emesis . The possible mechanism of pneumopericardium in this case could either be a severe bout of cough or a fistulous communication of pericardium with an infected contiguous organ . The rise in intra - alveolar pressure above atmospheric pressure due to increased bout of coughing may lead to rupture of alveoli and the released air moves to hilar area, mediastinum, and through pericardial reflections on the pulmonary vessels in the pericardial cavity . The parietal pericardium is reflected on the visceral pericardium near ostia of the pulmonary veins, the weakest histological area . Nectrotizing pulmonary process like tuberculosis in the setting of hiv could have resulted in the fistulous communication between the lung and pericardium leading to pneumopericardium in this patient . Pneumopericardium may be symptomatic or asymptomatic depending upon the quantity of air in the pericardium . The patient with a small pneumopericardium may be asymptomatic and the cardiac examination may be normal . It may only be diagnosed incidentally on a chest radiograph and the gas usually does not rise above the upper limit of the pericardium in the erect position, which differentiates the pneumopericardium from pneumomediastinum as in this patient . In large pneumopericardium the patient may be symptomatic and the note may be tympanic and heart sounds may be metallic . This complication is thought to be caused by a ball valve mechanism preventing air from leaving the pericardial space . Pneumopericardium unlike pneumothorax often does not require any specific treatment and is usually self - limiting as in the present case . Treatment is required in cases of large and symptomatic pneumopericardium or in patients of tension pneumopericardium, which can be a needle aspiration or tube decompression . Oxygen therapy in high concentrations as in management of pneumothorax can also be helpful in absorption of air.
Complication following fracture of a central venous catheter can be catastrophic to both the patient and the attending doctor . Catheter fracture has been attributed to several factors namely prolong mechanical force acting on the catheter, and forceful removal or insertion of the catheter . The tip of the catheter was notably missing, and an emergency chest radiograph confirmed our diagnosis of a retained fracture of central venous catheter . The retained portion was removed by the interventional radiologist using an endovascular loop snare and delivered through a femoral vein venotomy performed by the surgeon . Endovascular approach to retrieval of retained fractured catheters has helped tremendously to reduce associated morbidity and the need for major surgery . The role of surgery has become limited to instances of failed endovascular retrieval and in remote geographical locations devoid of such specialty . Werner forssman first reported the insertion of a central venous catheter (cvc) almost 86 years ago . Thirty years later, introduction and wide spread use of the seldinger technique made the procedure safer and easily reproducible (1). Patients with end stage renal impairment are often the most common subset of patients whom require insertion of central venous catheters . The catheter acts as a hemodialysis access point pending creation of a more permanent arterio - venous fistula (avf). It is also inserted in patients presenting with blocked avf, for the purpose of emergency hemodialysis (2). Central venous catheter related complications can be classified into mechanical events, infection and venous thrombosis (3). Since the first report of an embolised catheter fragment in 1954, overall complication rates have remained low and are only reported to occur in less than 1% of cases . Prior to introduction of endovascular approach, retrieval of a fractured segment almost always required high risk open surgery such as thoracotomy (5). Since the late 70's, combined endovascular and open approach has seen much success and gained popularity as first line treatment in the advent of a retained fractured central venous catheter (6). Limitation of the use of this approach would be in rural health care settings devoid of access to an interventional radiologist . We report our experience with a successful retrieval of a central venous catheter utilizing the combined endovascular and open approach . A third year medical resident noticed an incomplete catheter tip post removal of a right subclavian central venous catheter in a 56 year old man diagnosed to have end - stage renal disease . The patient was otherwise well and did not complain of chest pain or shortness of breath . The catheter had been earlier used as a temporary access for hemodialysis pending maturation of a more permanent brachiocephalic arterio - venous fistula (avf). During the visit to the nephrology outpatient clinic, it was decided that the avf was ready for use and that the central venous catheter was no longer needed . Emergency chest radiograph was requested, and it confirmed the diagnosis of retained fractured central venous catheter (figure 1). The case was discussed with the interventional radiologist, and a central venogram was performed to identify the exact location of the fractured segment and to plan for retrieval approach . The venogram noted the fractured catheter segment to lie at the junction of the right brachioceohalic vein and subclavian vein (figure 2). Following discussion with the vascular surgeons interventional radiologists, decision was made to proceed with combined endovascular and open approach of retrieval . Under image intensifier guidance, a 15 mm amplatz gooseneck snare (ev3 inc, usa) was inserted through the right femoral vein, and the fractured catheter fragment pulled - back all the way to the groin region . The surgical team then externalized the retrieved fragment through a femoral vein venotomy, all the while ensuring good proximal and distal control (figures 3, 4). The venotomy site was then closed using non - absorbable sutures in the usual manner . The patient was discharged the next day and continued with his hemodialysis through his matured brachiocephalic avf . Chest radiograph showing the right subclavian catheter prior to planned removal retained portion of catheter seen on venogram the tip of the retained part as being extracted from femoral vein via groin incision the fractured retained part of the catheter after being completely retrieved in one piece central venous catheters are increasingly used worldwide in critically ill patients for administration of intravenous resuscitation fluids and medication, parenteral nutrition, chemotherapy agents, and monitoring of central venous pressure . They have become ubiquitous in the intensive care unit and are available as single and multi - lumen variety . Despite being a commonly performed procedure, it is not devoid of risks and complications . Mechanical complications include hemorrhage, pneumothorax, cardiac arrhythmias, malposition, catheter fracture and distant embolization of the fractured fragment into cardiac chambers or pulmonary artery . Venous thrombosis and central vein occlusion have also been noted to occur with long - term central vein catheter placement (3,4) patients with a fractured catheter may remain asymptomatic or experience shortness of breath, chest pain and even syncope . Diagnosis is often incidental and made in the presence of a radiopaque fragment on routine chest radiograph . Incomplete catheter tip noted during the process of catheter removal may also give rise to clinical suspicion that would warrant further radiological confirmation . Mechanism of catheter fracture has been largely attributed to mechanical shearing forces acting on the catheter over a prolonged period . Although it is a less popular approach, the supraclavicular technique is actually a safer option as it avoids many of the complications otherwise noted in the infraclavicular counter - part . Complications such as guide wire kinking, catheter compression and catheter fracture are less frequently seen in supraclavicularly placed catheters . The reason for the apparent superiority has been studied and linked to the coaxial lie of the catheter itself within the vessel, and absence of need to maneuver through narrow musculo - osseous spaces (7). Besides being fractured, the retained portion of the catheter may also dislodge and embolise distally . Mortality rate associated with intravascular foreign body embolization has been reported to range anywhere from 24% to 60% (3,8). Retrieval of retained intravascular devices may be approached through the conventional open technique or percutaneous method . To date, there are no consensus or clear guidelines for management of a fractured and retained central venous catheter . Over the years, endovascular approach to retrieval of such intravascular foreign bodies has been reported with much success (6). The lack of need for major open surgery has propelled its popularity due to the obvious reduction in overall morbidity and mortality . Patients who were subject to the endovascular approach were also able to assume normal activity of daily living sooner and experience early return to work . Given such positive results, conventional surgical approaches have been limited to instances of failed endovascular approach or in rural areas devoid of access to an interventional radiologist . Percutaneous retrieval of intravascular foreign bodies was initially reserved for retained catheters and wire fragments . In recent years, the spectrum of intravascular device and objects has broadened significantly to include items such as vena cava filters, embolization coils, and endovascular stents . A wide variety of percutaneous tools they include snares, biopsy forceps, dormia baskets, and tip - deflecting wires (6). The amplatz gooseneck snare (ev3 inc, usa) used in our patient is a nitinol based micro snare that has gained tremendous popularity over the years . The snare's super - elastic construction makes it less likely to be kinked or deformed during introduction into the vessel of choice . The snare catheter also contains gold tungsten loops that provide excellent radiopacity for improved procedural precision (9). Given the possible catastrophic complications following fracture of an intravascular catheter or device, it has become prudent to ensure that all cases of catheter fracture or retained catheter fragments are treated as an emergency situation that mandates prompt removal . Due to the superiority and good safety profile, percutaneous retrieval technique should be the treatment of choice (8). Loop snares such as the amplatz gooseneck snare (ev3 inc, usa) should be used when and if available . In the advent of failure to retrieve the retained catheter using a loop snare, the likelihood of success with other percutaneous tools is low, and open surgery should be considered (8,9). This, however, does not rule out the need for surgery in case of failed endovascular therapy.
Whole - cell patch clamp recordings from the ca1 region of rat hippocampal slices were performed using electrode placement as mentioned . Slices were prepared from rats of both sexes, aged 25 to 40 days, and maintained using standard procedures . Rats were decapitated under halothane anesthesia, and the brains rapidly removed . Using a vibratome (model 820, spencer inc), hie slices were then placed in oxygenated artificial cerebrospinal fluid (acsf) at room temperature . Briefly, borosilicate glass electrodes (resistance 4 - 6 m) were filled with 100 mm potassium citrate, 20 mm kci, 1 mm cacl2, 3 mm mgcl2, 2 mm mgatp, 2 mm sodium guanosine 5' -triphosphate, 3 mm ethyleneglycotetraacetic acid, and 40 mm hepes . Recordings were made with an axoclamp 2a amplifier (axon instruments, burlingame, ca) and basic fastlab software (indec systems, sunnyvale, ca). Ascf contained (in mm): nacl 124; kcl 3.75; kh2po4 1.25; mgcl2 1.3; cacl2 3.5; nahco3 26; glucose 10; it was bubbled with 95% o2/5% co2 and maintained at 302c throughout the recordings . Detailed information on the methods are described in another article, where some of the results were originally published . Excitatory (epsp) and inhibitory (ipsp) postsynaptic potentials were evoked by stimulation of the alvear pathway (figure 2). In one set of experiments, the amplitudes of the epsp and ipsp were measured under baseline conditions, after systemic application of different nmda antagonists, and after washout . In a second set of experiments, a tetanus (20 stimuli of 100 hz) was applied after measurement of the baseline ipsp using the same pathway . During the tetanus, the recorded neuron was hyperpolarized to -85 mv, with dc current injection, to prevent the induction of glutamatergic ltp onto the recorded neuron itself . The peak baseline value of the ipsp was compared with peak values obtained continuously until 21 minutes after tetanus . After characterizing the ltp of recurrent inhibitory circuits, we finally tested the sensitivity of this ltp to nmda antagonists in comparison to excitatory, feed - forward ltps in the same slice, using a second stimulus electrode in the stratum radiatum . To examine the effect of a shift in the relationship between the strength of excitatory to inhibitory ltps and its impact on learning and recall, we used a computer model of a local neuronal circuit resembling the typical cell population of the hippocampus . In this model, the functional role of synaptic modification of the excitatory input to inhibitory interneurons was explored in a network biophysical simulation of cortical autoassociative memory function, containing 240 pyramidal cells and 58 inhibitory interneurons activating chloride and potassium currents . Starting parameters for some currents were derived from previous simulation of the piriform cortex and of region ca3 . The simulation of pyramidal cells contained three compartments, with a range of synaptic and voltage - dependent currents . Both dendritic compartments contained excitatory synaptic sodium currents, while the proximal dendritic compartment contained inhibitory synaptic potassium currents and the soma contained inhibitory synaptic chloride currents . These currents include a fast activating voltage - dependent sodium current (i na), a delayed rectifier potassium current (i k(dr)), voltage - dependent potassium currents (i k(a)) and (i k(m)), a high - threshold voltage - dependent calcium current (i ca), and a calcium - dependent potassium current (i k(ahp)). Excitatory synapses between pyramidal cells, and from pyramidal cells to the class of inhibitory interneurons activating chloride currents, were modified according to a hebbian learning rule . When a spike arrived along a presynaptic axon, the maximal conductance of the synaptic current was changed in proportion to the amount by which the average of the postsynaptic membrane potential exceeded a modification threshold . Further methodological details are explained in the legend of figure 6 below . Reproduced from reference 11: grunze hc, rainnie dg, hasselmo me, et al . Copyright 1996, society for neuroscience . In order to examine the effects of age and hormonal status on recurrent inhibition, in vitro experiments similar to those described above the examined groups of long - evans rats consisted of younger females aged 200 days, ovariectomized females aged 200 days, older females aged 380 days, and males aged 250 days . One hemisphere of each brain was used for electrophysiological studies while the contralateral one was processed for immunohistochemistry . Elevated levels of the endogenous partial nmda antagonist n - acetyl - l - aspartyl - l - glutamic acid (naag) are one of the striking findings in postmortem brains of schizophrenic patients . To mimic this condition and to characterize the effect of chronic low - dose nmda antagonist exposure during puberty, we injected mk-801 (0.02 mg / kg body weight) intraperitoneally in 52-day - old rats for 2 weeks (mk-801: n=6; saline controls: n=6) before harvesting the hippocampi . During this period whole - cell patch clamp recordings were performed from ca1 principal neurons 3 days after the last injection . In addition, a relative cell count was performed on 15- m cryostat slices triple - stained with antiparvalbumin, anticalretinin, and dapi, as markers for the interncuronal subpopulation and total cell number, respectively, on the hippocampus not used for electrophysiology . Whole - cell patch clamp recordings were obtained from pyramidal ca1 cells, manually held at -60 mv, and the ipsp amplitude in response to low frequency (0.05 hz, 0.5 -1 ma, 400 ms) was measured . Under drug - free baseline conditions, all nmda antagonists tested, both competitive and noncompetitive, decreased the ipsp amplitude in a dose - dependent manner . The following results are given as a percentage decrease in the baseline ipsp amplitude (se) for the largest series conducted with the competitive nmda antagonist 2-amino-5-phosphovaleric acid (apv) and the noncompetitive nmda antagonist pcp: apv- 0.4 m: -12 + 13%; 1 m: -816%; 1.5 m: -129%; 3 m: -153%; 5 m: -154%; 10 m: -3613%; 25 m: -31 9%; 50 m: -479% . Thus, apv significantly reduced the inhibition in the circuitry even at the smallest concentrations, whereas an effect on epsps was only seen at concentrations above 10 mm . Pcp- 10 m: -2421%; 25 m: -916%; 50 m: -466%; 100 m: -4819% . For 50 m pcp, the reduction in the ipsp amplitude in the presence of naloxone (10 m) was, at -40% of the same magnitude, verifying the response as nmda- and not o - receptor - related . In 4/8 neurons tested, 6,7-dinitroquinoxaline-2,3-dione (dnqx) (5 m) abolished the ipsp completely, and in the other 4 neurons partially (-8022% compared with the baseline value . These results indicate that nmda receptors on inhibitory interneurons may play a role not only in ltp as they do on excito - excitatory synapses, but may also have an impact on network excitability under resting membrane potential conditions . Thus, at low concentrations, they may increase network excitability and only at higher doses cause overall inhibition . In 12 out of 15 neurons tested, posttetanic potentiation (ftp) of the ipsp was observed followed by significant ltp (mean 5216%) of more than 20 minutes (p<0.005, mann- whitney u test, figure 4). Neither ftp nor ltp of the ipsp required gabab receptor activation, as both were insensitive to the gabab receptor antagonist saclofen (250 mol / l). When a tetanus was applied during apv (50 mol / l) superfusion, 4 out of 7 neurons showed ftp (mean 13%) but none showed ltp of the ipsp . In order to obtain more stable and lasting recordings and to compare the ltp of orthodromically evoked epsps with recurrent inhibition ltp population spikes (ps) of ca1 pyramidal neurons were evoked using a bipolar stimulating electrode placed in the stratum radiatum.this orthodromically (o) evoked ps could be reduced by applying an antidromic (a) stimulus via the alvcar pathway at an appropriate time interval prior to the orthodromic stimulus . The reduction in the orthodromic ps results from activation of axon collaterals of ca1 pyramidal cells that project onto recurrent inhibitory interneurons .' Ittic size of the orthodromic ps was compared with its size after antidromic - orthodromic stimulation (means of 10 recordings at 20-s intervals), and the ratio of ps[a - o]/ps[o] was determined . A tetanus (4 trains of 10 stimuli at 100 hz) was then applied via the alvear electrode . The ps[a - o]/ps[o] ratio, determined before tetanus, was compared with three time intervals (2, 10, and 20 minutes) after tetanus . In 92% (24/26) of the recordings, a clear reduction in the ps[a - o]/ps[o] ratio was observed 20 minutes after tetanus compared with baseline values (mean reduction: 18.711.7%;p<0.005,wilcoxon matched pair signed rank test). Figure 5 depicts a typical recording showing traces for baseline and 20 min after tetanus for orthodromic and antidromic - orthodromic stimulation . Figure 5b reproduced from reference 11: grunze hc, rainnie dg, hasselmo me, et al . No change in the ps[a - o]/ps[o] ratio was observed following tetanic stimulation in the presence of apv (50 (m, p<0.025). Pcp, applied during tetanus (n=6, p<0.025), mimicked the effect of apv . These data suggest that nmda receptor activation is required for this long - lasting enhancement of inhibition . Naag, 50 m, applied during tetanus, also attenuated the reduction in the ps[a - o]/ps[o] ratio by 28% compared with the control (n=1), and abolished it at a concentration of 100 m (n=2). The ability of apv and naag to suppress ltp of the recurrent inhibitory drive was compared with its influence on ltp of the excitatory drive onto pyramidal cells with this antidromic - orthodromic stimulus paradigm . The dose - response curve obtained (figure 5) shows a significant 10-fold increased susceptibility of recurrent inhibition ltp to nmda antagonists compared with the more resistant ltp of excitatory input . Besides counteracting hyperexcitability through excitatory ltp, it may contribute to filtering stimuli under physiological conditions . In a realistic biophysical model, we demonstrated that modification of excitatory input to inhibitory interneurons prevented interference between different stored patterns . As shown in figure 6, we tested the ability of the network to store two patterns of 40 neurons, each with an overlap of 8 neurons . Strengthening of the excitatory synapses between pyramidal cells mediated the auto - associative memory storage of the patterns in the network, allowing completion of missing elements of a degraded pattern . However, the overlap between patterns could cause interference during learning and recall, where activity- elicited by one input pattern would spread into neurons which were only part of the other pattern . This interference could be prevented by simultaneously enhancing the strength of excitatory transmission from pyramidal cells to inhibitory interneurons . The enhanced activation of inhibitory currents prevented the excessive spread of activity within the network during learning and recall . This effect may prevent the breakdown of function due to the development of excessive associations between different stored activity patterns . Deficits in learning new verbal associations in the verbal paired associate learning test, which also correlate with a reduced volume of the left posterior superior gyrus, have been demonstrated in schizophrenic patients . Impairment of recall may be reflected by the greater search activity for congruent sentence endings seen in the n400 studies conducted by mccarley et al . This triggers population bursts that may shape local functional connectivity in the cortex, and also strengthen the overall excitatory limbic input onto mesolimbic gabacrgic neurons . High synchronicity of the cortical glutamatergic input is more likely to overcome the dopaminergic inhibition, leading downstream from the mesolimbic projection neuron finally to an improvement in the thalamic filter function . Some neuroleptics may exert a dual action in balancing the relationship between dopaminergic and glutamatergic input, eg, haloperidol, which also has glutamatergic agonist properties byenhancing nmda receptor sensitivity . Another physiological correlate of the glutamatergic interplay between excitatory projection neurons and gabaergic interneurons is -range neural synchronization . In an auditory evoked potential paradigm, schizophrenic patients are distinguished from controls by a reduced eeg power at 40 hz, but not at lower frequencies of stimulation . They exhibit a delayed onset of entrainment, poorer synchronization, and any longer persistence of entrainment after the end of the 40-hz stimulation, consistent with impaired generation of -synchronization . To further examine the compatibility of our model with schizophrenia, we conducted similar in vitro experiments on the impact of age and gender on recurrent inhibition . Aged rats of both sexes showed decreased amplitudes of the ipsps (1.1 0.9 mv in rats of 9 to 12 months, compared with 6.7 0.5 mv in prepubescent rats). This suggests that recurrent inhibition has a more prominent role in shaping networks and limiting excitation in youth; in old rats, however, extrinsic modulation of hippocampal networks may be more decisive . This idea is supported by the findings of decreased nmda receptor density in the hippocampus of older rats . However, the functionality of the single nmda receptor complex increases with age and, thus, also their sensitivity to excitotoxic damage . Slices from old female rats exhibited a tendency toward higher ipsp amplitudes compared with males (2.20.4% versus 0.90.5%). This is in line with a previously demonstrated higher functionality of the nmda receptor in female rats . To examine whether female sex steroids exert a neuroprotective effect and/or male sex steroids impair recurrent inhibition, we also examined rats which had been castrated prior to puberty . In fact, the ipsp in these castrated rats was significantly increased compared with age - matched male controls (2.41.3 mv, p<0.25, mann- whitney u test). Furthermore, the m - glu agonist trans-1-amino-1,3-cyclopentadicarboxylic acid (acpd) increased the ipsp amplitude in aged animals whereas it had no effect in young rats . Taken together, these data suggest that inhibitory local circuits undergo age -dependent changes, possibly with an important modulatory role of sex steroids, and that activation of m - glu receptors can support ipsp generation in aged animals, whereas, in young animals, a maximum of ipsp amplitudes is already achieved by ampa and nmda receptor activation . Ipsp modulation and possible cell loss may result from chronic exposure to high levels of the endogenous nmda antagonist naag, which may play a pathogenetic role in schizophrenia . As puberty and early adolescence appear to be a vulnerable time for schizophrenia, we examined the effect of chronic, not acute toxic, lowdose application of mk-801 on electrophysiological and histological changes in two interneuronal subpopulations in the rat hippocampus . There was no difference in the mean membrane resting potential, action potential threshold and overshoot, gabaa reversal potential, or response to locally applied gaba between treated rats and saline controls . However, local inhibition evoked by alvear stimulation was significantly reduced in the mk-801 group (ipsp amplitude -1.61.3 mv versus -3.712 mv in controls, p<0.025, mann - whitney test) (figure 7). This finding is consistent with a reduced ratio of parvalbumin - positive (pv[+])/calretinin - positive (cr[+]) interneurons in the treated group (figure 8). The loss of pv[+] interneurons also seems to be related to chronic mk-801 application, as rats that received only one high dose of mk-801 (1 mg / kg bw) had no shift of the pv[+]/ cr[+] interneuron ratio . Whole - cell patch clamp recordings were obtained from pyramidal ca1 cells, manually held at -60 mv, and the ipsp amplitude in response to low frequency (0.05 hz, 0.5 -1 ma, 400 ms) was measured . Under drug - free baseline conditions, all nmda antagonists tested, both competitive and noncompetitive, decreased the ipsp amplitude in a dose - dependent manner . The following results are given as a percentage decrease in the baseline ipsp amplitude (se) for the largest series conducted with the competitive nmda antagonist 2-amino-5-phosphovaleric acid (apv) and the noncompetitive nmda antagonist pcp: apv- 0.4 m: -12 + 13%; 1 m: -816%; 1.5 m: -129%; 3 m: -153%; 5 m: -154%; 10 m: -3613%; 25 m: -31 9%; 50 m: -479% . Thus, apv significantly reduced the inhibition in the circuitry even at the smallest concentrations, whereas an effect on epsps was only seen at concentrations above 10 mm . Pcp- 10 m: -2421%; 25 m: -916%; 50 m: -466%; 100 m: -4819% . For 50 m pcp, the reduction in the ipsp amplitude in the presence of naloxone (10 m) was, at -40% of the same magnitude, verifying the response as nmda- and not o - receptor - related . In 4/8 neurons tested, 6,7-dinitroquinoxaline-2,3-dione (dnqx) (5 m) abolished the ipsp completely, and in the other 4 neurons partially (-8022% compared with the baseline value . These results indicate that nmda receptors on inhibitory interneurons may play a role not only in ltp as they do on excito - excitatory synapses, but may also have an impact on network excitability under resting membrane potential conditions . Thus, at low concentrations, they may increase network excitability and only at higher doses cause overall inhibition . In 12 out of 15 neurons tested, posttetanic potentiation (ftp) of the ipsp was observed followed by significant ltp (mean 5216%) of more than 20 minutes (p<0.005, mann- whitney u test, figure 4). Neither ftp nor ltp of the ipsp required gabab receptor activation, as both were insensitive to the gabab receptor antagonist saclofen (250 mol / l). When a tetanus was applied during apv (50 mol / l) superfusion, 4 out of 7 neurons showed ftp (mean 13%) but none showed ltp of the ipsp . In order to obtain more stable and lasting recordings and to compare the ltp of orthodromically evoked epsps with recurrent inhibition ltp population spikes (ps) of ca1 pyramidal neurons were evoked using a bipolar stimulating electrode placed in the stratum radiatum.this orthodromically (o) evoked ps could be reduced by applying an antidromic (a) stimulus via the alvcar pathway at an appropriate time interval prior to the orthodromic stimulus . The reduction in the orthodromic ps results from activation of axon collaterals of ca1 pyramidal cells that project onto recurrent inhibitory interneurons .' Ittic size of the orthodromic ps was compared with its size after antidromic - orthodromic stimulation (means of 10 recordings at 20-s intervals), and the ratio of ps[a - o]/ps[o] was determined . A tetanus (4 trains of 10 stimuli at 100 hz) was then applied via the alvear electrode . The ps[a - o]/ps[o] ratio, determined before tetanus, was compared with three time intervals (2, 10, and 20 minutes) after tetanus . In 92% (24/26) of the recordings, a clear reduction in the ps[a - o]/ps[o] ratio was observed 20 minutes after tetanus compared with baseline values (mean reduction: 18.711.7%;p<0.005,wilcoxon matched pair signed rank test). Figure 5 depicts a typical recording showing traces for baseline and 20 min after tetanus for orthodromic and antidromic - orthodromic stimulation . Figure 5b reproduced from reference 11: grunze hc, rainnie dg, hasselmo me, et al . No change in the ps[a - o]/ps[o] ratio was observed following tetanic stimulation in the presence of apv (50 (m, p<0.025). Pcp, applied during tetanus (n=6, p<0.025), mimicked the effect of apv . These data suggest that nmda receptor activation is required for this long - lasting enhancement of inhibition . Naag, 50 m, applied during tetanus, also attenuated the reduction in the ps[a - o]/ps[o] ratio by 28% compared with the control (n=1), and abolished it at a concentration of 100 m (n=2). The ability of apv and naag to suppress ltp of the recurrent inhibitory drive was compared with its influence on ltp of the excitatory drive onto pyramidal cells with this antidromic - orthodromic stimulus paradigm . The dose - response curve obtained (figure 5) shows a significant 10-fold increased susceptibility of recurrent inhibition ltp to nmda antagonists compared with the more resistant ltp of excitatory input besides counteracting hyperexcitability through excitatory ltp, it may contribute to filtering stimuli under physiological conditions . In a realistic biophysical model, we demonstrated that modification of excitatory input to inhibitory interneurons prevented interference between different stored patterns . As shown in figure 6, we tested the ability of the network to store two patterns of 40 neurons, each with an overlap of 8 neurons . Strengthening of the excitatory synapses between pyramidal cells mediated the auto - associative memory storage of the patterns in the network, allowing completion of missing elements of a degraded pattern . However, the overlap between patterns could cause interference during learning and recall, where activity- elicited by one input pattern would spread into neurons which were only part of the other pattern . This interference could be prevented by simultaneously enhancing the strength of excitatory transmission from pyramidal cells to inhibitory interneurons . The enhanced activation of inhibitory currents prevented the excessive spread of activity within the network during learning and recall . This effect may prevent the breakdown of function due to the development of excessive associations between different stored activity patterns . Deficits in learning new verbal associations in the verbal paired associate learning test, which also correlate with a reduced volume of the left posterior superior gyrus, have been demonstrated in schizophrenic patients . Impairment of recall may be reflected by the greater search activity for congruent sentence endings seen in the n400 studies conducted by mccarley et al . This triggers population bursts that may shape local functional connectivity in the cortex, and also strengthen the overall excitatory limbic input onto mesolimbic gabacrgic neurons . High synchronicity of the cortical glutamatergic input is more likely to overcome the dopaminergic inhibition, leading downstream from the mesolimbic projection neuron finally to an improvement in the thalamic filter function . Some neuroleptics may exert a dual action in balancing the relationship between dopaminergic and glutamatergic input, eg, haloperidol, which also has glutamatergic agonist properties byenhancing nmda receptor sensitivity . Another physiological correlate of the glutamatergic interplay between excitatory projection neurons and gabaergic interneurons is -range neural synchronization . In an auditory evoked potential paradigm, schizophrenic patients are distinguished from controls by a reduced eeg power at 40 hz, but not at lower frequencies of stimulation . They exhibit a delayed onset of entrainment, poorer synchronization, and any longer persistence of entrainment after the end of the 40-hz stimulation, consistent with impaired generation of -synchronization . To further examine the compatibility of our model with schizophrenia, we conducted similar in vitro experiments on the impact of age and gender on recurrent inhibition . Aged rats of both sexes showed decreased amplitudes of the ipsps (1.1 0.9 mv in rats of 9 to 12 months, compared with 6.7 0.5 mv in prepubescent rats). This suggests that recurrent inhibition has a more prominent role in shaping networks and limiting excitation in youth; in old rats, however, extrinsic modulation of hippocampal networks may be more decisive . This idea is supported by the findings of decreased nmda receptor density in the hippocampus of older rats . However, the functionality of the single nmda receptor complex increases with age and, thus, also their sensitivity to excitotoxic damage . Slices from old female rats exhibited a tendency toward higher ipsp amplitudes compared with males (2.20.4% versus 0.90.5%). This is in line with a previously demonstrated higher functionality of the nmda receptor in female rats . To examine whether female sex steroids exert a neuroprotective effect and/or male sex steroids impair recurrent inhibition, we also examined rats which had been castrated prior to puberty . In fact, the ipsp in these castrated rats was significantly increased compared with age - matched male controls (2.41.3 mv, p<0.25, mann- whitney u test). Furthermore, the m - glu agonist trans-1-amino-1,3-cyclopentadicarboxylic acid (acpd) increased the ipsp amplitude in aged animals whereas it had no effect in young rats . Taken together, these data suggest that inhibitory local circuits undergo age -dependent changes, possibly with an important modulatory role of sex steroids, and that activation of m - glu receptors can support ipsp generation in aged animals, whereas, in young animals, a maximum of ipsp amplitudes is already achieved by ampa and nmda receptor activation . Ipsp modulation and possible cell loss may result from chronic exposure to high levels of the endogenous nmda antagonist naag, which may play a pathogenetic role in schizophrenia . As puberty and early adolescence appear to be a vulnerable time for schizophrenia, we examined the effect of chronic, not acute toxic, lowdose application of mk-801 on electrophysiological and histological changes in two interneuronal subpopulations in the rat hippocampus . There was no difference in the mean membrane resting potential, action potential threshold and overshoot, gabaa reversal potential, or response to locally applied gaba between treated rats and saline controls . However, local inhibition evoked by alvear stimulation was significantly reduced in the mk-801 group (ipsp amplitude -1.61.3 mv versus -3.712 mv in controls, p<0.025, mann - whitney test) (figure 7). This finding is consistent with a reduced ratio of parvalbumin - positive (pv[+])/calretinin - positive (cr[+]) interneurons in the treated group (figure 8). The loss of pv[+] interneurons also seems to be related to chronic mk-801 application, as rats that received only one high dose of mk-801 (1 mg / kg bw) had no shift of the pv[+]/ cr[+] interneuron ratio . The cellular model of glutamatergic dysfunction presented here is based on the smallest cortical network loop, the local inhibitory feedback circuit . We demonstrated in vitro that both recurrent inhibition and its ltp can be selectively inhibited by low doses of nmda antagonists . The nature of this differential sensitivity of nmda receptors is still unclear, but may be related to the different subunit assembly of nmda receptors on interneurons compared with pyramidal cells, which also has consequences on the expression of nmda - gated currents . Selective impairment of local inhibition may lead to increased excitability- of the cortical network, causing intrinsic excitotoxic damage with greatest vulnerability of pv[+] interneurons, and to functional impairment, which may also include higher cognitive functions as learning and recall, as demonstrated in the computer model . Lack of synchronization of the cortical input onto mesolimbic cells may lead to a relative hyperfunction of the dopaminergic modulation of these cells, with consecutive impairment of the thalamic filter . Consistent with schizophrenia, we also demonstrated that the vulnerability of nmda - mediated recurrent inhibition may be most pronounced in adolescent animals and male animals, whereas female sex steroids mayhave a protective effect . In conclusion, this model supplies a glutamatergic basis of schizophrenia . The next step is now to examine its modulation by other neurotransmitters implicated in schizophrenia, such as serotonin and dopamine.
Caudal regression syndrome (crs) is a rare disorder of distal spinal segments affecting the development of the spinal cord, with attendant sequelae . The exact etiology is elusive, though maternal diabetes, genetic factors, and hypoperfusion might play roles . We report late presentation of crs in a 9 year old with scoliotic deformity with an asymptomatic cervical syrinx, in absence of any systemic abnormality . A 9-year - old girl was referred in view of a lateral deformity of dorso lumbar spine to explore the possibility of an underlying skeletal dysplasia . There was no history of trauma, associated weakness of any part of body, bowel and bladder involvement or protruding mass over the spine . Child was born of third degree consanguious mating at full term by normal vaginal delivery at home . According to mother she was of normal intelligence with intelligence quotient (iq) of 105 on developmental assessment scales for indian infants (dasii) score . Clinical examination revealed short stature with short trunk, the height being less than 3 centile with upper to lower segment ratio 0.9 . Examination revealed multiple hypo pigmented patches over the left ear lobule, left angle of mouth, and mid dorsum of right leg . Additional finding was evidence of dysgenesis at lumbo sacral region with abnormal orientation of sacrum [figure 1]. Magnetic resonance imaging (mri) spine revealed butterfly vertebrae at d10 level, the first three sacral segments were hypoplastic, distal sacrum and coccyx were absent . A syrinx was present in cervico - thoracic region opposite c5-t1 vertebrae [figure 2]. X - ray showing lumbosacral dysgenesis with abnormal orientation of sacrum saggital neuroimaging shows rudimentary disc at l1-l2, l3-l4, visualization of only s1 and s2 segments and non - visualization of distal sacral segments and coccyx crs is a rare congenital malformation characterized by varying degrees of developmental failure first described by duhmel in 1964 to explain the spectrum of sacrococcygeal malformations . The developmental defects include the lower extremities, the lumbar spine, the coccygeal and thoracic vertebrae, and corresponding segments of spinal cord . A male to female ratio of 2.7:1 has been reported . However, upto 22% cases of crs are associated with diabetes mellitus in the mother, diabetic women being 200 times to 400 times more likely to have a child with crs . Group 1 has blunt spinal cord termination above l1, and is the most severely affected . Group 2 has less severe dysgenesis with low - lying tapered spinal cord and tethered cord, which may be caused by tight filum, lipoma, etc ., thus, the best diagnostic clue for crs is dysgenetic lumbosacral vertebrae and abnormal distal spinal cord . The latter may explain or at least aggravate the scoliosis that was the only clinical sign in this child . The sacral dysgenesis below s2 suggests this is a group 2 case, therefore an associated tethering mechanism should be suspected . Alternatively, probably the scoliosis may be explained by vertebral anomalies at d10, l1 - 2 and l3 - 4 instead of a tethering mechanism, it is unlikely that a child with spinal cord tethering associated with a crs would merely present with a scoliosis . On the other hand, it is even more unlikely to presume there would be a scoliosis without an associated tethering mechanism, unless the scoliosis is related to the malaligned dysgenetic vertebra as seen in our case . The exact etiology of crs at embryonic level is thought to result from defect in induction of caudal elements in the embryo before the 4 week of gestation . The insult occurs at midposterior axis mesoderm, causing the absence of the development of the mesoblastic caudal bud . The proximity and interdependence of developing caudal neurons, spinal, hindgut and mesonephric elements involved in closure of the neural tube result, in the constellation of neural, distal vertebral, anorectal, renal, and genital abnormalities . The attenuation of bone morphogenetic protein signaling at the posterior primitive streak of embryos leads to the caudal dysmorphogenesis including the cloaca and fusion of both hind limbs . Hedgehog - responding cells derived from peri - cloacal mesenchyme contribute to the urogenital / reproductive organs . These findings indicate the existence of developmental programs for the coordinated organogenesis of urogenital / reproductive tissues based on growth factor function and crosstalk . Interestingly, structures that are developmentally separate from these caudal elements such as the brain, proximal spine and spinal cord, are generally spared by crs . The consequences of the disruption after the maturation of spinal cord's caudal portion ensue after the 4 week of gestation are different . This results in motor deficits and neurologic impairment, varying from incontinence of urine and feces to complete neurologic loss . Understandably, most children affected by crs- except the very mild cases - would have some problem with genito - urinary and/or anorectal anatomy and function while our case was surprisingly asymptomatic till 7 years of age . Short trunk, late onset scoliosis and absence of anomaly of any other system are the unusual manifestations of our case . The presence of an asymptomatic cervical syrinx in association with crs in our case is also intriguing . The overall radiological incidence of syringomyelia in patients with distal spinal abnormalities has varied from 19% to 22.5% in asymptomatic group to 48.5% in symptomatic group . If one were to consider the most accepted theories concerning the pathogenesis of syringomyelia in children with crs, chiari ii malformation, hydrocephalus and tethered cord favour the formation of a cavitating lesion of the spinal cord . In fact, a sizeable spina bifida population, screened with cranial and spinal ultrasound in infancy, showed the absence of syringomyelia . The syrinx may start after the 1 year of life, and remain asymptomatic for a variable amount of time . In our child it was interesting to note the presence of segmental vitiligo in our patient with crs, which might be a chance assoction . Neurosurgical consultation taken advocated stringent follow - up, but decided against prophylactic untethering in absence of neurological symptoms . Given the fact that genetic and environmental causes may produce a very similar phenotype, a sharp division between syndrome and association is nearly impossible and makes counselling for recurrence risk challenging . The term caudal regression is probably incorrect since more than caudal structures are involved and nothing regressed that was previously present.
The prevalence of alzheimer s disease or senile dementia, in citizens over 65 years old has been implicated to increase from 8.8% in 2010 to 9.7% in 2020, 11.2% in 2040, and 13.2% in 20501 . Currently, the understanding of dementia is increasing, and therapies and interventions are being developed . The concept of milieu therapy for dementia through therapeutic activities and activity therapy is increasingly gaining interest . Activity therapy, which can reduce the degeneration rate of cognitive function, which is a characteristic symptom of dementia, is being attempted, and its effectiveness has been reported . Regular exercise for a patient with dementia is an essential factor and an effective strategy to delay the onset of dementia2 . For the demented elderly, exercise was reported to not only be effective in reducing the fall risk by improving brain activation, increasing brain blood flow and neurotransmitter secretion, and enhancing muscle flexibility and the sense of balance, but also in enabling smooth gastrointestinal motility, increasing joint motion range by increasing physical strength, and providing an anti - depressant effect3 . In addition, exercise can reduce the early - onset rate of stroke and adult diseases such as hypertension, diabetes, hyperlipidemia, and obesity, which cause dementia, thus preventing dementia progression or delaying disease initiation . Further, exercise is known to prevent mental and physical malfunction, improving confidence and a sense of accomplishment in patients4, 5 . In particular, regular exercise results in improvements in cognitive function, as indicated by the reduced loss of frontal and temporal node tissue when the brain volume was measured using the magnetic resonance imaging6 . However, there is still a lack of direct research on how activities for health promotion change electroencephalogram (eeg) findings in the demented elderly . If a recreation program is provided for treatment and rehabilitation, it may enhance the life quality by changing eeg findings of the demented elderly . Therefore, the purpose of this study was to investigate how participation in a recreation program influences changes in eeg findings in the demented elderly . Subjects included male and female patients over 65 years old at a dementia nursing center and a municipal geriatric hospital . For the final analysis, 14 patients were included in the experimental group and 18 in the control group . Before the study, the purpose and process these subjects had no regular exercise habits and walked independently, and scored 1123 points on the mini - mental state examination, were diagnosed as the first and mid - term dementia, and thus had no apraxia and could communicate . To empirically verify the changes in eegs of the demented elderly for depression, sleep disorder, and life quality during their participation in the therapeutic recreation program, the subjects were classified into the experimental group that attended therapeutic recreation programs regularly for 3 months, and the control group that did not attended any therapeutic recreation programs . Owing to the characteristics of on - site research, the experimental and control groups could not be divided evenly, and exact matching of background variables between groups was impractical; therefore, the non - equivalent control group design, which is a quasi - experimental design method, was applied . The eeg test: the 2-channel wired eeg monitor (lxe 3202; cans 3000, laxtha inc .) And telescan, a data acquisition and analysis software, were used to measure eeg (sp) and erp (ep), respectively . To measure changes in eegs and to analyze the brain functions, balance index, attention index, emotion index, anti - stress index, and brain quotient (bq) amusement, social activity, and habit - cultural activity were selected, as the main focus of health - sports activities, to match the health of the demented elderly, and indoor environment activities were selected by referring to the classification criteria, such as leisure education, cooking activity, outdoor activity, hobby, etc . (table 1table 1.the protocol in the therapeutic recreation programsteptypedayprogramintroductionsocial activity1massage2game3sing a songdevelopmenthealth - sports activities49yoga game10music and dance111214sing a song and rec - dance15mini sports16habit - cultural activities17korean music1819paper art2021magic22read a bookclosingevaluation23recreation24). These were reconstructed by jeong7 in reference to the models regarding amusement, social activity, hobby - cultural activity, watching - viewing activity, health - sports activity, and tour activity, as classified by choi8 and the leisure education programs, as suggested by the leisure - related document9 . The therapeutic recreation program is implemented for 40 minutes, 2 times per week for 3 months, and is composed of 3 stages: introduction, development, and closing / evaluation . All experiments were reviewed and approved by the committee of the chungnam national university . The pre - intervention and post - intervention data were examined using the paired t - test within each group of subjects and the independent t - test between the groups . In order to compare changes in eeg between the experimental and controls groups, the pre- and post - program eeg values were measured, the average and the standard deviation were calculated for each group, and the differences in eeg values between the two groups were analyzed . Table 2table 2.changes in eeg values between groupseegsgrouppre - testpost - testbalance indexe71.2 (2.1)74.6 (1.0)c70.7 (6.3)71.6 (1.8)emotion indexe54.3 (4.6)59.9 (4.3)c55.5 (2.4)56.3 (3.4)attention indexe46.1 (3.2)48.1 (4.2)c44.3 (1.4)45.8 (4.0)anti - stress index.e56.7 (3.0)60.1 (4.6)c54.2 (3.5)55.1 (3.6)brain quotiente52.6 (1.2)55.3 (2.7)c50.4 (2.3)51.1 (4.0)experimental group + control group, mean sd, : p<0.05 shows the average and the standard deviation values between the pre- and post - program eeg values for each group, as well as differences in the values between groups . Experimental group + control group, mean sd, : p<0.05 table 2 displays the average pre- and post - program eeg values for each group . The differences in pre - program eeg values in the experimental group were minor, but those in post - program eeg values between groups were greater . In brief, eeg values were higher in the experimental group than in the control group, demonstrating that participation in a therapeutic recreation program enhances eeg values . However, minor differences in post - program eeg values were noted for all variables between the two groups, except for the anti - stress index and bq . First, the balance index of patients with dementia showed significant patterns in the left - right asymmetry compared to that in the normal control group, as determined using the spatio - temporal pattern analysis10, and the corpus callosum, which transmits information between the left and the right hemispheres, became atrophied11, and the hemispheric disconnection syndrome could be explained . A normal brain is the one showing balance between the left and right sides, which means that there must be mutual interchange through the corpus callosum, thus providing stability in emotional inclination . Therefore, in this study, the left - right hemispheric balance index displayed more changes after the experiment compared to that before the experiment, implying partial brain activation . Second, bryant and veroff12 analyzed mental health in the social and historical context and found that the mental health structure can be positive or negative, and that positive mental health is the psychological wellbeing and is composed of positive emotion and emotional relation, and negative mental health is psychological stress and is made up of anxiety, depression, and emotional loss of control . Chung13 compared the estimated values of self - reported emotion, motif, and positive personality traits in each frontal lobe by activation groups, and found that the relative left activation group showed a significantly higher level of positive affect, approach motivation, happiness, hope, and ego - resiliency than the relative right activation group . Moreover, for the personal affect type, the relative activation of the frontal cortex has relations with positive affect with respect to the left side14 . This study confirmed that the therapeutic recreation program enabled changes in brain activation and revealed the relation between the activity program and emotion via the anti - stress index . Third, the study on eeg changes with respect to attention and concentration following the yoga program by exercise intensity showed that absolute and relative alpha waves emitted during concentration or creative thinking decreased, consequently increasing attention and concentration . In this study, the appropriate intensity of exercise implemented through therapeutic recreation enabled creative thinking in the elderly, thus increasing their attention concentration . Fourth, catecholamine, one of the stress hormones, is not essential for life support, but is released rapidly when during stress conditions . Many studies have used surveys to measure cognitive function through motion stimulus in elderly patients with alzheimer s disease, but these previous studies on the approach of brain function are from the viewpoint of physiology . Nevertheless, the study on the effect of resistance training on the brain function index in the elderly with alzheimer s disease16, 17 demonstrated that the brain index increased after exercise both in the resistance training and gymnastics groups, thus supporting this study result.
Motor and sensory functioning, cognitive functioning, autonomic functioning, as well as emotional and social functioning all depend on anatomic and physiological integrity of the neural networks . This integrity may be disturbed by numerous factors, varying from intrinsic brain disease to mood disorders and from metabolic disturbances and general disease to exogenous intoxications . Intrinsic brain disease may be focal, multifocal or generalized, and examples from these categories are brain tumors, multiple sclerosis and encephalitis, respectively . But optimal functioning of the brain is also determined by an intact environment . Cerebral functioning in a person who is generally ill as a result of infectious disease or disseminated cancer may be severely impaired . Consciousness and cognition may be hampered by a disturbed liver function, by hypoglycemia or by acidosis . And exogenous intoxications by carbon monoxide or alcohol, but also by numerous sorts of medication, such as sedative drugs, antidepressants and anti - epileptic drugs, may have a strong impact on functioning of the brain . In patients with brain tumors, disorders of cerebral functioning are a major concern and it is important to realize that not only the tumor itself but also our broad array of therapeutic interventions and all kinds of metabolic, psychological and social factors contribute to the cerebral condition that determines how the patient is able to express his emotions, how he is able to think, how he functions in his work and in his social environment, and, finally, what his quality of life is . Brain tumors almost invariably cause severe symptoms, such as focal neurological deficits, cognitive deficits, and focal or generalized epileptic seizures . Apart from that, brain tumors may give rise to increased intracranial pressure, resulting in headache or impairment of consciousness . Brain tumors have this significant impact on the brain, since they force the non - tumoral brain tissue not only to adapt to the presence of an expanding mass, but also to the invasion of healthy brain tissue by infiltrating tumor cells . It is a great challenge to clarify the underlying mechanisms accounting for the changes in the brain s functional status resulting from the presence of this foreign entity and leading to cognitive impairments, to an alteration of emotional functioning, and to epileptic seizures . When reviewing the current knowledge in neuro - oncology, there is a strong need for theory regarding the complex relations between the tumor on the one hand, and epilepsy and cognitive and emotional status on the other hand . The study of these relations is complicated by the fact that patients with primary brain tumors undergo a large number of therapeutic interventions, starting with surgery, which is almost invariably followed by irradiation, and in a number of cases by chemotherapy . Apart from these anti - tumor treatment modalities, brain tumor patients may receive antiepileptic drugs, antidepressants and corticosteroids, all having their specific impact on cerebral functioning . Cognition is a collective term for a number of functions that can be subdivided into different domains . The six most important domains are (1) information processing, (2) psychomotor functioning, (3) attention, (4) verbal memory, (5) working memory, and (6) executive functioning . In order to get properly informed on the cognitive abilities of an individual patient, a number of cognitive tests should be performed, measuring these separate functions . The more extensive the test battery, the more detailed the information on the cerebral functioning of the patient . A number of studies on cognitive functioning in glioma patients have been published . Severe neuropsychological impairments have been found in up to 89% of patients with high - grade gliomas (hgg) [24]. A comparable percentage of patients with low - grade gliomas (lgg) display cognitive deficits [512]. The most striking collective finding of these studies is that cognitive deficits are not restricted to the area of the brain where the tumor is located and where it has caused local damage, but rather should be traced to a more global dysfunctioning of the brain: memory disturbances, loss of concentration, difficulties with planning and language, and psychomotor slowness . It appears that the rate of tumor growth is related to the degree of cognitive dysfunction: a fast - growing brain tumor causes more profound cognitive deficits than a slow - growing tumour [13, 14]. This is in accordance with the observation that brain tumor patients show remarkable preservation of cognitive functioning when compared to patients with acute lesions of the same size . When comparing the devastating effect of these acute lesions with that of slowly growing tumors, it is clear that plasticity plays a major role in the resilience to cognitive deficits in brain tumor patients . Epilepsy could be considered as one of the manifestations of brain dysfunction and is often the first manifestation of a brain tumor . Between 10 and 15% of adult patients presenting with epileptic seizures are diagnosed with a brain tumor as the underlying cause of the seizures [16, 17]. Conversely, most patients suffering from brain tumors develop epileptic seizures at some point during the course of their disease . This is the case in 85% of lgg patients, whereas about 50% of hgg patients will experience epileptic seizures at some point of their disease . We do not know why some patients develop epileptic seizures whereas others, suffering from the same tumor type in the same location, do not . Surgery, radiotherapy, anti - epileptic drugs, glucocorticoids and various chemotherapy regimens may have influence on cerebral functioning of brain tumor patients . But also the tumor itself, psychological distress, depression and fatigue have a certain impact, and usually a combination of all these factors eventually destines how the patient will function . This makes it difficult to sort out what the contribution of separate treatment modalities may be . The primary aim of surgery is to reduce tumor mass if the localization of the tumor allows this . Talacchi and coworkers (2011) reported a series of 29 glioma patients in whom extensive neuropsychological examination was performed both pre- and postoperatively . They found postoperative impairment in comparison to preoperative functioning in 38% of patients and the reverse (postoperative improvement in comparison to preoperative functioning) in 24% of patients . This illustrates that surgical treatment of brain tumors may be of benefit to cognitive function, since it reduces intracranial pressure and compression of brain tissue, but may also cause damage whether transient or more permanent . Another study in 23 patients harboring a lgg in the language areas stressed the importance of neuropsychological assessment before operation: the authors found transient worsening of verbal working memory immediately after surgery which recovered within three months . Improvement of language function after initial worsening following tumor resection was also reported by sanai et al . . Especially the first reports on cognitive damage in young children who had been treated with radiotherapy for brain tumors or acute leukemia were alarming . Later, radiation induced neurotoxicity was also reported in adults who had been treated with radiotherapy for primary brain tumors or brain metastases [22, 23]. Our findings suggested that a mean of 6 years after diagnosis the tumor itself, rather than the radiotherapy had the most deleterious effect on cognitive functioning; only high fraction dose radiotherapy (> 2 gy) resulted in significant added cognitive deterioration . In a follow - up study of the same cohort at a mean of 12 years after first diagnosis, however, it appeared that long - term survivors of lgg who did not have radiotherapy had stable cognitive status in contrast to patients who had received radiotherapy . It is important to realize that this attentional deficit developed independently of the localization of the tumor and that treatment in this patient group had been restricted to focal irradiation . This finding indicates that global cognitive dysfunctioning may result from local damage . In patients with hgg, it is more difficult to determine the long term role of radiotherapy because in these patients the duration of survival is much shorter than in lgg patients . Similar to surgical treatment, radiotherapy in hgg often leads to tumor response and this may result in improvement of cognitive functioning . A rapid decline of cognitive functioning occurs in almost all cases of glioma when tumor progression or tumor recurrence occurs . Epileptic seizures necessitate the prescription of anti - epileptic drugs, and especially the older or first - generation anti - epileptic drugs (phenytoin, carbamazepine and valproic acid) may have a negative influence on cognition . It is interesting and important to note that levetiracetam, a second generation drug, may have a positive influence, instead of a deteriorating influence on cognition . Glucocorticoids are usually very effective in the treatment of vasogenic edema, but may also induce new problems with regard to cognitive and emotional functioning . Nowadays, temozolomide is incorporated in the treatment of most newly diagnosed hgg s . The use of temozolomide is not associated with neurocognitive side effects and may even have a beneficial effect on seizure frequency, but it is very well possible that other drugs will become part of glioma combined treatment regimens in the future and some of these drugs may prove to have neurotoxic side effects . In conclusion, brain tumors may negatively affect cerebral functioning and various combinations of all therapeutic modalities, as summarized here, may have an additional negative, but in some situations also a positive impact . The study of brain functions and the way these functions are affected by tumor progression and by various treatment modalities is usually performed by means of measurement of function: neurological examination, a variety of neuropsychological function tests, performance status scales and quality of life questionnaires are examples of instruments to directly or indirectly assess brain function . All these functions, however, depend on the integrity of the cerebral network and direct measurement of network parameters would have obvious advantages . Electroencephalography (eeg) and magnetoencephalography (meg) are potential instruments to study these parameters . If it would become possible to identify certain characteristics of the human cerebral network, more insight into the effects of local lesions, such as brain tumors, and into the effects of systemic treatments on the integrity and plasticity of the cerebral network could result . We will give a short overview of network theory and the possibilities to study neural networks in the following paragraphs . Network or graph theory originates from both the fields of mathematics and sociology . Combining these two has led to a number of methods of analyzing different types of networks by representing them in an abstract, theoretical figure called a graph. The challenge of the study of networks including the study of neural networks is to find the optimal method of describing and defining all kinds of biological and social networks . Most optimal functioning networks have so - called small - world characteristics, referring to a locally clustered architecture in combination with long distance connections . This means that parts of the network that are virtually remote from each other can actually be linked through only a few steps . This co - existence of integration and segregation in complex networks has only recently been elucidated . Random networks were first described in the 20th century and seemed promising to model complex networks [30, 31]. However, these graphs did not meet up to the expectations of explaining the abovementioned small - world characteristics of networks . They proposed a very simple model of a one - dimensional network (see fig . 1). In the regular network, each node or vertex is only connected to its next, with likelihood p, connections or edges are chosen at random and connected to other nodes, also chosen randomly . With increasing p, more and more edges become randomly reconnected and finally, for p = 1, the network is completely random . This comprehensible model allows investigation of all types of networks, ranging from completely regular to completely random.fig . 1schematic drawing of a random, a regular, and a small - world network . In the regular network therefore, it has both a high clustering coefficient (c) and a long path length (l), while the random network (right) combines a low c and a low l. the intermediate of the two: the so - called small - world network (middle) can be achieved by relocating but a few long - distance connections from the regular network, which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds schematic drawing of a random, a regular, and a small - world network . In the regular network (left), each node is only connected to its neighbors . Therefore, it has both a high clustering coefficient (c) and a long path length (l), while the random network (right) combines a low c and a low l. the intermediate of the two: the so - called small - world network (middle) can be achieved by relocating but a few long - distance connections from the regular network, which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds the intermediate between random and regular architecture proved to be crucial to the understanding of the small - world phenomenon . Two measures appear to be important for the classification of a network on the continuum of regular to random . The first measure is the clustering coefficient (c), which can be defined as the likelihood that neighbors of a vertex are also connected . This is defined as the average of the shortest distance between pairs of nodes counted in the number of edges . Using these two measures, we can resume the theory as follows: regular networks are very clustered (high c) but it takes a lot of steps to get from one side of the graph to the other (high l). In contrast, random networks lack this high clustering (they have a low c) and the path length is short (low l). Neither of the two have small - world properties . However, these small - world properties occur already when p is only slightly higher than 0: now the path length l drops sharply, while the clustering coefficient (c) hardly changes . Thus networks with only a few (randomly) rewired connections combine both high clustering and a small path length: this phenomenon is referred to as the small - world phenomenon . These measures c and l make it possible to define the index of small - worldness . The discovery of these tools to describe small - world networks initiated a widespread interest in all kinds of complex networks and gave rise to a wide range of theoretical and experimental studies . One of the most intriguing, and certainly the most complex network is the human brain . Brain activity is commonly studied by making use of functional magnetic resonance imaging (fmri), eeg or meg . Eeg has been a routine instrument in the study of brain function for a number of decades . It measures electrical flow and is especially useful for the diagnosis of epilepsy and other pathological cerebral conditions, such as encephalitis . Meg is a more sophisticated method to measure brain activity and has a higher spatial resolution . Meg registers brain activity by measuring magnetic flow, and the scalp and the skull do not distort signal registration in contrast with eeg . Within the signal measured by eeg and meg, different frequency bands can be distinguished: delta (0.54 hz), theta (48 hz), lower alpha (810 hz), upper alpha (1013 hz), beta (1330 hz), lower gamma (3055 hz), and upper gamma band (5580 hz). A fundamental conception for the study of brain dynamics is synchronization or functional connectivity . The basic assumption of functional connectivity is that statistical interdependencies between time series of brain activity at separate areas reflect functional interactions between these brain regions . This functional connectivity can be calculated on the basis of the amount of synchrony of brain activity measured in two different areas . The conception is that multiple local networks are maintained by long - distance patterns of functional connectivity and this results in higher and complex brain functions, such as planning, memory, and executive functioning [3538]. Functional connectivity between brain areas may thus be used to construct graphs of the brain (see fig . 2 for an example in an meg recording). The two prerequisites of local segregation, referring to local specialization in specific tasks, and integration, combining information from lower - level networks at a higher and more global level, are thought to be crucial for optimal brain functioning [3941]. The small - world network is a highly adequate model of organization in the brain, because it supports both segregated as well as integrated information processing . Brain tumors may interfere with neuronal structures and the resulting disturbances of anatomical connectivity may lead to alterations of functional connectivity patterns [43, 44].fig . Synchronization of signals from different regions of the brain is a measure of connectivity of these regions part of a 151-channel meg recording . Synchronization of signals from different regions of the brain is a measure of connectivity of these regions during the last decade, we have shown that most lgg and hgg patients perform significantly worse than healthy controls on almost all cognitive tests . In order to better understand why glioma patients develop those global cognitive deficits, we investigated the correlation between resting - state meg - based functional connectivity and cognitive functioning in patients with lgg . We therefore studied correlations between time series of 126 meg channels and we used the so - called synchronization likelihood (sl) as one of the measures of synchronization (or functional connectivity). The sl varies between zero (no synchronization at all) and one (total synchronization). We found that lgg patients showed increased sl in the delta, theta, and lower gamma frequency bands in comparison to healthy controls, while connectivity in the lower alpha band was decreased . In another study we also found a correlation between cognitive functioning and network architecture in lgg patients . These findings partly confirmed our previous studies, in which we also found changes in functional connectivity and network architecture in brain tumor patients with various histologies [47, 48]. There were some contradictory details between the earlier publications and the results of the later papers, especially regarding differences between tumor patients and healthy controls in the pattern and the direction of changes in connectivity in the various frequency bands . Possible explanations for these differences in both functional connectivity and network topology may relate to the different patient samples: a homogeneous group of lgg patients versus a more heterogeneous sample of brain tumor patients . Despite these contradictory details, it is most likely that conceptions like functional connectivity and network topology are pivotal for cognitive functioning . Both cognitive performance and functional connectivity are disturbed in lgg patients, and correlations exist between these two phenomena . Brain tumor patients consistently show pathologically increased connectivity particularly in the lower frequency bands (delta, theta), which is related to poorer cognitive functioning . We further investigated network characteristics in glioma patients with epilepsy by means of meg registrations . Functional connectivity was calculated in six frequency bands, as were a number of network measures such as normalized clustering coefficient and path length . Increased theta band connectivity appeared to be related to a higher total number of seizures . Furthermore, higher number of seizures was related to a less optimal, more random brain network topology . These results indicate that (pathologically) increased theta band connectivity is related to a higher number of epileptic seizures in brain tumor patients, suggesting that theta band connectivity changes are a hallmark of tumor - related epilepsy . Furthermore, a more random brain network topology is related to greater vulnerability to seizures . Thus, functional connectivity and brain network architecture may prove to be important parameters of tumor - related epilepsy . Brain tumors almost invariably cause severe symptoms, such as focal neurological deficits, cognitive deficits, and focal or generalized epileptic seizures . Apart from that, brain tumors may give rise to increased intracranial pressure, resulting in headache or impairment of consciousness . Brain tumors have this significant impact on the brain, since they force the non - tumoral brain tissue not only to adapt to the presence of an expanding mass, but also to the invasion of healthy brain tissue by infiltrating tumor cells . It is a great challenge to clarify the underlying mechanisms accounting for the changes in the brain s functional status resulting from the presence of this foreign entity and leading to cognitive impairments, to an alteration of emotional functioning, and to epileptic seizures . When reviewing the current knowledge in neuro - oncology, there is a strong need for theory regarding the complex relations between the tumor on the one hand, and epilepsy and cognitive and emotional status on the other hand . The study of these relations is complicated by the fact that patients with primary brain tumors undergo a large number of therapeutic interventions, starting with surgery, which is almost invariably followed by irradiation, and in a number of cases by chemotherapy . Apart from these anti - tumor treatment modalities, brain tumor patients may receive antiepileptic drugs, antidepressants and corticosteroids, all having their specific impact on cerebral functioning . Cognitive functioning may be impaired in many conditions that affect the brain . But cognitive functioning as such is an ill - defined entity . Cognition is a collective term for a number of functions that can be subdivided into different domains . The six most important domains are (1) information processing, (2) psychomotor functioning, (3) attention, (4) verbal memory, (5) working memory, and (6) executive functioning . In order to get properly informed on the cognitive abilities of an individual patient, a number of cognitive tests should be performed, measuring these separate functions . The more extensive the test battery, the more detailed the information on the cerebral functioning of the patient . A number of studies on cognitive functioning in glioma patients have been published . Severe neuropsychological impairments have been found in up to 89% of patients with high - grade gliomas (hgg) [24]. A comparable percentage of patients with low - grade gliomas (lgg) display cognitive deficits [512]. The most striking collective finding of these studies is that cognitive deficits are not restricted to the area of the brain where the tumor is located and where it has caused local damage, but rather should be traced to a more global dysfunctioning of the brain: memory disturbances, loss of concentration, difficulties with planning and language, and psychomotor slowness . It appears that the rate of tumor growth is related to the degree of cognitive dysfunction: a fast - growing brain tumor causes more profound cognitive deficits than a slow - growing tumour [13, 14]. This is in accordance with the observation that brain tumor patients show remarkable preservation of cognitive functioning when compared to patients with acute lesions of the same size . When comparing the devastating effect of these acute lesions with that of slowly growing tumors, it is clear that plasticity plays a major role in the resilience to cognitive deficits in brain tumor patients . Epilepsy could be considered as one of the manifestations of brain dysfunction and is often the first manifestation of a brain tumor . Between 10 and 15% of adult patients presenting with epileptic seizures are diagnosed with a brain tumor as the underlying cause of the seizures [16, 17]. Conversely, most patients suffering from brain tumors develop epileptic seizures at some point during the course of their disease . This is the case in 85% of lgg patients, whereas about 50% of hgg patients will experience epileptic seizures at some point of their disease . We do not know why some patients develop epileptic seizures whereas others, suffering from the same tumor type in the same location, do not . Surgery, radiotherapy, anti - epileptic drugs, glucocorticoids and various chemotherapy regimens may have influence on cerebral functioning of brain tumor patients . But also the tumor itself, psychological distress, depression and fatigue have a certain impact, and usually a combination of all these factors eventually destines how the patient will function . This makes it difficult to sort out what the contribution of separate treatment modalities may be . The primary aim of surgery is to reduce tumor mass if the localization of the tumor allows this . Talacchi and coworkers (2011) reported a series of 29 glioma patients in whom extensive neuropsychological examination was performed both pre- and postoperatively . They found postoperative impairment in comparison to preoperative functioning in 38% of patients and the reverse (postoperative improvement in comparison to preoperative functioning) in 24% of patients . This illustrates that surgical treatment of brain tumors may be of benefit to cognitive function, since it reduces intracranial pressure and compression of brain tissue, but may also cause damage whether transient or more permanent . Another study in 23 patients harboring a lgg in the language areas stressed the importance of neuropsychological assessment before operation: the authors found transient worsening of verbal working memory immediately after surgery which recovered within three months . Improvement of language function after initial worsening following tumor resection was also reported by sanai et al . . Especially the first reports on cognitive damage in young children who had been treated with radiotherapy for brain tumors or acute leukemia were alarming . Later, radiation induced neurotoxicity was also reported in adults who had been treated with radiotherapy for primary brain tumors or brain metastases [22, 23]. Our findings suggested that a mean of 6 years after diagnosis the tumor itself, rather than the radiotherapy had the most deleterious effect on cognitive functioning; only high fraction dose radiotherapy (> 2 gy) resulted in significant added cognitive deterioration . In a follow - up study of the same cohort at a mean of 12 years after first diagnosis, however, it appeared that long - term survivors of lgg who did not have radiotherapy had stable cognitive status in contrast to patients who had received radiotherapy . It is important to realize that this attentional deficit developed independently of the localization of the tumor and that treatment in this patient group had been restricted to focal irradiation . This finding indicates that global cognitive dysfunctioning may result from local damage . In patients with hgg, it is more difficult to determine the long term role of radiotherapy because in these patients the duration of survival is much shorter than in lgg patients . Similar to surgical treatment, radiotherapy in hgg often leads to tumor response and this may result in improvement of cognitive functioning . A rapid decline of cognitive functioning occurs in almost all cases of glioma when tumor progression or tumor recurrence occurs . Epileptic seizures necessitate the prescription of anti - epileptic drugs, and especially the older or first - generation anti - epileptic drugs (phenytoin, carbamazepine and valproic acid) may have a negative influence on cognition . It is interesting and important to note that levetiracetam, a second generation drug, may have a positive influence, instead of a deteriorating influence on cognition . Glucocorticoids are usually very effective in the treatment of vasogenic edema, but may also induce new problems with regard to cognitive and emotional functioning . Nowadays, temozolomide is incorporated in the treatment of most newly diagnosed hgg s . The use of temozolomide is not associated with neurocognitive side effects and may even have a beneficial effect on seizure frequency, but it is very well possible that other drugs will become part of glioma combined treatment regimens in the future and some of these drugs may prove to have neurotoxic side effects . In conclusion, brain tumors may negatively affect cerebral functioning and various combinations of all therapeutic modalities, as summarized here, may have an additional negative, but in some situations also a positive impact . The study of brain functions and the way these functions are affected by tumor progression and by various treatment modalities is usually performed by means of measurement of function: neurological examination, a variety of neuropsychological function tests, performance status scales and quality of life questionnaires are examples of instruments to directly or indirectly assess brain function . All these functions, however, depend on the integrity of the cerebral network and direct measurement of network parameters would have obvious advantages . Electroencephalography (eeg) and magnetoencephalography (meg) are potential instruments to study these parameters . If it would become possible to identify certain characteristics of the human cerebral network, more insight into the effects of local lesions, such as brain tumors, and into the effects of systemic treatments on the integrity and plasticity of the cerebral network could result . We will give a short overview of network theory and the possibilities to study neural networks in the following paragraphs . Network or graph theory originates from both the fields of mathematics and sociology . Combining these two has led to a number of methods of analyzing different types of networks by representing them in an abstract, theoretical figure called a graph. The challenge of the study of networks including the study of neural networks is to find the optimal method of describing and defining all kinds of biological and social networks . Networks usually combine two seemingly opposing concepts: integration and segregation . Most optimal functioning networks have so - called small - world characteristics, referring to a locally clustered architecture in combination with long distance connections . This means that parts of the network that are virtually remote from each other can actually be linked through only a few steps . This co - existence of integration and segregation in complex networks has only recently been elucidated . Random networks were first described in the 20th century and seemed promising to model complex networks [30, 31]. However, these graphs did not meet up to the expectations of explaining the abovementioned small - world characteristics of networks . They proposed a very simple model of a one - dimensional network (see fig . 1). In the regular network, each node or vertex is only connected to its next, with likelihood p, connections or edges are chosen at random and connected to other nodes, also chosen randomly . With increasing p, more and more edges become randomly reconnected and finally, for p = 1, the network is completely random . This comprehensible model allows investigation of all types of networks, ranging from completely regular to completely random.fig . 1schematic drawing of a random, a regular, and a small - world network . In the regular network (left), each node is only connected to its neighbors . Therefore, it has both a high clustering coefficient (c) and a long path length (l), while the random network (right) combines a low c and a low l. the intermediate of the two: the so - called small - world network (middle) can be achieved by relocating but a few long - distance connections from the regular network, which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds schematic drawing of a random, a regular, and a small - world network . In the regular network (left), each node is only connected to its neighbors . Therefore, it has both a high clustering coefficient (c) and a long path length (l), while the random network (right) combines a low c and a low l. the intermediate of the two: the so - called small - world network (middle) can be achieved by relocating but a few long - distance connections from the regular network, which causes l to decrease drastically but preserves a high c. thus it combines the best of two worlds the intermediate between random and regular architecture proved to be crucial to the understanding of the small - world phenomenon . Two measures appear to be important for the classification of a network on the continuum of regular to random . The first measure is the clustering coefficient (c), which can be defined as the likelihood that neighbors of a vertex are also connected . This is defined as the average of the shortest distance between pairs of nodes counted in the number of edges . Using these two measures, we can resume the theory as follows: regular networks are very clustered (high c) but it takes a lot of steps to get from one side of the graph to the other (high l). In contrast, random networks lack this high clustering (they have a low c) and the path length is short (low l). Neither of the two have small - world properties . However, these small - world properties occur already when p is only slightly higher than 0: now the path length l drops sharply, while the clustering coefficient (c) hardly changes . Thus networks with only a few (randomly) rewired connections combine both high clustering and a small path length: this phenomenon is referred to as the small - world phenomenon . These measures c and l make it possible to define the index of small - worldness . The discovery of these tools to describe small - world networks initiated a widespread interest in all kinds of complex networks and gave rise to a wide range of theoretical and experimental studies . One of the most intriguing, and certainly the most complex network is the human brain . Brain activity is commonly studied by making use of functional magnetic resonance imaging (fmri), eeg or meg . Eeg has been a routine instrument in the study of brain function for a number of decades . It measures electrical flow and is especially useful for the diagnosis of epilepsy and other pathological cerebral conditions, such as encephalitis . Meg is a more sophisticated method to measure brain activity and has a higher spatial resolution . Meg registers brain activity by measuring magnetic flow, and the scalp and the skull do not distort signal registration in contrast with eeg . Within the signal measured by eeg and meg, different frequency bands can be distinguished: delta (0.54 hz), theta (48 hz), lower alpha (810 hz), upper alpha (1013 hz), beta (1330 hz), lower gamma (3055 hz), and upper gamma band (5580 hz). A fundamental conception for the study of brain dynamics is synchronization or the basic assumption of functional connectivity is that statistical interdependencies between time series of brain activity at separate areas reflect functional interactions between these brain regions . This functional connectivity can be calculated on the basis of the amount of synchrony of brain activity measured in two different areas . The conception is that multiple local networks are maintained by long - distance patterns of functional connectivity and this results in higher and complex brain functions, such as planning, memory, and executive functioning [3538]. Functional connectivity between brain areas may thus be used to construct graphs of the brain (see fig . 2 for an example in an meg recording). The two prerequisites of local segregation, referring to local specialization in specific tasks, and integration, combining information from lower - level networks at a higher and more global level, are thought to be crucial for optimal brain functioning [3941]. The small - world network is a highly adequate model of organization in the brain, because it supports both segregated as well as integrated information processing . Brain tumors may interfere with neuronal structures and the resulting disturbances of anatomical connectivity may lead to alterations of functional connectivity patterns [43, 44].fig . Synchronization of signals from different regions of the brain is a measure of connectivity of these regions part of a 151-channel meg recording . Synchronization of signals from different regions of the brain is a measure of connectivity of these regions during the last decade, we have shown that most lgg and hgg patients perform significantly worse than healthy controls on almost all cognitive tests . In order to better understand why glioma patients develop those global cognitive deficits, we investigated the correlation between resting - state meg - based functional connectivity and cognitive functioning in patients with lgg . We therefore studied correlations between time series of 126 meg channels and we used the so - called synchronization likelihood (sl) as one of the measures of synchronization (or functional connectivity). The sl varies between zero (no synchronization at all) and one (total synchronization). We found that lgg patients showed increased sl in the delta, theta, and lower gamma frequency bands in comparison to healthy controls, while connectivity in the lower alpha band was decreased . In another study we also found a correlation between cognitive functioning and network architecture in lgg patients . These findings partly confirmed our previous studies, in which we also found changes in functional connectivity and network architecture in brain tumor patients with various histologies [47, 48]. There were some contradictory details between the earlier publications and the results of the later papers, especially regarding differences between tumor patients and healthy controls in the pattern and the direction of changes in connectivity in the various frequency bands . Possible explanations for these differences in both functional connectivity and network topology may relate to the different patient samples: a homogeneous group of lgg patients versus a more heterogeneous sample of brain tumor patients . Despite these contradictory details, it is most likely that conceptions like functional connectivity and network topology are pivotal for cognitive functioning . Both cognitive performance and functional connectivity are disturbed in lgg patients, and correlations exist between these two phenomena . Brain tumor patients consistently show pathologically increased connectivity particularly in the lower frequency bands (delta, theta), which is related to poorer cognitive functioning . We further investigated network characteristics in glioma patients with epilepsy by means of meg registrations . Functional connectivity was calculated in six frequency bands, as were a number of network measures such as normalized clustering coefficient and path length . Increased theta band connectivity appeared to be related to a higher total number of seizures . Furthermore, higher number of seizures was related to a less optimal, more random brain network topology . These results indicate that (pathologically) increased theta band connectivity is related to a higher number of epileptic seizures in brain tumor patients, suggesting that theta band connectivity changes are a hallmark of tumor - related epilepsy . Furthermore, a more random brain network topology is related to greater vulnerability to seizures . Thus, functional connectivity and brain network architecture may prove to be important parameters of tumor - related epilepsy . These disturbances may consist of function loss (especially loss of complex functions), and epileptic seizures . It has been shown that both types of cerebral function disruption are associated with changes in functional connectivity and network architecture . Although we are only in the very beginning of unraveling the extremely complex network architecture of the brain, the findings in brain tumor patients may prove to be of great importance for our future strategies in the treatment of these patients . Possibilities to successfully combine brain tumor surgery and epilepsy surgery will improve, and we will be able to longitudinally study the influence of various treatment strategies on the network . In short, we will be able to study the plasticity of the brain in a direct and non - invasive way.
The prevalence of diabetes mellitus has continued to rise worldwide with the latest estimation by the international diabetes federation that, by the year 2040, one in 10 adults or 642 million people will have diabetes . Tackling its rise and the health burden arising from the disease is a major challenge to almost all health care systems . Hong kong, returned to the sovereignty of china as a special administrative region from the united kingdom, is no exception . Being the most developed region of china, its prevalence of diabetes is higher than the national average . The direct medical cost for managing diabetes and its complications was estimated to take up 3.9% of the total healthcare expenditure and 6.4% of the public health expenditure . Hong kong has a dual track system with public and private care along each other . For historical reasons, specialist and inpatient care is mainly provided by the public sector (90%), which also provides 29% of primary outpatient care through the general outpatient clinics (gopcs). However, in the, hitherto, absence of an universal health insurance or co - payment scheme, much of the primary care provided by the public sector actually involves caring for people with chronic diseases, since most of them find long term treatment of their conditions by private doctors difficult to afford . The hospital authority (ha) of hong kong is a statutory body set up to provide all public health care services in the territory . It manages 41 hospitals and institutions, 47 specialist outpatient clinics, which together provide inpatient and specialist care; and 73 gopcs where primary care for the early stages of chronic diseases for most patients is delivered . These hospitals and clinics are organised into seven geographical clusters to provide a continuum of primary and specialist services to the citizens . The expenditure of the ha is largely dependent on government funding which amounts to hk$50.76 billion (~usd 6.5 billion) and constitutes less than half of the 5.4% of the gross domestic product (gdp) that is spent on health care in hong kong (table 1). A recent bloomberg news coverage ranked the health care system of hong kong the most efficient in the world . Therefore, it is obvious that the ha has been running on a very tight budget . The prevalence of diabetes in hong kong is about 10% . With a population of 7.4 million, it is estimated that there are about 740 thousand diabetic patients, 40% to 50% of whom are undiagnosed . However, the number of diabetic patients under the care of the ha has been rising steadily from 397 thousand in 2009/2010 to 408 thousand in 2015/2016 (fig ., 33 to 35 thousand new diabetic patients are added to the public health care system (fig . The rise is likely contributed to by earlier diagnosis among the undiagnosed because of increased awareness of diabetes in the community; attraction of more patients from the private to the public sector; an increase in the prevalence of diabetes due to an increase in the incidence among younger people, etc . Thus, diabetes is a major burden to the ha because it is estimated that more than 90% of the known diabetic patients are under its care . With the background of forerunners in research and service development, the ha started a journey of a system approach to providing diabetes care across the whole public health care system in 2009 . The following strategies have been adopted: service data were captured and analysed through the electronic clinical management system.clinical modules in the clinical management system to facilitate clinical care for diabetes and capture diabetes specific clinical data.corporate clinical practice guideline (cpg) on the management of type 2 diabetes: first published 2013; currently being reviewed to be updated in 2017.large scale programmes to enhance diabetes care including a territory - wide multi - disciplinary risk assessment and management program for diabetes (ramp - dm) and a structured patient empowerment program (pep) in the gopcs.regular monitoring of performance indicators . Clinical modules in the clinical management system to facilitate clinical care for diabetes and capture diabetes specific clinical data . Corporate clinical practice guideline (cpg) on the management of type 2 diabetes: first published 2013; currently being reviewed to be updated in 2017 . Large scale programmes to enhance diabetes care including a territory - wide multi - disciplinary risk assessment and management program for diabetes (ramp - dm) and a structured patient empowerment program (pep) in the gopcs . The intent of the guideline was to describe the minimal level of care to be provided across the different care levels in the ha, with due consideration of available evidence and local systems factors . On the other hand, a service framework outlining the cycle of periodic assessment, risk stratification for different management and patient empowerment strategies and regular follow - up at the different care levels was included (fig . An algorithm was drawn to guide the use of the various glucose lowering agents (fig . The management of hypertension and dyslipidemia was discussed with a view to minimizing cardiovascular risk . Although the targets for glycosylated hemoglobin (hba1c), blood pressure and low density lipoprotein cholesterol (ldl - c) are to be individualized, those recommended by international guidelines in 2013 were adopted for suitable patients (table 2). Criteria for referral to specialists such as endocrinologists, ophthalmologists, orthopaedic, and vascular surgeons were outlined . A special note should be made regarding the use of drugs, including glucose lowering agents, in the ha . There is a corporate drug formulary in which drugs are classified into general, special, and self - financed items . General drugs can be freely used by all doctors; special drugs can only be used for specific clinical indications by designated specialists; self - financed items are purchased by their patients out - of - pocket if the clinical indications are not met or present . The formulary effectively governs that less expensive options have to be used first unless or until they are not tolerated or effective, which helps contain the drug expenditure . The formulary is periodically reviewed to take into consideration new clinical evidence, changes in prize and introduction of new drugs or formulations . Thus, metformin, sulphonylureas, and human insulin are general drugs, to which pioglitazone was recently added as its price has come down . Ddp-4 and sodium / glucose cotransporter 2 inhibitors are special drugs that can only be used when the combination of metformin and sulphonylureas with or without pioglitazone fail to control glycemia . Insulin analogues can only be used for patients with repeated hypoglycemia while on human insulin or patients with established cardiovascular and renal complications . Glucagon - like peptide-1 analogues are self - financed items because of the high prices . In 2009, the ha introduced a multidisciplinary ramp - dm to improve the quality of care for patients receiving diabetic care in the gopcs . All patients with type 2 diabetes mellitus who are independent in their activities of daily living and being followed up regularly at the gopcs are eligible for the ramp . Enrolled patients undergo a comprehensive risk assessment with checking of relevant clinical parameters including hba1c, blood pressure, ldl - c, and screening for diabetes - related complications according to a standardized protocol . After the assessment, patients are classified into different risk groups according to the joint asia diabetes evaluation (jade) criteria and are offered different management options to receive appropriate interventions and education provided by a team of multidisciplinary healthcare professionals . Low risk patients continue with the usual gopc care; medium risk patients are given additional intervention by a nurse with special training in diabetes; and high risk / very high risk patients are reviewed by a specialist family physician for intensification . About two - thirds of the diabetic patients under the care of the gopcs (277,309 in 2015/2016) have been enrolled . The effect and effectiveness of the ramp - dm has been evaluated by three prospective cohort studies . Thus, it was found that there was a significant decrease in hba1c (0.20%, p<0.01), systolic blood pressure (sbp; 3.62 mm hg, p<0.01), 10-year cardiovascular disease (cvd) risks (total cvd risk, 2.06%, p<0.01; coronary heart disease [chd] risk, 1.43%, p<0.01; stroke risk, 0.71%, p<0.01) at 12 months in a random sample of 1,248 patients enrolled to ramp - dm compared with an age-, sex-, and hba1c - matched group of unenrolled 1,248 patients under the usual primary care . There was a rise in the percentage of patients reaching treatment targets of hba1c (5.4%, p<0.01), and sbp / diastolic blood pressure (5.77%, p<0.01) and a lower cardiovascular events incidence (1.21% vs. 2.89%, p=0.003). More significantly, in another prospective cohort study of 18,188 propensity score matched ramp - dm participants and patients receiving the usual primary care with a median follow - up of 36 months (9,094 subjects in each group), there were significantly lowered adjusted hazard ratios (hrs) in the ramp - dm group compared with the usual care group in all - cause deaths (0.363; 95% confidence interval [ci], 0.308 to 0.428; p<0.001); chd (0.570; 95% ci, 0.470 to 0.691; p<0.001); stroke (0.652; 95% ci, 0.546 to 0.780; p<0.001); and congestive heart failure (0.598; 95% ci, 0.446 to 0.802; p=0.001). Further, a third study comparing ramp - dm participants with subjects under the usual primary care (14,835 in each group) with a median follow - up of 36 months, ramp - dm participants had a lower incidence of microvascular complications (760 vs. 935; adjusted hr, 0.73; 95% ci, 0.66 to 0.81; p<0.001) and lower incidences of all specific microvascular complications except neuropathy (adjusted hr, 0.94; 95% ci, 0.61 to 1.45; p=0.778). Adjusted hrs for the ramp - dm versus control group for end stage renal disease, sight threatening diabetic retinopathy or blindness, and lower - limb ulcers or amputation were 0.40 (95% ci, 0.24 to 0.69; p<0.001), 0.55 (95% ci, 0.39 to 0.78; p=0.001), and 0.49 (95% ci, 0.30 to 0.80; p=0.005), respectively . Although these are not randomized controlled trials, the careful matching, large numbers of patients and long duration of follow - up meant that a program of risk assessment and stratification followed by appropriate intervention is likely to be high effective in improving long term clinical outcomes . Life style modification through patient empowerment has long been considered an important and integral component of management of diabetes . Diabetic self - management education, whether delivered on an individual or group setting, has been shown to improve glycemic and cardiovascular risk factor control . In 2010, the hong kong hospital authority launched the pep as a territory - wide primary care component with the purpose of improving the quality of care . The curriculum of the pep, designed by a group of specialist diabetes nurse educators, was delivered in six sessions by trained healthcare workers in community centers run by non - government organizations . There was a generic component covering behavior modification, healthy diet and regular exercise habit, goal setting and problem solving skills, sharing on self - monitoring experience, stress coping management, psychosocial support and networking, and communications with healthcare professionals . A second, disease - specific component dealt with specific knowledge about diabetes, medications, and management of hypo- and hyperglycemia . In an observational matched cohort study comparing a random sample of pep participants with a group of matched control, each consisting of 1,141 subjects, the pep group had an average decrease of 0.138% in the hba1c level (95% ci, 0.252 to 0.024; p=0.017) more than the control group . The pep group also achieved a significant decrease in the mean ldl - c value (0.254 mmol / l, p<0.001), and the decrease was significantly more (0.136 mmol / l; 95% ci, 0.223 to 0.048; p<0.001) than that of the control group . More importantly, three prospective cohort studies, each comparing a group of more than 12,000 pep participants with a group of matched control of the same number of patients with a median follow - up of more than 21 months, have been published . Pep participants had a lower rate of all - cause mortality (hr, 0.564; 95% ci, 0.445 to 0.715; p<0.001), first cvd (hr, 0.807; 95% ci, 0.696 to 0.935; p=0.004), and stroke (hr, 0.702; 95% ci, 0.569 to 0.867; p=0.001) than those without pep . Pep participants were associated with a lower incidence of first microvascular event (hr, 0.85; 95% ci, 0.78 to 0.94; p=0.001) and nephropathy (hr, 0.71; 95% ci, 0.62 to 0.80; p<0.001) than non - pep participants, after adjusting for confounding variables . There were a significantly lower number of emergency department visits (incidence rate ratio, 0.903; p<0.001): 40.4 visits per 100 patients annually among the pep group versus 36.2 per 100 patients annually in the control group; and significantly fewer hospitalization episodes (incidence rate ratio, 0.854; p<0.001): 20.0 hospitalizations per 100 patients annually in the pep group versus 16.9 hospitalizations per 100 patients annually in the control group . To date, the pep is the first structured diabetic self - management education program shown to improve mortality, macro- and microvascular complications, and utilization of health services . In order to monitor the overall improvement in diabetic control, the percentage of all the patients under the care of the hospital authority with hba1c below 7% has been captured from the laboratory information system of the corporate electronic clinical management system . The percentage improved from 33% in 2008/2009 to over 50% in 2015/2016 (fig . 5), reflecting that the strategies have been effective in improving the overall control . This will probably translate into a decrease in the incidence of complications . The experience of hong kong demonstrated that a system approach, comprising a cpg to guide clinical care, a system for ensuring that the key care processes of assessment, risk stratification and appropriate multidisciplinary interventions are implemented, is required to tackle the problem . Charged with the responsibility for providing a health care safety net for all citizens of hong kong, the ha has to provide efficient and effective care for the majority of people with diabetes in the city within a very tight budget . However, since all the public hospitals and clinics are under its management, it has the advantage of being able to adopt a system approach to organize diabetes care across the different levels of health care settings for the whole continuum of the disease . The organizational cpg sets the standard of care, covering the following areas: (1) diagnosis; (2) minimal processes in outpatient management; (3) patient self - education as an integral component of management; (4) glucose lowering therapy; (5) control of cardiovascular factors; (6) regular screening for diabetic complications at 1 to 3 years' intervals; and (7) inpatient management of hyperglycemic emergencies and perioperative situations . A number of programs have been set up to support systemic and structured care, including a 6-class interactive pep for early disease run by non - governmental organizations; education through telephone calls by trained nurses; a structured complication assessment program; an insulin initiation program at the gopcs; control intensification at diabetes centers at hospitals . Various key performance and clinical indicators are regularly monitored across different hospitals and clinics to assure quality . The provision of diabetes care by the ha is an integral and representative component of the highly efficient and regarded public health care system of hong kong, which many economies, including mainland china, can learn from.
An unfortunate consequence of the childhood and adolescent obesity epidemic is the emergence of metabolic syndrome, a condition that was historically seen in adults . Thirty percent of obese adolescents in the united states meet criteria for metabolic syndrome and the prevalence is increasing . Consistent with adult rates, prevalence of metabolic syndrome in adolescents is highest among hispanics, followed by african americans when compared to non - hispanic whites, while type 2 diabetes is increasing among american indian adolescents more than any other racial / ethnic group in the united states . Weight loss through behavioral modification is an appropriate first step in the primary care treatment of metabolic syndrome . Nevertheless, almost 80% of pediatricians report frustration with their ability to make an impact on obesity and many providers feel they do not have the tools to effectively address lifestyle modification for weight loss . Schools, where children and adolescents spend the majority of their time, are promising venues for intervention work . A recently released institute of medicine report strongly endorsed this perspective, citing schools as a national focal point in efforts to address obesity in children and adolescents . As several systematic reviews demonstrate, however, there is a paucity of obesity intervention research in high school settings targeting older adolescents and even less in school - based health centers (sbhcs) [1012]. Located in the school setting, sbhcs have several compelling features for a successful lifestyle intervention program to treat overweight / obesity and prevent development of metabolic syndrome: (1) sbhc clinicians have more access to adolescents than community health care providers; (2) adolescents have better compliance and followup in school - based clinics (both of which are needed for weight loss and maintenance); and (3) sbhcs focus on early identification of high - risk problems [1318]. In response to the obesity epidemic and adolescents' interest in promoting physical activity and healthy eating, our research was built on a long - standing partnership between the university of new mexico school - based health center program and an urban school community . Through previous engagement with school - based clinicians and adolescent students, we reached consensus on collective interest to create a dvd to help adolescents achieve weight loss and a clinician tool kit to assist providers in their motivational interviewing efforts . This partnership led to the creation of a culturally and developmentally appropriate overweight / obesity intervention program, adolescents committed to improvement of nutrition and physical activity (action). Consistent with american academy of pediatric expert committee recommendations regarding the treatment of adolescent overweight / obesity, the action intervention featured eight sessions with a health care provider using motivational interviewing to encourage behavior change toward active living and healthful eating . This paper describes the use of an adaptive community based participatory research (cbpr) process through which formative research data were collected for development of an sbhc intervention for prevention of metabolic syndrome in multiethnic high school students . Formative research is used to understand people's beliefs, perceptions, and behaviors for the purpose of developing culturally appropriate behavioral change programs . Health researchers typically include a formative stage of research in their study designs to ensure that the identification of such cultural attributes and contextual factors can be integrated into intervention activities [2224]. Given our interest in developing intervention tools (dvd and toolkit) to help school - based providers and multiethnic adolescents, we designed this study to ground formative assessment through a cbpr partnership [25, 26]. Our review of the literature revealed that use of cbpr in formative research is quite limited and we have not found examples of other manuscripts reporting this type of approach in the context of an sbhc study . We provide further detail showing the link between cbpr principles and our research steps in table 1 . To maximize time for development and completion of the dvd and toolkit in one year, recruitment for semistructured interviews with overweight / obese adolescents and parents of overweight / obese adolescents was concurrent with recruitment of overweight / obese adolescents and their parents to the community advisory council (cac). Adolescents and parents were recruited from the two participating urban high schools with long - standing school - based health centers . Interviews with participants unknown to the cac were conducted to obtain fuller disclosure of sensitive information about living with obesity that the university research team felt may have been too sensitive to discuss openly among the cac members . In the second year of the study, the intervention was implemented using these materials . The study protocol was approved by the university of new mexico human research protections office and the urban schools' research, development, and accountability department . A purposeful sampling strategy that reflected the culturally diverse school population was used to recruit overweight / obese adolescent and parent dyads . Given the intent to understand perceptions of obesity and barriers / facilitators to weight loss, we focused recruitment efforts on overweight / obese adolescents . We also conducted extreme case sampling to identify adolescents who self - reported success in achieving weight loss (table 2). We believed that these more unusual cases would lead to insights relevant to our intervention development . All of the adolescents were referred to the research team by participating school - based health center providers and partnering school staff (e.g., counselors, school nurse, and teachers). The research team coordinated recruitment efforts with collaborators at the participating high schools including the activity directors, school health advisory council members, student senate members, and sbhc clinicians . Adolescent and parent interview guides were designed to assess concordance of perspectives in the following areas: media use and information seeking strategies, definitions of health, health concerns (weight related), strategies / approaches to weight loss, barriers / facilitators to health in the school and home environment, and suggested content and style for the dvd . The interview guide was designed to be neutral to potentially stigmatizing perspectives about adolescent obesity and contributing factors . Adolescents and parents were interviewed separately and interviews were conducted at times and locations convenient for the family, mostly in their homes and some at the university . Consent / assent forms were mailed in advance for review and respondents were formally consented at the start of each interview . Most interviews lasted approximately one hour and each respondent received a $20 reimbursement for their time . Following an iterative analytic process, the multidisciplinary research team including a medical anthropologist, adolescent medicine physician, primary care nurse practitioner, health communications researcher, and community based participatory research practitioner reviewed sets of 3 to 4 transcripts independently, identifying key themes relevant to the subsequent creation of the intervention materials . We specifically focused on responses between adolescents and parents, seeking both complementary and divergent perspectives on key elements for the dvd and provider toolkit materials including content, messaging, and context of use . Ongoing data collection and analysis continued until 15 interviews had been conducted (7 students, 8 parents; the sample consisted of 6 parent - child pairs), at which point the research team reached consensus that the full range of interpretive themes had been identified . At that point, the medical anthropologist (als) coded each interview in the qualitative data analysis software program nvivo 8 to facilitate text retrieval and create summary reports . Further demographic details of the student and parent interviewees are presented in table 2 . Following the intervention and as part of a broader process evaluation effort designed to examine a range of implementation issues, we assessed adolescent and parent views of acceptability and satisfaction with the materials developed in the formative phase . Intervention students were each asked to fill out questionnaires with likert scale ratings of the action dvd, toolkit handouts, and overall satisfaction with the intervention . Parent questionnaires focused on materials they received as part of the action intervention, including a parent newsletter as well as views of the toolkit handouts and overall satisfaction with their child's participation in action . Descriptive summaries of the questionnaires for each group were assessed . Once the interviews and a preliminary analytic summary had been developed, we convened the community advisory council (cac) as a forum in which to translate themes from the interviews into specific strategies and materials to be used in the intervention . Our goal was to form a group with similar demographic characteristics to the targeted study population: hispanic, african american, and american indian overweight / obese students and their parents . Given our expectation of attrition, we recruited a total of 16 participants (divided equally between students and parents, including four parent - child pairs). We began a series of six consecutive monthly meetings from december 2008 to may 2009 at the university of new mexico during which the dvd and toolkit were developed . The cac met on a less frequent basis throughout the second year of the study (august 2009 to may 2010) to provide implementation guidance and feedback . We provided a full dinner, free parking, and reimbursement for travel expenses ($10) for all participants immediately following each meeting . Cac members were also informed that all would receive the dvd resulting from their efforts as well as a personal dvd player . Sessions were typically comoderated by the dvd producer and study coinvestigator (als). However, when meetings were focused on topics of nutrition and/or physical activity, sessions were led by the research team registered dietician and fitness expert . This approach addressed a central cbpr principle in fostering information exchange between community members and the research team . The meetings were designed to be cyclical and iterative as a way to reach consensus and tailor evidence - based strategies to ensure the cultural and age appropriateness of the dvd and toolkit materials as well as stylistic elements (e.g., music and images). Given the flexible, participatory nature of these meetings, cac members shaped the process by volunteering to test potential data collection methods (e.g., maintaining a food diary) and requesting further information of the research team to facilitate colearning (connections between obesity and health). Overview . We have organized the presentation of results to reflect the sequential process of data collection leading to the creation of the action intervention materials . We first present the thematic findings from key informant interviews that provided guidance to the research team and the cac . We then describe how the cac translated this input into the dvd and provider toolkit . Lastly, we include process evaluation measures reflecting adolescent and parent views of these materials following the action intervention . Adolescents consistently indicated use of the internet as a primary source for both entertainment and information seeking . A lot of teenagers i know use the internet and look up information that way . If it's something that looks interesting they'll go look at it . A lot of teenagers i know use the internet and look up information that way . If it's something that looks interesting they'll go look at it . Further, most adolescents described using the internet as a way to gather health information about a range of issues, including diet and exercise . They viewed the internet as a reliable source of information that could be accessed in private . Another female adolescent described using the internet for health information and as a basis for discussion with her doctor: they have that pyramid, i'm not sure what it's called . And you can look at how many calories you're supposed to take in a day and i looked at those and i just started doing that last month with my doctor . They have that pyramid, i'm not sure what it's called . And you can look at how many calories you're supposed to take in a day and i looked at those (2) functional definition of health . In each of the interviews, we asked adolescents how they define health and what that means to them . In some cases, they identified the association between being overweight / obese and the future development of health problems such as diabetes and heart disease . Several of these teens were aware of these risks given family members dealing with these conditions . However, the most striking theme to result from these interviews related to how being overweight / obese limited adolescents activities in the present . Overall, they were less concerned with the long - term consequences and referred to a functional definition of health that focused on the degree to which their weight impacted the ability to engage in activities of interest . One male adolescent, echoing the response of several others, responded to the question what does it mean to be healthy? By stating (3) barriers to weight loss in the school . Not surprisingly, both teens and parents reported a wide range of challenges related to their struggles with food choices and physical activity in their weight control efforts . Teens consistently noted that healthful foods / drinks were not available in the school setting . Adolescents regularly purchased foods from vendor carts but were frustrated by the lack of healthful options such as water rather than soda . In addition to expressing a need for healthier snacks from vendors, the teens also pointed to unhealthful meal choices at school . As a potential way to address this problem, one teen stated mostly not sell as much pizza, cause like everywhere you go they sell pizza and burritos, like breakfast burritos and sometimes even regular burritos at lunch . Even if they change the menu up a little bit that'd be better because it wouldn't be the same thing every day just burritos, burritos or pizza, pizza, pizza . Mostly not sell as much pizza, cause like everywhere you go they sell pizza and burritos, like breakfast burritos and sometimes even regular burritos at lunch . Even if they change the menu up a little bit that'd be better because it wouldn't be the same thing every day just burritos, burritos or pizza, pizza, pizza . Parents consistently agreed with adolescents about the need for schools to offer foods that are both appealing and offer higher nutritional value . Another challenge identified by both the teens and the parents is the lack of opportunity for exercise in the school setting . Teens emphasized the need for more physical activities not related to formal participation on sports teams . Parents focused their concern on curricular issues such as the need to require a gym class for all four years of high school rather than just one semester . Several adolescents interviewed reported success in their efforts to lose weight . Although a range of strategies were described mostly involving diets and increases in physical activity adolescents expressed the importance of internal motivation as the essential element in weight loss initiation efforts . Consistent with the functional definition described above, students identified personal goals such as making a sports team or improving their appearance for a significant school event such as the prom as a reason to make physical activity and nutritional changes . Similarly, there was consensus among the parents that it was their obligation to serve as a principal source of motivation . As one parent saidi would have to say it (motivation) would have to be left on the parent's shoulders . I think if they see the parents motivated to want to exercise and take care of their body and eat healthy then the children are going to see that and that's going to help them . Mom's doing this, maybe we should do this, too .' I would have to say it (motivation) would have to be left on the parent's shoulders . I think if they see the parents motivated to want to exercise and take care of their body and eat healthy then the children are going to see that and that's going to help them . Okay, mom's doing this, maybe we should do this, too .' (5) parent views on changing home environment . While we mostly focused these interviews on the school environment, we specifically engaged parents about the challenges they confront in weight loss efforts at home . The goal of these discussions was to elicit strategies that could be included in the home - based component of the action intervention . Parents echoed the adolescents in recognizing the broad importance of motivation as a way to initiate health behavior changes . As one mother indicated some ideas on how to motivate your kids ideas on how to keep them wanting to live a good lifestyle, you know, healthy lifestyle and full of exercise . And then just trying to change their mentality, change their way of thinking so that it's stuck in their head . My goodness, it's hard . Some ideas on how to motivate your kids ideas on how to keep them wanting to live a good lifestyle, you know, healthy lifestyle and full of exercise . And then just trying to change their mentality, change their way of thinking so that it's stuck in their head . We also asked parents for suggestions of informational needs about nutrition and/or physical activity that would be useful to them at home . Another mother described interest in having a list of practical comparisons of food choices: i always find it interesting when they do comparisons like which is actually the healthier (food) and you're going, i don't know, they're actually both kind of bad, but which is actually the better choice?' I always find it interesting when they do comparisons like which is actually the healthier (food) and you're going, i don't know, they're actually both kind of bad, but which is actually the better choice?' (6) input on dvd content . Lastly, the interviews provided a rich opportunity to gather input on content and stylistic elements of the proposed dvd . Consistent with the theme of internal motivation, most adolescents requested that the dvd content did not scare or threaten by presenting adverse health consequences of overweight / obesity . Instead, they emphasized the importance of featuring teens in the dvd that were currently overweight / obese so that adolescents would relate more to the content: include overweight people and show that they're really working hard and then show them successfully doing it and get in a normal (weight) range . Include overweight people and show that they're really working hard and then show them successfully doing it and get in a normal (weight) range . Reflecting common elements of teen culture, most of the teens suggested using popular activities as a vehicle through which the messages could be promoted . This included differing forms of dance, kickboxing, use of appropriate music, and presenting these activities with careful attention to style through rapid cutting to different camera angles and shots . When asked what should be in the dvd, one male adolescent quickly responded dancing, i'm pretty sure cause everyone loves to dance; say if you want to learn how to fight and get in shape and defend yourself there's kickboxing, so pretty much those things . Dancing, i'm pretty sure cause everyone loves to dance; say if you want to learn how to fight and get in shape and defend yourself there's kickboxing, so pretty much those things . As the research team finalized the analysis of the parent and adolescent interviews, we convened the cac and held the first of six consecutive monthly sessions . The first session was primarily an orientation to the project and subsequent meetings were led by content expert moderators from the research team to identify and refine intervention content . Below, we provide a list of the major themes and the resulting strategies that were integrated into the dvd and toolkit . Cac participants confirmed this and agreed with a proposal to include websites in the intervention materials for adolescents to access nutritional and physical activity information . During one of the sessions, we reviewed potential websites for inclusion (ranging from purely informational to highly interactive) with the cac and our final selection was based on feedback pertaining to ease of use and clarity of presentation . Functional definition of health articulated during the adolescent interviews and the cac concurred with the preference to encourage individual autonomy and goal setting rather than emphasize negative outcomes associated with poor health . The cac adolescent participants were clear that the kids in the dvd needed to be real teens (both overweight / obese and not overweight / obese teens) dealing with challenges of weight . This strategy was incorporated into the dvd by having cac participants and friends appear in the dvd and briefly describe their struggles as well as the internal sources of motivation that inspired them to make changes . The group advocated that the dvd contain practical nutritional information to help them make healthier choices amongst likely food offerings (e.g., pizza, burritos, bagels, etc . ). Websites containing further information were also referenced in both the dvd and health care provider toolkit . As described above, teens indicated that any effort to achieve weight loss had to begin with an individual identifying some type of internal motivation and, ideally, having a supportive network of friends and family to sustain the effort . While the cac agreed with this perspective and specific strategies were included in the dvd to encourage recognition of this internal motivation, the group also advocated for the inclusion of physical activities to jumpstart the process . Therefore, following the input of both the interviewees and the cac, brief instructional segments featuring dance, kickboxing, and weight lifting were added to the dvd . (adolescents and parents in the cac agreed that it was not sufficient to only focus on the school environment . The group offered a series of strategies for involving parents in a practical and appropriate way during the action intervention . This included having the school health provider call the parent to provide regular updates, distribution of a short parent newsletter, and ideas for affordable and quick healthy recipes for busy families . (6) dvd content . Toward the conclusion of the six cac sessions in the first year of the study, the group reached consensus on three sections to feature on the dvd: (1) adolescent motivation for change, (2) strategies targeting energy balance and nutritional quality, and (3) physical aerobic dance and strength / resistance training instructional segments . The cac reviewed each of the elements to be included in the dvd as well as offered stylistic suggestions relating to background music and video editing techniques to appeal to adolescents . (iii) use of the action dvd and toolkit . At the conclusion of the six cac sessions, coordination and filming of the dvd were led by the video producer . Many of the cac members teens and parents participated in this process and appear in the final dvd either sharing their own personal stories or presenting the nutrition or physical activity strategies . The action toolkit contained a parent newsletter and adolescent session tools (e.g., goal setting, internet resources, activity / food journal, etc . ). (iv) action outcomes and satisfaction results . While the focus of this paper is to report on the process of developing the action intervention materials, we are also able to offer a brief overview of process evaluation findings related to participant satisfaction with these components as well as the primary outcome measure . Health outcome measures for the action study showed that students receiving the intervention had greater prepostimprovements in bmi percentile (p = 0.04) and waist circumference (p = 0.04) as compared to the standard care control group . Bmi median percentile decreased 0.3% in the intervention group while the standard care control group's bmi median percentile increased by 0.2% . Mean waist circumference in the intervention group remained unchanged and there was a 1.7 cm increase in the standard care group . While these outcome measures are important, we also conducted process evaluation aimed at elucidating factors that may help us to better understand how the use of this adaptive cbpr approach relates to these results . Students reported high levels of satisfaction with the materials used in the intervention . On a scale of 0 (not at all useful) to 5 (very useful), students were asked to rate the usefulness of the dvd (n = 26; mean score 3.1), the clinician toolkit handouts (n = 27; mean score 4.0), and their overall satisfaction with the intervention (n = 28; mean score 4.4). Parents favorably rated the usefulness of the parent publication (n = 18; mean score 3.6), the usefulness of the clinician toolkit handouts (n = 23; mean score 3.7), and their overall satisfaction with the intervention (n = 25; mean score 4.4). Lastly, process evaluation findings reveal high rates of retention among participants in both the intervention group (90%) and standard care control group (79%). We used formative assessment research guided by an adaptive cbpr approach to create an sbhc obesity intervention program . The student and parent interviews generated initial thematic findings that were used to guide discussions in the subsequent cac sessions toward the development of the intervention dvd and clinician toolkit . This approach enabled us to accomplish the following goals in the first year of this study: (1) gain a better understanding of the school setting with regard to barriers to physical activity and healthful food choices, (2) create strategies / materials to address these barriers, and (3) develop culturally appropriate intervention materials and approaches based on input from study participants . We identified several elements that may be useful in subsequent efforts to develop and/or further refine obesity intervention materials in school - based settings . Adolescents emphasized the importance of media, particularly use of the internet, as a primary source for most informational needs . We also observed that these culturally diverse (hispanic, american indian, and african american) adolescents expressed mostly similar views about the types of barriers and proposed solutions to addressing overweight / obesity both at home and in the school . There was strong consensus regarding a functional definition of health that focused on identifying internal sources of motivation to achieve goals and engagement in activities as opposed to one based on more standard biomedical measures of health (e.g., blood pressure or cholesterol levels). Partnering with these teens and adolescents was essential in order to ensure not only the appropriate content in the action intervention but also the stylistic elements that maximize appeal and likelihood of use . We believe that the high levels of satisfaction reported by action participants, as well as the promising results in the primary outcome measures, are attributable to this adaptive cbpr process . Over the past several years, increasing attention has been directed toward schools settings as promising venues for obesity prevention and treatment interventions . The recently released institute of medicine report identified schools as catalysts in our efforts to accelerate progress in obesity prevention . A growing body of literature supports the value of directing programmatic and research resources to school - based interventions . Prior research has demonstrated the efficacy of a quality improvement initiative aimed at enhancing sbhc provider implementation of obesity treatment guidelines . Another recently published study reports findings from an adolescent obesity screening program conducted through a school - based health center . In this study, following a medical evaluation, adolescents determined to be outside while these types of quality improvement and screening and referral projects are consistent with the need to engage schools in obesity prevention efforts, there is still a gap in the development and implementation of obesity interventional research through sbhcs . We believe the action study is the first to actually create and test such an intervention through a partnership with key stakeholders located in the school community . Conducting research in partnership with the sbhcs, the school administration, the students, and their families poses a set of logistical and study design challenges . The action study involved each of these key stakeholders in different ways and at different stages to both maintain relationships that preceded this project and establish new ones to sustain our efforts going forward . The adaptive cbpr design employed in the action study may offer useful guidance to other researchers conducting research in school settings . While much cbpr is oppositely structured forming the community advisory group first in this two - year study it was imperative that the formative research and production of the intervention materials (dvd and clinician toolkit) are completed by the start of the intervention, coinciding with the school calendar . Our goal was to incorporate the core principles of cbpr while operating in a compressed timeframe imposed by the research funding . As evidenced from both the process evaluation findings and health outcome measures, we believe that these adaptations were essential to create an authentic partnership while adhering to a timeline that may not have been feasible using a more traditional cbpr approach . While use of cbpr appears to enhance the effectiveness of interventions, there exists a lack of clarity regarding how different types of partnership configurations and processes contribute to these outcomes . The first relates to the limited generalizability of these findings to other populations and school - based settings . Our purpose in this paper, however, is to demonstrate the applicability of an adaptive cbpr process through which researchers can identify locally and contextually relevant factors in their school - based obesity interventions . The second limitation involves the relatively small sample size of interviews in the formative assessment phase . The iterative process of data collection and analysis led to data saturation at an early stage and the consistency of thematic responses enhanced the confidence of findings from ethnoracially diverse students and parents . Cbpr partnerships with school - based communities represent an important opportunity for researchers seeking to address a broad spectrum of adolescent health problems . There is an urgent need to find innovative, cost - effective, and sustainable strategies to reach underserved communities . We found that conducting formative assessment using an adaptive cbpr process with a range of school - based stakeholders was an effective way to develop culturally appropriate and tailored intervention materials and approaches in these multiethnic school settings . We demonstrated that researchers can adapt their study designs in ways that best maximize limited resources and time constraints without compromising the core principles of cbpr.
It is caused by trauma, stress, sepsis, adrenal tumors, anticoagulation, hemorrhagic disorders and pregnancy . Idiopathic adrenal hematoma. The symptoms vary from subclinical to clinical, such as nausea, abdominal pain, fever and hypotension due to circulatory collapse . Adrenal tumors associated with hemorrhage primarily include pheochromocytomas, adrenocortical cancers and metastatic lesions from other organs . From a clinical perspective, whether the lesion is benign or malignant is an important issue but is difficult to determine prior to surgery . A 59-year - old japanese man was referred to our hospital for evaluation of a 7.0 cm mass in the posterior segment of the liver or right adrenal gland in november 2009 . This mass was discovered when abdominal ultrasonography was obtained to evaluate a weight loss of 8 kg from may to november 2009 . His body temperature was 37.0c, his blood pressure was 124/73 mm hg, and his pulse was regular at 67/min . Hormonal examination revealed a slightly elevated metanephrine level (0.35 mg / day; normal range 0.040.19). Tumor marker levels, including serum -fetoprotein, carcinoembryonic antigen and carbohydrate antigen 19 - 9 were all within normal ranges . Only pivka - ii was slightly elevated at 53 mau / ml (normal <37). Abdominal computed tomography (ct) demonstrated a well - demarcated, 7.0 cm lesion in either the posterior segment of the liver or in the right adrenal gland . The peripheral region was slightly enhanced and the central part was hypovascular, indicating central necrosis (fig . There was no evidence of lymph node enlargement or distant metastasis . On magnetic resonance imaging (mri),, the mass showed arterial enhancement and a ct filling defect in the right superior and inferior adrenal arteries and arterial portography, respectively (fig . I - meta - iodo - benzylguanidine (mibg) scintigraphy demonstrated no aberrant accumulation in the mass, which ruled out the possibility of a pheochromocytoma . For the purposes of securing a definite diagnosis and treatment, an open right adrenalectomy was planned for march 2010 . The resected specimen was 7.0 5.5 cm in size, and the cut surface of the mass was heterogeneous and dark red and white in appearance (fig . Hematoxylin - eosin staining revealed hemorrhage, necrosis and hemosiderin deposit with normal adrenal tissue . The patient had an uneventful postoperative course and was discharged home on postoperative day 12 . Small adrenal branches from the three main adrenal arteries form a subcapsular plexus, and the gland is drained by relatively few venules . For example, stress increases adrenal vascularity and increases adrenal venous pressure due to vasoconstriction, resulting in intraglandular hemorrhage . Idiopathic adrenal hematomas are difficult to diagnose prior to surgery . In japan, koizumi et al . Described 14 cases of idiopathic adrenal hematomas from 1983 to 2010 . Of these 14 cases, 13 had been suspected to be adrenal tumors including malignant lesions preoperatively, but were then found to be idiopathic adrenal hematomas on pathological examination after surgery . Only 1 patient was diagnosed with an idiopathic adrenal hematoma without surgery . The adrenal hemorrhage was not suspected to be caused by an adrenal tumor on the basis of ct, hormonal assay and mibg results, and this mass spontaneously regressed after 2 weeks . Although imaging modalities such as ct and mri are helpful in detecting adrenal hemorrhage, it is difficult to determine whether the hemorrhage is associated with tumors or not . Adrenal hemorrhage associated with pheochromocytoma (48%) was the most frequently observed situation, and the second most frequently observed was a malignant lesion (20%) such as adrenocortical cancer or metastasis from another organ . Hematomas derived from pseudocysts or adenomas comprised 17% of all cases . Therefore, even if pheochromocytoma is ruled out by hormonal evaluations and imaging studies, the possibility that the mass may be a malignant lesion remains approximately 50% . Furthermore, the size of adrenal incidentalomas, which are adrenal gland masses discovered serendipitously on imaging, is an important factor in differentiating benign tumors from malignant lesions . Nieman recommended routine surgical resection for adrenal incidentalomas> 4 cm in diameter without a clear - cut diagnosis . In our case, metastatic liver tumors, hemorrhaged hepatic cellular carcinoma with central necrosis and adrenal tumor were raised as differential diagnoses at the time of the initial consultation because of ring enhancement with hypovascularity on enhanced ct and a slightly elevated pivka - ii level . Subsequently, abdominal angiography revealed the origin of the mass to be the right adrenal gland . Although vital signs, laboratory data and mibg scintigraphy ruled out the possibility of a pheochromocytoma, the possibility of another malignancy could not be completely ruled out . Therefore, surgery was strongly recommended for a definite diagnosis as well as for treatment . The reason for the patient's weight loss and the increase in the pivka - ii in our case was not clear, but may have been related to exhaustion during the summer season from may until november that kept him from eating sufficiently . In fact, his weight loss ceased after he consulted our hospital, and the pivka - ii value also normalized prior to surgery . In conclusion, we report herein a case of idiopathic adrenal hematoma . An accurate diagnosis of idiopathic adrenal hemorrhage is quite difficult to make prior to surgery . Some imaging modalities are useful in generating a differential diagnosis, but if the potential for malignancy is not excluded, surgical resection should be taken into consideration.
Perceived health is a subjective assessment that people make about their own health state and it is an indicator of overall health status.1 it is included as one of the world health organization (who) health targets . Perceived health status is not a substitute for more objective health outcomes but rather complements them.2 perceived health status accords well with objective health status.34 it is a useful and valid measure of population's overall well - being . This is because lower ratings of health status have been associated with increased morbidity and mortality.5 perceived health status is generally accepted as a valid measure of health status in population studies and understanding its correlates may help public health professionals prioritize health promotion and disease - prevention interventions.6 several factors related to demographic, socio - economic and lifestyle characteristics have been found to be related to perceived health status.78 chronic diseases and disability have also been found to influence one's perception of health.910 hypertension and diabetes mellitus are considered to be the most prevalent chronic diseases in both developing and developed countries . A large survey in the united states from 1996 - 2005 concluded that self - rated fair or poor health was three times more common among adults with diabetes than those without diabetes.11 existing studies on the perceived health and symptoms of elderly patients with diabetes have shown that diabetic patients rate their health more negatively and report more symptoms than do non - diabetic patients.12 duration of diabetes is also an important factor in perception of one's own health . It is a significant predictor of mortality in people with older onset diabetes but not in those with younger onset diabetes where physical health status is controlled.13 similarly, it is also well known that hypertension affects patient's quality of life . Self - perception of health status correlates well with hypertension and its co - morbid conditions . Individuals with two or more risk factors who rated their own health as not good were much more likely to have a stroke.14 these were gender specific and education sensitive . Men appeared to be at greater risk of stroke than women, as were well - educated people of both sexes.14 perceived ill - health amplifies the risk for development of coronary artery disease.15 perceived health status is also reflected in the number of visits to the health care system . Hypertensive patients with poor self - perceived health tend to have more visits.16 to our knowledge, there are currently no specific studies on perceived health status on hypertension and diabetes mellitus patients in oman . The aim of this study was to assess the impact of diabetes mellitus and hypertension as well as other demographic and clinical characteristics on perceived health status in primary health centers in oman . This was a retrospective cross - sectional study conducted in the wilayat of as - seeb (one of the six wilayats in the muscat region). At the time of the study, the primary health care service was provided by six health centers (al - maabela north, al - maabela south, al - shaadi, as - seeb, al - khoudh and al - mawaleh). These centers provide comprehensive primary health care services as prescribed by the ministry of health (moh) of oman . They also serve a considerable population (223, 267 patients).17 in addition, these centers run specialty clinics for chronic conditions including those for hypertension and diabetes . The target population for this study was all patients with hypertension and/or diabetes aged 18 years of age attending the aforementioned health centers during the study period (january 1 2007 till december 31 2007). Hypertension and diabetic registries in each of the 6 health centers were used to obtain the total number of patients registered in the study period (2,923 patients). To estimate an approximate prevalence rate of 20% for diabetes mellitus and hypertension patients with an error margin of 5% and 95% confidence intervals, a sample size of 227 patients was needed . To compensate for drop - outs, losses to follow - up, as well as missing information, the sample size was more than doubled to 450 . The patients were selected randomly on the day that they came for their follow up provided they matched the inclusion criteria (diabetes / or hypertension). The first was a checklist of indicators / outcomes for each disease (diabetes / hypertension) with socio - demographic details (form 1). The second was the 12-item short form health survey (sf-12) questionnaire that was used to assess the perceived heath status (form 2). The questionnaire, form 1, was administered by the investigators (ah & ns). Information was retrieved from the computerized healthcare system in each of the health centers (known as the al - shifaa). Some of the information regarding demographic variables was obtained by direct interview with the patients . This information included age, sex, marital status, financial status, educational status and lifestyle status (smoking, alcohol drinking). It also included history of any chronic illness or disability (asthma, osteoarthritis, cancer, depression, seizure, etc), duration of hypertension/ diabetes, number of visits, and drug history . Health indicators like blood pressure (bp), body mass index (bmi), glycated hemoglobin (hba1c) were recorded retrospectively . For the control of hypertension, bp value of <140/90 mmhg was considered to be good control . However, for patients with diabetes, bp value of <130/80 mmhg was considered as good control . With regards to hba1c, a value <recorded complications were retinopathy, nephropathy, neuropathy, coronary artery disease and cerebrovascular disease . Form 2 was the questionnaire sf-12, which is an internationally developed tool to assess perceived health status . The arabic version was obtained from the principal investigator (am) who had translated and published it in a previous study . The sf-12 questionnaire measures generic health concepts relevant across age, disease, and treatment groups . It provides a comprehensive psychometrically sound and efficient way to measure health from patient's point of view by scoring standardized responses to standard questions . The 12 questions assess eight dimensions of health: 1) physical functioning; 2) physical role; 3) bodily pain; 4) general health; 5) vitality; 6) social functioning; 7) emotional role; and 8) mental health . Two scores were given for each option, after which all scores were added up to give two measures of health status including the physical component summary (pcs) and the mental component summary (mcs) scores . The sf-12 questionnaire was administered by the authors themselves (ah, ns) who were trained in keeping consistency when asking the questions . Institutional review board approval was granted and differences between groups (hypertension, diabetes, both) were analyzed using univariate logistic regression . However, the relationship between the variables, finance and number of prescriptions, with clinic type was analyzed using fisher's exact test . For continuous variables, mean and standard deviation were used to present the data while analysis was performed using univariate ordinary least squares (ols) regression . For those continuous variables that are abnormally distributed, median and interquartile ranges were used to present the data while the analysis was performed using the non - parametric test, kruskal - wallis . The relationship between the various demographic, clinical and healthcare resource use characteristics and health - related quality of life as measured by sf-12 were evaluated using ols regression (categorical and continuous variables) and pearson's correlation co - efficient test (continuous variables). However, the relationship between duration and both physical and mental component scores of sf-12 were analyzed using spearman's correlation coefficient . An a priori two - tailed level of significance was set at the 0.05 level . Statistical analyses were conducted using stata version 11.1 (stata corporation, college station, tx). Descriptive statistics were used to describe the data . For categorical variables, frequencies and percentages were reported . Differences between groups (hypertension, diabetes, both) were analyzed using univariate logistic regression . However, the relationship between the variables, finance and number of prescriptions, with clinic type was analyzed using fisher's exact test . For continuous variables, mean and standard deviation were used to present the data while analysis was performed using univariate ordinary least squares (ols) regression . For those continuous variables that are abnormally distributed, median and interquartile ranges were used to present the data while the analysis was performed using the non - parametric test, kruskal - wallis . The relationship between the various demographic, clinical and healthcare resource use characteristics and health - related quality of life as measured by sf-12 were evaluated using ols regression (categorical and continuous variables) and pearson's correlation co - efficient test (continuous variables). However, the relationship between duration and both physical and mental component scores of sf-12 were analyzed using spearman's correlation coefficient . An a priori two - tailed level of significance was set at the 0.05 level . Statistical analyses were conducted using stata version 11.1 (stata corporation, college station, tx). Twenty - one percent (n = 95), 39% (n = 176), and 40% (n = 179) had hypertension, diabetes, and both hypertension and diabetes, respectively . Most of the participants were females (62%; n = 277), married (75%; n=339), literate (56%; n = 254), non smoker (91.1%; n = 410), had high bmi (30 5.7 kg / m), poor blood pressure control (81%; n = 364), poor hba1c control (88%; n = 390), had more frequent healthcare visits (monthly or less) (60%, n = 271), had higher number of prescriptions (4) (64%; n = 289) with a median disease duration of 7 (4 - 10) years (table 1). Demographic and clinical characteristics of the study participants those participants with diabetes alone were significantly younger compared to those with hypertension (49 vs. 54 years; p <0.001) or those with both hypertension and diabetes (49 vs. 56 years; p <0.001). Illiteracy was associated more with those that had both hypertension and diabetes than those with diabetes alone (50% vs. 33%; p = 0.005). Systolic (141 vs. 129 mmhg; p <0.001) and diastolic (84 vs. 80 mmhg; p <0.001) blood pressures were significantly higher in those with dual diseases compared to those with diabetes alone . Furthermore, blood pressure control was worse in those with dual disease when compared to those with hypertension (6% vs. 33%; p = 0.001) or diabetes (6% vs. 19%; p = 0.015) alone . With regards to blood pressure control, those with hypertension alone fared better when compared to those with diabetes alone (33% vs. 19%; p <0.001). Those that had dual disease had worse control of their hba1c (<7%) when compared to those that had diabetes alone (14% vs. 26%; p <0.001). Dual disease was also associated with higher number of prescription use compared to those with single diseases (88% vs. 50% vs. 45%; p <0.001). Moreover, those with dual disease were associated with higher median disease duration compared to those with both hypertension and diabetes alone (8 vs. 6 vs. 6 years; p <0.001). Dual disease was associated with lower physical scores of the sf-12 when compared to those with diabetes alone (37 vs. 42; p = 0.001) but only marginally lower when compared to those with hypertension alone (37 vs. 39; p = 0.066). No significant differences in mental components of the sf-12 quality - of - life instrument were noted across the different diseases (table 2). Self - perceived health status on the physical (pscc) and mental (mscc) health components of the sf-12 quality - of - life instrument table 3 outlines the relationships between the various demographic and clinical characteristics of participants against the physical components of the sf-12 . Specifically, older age was associated with lower physical scores (rho -338; p <0.001). Male (43 vs. 37; p <0.001), married (40 vs. 35; p <0.001), and literate (42 vs. 36; p <0.001) participants were associated with higher physical components of the sf-12 when compared to their opposite counterparts . Relationship between demographic and clinical characteristics against both physical (pscc) and mental (mscc) composite scores of the sf-12 (n=450) the table also indicates that higher monthly income was associated with higher physical scores (p = 0.002). Those who had monthly visits (or fewer) were associated with higher physical scores when compared to those with longer visits (40 vs. 37; p <0.001). Furthermore, those with higher number of prescriptions were associated with lower physical scores (p <0.001). Moreover, table 3 also outlines the relationships between the various demographic and clinical characteristics of participants against the mental components of the sf-12 . Male (46 vs. 43; p = 0.005) and married (45 vs. 42; p = 0.017) participants were associated with higher mental components compared to their opposite counterparts . The table also indicates that higher monthly income was associated with higher mental scores (p <0.001). Those who had monthly visits (or fewer) were associated with higher mental scores when compared to those with longer visits (46 vs. 42; p <0.001). Furthermore, those with higher number of prescriptions were associated with lower mental scores (p = 0.005). Higher scores of the physical component of perceived health was significantly associated with good control of systolic blood pressure (p = 0.002). There was marginally significant association between good blood pressure control in patients with hypertension and patients with diabetes and the physical component of the perceived health (p = 0.051). This is the first study of its kind in oman to assess the association between perceived health status and various socio - demographic factors, type and duration of disease in patients with diabetes and hypertension . The study found that patients with dual disease have lower physical score than those with diabetes or hypertension alone . One explanation for this finding is that patients with diabetes were at younger age compared to those with dual diseases . This is further supported by the findings that older age was associated with lower physical score . Another possible explanation could be that patients with dual diseases are using multiple medications and have longer disease history compared to those with diabetes only . However, there was no significant difference in the mental score between patients having diabetes and/or hypertension . However, this finding is expected given the omani culture with extended family concept which may play a role in securing the emotional status of elderly people . Such findings can be explained by the fact that men are having better overall perception of life compared to women.81924 similarly, married people have less frequent visits to health care facilities and are emotionally stable compared to the unmarried counterparts.1825 the findings of the current study are in the same line with those of other studies . Martin lindstrom and colleagues found in their study that never married and the divorced had significantly higher poor self - rated health than married group.18 on the other hand, financial status has a significant effect on the perceived health status as higher monthly income was associated with better self - perceived physical and mental scores . This is similar to the findings of the study done by wagstaff and van doorslaer, in which they found negative effect of low income on population health.26 this could be explained by the fact that people with higher income have better access to high quality health care services . Literacy had a positive effect on the physical component of the self - perceived health but not on the mental component . A recent study done by faresjo and rahmqvist concluded that educational level appears to be a vital factor for good perceived health.27 this can be explained as literate people read more and become more anxious about their health in our culture . Smoking was not shown to have any effect on the self - perceived health in this study as majority of the population was non - smokers . Furthermore, bmi was not shown to have significant effect on the perceived health status in our study . This is in contrary with other studies which have shown that obese as well as underweight subjects were more likely to have poor self - rated health.828 the finding in our study could be explained by the fact that the majority of the selected sample had high bmi . Interestingly, our study found that perceived health status is affected by the number of visits to a local health center . Those with frequent visits like monthly or less had higher scores of perceived physical and mental health status . This could mean that continuity of care has a positive influence on the self - perceived health status.29 this finding is similar to what finkelstein et al . Found in their study.16 as there are factors that reflect disease control and at the same time may have an impact on the perceived health status, the current study found interesting results . On the one hand however, presence of chronic diseases was found to be associated with poor self - rated health.11 on the other hand, the study showed that patients using four or more drugs reported lower physical and mental scores . This finding is similarly reported by some studies such as the one reported by moen et al.30 however, only marginal association was found between perceived physical health status and hba1c, which is considered as one of the indicator of disease control . This could be explained by the fact that physical symptoms of diabetes such as polyuria, polydipsia and nocturia affect patient's quality of life and hence link with the perceived physical health . Similarly, there was only marginal association between controlled blood pressure (<130/80 mmhg for patients with diabetes and <140/90 mmhg for hypertensive) and the perceived physical health (p = 0.051). In contrast, good control of the systolic blood pressure had significant positive effect on the physical score of the self - perceived health (p = 0.002). There was no association with the perceived mental health . However, no similar studies showed such results . This needs to be explored by further studies and on other chronic illnesses . In spite of our study findings the first limitation is that the sample was selected from one area in the capital region (muscat), which might not represent the whole community with regard to the demographics and clinical characteristics . Secondly, arabic self - perceived health status questionnaire was used for the first time in patients with diabetes and hypertension in oman or the region at large . However, this questionnaire has been used earlier in the omani general community,31 which showed it to be a valid and reliable instrument . Moreover, this questionnaire can be used in diabetes and hypertension since it covers all domains of health . It is also important to note that correlations, though statistically significant, were generally weak . Our study showed that there was significant association between perceived health status and other demographic, socio - economic and clinical characteristics of patients suffering from chronic illnesses . These findings reflect the importance of considering these factors in order to ensure providing comprehensive care . Furthermore, it reflects the importance of considering these factors during care delivery in order to improve the quality of life for patients suffering from such chronic illnesses . However, more research on this area is needed to corroborate our findings in oman or the region at large.
Wild animals are exposed to environmental contaminants of both natural and anthropogenic origin and are suitable bioindicators of environmental pollution (obrien et al . Concentrations of toxic compounds in body tissues are determined by the degree of exposure, the compound s bioavailability and half - life, as well as the type of tissue (nordberg et al . Pollutants can be transported over significant distances by air or water, and they are found even in regions situated remotely from industrial centers (ansara - ross et al . Therefore, due to the rapid development and wide diversity of human activities, animals are increasingly exposed to harmful effects of compounds of anthropogenic origin (obrien et al . 1993). With growing public awareness of environmental pollution, regular monitoring of pollutants in the environment is essential to ensure levels of pollutants are below harmful or recommended, legal values (nasreddine and parent - massin 2002; european commission 2006). Such monitoring should be carried out not only in industrialized areas but also in natural and agricultural ecosystems away from the source of emissions (nasreddine and parent - massin 2002; szkoda et al . 2012). Metals include toxic elements such as cadmium (cd) and lead (pb), as well as essential elements in which toxicity is determined by the dose, as copper (cu) and zinc (zn). Although these four elements occur naturally in the environment, they are supplied in large quantities through domestic and industrial pollution (bjerregaard and andersen 2011). Heavy metals are also persistent environmental contaminants whose physiochemical properties contribute to their mobility in the natural environment, posing a significant threat for living organisms and ecosystems (bjerregaard and andersen 2011). Previous studies reported essential and toxic metals in beaver (family: castoridae) tissues obtained from industrial areas, generally regarded as polluted (hillis and parker 1993; zalewski et al . 2012), agricultural regions (fimreite et al . 2001), and natural ecosystems (wren 1984; zalewski et al . 2012; giejewska et al . Beavers are semi - aquatic, territorial herbivorous rodents feeding on the bark, shoots, and leaves of a wide variety of woody plants including willow (salix sp .) And aspen (populus tremuloides), as well as on non - woody terrestrial and aquatic plants (haarberg and rosell 2006; krojerov - prokeov et al . Beavers catholic diet and sedentary behavior make them suitable bioindicators of local environmental pollution . In poland, the status of eurasian beavers (castor fiber) as a partially protected species (polish minister of the environment 2011) contributed to rapid population growth . It is currently estimated that 78,000 beavers occur in the country, and beaver populations continue spreading to new areas, including near human settlements, where they are likely to get exposed to anthropogenic pollutants (flis 2013). In this study, our aim was to use the eurasian beaver as a bioindicator organism (i) to assess trace element contents in a region away from major industrial centers and (ii) to evaluate the degree of contamination against legal cd and pb levels recommended by the european commission in farm animal edible tissues (european commission 2006) with regard to the lack of an official threshold for wildlife (giejewska et al . 2014). Specifically, we measured concentrations of cd, pb, cu, and zn in liver and kidney of wild beavers . We predicted (i) metal concentrations to differ between liver and kidney according to element - specific metabolism, (ii) no sexual difference in metal concentrations in this non - dimorphic species, and (iii) higher metal concentrations in adult than in juvenile individuals because of bioaccumulation processes . We compared our results with previous research, and we discuss the implications for future use of beavers as bioindicators for the monitoring of metal pollution . N), warmia and mazury region, northeastern poland, in summer 2012 . Warmia and mazury is a traditional agricultural region with extensive forests and numerous lakes . The region is an important recreational center for local and international tourists who seek activities in a natural environment . The sampling site was away from a major industrial complex, which could putatively be a source of pollutants in their vicinity . Animals were collected upon approval of the regional nature conservation authority (opinion no . Rdo-28-oop-6631 - 0007 - 638/09/10/pj 2010) and the local ethics committee (opinion no . All beavers were collected during daytime with a net and transported to the laboratory in cages specially adapted for this purpose (zalewski et al . 2010), and they were classified into two age classes: (i) juveniles, 2 months old, and (ii) adults,> 24 months old (table 1). Sex was determined based on the color of anal gland secretions (schulte et al . Beavers were anesthetized with xylazine and ketamine in doses appropriate to body weight, and body measurements taken.table 1biometric data of 10 eurasian beavers from wiajny municipality, northeastern poland, 2012individualsexage classbody weight (kg)body length (cm)m1juvenile2.562m2juvenile2.862f3juvenile2.864f4juvenile2.760m5adult18.6116m6adult22.6114f7adult17.3115f8adult21.1117f9adult22.7118f10adult22.0121 biometric data of 10 eurasian beavers from wiajny municipality, northeastern poland, 2012 then, beavers were sacrificed by decapitation under full anesthesia . Liver and kidney samples of c. 15 g each were collected and stored in polyethylene bags at 25 c until analysis (zalewski et al . Each sample was homogenized and two subsamples were prepared from each individual organ and analyzed twice each as replicates . All tissue samples were mineralized in the muffle furnace at 450 c for 24 h. ashes were dissolved in 1 m hno3 and deionized water was added to 25 ml (uczyska et al . We performed graphite furnace atomic absorption spectroscopy to determine cd and pb concentrations and flame atomic absorption spectroscopy to determine cu and zn concentrations (whiteside and milner 1984) using an ice 3000 series spectrometer (thermo scientific, uk). Analysis for each element was performed based on calibration curves plotted from working standards . For calibration, we used commercial stock solutions (1000 mg working standards were prepared by dissolving stock solutions with 0.1 m hno3 and deionized water . Calibration ranges were 0.00050.004 g / g for cd, 0.0010.01 g / g for pb, 0.050.8 g / g for cu, and 0.10.8 g / g for zn, respectively . Limits of detection were 0.002 g / g for cd, 0.001 g / g for pb, and 0.05 g / g for cu and zn . Quality control of analytical measurements was performed using certified reference material (bcr-185r bovine liver, irrm, belgium). Recoveries of all four elements were within the range 86109% and relative standard deviations were below 10% . Metal concentrations were expressed in micrograms per gram of wet weight, with precision consistent with limits of quantitation0.001 g / g for cd and pb and 0.1 g / g for cu and zn . Concentrations in beaver livers and kidneys were compared between age or sex classes using a non - parametric mann - whitney u test . Pair - wise comparisons between element concentrations in each organ were performed using a spearman rank correlation analysis . We report arithmetic means, standard deviations (sd), median, and range . Where necessary, data were transformed to meet statistical assumptions . Significance level was set at p <0.05, and all statistical analyses were run using r version 3.0.0 (r core team 2013). We captured six female (four adults, two juveniles) and four male (two adults, two juveniles) beavers (table 1). Mean concentrations of cd, pb, cu, and zn determined in liver and kidney samples are presented in table 2 and compared with the literature (table 3). Higher concentrations in liver than in kidney were found for cu (t = 4.534; df = 9; p = 0.001) and zn (t = 19.826; df = 9; p <0.001). Additionally, zn levels were significantly correlated to cd levels in kidney (n = 10, rs = 0.939; p <0.001).table 2summary statistics of the concentration (g / g wet weight) of four metals (cd, pb, cu, and zn) in liver and kidney of 10 eurasian beavers, ne poland, 2012metalmean sdmedianrangemaximum legal level liver cd0.21 0.440.060.031.440.5 pb0.08 0.030.060.050.120.5 cu9.2 4.56.95.216.4 zn35.7 3.536.231.440.6 kidney cd2.81 4.521.370.0214.881.0 pb0.08 0.030.070.050.140.5 cu3.7 1.13.53.03.8 zn21.5 2.720.219.027.9 maximum levels set by the european commission (2006) for contaminants of toxic elements in food of animal origin no regulationtable 3average concentrations (g / g wet weight) of cd, pb, zn, and cu in liver and kidney of beavers (family: castoridae) given in the literaturespeciesliverkidneysitesreferencecdpbcuzncdpbcuzn c. fiber 0.210.089.235.72.810.083.721.5unpollutedthis study c. fiber 0.880.117.930.06unpollutedgiejewska et al . (2001) c. canadensis 2.32.7880.818.83.49.399.315 km from ore smelterhillis and parker (1993) c. canadensis 1.42.190 km from ore smelterhillis and parker (1993) c. canadensis 0.192.929.61.44325.4175 km from ore smelterwren (1984) summary statistics of the concentration (g / g wet weight) of four metals (cd, pb, cu, and zn) in liver and kidney of 10 eurasian beavers, ne poland, 2012 maximum levels set by the european commission (2006) for contaminants of toxic elements in food of animal origin average concentrations (g / g wet weight) of cd, pb, zn, and cu in liver and kidney of beavers (family: castoridae) given in the literature we found no differences between female (n = 6) and male (n = 4) beavers for any of the four metal concentrations in liver or kidney (fig . 1). Contrastingly, we found significant differences in cd and cu concentrations between adult and juvenile beavers (fig . 2). Cadmium concentrations were significantly higher in adults (n = 6) than in juveniles (n = 4) in both liver (u = 0.0; z = 2.452; p = 0.014) and kidney (u = 0.0; z = 2.452; p = 0.014). In liver, cu levels were significantly lower in adults than in juveniles (u = 0.0; z = 2.452; p = 0.014), and in kidney, cu levels were significantly higher in adults than in juveniles (u = 2.0; z = 2.025; p = 0.043).fig . 1concentrations (g / g wet weight) of cd, pb, cu, and zn in a liver and b kidney of six female and four male eurasian beavers, ne poland, 2012 . No statistical significant difference in metal concentrations between sex classes was found (mann - whitney u test, all p> 0.5). Box and whisker plots show median (horizontal line within box), 25 and 75% percentiles (box), 1.5 interquartile range (whiskers). For clarity, extreme outliers 2concentrations (g / g wet weight) of cd, pb, cu, and zn in a liver and b kidney of six adult and four juvenile eurasian beavers, ne poland, 2012 . Significant statistical differences in metal concentrations between age classes were found in liver cd (u = 0.0; z = 2.452; p = 0.014) and cu (u = 0.0; z = 2.452; p = 0.014) and in kidney cd (u = 0.0; z = 2.452; p = 0.014) and cu (u = 2.0; z = 2.025; p = 0.043). Box and whisker plots show median (horizontal line within box), 25 and 75% percentiles (box), 1.5 interquartile range (whiskers), and statistical outliers (open circles). For clarity, extreme outliers are not shown concentrations (g / g wet weight) of cd, pb, cu, and zn in a liver and b kidney of six female and four male eurasian beavers, ne poland, 2012 . No statistical significant difference in metal concentrations between sex classes was found (mann - whitney u test, all p> 0.5). Box and whisker plots show median (horizontal line within box), 25 and 75% percentiles (box), 1.5 interquartile range (whiskers). For clarity, extreme outliers are not shown concentrations (g / g wet weight) of cd, pb, cu, and zn in a liver and b kidney of six adult and four juvenile eurasian beavers, ne poland, 2012 . Significant statistical differences in metal concentrations between age classes were found in liver cd (u = 0.0; z = 2.452; p = 0.014) and cu (u = 0.0; z = 2.452; p = 0.014) and in kidney cd (u = 0.0; z = 2.452; p = 0.014) and cu (u = 2.0; z = 2.025; p = 0.043). Box and whisker plots show median (horizontal line within box), 25 and 75% percentiles (box), 1.5 interquartile range (whiskers), and statistical outliers (open circles). For clarity, extreme outliers our study revealed the presence of cd, pb, cu, and zn in liver and kidney of all sampled beavers . No relationship was observed between metal concentrations and the beavers sex . Although we acknowledge the limitations of the tests due to a reduced number of individuals in each group, these results were expected as the species present no sexual dimorphism and no partitioning in diet (haarberg and rosell 2006; krojerov - prokeov et al . Contrastingly, some metal concentrations were affected by the animals age . Higher liver and kidney cd concentrations were found in adult beavers than in juveniles, indicating that this metal bioaccumulates over time (parker and hamr 2001; bilandi et al . Cd levels in all six adult beavers exceed the maximum threshold of 1.0 g / g in kidney recommended in farm animal edible tissues . In liver, however, only one adult female (f7) exceeded the recommended 0.5 g / kg cd limit (european commission 2006). Such differences in cd levels with age might be due to this metal accumulating over an animal s entire life span, especially in kidney, as previously described in other species (e.g., red fox (vulpes vulpes) and stone marten (martes foina): bilandi et al . 2010; african grass owl (tyto capensis): ansara - ross et al . 2013; penguins (pygoscelis spp . ): jerez et al . Average cd content in kidney was similar to that observed in 2003 in beavers from srokowo forest division, an uncontaminated area in northeastern poland (zalewski et al . 2012). However, our results were approximately four- to fivefold lower in liver and three- to fourfold lower in kidney than levels measured in earlier studies on beaver at unpolluted sites in poland (giejewska et al . 2014), in norway (fimreite et al . 2001), and in a putatively contaminated former air base in poland (zalewski et al . Contrastingly, cd concentrations in liver and kidney were higher than those reported in canadian beavers (castor canadensis) in habitats located 175 km from an ore smelter in ontario, canada (wren 1984). Cadmium concentration in wildlife tissue is attributed to its levels in the environment and additionally with animals age . Previous studies concerned with cd content in unpolluted areas presented values that could be attributed to metal transport over long distances from the pollution source as well as increased mobility due to soil and water acidification (wren 1984). Although metal concentrations in food sources typically consumed by beavers were not examined in the present study, it has been reported that beavers have a strong preference for aspen (p. tremuloides) and willow (salix sp . ), trees that accumulate high levels of cd particularly on acidic soils that increase the mobility of heavy metals (wren 1984). Cd is easily absorbed and accumulates even at low levels of chronic exposure (nordberg et al . Interspecific differences, intraspecific variations among populations, or physiological determinants of metal absorption and excretion can also contribute to the observed differences (nordberg et al . All values of pb found in beavers were below the 0.5 g / g threshold recommended for both organs in farm animal edible tissues (european commission 2006). Lead concentrations in kidney ranged from 0.052 g / g (f9) to 0.142 g / g (f10). Lead passes the placenta and young animals have a higher rate of absorption from the intestinal tract (nordberg et al . As this metal is not metabolically regulated in organisms, pb concentrations in animal tissues are related to its presence in the ecosystem (jerez et al . Soil intentionally or accidentally consumed by beavers could constitute an additional source of contamination as pb can be bound in soil and bottom sediments and secondarily released (rajaganapathy et al . Lead concentrations in our study were low compared to previous research (table 3). This could be attributed to the absence of major industrial sites close to the sample site, seasonality, or individual variability in diet (brekken and steinnes 2004). Copper absorption is regulated by homeostatic mechanisms in the liver and is mainly excreted through the bile . Absorbed zn is bound to plasma albumin and macroglobulins and distributed to the liver where it rapidly accumulates (parker and hamr 2001). Although cu has higher affinity than zn to metallothionein (low molecular weight metal - binding protein) which binds cu and zn at low levels of cd (hillis and parker 1993; nordberg et al . 2011a), hepatic metallothionein seems to have an important role in accumulation and storage of both cu and zn in the liver (parker and hamr 2001). Copper concentrations were higher in juvenile than in adult beavers in liver, indicating age - specific metabolism capabilities of this microelement (nordberg et al . Higher cu levels in juveniles liver can be attributed to increased requirements for this element in growing organisms (parker and hamr 2001; jerez et al . Our cu values in both liver and kidney were higher than those in previous studies (table 3). Copper as an essential microelement has effective regulation mechanisms of uptake and excretion, and beaver diet contains high amounts of cu and zn (brekken and steinnes 2004). Therefore, we assume that our values were at normal physiological levels (nordberg et al zinc concentrations did not differ between organs and sex or age classes in our study . However, excluding beavers collected in the vicinity of the ore smelter in ontario, canada, where zn concentrations reached 80.8 g / g in the liver (hillis and parker 1993), we found zn levels in liver higher than in previous studies (table 3). In kidney, zn levels were highly correlated to cd levels regardless of the beaver age or sex . Zinc and cadmium have similar chemical properties, and they are usually found as a mineral combination in the environment (nordberg et al . Metallothionein can bind cd, cu, and zn, and this protein will bind most of the cd and store it in kidney . Exposure to low cd levels may cause a redistribution of zn in the organism, increasing zn concentrations in kidney (nordberg et al . Higher kidney zn concentrations were reported in beavers from ontario (hillis and parker 1993). The highest zn concentrations (liver 40.6 g / g; kidney 27.9 g / g) we found in adult female f7 could also possibly be attributed to a diet rich in this microelement (brekken and steinnes 2004). General metal concentrations in beaver tissues from northeastern poland were similar to or lower than those found in other countries . However, the fact that high cd concentrations and pb were present in a putatively unpolluted area calls for a regular monitoring of environmental pollutants in agricultural and natural areas . Future investigation of trace elements in beaver tissues sampled from industrial regions is needed for comparison and to draw further conclusion about levels of contamination . Significant expansion of beaver populations in poland contributes to growing human - beaver conflict, increasing the amount of compensation paid to farmers and landowners . Although beaver is currently not considered as a consumptive species in poland, beaver is classified as a game species in six european countries (belarus, estonia, lithuania, latvia, norway, and sweden) as well as in canada and russia (jankowska et al . Hunting beavers for consumption has been suggested as a tool for population management, and beaver consumption could occur in the near future in poland . Research to ascertain tissues are safe from a toxicological point of view is therefore timely . In this context, the fact that cd concentrations in adult beavers exceeded limits recommended in farm animal tissues warrants caution . Also, it would be necessary to investigate levels of persistent organic pollutants (pops) in beaver tissues . Pops are hydrophobic and lipophilic persistent compounds that may accumulate along the food chain and can cause severe metabolic disturbance, such as endocrinal disturbance (jones and de voogt 1999). The presence of pops in remote areas such as the arctic is a major environmental issue (brault et al . Consequently, we recommend future research and regular monitoring to identify the source of contaminants in the ecosystem and possible mitigation measures, and to ensure that contamination levels are within a safe range.
Parturient with congenital heart block may be asymptomatic but can present with sudden vascular collapse, especially during labour . Few patients with congenital heart block may have sudden cardiac death (scd) for which there are no predictors . We present our experience of spinal anaesthesia in a 29-year - old female with congenital complete heart block for lower segment caesarean section (lscs). A 29-year - old parturient (gravida 3, para 0, abortion 2) was admitted to our hospital with 9 months of amenorrhea . She had undergone appendicectomy 8 years back under spinal anaesthesia, and the procedure was uneventful . Past obstetric history revealed two abortions and an episode of perioperative seizures during intravenous sedation for medical termination of pregnancy . The episode was associated with bradycardia non - responsive to atropine, during which she was evaluated as having a congenital complete heart block . She had been prescribed orciprenaline by a local practitioner and had received orciprenaline 10 mg bd for 2 months preceding the present pregnancy and till 10 days before presenting to the hospital . It was stopped by our cardiologist as it does not help to increase heart rate in complete heart block . Her general condition was stable; pulse rate was 46/min and blood pressure (bp) was 110/70 mmhg . Clinically, cardiorespiratory and central nervous system examinations were normal . Per - abdomen examination showed a foetus in cephalic presentation, uterine height at 38 weeks, foetal heart rate 136 beats / min and regular with clinical and ultrasonographic evidence of reduced liquor . Electrocardiography showed a complete heart block with an atrial rate of 80/min, ventricular rate of 46/min and a narrow qrs complex [figure 1]. She was accepted for anaesthesia under asa ii and was explained about the anaesthetic technique . Patient was kept nil oral for 8 h. tab . Metoclopramide 10 mg the next morning 2 h before the surgery . On the morning of the surgery, the patient was taken for cardiac catheterization in supine position with a wedge under the right buttock and temporary pacemaker insertion (ventricular, ventricle inhibition the position of the lead in the right ventricle was confirmed by fluoroscopy, with a lead shield cover over the abdomen of the patient . A pacemaker can also be inserted using electrocardiographic and echocardiographic guidance to avoid foetal exposure to ionizing radiation, but our cardiologist could not get enough window to do the same . Immediately after the procedure, a spinal anaesthesia was given in the l3-l4 interspace with a total of 1.5 ml, which is a combination of a 1.0 ml hyperbaric 0.5% bupivacaine with 0.5 ml fentanyl (25 g). Intra - operatively, the first episode of hypotension after the spinal anaesthesia was treated by increasing the pacing rate to 70 beats / min and the second episode was treated by 3 mg of intravenous (iv) ephedrine . A healthy male baby weighing 2.5 kg was born with an apgar score of 8/10 in the 1 minute and 9/10 in the 5 minute . Post - operatively, the pacemaker rate was changed to 60 beats / min and the patient was shifted to the post - operative ward for continuous monitoring . Post - operative pain relief was achieved with iv tramadol 50 mg and diclofenac 75 mg iv alternatively 8 hourly . The temporary pacemaker was removed after 24 h and the patient was haemodynamically stable after the removal . Her post - natal period was uneventful and she was discharged on the 5 day with an advice for the placement of permanent pacemaker as early as possible . Heart block may be congenital or acquired . Congenital heart block may occur alone or in association with other cardiac abnormalities . Conversely, in isolated complete heart block, 8590% of all births live beyond the neonatal period, even up to late adulthood . If congenital complete heart block occurs alone, then it is relatively benign, as the block to conduction is at the level of the av node . The ventricular pacemaker is proximal to the bifurcation of the bundle of his, and therefore the qrs complexes are narrow, and the ventricular conduction system intact . The rate is relatively high and can vary from 40 to 80 beats / min, and may increase with exercise, atropine or sympathomimetics . Acquired heart block in children or early adulthood is mostly secondary to cardiac surgery involving closure of perimembranous or infundibular ventricular septal defect (vsd) or muscle bundle resection near the conduction tissues, but can occur as an isolated condition also . In the chronic type, the atrio ventricular (av) junction or bundle branches are usually involved, the qrs complexes are wide and the heart rate is lower and is not increased by exercise or atropine . Prophylactic placement of a pacemaker is not indicated in an asymptomatic pregnant patient with complete heart block as it does not cause unusual problems. [35] if the patient is symptomatic during her first and second trimesters, then the placement of a permanent pacemaker is indicated . We used temporary pacing in this patient because the patient's heart rate was resistant to exercise and atropine, and the same cannot adapt to her changing bp . Increase in the heart rate during labour is essential to increase in the cardiac output and to maintain the haemodynamics, which is not possible in the patient . Hence, for a safe delivery, temporary pacemaker insertion was essential, which ideally we did before operative delivery . There are quite a few anaesthetic problems in patients with complete heart block undergoing incidental surgeries . The anaesthetic technique that least alters the cardiac stability should be wisely planned and executed for the procedure . General anaesthesia carries a potential risk to these patients because both the inhalational and the intravenous agents alter the haemodynamics to such an extent to put them in peril. [68] if general anaesthesia is planned, drugs with minimal effects in depressing the heart rate have to be preferred, such as ketamine for induction, pancuronium for relaxation and isoflurane for maintenance . Combined spinal epidural is another option, but we did not opt for it due to cost constraints and brevity of surgery . Modi et al . Successfully managed such a case with the epidural anaesthetic technique . In our patient, we opted for intrathecal opioids, especially fentanyl, which gives adequate anaesthesia with minimal effects on the cardiovascular system . We added 25 g of fentanyl with almost half the usual dose of hyperbaric 0.5% bupivacaine . We did use 3 mg of vasopressor ephedrine, but such a single low dose to maintain adequate haemodynamic stability was found satisfactory to us . We did insert a temporary pacemaker to compensate for any possible haemodynamic eventuality that can occur during anaesthesia, and the immediate post - partum period . The pacemaker rate was set at 50/min in order to preserve her native rhythm till her blood pressure is maintained in the intra - operative period . Long - term pacing suppresses the native rhythm, and our intention was to avoid it . Also, the fluoroscopy time for positioning the lead was minimized to less than 10 s. the patient was discharged with an advice for permanent pacemaker implantation (ppi). As per the recent guidelines, ppi is indicated for all congenital complete heart blocks (class 2a / b) as trials have shown that there is a subgroup of patients who may have scd for which there are no predictors available . To conclude, we successfully managed a case of congenital complete heart block for operative delivery with temporary pacemaker in situ with intrathecal low - dose bupivacaine
Ocular surface is protected by tears, and tear abnormalities or ocular surface disorders could result in dry eye syndrome (des). Des is a common and complex condition with reduced ocular comfort and impaired visual performance . Nowadays, des is regarded as one of the most common ocular disorders throughout the world . The treatment of des is based on local application of eye drops, including artificial tears and anti - inflammatory agents . Although most des patients treated with eye drops will feel more comfortable, it is hard to cure des . In addition, for some individuals, there are no satisfactory therapeutics that can provide relief from des ocular discomfort symptoms . Thus, better understandings of the risk factors and pathogenesis of des would help in primary prevention of des and effective therapy development . The generally recognized risk factors for des included elder age, reproductive factors, tobacco smoking, contact lenses use, ocular surgery, systemic diseases, and dry environment contact . Better understandings of risk factors and related lifestyle changes of des patients can be helpful in the des related discomforts management . Vitamin d is a fat soluble vitamin that is involved in a number of physiological and pathological processes . Vitamin d formulations have been widely used in different diseases and have been reported to be effective in the treatment or prevention of osteoporosis, cancers, and cardiovascular disorders [1012]. In advanced in - vivo and in - vitro studies, vitamin d has been reported to enhance immunity, relieve inflammatory reaction and regulate cell cycles [1315]. The application of vitamin d in treatment of ocular surface disorders has been reported . In a previous study, it was suggested that there is a possible role for vitamin d in modulating corneal wound healing, thus have important implications for therapeutic use of vitamin d at the ocular surface . In another study based on mouse, rabbit, and human samples, it was determined that 25-hydroxyvitamin d[(3)(25(oh)d(3)] and/or its active metabolite, 1,25-dihydroxyvitamin d[(3)(1,25(oh)(2)d(3)] can enhance corneal epithelial barrier function . Several epidemiological studies were conducted to evaluate the association between vitamin d or vitamin d deficiency and incidence rate of des [1821], however, no conclusions were reached . A cross - sectional study that included 17,542 adults from korea found that lower serum 25(oh)d levels were associated with incidence rates of des . However, in another cross - sectional study containing male patients, it was reported that vitamin d levels were not significantly associated with the presence or severity of des . The purpose of our case - control study was to determine the effect of serum 25(oh)d on des incidence . The study was also designed to determine whether serum 25(oh)d levels were associated with ocular parameters of des patients . Ethical approval of this current study was obtained from the human research ethics committee of changshu no 2 people s hospital . In this study, a total of 70 des patients and 70 matched controls were included in this study . All the des patients were diagnosed from may 2015 to january 2016 at the out - patient clinic of the department of ophthalmology, changshu no 2 people s hospital, changshu, people s republic of china . The inclusion criteria were: (1) matched to the diagnosis criteria of des, (2) aged over 18 years, and (3) signed the informed consents before inclusion in the study . The exclusion criteria contained: (1) unable to co - operate in the study handling, (2) sjogren syndrome, rheumatoid arthritis, lupus erythematosus, or any other immune diseases; (3) mental disorder or serious systemic disease, such cancer, hematological system disease, or hyperthyroidism; (4) pregnancy or breast - feeding in women; (5) participation in other ophthalmic clinical trials; (6) other ocular disease, such as ocular surgery history within the recent six months, use of any ophthalmic eye drops or contact lenses within recent one month, eyelid or eyelash abnormalities, nasolacrimal apparatus abnormalities glaucoma, uveitis, retinal hemorrhage, or optic neuritis . Patients with des were diagnosed using experts consensus about clinical diagnosis and treatment of dry eye (2013). The diagnostic criteria includes: (1) subjective symptoms of dryness, foreign body sensation, burning sensation, fatigue, discomfort, visual acuity fluctuation, and tear film breakup time (tbut) test 5 seconds or schirmer i test (without surface anesthesia) 5 mm/5 minutes; (2) the subjective symptoms of ocular discomfort and tbut test 510 seconds or schirmer i test (without surface anesthesia) 510 mm/5 minutes combined with corneal fluorescein staining positive . Clinical data, including body mass index (bmi, kg / m), smoking history, diabetes status, and blood pressure were obtained through questioning of the participants or reviewing their medical records . The ocular surface disease index (osdi) quantitation was conducted by trained investigators . The osdi scale questionnaire included 12 questions, containing three aspects of eye symptoms, visual function, and environmental stimulation . Detailed information was collected from each participant regarding ocular dryness, foreign body, visual fatigue, and other subjective discomfort symptoms . The response grades were divided into no symptoms (0 point), occasional presence (0.5 points), intermittent (1 point), and persistent discomfort (2 point). The examinations were conducted in the following order: tbut, keratoepitheliopathy examination, and schirmer i test . All the clinical data was collected and recorded in electronic tables . The longer half - life of 25(oh)d, allows it to remains stable in serum and as such it can be used to reflect the level of vitamin d. in this study, 25(oh)d was chosen as a main study parameter . Peripheral blood samples from each participant were collected and serum samples were frozen at 70c until 25(oh)d detection was conducted . Serum 25(oh)d levels were measured by a reverse phase liquid chromatography method (agilent technologies, ca, usa). The accuracy quality control of low, medium, and high quality was to be 85~115% with the accuracy value within 15% . The statistical analysis was performed using package for spss version 17.0 software (spss inc ., the data of each index is shown as the mean standard deviation (sd). Comparison of discontinuous variable parameters was done using the chi - square test or fisher s exact test . The differences in the continuous variables were detected using non - paired t - test . Pearson correlation analysis was used in the detection of the associations between different continuous variables . A total of 70 des patients and 70 matched controls were included in this study . All the des patients were diagnosed from may 2015 to january 2016 at the out - patient clinic of the department of ophthalmology, changshu no 2 people s hospital, changshu, people s republic of china . The inclusion criteria were: (1) matched to the diagnosis criteria of des, (2) aged over 18 years, and (3) signed the informed consents before inclusion in the study . The exclusion criteria contained: (1) unable to co - operate in the study handling, (2) sjogren syndrome, rheumatoid arthritis, lupus erythematosus, or any other immune diseases; (3) mental disorder or serious systemic disease, such cancer, hematological system disease, or hyperthyroidism; (4) pregnancy or breast - feeding in women; (5) participation in other ophthalmic clinical trials; (6) other ocular disease, such as ocular surgery history within the recent six months, use of any ophthalmic eye drops or contact lenses within recent one month, eyelid or eyelash abnormalities, nasolacrimal apparatus abnormalities glaucoma, uveitis, retinal hemorrhage, or optic neuritis . Patients with des were diagnosed using experts consensus about clinical diagnosis and treatment of dry eye (2013). The diagnostic criteria includes: (1) subjective symptoms of dryness, foreign body sensation, burning sensation, fatigue, discomfort, visual acuity fluctuation, and tear film breakup time (tbut) test 5 seconds or schirmer i test (without surface anesthesia) 5 mm/5 minutes; (2) the subjective symptoms of ocular discomfort and tbut test 510 seconds or schirmer i test (without surface anesthesia) 510 mm/5 minutes combined with corneal fluorescein staining positive . Clinical data, including body mass index (bmi, kg / m), smoking history, diabetes status, and blood pressure were obtained through questioning of the participants or reviewing their medical records . The osdi scale questionnaire included 12 questions, containing three aspects of eye symptoms, visual function, and environmental stimulation . Detailed information was collected from each participant regarding ocular dryness, foreign body, visual fatigue, and other subjective discomfort symptoms . The response grades were divided into no symptoms (0 point), occasional presence (0.5 points), intermittent (1 point), and persistent discomfort (2 point). The examinations were conducted in the following order: tbut, keratoepitheliopathy examination, and schirmer i test . The longer half - life of 25(oh)d, allows it to remains stable in serum and as such it can be used to reflect the level of vitamin d. in this study, 25(oh)d was chosen as a main study parameter . Peripheral blood samples from each participant were collected and serum samples were frozen at 70c until 25(oh)d detection was conducted . Serum 25(oh)d levels were measured by a reverse phase liquid chromatography method (agilent technologies, ca, usa). The accuracy quality control of low, medium, and high quality was to be 85~115% with the accuracy value within 15% . The statistical analysis was performed using package for spss version 17.0 software (spss inc ., the data of each index is shown as the mean standard deviation (sd). Comparison of discontinuous variable parameters was done using the chi - square test or fisher s exact test . The differences in the continuous variables were detected using non - paired t - test . Pearson correlation analysis was used in the detection of the associations between different continuous variables . Basic clinical characteristics of patients with des and healthy controls are presented in table 1 . In this study, 70 des cases (43 males and 27 females) and 70 matched controls (36 males and 34 females) were included . The mean bmi in the des group and the control group was 23.24.4 kg / m and 23.65.1 kg / m, respectively . There were no differences in smoking status, diabetes prevalence, and blood pressure between the des group and the control group . When the schirmer i test was considered, the des group (9.43.9 mm/5 minutes) demonstrated a significantly lower value compared with the control group (13.95.3 mm/5 minutes, p<0.001). In addition, a significantly reduced tbut was detected in the des cases (des group, 6.12.4 second; control group, 13.43.8 seconds, p<0.001). Compared with the control group, the mean osdi value was significantly higher in the des group (p<0.001). The serum 25(oh)d levels in patients with des and the control patients are shown in table 2 . Serum 25(oh)d levels were significantly lower in the des group than in the healthy control group (des group, 19.35.8; control group, 31.67.3, p<0.001). Furthermore, serum 25(oh)d levels in both male and female patients were lower compared with the healthy controls (p<0.05). When the participants were divided into different vitamin d status (25(oh)d, <10 ng / ml, 10~20 ng / ml, 20~30 ng / ml, and 30 ng / ml), deficiency of vitamin d was more common in the des group (p<0.001). Considering that serum 25(oh)d level was significantly lower in the des group, we conducted additional analyses on the association between serum 25(oh) d levels and des parameters . Through pearson correlation analysis, it was found that serum 25(oh)d level was associated with increased schimer test i (r=0.8248, p<0.001). In addition, there was an inverse correlation between serum 25(oh)d and odsi scores (r=0.3348, p=0.005) and tbut (r=0.6806, p<0.001). Basic clinical characteristics of patients with des and healthy controls are presented in table 1 . In this study, 70 des cases (43 males and 27 females) and 70 matched controls (36 males and 34 females) were included . The mean bmi in the des group and the control group was 23.24.4 kg / m and 23.65.1 kg / m, respectively . There were no differences in smoking status, diabetes prevalence, and blood pressure between the des group and the control group . When the schirmer i test was considered, the des group (9.43.9 mm/5 minutes) demonstrated a significantly lower value compared with the control group (13.95.3 mm/5 minutes, p<0.001). In addition, a significantly reduced tbut was detected in the des cases (des group, 6.12.4 second; control group, 13.43.8 seconds, p<0.001). Compared with the control group, the mean osdi value was significantly higher in the des group (p<0.001). The serum 25(oh)d levels in patients with des and the control patients are shown in table 2 . Serum 25(oh)d levels were significantly lower in the des group than in the healthy control group (des group, 19.35.8; control group, 31.67.3, p<0.001). Furthermore, serum 25(oh)d levels in both male and female patients were lower compared with the healthy controls (p<0.05). When the participants were divided into different vitamin d status (25(oh)d, <10 ng / ml, 10~20 ng / ml, 20~30 ng / ml, and 30 ng / ml), deficiency of vitamin d was more common in the des group (p<0.001). Considering that serum 25(oh)d level was significantly lower in the des group, we conducted additional analyses on the association between serum 25(oh) d levels and des parameters . Through pearson correlation analysis, it was found that serum 25(oh)d level was associated with increased schimer test i (r=0.8248, p<0.001). In addition, there was an inverse correlation between serum 25(oh)d and odsi scores (r=0.3348, p=0.005) and tbut (r=0.6806, p<0.001). In this study, it was shown that 25(oh)d levels were lower in patients with des than in healthy controls . When the 25(oh)d levels was stratified, vitamin d deficiency was more common in the des cases . In further analysis, it was found that there were statistical significant associations between serum 25(oh) d levels and the schimer test, tbut value and osdi scale . Des is a multifactorial disorder and various risk factors were associated with the incidence of des . Nowadays, there is more interest in the association between vitamin d and des cases . The major conclusions of this case - control study were consistent with previous cross - sectional study conclusions, in that low serum 25(oh)d levels were associated with des . In previous cross - sectional data analysis, it was found that low serum 25(oh)d levels were associated with des in korean adults . These results provide clues that vitamin d supplementation might be used in des treatment . In another case - control study with 34 patients with serum vitamin d deficiency and 21 control patients with normal vitamin d levels, it was found that vitamin d deficiency decreases the tbut and schirmer test values . In our study, significant lower vitamin d level was detected in des patients and it was found that significant associations were detected in both male and female groups . In addition, the advanced correlation analysis results showed more interesting and meaningful association between serum 25(oh) d and severity and ocular discomfort in des cases . Rather than considering vitamin d deficiency as a risk factor for des as in a previous study, more attention was focused on the association between vitamin d and ocular physical examination and indisposed symptoms in this current study . Positive correlation between serum vitamin d level and des severity suggests there is the potential for application of vitamin d for treatment or prevention of des . Ass shown in the first study on the effect of vitamin d on the des in chinese population, this study provides additional knowledge on the effect of vitamin d on the des development . However, even though the association of vitamin d and des was detected in different studies, the study of the detailed mechanism was lacking . There were several possible explains for the effect of vitamin d on the des development . It has been recognized that systemic autoimmune diseases or immune abnormalities could result in the increased risk of des . Considering the remarkable effect of vitamin d on the immune system, it could be conjectured that the immunoregulatory effect of vitamin d might influence the development of des . For instance, in a case - control study with 107 sjogren syndrome (ss) patients and 74 healthy controls, plasma vitamin d levels in ss patient group were significantly lower than in the control group . As dry eye was one of the most frequent symptoms of ss, and vitamin d deficiency was frequent found in patients with ss, vitamin d might be associated with the ss - related des incidence . Besides, there were several previous studies that reported on the associations between des and systemic autoimmune disorders, including rheumatism and lupus erythematosus . Abnormal autoimmune status might result in a higher incidence of des and the protective immunomodulatory effect of vitamin d in the regulation of immune cells might help in the prevention of des incidence . There was a previous study that reported that vitamin d deficiency decreased the tbut and schirmer test values and may be associated with dry - eye symptoms in non - sjogren syndrome, thus suggesting a potential mechanism beyond the autoimmune effect of vitamin d may exist . Chronic inflammation induced by autoimmune disorders or environmental stimulating factors in ocular surface cells has been considered to be involved in des . For instance, vitamin d could decrease the inflammatory response of cancer and inhibited the proliferation of cancer cells in - vitro . Considering that inflammation was one of the key pathogenesis and phenotype of des, higher level vitamin d would decreased the inflammatory response in the ocular surface and then improve the severity of des . The production of vitamin d in the metabolic system is from two main sources . In general, vitamin d is mainly produced by cutaneous synthesis upon skin exposure to ultraviolet light and partial vitamin d is gained from food sources . Because of the short half - life of 1,25(oh)d (57 days) and the long half - life of 25(oh)d (20~30 days), the serum 5(oh)d level is commonly used as the indicator of vitamin d status and it was used in the present study . Vitamin d receptor (vdr) is a member of the nuclear hormone receptor superfamily and plays an important role in vitamin d function . In a case - control study of 64 des cases and 51 controls, the single nucleotide polymorphisms (snps) in the vdr genes also varied between des cases and controls . Thus, the influence of snps in the vitamin d related gene might explain the association between serum vitamin d and des incidence . A major strength of our study was that the method used to assess serum vitamin d levels was sensitive and therefore might produce more robust conclusions . First, the sample size of this study was relatively small and it has been demonstrated that larger sample sizes are requirements for advanced study analysis . Considering that this was a hospital - based study, it was hard to include a large sample size in the participant selection process . Additionally, population - based studies are required for the detection of the association between vitamin d and des status . Second, even though 5(oh)d is the most commonly used index for vitamin d detection, there are other indices reflecting the intracellular effect of vitamin d that were not reported in this study . The association of these related indices might provide additional knowledge to aid in our understanding of the mechanism of vitamin d on des . Future prospective studies were needed to assess other serum markers of vitamin d in addition to 25(oh)d in the des cases . In advanced studies, the effect of dietary vitamin d intake, vitamin d supplementation, and ultraviolet radiation exposure on the development of des should be examined . A third limitation of our study was that that using a case - control study design, selection bias could influence the conclusions . A significant association between serum 25(oh)d level and des incidence was detected in this study . In addition, serum 25(oh)d was associated with the des parameters and ocular discomfort status . It could be conjectured that vitamin d might be a potentially favorable adjunctive option for patients with des . Considering the relatively small sample size of this study, larger studies with longer follow - up duration are needed.
Congenital scrotal anomalies are unusual and include penoscrotal transposition, bifid scrotum, ectopic scrotum, and accessory scrotum (as). Among these, as is the least frequent, with only 42 cases reported in the english literature . As is characterized by additional scrotal tissue lacking a testis, besides a normally developed scrotum . Various associated anomalies have been reported . In particular, contiguous subcutaneous tumor is the most frequently associated abnormality and is reported to be related to the etiology of as . Although prenatal screening techniques have advanced, most reported cases of congenital perineal mass have been identified after birth . A 28-year - old woman was referred to our hospital for the evaluation of a fetal perineal mass at a gestational age of 31 weeks . Prenatal ultrasonography and magnetic resonance imaging (mri) showed a mass of 1.0 1.2 cm located posterior to the scrotum in a male fetus . 1) the likely diagnosis was lipoma and the mass maintained a stable appearance until delivery . The male newborn was delivered vaginally at 38 weeks of gestation and his body weight was 2208 g. there were no specific symptoms after birth . Arrowheads indicate a 1.0 1.2 cm mass located posterior to the scrotum . On physical examination, the soft peduncular mass, measuring 2.0 cm in diameter, was attached to a midperineal skin tag . There was also a rugged pigmented swelling on the mass, measuring 0.7 cm in diameter, which resembled the scrotum . The perineal mass showed high signal intensity on t1- and t2- weighted images and the signal intensity was suppressed on fat - suppressed t1-weighted images . Mri revealed no associated abnormalities of the intraabdominal organs, musculoskeletal system, or genitourinary system . A. a soft peduncular mass with a rugged and pigmented swelling is located posterior to the normally developed scrotum . C. the mass is attached at the midperineum with a skin tag . The preoperative diagnosis was as with perineal lipoma, and we completely excised the mass under general anesthesia at one month of age . The postoperative course was uneventful, and the patient was discharged on the day after the operation . There was no recurrence or functional sequelae within a follow - up period of six months . A histological examination revealed that the peduncular mass consisted of mature adipose tissue . In this case, it was difficult to distinguish between lipoma and normal adipose tissue pathologically . However, our clinical diagnosis was lipoma because the peduncular mass was separated from normal perineal region by the skin tag . The rugged swelling on the peduncular mass showed smooth muscle fibers in the subcutaneous layer, which represented the tunica dartos . 3) the swelling was definitively diagnosed as as . Histological examination . B. the peduncular mass smooth muscle fibers in the subcutaneous layer of the rugged swelling represent the tunica dartos . These swellings appear at four weeks of gestation and migrate to the caudal portion after 12 weeks of gestation . Abnormal migration or early division of the labioscrotal swellings is possibly related to the etiology of congenital scrotal anomalies . The least frequent congenital scrotal anomaly is as, characterized by additional scrotal tissue without a testis, besides a normally developed scrotum . (table) characteristics of accessory scrotum various anomalies associated with as have been reported . In particular, contiguous subcutaneous tumor has a high incidence (72.5%) of association with as . Histologically, one case of subcutaneous tumor was lipoblastoma, three cases were hamartoma, and the others were consistent with lipoma . (table) it is assumed that the contiguous subcutaneous tumor is related to the etiology of as . Sule hypothesized that as develops when intervening mesenchymal tissue (i.e., the developing subcutaneous tumor) disrupts the continuity of the developing caudal labioscrotal swelling . However, the complete etiology of as is not explained by this hypothesis because as can occur with no contiguous tumor . Takayasu hypothesized that as develops from the early division and teratoid growth of pluripotential labioscrotal tissue elements . However, two cases associated with skeletal abnormalities were located in the pubic area, and one case was located on the distal penile shaft . Our case was detected at a gestational age of 31 weeks, and the other two reported cases were detected at 24 weeks and 32 weeks of gestation . A congenital perineal mass is unusual in itself, and most reported cases have been diagnosed after birth . However, detection is possible with careful prenatal screening . The differential diagnosis of a fetal perineal mass includes lipoma, lipoblastoma, infantile hemangioma, hamartoma, and choristoma . If a fetal perineal mass is detected during the antenatal period, it is important to look for any associated congenital anomalies . The prognoses of surgically treated patients are good, and only one has died, from an associated anomaly before surgery . The reported ages at surgery range from four days to 46 years (median, nine months), and three adult cases are recorded in the literature . Our patient was operated upon in the neonatal period because the mass was considered to be excisable without complications and the associated subcutaneous tumor had a low probability of malignancy . Although a fetal perineal mass is difficult to diagnose, it can be detected with careful prenatal screening . Many ass are associated with contiguous subcutaneous tumors, which are assumed to be related to the etiology of as.
Sanitation, immunization and antibiotics, and improved nutrition have increased life expectancy and dramatically changed the patterns of disease in many countries . Along with these benefits, however, development, including economic development, population growth, industrial development, urbanization, and increased use of motorized transportation, has been accompanied by global environmental deterioration, which poses a threat to current and future human health, especially in developing and underdeveloped countries . Economic disadvantage (areas with low socioeconomic status) augments the deleterious effects of air pollution on human health, a situation designated as environmental inequity (1). Environmental quality has been considered to be dependent on regional characteristics, among which social and economic factors play a pivotal role (2,3). Environmental disparities may increase as the result of the expansion of the interest of the automotive industry, predominantly diesel engine manufacturers, in the growing markets in latin america, africa, and asia, where environmental and health policies are not strongly consolidated (4). Due to their high toxicity, diesel emissions have been subjected to increasingly restrictive regulations over the last several decades at a cost of higher investments in fuel (low sulfur content) and engine technologies in developed countries (5). Sulfur is a naturally occurring component of crude oil and is found in both gasoline and diesel . When these fuels are burned, sulfur is emitted as sulfur dioxide (so2) or sulfate particulate matter (6). Since 1993, the european union (eu) has established standards to regulate the quality of automotive fuels and vehicle emissions to combat the atmospheric pollution caused by emissions from motor vehicles . A series of directives resulted in the progressive introduction of increasingly stringent standards (euro standards) that define the acceptable limits for exhaust emissions of vehicles sold in eu member states (7). Unfortunately, the benefits of cleaner technologies are lagging in less developed regions . In the usa, trucks sold after 2007 emit 0.01 g / bhp - hr pm, and in germany, trucks can emit only 0.02 g / kwh (defined in the euro v standard). In contrast, in brazil, india, and china, the allowed level of emissions is 0.1 g / kwh pm (euro iii standard) (8). These differences in emissions standards mean that heavy - duty diesel engines produced by the same manufacturer emit markedly different levels of pollutants depending on the regional market in which the engines are sold . Economic issues have been evoked to support the aforementioned regulatory differences in environmental emission standards . The trade - offs associated with these regulatory differences are relevant to the situation in brazil because the implementation of a policy requiring cleaner diesel technologies (cdt; euro iv standards for vehicles produced in 2009 and low sulphur diesel; diesel with 50 ppm of sulphur) was postponed until 2012 without a comprehensive analysis of the health consequences (9). To evaluate the health impact of the decision to delay implementation of the cdt policy, we estimated the monetary health costs of the non - abatement of ambient pm2.5 emissions due to this postponement in brazil . Pm2.5) as the estimator of exposure to air pollution . Diesel emissions contribute significantly to ambient pm2.5 concentrations in urban areas, and this class of pollutant exhibits a robust association with adverse health effects, especially lung cancer and cardio - respiratory diseases (10,11). During 2007 - 2008, we performed daily measurements of the ambient levels of pm2.5 in six brazilian metropolitan regions: curitiba, so paulo, belo horizonte, rio de janeiro, recife, and porto alegre . These cities have a combined population of 46,811,100 inhabitants (25% of the brazilian population) and generate 37.3% of the country's gross national product (12,13). The daily concentration of particulate matter (pm2.5) was obtained by a gravimetric method using a harvard sampler with a pm2.5 impactor (air diagnostics and engineering inc ., harrison, me, usa) at a flow rate of 10 l.min using a polycarbonate membrane . Each membrane was weighed before and after sampling using an ultra - microbalance (mettler toledo umx2, readability 1 g, zurich, switzerland) to determine the daily mass trapped by each membrane, allowing the calculation of the daily mean concentration of pm2.5 (13). The black carbon concentrations were measured using the reflectance method (14) using the same membranes, and 1/3 of the membranes were submitted to x - ray fluorescence spectrometry (15) and ion chromatography (16) analysis to determine the concentrations of the predominant chemical elements (na, al, si, p, s, k, ca, ti, v, fe, ni, cu, zn, and pb), nitrates and sulfates . The daily mean concentrations of pm2.5 were used to calculate the annual daily mean of pm2.5 as described by the who (30). All of the samplers were installed in central areas of the metropolitan regions at least 200 m from major traffic routes . Sources and their relative contributions to the ambient pm2.5 concentrations were identified using receptor modeling techniques based on the chemical characteristics of the particles collected at the receptor sites (in this case, the different metropolitan areas). In addition, pmf (positive matrix factorization), a factor analysis method, was applied to identify sources and determine the contribution of each identified source to the ambient concentration of pm 2.5 (17). The data used in this study were obtained from a recent research project conducted by our group (18). The non - abatement of ambient pm2.5 concentrations due to the delay in the adoption of more stringent emissions standards was estimated by considering the total emissions inventory agreed upon by the brazilian ministry of environment and truck manufacturers and oil companies operating in brazil (19). The projected reduction in the pm2.5 concentration that would have been achieved if the cdt policy had been implemented and those expected considering the delay are presented in figure 1 (20). The differences between the emissions curves presented in figure 1 are assumed to be proportional to the changes in the proportion of pm2.5 generated by diesel engines for each metropolitan area . The delay in the decline in the level of diesel - generated particles was computed until 2040 based on the present ambient concentrations of pm2.5 and the relative contribution of diesel to pm2.5 determined for each study site . The impact on morbidity of the additional pm2.5 due to the delay in the implementation of the cdt policy was estimated in terms of hospitalizations due to respiratory and cardiovascular events . The impact was determined separately for different age groups, employing coefficients from time series studies (table 1), favoring studies that were conducted locally (21 - 26). Functions associating increases in air pollution with adverse health effects may be derived based on short - term or long - term exposures . When there is no clear evidence of a chronic effect, the short - term dose response is preferable and more conservative . For pm2.5, therefore, we used the annual mean pm2.5 concentration, as recommended by the who (30). For mortality studies, we decided to consider the coefficients that associate chronic exposure to pm2.5 with all causes of mortality in adults to avoid possible bias in establishing the cause of death based on death certificates (who). The numbers of respiratory and cardiovascular hospital admissions in the public health system for each metropolitan area for 2007 were obtained from the brazilian health ministry database (27) and are presented in table 2 . We estimated the number of hospital admissions in the private system by extrapolation based on the health insurance coverage rate (28) of the private system for each metropolitan area (as presented in table 6- supplemental data). The number of hospital admissions in the private health system was computed based on the relationship described in the following equation (1): nps is the number of hospital admissions in the private system, npublic is the number of hospital admissions in the public system, and cr is the proportion of the population with access to private health insurance . After determining the baseline number of hospital admissions for the relevant health outcomes and the age groups of interest and after establishing the coefficients that relate changes in the pm2.5 concentration to hospital admissions, the projected morbidity effects of the delay in implementing the cdt policy were computed using the following equation (2): hadm (concyear) = hospital admissions due to the delay in reducing the ambient concentration of pm2.5 in a given year; = coefficient relating pm2.5 to hospital admissions (table 1); concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay; and totadm = total hospital admissions for a given year . Equation (2) was used to estimate the effects of the additional pm2.5 on the different health outcomes listed in table 2 . The value of hadm (concyear) was integrated over 40 years, as shown in figure 1 . The effects of the delay in implementing the cdt policy were also expressed in terms of mortality . Data on mortality due to natural causes in individuals over 40 years of age were obtained from datasus (29). The additional mortality burden due to the delay in implementing the cdt policy was estimated as suggested by the who (30) using the following equation: m (concyear) = number of deaths in a given year due to the delay in reducing the concentration of pm2.5; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay; 0.006 = relative risk of adult mortality for each 1.0 g / m of pm2.5; and totalm = the baseline mortality counts for each metropolitan area, as presented in table 3 . Morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs (productivity loss). The mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus (31). The cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo . The cost associated with lost productivity considers the number of days of absence due to hospitalization (data were obtained from datasus (31)) and the mean gross income for each age group (data obtained from the ibge (the brazilian statistical and geographic agency), which was responsible for the public economic census conducted in 2000 (32). The economic valuation of mortality was performed based on the disability - adjusted life years (daly) method (33), which combines an ambient factor, in this case the pm2.5 concentration, with a health indicator (mortality) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy . The number of (daly) (33) was calculated using an equation that estimates years of life lost (yll), the daly component that refers to time lost due to premature mortality . The yll was estimated based on the age at death and the life expectancy in southeastern brazilian . The values of the other parameters (the discount rate and the age - weighted modulation factor) were adopted as recommended by murray and lopez (33). The total years of life lost were converted into monetary values as described previously (34), and the values for the years of life lost (yll) were provided by externe (35). The life expectancy values were obtained from the ibge (the brazilian statistical and geographic agency) (12) for each metropolitan region . Pm2.5) as the estimator of exposure to air pollution . Diesel emissions contribute significantly to ambient pm2.5 concentrations in urban areas, and this class of pollutant exhibits a robust association with adverse health effects, especially lung cancer and cardio - respiratory diseases (10,11). During 2007 - 2008, we performed daily measurements of the ambient levels of pm2.5 in six brazilian metropolitan regions: curitiba, so paulo, belo horizonte, rio de janeiro, recife, and porto alegre . These cities have a combined population of 46,811,100 inhabitants (25% of the brazilian population) and generate 37.3% of the country's gross national product (12,13). The daily concentration of particulate matter (pm2.5) was obtained by a gravimetric method using a harvard sampler with a pm2.5 impactor (air diagnostics and engineering inc ., harrison, me, usa) at a flow rate of 10 l.min using a polycarbonate membrane . Each membrane was weighed before and after sampling using an ultra - microbalance (mettler toledo umx2, readability 1 g, zurich, switzerland) to determine the daily mass trapped by each membrane, allowing the calculation of the daily mean concentration of pm2.5 (13). The black carbon concentrations were measured using the reflectance method (14) using the same membranes, and 1/3 of the membranes were submitted to x - ray fluorescence spectrometry (15) and ion chromatography (16) analysis to determine the concentrations of the predominant chemical elements (na, al, si, p, s, k, ca, ti, v, fe, ni, cu, zn, and pb), nitrates and sulfates . The daily mean concentrations of pm2.5 were used to calculate the annual daily mean of pm2.5 as described by the who (30). All of the samplers were installed in central areas of the metropolitan regions at least 200 m from major traffic routes . Sources and their relative contributions to the ambient pm2.5 concentrations were identified using receptor modeling techniques based on the chemical characteristics of the particles collected at the receptor sites (in this case, the different metropolitan areas). In addition, pmf (positive matrix factorization), a factor analysis method, was applied to identify sources and determine the contribution of each identified source to the ambient concentration of pm 2.5 (17). The data used in this study were obtained from a recent research project conducted by our group (18). The non - abatement of ambient pm2.5 concentrations due to the delay in the adoption of more stringent emissions standards was estimated by considering the total emissions inventory agreed upon by the brazilian ministry of environment and truck manufacturers and oil companies operating in brazil (19). The projected reduction in the pm2.5 concentration that would have been achieved if the cdt policy had been implemented and those expected considering the delay are presented in figure 1 (20). The differences between the emissions curves presented in figure 1 are assumed to be proportional to the changes in the proportion of pm2.5 generated by diesel engines for each metropolitan area . The delay in the decline in the level of diesel - generated particles was computed until 2040 based on the present ambient concentrations of pm2.5 and the relative contribution of diesel to pm2.5 determined for each study site . The impact on morbidity of the additional pm2.5 due to the delay in the implementation of the cdt policy was estimated in terms of hospitalizations due to respiratory and cardiovascular events . The impact was determined separately for different age groups, employing coefficients from time series studies (table 1), favoring studies that were conducted locally (21 - 26). Functions associating increases in air pollution with adverse health effects may be derived based on short - term or long - term exposures . When there is no clear evidence of a chronic effect, the short - term dose response is preferable and more conservative . For pm2.5, therefore, we used the annual mean pm2.5 concentration, as recommended by the who (30). For mortality studies, we decided to consider the coefficients that associate chronic exposure to pm2.5 with all causes of mortality in adults to avoid possible bias in establishing the cause of death based on death certificates (who). The numbers of respiratory and cardiovascular hospital admissions in the public health system for each metropolitan area for 2007 were obtained from the brazilian health ministry database (27) and are presented in table 2 . We estimated the number of hospital admissions in the private system by extrapolation based on the health insurance coverage rate (28) of the private system for each metropolitan area (as presented in table 6- supplemental data). The number of hospital admissions in the private health system was computed based on the relationship described in the following equation (1): nps is the number of hospital admissions in the private system, npublic is the number of hospital admissions in the public system, and cr is the proportion of the population with access to private health insurance . After determining the baseline number of hospital admissions for the relevant health outcomes and the age groups of interest and after establishing the coefficients that relate changes in the pm2.5 concentration to hospital admissions, the projected morbidity effects of the delay in implementing the cdt policy were computed using the following equation (2): hadm (concyear) = hospital admissions due to the delay in reducing the ambient concentration of pm2.5 in a given year; = coefficient relating pm2.5 to hospital admissions (table 1); concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay; and totadm = total hospital admissions for a given year . Equation (2) was used to estimate the effects of the additional pm2.5 on the different health outcomes listed in table 2 . The value of hadm (concyear) was integrated over 40 years, as shown in figure 1 . The effects of the delay in implementing the cdt policy were also expressed in terms of mortality . Data on mortality due to natural causes in individuals over 40 years of age were obtained from datasus (29). The additional mortality burden due to the delay in implementing the cdt policy was estimated as suggested by the who (30) using the following equation: m (concyear) = number of deaths in a given year due to the delay in reducing the concentration of pm2.5; concyear = difference between the predicted concentration of pm2.5 that would have been achieved if the cdt policy had been implemented and the estimated concentration of pm2.5 given the delay; 0.006 = relative risk of adult mortality for each 1.0 g / m of pm2.5; and totalm = the baseline mortality counts for each metropolitan area, as presented in table 3 . Morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs (productivity loss). The mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus (31). The cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo . The cost associated with lost productivity considers the number of days of absence due to hospitalization (data were obtained from datasus (31)) and the mean gross income for each age group (data obtained from the ibge (the brazilian statistical and geographic agency), which was responsible for the public economic census conducted in 2000 (32). The economic valuation of mortality was performed based on the disability - adjusted life years (daly) method (33), which combines an ambient factor, in this case the pm2.5 concentration, with a health indicator (mortality) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy . The number of (daly) (33) was calculated using an equation that estimates years of life lost (yll), the daly component that refers to time lost due to premature mortality . The yll was estimated based on the age at death and the life expectancy in southeastern brazilian . The values of the other parameters (the discount rate and the age - weighted modulation factor) were adopted as recommended by murray and lopez (33). The total years of life lost were converted into monetary values as described previously (34), and the values for the years of life lost (yll) were provided by externe (35). The life expectancy values were obtained from the ibge (the brazilian statistical and geographic agency) (12) for each metropolitan region . Morbidity costs were estimated in terms of the direct costs of hospital admissions due to respiratory and cardiovascular diseases in both the public and private health systems and in terms of indirect costs (productivity loss). The mean cost of hospital admissions for respiratory and cardiovascular diseases for each age group was obtained from datasus (31). The cost of hospital admissions in the private health system was not available and was estimated to be three times higher than the public cost based on data from the hospital das clnicas of the faculdade de medicina da universidade de so paulo . The cost associated with lost productivity considers the number of days of absence due to hospitalization (data were obtained from datasus (31)) and the mean gross income for each age group (data obtained from the ibge (the brazilian statistical and geographic agency), which was responsible for the public economic census conducted in 2000 (32). The economic valuation of mortality was performed based on the disability - adjusted life years (daly) method (33), which combines an ambient factor, in this case the pm2.5 concentration, with a health indicator (mortality) to estimate the number of years of life lost through premature mortality relative to brazilian life expectancy . The number of (daly) (33) was calculated using an equation that estimates years of life lost (yll), the daly component that refers to time lost due to premature mortality . The yll was estimated based on the age at death and the life expectancy in southeastern brazilian . The values of the other parameters (the discount rate and the age - weighted modulation factor) were adopted as recommended by murray and lopez (33). The total years of life lost were converted into monetary values as described previously (34), and the values for the years of life lost (yll) were provided by externe (35). The life expectancy values were obtained from the ibge (the brazilian statistical and geographic agency) (12) for each metropolitan region . Our approach indicated that diesel sources contributed approximately 40% of the total mass of pm2.5, considering both primary and secondary aerosol formation . This estimate was consistent with that of the so paulo state sanitation agency (cetesb) and previous studies focused on the city of so paulo (37 - 38). Table 4 presents the estimated excess hospital admissions due to respiratory and cardiovascular diseases in the public and private health systems and the estimated excess mortality for the six metropolitan areas for the years 2009 to 2040 resulting from the delay in the implementation of the cdt policy . Table 5 presents the costs associated with the excess hospital admissions due to respiratory and cardiovascular diseases attributable to pm2.5 . Mortality costs due to respiratory and cardiovascular diseases attributable to pm2.5 were estimated based on the total years of life lost (ylls). For the period 2009 - 2040, the non - abatement of pm2.5 accounted for 162,878 ylls, which represents a total cost of us$11.4 billion . Our approach indicated that diesel sources contributed approximately 40% of the total mass of pm2.5, considering both primary and secondary aerosol formation . This estimate was consistent with that of the so paulo state sanitation agency (cetesb) and previous studies focused on the city of so paulo (37 - 38). Table 4 presents the estimated excess hospital admissions due to respiratory and cardiovascular diseases in the public and private health systems and the estimated excess mortality for the six metropolitan areas for the years 2009 to 2040 resulting from the delay in the implementation of the cdt policy . Table 5 presents the costs associated with the excess hospital admissions due to respiratory and cardiovascular diseases attributable to pm2.5 . Mortality costs due to respiratory and cardiovascular diseases attributable to pm2.5 were estimated based on the total years of life lost (ylls). For the period 2009 - 2040, the non - abatement of pm2.5 accounted for 162,878 ylls, which represents a total cost of us$11.4 billion . Our results indicate that considerable health and economic burdens resulted from the delay in implementing the cdt policy . Air pollution in urban areas has negative health effects, including difficulty breathing, wheezing, coughing, eye irritation, aggravation of pre - existing respiratory and cardiac diseases, and even premature death . Increases in hospital admissions and medication use in areas with moderate or high levels of air pollution are commonly reported in different parts of the world (40). The use of motorized transportation in brazil increases every year . According to denatran (national traffic department), the brazilian fleet has doubled in the last ten years; there are currently almost 65,000,000 registered vehicles in brazil, including cars, trucks, buses and motorcycles (41). The states of so paulo, minas gerais, and paran have the largest fleets, with nearly 20, 7, and 5 million cars, respectively . Although this growth in motorized transportation is a result of and an indicator of the growing economy and a higher standard of living, it is accompanied by environmental and social challenges, including air pollution and traffic congestion, which pose threats to the health and well - being of the residents . To address these problems, different countries have conducted economic valuations of public health endpoints to substantiate environmental and health politics (42). The translation of the social and health benefits of cleaner fuel and vehicle regulations into monetary amounts is necessary to direct the decision - making process regarding actions that have the potential to affect air quality in urban areas (43). Unfortunately, in brazil, the decision to postpone the adoption of the cdt policy was not based on any cost - benefit analysis . The displacement of the curve for the reductions in diesel emissions (figure 1) agreed upon by industries and the brazilian authorities was considered not to be significant . However, the more detailed analysis of the health consequences of this delay performed in our study indicated that because of the large number of exposed people, the delay in implementing the cdt policy will be responsible for a considerable number of hospitalizations and a presumable excess of approximately 14,000 premature deaths . Most likely, the authorities that approved the postponed implementation of the cdt policy would have made a different decision if the health consequences had been fully considered in the decision - making process . Because cost - benefit analyses may be highly influenced by regional characteristics, in the present investigation, we collected primary data to minimize uncertainties in the estimates . For example, the concentration response curves for morbidity preferentially used data from local studies . A comprehensive sampling and chemical analysis of the particle composition and source apportionment techniques were applied to determine the contribution of the present diesel technology to ambient particle levels in the areas of study . Vehicles, fuels, air pollution, and health should be treated and analyzed as a system . Changes in fuel composition can immediately impact air pollution and consequently influence air pollution control (44 - 45). There are many examples of how cleaner diesel fuel contributes to reductions in air pollution . In hong kong, a one - weekend intervention study that required all power plants and road vehicles to use fuel oil with a low sulfur content led to an immediate decrease in the level of ambient sulfur dioxide (so2) and provided direct evidence that this type of change has immediate and long - term health benefits, thus reducing mortality rates (46). Donald mccubbin and mark delucchi evaluated the health costs of a kilogram of various air pollutants, including co, nox (nitric oxides), pm2.5, sox (sulfur oxides), and vocs (volatile organic compounds). They estimated health costs based on such factors as hospitalization, chronic illness, asthma attacks, and lost work days for us urban areas . These researchers found that the ranges in health costs per kilogram were from us$0.01 to us$0.10 for co, us$1.59 to us$23.34 for nox, us$14.81 to us$225.36 for pm2.5, us$9.62 to us$90.94 for sox, and us$0.13 to us$1.45 for vocs (47). In the indian context, the health cost of urban air pollution was estimated to be us$1.4 billion (48). Our results are consistent with the results of previous studies and indicated that the sulfur content of diesel represents a considerable source of ambient fine particles and that considerable health benefits would result from implementing the cdt policy . Indeed, a previous study performed in so paulo indicated that the use of improved diesel technology exhibited the best cost - benefit ratio among all other policies considered to reduce the health effects of air pollution (49). Interestingly, although this report was fully available when the decision to delay the implementation of the cdt policy was made, the relevant authorities did not consider the recommendations presented in that report . This failure to consider published data - based recommendations indicates that the health consequences of industrial and fuel policies are not usually considered in situations in which economic pressures are high . In conclusion, our results indicate that it is necessary to increase the level of awareness about the health consequences of policies that regulate transportation and fuel technology and that human health must play a role in the environmental agenda in urban areas . We thank the following researchers and institutions for monitoring the pm2.5 concentration and for the sampling infrastructure: prof dr elisabeth neves from the department of anatomy, center for biological sciences (ufpe, federal university of pernambuco); prof dr walter zin from the respiratory physiology laboratory, carlos chagas filho institute of biophysics (ufrj, federal university of rio de janeiro); prof dr geraldo brasileiro filho from the pathology department, school of medicine (ufmg, federal university of minas gerais); prof dr orliney maciel guimares from the chemistry institute (ufpr, federal university of paran); and prof dr cludia ramos rhoden from the department of physiological sciences (ufcspa, porto alegre federal foundation for medical sciences).
Cancer vaccine concept have been encompasses a broad spectrum of platforms intended to trigger or increase an adaptive immune response against a malignant tumor . Thus cancer vaccines are aimed as active immunotherapeutic interventions mainly in an established disease state in which the malignancy is expressing all its functional capabilities or hallmarks . Understanding of relationship between structure and function in cancer vaccines is essential to interpret their opportunities to impact into prolonging survival and increasing quality of life in cancer patients . The structure of cancer vaccines is referred to a tumor associated (or specific) antigen shaped as peptides, recombinant vectors, whole tumor cells or antigen presenting cells linked or supported by an adequate immunopotentiating platform, which should be inserted in a context of therapeutic maneuvers . The expected function of cancer vaccines is basically to circumvent the immunologic tolerance in the tumor microenvironment inducing a lowest rate of tumor progression and increasing the host survival . So, cancer vaccines function is expected to be relevant at all levels of biological organization of the malignancy host, from the tumor microenvironment up to the whole organism . Then, to success with active immunotherapy strategy two items need to be considered: the role of target antigens in tumor biology (cancer hallmark related) and the immunogenic capacity of vaccine composition . Once selected the right antigen and the most adequate platform for immune potentiating, the battle field for cancer vaccines is the immunosuppressive microenvironment induced by the malignant machinery; the corner between structure and function in cancer vaccines is accurately the interaction between the tumors hallmarks and the host immune response . Prostate cancer has been proposed as a prototype for vaccine therapy scenario . Besides the increasing spectrum of therapeutic tools for the disease control at the advanced disease stage, meaning new androgen deprivation modalities such as abiraterone, advanced chemotherapeutic agents such as cabazitaxel and small tyrosine kinase inhibitors such as cabozantinib, a cancer vaccine appear in the prostate carcinoma treatments alternative . Evidences supporting that sipuleucel - t, a vaccine based on dendritic cells pulsed with prostatic acid phosphatase linked to granulocyte macrophage colony - stimulating factor, prolonged survival in minimal or non - symptomatic mcrpc patients and its fda approval (april, 2010), constitute the proof of concept of cancer vaccines application in the scenario of a solid tumor with long term disease course, characterized by well described tumor associated antigens, a defined biomarker (serum psa) and a good physicians tool to follow up the patients therapeutic response (halabi nomogram). Non- small cell lung cancer (nsclc) is the first cause of cancer associated death worldwide, the 60- 80% of patients are diagnosed at the advanced stages (iiib / iv) in which their life expectancy is not too far from 12 months median survival even second and emerging third line of onco - specific treatments . In this particular case the active immune intervention using cancer vaccines is strongly challenged by the tumor induced immunologic tolerance . There are several evidences of a marked infiltration of different types of immune cells in the nsclc microenvironment, and the distribution, tissue localization, and cell types are significantly associated with progression and patient s survival . The clinical relevance of the microenvironmental immunological milieu in this malignacy is highlighted by the facts that higher foxp3+/cd8 + ratio in tumor sites is an independent factor for poor response to platinum - based neoadjuvant chemotherapy in a setting of advanced stages patients . This review is aimed to focus on nsclc as an emerging and promising model for active immunotherapy and the challenges for its inclusion in the current clinical scenario, considering the increasing body of evidences of the interaction between inflammatory cells and tumor cells, facilitating the lung carcinogenesis and tumor hallmarks expression (angiogenesis and tumor cell migration among others), in which antigen - specific antitumor responses are overtly present . In this context, the clinical relevance of cancer vaccines should be predicted pointing out the emerging determinants of its structure and function relationships in the particular situation of nsclc malignancy hallmarks . The kinetics of the active immunizations encompasses a sequential cascade of clinical endpoints: starting by the activation of the immune system, followed by the antitumor response and finalizing with the consequential impact on patients overall survival . Today this cascade of clinical endpoints is the backbone for active immunization assessment and moreover the concept of cancer vaccines, applied in the nsclc setting, is just evolving as a complex therapeutic strategy, in which the opportunities for cancer vaccines start from the selection of the target cancer hallmark, followed by the vaccine formulation and its platforms for immune potentiating, also encompass the successful insertion in the standard of care, the chronic administration beyond progression disease, the personalization based on predictors of response and the potential combination with other targeted therapies . As an inherently condition of all malignant processes, nsclc carcinogenesis is resulting from the interaction between the epithelial mucosa genome and the lung microenvironment . In this context, the intrinsic cell capacity for dna repair, the nutrients accessibility, the angiogenesis state and the hypoxia levels, seems to be the common context of selective pressures for the cascade of mutations during tumor progression and coming out of the malignant cells phenotypes . Particularly, the continuing exposure of dna to metabolically activated carcinogens, throughout the exposition to air contaminants or cigarette- smoke, resulting in the formation of dna adducts and consequential genetic instability . The rate of dna injure, taking place day after day over many years, is fully consistent with multiple genetic changes in lung cancer and is associated with increasingly severe histopathological phenotypes . The importance of the target antigens to tumor biology is considered relevant for the success of active immunotherapy . Indeed, the selection of vaccine antigens must be focused mainly on those molecules significantly associated with the hallmarks of cancer, also called oncoantigens; those specific or tumor associated antigens that are highly correlated with the phenotypic consolidation of the malignancy and tumor progression [20, 21]. The egfr network induce a broad range of biological effects by controlling the cellular outcome, through a multiple ligands activation - dependent regulatory loops, which fall into time dependants temporal domains; early loops comprise protein modifications mechanisms, while delayed loops involve transcription regulation mechanisms; both became aberrant and without feedback control in malignant cells . During development and progression of lung neoplasms the egfr is overexpressed in the following proportions: 62% of all nsclc tumors, 89% of squamous cell tumors, 41% of adenocarcinomas and 80% of bronchoalveolar tumors . In the case of adenocarcinoma in the peripheral compartment of the lung (terminal bronquiole and alveoli) in never smokers, the egfr overexpression is associated with a lower methylation index of the p53 cancer suppressor gene through high proportion of transition mutations (ie, purine for purine or pyrimidine for pyrimidine). The degree of egfr expression has been reported as a predictive factor of response to biological therapy in nsclc patients . There are two classes of anti - egfr agents that have shown clinical activity in nsclc . These are either monoclonal antibodies directed at the extracellular domain of the egfr and inhibiting the binding of natural ligands to the receptor, or low molecular weight tyrosine kinase inhibitors (tkis) that inhibit the tyrosine kinase activity of egfr, generally by competing reversibly with atp for the atp binding site . Targeting the egfr with a cancer vaccine is a newly strategy currently in clinical scrutiny using the active immune deprivation of egf ligand as important tumor growth factor (cimavax egf) [27, 28]. Muc1 is over - expressed in almost all nsclc patients, being associated with bad prognosis . The role in tumor biology of the nglycosylated muc1c (muc1 c - terminal transmembrane subunit) seems to be associated to a functionally important extracellular bridge mediated by galectin 3 between this recognized mucin antigen and tyrosine kinases receptors, such as the egfr . Muc1 looks to be related not only with promoting cell growth and survival but also with t cell proliferation inhibition and accordingly, immunosuppression . In this context, other molecules became aberrant expressed in the cascade of events associated with the sustained proliferation and its negative regulation, apoptosis evasion and the limitless replicative potential . Tumor suppressor gene p53 receives stress signals from excessive dna damage and suboptimal nutrients triggering dna repairing mechanism or apoptosis . Loss of p53 function by mutations are frequent events in human oncogenesis, which leads to persistent aberrant expression in about 60% of nsclc, correlated with poor cht response and lowest overall sv of the patients . This conduced to exploration of a mutant p53 peptide pulsed dendritic cell vaccine in a phase ii clinical trial with stage iii nsclc patients . The specialized dna polymerase, named telomerase, which adds telomere repeat segments to the end of telomeric dna is nearly absent in nonimmortalized cells but expressed at functionally significant levels in 90% of human tumors . Telomerase- based vaccine gv1001 is starting the clinical scrutiny, supported also by the facts that its presence in sputum samples has been identified as a potential specific marker for early detection of lung cancer . The activation of cdk1-cyclin b1 triggers the mitosis progression and its translocation from the cytoplasm to the nucleus . Positive feedback loops regulate its biological activity and spatial localization, ensuring a rapid, complete, robust, and irreversible transition from interphase to mitosis . There is reported the aberrant expression of these regulators of the g2 m cell cycle checkpoint in many neoplasms, including nsclc and significant association between increased cyclin b1 expression and reduced patients survival in stage i/ ii nsclc has been described . Furthermore, cyclin b1 peptide - pulsed autologous dendritic cell vaccine is early of clinical evaluation in resectable nsclc patients . The ras genes, including h - ras, k - ras and n - ras, encode a family of proteins regulating cell growth, differentiation and apoptosis . Mutations in the k - ras gene, mainly in codons 12 and 13, have been found in 20 - 30% of nsclc tumor samples and occur most commonly, but not exclusively, in adenocarcinoma histology and in heavy smokers . Active immune strategies aimed at interfering with the ras - raf - mek pathway in nsclc are also incipient exploring the use of a ras peptide based vaccine . The continuous cascade of molecular events starting from at least a cell clone and characterized by unregulated cellular proliferation and clonal heterogeneity, is resulting in a characteristic nsclc malignant phenotype . Based only on histology nsclc comprises i.e. For adenocarcinoma, squamous - cell carcinoma and large - cell carcinoma subsets, while, based on the molecular subsets expressed in the resultant malignant phenotypes, nsclc drive a broad spectrum of diseases subsets: egfr, her2, kras, alk, braf, pik3ca, akt1, ros1, nras, map2k1 and the recently identified kif5b - ret fusion subset, each one with dissimilar pattern of incidence in a population . The personalization of cancer vaccines therapy is a pending chapter in this background, and the selection of the right vaccine for the right patient is becoming as challenge . The design of cancer vaccines attempt to harness the specificity and resistance potentials of the human immune system . With the aim to stimulate the immune system to recognize, attack and destroy tumor cells the personalization treatment is intrinsic for mhc context dependent antigens vaccines (peptides based vaccines) and for cell based cancer vaccines . In the context of the adaptive immune response one foremost challenge for clinical researchers is to classify the patient s population according the pre - existing immune response in potential responders or non- responders to the target antigen . It is widely accepted that the failure of the cancer vaccines in the nsclc scenario is related with its introduction in the advanced disease stages and poor performance status of the patients due to the combination of the tumor induced immunosuppression with the immune senescence . The facts that nsclc occur in subjects who have never smoked and have no evident cause for chronic inflammatory changes in the lung parenchyma, suggest that the interactions between the broncho - alveolar compartment and the in situ immune regulators are involved in facilitating tumor occurrence, growth, and spread . It is progressively ostensible that crosstalk between cancer cells and cells of tumor stroma is involved in the acquired capability for invasive growth and metastases . Tumor stroma has been described as pivotal in the established hostile immune environment through the secretion of immunosuppressive factors and the recruitment of suppressive immune cells of myeloid and lymphoid origin, while the tumor cells directly contributing to immune resistance through the secretion of immunosuppressive mediators, such as transforming growth factor (tgf) or natural killer (nk) cell receptor decoys, and through the expression of ligands for immune checkpoint (or co - inhibitory) receptors, such as programmed cell death ligand 1 (pdl1). The distribution, tissue localization, and cell types infiltrating lung tumor microenvironment are significantly associated with the disease progression and patient s survival . For example dendritic cell defects increases immature forms that may play an immunosuppressive role and facilitate cancer migration, invasion, and epithelial - to - mesenchymal transition (schneider et al . 2011). Also, genetic variations in the transforming growth factor - beta pathway are considered as predictors of survival in advanced non - small cell lung cancer and higher foxp3+/cd8 + ratio in tumor sites is described as an independent factor for poor response to platinum - based neoadjuvant chemotherapy in a setting of advanced stages patients . Summarizing, a variety of cellular immune abnormalities, antigen processing and presentation machinery defects, cytokine alterations and also individual conditions, increase the challenge for nsclc therapeutic vaccines . Vaccines candidates should activate the immune system and elicit a protective antitumor response even when chronic inflammation, the initiator of malignancy, is prevalent in tissues, modulate the tumor microenvironment and circumvent the dominant tolerance to tumor antigens to induce a tumor rejection . Overcoming the immunosuppressive microenvironment with cancer vaccines in nsclc, became an active immunotherapeutic intervention in an established malignant disease, which structurally imply the precise antigen selection, supported by at least two immune potentiating platforms; the correct immune system activation, and the accurate therapeutic maneuvers, aimed for induce the lowest rate of tumor progression and increase the host survival with ethical acceptable quality of life . Hopeful, certain immunomodulating agents, including a monoclonal antibody directed against ctla-4 (ipilimumab), pd-1 and pd - l1 blocking antibodies and talactoferrin, a dendritic cell activator, are just in scrutiny and have shown clinical activity in nsclc patients, highlighting the space of the immunotherapy as valid choice in this malignant condition [47, 48]. The well - known tumor associated antigens mage, muc1, ngc gm3ganglioside, cea and ny - eso-1 are overtly in the resultant malignant phenotype of nsclc, supporting an initial generation of tumor antigen - specific cancer vaccines presently under pressure for achieve highest level of clinical evidences . Vaccines as mage - a3, muc1 (emepepimut - s, tg4010) and racotumumab (anti - idiotipic mab ngc gm3 specific) are currently ongoing phase iii proof of efficacy clinical trials, whilst cea based cancer vaccines are just in ongoing phase i or ii studies . Other vaccines as ny - eso- 1 plasmid dna cancer vaccine have been explored in a phase i clinical trial . Cell - based cancer vaccines (cdx-1401, l - vax, dribble, mrc-5, tergenpumatucel - l, and belagenpumatucel - l) became as a second generation of active immunization approaches that becoming the scrutiny for clinically applicability in nsclc and belagenpumatucel - l or lucanix (allogeneic cells tgf - b2 antisense gene modification) seem to be the more advanced competitors with ongoing phase iii clinical trials . As shown, the strategies improved for overcome the tumor immunosuppressive machinery in lung cancer vaccines are supported on the primary platform comprising peptides (ras peptides, epemimut - s, tg4010 from muc-1 peptides and mage-12 peptides), proteins (cimavax egf and ny - eso-1), neo - antigen anti - idiotypes (racotumumab), recombinant vectors (mrc-5), whole tumor cells (dribble, l - vax, hyperacute, tergenpumatucel - l and belagenpumatucel - l) or antigen presenting cells (p53, cyclin b1, cdx-1401) [39, 52, 53]. The above primary structure of the immunization strategy is commonly linked or supported by an adequate secondary platform for the correct apc presentation and immune potentiation . It is the case of the autologous egf antigen coupled with non - self - molecule from neisseria meningitides, emulsified with an oil adjuvant (cimavax egf), or the case of a fusion protein between the ny - eso-1 tumor antigen and a fully human monoclonal antibody specific for the dendritic cell receptor dec-205 (cdx-1401 vaccine). Other examples are the emepepimut - s vaccine that targets the exposed core peptide of muc1 by a liposomal formulation and racotumumab anti - idiotipic vaccine, which act as a surrogate of neugm3 generating specific autologous antibodies [54, 55]. Once selected the right antigen and the most adequate platform for immune potentiating, those primary and secondary platforms, are not in condition to be winners in the battle field of the immunosuppressive microenvironment induced by the malignant machinery, and should be additionally inserted in a context of therapeutic maneuvers such as the priming and boosting strategies, the pre - treatment with a single low dose of cyclophosphamide (cimavax egf, epemimut - s), the chemotherapy induced lymphopenia and combinations with gm - csf, il-2, resiquimod and bcg, among others, for improve a tertiary platform for immune potentiation (table 1) [56, 57]. Nsclc vaccines should be able to elicit both potent cd4 + and cd8 + t - cells to achieve an antitumor response . The most useful reaction of the immune system against tumors is to kill the abnormal cells using ctls . But a broad spectrum of mechanism of action, from cellular to humoral effector, is present in more advances vaccines candidates, mainly depending on targeted antigen . Success of cimavax egf vaccine, which is oriented to castrate the autologous egf from the serum to subtract this growth factor from the tumor microenvironment, is obtained by inducing high titers of sequestering antibodies . In this case the cellular response seems to be not relevant for the vaccine functionality . Using cimavax egf higher anti - egf antibodies titers correlate with lowest serum egf and longer patient s survival . Mage - a3-vaccine, based on a fusion protein containing the mage - a3 antigen and protein d, use a second - generation adjuvant system as02b, a saponin based adjuvant containing monophosphoryl lipid a. this vaccine induces both humoral and cellular immune response, but appears to be effective in patients expressing hla - a1 allele, only present in the 20% of patients, by the induction of specific ctl response . The use of the adjuvant have been proved to be a prerequisite for cellular response to l - blp25 vaccine (stimuvax), based on muc1 lipopeptide (25 aminoacids), use a liposomal delivery system and monophosphoryl lipid a, added to enhance the immune response . Vaccination is assumed to provoke internalization mediated by liposomal structure inducing cd4 + and cd8 + t cells, and also b cell response . Induced antibodies mediate antibody dependent cellular cytotoxicity (adcc) against tumor . Up to date the clinical insertions of cancer vaccines as therapeutic interventions in nsclc start in the more advanced disease settings in which the malignancy is already established and acquired all its functional capabilities, while the scenario in which the tumor is surgically removed is fewer explored . It is important to point out that in this setting the immunosupresive machinery of the malignancy is extremely developed . Additionally cancer condition is most common in elderly persons in which the immune response could be modified by the natural immunosenescence mechanisms . This situation is very controversial and should be carefully considered to design the immunotherapeutic intervention . Cancer vaccines for nsclc have been focused as a therapeutic option based on the identification of a tumor hallmark and the active immunization with the related molecules that triggers cellular and/or humoral responses that consequently destroy or delay the rate of malignant progression . This therapeutic intervention in an established disease state has been aimed to impact into prolonging patients survival with ethically accepted quality of life . Currently mage - a3, stimuvax, lucanix, tg4010, racotumumab and cimavax egf are just in advanced proof of efficacy phase iii clinical trials in nsclc . While mage - a3 improve a trend to benefit the disease free interval in the post - resection of early disease stages pib to ii (hr 0.73; p= 0.109), the advanced disease setting remain the most clinically explored . In this disease setting tg4010 showed a trend to benefit pfs at 6 months with rates of 44% for vaccinated versus 35% for controls (p= 0.13) and in terms of trends for benefit patient s overall survival stimuvax show a hr 0.74 with p = 0.112, lucanix achieve a clinical response of 15% and better survival in the high - dose group, racotumumab achieve numerical differences of 9.93 months for vaccinated group vs. 4.53 months for control group and cimavax egf achieve 3.5 month of benefit for the subset of 60 years old and younger patients (table 2). Despite first, second and emerging third line of onco - specific treatments the life expectancy for nsclc patients diagnosed at advanced stages is surrounding the 12 months of median survival and in facts the today real circumstances are extremely demanding for the success inclusion of cancer vaccines as therapeutic choice in the clinical scenario . As corollary of the described development, an increasing interest in utilizing the multiple new agents that show activity in nsclc and have a tolerable side - effect profile, to maintain response to initial therapy after treatment with platinum - based doublets is rising . However considerable controversy, maintenance therapy has been considered a suitable treatment alternative and becoming a plausible therapeutic space for cancer vaccines, in the two proposed modalities; continuous maintenance or switch maintenance . In both cases the goal is to delay the second line therapy without treatment holidays for the malignancy . The incorporation of the active vaccination in the current standard of care based generally in chemotherapeutic agents is yet poorly exploit in the clinical setting . There is accumulating evidence that conventional therapies may profit the antitumor effects from the participation of the immune system by several mechanisms such as the immunogenic tumour - cell death . In a phase i / ii clinical trial, 20 patients diagnosed with advanced nsclc, received a schedule of two vaccinations of cimavax egf before the platinum based first line chemotherapy and subsequent monthly vaccinations were improved once concluded the chemotherapy cycles . This small study evidenced that anti - egf antibody titers were not affected and correlate with a reduction of serum egf concentration, while the patients overall survival also correlate with the highest anti - egf antibody titers without modification of the safety profile . The therapeutic vaccine tg4010 was added to nsclc first - line chemotherapy and used until documentation of progression disease in a phase iib, open - label, controlled trial, recruiting 148 chemotherapy - naive patients with diseases stages iiib or iv . This study show that 43.2% of patients who receiving the combination therapy were progression free, compared with 35.1% of the chemotherapy - alone group at 6 months of follow up . These results assessed the addition of a therapeutic cancer vaccine to first - line chemotherapy in advanced nsclc as another choice for insertion in the current standard of care . The emergency during the last five years of a methodological framework to enhance the clinical success of cancer immunotherapy is strong - minded the therapeutic insertion of cancer vaccines in nsclc . From this perspective, the kinetics of active immunizations is understood as a sequential cascade of clinical endpoints . Starting by the initiation of vaccinations that leads to activation of the immune system which results in a cellular immune response developed in days to weeks; followed by the antitumor response that becomes evident weeks to months after first immunization and finalizing with the consequential impact on patients overall survival after several months of treatment . The immunotherapists and oncologists communities are recognizing this cascade of clinical endpoints as a backbone for active immunization assessment and each one of these three clinical ends points implies their own specific challenges and opportunities . Connected with the circumstances of the currently clinical scenario, the exactly position of cancer vaccines in the advanced nsclc setting is foremost challenging the current criteria for clinical evaluation of tumor response, because the possibility of pseudo- progression induced by immunotherapy, due to the elicited antitumor cd4 + and cd8 + t - cells infiltration, justifying the forthcoming immune related criteria of tumor progression and therefore the immunization beyond disease progression . Cimavax egf have been explored after first line therapy and administered beyond diseases progression in a referred phase ii, open, controlled trial in 80 patients after first line platinum based treatment with promising results . The metanalysis of 58 patients who were vaccinated monthly for more than one or two years evidenced that long term vaccination was feasible and safe, without evidences of cumulative toxicity or immune response exhaustion . The results of the planned interim analysis with the first 226 patients recruited in the ongoing randomized phase iii trial, vaccinating the patients who respond to first line chemotherapy with at least stable disease, in the modality of switch maintenance, beyond progression disease and until change their performance status as per physician criteria, confirm previous results in benefit the patient s overall survival by intent to treat analysis (manuscript in preparation). So, chronic vaccination improved beyond progression disease in advanced stages; seems to be one of the transitions points for positioning cancer vaccines in the current and imminent nsclc therapeutic scenario . Regarding the insertion of cancer vaccines in the standard of care for advanced nsclc is important to highlight the appearance of an algorithm proposed for choose a first line chemotherapy alternative . In this algorithm the presence of positive egfr mutations or alk fusions, the clinical conditions of the patients (performance status) and the tumor histological type (non squamous or squamous), are driving the selection of erlotinib, crizotinib, bevacizumab combined or not with platinum / pemetrexed, platinum / gemcitabine doublets or single- agent chemotherapy . Besides, another transition points for the inclusion of cancer vaccines in the current nsclc therapeutic context is the opportunity to personalize this immunotherapy, based on biomolecules that will be predictive of the potential efficacy for a predetermined subset of patients . Up to now during active immunization some patients become not responders due to their specific pattern of immune response . For instance, the case of cimavax egf in which those patients who reach the super responder condition, based on their anti- egf antibodies titers, achieve significant benefit in overall survival, but so far there is not predictable this subset of patients before start immunizations . On the other hand, due to the intrinsic relation with the vaccine mechanism of action, intended to induce a humoral immunity that sequestering the circulating egf and its possible effect on patients outcome, today the correlation between the basal serum egf concentrations and the patients survival after vaccine treatment, is been exhaustive evaluated for today is widely accepted that the blockade of a single hallmark is not enough to achieve clinical noteworthy benefit on patients survival . Because the complexity and redundancy of the molecular pathways that promote tumour growth and maintenance, the generation of potent anti - tumour immune responses requires the blockade of multiple steps . An additional transition points for the successful inclusion of cancer vaccines in the current nsclc clinical scenario is the combination with other targeted therapies or the called combinatorial immunotherapy . This concept supports the criteria that optimizing immunotherapy requires treatments that affect multiple aspects of the immune response . A small trial combining autologous granulocyte macrophage colony - stimulating factor (gm - csf)-secreting tumour cell vaccines with ctla4 blockade found increased inflammatory infiltrates and tumour regression, suggesting that vaccine - induced anti - tumour t cells were present within the tumour but anergized owing to ctla4 co - inhibition . The combinatorial approach was explored in prostate cancer preclinical models using anti - ctla-4 mab in combination with rv - cea - tricom cancer vaccine to augment antigen - specific t - cell responses and an independent prognostic effect of ctla-4 overexpression in radically resected nsclc has been recently described pointing out the potentials of this checkpoint modulation in this malignancy [70, 71]. The clinical development of combinatorial approaches has been moved forward after the fda approval of antictla4 therapy, quickly followed by reports of encouraging preliminary clinical data for antipd1 therapies [47, 48]. Immune checkpoints are a tiny fraction of the receptors and ligands that have been defined by genetic and biological analyses to inhibit specific types of immune responses at various levels and its immunomodulatory manipulation may results in enhancing efficacy of therapeutic vaccinations . Thus, the blockade of immune checkpoints promises broad and diverse opportunities to enhance antitumour immunity with the potential durable clinical responses . Moreover combinatorial immunotherapies such as bispecific t cell engagers (bites) and immunotoxins fc - fusion proteins are entering clinical testing in acute lymphoblastic leukemia and breast cancer patients respectively; opening a new era for cancer active immunotherapy [73, 74]. Targeted therapies and immunotherapy offer a number of possible synergies in treatment when used together; however, these combinations should be more intensive studied and dose optimization, timing and sequence will required rationally design in future clinical trials with the aim of maximize anti - tumour efficacy whereas minimizing every possible immunosuppressive adverse reactions . The critical obstacles for cancer immunotherapy have been defined, so, for the medical positioning of the cancer vaccines in the current nsclc clinical scenario is important to understand that this concept is evolving into a complex therapeutic strategy . Support the use of therapeutic vaccines in nsclc is essential to consider the following: insertion in the standard of care . During past two decades the onco - specific treatments advanced from the unique option of platinum doublets to the routine use of first - line, second - line, and even third - line treatment . Second; immunotherapies may induce tumor pseudo- progression, challenging current criteria for clinical evaluation of tumor response and allowing the forthcoming immune related criteria of tumor progression . Personalization based on predictors of response . Targeted drugs such as erlotinib and crizotinib are inserted in the therapeutic context of nsclc using specific biomolecules indicatives of therapeutic efficacy for selected subset of patients . The blockade of a single hallmark is not enough to achieve clinical noteworthy benefit on patients survival . Combinations may achieve a synergism, strength of antitumor effects and also avoid or overcome the tumor mechanism of resistance . = antigen presenting cells = eastern cooperative oncology group = epidermal growth factor = epidermal growth factor receptor = non- small cell lung cancer = metastatic castration resistant prostate cancer = p64k carrier protein from neisseria meningitides = performance status = randomised controlled trial = tirosine kinases inhibitors
The recognition that elevated intracranial pressure (icp) is transmitted through the optic nerve and its sheath has been known for many years . This physiological process is the basis for the physical exam finding of papilledema on fundoscopic examination . Recently, interest has turned to measurement of the optic nerve sheath diameter (onsd) through non - invasive imaging technologies to provide surrogate markers for early elevated icp . In this issue of critical care, geeraerts and colleagues present their research correlating magnetic resonance imaging (mri) measurements of onsd with icp . In a retrospective review of 38 patients with traumatic brain injury requiring both invasive icp monitoring and mri, they found a significant positive relationship between onsd measured by mri and icp (r = 0.71). The best cut - off value to detect an icp> 20 cm h2o based on a receiver operating characteristic curve was found to be onsd = 5.82 mm with a sensitivity of 90% and a specificity of 92% . The optic nerve is surrounded by cerebrospinal fluid (csf), which is contiguous with intracranial csf . Increased icp is transmitted through this subarachnoid space causing distention of the dural optic nerve sheath, especially the retrobulbar segment . The optic nerve and its surrounding sheath can be imaged and measured on mri using a fat - suppressed t2-weighted sequence . Mri has been used to demonstrate increased onsd in idiopathic intracranial hypertension, and interestingly, decreased onsd in csf hypotension . The onsd has also been shown on mri to decrease after drainage of subdural hematomas . The research presented by geeraerts and colleagues is unique in its comparison of onsd with simultaneous direct measurements of icp through invasive monitoring . Their findings generally correlate with a growing body of research using bedside ultrasound measurements of onsd to detected elevated icp . Original research with lumbar intrathecal infusions performed by hansen and helmke demonstrated rapid changes in the onsd with alteration of csf pressures . In emergency department patients with traumatic brain injury, the onsd correlates with signs of elevated icp on computed tomography scans . More recently, researches have compared bedside ultrasound measurements of onsd to invasive icp [11 - 13]. While there is some variation in the optimal cut - off value, the correlation between onsd and icp remains consistent . In their current article, geeraets and colleagues provide further evidence of this physiological relationship and an intriguing possibility for non - invasive assessment of icp using mri . The obvious drawbacks to mri include its expense, long acquisition times, need for patient transport, and limited availability . However, some research has shown that mri may provide more precise measurements then ultrasound . Geeraerts and colleagues used a conventional t2 sequence with relatively large slice thickness and interslice spacing, resulting in an overall feasibility of measuring the onsd in 95% of patients . Greater accuracy and reliability would be expected in coronal t2 slices with thinner slices . As mri becomes more accessible and faster, non - invasive mri measurements may prove to be useful in certain clinical settings and as a potential reference standard for further research . Continued research with larger studies is required to confirm the precision and accuracy of mri measurements of onsd, as well as the optimal measurement technique . Additionally, the time course of onsd distention and reduction needs to be further delineated . Currently, non - invasive assessments of icp do not obviate the need for invasive icp monitoring . Invasive monitoring detects minute to minute variations in icp and, in the case of intraventricular drains, can also be therapeutic . However, non - invasive screening tests may be useful in select populations who would not otherwise require invasive monitoring and could undergo mri scans, such as patients with liver failure, meningitis, stroke, and moderate traumatic brain injury . In summary, the study by geeraerts and colleagues adds to a growing body of research demonstrating a correlation between increased onsd and elevated icp . By demonstrating the correlation of mri measurements of the onsd with invasive icp monitoring, they illustrate the potential of yet another non - invasive method to screen for elevated icp . While this technique will not replace invasive icp monitoring, it may be useful in select patient populations that would not otherwise have invasive monitoring but are at high risk for elevated icp . Further research is required before we can use measurements of the onsd to predict exact values of icp, but it may be useful as a screening test to estimate the probability of elevated icp . Csf: cerebrospinal fluid; icp: intracranial pressure; mri: magnetic resonance imaging; onsd: optic nerve sheath diameter.
There is no doubt that dog is the most favorite pet animal throughout the world . Further, due to its physiological similarity with human being, it has turned out to be an ideal model for studying human diseases like diabetes and cancer . Therefore, a better understanding about the normal physiological events of dogs might have paramount significance for clinicians, researchers, and pet owners . However, in many cases it is very difficult to have an accurate age estimation of a dog . This is because: (i) dog owners fail to keep the birth record of their pets, and it is not possible in stray dogs, (ii) dogs like any other companion animal is sold and resold more than once during their lifetime; hence, change of ownership may cause loss of data regarding age, (iii) normal available techniques are not very accurate and/or affordable, and (iv) very less information is available about age - related biomarkers in dogs . In a dog, denture wear, loss of elasticity of skin, rough hair coat, and so on are the common external appearances in old age . During this period, hematological alterations like anemia, lowered pcv (packed cell volume), and so on and biochemical changes like decreased serum protein, increased cholesterol, and bun (blood urea nitrogen) level, and so on are evident which can be correlated with dysfunction of different organs like liver, kidney, heart, spleen, nervous, and musculoskeletal system, and so on . Many disease conditions and/or diseases are also common in geriatric dogs like glaucoma, arthritis, skin disease, arteriosclerosis, prostrate hyperplasia, brown pigmentation of internal organs, and so on . However, all these age - related alterations are not very specific for age determination . Importantly, most of these only indicate about the presence or absence of old age . They do not give an idea about the intermediate stages of age progression from young to old age . Therefore, efforts should be made to identify novel age - related biomarkers . In many organs, somatic cells undergo a state of permanent growth arrest and altered function after a finite number of divisions, and this is known as replicative senescence . The process of replicative senescence was being established by using the human cells, which showed limited number of doublings before arrest of cell cycle . Senescent cells are resistant to apoptosis for a long period of time but are metabolically active . There is also evidence that senescent cells can accumulate in renewable tissues with age and at sites showing age - related pathologies like osteoarthritis and atherosclerosis . By only looking at cell morphology, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues . Various studies have confirmed that senescent - associated gal (sa-gal) is same as normal lysosomal gal . However; in senescence, overproduction of this protein increases the overall activity of this enzyme . The normal lysosomal gal is active at ph 4, but sa-gal activity can be easily detected at ph 6 . This might be due to presence of high concentration of sa-gal enzymes in senescent cells . On the basis of this observation that at a higher ph, sa-gal activity can be detected, various protocols have been adapted to detect sa-gal activity in tissues and cell cultures . Particularly, the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d - galactosidase (x - gal) has been extensively used to detect the sa-gal activity in vivo . Gal cleaves x - gal and produce an insoluble blue compound, which is detectable in situ . The x - gal staining is a very useful assay to design a reliable protocol for the identification of senescent cells quickly with simple sectioning and staining procedure . Importantly, an increase in sa-gal activity detected in human skin tissues correlated with the old age . We were able to detect sa-gal activity successfully in skin samples obtained through rapid necropsy of dead dogs . Gluteraldehyde, 50% solution, reagent grade and magnesium chloride; anhydrous; high purity grade was obtained from amresco (solon, ohio, usa). Potassium ferrocyanide (extrapure analytical reagent), potassium ferricyanide, and egta (ethylene glycol tetraacetic acid) were obtained from sisco research lab pvt ltd (mumbai, india). The fixative solution was prepared with gluteraldehyde (0.50%), egta (1.25 mm; ph 6 or 7.3), mgcl2 (2.00 mm) and 1 phosphate buffered saline (pbs) (ph 6 or 7.3). The wash buffer was made up with mgcl2 (2 mm), np-40 (0.02%) and 1 pbs . And the x - gal stain was prepared with x - gal (0.6 mg / ml), k ferrocyanide (4 mm), k ferricyanide (4 mm), and wash buffer . All the tissue samples used in this study were collected during necropsy of dogs that have died just 10 - 15 min before the necropsy procedure . Particularly, we collected sample from those dogs whose exact birth record was available with the owner . We also restricted us in collecting and using samples from dogs that have died not due to any chronic disease . Most of the samples were collected from dogs that encountered accident and presented at the college of veterinary science and animal husbandry, orissa for treatment, but later on succumbed to death . These cases were referred to the department of veterinary pathology for postmortem / necropsy . In this way, we were able to collect samples from three dogs (two old and one young). Further tissue storage and processing was done at the institute of life sciences, bhubaneswar . As samples were collected from dead animals and used in a nonprofitable research study however, before collecting the samples, we took the prior consent of the corresponding owners . Previous studies have shown that storage of snap frozen tissues might reduce the activity of sa-gal activity . Therefore, the snap frozen tissues were processed on the same day for the x - gal staining . Out of curiosity, we also kept one fragment of the snap frozen tissue in -80c and 7 days later processed for further sectioning and staining as described below . Tissues were taken out of liquid nitrogen or -80c and immediately embedded in tissue freezing medium (leica microsystems nussloch gmbh). Tissue sections of 7 - 15 micron thickness were made by using cryomicrotome (leica cm1850). After sectioning, tissues were fixed over the charged slides . Until beginning of the staining procedure, for the x - gal staining procedure all the working solutions were made fresh from corresponding stocks . This was followed by incubation of slides with x - gal staining solution for more than 12 h in 37c incubator . Two beakers containing water were kept inside the incubator for maintaining the humidity and protecting from drying of staining solution (x - gal). Last, slides were mounted with aqueous mounting solution (vectamount aq; vector laboratories, inc . ; a consecutive frozen slide was stained using h and e. all slides were observed under leica dm500 light microscope and representative photographs were taken . Gluteraldehyde, 50% solution, reagent grade and magnesium chloride; anhydrous; high purity grade was obtained from amresco (solon, ohio, usa). Potassium ferrocyanide (extrapure analytical reagent), potassium ferricyanide, and egta (ethylene glycol tetraacetic acid) were obtained from sisco research lab pvt ltd (mumbai, india). The fixative solution was prepared with gluteraldehyde (0.50%), egta (1.25 mm; ph 6 or 7.3), mgcl2 (2.00 mm) and 1 phosphate buffered saline (pbs) (ph 6 or 7.3). The wash buffer was made up with mgcl2 (2 mm), np-40 (0.02%) and 1 pbs . And the x - gal stain was prepared with x - gal (0.6 mg / ml), k ferrocyanide (4 mm), k ferricyanide (4 mm), and wash buffer . All the tissue samples used in this study were collected during necropsy of dogs that have died just 10 - 15 min before the necropsy procedure . Particularly, we collected sample from those dogs whose exact birth record was available with the owner . We also restricted us in collecting and using samples from dogs that have died not due to any chronic disease . Most of the samples were collected from dogs that encountered accident and presented at the college of veterinary science and animal husbandry, orissa for treatment, but later on succumbed to death . These cases were referred to the department of veterinary pathology for postmortem / necropsy . In this way, we were able to collect samples from three dogs (two old and one young). Further tissue storage and processing was done at the institute of life sciences, bhubaneswar . As samples were collected from dead animals and used in a nonprofitable research study, we did not seek any animal ethical committee approval . However, before collecting the samples, we took the prior consent of the corresponding owners . Previous studies have shown that storage of snap frozen tissues might reduce the activity of sa-gal activity . Therefore, the snap frozen tissues were processed on the same day for the x - gal staining . Out of curiosity, we also kept one fragment of the snap frozen tissue in -80c and 7 days later processed for further sectioning and staining as described below . Tissues were taken out of liquid nitrogen or -80c and immediately embedded in tissue freezing medium (leica microsystems nussloch gmbh). Tissue sections of 7 - 15 micron thickness were made by using cryomicrotome (leica cm1850). After sectioning, tissues were fixed over the charged slides . Until beginning of the staining procedure, slides with the tissue were kept on ice . For the x - gal staining procedure initially, slides were placed in fixative for 30 min at 4c . Then slides were rinsed with wash buffer for 4 5 min . This was followed by incubation of slides with x - gal staining solution for more than 12 h in 37c incubator . Two beakers containing water were kept inside the incubator for maintaining the humidity and protecting from drying of staining solution (x - gal). Last, slides were mounted with aqueous mounting solution (vectamount aq; vector laboratories, inc . ; a consecutive frozen slide was stained using h and e. all slides were observed under leica dm500 light microscope and representative photographs were taken . As discussed above, the key to distinguish a lysosomal gal activity from sa-gal activity is staining at different ph - range (4 or more than 6). In the current study, we noticed x - gal staining both at ph 6 and ph 7.3 . As reported earlier, we observed a reduction of sa-gal activity after freezing of tissues in -80c . However, even after 5 - 6 days of storage in -80c, we were able to detect x - gal staining; though of lower intensity . This observation is not in accordance with a previously reported statement that even overnight storage of tissue samples at -80c can destroy the enzyme activity . The staining was detectable after minimum of 10 h incubation and staining intensity went on increasing with an increase in incubation time . Staining was mostly noticed at the inner epithelial layers of hair follicles and sebaceous glands [figure 1a and b]. Further, the x - gal staining intensity was more intense at ph 6.0 than ph 7.3 [figure 2a and b]. Overall, the number of hair follicles positive for x - gal was more at ph 6.0 than ph 7.3 . We did not have enough samples to make a comparison of x - gal positive cells (%) in young and old age animals; however, in the tissue of only one young dog available with us, we did not see any staining even after 20 h of incubation (data not shown). Histology of a dog skin tissue stained with h and e or x - gal (a) h and e stained dog skin tissue shows presence of various cellular components of epidermis and dermis . Arrows (yellow) indicate follicular epithelia cells (compound follicle), stars indicate stromal cells, and triangle indicates epithelial cells of a sebaceous gland . For h and e staining, frozen skin sample of a greatdane dog (~5 years old) was cryosectioned and processed by usual h and e staining procedure . (b) x - gal stain of a corresponding tissue shows clear x - gal staining (red arrow) in the region of follicular epithelium and sebaceous gland . The tissue was from the same dog and processed for x - gal staining at ph 7.3 . Note: tissue used in this experiment was preserved at -80c for 5 days after its snap freezing in liquid nitrogen x - gal staining at different ph (a) gross appearance of canine skin tissues stained with x - gal . All the staining regents were adjusted to ph 6 or 7.3 . Cryosectioned skin tissues were stained for x - gal at different ph, and after overnight (12 h) staining, photos of those slides were taken by normal digital camera . Staining at ph 6 produced more intense color (blue) than at ph 7.3 . (b) microscopic images of tissues stained at different ph of staining solutions . More intense x - gal staining (blue) was observed at ph 6 than ph 7.3 knowledge about the age of a dog might have multiple uses in clinic and research . Currently, available techniques for the detection of a dog's age are not very reliable . The measurement of gal activity has been proposed to be a marker for the estimation of a human age . However, not much effort has been made to detect the expression of this protein in other species like dog . To best of our knowledge, the current study is the first report regarding possible detection of endogenous gal activity in dog skin tissue . Detection of gal activity in skin tissues obtained through rapid necropsy showed the feasibility of detecting this molecule's activity from just died and most potentially from live animal's skin . Further, detection of x - gal staining efficiently in the epidermis of skin suggests requirement of a small amount of superficial skin for carrying out this analysis . X - gal staining of canine tissues might be instrumental in estimating the proper age of a dog it has also potential use in forensic science including wildlife forensic, where age estimation is warranted . For better use of this technique, an extensive analysis is required to get the actual idea about sa-gal activity in different age, sex, and breed of dogs . We also believe that skin tissues from different regions of the same animal might have different number of senescent cells; therefore, a more in - depth study is required before the adaptation of x - gal staining as a marker for old age of dogs . Lac - z, the bacterial gal, has been widely used as a reporter gene in many animal models including dogs . In most of the gene therapy studies in dog, lac - z has a similarity with endogenous gal of other species; therefore, an idea about expression level of endogenous gal expression / activity in different organs of this animal, might help to carry out x - gal staining in a more careful fashion and proper interpretation of the results . In various literatures, most of the protocols recommend ph 7.3/7.4 for lac - z staining; however, at this ph we were also able to detect endogenous -gal activity . Therefore, during lac - z staining of canine tissues, tissue from the control animals should be used to avoid confusion of background staining . Furthermore, detection of senescent cells through x - gal staining in canine tissues will help in checking the coexistence and functional significance of these cells in different chronic diseases like cancer . Taken together, we believe that our current report has shown the possibility of carrying out x - gal staining with canine tissues and has created an opportunity to exploit this technique for several clinical and research studies . On the basis of the current study, following conclusions are made: sa-gal activity in dog is detectable through x - gal staining in situ;use of x - gal staining reagents with higher ph (7.3) might help in detecting more specifically senescent cells than previously suggested ph (6);x - gal staining is possible with tissues collected immediately after death;storage of tissues at -80c reduce sa-gal activity; however, x - gal staining is still feasible in those frozen tissues;x - gal staining of dog skin tissue has the potential to be explored as a marker of senescence or old age . Sa-gal activity in dog is detectable through x - gal staining in situ; use of x - gal staining reagents with higher ph (7.3) might help in detecting more specifically senescent cells than previously suggested ph (6); x - gal staining is possible with tissues collected immediately after death; storage of tissues at -80c reduce sa-gal activity; however, x - gal staining is still feasible in those frozen tissues; x - gal staining of dog skin tissue has the potential to be explored as a marker of senescence or old age.
Three - dimensional (3d) image analysis of the mandible is possible due to the wide availability of computed tomography (ct) imaging technology . Accordingly, there have been several anatomical analyses of the mandibular foramen (mnf),1234 most involving distance - only measurement methods . Moreover, the analytical data for the mandible are limited with respect to their relationship with skeletal malocclusion, the size of the mandible, and the measurement planes and reference points for locating the position of the mnf . The position of the mnf has been studied for mandibular treatment (i.e. Ramal surgery, including osteotomy; inferior alveolar nerve block).56 the present study used 3d images of the mandibles of patients diagnosed with skeletal class iii malocclusion, and analyzed the position of the mnf (i.e. Its proportional position in the mandible) using measurement planes and anatomical reference points . The purpose of this study was to produce a useful guide of mnf positions in patients diagnosed with skeletal class iii malocclusion . Specifically, the study used ct images of mandibles that were taken during preoperative diagnosis work - ups . The ct images were taken with a siemens sensation 64 system (1.0 mm slice thickness, siemens sensation 64 ct scanner; siemens ag, erlangen, germany) using a pixel size of 0.4375 mm and a field - of - view of 22.40 cm . We excluded ct data from facial asymmetry patients with a mandibular shift greater than 5 mm (considered more than a moderate state of asymmetry).7 the ct data were collected for a total of 85 patients, and both right and left mnf images were analyzed . Simplant version 14.0 (materialise dental, leuven, belgium) was used to reconstruct the ct images into 3d images . The anatomical reference points, lines, and planes were then set onto the image of the mandible . The 3d position of the mnf was measured based on the established reference points and planes . This process was intended to help the surgical team locate the mnf during mandibular ramus osteotomy . The anatomic points, lines, and planes that were used, as well as the measurements, are described below . The mnf, coronoid process, sigmoid notch, condyle, gonion, antegonial notch, menton, mesiobuccal cusp of the mandibular first molar, and the most superior incisal contact point of the lower central incisor were identified in the images . In addition, the points of the anterior and posterior ramus were defined (table 1). The anatomic lines used for defining the reference planes in this study are shown in table 2 . Three vertical lines were constructed: one connecting the sigmoid notch points and antegonial notch points, termed the sn - agn line; another connecting a sigmoid notch point and gonion point, termed the sn - gn line; and the other connecting a condyle point and a gonion point, termed the cond - gn line . In addition, five horizontal lines were constructed that connected each of the anterior ramus points and each of the posterior ramus points . The occlusal plane, mandibular plane, and five reference horizontal planes were defined (table 3, fig . Five vertical coronal planes were also defined, as shown in table 3 and figure 1b . The vertical heights of the anatomic points were measured according to each of the five horizontal planes (fig . The vertical distances from the coronoid process, sigmoid notch, and condyle to each horizontal plane were added to the vertical distances from the gonion to each horizontal plane, and these distances were set as the vertical heights of the coronoid process, sigmoid notch, and condyle in the mandible for each horizontal plane (table 4). The vertical distances from the gonion to the horizontal planes were set as the heights of the mnf in the mandible, according to each horizontal plane . Based on each of the five horizontal planes that pass through the mnf, the distance on each plane from the anterior ramus point to the posterior ramus point was designated the anteroposterior horizontal distance of the ramus (table 4). The perpendicular distance from each anterior ramus point to each vertical plane was designated the horizontal distance from the anterior ramus to the mnf (fig . 3). To investigate the positional relationship of the mnf in the mandible, regression analysis was used to examine the rational relationships between the vertical heights of the coronoid process, sigmoid notch, and condyle points and the vertical height of the mnf . Regression analysis was also used to determine the rational relationship between the anteroposterior horizontal distance of the ramus from each of the five horizontal planes passing through the mnf and the horizontal distance from the anterior ramus point to the mnf ., armonk, ny, usa), and statistical significance was established at p<0.05 . The intra - observer error for anatomic point determination was calculated via repeated measurement (10 measurements) of the mnf . Simplant version 14.0 (materialise dental, leuven, belgium) was used to reconstruct the ct images into 3d images . The anatomical reference points, lines, and planes were then set onto the image of the mandible . The 3d position of the mnf was measured based on the established reference points and planes . This process was intended to help the surgical team locate the mnf during mandibular ramus osteotomy . The anatomic points, lines, and planes that were used, as well as the measurements, are described below . The mnf, coronoid process, sigmoid notch, condyle, gonion, antegonial notch, menton, mesiobuccal cusp of the mandibular first molar, and the most superior incisal contact point of the lower central incisor were identified in the images . In addition, the points of the anterior and posterior ramus were defined (table 1). The anatomic lines used for defining the reference planes in this study are shown in table 2 . Three vertical lines were constructed: one connecting the sigmoid notch points and antegonial notch points, termed the sn - agn line; another connecting a sigmoid notch point and gonion point, termed the sn - gn line; and the other connecting a condyle point and a gonion point, termed the cond - gn line . In addition, five horizontal lines were constructed that connected each of the anterior ramus points and each of the posterior ramus points . The occlusal plane, mandibular plane, and five reference horizontal planes were defined (table 3, fig . Five vertical coronal planes were also defined, as shown in table 3 and figure 1b . The vertical heights of the anatomic points were measured according to each of the five horizontal planes (fig . The vertical distances from the coronoid process, sigmoid notch, and condyle to each horizontal plane were added to the vertical distances from the gonion to each horizontal plane, and these distances were set as the vertical heights of the coronoid process, sigmoid notch, and condyle in the mandible for each horizontal plane (table 4). The vertical distances from the gonion to the horizontal planes were set as the heights of the mnf in the mandible, according to each horizontal plane . Based on each of the five horizontal planes that pass through the mnf, the distance on each plane from the anterior ramus point to the posterior ramus point was designated the anteroposterior horizontal distance of the ramus (table 4). The perpendicular distance from each anterior ramus point to each vertical plane was designated the horizontal distance from the anterior ramus to the mnf (fig . To investigate the positional relationship of the mnf in the mandible, regression analysis was used to examine the rational relationships between the vertical heights of the coronoid process, sigmoid notch, and condyle points and the vertical height of the mnf . Regression analysis was also used to determine the rational relationship between the anteroposterior horizontal distance of the ramus from each of the five horizontal planes passing through the mnf and the horizontal distance from the anterior ramus point to the mnf ., armonk, ny, usa), and statistical significance was established at p<0.05 . The intra - observer error for anatomic point determination was calculated via repeated measurement (10 measurements) of the mnf . The sex ratio and age of the 85 patients with skeletal class iii mandibular prognathism were 46: 39 (men: women) and 22.25.5 years . The condyle height relative to the mnf - mandibular plane was the smallest (53.456.45 mm). The heights of the coronoid process and the mnf were the greatest when measured relative to the mnf - mandibular plane (60.095.96 mm and 21.313.83 mm, respectively). The height of the sigmoid notch was the greatest when based on the mnf - sn / gn p plane (43.895.02 mm). The height of the condyle was the greatest when based on the mnf - cond / gn p plane (63.805.79 mm). The heights of the mnf, coronoid process, and sigmoid notch were the smallest when based on the mnf - cond / gn p plane (18.604.56 mm, 50.086.89 mm, and 40.505.45 mm, respectively). The anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were the longest when they were based on the mnf - mandibular plane (42.985.17 mm and 27.604.22 mm, respectively; table 5). The heights of the coronoid process, sigmoid notch, and condylar head on the five horizontal planes were all significantly associated with the height of the mnf according to the regression analysis . The regression relationship was the strongest for the coronoid process, sigmoid notch, and condylar head when the measurements were based on the mnf - mandibular plane (coefficients of determination (r) of 0.424, 0.597, and 0.604 respectively). The heights of the coronoid process and the mnf were significantly associated according to the regression analysis (table 6). The non - standardized regression coefficients were significant for the relationships between the heights of the coronoid process and the mnf based on all the horizontal reference planes . The highest coefficients, based on the mnf - mandibular plane, were a standardized regression coefficient of 0.654 and determination coefficient (r) of 0.424 . The heights of the sigmoid notch and the mnf were significantly associated in the regression analysis (table 7). The non - standardized regression coefficients for the relationships between the heights of the sigmoid notch and the mnf were significant for all the horizontal reference planes . The regression constant was highly significant when it was based on the mnf - mandibular plane (p=0.002). The highest coefficients were a standardized regression coefficient of 0.774 and determination coefficient of 0.597, and these were based on the mnf - mandibular plane . The heights of the condylar head and the mnf were significantly related in the regression analysis (table 8). For all the horizontal reference planes, the non - standardized regression coefficients for the relationships between the heights of the condylar head and the mnf were significant . The regression constant was significant when it was based on all of the horizontal planes, except for the mnf - sn / gn p plane . The highest coefficients were a standardized regression coefficient of 0.779 and a determination coefficient of 0.604, and were based on the mnf - mandibular plane . Using the five horizontal planes as references, the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf for all the horizontal reference planes, the non - standardized regression coefficients were significant for the relationships of the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf . The regression constants were significant when the mnf - mandibular plane and mnf - sn / gn p plane were used as references . The standardized regression coefficients for all five horizontal planes used as references were greater than 0.73, and the coefficient was 0.877 using the mnf - mandibular plane and 0.901 using the mnf - sn / gn p plane . The determination coefficient was 0.766 using the mnf - mandibular plane and 0.811 using the mnf - sn / gn p plane . Notably, there were 53 missing values for measurements using the mnf - mandibular plane and 3 missing values for the mnf - sn / gn p plane . For the mnf - mandibular plane, which is parallel to the mnf, there were missing values when the anterior part of the plane sloped downward, thus leading directly to the mandibular body instead of the anterior ramus . Similarly, there were missing values for the mnf - sn / gn p plane, although there were fewer than for the mnf - mandibular plane . The heights of the coronoid process, sigmoid notch, and condylar head on the five horizontal planes were all significantly associated with the height of the mnf according to the regression analysis . The regression relationship was the strongest for the coronoid process, sigmoid notch, and condylar head when the measurements were based on the mnf - mandibular plane (coefficients of determination (r) of 0.424, 0.597, and 0.604 respectively). The heights of the coronoid process and the mnf were significantly associated according to the regression analysis (table 6). The non - standardized regression coefficients were significant for the relationships between the heights of the coronoid process and the mnf based on all the horizontal reference planes . The highest coefficients, based on the mnf - mandibular plane, were a standardized regression coefficient of 0.654 and determination coefficient (r) of 0.424 . The heights of the sigmoid notch and the mnf were significantly associated in the regression analysis (table 7). The non - standardized regression coefficients for the relationships between the heights of the sigmoid notch and the mnf were significant for all the horizontal reference planes . The regression constant was highly significant when it was based on the mnf - mandibular plane (p=0.002). The highest coefficients were a standardized regression coefficient of 0.774 and determination coefficient of 0.597, and these were based on the mnf - mandibular plane . The heights of the condylar head and the mnf were significantly related in the regression analysis (table 8). For all the horizontal reference planes, the non - standardized regression coefficients for the relationships between the heights of the condylar head and the mnf were significant . The regression constant was significant when it was based on all of the horizontal planes, except for the mnf - sn / gn p plane . The highest coefficients were a standardized regression coefficient of 0.779 and a determination coefficient of 0.604, and were based on the mnf - mandibular plane . Using the five horizontal planes as references, the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf were significantly associated according to the regression analysis (table 9). For all the horizontal reference planes, the non - standardized regression coefficients were significant for the relationships of the anteroposterior length of the ramus and the distance from the anterior ramus point to the mnf . The regression constants were significant when the mnf - mandibular plane and mnf - sn / gn p plane were used as references . The standardized regression coefficients for all five horizontal planes used as references were greater than 0.73, and the coefficient was 0.877 using the mnf - mandibular plane and 0.901 using the mnf - sn / gn p plane . The determination coefficient was 0.766 using the mnf - mandibular plane and 0.811 using the mnf - sn / gn p plane . Notably, there were 53 missing values for measurements using the mnf - mandibular plane and 3 missing values for the mnf - sn / gn p plane . For the mnf - mandibular plane, which is parallel to the mnf, there were missing values when the anterior part of the plane sloped downward, thus leading directly to the mandibular body instead of the anterior ramus . Similarly, there were missing values for the mnf - sn / gn p plane, although there were fewer than for the mnf - mandibular plane . The size of the human mandible varies greatly according to age, ethnicity, and sex.2 accordingly, the present study investigated the relative position of the mnf rather than simply measuring its position within the surrounding anatomical structures . Using specific anatomic planes as references, we measured the position of the mnf in patients diagnosed with skeletal class iii malocclusion to determine the 3d position in the mandible . We determined the position of the mnf was related vertically to the heights of the mandibular condyle, sigmoid notch, and coronoid process and horizontally to the anteroposterior length of the mandibular ramus . Further, we found that the mandibular plane was best for determining the relationships of the heights of the mandibular condyle, sigmoid notch, and coronoid process with the height of the mnf . This suggests that the mandibular plane can be utilized in clinical cases, not only for analyzing maxillofacial malformations but also for tracing the position of the mnf . During mandibular prognathism surgery, the vertical position of the mnf can be estimated using the exposed sigmoid notch area . With reference to the mandibular plane, the height (in millimeters) of the mnf can be estimated with the following equation: 0.621(the height of the sigmoid notch) - 5.318 . As an alternative to the mandibular plane, one can use the horizontal plane perpendicular to the sn - gn line that connects the sigmoid notch and gonion; this plane (the mnf - sn / gn p plane) is second to the mandibular plane in terms of usefulness for estimating the height of the mnf . When using the condyle height, the occlusal plane can be a useful alternative for estimating the height of the mnf . Vertical ramus osteotomy is a surgical procedure that can be used for patients with mandibular prognathism when the mandibular ramus posterior to the mnf is cut . The relationship between the anteroposterior length of the mandibular ramus and the position of the mnf was best shown when the horizontal mnf - sn / gn p and mnf - mandibular planes were used as references . The mandibular ramus area at the mnf level is exposed during mandibular prognathism surgery . Therefore, the relationship of the horizontal position of the mnf with the anteroposterior length of the ramus was investigated . Using the mnf - sn / gn p plane as a reference, the horizontal length (in mm) from the anterior ramus to the mnf can be estimated with the following equation: 0.728(the anteroposterior distance of the mandibular ramus)- 3.948 . Using the mnf - mandibular plane as the reference, the horizontal length (in millimeters) from the anterior ramus to the mnf can be estimated with the following equation: 0.716(the anteroposterior distance of the ascending ramus)- 3.148 . However, there are cases in which the mandibular plane leans in the anteroinferior direction, and the mnf - mandibular plane cannot pass through the anterior ascending ramus area . In the present study, there were 53 missing values for the anterior ramus point when using the mnf - mandibular plane as the reference, and there were 3 missing values when using the mnf - sn / gn p plane . Missing values may limit the usefulness of these calculations since they may occur in actual clinical cases . If this happens, it is recommended that the mnf - sn / gn p plane be used, and the use of the occlusal plane may be more helpful for determining the horizontal position of the mnf when the anterior ramus point cannot be defined using the mnf - mandibular plane . The vertical height of the mnf from the gonion ranged from 18 mm to 21 mm, and the anteroposterior length of the ramus was about 33 mm to 42 mm . The horizontal distance from the anterior ramus point to the mnf was about 20 mm to 27 mm . The wide availability of ct scans has resulted in useful 3d images of the mandible . In a study that analyzed the 3d position of the mnf using cone - beam ct, the average distance from the gonion to the mnf was about 24 mm and the average distance from the anterior ramus to the mnf in the axial plane was about 15 mm.2 the difference between the results of that study versus the present study is likely due to the use of the axial and sagittal planes of the ct images in the former . In another study that analyzed the 3d position of the mnf using multiple detector ct, the average height from the gonion to the mnf relative to the occlusal plane was about 18 mm, the average anteroposterior length of the ascending ramus was 37 mm, and the average distance from the anterior ramus to the mnf on a horizontal plane (parallel to the occlusal plane that passes through the mnf) was about 22 mm.8 these results are similar to those of the present study, although there are minor differences because the present study only examined mandibles with skeletal iii class malocclusion, and the horizontal length from the anterior ramus to the mnf was measured as the perpendicular distance projected onto each vertical plane that passed through the mnf . Simply measuring the vertical and horizontal distances may not be that beneficial in actual clinical cases because human mandibles vary in size . However, assessing the positional relationships using 3d planes can be useful clinically, as this shows the objective relationship with respect to the reference planes used in the analyses . Image - assisted surgical techniques are increasingly used for maxillofacial operations; accordingly, the present study is valuable in that it provides 3d image data that can be utilized in such surgical procedures . This study's research methods and results, with respect to the positional 3d analysis of the mnf, will be useful for computer - assisted surgical methods, such as surgical simulation, navigation, augmented reality models, and robotic surgery . Previous studies of the mandible were performed on cadavers,910 and often just a few distances were measured in 3d reconstructed images even though ct images were examined.234 importantly, previous studies performed quantitative analyses without considering the different sizes of the mandibles . Moreover, even when 3d reconstruction images were used, few studies performed vertical and horizontal assessments using 3d reference planes . The present study found that the position of the mnf was related to the vertical heights of the sigmoid notch, coronoid process, and condyle and to the horizontal anteroposterior length of the ascending ramus . These findings indicate that the mnf, at least to some extent, maintains its relative position with the growth of the rami . In addition, the results indicate that the mnf is posterior to the ascending ramus . We expect that future studies will contribute further knowledge about the position of the mnf in the mandible via 3d image analysis of mandibles with skeletal class ii malocclusion for ramus surgery . In conclusion, the 3d mandibular image analysis data reported in this study will be useful for defining and describing the position of the mnf in mandibles of different sizes and shapes and in different ethnic groups . The present study showed that the proportional position of the mnf was significantly related to the vertical heights of the sigmoid notch, coronoid process, and condyle and to the horizontal anteroposterior length of the ascending ramus . Furthermore, the methods used here and the positional 3d analyses of the mnf can be used clinically for computer - assisted surgical methods, especially for ramal surgery in patients with skeletal class iii mandibular prognathism.
Dengue has become the most common mosquito - borne viral disease worldwide . According to estimates by the world health organization (who), the global incidence of dengue infections ranges between 50 million and 200 million every year . However, the real disease burden of dengue may be underestimated because of inadequate disease surveillance, misdiagnosis, and low levels of reporting . One recent study, based on 1780 country - years of mortality data from 130 countries and 1636 country - years of dengue case reports from 76 countries, estimated an average of 9221 dengue deaths per year between 1990 and 2013 . Taiwan is no exception, and several dengue infection outbreaks have recently been reported here . In 2015, a large outbreak of dengue infections caused by dengue serotype 2 developed in tainan, taiwan, and a significant portion of patients with severe dengue infections required intensive care unit (icu) admission . Because the outcomes of adult patients with dengue infections requiring icu admissions have rarely been investigated, we did this retrospective study to assess the clinical manifestations and the prognostic factors of patients critically ill with severe dengue infections . This study was done at chi mei medical center, a tertiary referral hospital with 96 adult icu beds . In this retrospective study, all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31, 2015, during the large outbreak period . The data were routinely collected and the analyses were done retrospectively . Therefore, the study was approved by the institutional review board of chi mei medical center (irb10503 - 005), and informed consent was waived . The following information was collected: age, gender, and severity scores the acute physiology and chronic health evaluation ii (apache ii) score, therapeutic intervention scoring system (tiss), and glasgow coma scale (gcs) score . Underlying comorbidities included congestive heart failure, chronic lung diseases, end - stage renal disease, liver cirrhosis, diabetes mellitus, and cancer . We also measured immunocompromised conditions, associated infections and organisms, the number of multiorgan failures, the use of a mechanical ventilator and continuous renal replacement therapy (crrt), associated laboratory data at or during admission, symptoms presented in the emergency room, and patient outcomes . Laboratory - confirmed dengue cases included those with at least one of the following positive laboratory results: nonstructural protein 1 antigen test, dengue immunoglobulin - m, dengue immunoglobulin - g, a dengue polymerase chain reaction, or viral isolation . Thoracic failure was defined as the ratio of the partial pressure of oxygen in the patient's arterial blood to the fraction of oxygen in the inspired air less than 200 . Cardiovascular failure was defined as a systolic blood pressure (sbp) of 90 mm hg or a mean arterial pressure (map) 65 mm hg for at least 1 hour despite adequate fluid resuscitation; or the need for vasoactive agents (dopamine 5 mg / kg / min) to maintain sbp 90 mm hg or map 65 mm hg . Metabolic acidosis was defined as a ph 7.30 or a base deficit 5.0 meq / l and a plasma lactate level> 3 mmol / l . Hematologic failure was considered a platelet count <80,000/mm or a 50% decrease in the platelet count from the highest value recorded over the previous 3 days . Kidney failure was considered oliguria with an average urine output <0.5 ml / kg / h for 4 hours despite adequate fluid resuscitation or a creatinine level 2 mg / dl . Hepatic failure was defined as a markedly increased serum bilirubin level 4 mg / dl . According to the who 2009 criteria, patients were classified as having dengue with warning signs if there were reports of abdominal pain or tenderness, vomiting, clinical fluid accumulation, mucosal bleeding, lethargy or restlessness, hepatomegaly, and a rise in hematocrit concurrent with a rapid drop in the platelet count . Severe dengue (group c) criteria included the following symptoms: severe plasma leakage leading to shock or fluid accumulation with respiratory distress, severe bleeding, severe organ involvement, or transaminase levels> 1000 those who were diagnosed with severe dengue by emergency physicians were admitted to the icu and included in this study . The focus of bacterial infections was diagnosed on the basis of clinical, laboratory, and radiologic findings . Patients with bacteremia were divided into 2 subgroups: secondary bacteremia caused by another primary focus, such as a respiratory tract or urinary tract infection, and if no primary focus could be identified, the bacteremia was classified as primary . Continuous variables are expressed as means standard deviation . The differences between groups, and between survivors and nonsurvivors at hospital discharge, continuous data were compared using student t test or the wilcoxon rank - sum test . Patients significantly associated with in - hospital mortality in univariate analysis (p <this study was done at chi mei medical center, a tertiary referral hospital with 96 adult icu beds . In this retrospective study, all of the patients with laboratory - confirmed severe dengue infections and admitted to the icu were enrolled between july 31 and november 31, 2015, during the large outbreak period . The data were routinely collected and the analyses were done retrospectively . Therefore, the study was approved by the institutional review board of chi mei medical center (irb10503 - 005), and informed consent was waived . The following information was collected: age, gender, and severity scores the acute physiology and chronic health evaluation ii (apache ii) score, therapeutic intervention scoring system (tiss), and glasgow coma scale (gcs) score . Underlying comorbidities included congestive heart failure, chronic lung diseases, end - stage renal disease, liver cirrhosis, diabetes mellitus, and cancer . We also measured immunocompromised conditions, associated infections and organisms, the number of multiorgan failures, the use of a mechanical ventilator and continuous renal replacement therapy (crrt), associated laboratory data at or during admission, symptoms presented in the emergency room, and patient outcomes . Laboratory - confirmed dengue cases included those with at least one of the following positive laboratory results: nonstructural protein 1 antigen test, dengue immunoglobulin - m, dengue immunoglobulin - g, a dengue polymerase chain reaction, or viral isolation . Thoracic failure was defined as the ratio of the partial pressure of oxygen in the patient's arterial blood to the fraction of oxygen in the inspired air less than 200 . Cardiovascular failure was defined as a systolic blood pressure (sbp) of 90 mm hg or a mean arterial pressure (map) 65 mm hg for at least 1 hour despite adequate fluid resuscitation; or the need for vasoactive agents (dopamine 5 mg / kg / min) to maintain sbp 90 mm hg or map 65 mm hg . Metabolic acidosis was defined as a ph 7.30 or a base deficit 5.0 meq / l and a plasma lactate level> 3 mmol / l . Hematologic failure was considered a platelet count <80,000/mm or a 50% decrease in the platelet count from the highest value recorded over the previous 3 days . Kidney failure was considered oliguria with an average urine output <0.5 ml / kg / h for 4 hours despite adequate fluid resuscitation or a creatinine level 2 mg / dl . Hepatic failure was defined as a markedly increased serum bilirubin level 4 mg / dl . According to the who 2009 criteria, patients were classified as having dengue with warning signs if there were reports of abdominal pain or tenderness, vomiting, clinical fluid accumulation, mucosal bleeding, lethargy or restlessness, hepatomegaly, and a rise in hematocrit concurrent with a rapid drop in the platelet count . Severe dengue (group c) criteria included the following symptoms: severe plasma leakage leading to shock or fluid accumulation with respiratory distress, severe bleeding, severe organ involvement, or transaminase levels> 1000 those who were diagnosed with severe dengue by emergency physicians were admitted to the icu and included in this study . The focus of bacterial infections was diagnosed on the basis of clinical, laboratory, and radiologic findings . Patients with bacteremia were divided into 2 subgroups: secondary bacteremia caused by another primary focus, such as a respiratory tract or urinary tract infection, and if no primary focus could be identified, the bacteremia was classified as primary . Continuous variables are expressed as means standard deviation . The differences between groups, and between survivors and nonsurvivors at hospital discharge, continuous data were compared using student t test or the wilcoxon rank - sum test . Patients significantly associated with in - hospital mortality in univariate analysis (p <during the study period, there were 4787 patients with dengue infections, 143 (2.99%) of whom were critically ill and required icu admission (mean age: 69.7 years; age range: 694 years) (table 1). Thirty - three of these patients died (mortality rate: 23.1% for icu patients and the average apache ii, tiss, and gcs scores were 17.9, 22.8, and 12.0, respectively . Hypertension (n = 90; 62.9%) and diabetes mellitus (n = 70; 49.0%) were the 2 most common comorbidities . Eighty patients had cobacterial infections: the lungs (pneumonia) (n = 40) and the urinary tract (n = 33) were the most common sites . Thirty - three of the 80 patients had cobacteremia: 11 primary bacteremia cases and 22 secondary bacteremia cases; 59 (41.3%) had multiorgan failure: the hematologic system failed most frequently, followed by thoracic and cardiovascular systems . Overall, there were 50 (35.0%) patients who required mechanical ventilation and 20 (14.0%) who required crrt . The patients who died in hospital had significantly higher apache ii, tiss, and gcs scores, and more hypertension, bacteremia, pneumonia, and organ failure (cardiovascular, thoracic, metabolic, renal and hepatic failures, and multiorgan failure) and needed more mechanical ventilation and crrt than did survivors . . The levels of procalcitonin and c - reactive protein (crp) were 12.3 (34.7) g / l and 49.0 (72.8) mg / l, respectively (table 2). The mean levels of the n - terminal prohormone brain natriuretic peptide and of lactate were 4788.5 (7492.8) and 3.4 (4.1), respectively . The lowest platelet count was 35,000 (43,000)/mm, and it recovered to 151,000 (107,000)/mm before the patients were discharged from the icu . The highest levels of aspartate aminotransferase (ast / got) and alanine aminotransferase (alt / gpt) were 1126.4 (2488.5) the patients who died had significantly higher activated partial thromboplastin time, lactate, ast / got, and alt / gpt levels than did survivors . The patients who died, however, had significantly lower albumin, hemoglobin, and hematocrit, and lower platelet counts in the trough and recovery stage before they were discharged from the icu than did survivors . Fever was the most common presentation symptom (n = 112; 78.3%), followed by anorexia (n = 47; 32.9%) and abdominal pain (n = 46; 32.2%) (table 3). The gastrointestinal tract (n = 45; 31.5%), the genitourinary tract (n = 26; 18.2%), and the gums (n = 3; 2.1%) were the most common sites, in that order, for bleeding . There were no significant differences in the symptoms, the overall icu 7.8 (10.3) and hospital - stay 14.7 (16.1) days, or hospital costs between survivors and patients who died . Mortality was highest for 60- to 69-year - olds (8 died [36.4%]; 14 survived), 70- to 79-year - olds (16 died [24.2%]; 50 survived), and 80- to 89-year - olds (7 died [23.3%]; 23 survived) (fig . Multiple logistic regression analysis showed that icu mortality was significantly positively associated with the following independent predictors: lower gcss, lower platelet counts, and more organ failures before icu discharge (table 4). This is the first study that focuses on dengue patients in taiwan who require icu admission and is one of few studies that investigates this topic anywhere in the world . First, we found that the mortality of this specific group remains as high as 23.1%, which is higher than reported by other studies . In 1 study of 72 critically ill patients with dengue in northern india, 8 patients died (mortality: 11.1%). In another of 198 icu patients with dengue in new delhi, 12 patients died (mortality: 6.1%). In contrast, another multicenter study of 42 adults with dengue hemorrhagic fever or dengue shock syndrome admitted to icu in india reported 8 in - icu deaths (mortality: 19%). A study in brazil of 97 adult patients with dengue admitted to the icu reported in - icu and in - hospital mortality rates of 18.6% and 19.6%, respectively . The differences between our study and others might be the differences in disease severity . In our study, the mean apache ii score was 17.9 9.6, higher than the 7.5 and the 11 in 2 of the others . In addition, only group c (severe dengue patients with warning signs and organ failure) were enrolled in our study, which probably explains why the outcomes of our study were worse . In addition, the mean age of our enrolled patients was about 70 years, older than 2 of the other studies; age itself might also partially contribute to the higher mortality rate . Second, univariate analysis showed that the patients who died had cases of dengue that were more severe, based on apache ii, tiss, and gcs scores, than did survivors . This is similar with other reports that in - hospital mortality was associated with apache ii scores and sequential organ failure assessment scores . In contrast, after multivariate analysis, only the association between lower gcs scores and in - hospital mortality remained significant in the present study . Our finding might indicate that gcs scores, in addition to other commonly used severity score systems, are good predictors of the outcomes of patients with severe dengue . Third, a multivariate analysis showed that a higher number of organ failures was independently associated with mortality in the present study . Like another study which reported that some common etiologies of multiple organ dysfunction dengue shock syndrome and multiple organ dysfunction syndrome directly related to the organ failure index present a significant risk of mortality, we found that patients who died had more thoracic (72.2% vs 20.0%), cardiovascular (72.7% vs 17.3%), and multiorgan (93.9% vs 25.5%) failures than did survivors (both p <0.0001). Other studies have reported that patients who die were more likely to have cardiovascular (100% vs 12%) or respiratory (88% vs 12%) failures than were survivors (both p <0.01), that a dengue viral infection with an acute respiratory failure is a significant mortality risk, and that a lower platelet count before being discharged from the icu was associated with higher in - hospital mortality . Finally, we found that 80 (55.9%) of our patients developed bacterial infections in the icu . Moreover, 15 (45.5%) of the 33 patients who died had pneumonia, and 13 (39.4%) had bacteremia . In another study, 45 (46.4%) patients had had been treated with antibiotics, and 7 (36.8%) of 19 deaths were presumably attributed to bacterial infections associated with dengue . In the present study, the most common pathogen in the 33 cases of confirmed bacteremia was escherichia coli, followed by staphylococcus aureus (s aureus), and streptococcus pp . Despite our finding that s aureus was the most commonly reported copathogen, different from that of other reports, the present study and other reports might indicate the clinical significance of bacterial infections in patients with severe dengue . It should at least emphasize the importance of an early diagnosis of cobacterial infections and prompt antibiotic treatment of patients critically ill with dengue . In the present study, crp levels were nonsignificantly higher in patients who died (66.9) than in survivors (43.4), which does not support the hypothesis that crp is an early predictor of dengue severity in adult patients . We enrolled only patients with severe dengue in the present study and found that crp was a limited outcome predictor in patients with severe dengue . We also found that albumin levels were significantly lower (2.8 0.6 vs 3.1 0.5) and lactate significantly higher (6.2 6.7 vs 2.4 1.9) in patients who died than in survivors, which was consistent with other studies that low serum albumin levels on icu admission were associated with worse outcomes . Although a multivariate analysis eliminated the significance in our study, it still indicates an association between albumin levels and the outcomes in patients with severe dengue . First, because it is a single - center study, the number of cases is limited . Second, because of the study design of a retrospective investigation, our data collection might have been inadequate . Third, we did not test the serotypes of dengue virus because our laboratory cannot do that type of examination . However, several studies have reported the association between dengue virus serotypes and the severity of dengue infection . In addition, the predictors of icu mortality by multiple logistic regression analysis should be considered as the association with mortality but not the biological causes of death . Finally, despite several reports showed the association between different medications, such as n - acetyl cysteine, factor vii, diuretic, and fluid administration, and the outcome of dengue patients the mortality of severe dengue patients admitted to the icu remains high, and the mortality was associated with lower gcs scores, lower platelet count before being discharged from the icu discharge, and more organ failures . In addition, a significant portion of patients with severe dengue who die in the icu have bacterial infections.
Implant - supported fixed dental prosthesis is a standardized restoration method that is often used when an aesthetically critical anterior single tooth is missing . It has similar 5-year survival rates as tooth - supported fixed dental prosthesis.12 although the success rate of an all - ceramic crown was reported to be lower than that of a metal - ceramic crown, all - ceramic crowns displayed similar success rates in both tooth - supported fixed dental prosthesis and implant - supported fixed dental prosthesis.234 titanium is a stable implant material, and titanium abutments have the advantage of supporting gingival health and preventing galvanic reaction between the fixture and abutment.56 however, when a titanium abutment is used in thin peri - implant mucosa in the anterior area, the metal part can be detected through the mucosa . For this reason, studies on alumina and zirconia, which are high - strength ceramics, have been conducted, and the study on implant abutments using zirconia has advanced, showing excellent material properties and biocompatibility . 78 the influence of the type of connection between a zirconia abutment and a titanium fixture were addressed previously . The types of implant - abutment connection can be divided into two major groups - internal connection and external connection . Sailer et al.910 reported that the internal implant - abutment connection type, including zirconia abutment with titanium insert, showed the highest strength, followed by external implant - abutment connection type and 1-piece internal type, when artificial aging was not conducted . However, in the above - mentioned experiment, only the implant - abutment fracture load was measured, without involvement of the crowns . Therefore, the fracture load was measured under non - physiological conditions, and intra - oral temperature changes and dynamic functional load were not applied . They showed that the fracture load was dependent on the types of connection, which was the same result as that of the former studies, while the strength was lower after artificial aging . However, that study faced the limitation that it cannot be applied to actual clinical situations, because the fracture load of the zirconia abutment was measured without crown restoration, and the fracture loads before and after artificial aging were not examined . Due to the development of cad / cam systems (computer - aided design / computer - aided manufacturing system), the prosthesis using zirconia has replaced the conventional ceramic restoration and is now additionally being applied to implant abutments.1213 park et al.14 investigated the fit of a customized zirconia abutment manufactured by the cad / cam system . They reported that while the fit was less precise than that of a prefabricated zirconia abutment, it was within the clinically acceptable range . The strength of the customized zirconia abutment was significantly higher than that of the prefabricated zirconia abutment . When restoring all - ceramic implant crowns, shoulder or deep chamfer preparation of zirconia abutment koutayas et al.15 measured the fracture loads of 1-piece internal full zirconia abutments with three preparation depths (0.5 mm, 0.7 mm, and 0.9 mm) that were restored using lithium disilicate . They reported that the fracture loads of all three specimens were higher than physiologic masticatory force, but preparation depths of over 0.7 mm were not recommended . Subsequent to that report, mitsias et al.16 reported measurement of the fracture load of 1-piece internal full zirconia abutment with artificial aging . Preparation depths under 0.9 mm were found to have had no influence on the fracture load . Many literature reports have stated that the optimum preparation depth of zirconia abutment ranged from 0.5 mm to 1.0 mm.171819202122 however, further studies regarding standard indicators are still needed . Therefore, the purpose of this study was to investigate the change of fracture load of customized zirconia abutments with titanium insert according to the different preparation depths, with aging and chewing simulation . The null hypotheses were that the fracture loads of customized zirconia abutment with titanium inserts will not change depending on the various preparation depths, or as a result of aging and chewing simulation . A commercial titanium fixture of 4.5 mm in diameter and 10 mm in length (anyridge, megagen, gyeongsan, korea) was used . The fixture was embedded in acrylic resin (orthoresin dentsply, weybridge, surrey, uk) according to the iso - normed protocol [iso 14801].23 prefabricated titanium inserts that fit into the titanium fixture (prop abutment, megagen, gyeongsan, korea) were used, and the zirconia supra - structures to be attached to the insert were fabricated with zirconia blocks (zenostarzr translucent, wieland, germany) (table 1). The preparation depths of the zirconia suprastructure were designed to 0.5 mm, 0.7 mm and 0.9 mm using the cad system (3shape dental designer premium 2013, 3shape, denmark), and the same - sized zirconia structures were manufactured using the cam system (zenotec t1, wieland dental + technik gmbh, pforzheim, germany). The customized zirconia suprastructures and prefabricated titanium inserts were then bonded using dual cure self - adhesive resin cement (rely x unicem, 3 m espe, st . Crowns fabricated to replace a maxillary right central incisor in asian adult (8.6 mm in width and 11.9 mm in length) were made using lithium disilicate (ips e.max press, ivoclar - vivadent, schaan, liechtenstein) (table 1).24 crowns were designed to fit over the customized zirconia abutments using the cad system (3shape dental designer premium 2013, 3shape, denmark). Rapid prototype model of crowns with the same overall size and different preparation depths were fabricated by 3d printer (digital dental printer, envisiontec, gladbeck, germany). 1). Customized zirconia abutments with titanium insert were sandblasted for 30 seconds under the pressure of 0.5 bar using 50 m al2o3 particles (cobra, renfert, germany). After the surface processing, ultrasonic cleansing was performed on every specimen for 10 minutes using acetone and alcohol, after which the specimens were dried at room temperature . Next, zirconia primer (metal / zirconia primer, ivoclar - vivadent, schaan, liechtenstein) was applied to the bonding surface . Following the manufacturer's recommendations, the inner surfaces of the lithium disilicate crowns were etched with hydrofluoric acid and silane finished (monobond - s, ivo c l a r - vivadent, schaan, liechtenstein) for 1 minute . The fixture and abutment were then tightened with a torque of 30 ncm, and the inside of the abutment was filled with cotton and temporary restorative material (caviton, gc, tokyo, japan). The abutment and crown were then bonded using dual cure self - adhesive resin cement (rely x unicem, 3 m espe, st . Paul, mn, usa), and finger pressure was applied for 5 minutes . Half of the samples (n=18) were put into distilled water at 37 or 24 hours, after which thermocycling was conducted in water baths set to 5 0.5 and 55 0.5, according to the international standard iso normed protocol (iso 10477).25 thermocycling were conducted to obtain 5-year artificial aging, based on previous reports (fig . 2).2627 after thermocycling, the samples were placed at a 45 degree angle on a chewing simulator (chewing simulator cs-4, sd mechatronic gmbh, germany) using a customized jig . Chewing simulation was performed 1,200,000 cycles at 1.67 hz and 49 n, which corresponds to 5-year artificial aging.27 a cobalt - chrome steel indenter with a rounded tip (8 mm in diameter) was used as an antagonist . The fracture load of the abutment was measured using the universal testing machine (rb model 301 unitech m, r and b, korea). The maximum fracture load was determined by applying load on the palatal 2 mm of the incisal part of the abutment at the cross head speed of 0.5 mm / min . Following the a previously published method, 0.5 mm of aluminum foil was inserted between the sample and the testing machine for even distribution of the load.1128 depending on the preparation depths and performance of artificial aging, the specimens were classified into 6 groups, including three non - artificial aging groups (n5, n7, n9) and three artificial aging groups (a5, a7, a9) (table 2). To evaluate the fracture mode, 3). The fracture loads were processed statistically using the spss program (spss version 21.0, spss inc ., after confirmation of the equal variance assumption by levene's test, two - way anova for analysis and tukey hsd post - hoc tests were conducted to determine the interactions between the changes in preparation depth and artificial aging on the fracture load of the customized zirconia abutment with titanium insert (p>.05). Under the equal variance assumption, one - way anova was performed to analyze the fracture load depending on the preparation depths of the samples with artificial aging (p<.05), while the non - parametric kruskal - wallis test was used in groups without artificial aging because there was no assumption of equal variance (p<.017). The differences in fracture load between the artificial aging group and non - artificial aging group at each preparation depth were statistically analyzed using the independent t - test (p<.05). Multiple linear regression analysis was conducted to determine the relationships between variables and the interaction between artificial aging and changes in preparation depths or fracture load (p<.05). The means and standard deviations of the fracture loads of whole specimens are shown in table 3 . One sample in group a5 was broken during the chewing simulation, as it could not resist the masticatory force . Therefore, there were 35 valid samples, for which each fracture load was measured . The mean fracture loads in groups n5, n7, and n9 (non - artificial aging groups) were 539.28 63.11 n, 406.56 28.94 n, and 366.66 30.19 n, respectively . The fracture area of this group, without artificial aging, was found to be located in the implant - abutment titanium insert internal connection area, and all specimens showed the typical fracture or deformation . 3a) the kruskal - wallis test indicated that the fracture load, depending on preparation depth, was higher in the n5 group than in the n7 and n9 groups (p<.017), while no significant difference in fracture load was observed between groups n7 and n9 (p>.017) (fig . 4). The fracture loads of groups a5, a7, and a9 (with artificial aging) were 392.616 50.57 n, 317.94 30.05 n, and 292.74 37.15 n, respectively . Like the groups without artificial aging, the fracture areas of these groups were also located in the implant - abutment titanium insert internal connection area, and all specimens showed the typical fracture or deformation (fig . 3b) according to one - way anova, there were significant differences in the fracture load depending on the preparation depth (p<.05). Tukey hsd post hoc test indicated that the fracture load, depending on preparation depth, was significantly higher in the a5 group than the a7 and a9 groups, while no meaningful difference was found in the fracture load between groups a7 and a9 (p>.05) (fig . 5). The difference in fracture load was evaluated between each paired group (n5/a5, n7/a7, n9/a9), depending on whether artificial aging was performed or not . Significant differences in the fracture load were observed for every pair of preparation depth through independent t - test (p<.05) (fig . Multiple linear regression analysis was conducted to predict the changes in fracture load depending on the three preparation depths and the performance of artificial aging, as well as to examine the influence of fracture load according to each variable . The regression model had a power of explanation of 73.4%, and was significant when assumed through analysis of variance (p<.05). Coefficient analysis revealed that fracture load decreased by 102.456 n in the case of artificial aging, and by about 68.56 n with increase in the preparation depth of 0.2 mm . Overall, changes in the preparation depth were found to have more influence on the fracture load than artificial aging (table 4). In the present study, the fracture loads of customized zirconia abutments with titanium inserts were investigated according to preparation depths and the performance of artificial aging (thermocycling & chewing simulations). Moreover, for representation of the' crown - abutment - fixture complex', lithium disilicate crowns were fabricated, and the optimum preparation depth of the crown was also assessed . The results indicated that the fracture load was significantly influenced by the variance of preparation depth, as well as by artificial aging . The fracture load of preparation depth 0.5 mm (groups n5 and a5) was significantly higher than those of the other preparation groups (groups n7, n9, a7, and a9) regardless of artificial aging . After artificial aging and chewing simulations, all groups were significantly weakened . The width and length ratio of the maxillary anterior crown is known to be an important factor for anterior esthetic restorations . Reported that the average size of the central incisor in adult asians was 8.6 mm in width and 11.9 mm in length.24 culp and mclaran reported the use of lithium disilicate crowns with a variety of transparencies for maximization of esthetics.29 many studies in the literature reported that the 6-degree angle of axial preparation offered adequate mechanical retention of the crowns and improved the adaptation of the crown margins . 30313233 in the present experiments, the crowns and abutments were fabricated based on the reported values . For restoration of all - ceramic implant crowns, zirconia abutments require shoulder or deep chamfer margins . Selected three preparation depths which were thinner than the natural teeth.15 although such specific limitation of the preparation depth resulted in a restricted space for the veneering materials, this disadvantage was outweighed by the favorable color of the underlying zirconia abutment . Koutayas et al . Also stated that when static load was applied to the implant axis at 135 degrees, the thinnest portion of the abutment and fixture connection was fractured due to the levering effect within the internal connection of the 1-piece zirconia abutment.15 they thus named the thinnest point as the " weakest link " . Mitsias et al . Also reported that all specimen of 1-piece zirconia abutment presented the typical fracture at the implant - abutment internal connection after dynamic loading.16 substituting the link with metal materials may allow strengthening of the fractured joint.11 however, despite replacement of the link, all specimens in the present study presented fractures in the same area as the previous reports . It should be noted that fracture did not occurin all samples, as titanium insert and screw deformation appeared in some of the specimens . The physiological force during mastication and the swallowing of food ranges from 10 - 120 n, with the maximum masticatory force of between 108 - 299 n.3435 herein, except for some specimens in the a7 and a9 groups, most specimens demonstrated tolerance of the physiological and maximum masticatory force . Exposure of ceramic materials to mechanical stress and moisture results in low - temperature degradation,36 and artificial aging has an adverse impact on the mechanical properties of zirconia material . Moreover, the phase of zirconia could be transformed from tetragonal to monoclinic.37 mitsias et al . Reported that the fracture strength and durability of 1-piece full zirconia abutment was increased after dynamic loading in the chewing simulator . However, they stated that such results could not be supported by evidence - based scientific data.16 in the present study, the fracture loads of all preparation groups were decreased after artificial aging and chewing simulation . Therefore, unlike the results of the abovementioned study, the fracture load of zirconia abutment with titanium insert examined herein was reduced after the 5-year cyclic loading . However, it could be expected that 5-year cyclic loading might weaken the physical properties of the zirconia abutment with titanium insert . Therefore, in order to determine the exact mechanisms involved, more advanced study will be needed . For the zirconia, phase aging and chewing simulation, as well as xrd (x - ray diffusion) analyses will be required . In order to evaluate the correlation of the independent variables (preparation depth and artificial aging) on the dependent variable (fracture load), multiple linear regression analysis was performed . The results revealed that increase of the preparation depth by 0.2 mm tended to decrease the fracture load by 68 n, while the aging and chewing simulations tended to cause decreases of 102 n. changes in the preparation depth of 0.2 mm had a higher effect than the aging and chewing simulations . Thus, appropriate setting of the preparation depth of zirconia abutment is critical to the survival rate, and hence, the clinical applicability . Within the limitations of this in vitro study, the following conclusions could be drawn: regardless of chewing simulation, the circumferential preparation depth of 0.5 mm of zirconia abutments had a significantly higher fracture load than other groups . Artificial aging caused a significant decrease of the fracture load for all groups of different preparation depths . The change of preparation depth was more influential than the chewing simulation on the fracture load of the customized zirconia abutment with titanium insert . Single implants restored with lithium disilicate crowns and zirconia abutments with titanium insert could with - stand maximum masticatory force in the incisive area when the preparation depth was 0.5 mm.
According to the paradigm of antigen processing by the antigen presenting cells (apc), peptides arising from intracellular proteins are presented via class i mhc molecule . This occurs after they are degraded via proteasome and transported by transporters associated with antigen processing (taps). Differently, peptides that originate from extracellular antigens are delivered to the late endosomal / lysosomal compartment, where they are degraded by the lysosomal proteases and then presented in association with class ii mhc . In the lysosomes, the invariant chain, which blocks class ii mhc, is also degraded, rendering the mhc molecules available for peptide loading . Besides the differences in the sites of origin and processing, peptides presented via class i or ii mhc activate different populations of t lymphocytes, which are cd8 or cd4, respectively . However, exceptions to this model have been described and, for example, dendritic cells (dcs), the most powerful apc, are able to present extracellular antigens also via class i mhc . This event, known as cross - presentation, allows dcs to activate both cd4 and cd8 t cells, in response to an extracellular antigen . On the other hand, antigens of intracellular origin can be presented also in class ii mhc, after being delivered to the lysosomes, through double membrane vesicles, called autophagosomes . Autophagosomes are formed during the induction of autophagy, a self - eating mechanism through which cells recycle their own constituents and survive in stressful conditions . Some of them, such as atg1, atg11, and atg13, regulate autophagosome formation; others (e.g., atg2, atg9, and atg18) are required for membrane flow to the expanding phagophore . The vesicle nucleation is instead dependent on the class - iii phosphatidylinositol 3 kinase (ptdins3k) complex formed by vps34, vps15, vps30/atg6, and atg14 . Atg6, also named beclin 1, represents a protein with a pivotal role in the autophagy induction and the microtubule - associated protein light chain 3 (lc3), or atg8, is a marker of the autophagic vacuoles . Lc3 is expressed as full - length cytosolic protein and upon autophagy induction is cleaved by atg4 to form lc3i, which is then conjugated to phosphatidylethanolamine (pe), generating lc3ii . This molecule, whose formation indicates autophagy induction, is associated with the internal and external membrane of autophagosome and, by interacting with adaptor molecules, such as p62/sqstm1, promotes uptake and degradation of both cargo and adaptors into the lysosomes . Indeed, autophagosomes fuse with lysosomes transporting intracellular proteins in the last autophagic steps . Among them, also viral antigens can end up in the lysosomes, in virally infected cells . Here peptides derived from their degradation may be complexed and presented in association with class ii mhc molecules . Another important role of autophagosomes is to transport entire viral particles to the endosomal / lysosomal compartment, to be degraded and eventually eliminated, a process known as xenophagy . After entering the cells, alternatively, phagosomes as well as free viral particles can be engulfed by the double membrane vesicles of autophagosomes, before reaching the lysosomes . The latter process is under the control of the atg genes, even if it represents a particular form of autophagy aimed at the antimicrobial defense . In addition, autophagy facilitates the pathogen engagement of intracellular tlrs and the consequent release of cytokines, such as type i ifn . The binding of tlrs as well as other molecules involved in the immune response, for example, cd46, by ligands or pathogens that use them as receptors, also triggers autophagy . This has been reported for epstein - barr virus (ebv) that binds tlrs and for measles virus that uses cd46 as receptor . Intriguingly, since cd46 represents the cellular receptor also for human herpes - virus 6 (hhv6), it would be interesting to investigate the impact on autophagy of cd46 engagement by hhv6 . As if the role of autophagy in the immune response was not enough important, it has been recently reported that autophagy is induced by gm - csf in monocytes and that it stimulates monocyte differentiation into functional macrophages and dcs . Besides that, autophagy also plays an essential role in preventing apoptosis of these cells . Among the strategies that allow viruses to escape from the immune control, the impairment of monocyte differentiation into functional dcs represents a common one [1719]. Based on the recent finding, suggesting that autophagy is involved in monocyte differentiation in macrophages and dcs, the interference with the autophagic process could represent one of the underlying mechanisms responsible for such impairment . This strategy has been recently reported to be exploited by human hepatitis c virus (hcv), whose infection of human monocytes results in a reduction of lysosomal cathepsins, in an autophagic block at the late steps, and, as a consequence, in an impairment of dc differentiation . Hcv - mediated reduction of lysosomal acidification, as result of cathepsin release, has been reported also in other cell types . However, autophagy can contribute to hcv replication since the autophagosomal membranes can be used for viral production . Given the importance of autophagy in the immune response, it is not surprising that viruses have evolved strategies to interfere with it, in order to avoid their elimination into the lysosomes, impair the production of antiviral cytokines, reduce the presentation of their antigens, and, as described for hcv, alter dc differentiation . This is a must for viruses to persist in the infected host, sometimes with pathological consequences . All the steps of the autophagic process, from the autophagosome formation to the lysosomal degradation of their content, can be manipulated by viruses [24, 25] (figure 1). It has been described that it depends on the virus types, on the phase of their life cycle, and on the host cell that they infect . For example, human immunodeficiency virus, a single - stranded rna lentivirus belonging to the retroviridae family, is able to induce the initial phases of autophagy by env, an envelope fusogenic protein . On the other hand, it blocks the late autophagic phases to enhance viral production by expressing nef, an accessory protein that interferes with the autophagosomal maturation [24, 25]. During the replicative phase of their life cycle, also herpesviruses such as ebv and kaposi's sarcoma human herpesvirus (kshv) can promote autophagy and exploit the autophagic machinery to enhance their replication [2628]. To do so, they promote the first autophagic steps and block the last ones . This strategy allows them to avoid being delivered into the destructive environment of the lysosomes and to usurp the autophagic machinery for viral transportation through the cell cytoplasm [22, 26, 29]. Pathways involved in the autophagy induction, such as pkr / eif2 alpha and m - tor, can be also targeted by viruses belonging to the herpesvirus family, for example, herpes simplex virus-1 (hsv-1) [3032] and cytomegalovirus (hcmv). Also beclin 1, a key molecule involved in several steps of the autophagic process, can be bound and altered in its function by viral proteins such as vbcl2 of kshv or icp 34.5 of hsv-1 [3436]. All these strategies allow viruses to block autophagy induction during infection of the host cells . Differently, another herpesvirus, varicella - zoster virus (vzv), has been recently reported to successfully infect target cells without blocking the autophagic process . Indeed icp 34.5 or us11, the two proteins responsible for the block of autophagy by hsv1, are not present in vzv although both viruses belong to the same subfamily . Herpesviruses are large, double strand dna viruses having a common particle structure . To date eight human herpesviruses have been identified and classified into three subfamilies (alpha, beta, and gamma) based on their growth characteristics and tissue tropism . The -subfamily includes the neurotropic viruses herpes simplex viruses (hsv) 1 and 2 and varicella - zoster virus (vzv). Human cytomegalovirus (hcmv) and the human herpesviruses 6 and 7 are members of the -subfamily, while ebv and kshv are members of the -subfamily . A common feature shared by all human herpesviruses is the viral persistence into host and the possibility to undergo two alternative life cycle programs, namely, latency and lytic replication . During latency the viral genome is retained as a circular episome in the nucleus and no viral progeny is produced . Furthermore, in the course of latent infection a limited set of genes is expressed in order to reduce immune recognition . In contrast to latency, the herpesvirus lytic program is characterized by a regulated cascade of viral gene expression accompanied by viral production and killing of infected cells . Although most herpesviruses have been reported to interfere with the autophagic process, the next part of this review will focus on the interplay between autophagy and -herpesviruses, given that our laboratory has for long time worked on the virus - host interaction of these viruses associated with several human cancers . As for other herpesviruses, ebv infection can be latent or lytic [41, 42]. During latency the ebv genome is retained as a circular episome in the cell nuclei and upon appropriate stimuli the virus switches into the lytic replication program . It is known that ebv infects primarily human b lymphocytes and epithelial cells; however, its infection can also occur in other cells with a central role in the immune response, such as monocytes and dcs [44, 45]. The in vitro infection of monocytes induces apoptosis and results in an impairment of dc development [45, 46], although the underlying mechanisms have not been investigated in these studies . Therefore, the autophagic pathway could be investigated in these cells since, as previously described, a block of autophagy can switch cell differentiation into cell death in monocytes dcs . These are the most powerful cells in the priming of the cd8 t cell response, playing a pivotal role in control of the ebv infection . Besides cytotoxic t cells, dcs are able to activate a cd4 t cell - mediated immune response against the ebv antigens, such as ebna1, through the mechanism of cross - presentation . Autophagy has been shown to be essential for class ii mhc presentation of ebna1 protein . The block of autophagy resulted in an accumulation of this protein in the intracellular autophagosomes and, more importantly, in a reduction of ebna1 recognition by ebna1 specific cd4 t cells . The involvement of autophagy in ebna 1 presentation was later confirmed by a more recent study . Regarding the ebv interaction with plasmacytoid subpopulation of dcs (pdcs), the main type i ifn producing cells, it has been reported that autophagy is essential for ifn release in response to the ebv infection . Autophagy is stimulated by the virus and facilitates its interaction with tlr7 and tlr9 pprs, both located in the endosomal / lysosomal compartment and essential for ebv recognition by these cells . However, although ebv stimulates autophagy and ifn release, its infection results in an impairment of pdc maturation . Since it has been reported that ebv downregulates tlr 9 in b cells and that this effect is mediated by two ebv proteins, namely, latent membrane protein 1 (lmp1) and bglf5, it would be interesting to investigate tlr9 expression levels in the pdcs ebv - infected versus uninfected control cells . Ebv is associated with several different human cancers of b and epithelial cell origin, such as posttransplant lymphoproliferative disorder (ptld), hodgkin and non - hodgkin lymphomas, nasopharyngeal carcinoma (npc), and some forms of gastric carcinoma . Besides its strong association with some human cancers, ebv infects and establishes a life - long asymptomatic infection in 95% of adult healthy population, reducing to the minimum or not expressing any protein, in order to escape from the immune recognition . Among the viral antigens, only the ebv latent nuclear antigen 1 (ebna1) expression is required for the maintenance of the viral episome in the ebv infected human b cells and this is the only protein always expressed in ebv - associated malignancies . This protein has been demonstrated to activate both cd4 t cells, being presented via class ii mhc and cd8 t cells, although for long time it has been considered invisible to the immune system . As previously described, autophagy is essential for ebna1 antigen presentation via class ii mhc while, interestingly, it does not seem to play a role in the class ii mhc presentation of two other ebv latent nuclear proteins, ebna2 and ebna 3c, both expressed only in pathological conditions . The most oncogenic ebv latent protein, lmp1, has been reported to regulate its own expression by inducing or inhibiting autophagy . When lmp1 is highly expressed in b cells, autophagy is stimulated and promotes degradation . Given that lmp1 plays an important role in ebv - induced oncogenesis, the stimulation of autophagy could be used as a strategy to reduce the expression of this protein and consequently to affect the ebv - driven tumorigenesis . Kshv or human herpesvirus-8 (hhv-8) is the last human -herpesvirus identified to date . Generally, latency is the kshv default program within 4872 h postinfection depending on target cells . In vivo, kshv mainly infects endothelial and b cells and establishes a lifelong latency in b lymphocytes of infected individuals, escaping from the host immune response [65, 66]. Several kshv latent and lytic proteins are involved in immune evasion, in preventing apoptosis and blocking autophagy as well as in transformation . Kshv is the etiologic agent of kaposi's sarcoma (ks), a multifocal angioproliferative disorder arising from kshv - infected endothelial cell, and of lymphoproliferative disorders, such as primary effusion lymphomas (pels), a non - hodgkin b cell lymphoma localized in body cavities, and the plasma cell variant of multicentric castleman disease (mcd). It is important to underline that, although kshv is detectable in all ks lesions, regardless of disease stage or clinical variant, the virus is necessary but not sufficient for the development of this tumor [67, 68]. The majority of spindle cells are latently infected by kshv and only a small percentage of these cells express viral lytic antigens [68, 70]. However, it is currently believed that lytic replication is necessary to support ks lesion formation and maintenance [68, 7174]. In addition, viral replication allows the secretion of proinflammatory and/or proangiogenic factors that create the inflammatory microenvironment essential in ks pathogenesis [68, 75]. Monocytes are among the inflammatory cells found in ks lesions and a study, based on immunohistochemical staining and in situ hybridization, reported that these cells are kshv infected . The virus has also been detected in peripheral t - cells and macrophages [77, 78]. As other herpesviruses, kshv has to cope with innate and adaptive immunity in order to establish a persistent infection in immunocompetent host . Latency is one of the strategies used by kshv since viral gene expression is reduced . Conversely, during its lytic cycle, a high number of immunogenic proteins are produced [71, 79]. The relevance of the equilibrium between kshv lytic cycle and host immunity is indicated by the increased viral replication and the development of kshv - related malignancies in immunosuppressed patients [8082]. About 25% of kshv proteins are involved in kshv immune evasion mechanisms and, although most of these belong to lytic cycle, both latent and lytic proteins are able to hijack innate and adaptive immune response . They can indeed inhibit complement - mediated lysis of infected cells and the ifn i signaling, deregulate the inflammatory cytokine / chemokine networks, and interfere with antigen presentation [67, 79, 8385]. Pdcs, monocyte - derived dendritic cells, and monocytes are among the cell types infected by kshv [19, 86, 87]. As consequence of the infection, a reduction of costimulatory molecules as well as a deregulation of the cytokine release and an impairment of allostimulatory capacity can occur [19, 76, 8790]. In vivo, dc functional impairment has been reported in patients with classical ks and a reduction of pdcs was observed in aids - ks in comparison to ks negative hiv-1 infected individuals . Moreover, the number of langerhans cells is also decreased in ks lesions compared to normal skin . Furthermore, it has been reported that the signal transducer and activator of transcription 3 (stat3) activation in noninfected monocytes / macrophages leads to a block of autophagy and consequent dysfunction, due to the cytokines released during hiv infection . We showed that khsv is able to activate stat3 pathway in dcs by binding to its receptor on these cells, namely, the type ii c - type lectin, dendritic cell - specific icam-3 grabbing nonintegrin (dc - sign; cd209) [87, 96]. Besides, we showed that stat3 activation led to a block of the autophagic flux, as demonstrated by the reduced expression of lc3ii and the increased level of p62 . In addition, looking for a possible mechanism responsible for the block of autophagy mediated by stat3 activation in dcs exposed to kshv, we found an upregulation of mcl-1 [96, 97]. This is one of the proteins able to bind and sequester beclin 1, hampering its essential role in autophagosome formation . Remarkably, stat3 inhibition by ag490 was able to prevent the effects on p62, lc3ii, and mcl-1 . In agreement, a previous paper has shown that the inhibition of stat3 by sorafenib resulted in a downregulation of mcl-1, disruption of beclin 1-mcl-1 complex, and reversion of the autophagic block in hepatocarcinoma cells . Of note, the autophagic block occurred also in the presence of uv - inactivated kshv, indicating that neither vbcl2 nor vflip expression was required [34, 96, 99, 100]. Concomitantly to the autophagic block induced by kshv - mediated stat3 activation, we also observed a reduction of il12p70 release in response to lps stimulation and a higher production of il-10, il-6, and il-23 . This cytokine pattern skews the th1/th2 profile towards th2 and/or th17, promoting immunosuppression and inflammation . Therefore, stat3 activation could be one of the molecular mechanisms underlying kshv - mediated immunosuppression in dcs . Indeed, stat3 activation correlates with an immunosuppressive phenotype and dc dysfunction in the ks microenvironment and in the peripheral blood of tumor bearing patients [103, 104]. Several papers have investigated the interplay between kshv and the autophagic pathway . During the latent phase of its life - cycle, kshv expresses the fadd - like interleukin-1 beta - converting enzyme inhibitory protein (v - flip), a truncated homolog of the cellular flip, which besides having an antiapoptotic activity [105, 106] has been recently shown to block the autophagic flux . It competes with lc3 for binding to atg3, thereby preventing lc3 binding and processing during autophagosome biogenesis . Furthermore, the inhibition of vflip binding to atg3 reduced the size of kshv positive tumor in mice . It has also been demonstrated that v - flip suppression of autophagy counteracts v - cyclin - induced cellular senescence [107, 108]. V - cyclin is a kshv latent protein that deregulates cell cycle, causes aberrant host dna replication, and triggers the dna damage responses (ddrs). Besides vflip, two kshv proteins expressed during the lytic cycle, vbcl2 and k7, can interfere with autophagy . Vbcl2 is a homolog of cellular bcl-2 that inhibits both apoptosis [99, 110] and autophagy [34, 35]. As its cellular counterpart, vbcl2 negatively regulates autophagy by binding beclin 1 . Always within the context of the lytic cycle, recent evidence suggests that kshv k7 protein prevents autophagosome maturation, by interacting with rubicon autophagic protein . K7 is involved in apoptosis suppression [112, 113] while rubicon is a subunit of the beclin 1/uvrag / vps34 autophagy complex, which regulates the autophagosome maturation and the endocytic trafficking [114116]. As shown by authors, k7 transfection in epithelial cell lines promoted a greater interaction between rubicon and beclin 1/uvrag / vps34 complex resulting in a block of vps34 enzymatic activity and, consequently, of autophagosome maturation . Interestingly, during the lytic cycle kshv exploits autophagy to enhance its reactivation mediated by rta immediate lytic protein . Altogether, the data here reviewed suggest that kshv has evolved several strategies to circumvent autophagy - mediated immune responses, to persist in infected hosts . Moreover, the autophagic block during latency and the autophagy induction during the lytic cycle may contribute to the pathogenesis of kshv associated malignancies . A better understanding of how autophagy manipulation could influence antiviral immune response and control virus - induced tumorigenesis might help to discover strategies improving the outcome of the treatments of virus - associated malignancies . Moreover, since autophagy is activated and is involved in ebv and kshv replication [20, 27], its manipulation could also affect the viral particle release that plays a role in -herpesvirus - associated cancers.
Seven able - bodied male subjects (age 30.7 4.2 yrs; height 178.9 10.2 cm; mass 73.9 12.2 kg, mean sd) participated in this study the study was approved by the local ethics committee (ethics committee of the swiss canton of bern, kek bern). The knee dynamometer is a custom made measurement device, which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension . The lower leg is fixed with a brace to an aluminium load cell (lcb130, me - mesysteme gmbh, germany), which moves, via a lever arm, a chain drive system connected to a magnetostrictive torque sensor (s-2220 - 75, ncte ag germany). The torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . A brushless motor (ec45, 250 w, maxon motor ag, switzerland) is used with a planetary gear head (gear ratio: 156:1, gp42, maxon motor ag, switzerland). The actuator can generate a maximum continuous torque of 90 nm . A position sensor (vert - x 28, contelec gmbh, switzerland) is used for angle measurement with a resolution of 0.648 deg to control the motor torque . The measurements were performed at a constant angular velocity of ~110 /s, which is equivalent to a cycling cadence of 50 rpm, and the range of motion was set from 45 to 130 knee extension (where 180 is full extension). The electrical pulses were generated with an eight channel stimulator (rehastim, hasomed gmbh, germany). Co., ltd, usa) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point (fig . 1). The skin was cleaned and the body hair shaved at the position of the electrodes . Muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen (motor point pen, compex, switzerland). Subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma . Current was applied using an sdss electrode setup, which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode (9 5 cm) with the same total area . Each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90, which corresponds to an overall stimulation frequency of 35 hz . Two different sdss electrode placements were investigated: distal versus proximal sdss electrodes . In both cases the active part the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup . Each subject participated in two sessions with only one electrode placement tested for each leg within each session . Between the two measurements in each session, subjects had a break of 15 minutes . Stimulation setup order and the leg were chosen randomly . Before each measurement subjects were placed on the dynamometer and individual adjustments to body proportions were made . Then a two - minute phase was started in which the measured leg was moved by the device without stimulation (non - stimulation [ns] phase). Then the pulse width was manually increased after every third extension, starting at 0 s . Pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed . 80% of this maximal pulse width (pwmax) was then used for the test measurements . After a rest period of 10 minutes, the measurement started with an ns - phase of two minutes followed by a stimulation phase (st - phase) of 6 minutes . Each session was conducted on a different day with at least one day of rest in between . Electrode positions were marked to ensure identical placement each day only the extension phase of joint motion was evaluated, as the setup was to simulate cycling motion . The measured torque (), together with the angular speed was used to calculate the instantaneous output power (pm). The power used to move the leg during the ns - phase was denoted as pns . The net effective power output of one stimulation cycle is thus pstim = pns pm . For every knee extension the following outcomes were calculated: (a) mean power output during one extension (pmean), (b) peak power output (ppeak) and (c) the time from onset of the stimulation to 80% of ppeak (tpeak80). To compare the different stimulation setups, pstim was scaled using a reference pulse width of 100 s (pstim, s), e.g. Subject a had a pulse width of 80 s, so the scaled mean power output pmean, s of that subject is pmean (100/80) and the scaled peak power output ppeak, s is ppeak (100/80). All outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions . A fatigue index (fi) describes the percentage reduction in pmean from the initial phase (pinit) to the final phase (pfinal): fi=1-(pinit - pfinal)/pinit . The higher the value, the higher the fatigue resistance; fi=1 means no fatigue . The data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means . Seven able - bodied male subjects (age 30.7 4.2 yrs; height 178.9 10.2 cm; mass 73.9 12.2 kg, mean sd) participated in this study the study was approved by the local ethics committee (ethics committee of the swiss canton of bern, kek bern). The knee dynamometer is a custom made measurement device, which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension . The lower leg is fixed with a brace to an aluminium load cell (lcb130, me - mesysteme gmbh, germany), which moves, via a lever arm, a chain drive system connected to a magnetostrictive torque sensor (s-2220 - 75, ncte ag germany). The torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . A brushless motor (ec45, 250 w, maxon motor ag, switzerland) is used with a planetary gear head (gear ratio: 156:1, gp42, maxon motor ag, switzerland). A position sensor (vert - x 28, contelec gmbh, switzerland) is used for angle measurement with a resolution of 0.648 deg to control the motor torque . The measurements were performed at a constant angular velocity of ~110 /s, which is equivalent to a cycling cadence of 50 rpm, and the range of motion was set from 45 to 130 knee extension (where 180 is full extension). The electrical pulses were generated with an eight channel stimulator (rehastim, hasomed gmbh, germany). Co., ltd, usa) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point (fig . 1). The skin was cleaned and the body hair shaved at the position of the electrodes . Muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen (motor point pen, compex, switzerland). Subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma . Current was applied using an sdss electrode setup, which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode (9 5 cm) with the same total area . Each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90, which corresponds to an overall stimulation frequency of 35 hz . Two different sdss electrode placements were investigated: distal versus proximal sdss electrodes . In both cases the active part was placed on the previously detected motor point . The mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup . Each subject participated in two sessions with only one electrode placement tested for each leg within each session . Between the two measurements in each session, subjects had a break of 15 minutes . Stimulation setup order and the leg subjects were placed on the dynamometer and individual adjustments to body proportions were made . Then a two - minute phase was started in which the measured leg was moved by the device without stimulation (non - stimulation [ns] phase). Then the pulse width was manually increased after every third extension, starting at 0 s . Pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed . 80% of this maximal pulse width (pwmax) was then used for the test measurements . After a rest period of 10 minutes, the measurement started with an ns - phase of two minutes followed by a stimulation phase (st - phase) of 6 minutes . Each session was conducted on a different day with at least one day of rest in between . Only the extension phase of joint motion was evaluated, as the setup was to simulate cycling motion . The measured torque (), together with the angular speed was used to calculate the instantaneous output power (pm). The power used to move the leg during the ns - phase was denoted as pns . The net effective power output of one stimulation cycle is thus pstim = pns pm . For every knee extension the following outcomes were calculated: (a) mean power output during one extension (pmean), (b) peak power output (ppeak) and (c) the time from onset of the stimulation to 80% of ppeak (tpeak80). To compare the different stimulation setups, pstim was scaled using a reference pulse width of 100 s (pstim, s), e.g. Subject a had a pulse width of 80 s, so the scaled mean power output pmean, s of that subject is pmean (100/80) and the scaled peak power output ppeak, s is ppeak (100/80). All outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions . A fatigue index (fi) describes the percentage reduction in pmean from the initial phase (pinit) to the final phase (pfinal): fi=1-(pinit - pfinal)/pinit . The higher the value, the higher the fatigue resistance; fi=1 means no fatigue . The data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means . Figure 2 shows the development of pmean, s, ppeak, s and tpeak80 over the 6-minute stimulation phase . The corresponding outcome measures for the initial and final stimulation phases are summarised in tab . 1 . No significant differences between distal and proximal electrode placement were found for any outcome measures during the initial stimulation phase . In the final stimulation phase, ppeak and ppeak, s showed significantly higher values for the distal sdss setup: 25.4 8.1 w vs. 28.2 6.2 w, p=0.0062 and 34.8 9.5 w vs. 38.9 6.7 w, p=0.021, respectively . In the final phase 3) and pmean, s with the distal sdss placement (11.8 3.8 w vs. 12.7 3.3 w, p=0.071 and 16.2 4.5 w vs. 17.4 3.4 w, p=0.14), and of longer tpeak80 for distal sdss (347.6 29.2 ms vs. 359.4 38.2 ms, p=0.096). The modestly higher mean power output in the final stage with distal sdss, and a lower dispersion of power values, can be conveniently visualised (fig . Fatigue resistance was not different between the two stimulation setups (fi 0.61 0.14 vs. 0.64 0.9, p=0.38). The aim of this study was to compare the power output, fatigue and activation properties of proximally versus distally placed sdss electrodes in an isokinetic knee extension task simulating knee movement during recumbent cycling . Overall, especially in the final phase of stimulation, the distal sdss setup showed higher power outputs (the only exception was pmean, s in the initial phase, which was minimally lower for distal sdss). This might be regarded as surprising, since the active electrode was placed exactly on the motor point in the proximal sdss setup . Splitting one large electrode into four small ones of the same overall size, and using a sequential stimulation strategy, was previously shown to give increased power output and better fatigue resistance compared to a standard electrode setup . The temporal and spatial shift had a significant impact on muscle activation in a dynamic knee extension task . Using a standard electrode setup (ses), it should not make a difference which electrode (active, dispersive) is positioned at the motor point: the electrical field is the same and the current direction should not activate the muscle fibres differently or change the number of recruited motor units . On the other hand, the electrical field changes with the size of the electrodes, and the distance between the active and dispersive electrodes has a substantial influence on the torque . To control for these factors in the two setups investigated in the present study, the distance was not changed and the active electrode was placed distally . That no substantial or significant difference was observed for any power output parameter in the initial phase of the task may indicate that both setups recruited and activated a similar number of motor units . The similar development over time of pmean and ppeak, as well as the different development of tpeak80 (fig . 2a) between setups, shows that the muscle fibre recruitment and the power curve of a single extension is not the same over time . Although fi was not significantly different, the examination of power development over time (fig . 2b, c) shows that distal placement seems to have a slower power decrease and a higher power output in the final stimulation phase . Smaller electrodes increase the current density compared to larger electrodes using the same amplitude and pulse width . Thus, the non - equal size of the electrodes (active, dispersive) leads to an asymmetric electrical field, which seems to influence muscle activation . In contrast to the distal placement, where four small active electrodes are placed around the motor point, in the proximal setup one large active electrode is placed exactly on the motor point . In consequence, the change of the electrical field at this sensitive position is lower in this setup . Less change over time in the pattern of activation has been shown to favourably affect fatigue and power output development (13, 14). Activation time, i.e. The time from stimulation onset to 80% of peak power, plays a crucial role when electrical stimulation is used to produce a functional movement . Usually, more than just one muscle group is involved, so that both the coordination of the force and the activation of the different muscles are of importance . In fes - cycling, often only three major muscle groups are involved (m. quadriceps, m. hamstrings and m. gluteus) and the coordination of these muscles is one factor for achievement of high power output . During repetitive activation, muscles not only fatigue, but tpeak80 for the proximal placement starts to flatten out after about 30 extensions, whereas tpeak80 for the distal placement is still increasing at this time and only begins to flatten out after about 65 extensions (fig . The mean difference for tpeak80 of 11.8 ms in the final phase corresponds to a phase shift of 6 when cycling 50 rpm, which might have an influence on the overall cycling performance . By positioning the electrodes more precisely in relation to the motor points (proximal setup), the activation becomes more efficient and the muscle activation time is less affected by the duration of the task . In conclusion, the sdss approach to muscle stimulation seems to provide substantial performance benefits, but the placement of the electrodes is still a crucial factor . Distal placement of the sdss electrodes showed higher power output values in the final stimulation phase but also a slightly increased activation time . The development of new array electrodes, specifically for sdss, where the initial pulse is applied directly on the motor point and the following pulses are randomly distributed, may combine the positive effects of the proximal and distal electrode placements . Based on the evidence presented here, for practical fes applications, distal placement of the sdss electrodes appears to be preferable.
Bipolar disorder is a psychiatric disease state manifesting as two different mood extremes, mania and depression . Manic symptoms often include hyperactivity, hyper - talkativeness, decreased need for sleep, grandiosity, increased risk taking, and being easily distracted (apa 2000). Depression symptoms typically consist of a depressed mood, decreased energy, feelings of guilt, hopelessness, helplessness, crying, and even suicidal ideations (apa 2000). Symptoms of bipolar disorder typically present during late adolescence between the ages of 15 and 19 years, however it is not uncommon for proper diagnosis to be delayed for 510 years (bowden et al 2002). Bipolar i disorder is estimated to be prevalent in 1% of the us population, however recent findings from a large - scale survey suggest an adjusted prevalence rate near 3.7% (bowden et al 2002; hirschfeld, calabrese et al 2003). Additionally, it has been found through survey that there is often a gap of up to 10 years between the first visit and subsequent proper diagnosis, with the majority either not being diagnosed at all or inappropriately diagnosed with unipolar depression (hirschfeld, calabrese et al 2003; hirschfeld, lewis et al 2003). Identification of which type of bipolar disorder is present is complicated by the fact that both bipolar i and bipolar ii patients spend more time symptomatic in the depressed phase than the manic or hypomanic states (judd et al 2002). This is significant when considering the potential risk of switching a bipolar patient from a depressed state to manic state with antidepressant therapy . Contrary to this is that failure to address a depressive episode of bipolar disorder presents a dramatic increase in suicide risk . Additional things to consider when treating a patient with bipolar disorder are the high rates of concurrent substance abuse and anxiety disorders (bowden et al 2002; mcelroy 2004). Several hypotheses have been proposed concerning the mechanism of action in epilepsy and bipolar disorder . The most well studied and understood mechanism of valproate is its ability to potentiate or mimic the effects of the inhibitory neurotransmitter, gamma - aminobutyric acid (gaba) (loscher 2002; casey et al 2003; salloum et al 2005). Indirectly, this potentiation of gaba has been hypothesized to produce inhibitory effects on central dopamine (casey et al 2002; loscher 2002). Some of the other and less well understood mechanisms involve the inhibition of neuronal excitability and a resultant anti - kindling effect (loscher 2002). One specific area of study has focused on the inhibition of protein kinase c epsilon (pkc - epsilon) (brunello 2004; toth 2005). Pkc - epsilon has been linked to the stimulation of intracellular calcium release and an increase in cortical excitation and instability . Valproate has also exhibited effects producing the blockade of voltage - dependent sodium channels (loscher 2002; owens and nemeroff 2003). Another proposed mechanism, though controversial, is one likening valproate to lithium as a potential inhibitor of inositol synthesis through inhibition of myo - inositol-1phosphate (mip) (harwood and agam 2003; shaltiel et al 2004; harwood 2005). It is not well understood if valproate inhibits mip directly, but it has been shown to deplete inositol (harwood and agam 2003; shaltiel et al 2004; harwood 2005). Delayed - release divalproex (dr) is an enteric - coated compound consisting of sodium valproate and valproic acid in a 1:1 molar ratio (abbott laboratories 2006). Divalproex is metabolized in the liver, primarily via glucuronidation to active metabolites, with the major active metabolite being trans-2-en - valproate (loscher 2002). The estimated half - life of divalproex ranges from 9 to 16 hours (abbott laboratories 2006). The usual dosing regimen for dr is on a two or three times daily schedule, often with the larger dose given at bedtime . The therapeutic range for divalproex sodium in acute mania according to primary literature suggests improvement is greatest at concentrations above 50 g / ml and that adverse effects increase significantly at concentrations above 125 g / ml (bowden et al 2002). The more common side effects of dr are transient nausea (31%), asthenia (20%), somnolence (17%), dyspepsia (13%), dizziness (12%), diarrhea (12%), vomiting (11%), and tremor (9%) (abbott laboratories 2006). Some long - term side effects that have been associated with dr use are alopecia and weight gain . Therapeutic drug monitoring for valproic acid or any formulation of divalproex requires periodic monitoring of liver enzymes as serious liver toxicity has been reported, especially with use in children under the age of two and in patients receiving multiple antiepileptic medications . Valproic acid / divalproex have also been shown to produce a dose - related thrombocytopenia, thus periodic monitoring of platelets is also suggested . The primary advantage of the dr formulation over immediate release valproic acid (ir) is the enteric coating which helps reduce the incidence of gastrointestinal complaints . Prevalence of hepatic enzyme elevation, tremor, ataxia, increased appetite, weight gain, and alopecia have not been shown to be substantially different in clinical trials between dr and ir preparations . The newest formulation of divalproex is in the form of an extended - release tablet, depakote er (er), which provides a once - daily administration option for the treatment of acute manic or mixed episodes of bipolar disorder, independent of the presence of psychotic features (abbott laboratories 2006). The er formulation uses a hydrophilic polymer matrix controlled - release tablet system to provide controlled continued release of medication . After oral administration and entry into the stomach, the outer coating of the tablet dissolves and exposes the polymer matrix . The outer layer of the matrix becomes hydrated and forms a gel layer from which drug is released . In addition to bipolar disorder, er is also approved for the prophylactic management of migraine headaches in adults, as well as monotherapy and adjunctive therapy in adults and children 10 years of age and older with complex partial seizures, adults and children 10 years of age or older with simple and complex absence seizures, and adults and children 10 years of age and older with multiple seizure types including absence seizures (abbott laboratories 2006). The two primary kinetic differences are that the er preparation results in an average bioavailability of 81%89% relative to dr and er has produces a 10%20% lower fluctuation in peak serum concentration as compared to dr (abbott laboratories 2006). These findings suggest that when converting patients from dr to er that the er dose may need to be increased between 8% and 20% to produce an equivalent serum concentration . Further evidence for pharmacokinetic differences were supported in a study using a healthy adult population and comparing the bioavailability of unequal doses of dr and er (dutta et al 2002). The results from this study determined that daily doses of er dosed 14% and 20% higher than dr (1000 mg / day er vs 875 mg / day dr and 1500 mg / day er vs 1250 mg / day dr) produced equivalent serum concentrations as determined by area - under - the - curve (auc) (dutta and et al 2002). Peak - to - trough a higher cmin fluctuations were 42%48% lower for the er preparation (dutta et al 2002). This study also showed that the increase in dose required with the er dosage form was well tolerated (dutta et al 2002). While evidence is increasing, there continues to be a relative paucity of information available describing the use of er in treating mood related symptoms within the psychiatric population . Delayed - release divalproex (dr) is an enteric - coated compound consisting of sodium valproate and valproic acid in a 1:1 molar ratio (abbott laboratories 2006). Divalproex is metabolized in the liver, primarily via glucuronidation to active metabolites, with the major active metabolite being trans-2-en - valproate (loscher 2002). The estimated half - life of divalproex ranges from 9 to 16 hours (abbott laboratories 2006). The usual dosing regimen for dr is on a two or three times daily schedule, often with the larger dose given at bedtime . The therapeutic range for divalproex sodium in acute mania according to primary literature suggests improvement is greatest at concentrations above 50 g / ml and that adverse effects increase significantly at concentrations above 125 g / ml (bowden et al 2002). The more common side effects of dr are transient nausea (31%), asthenia (20%), somnolence (17%), dyspepsia (13%), dizziness (12%), diarrhea (12%), vomiting (11%), and tremor (9%) (abbott laboratories 2006). Some long - term side effects that have been associated with dr use are alopecia and weight gain . Therapeutic drug monitoring for valproic acid or any formulation of divalproex requires periodic monitoring of liver enzymes as serious liver toxicity has been reported, especially with use in children under the age of two and in patients receiving multiple antiepileptic medications . Valproic acid / divalproex have also been shown to produce a dose - related thrombocytopenia, thus periodic monitoring of platelets is also suggested . The primary advantage of the dr formulation over immediate release valproic acid (ir) is the enteric coating which helps reduce the incidence of gastrointestinal complaints . Prevalence of hepatic enzyme elevation, tremor, ataxia, increased appetite, weight gain, and alopecia have not been shown to be substantially different in clinical trials between dr and ir preparations . The newest formulation of divalproex is in the form of an extended - release tablet, depakote er (er), which provides a once - daily administration option for the treatment of acute manic or mixed episodes of bipolar disorder, independent of the presence of psychotic features (abbott laboratories 2006). The er formulation uses a hydrophilic polymer matrix controlled - release tablet system to provide controlled continued release of medication . After oral administration and entry into the stomach, the outer coating of the tablet dissolves and exposes the polymer matrix . The outer layer of the matrix becomes hydrated and forms a gel layer from which drug is released . In addition to bipolar disorder, er is also approved for the prophylactic management of migraine headaches in adults, as well as monotherapy and adjunctive therapy in adults and children 10 years of age and older with complex partial seizures, adults and children 10 years of age or older with simple and complex absence seizures, and adults and children 10 years of age and older with multiple seizure types including absence seizures (abbott laboratories 2006). The two primary kinetic differences are that the er preparation results in an average bioavailability of 81%89% relative to dr and er has produces a 10%20% lower fluctuation in peak serum concentration as compared to dr (abbott laboratories 2006). These findings suggest that when converting patients from dr to er that the er dose may need to be increased between 8% and 20% to produce an equivalent serum concentration . Further evidence for pharmacokinetic differences were supported in a study using a healthy adult population and comparing the bioavailability of unequal doses of dr and er (dutta et al 2002). The results from this study determined that daily doses of er dosed 14% and 20% higher than dr (1000 mg / day er vs 875 mg / day dr and 1500 mg / day er vs 1250 mg / day dr) produced equivalent serum concentrations as determined by area - under - the - curve (auc) (dutta and et al 2002). Peak - to - trough a higher cmin fluctuations were 42%48% lower for the er preparation (dutta et al 2002). This study also showed that the increase in dose required with the er dosage form was well tolerated (dutta et al 2002). While evidence is increasing, there continues to be a relative paucity of information available describing the use of er in treating mood related symptoms within the psychiatric population . There have been a number of recent additions to the list of approved medications for the treatment of bipolar disorder (see table 1). Despite these new advancements in drug delivery and efficacy for the treatment of bipolar disorder, dr continues to be a highly utilized mood stabilizer for the treatment of acute mania (abbott laboratories 2006). In many instances, bipolar disorder treatment guidelines recommend valproate as a preferred agent, with dr usually being the suggested formulation of valproate / valproic acid (divalproex) due to the presumed improved tolerability (zarate et al 1999; bowden et al 2002; keck et al 2004; suppes et al 2005). The disease states where evidence based guidelines support the use of valproate as a first - line choice include: acute manic exacerbations, euphoric mania, dysphoric or mixed mania, and in patients being treated for rapid cycling bipolar disorder (see table 2) (bowden et al 2002; keck et al 2004; suppes et al 2005). Valproate is also considered an appropriate agent for maintenance therapy, though it lacks the fda maintenance indication (taylor and goodwin 2006). Other fda approved indications for dr include the treatment of complex partial, simple absence, and complex absence seizures in addition to prophylaxis of migraine headaches (abbott laboratories 2006). Dr is not fda approved for aggression, though it is routinely used as monotherapy and is considered appropriate adjunct therapy to reduce hostility associated with schizophrenia (citrome, casey et al 2004; lehman et al 2004). In order to identify published, primary literature studying the er formulation in bipolar disorder we conducted an ovid medline search . Our search terms included bipolar disorder, mania, depression, extended - release divalproex, delayed - release divalproex, divalproex, and schizophrenia . We did not include any information that was not published in primary literature form so any data on file with abbott laboratories that is not published is not included in this review . Our search yielded a small number of clinical trials reporting on the use of extended - release divalproex in bipolar disorder . Three of the trials are open - label with a study enrollment ranging from 10 to 55 patients . One additional study was included that examined the use of extended - release divalproex for mood stabilization and antipsychotic augmentation in schizophrenia . The first trial discussed is an open - label, seven day study evaluating the efficacy and safety of converting psychiatric patients from dr to er (horne and cunanan 2003). The majority of participants carried a diagnosis of bipolar disorder or major depression, 36% and 27% respectively . A total of 55 patients were included in the conversion, 75% outpatients and 25% inpatients hospitalized for acute symptoms . Participants had been treated with dr from 2 days to> 4 years at doses of 500 to 5,000 mg / day . Concomitant medications were described and included agents such as antipsychotics, antidepressants, and anxiolytics . Following baseline measurements of plasma valproic acid concentrations, study participants were switched to er at a dose equal to their total daily dose of dr . Subsequent therapeutic drug monitoring included assessment of plasma valproic acid concentrations which were obtained 10 to 12 hours after the last dose on study days 3, 5, and 7 . Over half the patients in the study (58%) experienced an increase in valproic acid plasma concentration when switched from dr to er dosage forms . In all but three cases, plasma concentrations remained within the therapeutic range of 50125 g / ml . In two of the cases the plasma levels increased following the initiation of the er dosage form with no signs of toxicity and returned to values within therapeutic range with dosage reduction . In the third case, the patient s serum valproic acid level decreased below the lower limit of normal but increased following dosage titration . Efficacy was assessed with the positive and negative syndrome scale (panss) at baseline and endpoint . Upon analysis of the total patient population, a statistically significant improvement was observed in mean panss total score, positive subscale and general psychopathology subscale from baseline to endpoint . Mean total panss scores at baseline were 71.5 21.4 with a mean change of 4.3 11.1 at endpoint . While a statistical change was appreciated, clinical impact of improvement was most likely small . Adverse events were assessed with the udvalg for kliniske undersogelser (uku) side effect rating scale . Patients reported a decrease in the number and severity of adverse effects at endpoint . Following the cessation of the study, this design characteristic becomes less of an issue for outcome measures in which statistical improvement is observed . Overall, conversion from dr to er in this inpatient and outpatient psychiatric population was not associated with deterioration in mental status . In addition, a reduction in both the incidence and severity of adverse effects was appreciated with the er dosing formulation, most likely thought to be the result of lower peak concentrations . A second published, open - label study highlighted the conversion of dr to er in ten subjects over a four - week time period (stoner et al 2004). Subjects were deemed eligible if they had been on dr at least 8 weeks and were considered subjects were also required to be experiencing two mild adverse events or one moderate adverse event that was considered a potential side effect of dr . All subjects were using dr for mood or behavioral related symptoms, with most diagnosed with schizophrenia, bipolar disorder, or schizoaffective disorder, bipolar type . Seven of the ten subjects were converted on an equal milligram - per - milligram basis, while the other three received a 250 mg dose increase to 500 mg as at the time of the study only 500 mg er tablets were available . The mean dr dose at baseline was 2,475 1,010 mg / day with a slightly higher mean dose of er observed at study endpoint, 2,550 985 mg / day . The study group included six males and four females with an average age of 39.4 years and an average length of mental illness of 21.4 years . Subjects were diagnosed primarily with schizophrenia (n = 4), bipolar disorder (n = 2), and schizoaffective disorder (n = 2). The primary outcome measure in this study was the 18-item brief psychiatric rating scale (bprs), selected to identify any changes in psychiatric, behavioral, or mood related symptoms . The bprs was completed at baseline and then at days 7, 14, 21, and 28 . Of particular interest, no significant changes were appreciated in weekly bprs scores, although numerically the mean scores improved from baseline (29.10 6.28) to endpoint (26.5 7.14, p = 0.208). No individual bprs items showed a statistically significant change, however a trend suggested a decrease in somatic complaints (p = 0.057). Eleven - hour post - dose valproic acid serum concentrations were collected at days 14 and 28 . No statistically significant difference was found in dr baseline serum valproic acid concentrations (90.5 29.11 g / ml) and day 28 11-hour post - dose er valproic acid serum concentrations (95.50 13.68; p = 0.493). Additional monitoring parameters included assessment of weight changes, and collection hematologic, renal, hepatic, electrolyte, lipid, and glucose labs at baseline and study endpoint . Serum chemistry monitoring showed statistically significant decreases in ldl cholesterol and potassium, although the decline in potassium was not clinically significant . There were no significant changes in platelet counts observed during the course of the study . Tolerability and adverse event assessment was as measured by the systematic assessment for treatment emergent effects (saftee) at baseline and then days 7, 14, 21, and 28 . Saftee results showed statistically significant reductions in complaints of sedation, stomach and abdominal discomfort, and tremor from baseline to study endpoint . This study was limited by a small sample size and the inclusion of only stable patients, thus not allowing the clinical application the er findings to the acute phase of treatment . Additionally, the study subjects showed a broad variation of axis i diagnosis, not limited to bipolar disorder . Despite these limitations, this study does provide some level of evidence that er can be used in place of dr to help maintain psychiatric stability . A third open - label, six day study was designed to compare the conversion of stable bipolar i or ii or schizoaffective subjects (n = 12) from the dr to the er formulation (centorrino et al 2003). Upon entry into the study, subjects were required to have baseline serum valproic acid levels within the therapeutic range of 50120 g / ml and had to have been receiving stable does of medications for at least four weeks prior to study initiation . Participants were switched to er with a goal of maintaining stable valproic acid serum concentrations . As the er formulation was only available in the 500 mg tablet dosage form at the time of the study, doses were rounded to the nearest 500 mg / day . Serum valproic acid levels were collected at baseline, day 7, week 6, and one week following a medication adjustment . In this cohort of patients, it was observed that er doses needed to be 20.7% higher than the previous dr doses to maintain serum valproic acid levels, a finding consistent with package labeling for er . Numerous efficacy measurements were evaluated at baseline and weekly thereafter and included the young mania rating scale (ymrs), the 17-item hamilton depression rating scale (ham - d 17), clinical global impression of severity (cgi - s) and improvement (cgi - i), global assessment of functioning scale (gaf), and the 17-item brief psychiatric rating scale (bprs). The mean baseline ymrs score was 3.00 3.86 and at endpoint it increased to 3.42 2.53 . The baseline mean ham - d 17 score was 11.2 9.3 and at endpoint the mean improved to 7.67 6.97 . The mean cgi of severity score at baseline was 2.58 0.79 and at endpoint was 2.75 0.65 . The mean baseline gaf score was 68.3 6.2 and at endpoint improved marginally with a value of 69.2 6.0 . The baseline mean bprs score was 39.8 10.2 and at endpoint was 37.8 7.82 . None of the observed changes were deemed to be significant in regards to psychiatric stability . The most commonly reported adverse effects at both baseline and endpoint were impaired concentration, fatigue, depression, and decreased salivation . The only statistically significant adverse event that was seen more frequently with the er dosage form as compared to the dr was an increase in polyuria - polydipsia . All participants elected to continue treatment with the er dosage formulation at the conclusion of the study . This study was also not without limitations, most notably a small sample size and the inclusion of only stable patients . Three small studies which have been published as letter to the editors are useful in reporting conversions from dr to er dosage formulations in psychiatric outpatients (longo 2005; minirth and veal 2005; jackson et al 2006). In the first of these studies, a small, 12-week, open - label, pilot trial examined outpatients diagnosed with bipolar i or ii disorder or schizoaffective disorder (longo 2005). The patients described in this study were being treated with the dr formulation, but were reporting associated adverse events . The dose conversion was carried out per package labeling which recommends up to a 20% dose increase in converting from dr to er . The primary outcome measures were the clinical global impression scale (cgi) and the global assessment of functioning scales (gaf). According to these outcome measures, 9 of 10 patients were considered to exhibit no change or slight improvement in their symptoms, while 5 of 10 reported improvements in adverse events . Baseline psychometric assessment scoring, dosing information, length of prior treatment, and therapeutic drug monitoring parameters were collected, however none of the values were reported . This study possesses several limitations, however it does provide some information regarding practical experience with converting patients . Publications described a retrospective chart review that focused on the evaluation of efficacy, tolerability, and impact on adherence when switching patients from dr to er (minirth and veal 2005). Psychiatric patients, including those diagnosed with bipolar disorder types i and ii, were included . Participants eligible for enrollment had to have been taking dr for at least three months prior to the switch . Patients were evaluated using the cgi - s scale on the day of the switch from dr to er and again during the follow - up visit . Additional secondary assessments which were evaluated at baseline and endpoint included self - rating report of symptoms and review of clinician s notes regarding patient symptomatology . The study was conducted at a single study site and included the records of 32 patients . Doses of divalproex sodium dr ranged from 125 to 1000 mg / day and following the milligram - for - milligram switch patients were maintained on 5002000 mg / day of er . While this report suggests clinical improvement, the ability to critically evaluate the study is limited by a lack of data showing baseline and endpoint scores for the outcome measures . Therapeutic drug monitoring, particularly serum valproic acid levels, were not mentioned in the design of the study and were not reported . Adherence, one of the secondary outcome measures, was patient rated and evaluated by a telephone interview conducted by the raters . Briefly described the conversion of 52 patients stabilized on dr who were converted on an equal milligram - per - milligram basis to the er formulation for up to 24 weeks (jackson et al 2006). Psychometric assessment measures included the ham - d 21 (21-item scale) and ymrs . Utilizing the statistical method of repeated measures analysis, statistically significant improvement was noted from the time of conversion to study endpoint with both the ham - d 21 and ymrs . No significant changes were identified in therapeutic drug monitoring lab values and overall patients reported improved tolerability of the er formulation . The use of er in the management of psychiatric symptoms has not been limited to bipolar disorder alone . One 4-week, open - label dr to er conversion study included thirty patients diagnosed with schizophrenia (citrome, tremeau et al 2004). To be included in the study, patients had to be on a stable dose (1,000 to 3,000 mg / day) of dr for at least 4 weeks . Patients were converted at a 1:1 mg ratio (n = 12) for dr to er if the baseline serum concentration of valproate was 85 g / ml and 1:1.2 mg ratio (n = 18) for dr to er if the baseline serum concentration of valproate was <85 g / ml . Dosing required the use of 500 mg increments due to the lack of availability of the 250 mg tablet at the time of the study . The bprs was the primary outcome measure with side effects assessed with the uku side effect rating scale . The baseline mean bprs total score was 37.9 9.2 (n = 30) and the mean endpoint bprs score was 35.7 11.2 (n = 29), producing a significant mean reduction of 2.3 5.4 points (p = 0.0322). Significant improvement was noted for the 1:1 mg conversion group (p = 0.0561), but not for the 1:1.2 mg group (p = 0.2223). Mean uku scores also showed significant improvement, dropping from a mean of 8.8 6.7 (n = 29) at baseline to 7.5 5.8 (n = 28) at endpoint, though for patients with evaluable baseline and endpoint scores the mean change was a reduction of 2.2 4.1 (n = 27, p = 0.0111). The mean dr dose at study entry was 1,592 mg 498 mg / day which produced a mean baseline trough (12-hours post - dose) valproate concentration of 80.1 20.4 g / ml . The er dosing at study endpoint was 1,950 mg 592 mg / day which produced a mean trough concentration (24-hours post - dose) of 73.1 24.2 g / ml . The 1:1 mg conversion group produced trough levels that were significantly lower at endpoint compared to baseline (p = 0.0006), though the difference in baseline and endpoint levels for the 1:1.2 conversion group were not (p = 0.7102). The conversion of dr to er was not associated with any reports of psychiatric decompensation . While the bprs was noted to improve across the group, the small reduction should not be interpreted as being more effective as this study is limited by being open - label, small sample size, short duration, and the actual improvement was only a 6% reduction in total bprs score . The improvement of uku scores are consistent with other reports of improved tolerability associated with the er formulation . Current literature evaluating the use of er in psychiatric patients with bipolar disorder suggests the er formulation has advantages over the dr formulation . In contrast to dr, the er formulation provides a dosage form of divalproex that is pharmacokinetically supported for once - daily dosing . While the studies included in this review were not specifically designed to examine differences in medication compliance rates, a correlation between once daily dosing and medication adherence has been established . Of additional benefit, er had improved tolerability over dr, likely the result of lower peak plasma concentrations without apparent risk of lost efficacy . The studies included in this review article are greatly limited when utilizing a strict evidence based medicine review procedure . The studies included are not blinded or controlled, they are limited by small sample sizes, they are of short - duration, and are not powered . There is broad variation in the samples making cross - comparisons unreasonable . From a statistical standpoint, there is potential for a beta error, a declaration of no difference when a difference indeed exists, could result in a study not adequately designed to evaluate for mathematical significance . Despite these severe limitations, there is value in the information that has been reported in these study findings . These studies provide practical practice information on converting from dr to er in clinical settings and what can be expected in terms of efficacy, tolerability, and therapeutic drug monitoring . While there is no data indicating that the er formulation provides any enhanced efficacy over dr there is also no compelling data suggesting there is a risk to using the er over the dr . Formulation . The current treatment guidelines included in this review do not specifically recommend the er formulation and from an evidence perspective one cannot say there is strong enough evidence to make it a first - line choice over other treatment options . However, given that the molecule is the same (divalproex) and that the primary differences are rate of release and a reduced peak concentration, the er formulation should be considered a safe alternative to dr, particularly when there is a history or concern of dose related (peak concentration) adverse events or when once a day dosing is preferred in an effort to enhance compliance.
Children spend from 31 to 60% of their school day either writing or performing other fine motor tasks2 . The preparatory skills for writing are coordination of multiple joints, visual perception, vision - motor integration, and proprioception3 . Together with stereognosis, proprioception allows smooth joint movement when vision is impaired or absent5 . Children with poor proprioception have problems with handwriting legibility because their grip on a pen is too strong or too weak4 . Generally, proprioception can be measured with joint position sense (jps) and kinesthetic sense (ks)6 . While there are many studies investigating children s handwriting, quantitative studies on the association between handwriting and proprioception involving jps and ks are lacking7 . The aim of this study was to investigate the association between proprioception, involving jps and ks, and handwriting legibility in children . Nineteen healthy children (15 boys and 4 girls) with an average age of 9.7 0.36 years participated in this study . Prior to the study, the children and their parents were informed about the purpose of the study and the general procedures to be undertaken . All children and the study was approved by the kaya university of human health science studies committee . The investigator moved the child s right arm passively through 80 of flexion at the elbow (from 30 to 110). The children were then directed to repeat this motion 10 times following a metronome set at 1 s intervals . Their movements were recorded by a compact measuring system (cms) 10 for 3d motion analysis (using the winarm software zebris medical gmbh, germany). Cms markers were placed at the greater tubercle and lateral epicondyle of the right humerus and at the right wrist . Angles of deviation from the targeted range of flexion were analyzed using matlab version 2014a (the match works inc ., 2014). Ks was measured by the kinesthesia item in the sensory integration and praxis tests (sipt, wps, torrance, ca, usa). The investigator moved a finger of the subject passively along a line from beginning to end, allowing the child to learn the line direction and distance . Then, blindfolded and after one trial, the children were directed to trace five lines for each hand . Only the results of the right hand were analyzed in this study . The investigator then measured the distance between the test endpoints and the real endpoints of the lines . Legibility was evaluated using form, alignment, space, size, and slope8 . The legibility score was calculated as the ratio of the number of clearly written words to the total number of words (score for legibility (%) = number of letters that received 5 points/30 100). All data were analyzed using ibm spss statistics 20.0 (ibm corp ., armonk, ny, usa). Spearman s rank correlation was used to determine the relationships between legibility of handwriting and jps and ks, with significance defined as p<0.05 . Values obtained for jps and ks in relation to handwriting legibility are shown in table 1table 1.handwriting legibility and proprioception test results (n=19)mean sdjoint position sense 110 (degree)22.530 10.063joint position sense 30 (degree)14.256 10.408kinesthetic sense (cm)2.044 0.703handwriting legibility (%) 33.521 15.852standard deviation . There was no correlation between writing legibility and jps at either 30 or 110 of elbow flexion (p>0.05). A high ks correlated significantly with legible handwriting (p<0.05, table 2table 2.correlation between handwriting legibility and proprioception (n=19)handwriting legibility (%) joint position sense 110 (degree)0.016joint position sense 30 (degree)0.009kinesthetic sense (cm)0.370p<0.05). This study investigated the association between the legibility of handwriting and jps and ks in young children . Children aged between 7 and 8 years are expected to be proficient in building up their speed of handwriting, ensuring consistency in size and proportions of letters as well as the spacing between letters and words9 . The average age of the participants in this study was 9.7 years old . Our results showed that a highly accurate ks was associated with higher legibility scores . Ks provides ongoing error information and memory storage to be recalled when writing is repeated . A high ks leads to programmed error correction, and the upgraded program generates better writing legibility . The results of this study support the hypothesis that ks reinforces the linkage between visual and motor control required for clear handwriting15 . We studies jps at the elbow joint because proprioception at the elbow joint is necessary for performing fine manipulative tasks including handwriting16 . Studies have reported that proprioception at the wrist joint17 and finger joints18 influences handwriting legibility . We looked at children within a limited age range between 9 and 10 years of age . Further, we did not control for other factors that affect writing legibility, such as fine motor control and visual - motor integration19,20,21 . Finally, although handwriting quality is measured in terms of legibility and speed, this study investigated only writing legibility.
It includes hidradenitis suppurativa, acne conglobata, dissecting cellulitis of the scalp and pilonidal sinus . Though each of these conditions are commonly encountered on their own, as a symptom complex follicular occlusion tetrad has rarely been reported in the literature . Here we present a case of hidradenitis suppurativa in a 36-year - old male patient who also had the above mentioned associations . A 36-yeasr - old male patient presented to us with a history of recurrent boils since18 years . They initially started in the groin and buttocks with boils and gradually broke down to ooze pus . The boils healed with scarring after weeks to months . The process progressively started to involve the axillae and then the chest . The patient was treated with multiple antibiotics for over a decade that gave only symptomatic relief . The patient was diagnosed with pilonidal sinus at the age of 28 and he underwent surgery for the same . There was no family history of similar complaints, though his father suffered from psoriasis . On general examination, there were multiple areas of cicatricial alopecia on the scalp [figure 1] and nodular scars on lower half of the face [figure 2]. On query, the patient gave history of severe acne with nodules and pustules healing with scars on the face . He also had patchy swellings over the scalp region associated with pain in the past, which was diagnosed as folliculitis by a physician . Patch of cicatricial alopecia on the occiput region of the scalp pustules, cysts and nodule - reminiscent of acne conglobata - on the lower half of the face on local examination, there were skin colored - to - erythematous nodules, grouped comedones and band like scars in the groin, buttocks, axillae and chest [figure 3 and 4]. The patient had difficulty in lifting arms due to pain and scars in the axillae . Multiple nodules, cysts and band like scars in the axillary region multiple nodules and pustules over the buttocks with surgical scar (done for pilonidal sinus) seen in the upper part of gluteal cleft a thorough general examination was followed by lab work - up . Liver function tests and renal function tests were within normal limits and so was fbs, ppbs and fasting lipid profile . As the patient was obese, he was advised to follow a strict diet chart to lose weight . We also started him on oral isotretinoin 0.5 mg / kg body weight once daily . The patient came back after six weeks for prescription refill but was thereafter lost for follow - up . Follicular occlusion tetrad is a condition that includes hidradenitis suppurativa (hs), acne conglobata, dissecting cellulitis of the scalp and pilonidal sinus . Hidradenitis suppurativa was first described in 1839 by velpeau; verneuil gave it its name in 1854 and associated it with the sweat glands . Later, hs was classified as a member of the follicular occlusion triad, along with acne conglobata and dissecting cellulitis of the scalp . In 1975, pilonidal cyst was added as a member to this triad, forming the follicular occlusion tetrad . In 1989, plewig and steger introduced the term acne inversa to substitute the term hs . Hidradenitis suppurativa or acne inversa is a chronic, inflammatory, recurrent, debilitating skin disease that usually presents after puberty with painful, deep - seated, inflamed lesions in the apocrine gland - bearing areas of the body, most commonly the axillae, inguinal and anogenital regions . The exact pathogenesis of this group of diseases is unknown but evidence suggests that they share the same pathological process initiated by follicular occlusion . The pathogenesis and aetiology are unknown, but is thought to originate from poral occlusion of the pilosebaceous units . Diagnostic criteria of hidradenitis suppurativa (adopted by the 2 international conference on hidradenitis suppurativa, march 5,2009, san francisco, ca us). Typical lesions, i.e. Deep - seated painful nodules: blind boils in early lesions; abscesses, draining sinus, bridged scars and tombstonedouble - ended pseudo - comedones in secondary lesionstypical topography, i.e. Axillae, groins, perineal and perianal region, buttocks, infra and inter mammary foldschronicity and recurrences . Blind boils in early lesions; abscesses, draining sinus, bridged scars and tombstonedouble - ended pseudo - comedones in secondary lesions typical topography, i.e. Axillae, groins, perineal and perianal region, buttocks, infra and inter mammary folds chronicity and recurrences . Dissecting cellulitis(also called perifolliculitis capitis abscedens et suffodiens) manifests with perifollicular pustules, nodules, abscesses and sinuses that evolve into scarring alopecia . It predominantly occurs in african american men between 20 - 40 years of age, but can occasionally affect other races and women too the tendency of dissecting cellulitis to cause severe alopecia, fluctuant nodules, and sinus tracts helps to distinguish it from acne keloidalis nuchae . Culture of dissecting cellulitis does not produce a positive fungal culture and nuchal palpation does not reveal palpable lymph nodes, though reports have noted an inflammatory tinea capitis (kerion) that mimicked dissecting cellulitis in adolescents . Its follicular papules and pustules spread peripherally, leaving central scarred patches of alopecia without nodules or sinuses . Tufted folliculitis resolves with patches of scarring alopecia within which multiple hair tufts emerge from dilated follicular orifices . Acne conglobata is an uncommon nodulocystictype of acne vulgaris that is often resistant to therapy . This disorder typically begins in adulthood and presents as numerous comedones, papules, pustules, nodules, abscesses, and draining sinus tracts involving the chest, back and buttocks . These lesions frequently become secondarily infected with gram - positive bacteria and often heal with scarring . Pathology usually reveals inflammatory infiltrate around follicles, which can often disrupt the normal dermal architecture . Acne conglobata is particularly disfiguring and socially detrimental to patients because of its chronicity, severity, and treatment challenge . Acne conglobata (ac) resembles acne fulminans because both cause numerous inflammatory nodules on the trunk . Unlike acne conglobata, large nodules of acne fulminans tend to become painful ulcers with overhanging borders surrounding exudative necrotic plaques that become confluent . The therapy of choice for ac includes isotretinoin 0.5 - 1 mg / kg for 4 - 6 months . Simultaneous use of systemic steroids such as prednisone 1 mg / kg / d for 2 - 4 weeks may also prove beneficial, particularly if systemic symptoms are evident . Oral tetracycline antibiotics should not be combined with oral isotretinoin because of an increased risk of pseudotumor cerebri . For treatment - resistant cases, dapsone 50 - 150 mg / d is recommended; this treatment should be carefully monitored . Pilonidal sinus was described as far back as 1833 when mayo described a hair - containing cyst located just below the coccyx . No specific laboratory studies or tests are needed to diagnose pilonidal disease and its sequelae or differentiate it from other disease entities; it is a clinical diagnosis best elicited by history and physical examination findings . Treatment of infection or abscess with oral antibiotics followed by surgical excision of the sinus is practiced.
A consecutive series of 569 patients with shoulder pain who underwent ultrasonography between december 2008 and january 2010 were included retrospectively . A single orthopaedic surgeon performed ultrasonographic examination in cases where the authors suspected specific shoulder diseases on plain radiograph and physical examinations, such as rotator cuff tear, frozen shoulder, calcific tendinitis, or biceps tendinitis . The exclusion criteria were as follows: patients under 18 years of age, a history of fracture and shoulder surgery, a history of intra - articular injection before ultrasonographic examination, and biceps deformation including partially or completely ruptured blht . The patients' average age was 57.22 years (range, 26 to 88 years). Men comprised 37.95% (n = 115) of the patients studied, whereas women comprised 62.05% (n = 188). For the diagnosis of rotator cuff tear, the authors first used ultrasonography and then confirmed the final diagnosis by magnetic resonance imaging (mri). On ultrasonographic examination, a hyper - echogenic signal change or focal decrease in the thickness of the tendon was considered to be a partial - thickness rotator cuff tear, and a hyper - echogenic gap in the tendon substance with retraction was considered to be a full - thickness rotator cuff tear . For the diagnosis of calcific tendinitis, the authors made the diagnosis by plain radiograph . On standard anteroposterior radiographs, radio - opaque lesions visible in the rotator cuff area were considered to be calcific tendinitis . Furthermore, the authors checked the rotator cuff integrity on ultrasonography to rule out rotator cuff tearing . The authors considered the diagnosis of biceps tendinitis in patients with point tenderness in the bicipital groove and positive findings on speed's test . Furthermore, the authors performed mri in these patients to confirm biceps tendinitis and to rule out other pathologic conditions, including rotator cuff tear . On mri, biceps tendinitis demonstrated a signal change within the tendon or an irregular margin and a change in the diameter of the tendon . Adhesive capsulitis is well known to be characterized by a functional restriction of both active and passive shoulder motions, and the radiographs of the glenohumeral joint in this condition are essentially unremarkable.13) however, there is no confirmed definition of the degree of shoulder motion restriction . In the present study, the authors defined shoulder stiffness as forward flexion that was less than 120 on passive motion, or external rotation with the arm at the side of less than 30 on passive motion, or internal rotation at the back that was lower than the third lumbar vertebral level passively.14) the authors performed ultrasonography and mri on all patients with adhesive capsulitis to rule out combined shoulder lesions . If shoulder stiffness was combined with other lesions, it was categorized as part of the main shoulder disease, and not as adhesive capsulitis; for example, a rotator cuff tear with stiffness was considered part of the rotator cuff tear group, and not as part of the adhesive capsulitis group . Ultrasonographic examination was performed by a single orthopaedic surgeon experienced in ultrasonography and blinded to the clinical test . A philips hd11 xe ultrasound machine (amsterdam, the netherlands) was used with a high - frequency 12 to 5 mhz linear - array transducer . The patients were seated in the upright position with the arm in the neutral position, the elbow flexed to 90 and the palm face up . The ultrasonographic transducer was placed in the short axis of the blht at about 1 cm inferior to the coracoid process . If low - echogenic fluid was detected around the blht in the short axis view, the authors considered that there would be an appreciably sized glenohumeral joint effusion present . We measured the amount of effusion within the blht sheath as the length of effusion in the short axis view, that is, the longest distance from the sheath to the tendon margin was evaluated (fig . Zubler et al.12) reported that in their ultrasonographic study, the fluid collection in the blht sheath was 0.6 mm and that it increased to more than 0.9 mm after an injection of 8 to 12 ml fluid into the glenohumeral joint . Therefore, we considered shoulders showing an effusion of more than 0.9 mm to indicate effusion within the blht sheath . The clinical data and functional scores were checked at the same time of the ultrasonographic examination by another single orthopaedic surgeon . The clinical data were assessed by the visual analogue scale (vas) for pain and the passive range of motion of the affected shoulder: forward flexion with fixed scapular motion, external rotation in 90 abduction, external rotation at the side, and internal rotation at the back . The vas for pain ranged from 0 to 10, with 10 being the worst pain . The passive range of motion was measured with a full - circle manual goniometer for forward flexion and external rotation . The internal rotation level was checked by an indirect method, where the hand was passively placed behind the back and the vertebral level that was reached by the tip of the extended thumb was recorded . For easy statistical analysis, we converted the internal rotation levels into contiguously numbered groups: 0 for sacral level, 1 to 5 for l5 to l1, and 6 to 12 for t12 to t6 . The functional scores, including the american shoulder and elbow surgery (ases) score, simple shoulder test (sst) score and the korean shoulder score (kss) were also evaluated at the time of the ultrasonographic examination . Mary's hospital, college of medicine, the catholic university of korea (study no . 22.0 (ibm co., armonk, ny, usa), and p - value <0.05 was considered statistically significant . The one - way analysis of variance and student t - test were performed to determine the difference in the amount of effusion within the blht sheath between the diseases . Pearson correlation coefficients (pcc) were calculated to determine the correlation between the amount of the effusion within the blht sheath and the functional outcome . A consecutive series of 569 patients with shoulder pain who underwent ultrasonography between december 2008 and january 2010 were included retrospectively . A single orthopaedic surgeon performed ultrasonographic examination in cases where the authors suspected specific shoulder diseases on plain radiograph and physical examinations, such as rotator cuff tear, frozen shoulder, calcific tendinitis, or biceps tendinitis . The exclusion criteria were as follows: patients under 18 years of age, a history of fracture and shoulder surgery, a history of intra - articular injection before ultrasonographic examination, and biceps deformation including partially or completely ruptured blht . The patients' average age was 57.22 years (range, 26 to 88 years). Men comprised 37.95% (n = 115) of the patients studied, whereas women comprised 62.05% (n = 188). For the diagnosis of rotator cuff tear, the authors first used ultrasonography and then confirmed the final diagnosis by magnetic resonance imaging (mri). On ultrasonographic examination, a hyper - echogenic signal change or focal decrease in the thickness of the tendon was considered to be a partial - thickness rotator cuff tear, and a hyper - echogenic gap in the tendon substance with retraction was considered to be a full - thickness rotator cuff tear . For the diagnosis of calcific tendinitis, the authors made the diagnosis by plain radiograph . On standard anteroposterior radiographs, radio - opaque lesions visible in the rotator cuff area were considered to be calcific tendinitis . Furthermore, the authors checked the rotator cuff integrity on ultrasonography to rule out rotator cuff tearing . The authors considered the diagnosis of biceps tendinitis in patients with point tenderness in the bicipital groove and positive findings on speed's test . Furthermore, the authors performed mri in these patients to confirm biceps tendinitis and to rule out other pathologic conditions, including rotator cuff tear . On mri, biceps tendinitis demonstrated a signal change within the tendon or an irregular margin and a change in the diameter of the tendon . Adhesive capsulitis is well known to be characterized by a functional restriction of both active and passive shoulder motions, and the radiographs of the glenohumeral joint in this condition are essentially unremarkable.13) however, there is no confirmed definition of the degree of shoulder motion restriction . In the present study, the authors defined shoulder stiffness as forward flexion that was less than 120 on passive motion, or external rotation with the arm at the side of less than 30 on passive motion, or internal rotation at the back that was lower than the third lumbar vertebral level passively.14) the authors performed ultrasonography and mri on all patients with adhesive capsulitis to rule out combined shoulder lesions . If shoulder stiffness was combined with other lesions, it was categorized as part of the main shoulder disease, and not as adhesive capsulitis; for example, a rotator cuff tear with stiffness was considered part of the rotator cuff tear group, and not as part of the adhesive capsulitis group . Ultrasonographic examination was performed by a single orthopaedic surgeon experienced in ultrasonography and blinded to the clinical test . A philips hd11 xe ultrasound machine (amsterdam, the netherlands) was used with a high - frequency 12 to 5 mhz linear - array transducer . The patients were seated in the upright position with the arm in the neutral position, the elbow flexed to 90 and the palm face up . The ultrasonographic transducer was placed in the short axis of the blht at about 1 cm inferior to the coracoid process . If low - echogenic fluid was detected around the blht in the short axis view, the authors considered that there would be an appreciably sized glenohumeral joint effusion present . We measured the amount of effusion within the blht sheath as the length of effusion in the short axis view, that is, the longest distance from the sheath to the tendon margin was evaluated (fig . Zubler et al.12) reported that in their ultrasonographic study, the fluid collection in the blht sheath was 0.6 mm and that it increased to more than 0.9 mm after an injection of 8 to 12 ml fluid into the glenohumeral joint . Therefore, we considered shoulders showing an effusion of more than 0.9 mm to indicate effusion within the blht sheath . The clinical data and functional scores were checked at the same time of the ultrasonographic examination by another single orthopaedic surgeon . The clinical data were assessed by the visual analogue scale (vas) for pain and the passive range of motion of the affected shoulder: forward flexion with fixed scapular motion, external rotation in 90 abduction, external rotation at the side, and internal rotation at the back . The vas for pain ranged from 0 to 10, with 10 being the worst pain . The passive range of motion was measured with a full - circle manual goniometer for forward flexion and external rotation . The internal rotation level was checked by an indirect method, where the hand was passively placed behind the back and the vertebral level that was reached by the tip of the extended thumb was recorded . For easy statistical analysis, we converted the internal rotation levels into contiguously numbered groups: 0 for sacral level, 1 to 5 for l5 to l1, and 6 to 12 for t12 to t6 . The functional scores, including the american shoulder and elbow surgery (ases) score, simple shoulder test (sst) score and the korean shoulder score (kss) were also evaluated at the time of the ultrasonographic examination . Mary's hospital, college of medicine, the catholic university of korea (study no . 22.0 (ibm co., armonk, ny, usa), and p - value <0.05 was considered statistically significant . The one - way analysis of variance and student t - test were performed to determine the difference in the amount of effusion within the blht sheath between the diseases . Pearson correlation coefficients (pcc) were calculated to determine the correlation between the amount of the effusion within the blht sheath and the functional outcome . There were 157 shoulders with rotator cuff tear, 104 shoulders with adhesive capsulitis, 39 shoulders with calcific tendinitis, and three shoulders with biceps tendinitis . All patients with calcific tendinitis showed no combined rotator cuff tear, and rotator cuff tear combined with small calcification less than 2 mm were categorized to rotator cuff tear group . All shoulders with biceps tendinitis showed signal changes with the fraying of the biceps tendon on mri . The average of the vas for pain was found to be 4.8 2.7 in the rotator cuff tear group, 5.3 2.9 in the adhesive capsulitis group, 4.7 2.8 in the calcific tendinitis group, and 4.5 in the biceps tendinitis group . The overall results for the ases score were 58.5 points in the rotator cuff tear group, 58.8 points in the adhesive capsulitis group, 58.1 points in the calcific tendinitis group, and 53.3 points in the biceps tendinitis group . For the sst score, the results were as follows: 58.0 points in the rotator cuff tear group, 45.6 points in the adhesive capsulitis group, 51.9 points in the calcific tendinitis group, and 58.3 points in the biceps tendinitis group . For the kss score, the results were as follows: 42.4 points in the rotator cuff tear group, 40.8 points in the adhesive capsulitis group, 41.8 points in the calcific tendinitis group, and 41.0 points in the biceps tendinitis group . Effusion within the blht sheath was detected in 58.42% (n = 178) of the patients in this study: 69.23% (n = 72) of patients with adhesive capsulitis, 56.69% (n = 89) of patients with rotator cuff tear, 41.03% (n = 16) of patients with calcific tendinitis, and 33.33% (n = 1) of patients with biceps tendinitis (fig . The average amount of the effusion within the blht sheath was 1.7 1.6 mm: 2.0 1.6 mm in patients with adhesive capsulitis, 1.6 1.6 mm in patients with rotator cuff tear, 1.2 1.6 mm in patients with calcific tendinitis, and 1.1 1.8 mm in patients with biceps tendinitis . The amount of the effusion within the blht sheath in patients with adhesive capsulitis was significantly larger than that in patients with calcific tendinitis (p = 0.03). However, there were no statistically significant differences between the other shoulder diseases (fig . The amount of the effusion within the blht sheath overall had a negative correlation with the range of motion in all patients: forward flexion (pcc = -0.491; p <0.05), external rotation in 90 abduction (pcc = -0.381; p <0.05), external rotation at the side (pcc = -0.356; p <0.05), and internal rotation (pcc = -0.486; p <0.05). In patients with adhesive capsulitis, pcc was -0.349 with forward flexion (p <0.05), -0.310 with external rotation in 90 abduction (p <0.05), -0.280 with external rotation at the side (p <0.05), and -0.385 with internal rotation (p <0.05). In patients with rotator cuff tear, the pcc was -0.509 with forward flexion (p <0.05), -0.379 with external rotation at 90 abduction (p <0.05), -0.364 with external rotation at the side (p <0.05), and -0.506 with internal rotation (p <0.05). In patients with calcific tendinitis, pcc was -0.769 with forward flexion (p <0.05), -0.456 with external rotation in 90 abduction (p <0.05), -0.432 with external rotation at the side (p <0.05), and -0.537 with internal rotation (p <0.05) (table 1). The amount of the effusion within the blht sheath had a negative correlation with the functional score in each disease group . However, the correlation coefficients were relatively low: in patients with adhesive capsulitis, ases pcc = -0.311 (p <0.05), sst pcc = -0.268 (p <0.05), and kss pcc = -0.342 (p <0.05); in patients with rotator cuff tear, ases pcc = -0.449 (p <0.05), sst pcc = -0.402 (p <0.05), and kss pcc = -0.380 (p <0.05); and in patients with calcific tendinitis, ases pcc = -0.400 (p <0.05), sst pcc = -0.275 (p <0.05), and kss pcc = -0.343 (p <0.05). Furthermore, the vas for pain showed a weak positive correlation with the amount of the effusion within the blht sheath in each shoulder disease: adhesive capsulitis pcc = 0.109 (p <0.05), rotator cuff tear pcc = 0.183 (p <0.05), and calcific tendinitis pcc = 0.188 (p <0.05) (table 2). There were 157 shoulders with rotator cuff tear, 104 shoulders with adhesive capsulitis, 39 shoulders with calcific tendinitis, and three shoulders with biceps tendinitis . All patients with calcific tendinitis showed no combined rotator cuff tear, and rotator cuff tear combined with small calcification less than 2 mm were categorized to rotator cuff tear group . All shoulders with biceps tendinitis showed signal changes with the fraying of the biceps tendon on mri . The average of the vas for pain was found to be 4.8 2.7 in the rotator cuff tear group, 5.3 2.9 in the adhesive capsulitis group, 4.7 2.8 in the calcific tendinitis group, and 4.5 in the biceps tendinitis group . The overall results for the ases score were 58.5 points in the rotator cuff tear group, 58.8 points in the adhesive capsulitis group, 58.1 points in the calcific tendinitis group, and 53.3 points in the biceps tendinitis group . For the sst score, the results were as follows: 58.0 points in the rotator cuff tear group, 45.6 points in the adhesive capsulitis group, 51.9 points in the calcific tendinitis group, and 58.3 points in the biceps tendinitis group . For the kss score, the results were as follows: 42.4 points in the rotator cuff tear group, 40.8 points in the adhesive capsulitis group, 41.8 points in the calcific tendinitis group, and 41.0 points in the biceps tendinitis group . Effusion within the blht sheath was detected in 58.42% (n = 178) of the patients in this study: 69.23% (n = 72) of patients with adhesive capsulitis, 56.69% (n = 89) of patients with rotator cuff tear, 41.03% (n = 16) of patients with calcific tendinitis, and 33.33% (n = 1) of patients with biceps tendinitis (fig . The average amount of the effusion within the blht sheath was 1.7 1.6 mm: 2.0 1.6 mm in patients with adhesive capsulitis, 1.6 1.6 mm in patients with rotator cuff tear, 1.2 1.6 mm in patients with calcific tendinitis, and 1.1 1.8 mm in patients with biceps tendinitis . The amount of the effusion within the blht sheath in patients with adhesive capsulitis was significantly larger than that in patients with calcific tendinitis (p = 0.03). However, there were no statistically significant differences between the other shoulder diseases (fig . The amount of the effusion within the blht sheath overall had a negative correlation with the range of motion in all patients: forward flexion (pcc = -0.491; p <0.05), external rotation in 90 abduction (pcc = -0.381; p <0.05), external rotation at the side (pcc = -0.356; p <0.05), and internal rotation (pcc = -0.486; p <0.05). In patients with adhesive capsulitis, pcc was -0.349 with forward flexion (p <0.05), -0.310 with external rotation in 90 abduction (p <0.05), -0.280 with external rotation at the side (p <0.05), and -0.385 with internal rotation (p <0.05). In patients with rotator cuff tear, the pcc was -0.509 with forward flexion (p <0.05), -0.379 with external rotation at 90 abduction (p <0.05), -0.364 with external rotation at the side (p <0.05), and -0.506 with internal rotation (p <0.05). In patients with calcific tendinitis, pcc was -0.769 with forward flexion (p <0.05), -0.456 with external rotation in 90 abduction (p <0.05), -0.432 with external rotation at the side (p <0.05), and -0.537 with internal rotation (p <0.05) (table 1). The amount of the effusion within the blht sheath had a negative correlation with the functional score in each disease group . However, the correlation coefficients were relatively low: in patients with adhesive capsulitis, ases pcc = -0.311 (p <0.05), sst pcc = -0.268 (p <0.05), and kss pcc = -0.342 (p <0.05); in patients with rotator cuff tear, ases pcc = -0.449 (p <0.05), sst pcc = -0.402 (p <0.05), and kss pcc = -0.380 (p <0.05); and in patients with calcific tendinitis, ases pcc = -0.400 (p <0.05), sst pcc = -0.275 (p <0.05), and kss pcc = -0.343 (p <0.05). Furthermore, the vas for pain showed a weak positive correlation with the amount of the effusion within the blht sheath in each shoulder disease: adhesive capsulitis pcc = 0.109 (p <0.05), rotator cuff tear pcc = 0.183 (p <0.05), and calcific tendinitis pcc = 0.188 (p <0.05) (table 2). The effusion within the blht sheath was detected in 58.42% of all patients and the average amount was 1.7 1.6 mm in all patients of the present study . Among them, patients with adhesive capsulitis showed effusion within the blht sheath most frequently, and the average amount of that group was larger than that of the other shoulder diseases . Because the effusion within the blht sheath looks like a fried egg, the amount of effusion within the blht sheath showed a negative correlation with the range of motion and functional scores in each group . In particular, the forward flexion and internal rotation levels were most closely correlated with the amount of effusion within the blht sheath in each group . The glenohumeral joint cavity communicates with the blht sheath; thus, profuse effusion of the glenohumeral joint may lead to an increase in the effusion within the tendon sheath . Zubler et al.12) reported that in their ultrasonographic study, the width of the anechoic fluid collection in the blht sheath was increased after 8 to 12 ml of fluid was injected into the glenohumeral joint in most cases . These findings suggest that the amount of hypoechogenic fluid in the blht sheath may represent the ratio of the amount of glenohumeral joint effusion to the volume of the glenohumeral joint cavity . In the other words, however, zubler et al.12) did not evaluate the correlation between the effusion within the blht sheath and clinical outcomes, they suggested that the effusion within the blht sheath might represent glenohumeral joint effusion . In the present study, the effusion within the blht sheath was more frequently observed in adhesive capsulitis and the amount of effusion within blht sheath showed a negative correlation with the range of motion . The axillary fold is contracted, and that reduces the joint volume to below 15 ml.15) furthermore, joint shrinkage might push the joint fluid to another space, the blht sheath . Therefore, the more the joint shrinks, the more joint fluid is collected in the blht sheath . The amount of effusion within the blht sheath showed a higher correlation with the range of motion than with the variable functional score or shoulder pain . The effusion within the blht sheath was thought to be related to the status of synovitis or capsulitis . Several studies with synovial or capsular biopsy specimens from patients with adhesive capsulitis have demonstrated that variable proinflammatory cytokines and neovascularizations are involved in synovial hyperplasia and capsular fibrosis.1617) adhesive capsulitis, initially described by neviaser,18) is thought to be a combination of synovial inflammation and capsular fibrosis.15) thus, the limitation of the range of motion is thought to be closely correlated with joint synovitis, and in the present study, the range of motion was also closely correlated with the amount of effusion within the blht sheath . Therefore, in adhesive capsulitis, combined synovitis and capsulitis can lead to increased joint effusion and joint cavity shrinkage, and these conditions might produce the appearance of the effusion within the blht sheath in shoulders with adhesive capsulitis . Functional scores and shoulder pain are mainly affected by the severity of each disease; however, in this study, we aimed to evaluate the clinical meaning of the effusion within the blht sheath, something we frequently encountered during ultrasonographic examinations . Effusion within the blht sheath was detected in 56.69% of patients with rotator cuff tear . Several studies showed that low- to mild - grade synovial inflammation is present in shoulders with rotator cuff tears.31920) the expression levels of interleukin 1-beta and several degradative enzymes were highly upregulated in the synovium of patients with rotator cuff lesions, and these findings suggest that chronic synovitis is associated with rotator cuff tears.19) therefore, chronic synovitis combined with rotator cuff tears might lead to an increase in joint effusion, and these findings would be detected as effusion within the blht sheath . In calcific tendinitis, there was a stronger correlation between the amount of effusion within the blht sheath and range of motion than in rotator cuff tears . Calcific tendinitis can be divided into three distinct stages: precalcific, calcific, and postcalcific.21) among these stages, the postcalcific stage usually shows milky or creamy calcific deposits which frequently induce acute sharp pain, combined with acute synovitis or bursitis . Thus, acute synovitis in calcific tendinitis might induce increased joint effusion and severely painful limited range of motion . This may be the reason for the stronger correlation between the amount of effusion within the blht sheath and the range of motion in calcific tendinitis . Several studies have suggested that a fluid collection in the biceps tendon sheath may be induced by biceps tendinitis.2223) in the present study, only three patients had biceps tendinitis, and among them, only one patient showed effusion within the blht sheath . We had only three cases of biceps tendinitis, because our categorization, exclusion criteria, and low incidence of ultrasonographic examination in isolated biceps lesions . Because of low sample size of biceps lesions in the present study, the authors could not evaluate this lesion to a statistical degree . However, it is believed that the fluid collection in the biceps tendon sheath may be influenced by joint effusion, rather than by the condition of the local bicep . Although, the final diagnosis was confirmed at the last follow - up, the authors did not trace the patients' symptoms and functional outcomes after the ultrasonographic examination . Furthermore, because patients underwent different treatments according to their individual diagnosis, the correlation between the effusion within the blht sheath and disease prognosis could not be evaluated . Second, in the present study, if shoulder stiffness was combined with another shoulder disease, it was categorized as the main shoulder disease, and not as adhesive capsulitis . We simply categorized the rotator cuff tear patients with stiffness into the rotator cuff tear group, for example . However, it is well known that there are many differences between rotator cuff tear with stiffness and rotator cuff tear without stiffness, including symptoms and treatment . We did not evaluate normal shoulders as control group . Because, there would be asymptomatic effusion within blht sheath, the percentage of' fried egg sign' in each disease and conclusion of the present study might be different if the results were compared with control group . Lastly, we evaluated the effusion within the blht sheath using ultrasonography, so a bias might occur according to direction and location of ultrasonographic transducer (the intraobserver reliability was 0.80). The location and thickness of the biceps tendon within the blht sheath also could provide a bias . To reduce the variance, the examiner tried to locate the transducer at the same location in the same shoulder position and shoulders with biceps deformation, especially with biceps tendinitis or biceps tear, were excluded in the present study . In conclusion, the effusion within the blht sheath is closely related to the range of motion and clinical scores in patients with painful shoulders . The ultrasonographic detection of the effusion within the blht sheath might be useful to evaluate shoulder functions . However, further studies are required to understand the correlation between the effusion within the blht sheath and disease prognosis.
This was a retrospective cohort study approved by our institutional review board as a quality improvement research . The records of catheter days 1 year before the introduction of midlines (group a: august 1, 2011, to july 31, 2012) and catheter days of 1 year after the regular use of midlines (group b: november 1, 2012, to october 31, 2013) in the ventilator unit were collected (table 1). During the period from august 2012 to november 2012, internal medicine residents and nursing staff underwent training on the use and insertion of midline catheters and their care . This dedicated team was responsible for replacing cvcs by midlines as per our new practices . The midlines that we used were powerwand (access scientific, san diego, calif). Before midline catheter, patient days: number of days the patient was in the ventilator unit; catheter days: number of days the patient had a central venous catheter and after midline catheter, patient days: number of days the patient was in the ventilator unit; catheter days: number of days the patient had a midline catheter we chose this unit because it has a constant denominator of the number and type of patients . Most of the patients in this unit are ventilator dependent and being treated for problems such as health care associated pneumonia, urinary tract infections, wound and pressure ulcer infections, or other long - term conditions requiring acute care that cannot be administered at a nursing home facility . The patients here have longer length of stay compared with other units and generally have difficult venous access . The most common reason for cvc use in this unit is for difficult and long - term intravenous access . Indications of cvcs in these patients included antibiotic therapy, infusion therapy, diagnostic procedures, transfusions, and blood draws . The department of infection control of the hospital reports all the clabsi per 1000 catheter days in the hospital, categorized by location and unit . The number of clabsi during this period along with the type of bacteria and the date of culture was obtained from the microbiology department . We compared the clabsi rates between group a and group b to see whether it decreased through the use of midlines in comparison with central lines . We trained a team of residents for a period of 3 months in the insertion of ultrasound - guided midline catheters, which would replace cvcs by midline catheters whenever possible . The same antiseptic precautions used in cvc catheter placements under ultrasound guidance were applied to the placement of midlines . The policy for replacing the central lines included the following guidelines: any patient with a cvc in a femoral vein had their line removed and replaced by a midline catheter . All patients who were not on an ionotropic agent or total parenteral nutrition had their cvc replaced by midlines . A midline catheter replaced any cvc in place longer than a week . A patient on antibiotic therapy being sent to nursing home for further management received a midline catheter . The total number of catheter days was compared with the rate of clabsi in the 2 groups . Catheter days were calculated as the number of central line catheters on the unit every day . Adding the total number of catheters on the unit per day and adding this daily number for the length of time of the study help calculate catheter days . Central line associated bsi was reported as rate per 1000 catheter days and can be calculated as follows: (total number of clabsi / total number of catheter days) 1000 . We used a test to compare the number of catheter days per patient days in the 2 groups as well as to compare infections based on the number of catheter days in each group (table 2). Comparing the number of catheter days per patient days in the 2 groups, as well as comparing infections based on the number of catheter days in each group there was a significant decrease in the total number of catheter days on the ventilator unit in group a from 2408 catheter days in the 1 year (august 1, 2011, to july 31, 2012) before the introduction of midline catheters to 1521 catheter days in group b in the following year (november 1, 2012, to october 31, 2013; p <0.05 in both groups). The total number of clabsi infections in these periods was also significantly decreased from 8 to 0 (table 3). This calculates to 3.32 clabsi per 1000 catheter days and 0 clabsi per 1000 catheter days, respectively (add). The number of inpatient days during these periods was 3058 and 2948 days (table 4 and 5). Continuation of the test test: observed frequency test: expected (theoretical) frequency, asserted by the null hypothesis there were no bsis associated with midlines in this study . The centers for disease control and prevention has put in place guidelines to reduce the incidence of clabsi . These guidelines brought about by numerous studies for years have helped bring down the rate of clabsi . These measures include the following: reducing the number of dwell days by removing catheters as early as possible; reducing the use of central lines by using other means of venous access; proper care and technique for cvcs; maximum sterile barrier precautions during insertion; use of chlorhexidine for skin disinfection before catheter insertion; avoidance of the femoral insertion site; and use of recommended insertion site dressing care practices . Prolonged dwell time has been shown to increase clabsi rates rapidly after 9 catheter days . Replacing central lines with midlines decreases the dwell time and also the total number of catheter days . It may be argued that the decrease in the use of central lines and thereby reducing the number may be responsible in decreasing clabsi; however, our denominator is constant for comparison . The use of midlines reduces the use of and the dwell days for central lines, and with this study, we show that it can be a factor in reducing clabsi as well . Another study showed that clabsi rates were higher in patients who had central lines for longer than 7 days . In our study, by replacing central lines with midlines in patients, we essentially decreased risk factors such as dressing changes, catheter care, and duration of central line use . A more obvious outcome is the decrease in use of central lines itself, causing a drop in the infection rates . The fall in infection rate may be attributed to the fewer number of catheters in place for longer than 7 days . Before the introduction of midlines, difficult intravenous access in these chronically ill patients mandated the use of central lines . There are other complications of central lines, including inadvertent arterial puncture (3%), hemothorax, or pneumothorax (1%-2%). Central lines and picc lines require x - ray confirmation of tip placement, exposing the patient to radiation . Midlines have complications as well; our most common problem was the loss or malfunction of the midline including extravasations in 2 patients . The bsi rate of midlines in various studies has been reported to be between 0% and 0.9% . The combination of better available products and the increasing use of ultrasound guidance for intravenous catheter placement has renewed interest in midline catheters . Some limitations of midline catheters are inability to use for vasopressors, total parenteral nutrition or when large peripheral veins are not available such as in amputates and patients with arteriovenous fistulas . We conclude that the use of midline catheters to replace central lines for difficult intravenous access decreases the rate of clabsi in a ventilator unit in a community hospital.
Additional information about study design, ethical considerations, and laboratory methods can be found in supplementary materials . We studied 72 children (n = 21) and adults with type 1 diabetes without known renal impairment (estimated glomerular filtration rate <60 ml / min/1.73 m) (supplementary tables 1 and 2). Patients underwent a standard mmtt (1). Additional samples were taken at 30, 60, and 120 min in pediatric patients (n = 18), allowing area under the curve (auc) to be calculated . Urine was collected as a fasting second morning void immediately before the start of the mmtt (0 min) and after 120 min . Nmol / l, in accordance with the diabetes control and complications trial (8). Urine was collected in boric acid 120 min after the evening meal following a premeal void . Adult patients collected further home urine samples 120 min after a standard 60-g carbohydrate breakfast and following the patients own lunch . Urine samples were brought to the research center within 24 h, measured in aliquots, and frozen at 80c . We assessed the association between 90-min scp (1) and both the mmtt 120-min ucpcr and after the home evening meal (spearman rank correlation coefficient). In the pediatric cohort, correlations were also determined between auc scp and 120-min ucpcr . Ucpcr (120 min) following a home evening meal was compared with that after a mmtt (wilcoxon test for paired samples). Additional samples were taken at 30, 60, and 120 min in pediatric patients (n = 18), allowing area under the curve (auc) to be calculated . Urine was collected as a fasting second morning void immediately before the start of the mmtt (0 min) and after 120 min . Nmol / l, in accordance with the diabetes control and complications trial (8). Urine was collected in boric acid 120 min after the evening meal following a premeal void . Adult patients collected further home urine samples 120 min after a standard 60-g carbohydrate breakfast and following the patients own lunch . Urine samples were brought to the research center within 24 h, measured in aliquots, and frozen at 80c . We assessed the association between 90-min scp (1) and both the mmtt 120-min ucpcr and after the home evening meal (spearman rank correlation coefficient). In the pediatric cohort, correlations were also determined between auc scp and 120-min ucpcr . Ucpcr (120 min) following a home evening meal was compared with that after a mmtt (wilcoxon test for paired samples). Mmtt 120-min ucpcr was highly correlated with the 90-min scp (r = 0.97; p <0.0001). Nmol / l) was 0.53 nmol / mmol, with 94% sensitivity and 100% specificity (fig . A strong correlation was also seen between auc for scp and 120-min ucpcr during the mmtt (r = 0.96; p <0.0001). Scatter diagram showing the relationship between 90-min scp and 120-min ucpcr in the mmtt (a) and following the patients own evening meal at home (b). A: 120-min ucpcr is well correlated with 90-min scp in the mmtt (r = 0.97; p <0.0001). B: 120-min postprandial ucpcr is well correlated with 90-min scp in the mmtt (r = 0.91). Home postprandial evening meal ucpcr (120 min) was well correlated with 90-min scp (r = 0.91; p <0.0001) (fig . The equivalent ucpcr cutoff was 0.37 nmol / mmol, with 84% sensitivity and 97% specificity (fig . 1b). Using the ucpcr cutoff 0.53 nmol / mmol in the home evening meal samples yielded lower levels of sensitivity (71%) and specificity (97%) for significant endogenous insulin secretion . This is probably explained by a lower stimulus, as shown by the lower 120-min ucpcr in the home postprandial samples than in those in the mmtt (0.16 nmol / mmol [interquartile range 0.010.76] vs. 0.35 nmol / mmol [0.041.41]; p <0.0001). The correlations were similar in adults and children when analyzed separately (supplementary tables 4 and 5). Result tables for combined (supplementary table 3) and separate analysis of adults (supplementary table 4) and children (supplementary table 5) are given in the supplementary materials . Mmtt 120-min ucpcr was highly correlated with the 90-min scp (r = 0.97; p <0.0001). Nmol / l) was 0.53 nmol / mmol, with 94% sensitivity and 100% specificity (fig . A strong correlation was also seen between auc for scp and 120-min ucpcr during the mmtt (r = 0.96; p <0.0001). Scatter diagram showing the relationship between 90-min scp and 120-min ucpcr in the mmtt (a) and following the patients own evening meal at home (b). A: 120-min ucpcr is well correlated with 90-min scp in the mmtt (r = 0.97; p <0.0001). B: 120-min postprandial ucpcr is well correlated with 90-min scp in the mmtt (r = 0.91). Home postprandial evening meal ucpcr (120 min) was well correlated with 90-min scp (r = 0.91; p <0.0001) (fig . The equivalent ucpcr cutoff was 0.37 nmol / mmol, with 84% sensitivity and 97% specificity (fig . 1b). Using the ucpcr cutoff 0.53 nmol / mmol in the home evening meal samples yielded lower levels of sensitivity (71%) and specificity (97%) for significant endogenous insulin secretion . This is probably explained by a lower stimulus, as shown by the lower 120-min ucpcr in the home postprandial samples than in those in the mmtt (0.16 nmol / mmol [interquartile range 0.010.76] vs. 0.35 nmol / mmol [0.041.41]; p <0.0001). The correlations were similar in adults and children when analyzed separately (supplementary tables 4 and 5). Result tables for combined (supplementary table 3) and separate analysis of adults (supplementary table 4) and children (supplementary table 5) are given in the supplementary materials . Ucpcr measured during an mmtt or after a home meal is highly correlated with mmtt scp . Our results showed strong correlations between stimulated ucpcr and serum c - peptide (r = 0.910.97) during an mmtt . Because ucpcr is stable at room temperature for 3 days in boric acid preservative (7), home samples could be collected following a liquid mixed meal or the patients own home meal and a spot urine sample collected and posted for analysis directly . This would allow assessment to be done at home and to be noninvasive a particular advantage for children . As would be predicted, ucpcr values were lower after a meal compared with the standard mmtt, and so a lower concentration of ucpcr was required to suggest clinically significant insulin deficiency . The slight loss of precision compared with the standard mmtt needs to be balanced by the practicality of this approach because it would remove the need for inpatient testing and allow widespread screening . The strong correlation of ucpcr with serum c - peptide in the mmtt is supported by previous studies that have shown that timed measures of urinary c - peptide are a useful marker of endogenous insulin secretion (7,913). This allowed spot samples to be taken rather than sampling over 24 h, in which case complete collection is difficult . This is similar to the practical reason why spot albumin creatinine ratio is used as opposed to 24 h urine collections in the assessment of renal protein excretion . A strong correlation between auc c - peptide and 120-min ucpcr was demonstrated (r = 0.96); however, numbers were small (n = 18) and further work is needed to explore this . The test can be difficult in young children, especially those who are still in nappies . The ease of use means that, if used in conjunction with formal mmtt, ucpcr may be useful for screening patients for initial inclusion and also follow - up during intervention trials . In conclusion, our study demonstrates that in children and adults with type 1 diabetes, ucpcr may be a practical noninvasive alternative to the mmtt for use in routine clinical practice . Our results showed strong correlations between stimulated ucpcr and serum c - peptide (r = 0.910.97) during an mmtt . Because ucpcr is stable at room temperature for 3 days in boric acid preservative (7), home samples could be collected following a liquid mixed meal or the patients own home meal and a spot urine sample collected and posted for analysis directly . This would allow assessment to be done at home and to be noninvasive a particular advantage for children . As would be predicted, ucpcr values were lower after a meal compared with the standard mmtt, and so a lower concentration of ucpcr was required to suggest clinically significant insulin deficiency . The slight loss of precision compared with the standard mmtt needs to be balanced by the practicality of this approach because it would remove the need for inpatient testing and allow widespread screening . The strong correlation of ucpcr with serum c - peptide in the mmtt is supported by previous studies that have shown that timed measures of urinary c - peptide are a useful marker of endogenous insulin secretion (7,913). This allowed spot samples to be taken rather than sampling over 24 h, in which case complete collection is difficult . This is similar to the practical reason why spot albumin creatinine ratio is used as opposed to 24 h urine collections in the assessment of renal protein excretion . A strong correlation between auc c - peptide and 120-min ucpcr was demonstrated (r = 0.96); however, numbers were small (n = 18) and further work is needed to explore this . The test can be difficult in young children, especially those who are still in nappies . The ease of use means that, if used in conjunction with formal mmtt, ucpcr may be useful for screening patients for initial inclusion and also follow - up during intervention trials . In conclusion, our study demonstrates that in children and adults with type 1 diabetes, ucpcr may be a practical noninvasive alternative to the mmtt for use in routine clinical practice.
Metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus (tcr c-17317 and tcr v-rp24 - 74e19), the igh locus (igh v-224m14), igh c (from c1- 3of c) and a telomere - repeat specific peptide nucleic acid (pna) probe (applied biosystems). Three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described . Confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat (n.a . 1.4) objective lens with 0.07 m x - y pixel sampling, optical slice thickness of 0.8 m, and z - step size of 0.2 m . Volumes of interest (vois) were drawn around fish spots to be measured . Within the individual vois, the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software (mipav, cit / nih). Tetraspeck fluorescent microspheres (molecular probes / invitrogen) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope, fluorescence channel alignment, and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object . The minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . For distance measurements the following tcr probe sets were used: rp24 - 334b8 and rp23 - 255n13 (proximal), rp24 - 74e19 and 17317 (middle), rp23 - 304l21 and rp23 - 10k20 (distal) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d . Thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4, cd8, tcr (h57 - 597), tcr (gl3) (bd pharmingen, san diego, ca). Genomic dna was isolated from thymocytes using a qiagen dna isolation kit (qiagen, valencia, ca). 50ng dna was pcr amplified with a combination of gene - specific primers (supplementary table 3) using platinum taq polymerase (invitrogen, carlsbad, ca) for standard, or the power sybr green master mix kit (applied biosystems, foster city, ca) for qpcr . Real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software (applied biosystems, foster city, ca). For each assay, aliquots of dna were analyzed for a control, non - rearranging dna 3 of j2 . The cycle threshold numbers for each primer combinations (ct) and for the control amplification (ct) were used to calculate the absolute amount of pcr signal . The relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . For sequence analysis, 150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit (invitrogen). Amplify tcr v - dj coding joint sequences, v primers were used as forward primers with the primer 3j1 as reverse primer (supplementary table 4). Junctions from tcr mice were amplified using the indicated primers (supplementary table 4). Briefly, 10 g of total thymus genomic dna was digested with stui (new england biolabs). The digest products were run out on a 1% agarose tae gel, transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends . One g of genomic dna was treated with terminal deoxynucleotidyl transferase (new england biolabs) per manufacturer s protocol at a final concentration of 5 m datp . Two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers . Conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute, 57c for 45 seconds, and 72c for 45 seconds and then a final 5 minute extension step at 72c . Two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water . One l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii . Reaction conditions were the same as for the primary reactions, but the total number of cycles was increased to 30 cycles total . 15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane (bio - rad). Loading control pcr reactions, aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described . 10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane (bio - rad). The oligonucleotides d1, d2, univ - bglii, t17-univ and ds850 are listed in supplementary table 4 . Thymi were fixed in buffered 10% formalin, and paraffin sections were stained with hematoxylin - eosin . Metaphase spreads from cultured lymphocytes were prepared and analyzed by fish using bac probes containing the tcr locus (tcr c-17317 and tcr v-rp24 - 74e19), the igh locus (igh v-224m14), igh c (from c1- 3of c) and a telomere - repeat specific peptide nucleic acid (pna) probe (applied biosystems). Three dimensional fish on freshly isolated thymocytes using tcrv and tcrc locus specific probes was performed as described . Confocal image stacks were collected using a zeiss lsm510 meta microscope equipped with a 63 plan - apochromat (n.a . 1.4) objective lens with 0.07 m x - y pixel sampling, optical slice thickness of 0.8 m, and z - step size of 0.2 m . Volumes of interest (vois) were drawn around fish spots to be measured . Within the individual vois, the center of intensity mass was calculated for each fish spot and the 3-dimensional distance between red and green spots was measured using customized software (mipav, cit / nih). Tetraspeck fluorescent microspheres (molecular probes / invitrogen) 0.1 m and 0.5 m in diameter were imaged using the same microscope parameters and used to model the point spread function of the microscope, fluorescence channel alignment, and to determine the validity of the mipav software to accurately measure the center of intensity mass of a fluorescence object . The minimum measurable distance between the two fluorescence points was 70 nm in the lateral dimension . For distance measurements the following tcr probe sets were used: rp24 - 334b8 and rp23 - 255n13 (proximal), rp24 - 74e19 and 17317 (middle), rp23 - 304l21 and rp23 - 10k20 (distal) and as control rp23 - 309a8 and rp24 - 336f10 which are separated by 1 mb and map to mouse chromosome 1d . Thymocytes were washed twice in hbss containing 0.1% bsa and 0.1% nan3 and stained with antibodies specific for cd4, cd8, tcr (h57 - 597), tcr (gl3) (bd pharmingen, san diego, ca). Genomic dna was isolated from thymocytes using a qiagen dna isolation kit (qiagen, valencia, ca). 50ng dna was pcr amplified with a combination of gene - specific primers (supplementary table 3) using platinum taq polymerase (invitrogen, carlsbad, ca) for standard, or the power sybr green master mix kit (applied biosystems, foster city, ca) for qpcr . Real - time qpcr was performed in duplicate and data were collected on an abi 9700 sequence analyzer and analyzed using the sds 2.2 software (applied biosystems, foster city, ca). For each assay, aliquots of dna were analyzed for a control, non - rearranging dna 3 of j2 . The cycle threshold numbers for each primer combinations (ct) and for the control amplification (ct) were used to calculate the absolute amount of pcr signal . The relative ratios of each rearrangement were averaged and plotted together with the standard error of the mean . For sequence analysis, 150 ng of thymocyte dna was used to amplify coding joints which were cloned using a ta cloning kit (invitrogen). Amplify tcr v - dj coding joint sequences, v primers were used as forward primers with the primer 3j1 as reverse primer (supplementary table 4). Junctions from tcr mice were amplified using the indicated primers (supplementary table 4). Briefly, 10 g of total thymus genomic dna was digested with stui (new england biolabs). The digest products were run out on a 1% agarose tae gel, transferred onto zeta - probe gt membrane and probed with the c-i for coding joints and coding ends . One g of genomic dna was treated with terminal deoxynucleotidyl transferase (new england biolabs) per manufacturer s protocol at a final concentration of 5 m datp . Two percent of the total volume of each poly - adenylation reaction was then used for primary amplification using d1 and t17-univ primers . Conditions were 95c for 5 minutes followed by 15 cycles of 95c for 1 minute, 57c for 45 seconds, and 72c for 45 seconds and then a final 5 minute extension step at 72c . Two percent by volume of each primary amplification reaction was then serially diluted 5-fold in water . One l of the original primary reaction and of each serial dilution were used as template for a secondary amplification step using primers d2 and univ - bglii . Reaction conditions were the same as for the primary reactions, but the total number of cycles was increased to 30 cycles total . 15l of each secondary reaction was run out on a 1% agarose tbe gel and then transferred onto zeta - probe gt membrane (bio - rad). The membrane was subsequently hybridized with p - labeled ds850 oligonucleotide . For il-2 loading control pcr reactions, aliquots of the dna samples used for poly - adenylation were serially diluted 5-fold and amplified as previously described . 10l of each pcr product was run out on a 1% agarose tbe gel and then transferred overnight onto zeta - probe gt membrane (bio - rad). The oligonucleotides d1, d2, univ - bglii, t17-univ and ds850 are listed in supplementary table 4 . Thymi were fixed in buffered 10% formalin, and paraffin sections were stained with hematoxylin - eosin.
It integrates signals from cortical areas and subserves important functions like motor control (tecuapetla et al ., 2014, kravitz et al ., 2010) and reinforcement / punishment coding (kravitz et al ., 2012). Striatal neurons comprise gabaergic spiny projection neurons (spns, 95%) and interneurons (5%). Spns are classified into direct pathway spns (dspns), which project to the substantia nigra reticulata and external and internal segments of the globus pallidus (gpe and gpi, respectively), and indirect pathway spns (ispns), which project to gpe (bolam et al ., 2000, wu et al ., 2000 striatal interneurons include large aspiny cholinergic neurons and different populations of gabaergic interneurons (kawaguchi et al ., 1995). Excitatory glutamatergic neurons from virtually all cortical areas send projections to the striatum (mcgeorge and faull, 1989), and several studies suggest their recruitment during action selection (xiong et al ., 2015, znamenskiy and zador, 2013, koralek et al ., 2012). In contrast, cortical gabaergic neurons projecting to the striatum were not considered to be a component of the canonical corticostriatal network because they were identified only in the prefrontal, somatosensory, and retrosplenial cortices (lee et al ., 2014, jinno and kosaka, 2004). Only recently were direct gabaergic neurons projecting to the striatum also described in the motor and auditory cortices (rock et al ., 2016). The authors identified the long - range projecting neurons as somatostatin - positive (som) and, furthermore, reported that inhibition conveyed by these neurons was onto both dspns and ispns . We previously showed that long - range gabaergic neurons connecting several brain structures comprise different molecular subtypes . For instance, connectivity between the hippocampus and medial entorhinal cortex is supported by parvalbumin - positive (pv) and som neurons (melzer et al ., 2012). Moreover, projections from the septum to the medial entorhinal cortex are pv and calbindin, and they inhibit specific interneurons differentially (fuchs et al ., 2016). Hence, we wondered whether long - range gabaergic projecting neurons from the motor cortex to the striatum are diverse with respect to their molecular identity, target specificity, and function at the behavioral level . Based on virus - mediated tracing, optogenetics, patch - clamp recordings in vitro, and behavioral essays, we identified two distinct populations of long - range projecting gabaergic neurons in the primary (m1) and secondary (m2) motor cortex targeting the dorsal striatum and established that these two populations exhibit target cell preference in the striatum and affect locomotion differentially . Som cells are a major source of intracortical and corticofugal long - range gabaergic projections (tomioka et al ., 2005, rock et al ., 2016). To substantiate and extend these studies focusing on long - range gabaergic neurons connecting the motor cortex and the striatum, we injected adeno - associated virus (aav) double - floxed inverse open reading frame (dio) chr2-mcherry into the m1 and m2 area of som mice . This resulted in labeling of a subpopulation of gabaergic neurons (figures 1a and 1b; figure s1a) and revealed projections in several ipsilateral cortical and subcortical areas and, to a lesser extent, in contralateral cortices (table s1). There was consistent innervation of the ipsilateral dorsal striatum (figure 1b; table s1). Motor cortex som neuron projections traversed the dorsal striatum and branched preferentially ventro - laterally, sparing the most rostral and caudal part of the dorsal striatum (figure 1b).figure 1motor cortex som gabaergic neurons innervate the striatum(a) schematic drawing of the injection site and the location of long - range projections in the striatum shown in (b). Viral constructs encoding chr2-mcherry were injected into the motor cortex of som mice. (b) bright - field images of dab - stained sections showing the injection site in the motor cortex (left) and mcherry - labeled axons in the striatum (center) following aav dio chr2-mcherry injection into the motor cortex of som mice . A higher magnification of the boxed area is shown on the right. (c and d) confocal images showing a retrogradely labeled area (c) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex, visualized via fish for sst and gad1/2 (d). (e h) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva) rabies virus . Tcb was expressed cre - dependently in the motor cortex of som mice, and rabies virus was injected into the striatum . (e) shows differential interference contrast (dic) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in (f). (g) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in (h).str, striatum . Motor cortex som gabaergic neurons innervate the striatum (a) schematic drawing of the injection site and the location of long - range projections in the striatum shown in (b). (b) bright - field images of dab - stained sections showing the injection site in the motor cortex (left) and mcherry - labeled axons in the striatum (center) following aav dio chr2-mcherry injection into the motor cortex of som mice . (c and d) confocal images showing a retrogradely labeled area (c) following injection of the retrograde tracer ctb647 into the striatum and a retrogradely labeled gabaergic som neuron in the motor cortex, visualized via fish for sst and gad1/2 (d). (e h) som projecting neurons were identified by retrograde tracing with sadg - egfp(enva) rabies virus . Tcb was expressed cre - dependently in the motor cortex of som mice, and rabies virus was injected into the striatum . (e) shows differential interference contrast (dic) and epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex with the corresponding firing pattern shown in (f). (g) shows a confocal image of a retrogradely labeled tcb neuron in m1 immunostained for egfp and som with the corresponding morphological reconstruction shown in (h). See also figure s1 and tables s1 and s2 . To further substantiate the presence of gabaergic corticostriatal projections, we performed retrograde labeling . We injected cholera toxin b (ctb) subunit 647 into the ventro - lateral part of the dorsal striatum and analyzed retrogradely labeled cells in the m1 region (figures s1b and s1c). As expected, a dense band of retrogradely labeled cells became visible in cortical l5 (figure 1c); i.e., in the layer that is the major source of corticostriatal excitatory projections (wilson, 1987, cowan and wilson, 1994). To visualize gabaergic cells among the m1 retrogradely labeled cells, we performed multi - fluorescence in situ hybridization (fish) for sst (encoding som) and gad1/2 (encoding gad67/65). We found 13 retrogradely labeled cells in m1 that were clearly positive for gad1/2 (n = 3,582 ctb cells and 5,064 gad1/2 cells, cell counts across all layers in 35 slices from 4 hemispheres in 4 mice), 8 of which co - labeled for sst (figure 1d). To confirm a direct long - range gabaergic connection between the motor cortex (m1/m2) and the dorsal striatum, we performed retrograde monosynaptic tracing with rabies virus (wickersham et al ., 2007). We injected aavs encoding cre - dependent avian virus receptor (avian tumor virus receptor a mcherry [tcb]; weissbourd et al ., 2014) and rabies glycoprotein (rg) into the striatum of a2a - cre mice that express cre recombinase specifically in ispns (gong et al ., 2003). Subsequent injection of rg - deleted envelope protein from avian aslv type a (enva)-pseudotyped rabies virus (sadg - egfp(enva)) into the striatum resulted in transsynaptically retrogradely labeled cells in the cortex (figures s1e and s1f). Fish for rabies virus - specific mrna (rabv - gp1) and gad1/2 revealed double - positive neurons in the motor cortex (7 cells in 34 slices from 4 hemispheres in 4 mice; figure s1 the average number of labeled cells per slice was lower than after ctb647 injections, suggesting that ispns were not the only striatal target cells of gabaergic projecting neurons and/or reflecting lower efficiency of transsynaptic tracing (marshel et al .,, we expressed tcb cre - dependently in the motor cortex (m1/m2) of som mice and injected sadg - egfp(enva) rabies virus into the striatum . Tcb retrogradely labeled cells in the motor cortex had a classical or burst accommodating firing pattern (n = 11 cells from 5 hemispheres in 4 mice; figures 1e and 1f; figure s1h) similar to non - retrogradely labeled tcb cells (table s2). Reconstructed cells had a martinotti cell - like morphology (wang et al ., 2004) with axonal projections extending over all cortical layers (three reconstructions from three hemispheres in two mice; figures 1 g and 1h; figures s1i we next tested whether som projecting neurons form functional synapses onto striatal neurons and whether the connectivity exhibits target specificity . We injected aav dio chr2-mcherry into m1/m2 of som mice and combined optogenetic stimulation of long - range projections with patch - clamp recordings of putative postsynaptic cells in the striatum (figure 2a). All injections (n = 36 hemispheres) resulted in labeled axons that projected to the dorsal striatum . Of 305 patched neurons (in 27 mice), 50 responded with short - latency postsynaptic currents (pscs) to 5-ms photostimulation of motor cortex som neuron projections (figure 2b). As a specificity control, we repeated the experiment in wild - type mice injected with aav dio chr2-mcherry and found neither mcherry fibers in the dorsal striatum nor a response after photostimulation (n = 58 cells in 2 mice). Responses in som mice could not be blocked with 6-cyano-2,3-dihydroxy-7-nitro - quinoxaline (cnqx) and d-2-amino-5-phosphonovaleric acid (d - ap5) (165.6 32.7 pa baseline versus 169.5 31.1 pa with drugs [mean sem], paired t test, t(14) = 0.49, p = 1, n = 15 cells in 11 mice; figure s2a) but with gabazine (117.8 [134.3] pa versus 1.6 [1.6] pa [median interquartile range (iqr)], wilcoxon signed - rank test, w = 210, p = 0.0002, n = 20 cells in 15 mice; figures s2a and s2b). Responses reversed around the reversal potential of gabaergic receptors (n = 14 cells in 9 mice; figure 2b), thus confirming the gabaergic nature of motor cortex som neuron projections.figure 2motor cortex som neuron projections form functional synapses on striatal output and cholinergic neurons(a) schematic drawing indicating the injection site (left) and location of an exemplary patched neuron in the striatum (right). Aav dio chr2-mcherry was injected into the motor cortex of som mice, and target cells were patched in the striatum (dic image). (b) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation (blue ticks) of motor cortex som neuron projections . Responses were blocked with gabazine but not d - ap5/cnqx. (c) firing pattern (membrane potential upon 50-pa current injection and at the action potential [ap] threshold) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections. (d) firing pattern (spontaneous activity and maximal firing frequency) and dic image of a representative cholinergic interneuron (arrow) that was responsive to photostimulation of motor cortex som neuron projections. (e) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections (from top to bottom: maximal firing frequency, ap threshold, and 50-pa current injection). (f) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections . Number of mice: spns, 19; cholinergic, 11; gabaergic interneurons, 19. (g) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice. (h) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp (left) and som / drd2-egfp (right) mice exhibiting differential egfp expression in the globus pallidus (arrow) and substantia nigra (arrowhead). (i) exemplary traces of dspn and ispn responses to photostimulation (blue ticks) of m1 and m2 som neuron projections using cs - based low cl intracellular solution (from top to bottom: 0 mv, reversal potential, and 95 mv holding potential). (j) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections . Number of mice: m1-dspns, 7; m1-ispns, 16; m2-dspns, 8; m2-ispns, 17; m1-cholinergic, 19; m2-cholinergic, 13. (k) schematic drawing indicating the localization of responding cells in a coronal (left) and sagittal (right) cross - section . Color code: orange, m1 to dspn; yellow, m2 to dspn; dark green, m1 to ispn; light green, m2 to ispn; purple, m1 to cholinergic interneuron; red, m2 to cholinergic interneuron.hp, hippocampus; ach, cholinergic interneuron . Som neuron projections form functional synapses on striatal output and cholinergic neurons (a) schematic drawing indicating the injection site (left) and location of an exemplary patched neuron in the striatum (right). Aav dio chr2-mcherry was injected into the motor cortex of som mice, and target cells were patched in the striatum (dic image). (b) pscs recorded in a striatal neuron at the indicated holding potentials following 5-ms photostimulation (blue ticks) of motor cortex som neuron projections . (c) firing pattern (membrane potential upon 50-pa current injection and at the action potential [ap] threshold) of a representative spn that was responsive to photostimulation of motor cortex som neuron projections . (d) firing pattern (spontaneous activity and maximal firing frequency) and dic image of a representative cholinergic interneuron (arrow) that was responsive to photostimulation of motor cortex som neuron projections . (e) firing pattern of a striatal gabaergic interneuron that was responsive to photostimulation of motor cortex som neuron projections (from top to bottom: maximal firing frequency, ap threshold, and 50-pa current injection). (f) percentage of striatal neurons responding to photostimulation of motor cortex som neuron projections . Number of mice: spns, 19; cholinergic, 11; gabaergic interneurons, 19 . (g) schematic drawing indicating injection sites of aav dio chr2-mcherry in som / drd1a - egfp and som / drd2-egfp mice . (h) confocal image of egfp - immunostained sagittal sections of som / drd1a - egfp (left) and som / drd2-egfp (right) mice exhibiting differential egfp expression in the globus pallidus (arrow) and substantia nigra (arrowhead). (i) exemplary traces of dspn and ispn responses to photostimulation (blue ticks) of m1 and m2 som neuron projections using cs - based low cl intracellular solution (from top to bottom: 0 mv, reversal potential, and 95 mv holding potential). (j) percentage of striatal neurons responding to photostimulation of m1 and m2 som neuron projections . Number of mice: m1-dspns, 7; m1-ispns, 16; m2-dspns, 8; m2-ispns, 17; m1-cholinergic, 19; m2-cholinergic, 13 . (k) schematic drawing indicating the localization of responding cells in a coronal (left) and sagittal (right) cross - section . Color code: orange, m1 to dspn; yellow, m2 to dspn; dark green, m1 to ispn; light green, m2 to ispn; purple, m1 to cholinergic interneuron; red, m2 to cholinergic interneuron . Hp, hippocampus; ach, cholinergic interneuron . See also figures s2 and s3 and tables s3 and s4 . To scrutinize target specificity, striatal neurons were sorted into spns and cholinergic and gabaergic interneurons based on their electrophysiological properties and cell soma shape (planert et al ., 2013, gertler et al ., 2008, kawaguchi, 1992, bennett and wilson, 1999, kawaguchi et al ., 1995; experimental procedures; figures 2c2e; table s3). We found that 22.6% of spns, 33.3% of cholinergic cells, and only 2% of gabaergic interneurons responded to 5-ms photostimulation of motor cortex som neuron projections (figure 2f; figures s2c and s2d; table s3). Together, these data indicate that spns and cholinergic cells are the main target of motor cortex som neuron projections . To answer whether dspns and ispns are differentially targeted by motor cortex som projecting neurons, we cross - bred som mice to drd1a - egfp and drd2-egfp mice in which dspns and ispns, respectively, are selectively labeled (gong et al ., 2003; we injected aav dio chr2-mcherry into m1 or m2 of som / drd1a - egfp or som / drd2-egfp mice (figure 2 g; figure s2e) and combined photostimulation of m1 or m2 som neuron projections with patch - clamp recordings of striatal neurons (figure s2f). We found that m1 som projecting neurons innervated a comparable proportion of dspns (29%), ispns (22%), and cholinergic cells (40%) (figures 2i and 2j; fisher s exact test, p = 0.19). M2 som projecting neurons targeted dspns (25%) and ispns (16%) to a similar extent, whereas cholinergic cells tended to be innervated less frequently (4%) (figure 2j; fisher s exact test, p = 0.08). Inhibition of cholinergic cells by m2 was significantly less frequent than by m1 (figure 2j; fisher s exact test, m1 dspns versus m2 dspns: p = 1; m1 ispns versus m2 ispns: p = 1; m1 cholinergic versus m2 cholinergic: p = 0.006). Responses had a latency of 2.5 (1.3) ms (median [iqr]; n = 47 responding cells; see figure s3a and table s4 for more details) and a reversal potential of 58.0 1.4 mv (mean sem; n = 22 responding cells; see figure s3b and table s4 for more details). The strength of m1 and m2 som neuron connections to spns was similar (table s4). However, when comparing inputs with dspns and ispns both from m1 and m2, the response amplitudes were significantly larger for ispns (amplitudes at 0 mv with cesium [cs] internal solution: 15.7 pa versus 44.0 [36.5] pa [median (iqr)] in dspns and ispns respectively; mann - whitney u test, u = 102, p = 0.02, n = 14 and 26 responding cells, respectively; figure s3c). Detected target cells were located preferentially in the ventro - lateral part of the dorsal striatum (figure 2k; figures s3d targeted dspns and ispns were intermingled and localized 2.4 (0.7) mm lateral, 0.1 (0.7) mm posterior to the bregma and 4 (0.7) mm deep (median [iqr]). Spns targeted by m1 projections were more lateral than spns targeted by m2 projections (mann - whitney u test, u = 142, p = 0.04; table s4). The retrograde labeling experiments suggested that som neurons are not the only m1 gabaergic population projecting to the striatum . Pv neurons appeared to be attractive candidates because we identified them before as a major class of neurons providing long - range inhibition in the entorhinal cortex - hippocampal formation (melzer et al ., 2012). Testing for the presence of gabaergic long - range projecting pv cells based on virus tracing in pv mice may be complicated by the fact that a fraction of cortical pv cells are glutamatergic (jinno and kosaka, 2004). Hence, we first tested the gabaergic nature of pv cells in m1/m2 by counting the number of double - positive cells in injected pv / gad67 mice . 99.5% of pv cells were gad67 (759 of 763 pv cells from 9 slices in 3 mice). This result is in agreement with previous evidence showing the absence of pv glutamatergic neurons in the motor cortex (jinno and kosaka, 2004). In the mouse neocortex, pv and som neurons constitute two non - overlapping neuronal entities (xu et al ., 2010, tasic et al ., 2016). Thus, we injected aav dio chr2-mcherry into m1 of pv mice, and, indeed, we detected labeled axons in the dorsal striatum that branched preferentially ventro - laterally (in 12 of 17 injected hemispheres). In contrast, injections into m2 of pv mice were less likely to result in labeled striatal projections (5 of 12 injections), and projections when present were sparser than the ones detected after m1 injections . Thus, for a more detailed analysis of innervation patterns, we focused on projections from m1 . We injected aav dio chr2-mcherry into m1 of pv / drd1a - egfp or pv / drd2-egfp mice (figure 3a) and found that 31% of dspns, 6% of ispns, and 6% of cholinergic and 0% of gabaergic interneurons responded to photostimulation of m1 pv neuron projections (figures 3b and 3c). Thus, pv projecting neurons preferentially innervated dspns (fisher s exact test; dspns versus ispns: p = 0.009, dspns versus cholinergic: p = 0.04, ispns versus cholinergic: p = 1; figure 3b). Spns targeted by m1 pv projecting neurons had a response amplitude comparable with that following stimulation of m1 som projections (figure s3c; table s4). Although responses could not be blocked by cnqx / d - ap5 (although one of five cells showed a clear decrease in amplitude), subsequent application of gabazine led to a significant amplitude decrease (68.9 [147.3] pa baseline versus 70.3 [86.1] pa with cnqx / d - ap5 versus 0.5 [3.2] pa with gabazine, friedman test, p = 0.02, post hoc conover s test with bonferroni correction, baseline versus cnqx / d - ap5: p = 1, baseline versus gabazine: p = 0.004, cnqx / d - ap5 versus gabazine: p = 0.009, n = 5 cells in 4 mice; figure 3d; figures s3 g and s3h). The reversal potential of the responses was 60.3 2.5 mv (mean sem, n = 4 cells in 4 mice; figure s3b; table s4), reconfirming the gabaergic nature . Spns targeted by m1 pv projecting neurons were located 0.6 (0.7) mm posterior to the bregma, 3.0 (0.6) mm lateral, and 3.7 (0.8) deep (median [iqr]) (figure 3e; figures s3d s3f; table s4).figure 3m1 pv neuron projections preferentially target dspns in the striatum(a) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice. (b) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections . Number of mice: dspns, 8; ispns, 7; cholinergic, 13; gabaergic interneurons, 9. (c) firing pattern (upon 50 pa current injection and at the ap threshold) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections. (d) responses of the dspn shown in (c) at 70 mv holding potential using high cl intracellular solution . Responses were blocked with gabazine but not d - ap5/cnqx. (e) schematic drawing indicating the localization of responding cells in a coronal (left) and sagittal (right) cross - section . Color code: orange, m1 to dspn; yellow, m1 to ispns; gray, unidentified responding neurons. (f and g) epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex (arrow) (f) with the corresponding firing pattern (g) identified by retrograde tracing with sadg - egfp(enva) rabies virus . Tcb was expressed cre - dependently in the motor cortex of pv mice, and rabies virus was injected into the striatum. (h) dot plot of the action potential half - width for retrogradely labeled som (n = 11 cells from 5 hemispheres in 4 mice) and pv neurons (n = 3 cells from 3 hemispheres in 2 mice).see also figure s3 and tables s2 and s4 . M1 pv neuron projections preferentially target dspns in the striatum (a) schematic drawing indicating the injection site of aav dio chr2-mcherry in pv / drd1a - egfp and pv / drd2-egfp mice . (b) percentage of striatal neurons responding to photostimulation of m1 pv neuron projections . Number of mice: dspns, 8; ispns, 7; cholinergic, 13; gabaergic interneurons, 9 . (c) firing pattern (upon 50 pa current injection and at the ap threshold) of a representative dspn that was responsive to photostimulation of m1 pv neuron projections . (d) responses of the dspn shown in (c) at 70 mv holding potential using high cl intracellular solution . (e) schematic drawing indicating the localization of responding cells in a coronal (left) and sagittal (right) cross - section . Color code: orange, m1 to dspn; yellow, m1 to ispns; gray, unidentified responding neurons . (f and g) epifluorescent images of a retrogradely labeled tcb neuron in the motor cortex (arrow) (f) with the corresponding firing pattern (g) identified by retrograde tracing with sadg - egfp(enva) rabies virus . Tcb was expressed cre - dependently in the motor cortex of pv mice, and rabies virus was injected into the striatum . (h) dot plot of the action potential half - width for retrogradely labeled som (n = 11 cells from 5 hemispheres in 4 mice) and pv neurons (n = 3 cells from 3 hemispheres in 2 mice). The different innervation patterns of som and pv projecting neurons suggested that the two cell types represent distinct subpopulations in the motor cortex . We hence analyzed whether the firing pattern of pv projecting neurons differed from that of som projecting neurons and resembled that of classical pv interneurons . The detection of projecting neurons was assisted by retrograde tracing with sadg - egfp(enva) rabies virus injections into the striatum of pv mice expressing cre - dependent tcb in motor cortex neurons . Indeed, all tcb / rbv - egfp non - pyramidal neurons in the motor cortex exhibited a fast spiking firing pattern with faster action potentials than som projecting neurons (figures 3f3h; figure s3i; table s2). To investigate the behavioral effect of activating the newly discovered motor cortex som and pv projecting neurons (striatal targeting is summarized in figure 4a), we implanted optic fibers bilaterally into the dorsal striatum of pv and som mice that were injected with aav dio chr2-mcherry into the motor cortex (figures 4b4d). We compared the performance of four groups of animals: pv mice injected in m1 (pv - m1), som mice injected in m1 (som - m1), som mice injected in m2 (som - m2), and control mice (pv and som mice injected in m1 or m2 with aav dio eyfp and wild - type mice injected in m1 or m2 with aav tomato). We first asked whether activation of motor cortex pv and som neuron projections in the striatum modulated spontaneous locomotion . Mice were allowed to explore a circular arena, and locomotion was measured before and during light stimulation of corticostriatal projections (5-ms pulses at 20 hz; power, 3 mw). We next calculated the difference between motion (defined as the distance moved in 5 s) during and before light stimulation on four epochs of different durations; i.e., 10, 30, 60, and 120 s, starting at light stimulation onset . The performance of control mice was similar before and during light stimulation (figures 4e4 g) (the slight increase observed during the 10-s epoch was not significantly different from zero; one - sample t test (null hypothesis [h0] = 0), t(12) = 2.1, p = 0.06). Conversely, the performance of som - m1, pv - m1, and som - m2 changed upon photostimulation (figures 4e and 4f). During the 10-, 30-, and 60-s epochs, som - m1 mice showed a significant motion increase, whereas pv - m1 and som - m2 mice showed a significant motion decrease with respect to control mice (figures 4e and 4f; one - way anova followed by post hoc comparisons, 10 s: f(3, 31) = 7.71, p = 0.0005, control versus pv - m1: p = 0.03, control versus som - m1: p = 0.02, control versus som - m2: p = 0.04; 30 s: f(3, 31) = 10.48, p = 0.0001, control versus pv - m1: p = 0.04, control versus som - m1: p = 0.002, control versus som - m2: p = 0.05; 60 s: f(3, 31) = 12.52, p = 0.0001, control versus pv - m1: p = 0.002, control versus som - m1: p = 0.01, control versus som - m2: p = 0.008). For the 120-s epoch, only pv - m1 mice still showed a motion decrease with respect to control mice, whereas som - m1 and som - m2 performance could not be distinguished from that of control mice (figures 4e and 4f; one - way anova followed by post hoc comparisons, f(3, 31) = 3.96, p = 0.01, control versus pv - m1: p = 0.01, control versus som - m1: p = 0.42, control versus som - m2: p = 0.13).figure 4motor cortex som and pv projecting neurons mediate locomotion change(a) schematic drawing summarizing the newly identified corticostriatal gabaergic projections . M1 som neurons innervate dspns, ispns, and cholinergic interneurons (blue); m2 som neurons preferentially innervate dspns and ispns (green); m1 pv neurons preferentially innervate dspns (pink); m2 pv neuron projections are scarce (purple). D1, dspns; d2, ispns; in, gabaergic interneuron. (b) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice (top) and of optic fiber implantation in the striatum (bottom). (c and d) bright - field images of dab - stained sections showing m1 and m2 injection sites (c), optic fiber position, and mcherry - labeled axons in the striatum (d). (e) exemplary locomotion traces (in cm/40 ms) of control, som - m1, pv - m1, and som - m2 mice before (black) and during light stimulation (blue) (5-ms pulses delivered at 20 hz; light power, 3 mw). (f) mean sem difference between motion levels (in cm/5 s) during and before light stimulation for 10-, 30-, 60-, and 120-s epochs, starting at light stimulation onset. (g j) mean sem cumulative relative frequency of running speed (top), and mean sem number, speed (in cm / s), and duration (in seconds) of mobility and immobility bouts 60 s before (black lines and white bars) and during 60-s light stimulation (blue lines and bars) for control mice (g) and som - m1 (h), pv - m1 (i), and som - m2 (j) mice . Control, n = 13 mice; som - m1, n = 7 mice; pv - m1, n = 9 mice; som - m2, n = 6 mice . Motor cortex som and pv projecting neurons mediate locomotion change (a) schematic drawing summarizing the newly identified corticostriatal gabaergic projections . M1 som neurons innervate dspns, ispns, and cholinergic interneurons (blue); m2 som neurons preferentially innervate dspns and ispns (green); m1 pv neurons preferentially innervate dspns (pink); m2 pv neuron projections are scarce (purple). (b) schematic drawing indicating sites of aav dio chr2-mcherry injection in som and pv mice (top) and of optic fiber implantation in the striatum (bottom). (c and d) bright - field images of dab - stained sections showing m1 and m2 injection sites (c), optic fiber position, and mcherry - labeled axons in the striatum (d). (e) exemplary locomotion traces (in cm/40 ms) of control, som - m1, pv - m1, and som - m2 mice before (black) and during light stimulation (blue) (5-ms pulses delivered at 20 hz; light power, 3 mw). (f) mean sem difference between motion levels (in cm/5 s) during and before light stimulation for 10-, 30-, 60-, and 120-s epochs, starting at light stimulation onset . (g j) mean sem cumulative relative frequency of running speed (top), and mean sem number, speed (in cm / s), and duration (in seconds) of mobility and immobility bouts 60 s before (black lines and white bars) and during 60-s light stimulation (blue lines and bars) for control mice (g) and som - m1 (h), pv - m1 (i), and som - m2 (j) mice . Control, n = 13 mice; som - m1, n = 7 mice; pv - m1, n = 9 mice; som - m2, n = 6 mice . We characterized the photostimulation - induced motion changes occurring during the 60-s epoch in more detail . We first analyzed the cumulative frequency distribution of running speed 60 s before and for 60 s during photostimulation (figures 4g4j). As expected, in control mice, no difference was found between the cumulative distribution curves before and during photostimulation (figure 4 g, top; wilcoxon matched - pairs signed - rank test, w = 193, p = 0.15). In som - m1 mice, the curve during photostimulation was shifted to the right, and, accordingly, the median running speed increased significantly upon photostimulation (figure 4h, top; wilcoxon matched - pairs signed - rank test, w = 215, p = 0.01). In pv - m1 and som - m2 mice, the cumulative distribution curve during photostimulation was shifted to the left, and the median running speed was significantly lower during than before photostimulation (figures 4i and 4j, top; wilcoxon matched - pairs signed - rank test, pv - m1: w = 113, p = 0.005; som - m2: w = 90, p = 0.02). Finally, based on running speed, we defined mobility and immobility bouts (experimental procedures) and measured their number, speed, and duration before and during photostimulation . In control mice, the properties of mobility and immobility bouts did not change upon photostimulation (figure 4 g; paired t test, number of bouts (nr) mobility: t(12) = 0.76, p = 0.46; speed mobility: t(12) = 1.33, p = 0.21; nr immobility: t(12) = 0.51, p = 0.62; speed immobility: t(12) = 0.64, p = 0.53; wilcoxon matched - pairs signed - rank test: duration mobility: w = 19, p = 0.54; duration immobility: w = 6, p = 0.83). In som - m1 mice, photostimulation elicited significant changes in mobility bouts: the number decreased, whereas the speed and duration increased (figure 4h; paired t test: nr: t(6) = 3.17, p = 0.02; speed: t(6) = 2.97, p = 0.02; wilcoxon matched - pairs signed - rank test, duration: w = 21, p = 0.03). Immobility bouts remained unaffected (figure 4h; paired t test: nr: t(6) = 1.46, p = 0.19; speed: t(6) = 0.97, p = 0.37; wilcoxon matched - pairs signed - rank test, duration: w = 9, p = 0.44). In pv - m1 mice, the duration of mobility bouts decreased significantly, and the duration of immobility bouts increased significantly upon photostimulation (figure 4i; wilcoxon matched - pairs signed - rank test, duration mobility: w = 35, p = 0.04, duration immobility: w = 39, p = 0.02), whereas their number and speed remained unchanged (figure 4i; paired t test: nr mobility: t(8) = 0.97, p = 0.36; speed mobility: t(8) = 1.57, p = 0.14; nr immobility: t(8) = 1.62, p = 0.19; speed immobility: t(6) = 0.78, p = 0.46). In som - m2 mice, the speed of mobility bouts was significantly reduced upon photostimulation (figure 4j; paired t test: t(5) = 3.48, p = 0.02), whereas all other variables remained unchanged (paired t test: nr mobility: t(5) = 0.14, p = 0.89; nr immobility: t(5) = 0.03, p = 0.97; speed immobility: t(5) = 0.26, p = 0.8; wilcoxon matched - pairs signed - rank test, duration mobility: w = 1, p = 0.99; duration immobility: w = 11, p = 0.31). In sum, we conclude that stimulation of striatal long - range projections of m1 som neurons increased locomotion by increasing the duration and speed of mobility bouts, that stimulation of striatal long - range projections of m1 pv neurons reduced locomotion by increasing the duration of immobility bouts, and that stimulation of striatal long - range projections of m2 som neurons reduced locomotion by decreasing the speed of mobility bouts . It has been proposed that movement control and reinforcement coding are mediated by common corticostriatal circuits (kravitz and kreitzer, 2012). Thus, we next asked whether stimulation of motor cortex pv and som neuron projections in the striatum also affect reinforcement / punishment coding . We tested the mice in a place preference task (figure s4). The task lasted 3 days, during which we recorded the time mice spent in each compartment . During the first and second days (habituation and baseline, respectively), place preference was measured without photostimulation . During the third day (test), one of the compartments (stimulation side) was paired with photostimulation (5-ms pulses at 20 hz; power, 3 mw), and place preference was measured . We calculated a difference score as the percentage of time spent on the stimulation side during baseline minus the percentage of time spent on the same side during the test . We found that the difference score obtained for pv - m1, som - m1, and som - m2 mice was similar to that of control mice (figure s4b; one - way anova: f(3,21) = 0.67, p = 0.58). Hence, stimulation of striatal long - range projections of motor cortex pv and som neurons did not elicit place preference by employing this paradigm . Here we show that distinct long - range gabaergic neurons connect m1 and m2 with the dorsal striatum . The newly identified long - range gabaergic neurons express either som or pv and differ with respect to target cell preference and the modulatory effect on motor activity . Our results indicate that both m1 and m2 harbor long - range gabaergic neurons that target the dorsal striatum and thus extend recent findings by rock et al . Furthermore, we report the following new findings . First, m1 and m2 contribute differentially to gabaergic corticostriatal projections . Second, we identified and characterized an additional projection formed by pv neurons that differs significantly from that formed by som neurons . Multiple reasons can explain why these connections have not been noticed until recently (rock et al ., 2016). First, the scarcity of long - range gabaergic neurons constitutes a challenge as to their detection by anterograde or retrograde labeling, considering the high number of excitatory neurons that are also labeled in the same area with their axons extending along a similar path . Second, most studies focused on more dorso - anterior areas of the striatum . Hence, retrograde tracer injections are unlikely to reveal gabaergic projecting neurons in motor cortices because their axons target preferentially more lateral, posterior, and ventral parts of the dorsal striatum . However, jinno and kosaka (2004) did not detect motor cortical long - range gabaergic neurons even though injections included target areas that were innervated in our study . A possible reason may be low uptake efficiency and transport of the tracer fluorogold in gabaergic neurons . First, there was robust axon labeling in the striatum even with regionally restricted minimal anterograde injections . Second, long - range gabaergic neurons were retrogradely labeled with ctb from the striatum . Third, long - range gabaergic neurons were transsynaptically retrogradely labeled with rabies virus that infected only striatal starter cells . Finally, the electrophysiological and pharmacological results provide strong evidence for the gabaergic nature of pv and som projecting neurons . Although we have no indication for any glutamatergic inputs deriving from som cells, based on our immunocytochemistry, pharmacology, and rabies virus tracing, we cannot exclude that glutamatergic transmission has a minor contribution to the behavioral effects seen upon stimulation of striatal long - range projections from m1 pv neurons . It is important to note that the number of long - range gabaergic neurons presented in this study remains an underestimation because quantitative evaluations are currently hampered by a number of technical constraints . First, experiments entail conservative / limited virus injection to prevent viral spread beyond the target area . Second, labeling by retrograde tracing is strongly dependent on the tracer and cell type; e.g., pv neurons could be detected following transsynaptic virus - mediated tracing but not by ctb labeling . For quantitative studies, it would be highly desirable to identify markers / promoters for long - range gabaergic neurons . Characterization of motor cortex gabaergic projecting neurons revealed that m1 and m2 som and pv cells differentially innervate striatal neurons . Moreover, bilateral stimulation of corticostriatal long - range gabaergic projections modulates motor activity in spite of the scarcity of gabaergic corticostriatal neurons and the relatively small amplitude responses of targeted striatal neurons . Thus, stimulation of m1 som neuron projections, targeting dspns, ispns, and cholinergic cells, leads to an increase in locomotion . In contrast, stimulation of m2 som neuron projections, targeting preferentially dspns and ispns, and of m1 pv neuron projections, targeting preferentially dspns, leads to a decrease in locomotion . Decreased locomotion upon preferential inhibition of dspns (pv - m1) is in line with previous studies showing either bradykinesia upon deletion of dspns (drago et al ., 1998) or increased locomotion upon stimulation of dspns (kravitz et al . Notably, there was a similar effect on locomotion upon preferential inhibition of dspns (pv - m1) or of both dspns and ispns (som - m2). Comparable effects were also reported when optogenetically silencing either dspn or both dspns and ispns (tecuapetla et al ., 2014). Increased locomotion upon stimulation of m1 som neuron projections most likely reflects the participation of a larger fraction of cholinergic cells . These striatal interneurons, constituting 1%3% of all striatal neurons, are tonically active and provide powerful feedforward inhibition to spns (english et al ., 2011, nelson et al ., 2009), and enhance dopamine release (threlfell et al ., 2012). Direct activation or inhibition of cholinergic striatal interneurons in the dorsal anterior striatum had no effect on locomotor activity (maurice et al ., 2015). However, based on our results, it is tempting to speculate that cholinergic cells in more ventral and posterior striatal areas receiving input from m1 som neurons are involved in motor control . At present, we cannot resolve whether the observed change in locomotor activity results from long - range axon activation in the striatum only or whether activation of putative collaterals via back - propagating action potentials also plays a role . In either case, our results show that the activity of som and pv projecting neurons in the motor cortex differentially modulates locomotor activity . This study is also relevant when interpreting data regarding silencing of cortical areas by manipulating gabaergic neurons because the effects may also involve long - distance targets . It has been proposed that motor control and reward coding are mediated by common corticostriatal circuits (kravitz and kreitzer, 2012). Our data indicate that, although activation of motor cortex pv and som neuron projections in the dorsal striatum affected locomotor activity, it did not affect place preference, although we cannot exclude their implication in reward coding more generally . On the other hand, stimulation of gabaergic projections from the prefrontal cortex to the ventral striatum induces avoidance behavior, suggesting that they are involved in the coding of punishment (lee et al ., 2014). Further experiments will be required to elucidate whether, and, if so, which, corticostriatal gabaergic projections mediate both locomotion and reward coding . This study adds to the increasing evidence that long - range gabaergic neurons are more frequent than previously thought . The heterogeneity of long - range gabaergic neurons described here is in line with previous studies indicating neurochemical diversity of long - range gabaergic neurons in the cortex and hippocampus (jinno et al ., 2007, higo et al ., 2007, lee et al ., 2014, notably, we demonstrate that distinct long - range gabaergic neurons exhibit specific functional properties and differential connectivity . Finally, it will be of interest to study long - range gabaergic neurons in the context of movement disorders that are thought to be caused by an imbalance of dspn and ispn activity . Thus, parkinsonian - like movements can be reproduced by increased ispn activity (kravitz et al ., 2010) and can be reduced by selective inhibition of striatal cholinergic interneurons (maurice et al ., huntington s disease is marked by an early degeneration of ispns (vonsattel et al ., 1985, mitchell et al ., 1999) and an imbalance of excitation and inhibition of dspns and ispns (andr et al ., 2011). In light of our findings, it is tempting to speculate that motor cortex gabaergic projections to the striatum might be a potential target for restoring the balance of striatal output . All experiments were performed in 8- to 20-week - old male mice and were approved by the regierungsprsidium karlsruhe in compliance with the european guidelines for the care and use of laboratory animals (licenses g74/13 and g248/14). For anterograde tracing experiments, in vitro patch - clamp recordings, and behavioral experiments, aav dio chr2-mcherry was injected into the primary and/or secondary motor cortex of som (melzer et al ., 2012), pv (hippenmeyer et al ., 2005), pv / gad67-egfp (tamamaki et al ., 2003), som / drd1a - egfp, pv / drd1a - egfp, som / drd2-egfp (gong et al ., 2003), and pv / drd2-egfp mice with a c57bl/6 background . For retrograde tracing experiments, ctb 647 was injected into the dorsal striatum of wild - type mice . For retrograde transsynaptic rabies virus tracing, aav - cag - flex - tcb, aav - cag - flex - rg, and sadg - egfp(enva) were injected into the dorsal striatum of a2a - cre mice . For monosynaptic retrograde rabies virus tracing, aav - cag - flex - tcb was injected into the motor cortex of som (sst, jackson laboratory) and pv mice, followed by sadg - egfp(enva) injection into the dorsal striatum . In all cases, anesthesia was induced and maintained with isoflurane (1%2.5%), and the virus was delivered through a small craniotomy at the appropriate coordinates by a glass micropipette . For behavioral experiments, immunofluorescence and dab staining were performed on sagittal and coronal brain sections using standard protocols . Fresh - frozen 20-m sections were stained with fish using the rnascope fluorescent multiplex kit (advanced cell diagnostics). Mice were deeply anesthetized with isoflurane, transcardially perfused with 30 ml ice - cold sucrose solution, and 300-m - thick brain sections were cut . Chr2-expressing long - range axonal fibers were stimulated with 5-ms photostimulation (473 nm, 120 mw / mm laser intensity). Pscs were measured at 0-mv holding potential (using cs - based intracellular solution) or at 70-mv holding potential (using k - based, high cl intracellular solution). For firing pattern analysis, incrementally increasing currents of 1-s duration were injected in current clamp mode starting at 50 or 200 pa . Series resistances of 37 megohm were accepted for analysis of pscs . Stimulus delivery and data acquisition were performed using pulse software . Mice were video - tracked at 25 frames / s, and their movements were subsequently analyzed using a position tracking system (ethovision xt9, noldus). The implanted optic fiber cannulas were connected to two optic fibers attached to a rotary joint (doric lenses). A patch cord connected the optic fibers to a diode - pumped, solid - state, 473-nm laser (crystalaser). We used a pulse generator (master 8) and a transistor - transistor logic (ttl) control box (universal serial bus input / output [usb - io] box, noldus) to automatically control the photostimulation (5-ms pulses delivered at 20 hz; laser power, 3 mw). Evaluation of locomotor activity was performed in a circular arena (40 40 cm) placed in a dimly lit room where mice were allowed to run freely for 21 min . It lasted 2 min and was repeated three times with an inter - stimulation period of 4 min . Brown - forsythe and f tests were used to test the homogeneity of variances . For non - pairwise comparisons, unpaired t tests (either for equal or unequal variance) or mann - whitney u tests were used . For pairwise comparisons, paired t tests or wilcoxon matched - pairs signed - rank tests were used . One - way anova followed by tukey s multiple comparisons tests was used to compare motion differences . Performed in vitro electrophysiology, analysis, immunohistochemistry, reconstructions, and retrograde tracing studies.
Pectus excavatum (pe), characterized by posterior displacement of the sternum and cartilaginous - rib attachments, is one of the most common congenital chest wall deformities.1)2) the physiological impact of pe varies . Symptomatic patients frequently complain of dyspnea with exertion, progressive loss of endurance, tachycardia, palpitations and chest discomfort.3 - 5) in a critical location, even small depressions of the chest can create significant cardiac dysfunction when the right heart and pulmonary outflow tract are compressed to varying degrees by the depressed sternum.5 - 7) an index of severity can be calculated by measuring the inner width of the chest (at the lowest level of the pectus defect) and dividing it by the distance between the posterior surface of the sternum (at the lowest part of the defect) and the anterior surface of the spine (with normal being approximately 2.52).2)3) in general, an index of 3.2 or greater is considered severe however symptoms do not necessarily correlate with severity of index.2 - 4) echocardiography plays a significant role in the evaluation of patients with pe . The degree of right heart compression however is often difficult to assess by transthoracic echo (tte) especially with severe deformities that prevent the operator from obtaining the normal transthoracic views.7 - 9) a 32-year - old woman presented to out - patient clinic for further evaluation of a 1-year history of progressive chest pain, fatigue, dizziness, paroxysmal tachycardia and dyspnea with exertion . With even mild exertional effort she experienced sharp, " stabbing " chest pain along the left lower and mid - sternum . A 12-lead electrocardiogram demonstrated a right - bundle branch block with left posterior fascicular block . She had a history of severe pe (index of> 4) for which she had undergone operative repair 18 months prior by an open resection of cartilage attachments and sternal " flip " as described by hawkins & colleagues.10) prior to her first correction, she had some dyspnea with exertion, however, cardiac work - up, including echocardiogram had been reported to not show any abnormalities . She noted onset of her current symptoms approximately 6 months after her pe repair . A subsequent operation with superficial anterior remodeling of the chest wall cartilage on the left side of the sternum had failed to relieve her progression of pain and symptoms . She exhibited post - operative abnormal remodeling of the chest wall with residual as well as some recurrent pe . The sternum protruded anterior with bilateral depression of the costal - sternal attachments creating a " wave - like " appearance (fig . Close attention to the anteroposterior planes (best seen from the apical four - chamber views) clearly demonstrated extrinsic compression and deformation of the lower mid - right ventricle (rv) by the chest wall which is more obvious during diastole (fig . 3b). Biplane and live 3-d images of the preoperative transesophageal echocardiogram (tee) improved the visualization and localization of extrinsic compression of the right ventricle (fig . Tricuspid valve prolapse is seen likely due to partial compression of the rv resulting in distortion of the tricuspid annulus (fig . Open revision of her chest wall deformity was performed with placement of two stainless steel support bars (lorentz surgical, jacksonville, fl, usa) and a trabecular metal implant (zimmer, inc ., minneapolis, mn, usa) which elevated the sternum and depressed regions 3 - 4 centimeters anterior to the rv with good cosmetic results (fig . Symptomatic pe patients often present for cardiovascular evaluation with tte being the most commonly utilized diagnostic modality . Documenting the effects of a depressed chest wall on the right heart and outflow tract as well as any associated interference with diastolic filling is critical for decision making in patient treatment and the need for potential surgical intervention . Modifying standard views such as biplane transthoracic and transesophageal views may be necessary in some patients due to limitations from the abnormal anatomy of the deformed anterior chest wall . In some cases, rv compression is often difficult to assess by tte especially with severe deformities that prevent the operator from obtaining the normal transthoracic window views . Technical tips for assessment of the rv include 1) narrowing the 2-d sector width to optimize only rv structures; 2) use of harmonic imaging and adjustment of gain and compression for good contrast and endocrinal edge detection; 3) making all measurements at end - diastole or the frame demonstrating the largest chamber dimension; and 4) for 2-d measurements, obtain all acquisitions during quiet respiration or full - expiration . Apical four - chamber views when seen clearly can usually visualize any extrinsic compression to the rv of the heart . Use of all three possible traditional acoustic windows (parasternal short - axis, apical and subcostal) may be necessary . In an effort to minimize foreshortening of the rv, the transducer can be positioned down an intercostal space and laterally until the rv apex is clearly seen . In some patients a transesophageal and transgastric view may be necessary to better evaluate the right heart chambers and rv outflow obstruction . Many patients with pe have associated alterations in rv morphology and function . Assessment for localized sacculation of the rv wall, global dilation of the ventricle, prominent trabeculae, and/or hypertrophy of the moderator band is important . Identifying abnormalities, including mitral valve prolapse and aortic root measurements are especially critical in suspected or confirmed cases of marfan syndrome . Resolution of mitral valve prolapse with release of the chest wall entrapment is seen in more than half of patients after pe correction.7)11)12) more importantly, many patients improve their symptoms such as exertional dyspnea and chest pain which may be related to extrinsic compression of the rv and reduced preload.4)5)7) further studies are needed to better understand the pathophyiology of symptoms in relation to cardiac and chest wall function in patients with pe . In conclusion, echocardiographic evaluation is critical in patients with symptomatic pe to assess the degree of rv compression . We presented a case of a pe in a patient with symptoms including severe chest pain with exertion that was diagnosed with the expertise of echocardiography and subsequently surgically corrected . Subtle abnormalities in cardiac structure and chest wall compression may result in debilitating symptoms in a small population of patients.2 - 4) when astutely performed, echocardiography can accurately provide clinically relevant information about cardiac size and any hemodynamic compromise in patients with pe.
The adenoid cystic carcinoma (acc) is a relatively rare epithelial tumor of the salivary glands . It accounts for about 5 - 10% of all salivary gland neoplasms, representing 2 - 4% of malignant occurrences of the head and neck area . Approximately, 31% of lesions affect minor salivary glands, particularly the palate, although they can also be observed in the sub - mandibular and parotid glands . The frequency reported in the tongue is 19.8%, with 85% observed at the base of the tongue . We report one such rare case of tongue neoplasm which turned out to be acc in a middle aged lady . A 45-year - old - female patient presented with an asymptomatic growth of the tongue, which was perceived just 2 weeks before consultation . The intra - oral examination at that time revealed a mass in the dorsum of the tongue with light pain to pressure, without any evidence of cervical lymphadenopathy . The mass was firm, same color as that of the surrounding mucosa and asymptomatic otherwise [figure 1]. As a pre - operative assessment of the lesion, a fine needle aspiration was done and the smear revealed a salivary neoplasm consisting of well delineated, tightly cohesive clusters of basaloid cells surrounding mucoid, hyaline globules, or clear spaces also forming honeycomb (cribriform) pattern [figure 2]. At places dense aggregates of monomorphic small cells with uniform round to oval hyperchromatic nuclei and scanty cytoplasm were seen . Smears also showed individual tumor cells with high n: c ratio and nuclear moulding . Macroscopically, the mass had firm consistency with an irregular form and surface, brown color and measured 2.5 1.5 1.0 cm . The histopathologic study revealed multiple pseudocystic spaces of variable sizes surrounded by cuboidal cells with scarce cytoplasm and oval nuclei, filled with eosinophilic material and hence was consistent with the diagnosis of acc [figure 3]. However, there was no evidence of perineural infiltration on serial sections . Clinical photograph showing a swelling on the dorsum of the tongue cytological smears show well - delineated clusters of basaloid cells surrounding hyaline globules with uniform round to oval hyperchromatic nuclei and scanty cytoplasm (papanicolaou stain, 400) histopathological section showing multiple pseudocystic cavities of variable size composed of cuboidal cells with scarce cytoplasm and oval nuclei (h and e, 400) minor salivary gland neoplasms occur less commonly than the major salivary gland tumors and tongue is a relatively uncommon site for salivary gland neoplasms acc is a malignant neoplasm that originates in both the minor and major salivary glands, characterized by slow growth, diffuse invasion, and potential to produce distant metastases, mainly to the lungs and bones . It is an infrequent lesion, as it represents approximately 1 - 2% of all malignant neoplasms of the head and neck, and up to 10 - 15% of all malignant salivary gland neoplasms . The most common intra - oral site for minor salivary gland tumors is the hard palate, followed by the base of the tongue where up to 96% of all tumors are malignant, and acc represents 30% of them . On the other hand, one of the least frequent sites of presentation for acc is the mobile tongue, as several authors have reported an incidence of only approximately 3% of the cases ., analyzed 178 cases of salivary gland tumors, out of which only six cases were located on the tongue . Cytologically, cribriform variety of acc can be diagnosed by hypercellular smears composed of clusters of small, relatively monomorphic epithelial cells with hyperchromatic nuclei . These appear bright magenta in may - grunwald giemsa mgg stains and pale blue with papanicolaou stain . Finger - like process of similar material can also be found in between the groups of cells in tubular variety . The solid variant of acc also exhibits the same material and the cells resemble that of small cells of anaplastic carcinoma . The globules of amorphous material surrounded by the monomorphic hyperchromatic cells was a clue to the diagnosis of acc in our case, but since the hyaline globules are also found in other tumors like basal cell adenoma, pleomorphic adenoma, polymorphous low grade adenocarcinoma, epithelial myoepithelial carcinoma etc . It is important to distinguish the adenomas from acc because of the conservative mode of management in case of adenomas . We ruled out the adenomas because of the nature of the globules and the cytological morphology . Unlike the adenomas, the hyaline globules were dense and stained intensely with mgg and the cells were relatively monomorphic, hyperchromatic with coarse chromatin and irregular nuclear membrane like that of acc . Of three histologic variants - tubular, cribriform and solid; in our case cribriform pattern was the dominant one without any evidence of perineural infiltration . The main factors associated with patient survival were tumor location, clinical stage, and the observed histologic variable . Conversely, spiro et al ., have not found histologic classification to be of any benefit, and deny a correlation between microscopic appearance and prognosis . Due to the slow growth pattern of the tumor however, due to local recurrence and late metastasis, surgery remains the mainstay of management with or without radiotherapy.
It has been proven to be very effective and became a standard of care in the treatment of most cases with severe prolapsus . Sacropexy is a surgical repair technique that restores pelvic anatomy by attaching synthetic graft material into the vagina and sacrum . Erosion of vaginal wall or bowel can be seen as a long term complication . In this paper, we present a patient suffering from mesh migration into the rectum after abdominal sacral colpopexy . A 69-year - old woman was admitted to the hospital with a complaint of sensation of fullness and a feeling of a foreign material protruding during defecation . She had been diagnosed with uterine prolapsus and stress incontinence in 2008 and underwent total abdominal hysterectomy, bilateral salpingo - oophorectomy and sacral colpopexy with prolene mesh . The patient was admitted to our clinic five years after the procedure with complaints mentioned above . There was a foreign material palpated in rectum with digital examination . Prolene mesh was detected in sacral region but resection of the mesh could not be conducted because of high levels of adhesions in that region . Genital prolapse or genital hernia is described as the protrusion of pelvic organs along the vagina . It is one of the common gynecological conditions that affect the quality of life in women . It may be seen in up to 50% of multipara women, and its incidence increases with age . High rates of recurrence with traditional techniques led to the development of new surgical techniques . The use of synthetic mesh has become more popular surgical approach in cystocele and rectocele repair . Mesh migration is a well - known clinical pathology and have been reported in literature . Yolen and grossman suggested that intra - abdominal foreign bodies (like mesh) transmigrate into the small or large bowel by triggering an inflammatory reaction . Persistent inflammatory reaction causes an opening into a hollow organ assisted by the peristaltic movement of the bowel . Insufficient fixation of a mesh is another factor for migration of synthetic materials in some patients . Larger pores greater than 75 nm permit the migration of macrophage and leukocyte migration and reduce the infection rate . Large pores also improves flexibility of the mesh and cause tissue ingrowths and healthy collagen deposition . Complications reported after sacropexy include ileus, intraoperative vessel injury, ureter injuries, recurrent descensus and mesh tearing . The use of a mesh as a graft material results in higher success rates but also causes a higher number of complications, such as mesh erosions or chronic infections . Taoka reported a case of rectal migration of mesh in a 64-year - old woman who presented with a recto - cutaneous fistula 11 months after a tension - free vaginal (tvm) repair; the patient was treated by removal of the infected mesh and closure of the rectal wall defect under cover of a temporary colostomy . By contrast with the troublesome symptoms reported in such patients in the literature, the only presenting complaint of our patient was protrusion of foreign material from the rectum . In conclusion, mesh migration is a serious complication after sacral colpopexy . Sometimes surgical resection of migrated mesh with laparotomy can be difficult due to dense adhesions . Scu was responsible for writing, conception and design of the study; ob contributed toward analysis and interpretation of data; nas, oa, aa- performed acquisition of data; bk drafted the manuscript.
Congenital seminal vesicle cysts associated with abnormalities of the upper urinary tract are uncommon.1 they can be asymptomatic and discovered incidentally or can be associated with dysuria, urinary tract infections, and infertility . Diagnosis is more frequently made in adult life during the period of the greatest sexual or reproductive activity.1 2 we present a case of a congenital seminal vesicle cyst associated with ipsilateral renal agenesis incidentally discovered on ultrasound at the age of 4 years and its natural evolution until adolescence . The association of congenital seminal vesicle cyst with ipsilateral renal malformations is rare and was first described by zinner in 1914 . It is reported in literature as zinner syndrome.3 this condition is considered as the male equivalent of mayer - rokitansky - kustner - hauser (mrkh) syndrome described in females.4 a 4-year - old boy with known right renal agenesis discovered on antenatal ultrasound, presented in our radiology department for his annual control . The cyst was anechoic, measured 8 mm, and was not present on previous ultrasounds . It was initially considered as an ureterocele or bladder diverticula, however, neither of these diagnosis was confirmed on intravenous urography performed at that time . The cyst remained unchanged on size and aspect during 11 years of follow - up . However, at the age of 15 years its aspect on the ultrasound had changed: its content was hyperechoic, it had increased in size (measuring 1.9 cm), and protruded in the bladder . It was associated to magma of some echogenic, round retrovesical masses, initially considered as lymph nodes (fig . 1b, c). Magnetic resonance imaging (mri) of the urinary tract and pelvis was further performed (coronal, axial, and sagittal t2 spin - echo and axial t1 with fat saturation before and after gadolinium injection images). It revealed a dilatation of the seminal vesicles, ipsilateral to renal agenesis (corresponding to the magma of retrovesical round masses seen on ultrasound), ending to a relatively small seminal vesicle cyst (diameter of 2 cm) that protruded in the bladder (fig . 2 a c). Ultrasound (a) at the age of 4 years detects a right retrovesical anechoic cyst (+) (b) at the age of 15 years the cyst has increased in size and it has become hyperechoic (white arrow). (c) it is associated to magma of round retrovesical masses (black arrow). (b) t2 spin - echo axial and (c) sagittal images of the pelvis show dilatation of the right seminal vesicles (white arrow) ending to a seminal vesicle cyst (*) that protrudes in the bladder . So far, the patient was asymptomatic and had never presented with dysuria, signs of bladder obstruction or urinary tract infection . He had neither any perineal discomfort nor pain, he described normal ejaculation without pain, no hematospermia, and had never experimented any urinary infection or prostatitis and also he was not sexually active . In this particular case, in the absence of symptoms and because of the relatively small size of the cyst a conservative treatment was decided, with an annual clinical and ultrasonographic control, at least until the first symptoms appear . The association of congenital seminal vesicle cyst with ipsilateral renal malformations is rare and there are approximately 200 cases reported in the literature.4 this association is explained by the common embryologic origin of both the organs (kidneys seminal vesicles) from the mesonephric duct1 and is due to an insult in embryogenesis between the 4th and 13th week of gestation.4 the ureteral bud arises from the dorsal part of the distal mesonephric duct and extends dorsocranially to meet and induce differentiation of the metanephric blastema, from which the kidney will develop . The mesonephric duct will differentiate to epididymis, ejaculatory duct, vas deferens, seminal vesicle, and hemitrigone . Complete failure of the mesonephric duct results to absence of ipsilateral kidney, ureter, hemitrigone, and seminal vesicle . Anomalous development of the distal mesonephric duct results to atresia of the ejaculatory ducts and abnormal ureteral budding; the former leads to obstruction and cystic dilatation of the seminal vesicles with development of seminal cysts, the latter leads to renal agenesis or dysplasia.2 5 zinner syndrome could be considered as the male equivalent of mrkh syndrome described in females.4 the cysts are present since birth but enlarge and become symptomatic on late adolescence or adult life, usually in the third to fifth decade, at the period of greatest reproductive or sexual activity.1 2 at this moment the accumulation of secretions in the seminal vesicle due to atresia of the ejaculatory ducts and consequently insufficient drainage leads to formation of cysts in the seminal vesicle.2 cysts smaller than 5 cm are usually asymptomatic and are discovered incidentally . Larger cysts can irritate the bladder and be related to pain, dysuria, frequency, hematuria, urinary tract infection, epididymitis or prostatitis, infertility, hemospermia, bladder outlet, or colonic obstruction.1 2 6 malignant degeneration of the cysts has been also reported.2 4 this progressive dilatation of seminal vesicle remains often asymptomatic until the dilatation increases to 4 to 5 cm,4 5 unless the obstruction triggers vas deferens dilatation at younger age,5 which was not the case for our adolescent . This dilatation develops slowly, by accumulation of secretions in the seminal vesicle, for many years after puberty, due to congenital atresia of the ejaculatory ducts and consequently insufficient drainage2: literature reports a majority of cases becoming symptomatic around the third or fourth decade of life . Diagnosis can also be made earlier in front of infertility linked to contralateral distal ejaculatory pathway compression by this cystic seminal vesicle.3 in our case, the cyst was first seen at the age of 4 years, mimicking an ureterocele on ultrasound . In adolescence it increased in size, but remained small and, for this reason, asymptomatic . Diagnosis was made on mri, based on its anatomic connection and to the similar signal intensity with the seminal vesicles . Differential diagnosis includes other pelvic cystic masses such as utricular or mullerian cyst, ureterocele, dilated ureter, abscess or lymph nodes,2 4 and acquired seminal vesicle cysts . Acquired cysts are usually bilateral and concern older patients with chronic prostatitis or postprostate surgery . Ultrasound raises the suspicion of this malformation but the final diagnosis is usually made by mri (or computed tomography, with this last method being irradiating). In adults more invasive methods as cystoscopy and vasovesiculography are also used for diagnosis.2 there is a medical consensus to propose conservative follow - up for asymptomatic or minimally symptomatic cysts.1 4 7 treatment is considered only for symptomatic patients and is surgical . Different surgical options exist, from the less invasive transrectal or transperineal aspiration of the cyst which gives a transitory relief of the symptoms,1 to more aggressive technics . Transurethral unroofing of the cyst by transurethral resection of the ejaculatory duct provides improvement of the quality of semen and improves the paternity rate,3 but this procedure can create injury to rectum, bladder neck, and external sphincter, and induce consequent retrograde ejaculation, and epididymitis . Radical treatment, vesiculectomy with resection of ureteral or renal remnants if present, offers the better outcome . It was traditionally performed by open exploration but is now well described laparoscopically, with low morbidity and good results in term of symptoms relief and semen parameters.1 4 7 8 9 10 11 few children with surgical indication benefited from this approach.12 13 concerning fertility, for patients presenting with difficulty to procreate, with low volume of semen and poor quality of spermogram, treatment shows semen quality improvement, and paternity obtained without other medical help.3 4 assisted reproductive procedures should be kept for patients with infertility persisting after successful surgical procedure.4 our case is original as it shows the natural history and evolution of this malformation, from childhood to adolescence . Cases reported in literature concern, in their high majority the adult population (or late adolescence) and these cysts are rarely detected at a younger age . Congenital seminal vesicle cysts in patients with ipsilateral renal agenesis are rare but this association is known and should be considered in the differential diagnosis of cystic pelvic masses in males with renal agenesis or dysplasia . Ultrasound is useful for diagnosis but mri provides a more detailed analysis and accurate diagnosis . Congenital seminal vesicle cysts in patients with ipsilateral renal agenesis are rare but this association is well known . Pediatric surgeons should be aware and consider this entity in the differential diagnosis of cystic pelvic masses in males with renal agenesis or dysplasia . Ultrasound is useful for diagnosis but magnetic resonance imaging provides a more detailed analysis and accurate diagnosis.
Tori and exostoses are nodular protuberances of calcified bone and are designated according to their anatomic location.1 torus palatinus (tp) and torus mandibularis (tm) are the two most common types of intraoral osseous overgrowths.2,3 tp is a sessile, nodular bony mass commonly seen on the midline of the hard palate . Tm is bony protuberance found on the lingual aspect of the mandible, in the canine and premolar region . Buccal and palatal exostoses are multiple bony nodular masses found less frequently than tori.1,2,4 buccal exostoses occur as bilateral, smooth bony growth along the facial aspect of the maxillary and/or mandibular alveolus . Commonly found to appear in the premolar - molar region . On palpation, ulcerations may be seen as a result of trauma or any injury to the mucosa . Their size may increase to several centimeters thus contributing to periodontal disease of adjoining teeth by retaining food during chewing instead of flushing away . Usually no treatment is required, but for those possibly affecting the periodontal condition, or when the protruberances cause pain or discomfort to the patient, or when these bony enlargements cause pseudo swelling over the lip, then conservative surgical excision can be performed . Some of the suggested causes include genetic factors, environmental factors, masticatory hyperfunction and continued jaw bone growth.5,6 the histologic features of tori and exostoses are identical.2 a very small exostosis and tori consist entirely of compact bone but when large and nodular, it consists of cancellous bone surrounded by cortical bone . The diagnosis of a buccal exostosis is based on the clinical examination along with radiographic interpretations . Clinically, the torus may appear as numerous rounded protruberanes or calcified multiple lobules, whereas the exostosis is a single, smooth broad - based mass, may have a sharp, pointed bony projection producing tenderness just beneath the mucosa.8 lesions may slowly enlarge up to 3 - 4 cm in greatest diameter, but it does not have malignant transformation potential . Buccal exostoses are usually found only on the facial surface of the maxillary alveolar bone, especially in the posterior segment . Radiographically, exostosis appears as well - defined round or oval calcified structure superimposing the roots of teeth . The patients are having multiple bony growths or lesions which are not in the classic torus or buccal exostosis locations should be evaluated for gardner syndrome . This autosomal dominant syndrome shows other features such as intestinal polyposis and cutaneous cysts or fibromas.2,5 no bony exostosis or tori requires treatment unless it becomes large enough to interfere with periodontal health, denture placement, or cause recurrent traumatic ulcerations . When treatment is elected, the lesions should be cut - off or removed from the cortex using bone cutting bur or hand instruments . The diagnosis of a buccal exostosis is based on the clinical examination along with radiographic interpretations . Clinically, the torus may appear as numerous rounded protruberanes or calcified multiple lobules, whereas the exostosis is a single, smooth broad - based mass, may have a sharp, pointed bony projection producing tenderness just beneath the mucosa.8 lesions may slowly enlarge up to 3 - 4 cm in greatest diameter, but it does not have malignant transformation potential . Buccal exostoses are usually found only on the facial surface of the maxillary alveolar bone, especially in the posterior segment . Radiographically, exostosis appears as well - defined round or oval calcified structure superimposing the roots of teeth . The patients are having multiple bony growths or lesions which are not in the classic torus or buccal exostosis locations should be evaluated for gardner syndrome . This autosomal dominant syndrome shows other features such as intestinal polyposis and cutaneous cysts or fibromas.2,5 no bony exostosis or tori requires treatment unless it becomes large enough to interfere with periodontal health, denture placement, or cause recurrent traumatic ulcerations . When treatment is elected, the lesions should be cut - off or removed from the cortex using bone cutting bur or hand instruments . A 20-year - old female patient reported to the department of periodontology, maharana pratap college of dentistry and research centre, gwalior (madhya pradesh) with bilateral masses just above the premolar and molar region in maxilla interfering in her smile and aesthetics (figure 1). She had noticed slow, but steady enlargement of the masses over the past 5 years . Physical examination of the oral cavity revealed large, bilateral overgrowths located on the buccal aspect of the maxilla in the premolar and molar areas (figure 2). The overlying mucosa was thin and blanched, and generalized moderate gingivitis with minimal bone loss was present . The exostoses were oblong in shape, measuring approximately 1.7 cm 1 cm on the right side and 2 cm 1 cm on the left side . Radiographic examination showed a well - defined radiopaque area covering the roots of the premolar and molar teeth . These bony protruberance caused by the thickening or enlargement of the cortical plate of the facial surface of the maxilla without any systemic abnormality helped to reach to diagnosis that it was multiple buccal exostoses . Bilateral enlargement seen in premolar and molar region . Generally, no treatment for buccal exostoses is required but as a patient was not happy with the bony masses seen during speech and smile . The full thickness flap was raised to expose the exostoses adequately (figures 3 and 4). The bony growth was cut with bone cutting carbide bur, no 702 ss white bur under continuous saline irrigation (figure 5). Smoothening of the rough surface was carried out with bone file and granulation tissue were curetted . The surgical site was washed thoroughly with a solution of povidine iodine and saline in 1:1 proportions and flap was closed . A follow - up appointment was scheduled after 10 days of surgery to check the site and for suture removal . The tissue appeared healed, and the patient was totally asymptomatic after 10 days (figure 6). Flap reflection revealing the bony mass of 2 cm 1 cm on left side flap reflection revealing bony mass of 1.7 cm 1 cm on right side . The multiple masses in the maxilla are consistent with multiple buccal exostoses, which are bony protuberances that arise from the cortical plates in the maxilla and mandible . They usually occur in the late teens and early adult years, and many continue to enlarge slowly over time . The etiology of the multiple exostoses remains unknown, although it has been suggested to be the outcome of a mild, chronic periosteal inflammation . Furthermore, the patients with multiple bony growths, not in the classic buccal exostoses locations should be evaluated for gardner s syndrome . Intestinal polyposis and cutaneous cysts or fibromas are other common features of the autosomal dominant gardner s syndrome.2,5 neither the torus nor the bony exostosis require treatment unless it becomes large enough to interfere with function, denture placement, cause recurring traumatic surface ulceration (usually from sharp food such as potato chips or fish bones) or as used to get autograft as it is a potent donor site.7 when treatment is elected, the bony mass may be removed using bone cutting bur or chiseled off through the base of the lesion . The case report presented above illustrates a unique and rare presentation of exostoses on the buccal side of the maxillary premolar - molar region, bilaterally . Exostosis is rarely found on the facial surface of maxilla thus should not be ignored and should be carefully differentially diagnosed.
Sleep can be disturbed by several factors such as illness, stress, and noise . Over time these include poor memory, slower reaction time, emotional disturbances, and changes in the immune response (1, 2). However, administration of these drugs is accompanied by side effects including psychomotor impairment, drug dependence, tolerance, amnesia, rebound insomnia, and the potentiating effects of other central depressant drugs (3). Moreover, some patients with sleep disorders do not respond effectively to current therapeutics . Therefore, studies to find new hypnotic agents with fewer side effects and more efficacy have continued . Herbal agents have long been a valuable source for developing new therapeutics for disease treatment . Ocimum basilicum (o. basilicum) is an annual herb belonging to the lamiaceae family and grows mostly in tropical regions such as india, africa, and south asia (4). In traditional medicine, o. basilicum is used to treat many disorders such as anxiety, diabetes, cardiovascular diseases, headaches, neurological pain, and seizures (5, 6). It has been demonstrated that the ethyl acetate fraction (eaf) of o. basilicum decreases ischemia - induced oxidative stress in the brain and improves short - term memory and motor coordination (5). It was reported in some traditional medical texts that o. basilicum leads to sleep and a sedative state . Also, some biological activities of o. basilicum were screened by ismail, and it was found that the essential oil of o. basilicum induces anticonvulsant and hypnotic activities (7). In folk medicine, maceration is the most widely used form of o. basilicum preparation . However, there is no pharmacological evidence for the sedative - hypnotic effect of o. basilicum macerated extract . The present study was designed to evaluate the sleep - prolonging effects of hydro - alcoholic extract (hae) of o. basilicum) and its fractions . Dimethyl sulfoxide (dmso, code d4540), penicillin - streptomycin (code p4333), sodium pentobarbital (code p3761), and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2h - tetrazolium bromide (mtt, code m-5655) were bought from sigma (st . Dulbeccos modified eagles medium (dmem, code 12800 - 082) and fetal bovine serum (fbs, code 10270 - 106) were purchased from gibco life technologies (grand island, ny, usa). The leaves of o. basilicum were collected from mashhad, iran in the month of july and dried in the shade . A voucher sample was preserved for reference in the herbarium of the department of pharmacognosy, school of pharmacy, mashhad university of medical sciences (herbarium number: 12937 obl). Hae was prepared using the maceration method (8). The powder of the leaves of o. basilicum (100 g) was macerated in 800 ml of 70% ethanol for 48 hours . The extract was then filtered through a 0.106 mm filter and dried in a water bath . The yield of the dried extract as related to the weight of the dried leaves was 21% . For the preparation of different fractions from hae, 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . Using solvent - solvent extraction, the eaf and n - butanol fraction (nbf) were separated to obtain water fraction (wf) (9, 10). All the fractions were dried in a water bath (memmert, germany) and stored at -20c until used . Then, the wf was dissolved in saline, and the eaf and nbf were dissolved in distilled water containing 10% dmso . Male albino mice weighting 20 - 30 g were maintained at a controlled temperature (22 1c) with a 12 hours light / dark cycle and free access to water and food . The study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences (code 910240, 2012.07.05). First, to determine if hae has a sleep - prolonging effect, the animals received saline (control group) and diazepam (as positive control) or different doses of hae . In the second experiment, to determine the most effective fraction of hae, animals were treated with wf, eaf, nbf, or 10% dmso (vehicle for eaf and nbf). The hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . A single dose of hae (25, 50, 100 mg / kg), fractions of hae, diazepam (3 mg / kg), or other vehicles were injected intraperitoneally (ip) into the mice . After 30 minutes, pentobarbital (30 mg / kg ip) was administrated to induce sleep (11 - 13). Flumazenil (1 mg / kg) was administrated 30 minutes before diazepam or hae (12). All materials were injected with a volume of 10 mg / kg (11). The onset of sleep is the time that the mice stayed immobile and lost their righting reflex . The time interval between administration of pentobarbital and onset of sleep was considered sleep latency . Nine groups, each containing two mice were used for determination of ld50 of hae . Groups 1 - 8 were injected ip with 25, 50, 100, 200, 400, 800, 1,600, and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle . The highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded . The mean of these two doses was considered the median lethal dose (14, 15). The possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells (pc12) and murine fibroblast cells (l929). Pc12 cells have several neuronal characteristics and, therefore, are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts . Also, the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia (usp) and is frequently used for testing the possible toxic effects of materials . The cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs, penicillin (100 units / ml), and streptomycin (100 g / ml). Then, the culture medium was changed to a fresh one containing the vehicle (dmso 1%) or hae (50, 100, 200, 400, and 800 g / ml). The cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 . Then, cell proliferation was evaluated using the mtt assay as previously described (16). Briefly, the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours . Statistical analysis was performed using one - way analysis of variance (anova) followed by tamhane s t2 post hoc test using the instat software package (graphpad, san diego, ca). Dimethyl sulfoxide (dmso, code d4540), penicillin - streptomycin (code p4333), sodium pentobarbital (code p3761), and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2h - tetrazolium bromide (mtt, code m-5655) were bought from sigma (st . Dulbeccos modified eagles medium (dmem, code 12800 - 082) and fetal bovine serum (fbs, code 10270 - 106) were purchased from gibco life technologies (grand island, ny, usa). The leaves of o. basilicum were collected from mashhad, iran in the month of july and dried in the shade . A voucher sample was preserved for reference in the herbarium of the department of pharmacognosy, school of pharmacy, mashhad university of medical sciences (herbarium number: 12937 obl). Hae was prepared using the maceration method (8). The powder of the leaves of o. basilicum (100 g) was macerated in 800 ml of 70% ethanol for 48 hours . The extract was then filtered through a 0.106 mm filter and dried in a water bath . The yield of the dried extract as related to the weight of the dried leaves was 21% . For the preparation of different fractions from hae, 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . Using solvent - solvent extraction, the eaf and n - butanol fraction (nbf) were separated to obtain water fraction (wf) (9, 10). All the fractions were dried in a water bath (memmert, germany) and stored at -20c until used . Then, the wf was dissolved in saline, and the eaf and nbf were dissolved in distilled water containing 10% dmso . Male albino mice weighting 20 - 30 g were maintained at a controlled temperature (22 1c) with a 12 hours light / dark cycle and free access to water and food . The study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences (code 910240, 2012.07.05). First, to determine if hae has a sleep - prolonging effect, the animals received saline (control group) and diazepam (as positive control) or different doses of hae . In the second experiment, to determine the most effective fraction of hae, animals were treated with wf, eaf, nbf, or 10% dmso (vehicle for eaf and nbf). The hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . A single dose of hae (25, 50, 100 mg / kg), fractions of hae, diazepam (3 mg / kg), or other vehicles were injected intraperitoneally (ip) into the mice . After 30 minutes, pentobarbital (30 mg / kg ip) was administrated to induce sleep (11 - 13). Flumazenil (1 mg / kg) was administrated 30 minutes before diazepam or hae (12). All materials were injected with a volume of 10 mg / kg (11). The onset of sleep is the time that the mice stayed immobile and lost their righting reflex . The time interval between administration of pentobarbital and onset of sleep was considered sleep latency . Nine groups, each containing two mice were used for determination of ld50 of hae . Groups 1 - 8 were injected ip with 25, 50, 100, 200, 400, 800, 1,600, and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle . The highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded . The mean of these two doses was considered the median lethal dose (14, 15). The possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells (pc12) and murine fibroblast cells (l929). Pc12 cells have several neuronal characteristics and, therefore, are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts . Also, the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia (usp) and is frequently used for testing the possible toxic effects of materials . The cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs, penicillin (100 units / ml), and streptomycin (100 g / ml). Then, the culture medium was changed to a fresh one containing the vehicle (dmso 1%) or hae (50, 100, 200, 400, and 800 g / ml). The cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 . Then, cell proliferation was evaluated using the mtt assay as previously described (16). Briefly, the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours . Statistical analysis was performed using one - way analysis of variance (anova) followed by tamhane s t2 post hoc test using the instat software package (graphpad, san diego, ca). Sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes (figure 1). As expected, the reference drug diazepam was able to increase the duration of sleep (42 4.6 minutes, p <0.001 vs. control). The hae at doses of 25, 50, and 100 mg / kg could significantly increase sleep duration to 51 3 minutes (p <0.001), 79 3 minutes (p <0.001), and 46 5 minutes (p <0.001), respectively . The effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg (p <0.001). The animals were treated with saline (control), diazepam (3 mg / kg), or hydro - alcoholic extract (hae) 30 minutes before administration of pentobarbital (30 mg / kg ip). * * * p <0.001 vs. control; ###p <0.001 vs. groups of 25 and 100 mg / kg . As expected, pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam (42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam, respectively, p <0.001). Similarly, the effect of hae on sleep duration was significantly inhibited by flumazenil (79 3 minutes and 30 2 minutes for hae and hae plus diazepam, respectively, p <0.001). The animals were treated with saline (control) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract (hae) before injection of pentobarbital (30 mg / kg ip). Flumazenil (1 mg / kg) was administrated 30 minutes before diazepam or hae . * * * p <0.001 vs. control; ###p <0.001 vs. hae; p values are presented as mean sem (n = 8). As shown in figure 3, all three fractions of hae exhibited sleep - prolonging activity . Duration of sleep in groups receiving 50 mg / kg of wf, eaf, and nbf was 40 4.5 minutes (p <0.001 vs. control), 30 2 minutes (p <0.001 vs. vehicle) and 42 2 minutes (p <0.001 vs. vehicle), respectively . Among the fractions, nbf induced the maximum prolongation of sleep . The animals were treated with 10% dmso (vehicle) or 50 mg / kg of water fraction (wf), ethyl acetate fraction (eaf) or n - butanol fraction (nbf) before administration of pentobarbital (30 mg / kg, ip). * * * p <0.001 vs. control and vehicle; #p <0.05 vs. eaf group; values are presented as mean sem (n = 8). Figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . When compared to the control (6.7 0.4 minutes), diazepam significantly decreased sleep latency to 3.5 0.4 minutes (p <0.001). The latency time in animals receiving 25, 50, and 100 mg / kg of hae was 4.2 0.2 minutes (p <0.001), 4.5 0.5 minutes (p <0.01), and 4.4 0.4 minutes (p <0.01), respectively . The effect of diazepam was not statistically different from that observed with 25, 50, or 100 mg / kg of hae . The animals were treated with saline (control) and diazepam (3 mg / kg) or hydro - alcoholic extract (hae). * * p <0.01 vs. control; * * * p <0.001 vs. control; b, mice were treated with 10% dmso (vehicle) or 50 mg / kg of water fraction (wf), ethyl acetate fraction (eaf) or n - butanol fraction (nbf). * * * p <0.01 vs. control; ###p <0.01 vs. wf and nbf . Values are presented as mean sem (n = 8). Among the different fractions of hae, however, nbf significantly decreased the latency time to 4.7 0.3 minutes (p <0.001). Although wf could decrease sleep latency from the control level (6.7 0.4 minutes) to 5.2 0.2 minutes, this effect was not statistically significant . The highest dose, which did not kill any mice, and the lowest dose, which led to the death of one mouse, were 1.6 and 3.2 g / kg, respectively . The mean of these two doses (2.4 g / kg) was considered as ld50 . The possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells (figure 5). It was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . In the presence of 50, 100, 200, 400, and 800 g / ml of the extract, cell viability was 100 3, 102 2, 103 2, 110 3, and 113 3%, respectively, as compared to the vehicle (100 2.5%). Regarding l929 cells, the viability was 106 1.5, 108 2, 112 2, 113 1, and 123 2.5% in the presence of 50, 100, 200, 400, and 800 mg / ml, respectively . Again, there was no significant difference in the cell viability when compared to the vehicle (100 2%). The cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum . Sleep duration in the negative control group that received normal saline before pentobarbital was 20 2 minutes (figure 1). As expected, the reference drug diazepam was able to increase the duration of sleep (42 4.6 minutes, p <0.001 vs. control). The hae at doses of 25, 50, and 100 mg / kg could significantly increase sleep duration to 51 3 minutes (p <0.001), 79 3 minutes (p <0.001), and 46 5 minutes (p <0.001), respectively . The effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg (p <0.001). The animals were treated with saline (control), diazepam (3 mg / kg), or hydro - alcoholic extract (hae) 30 minutes before administration of pentobarbital (30 mg / kg ip). * * * p <0.001 vs. control; ###p <0.001 vs. groups of 25 and 100 mg / kg . As expected, pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam (42 4.5 minutes and 20 3 minutes for diazepam and flumazenil plus diazepam, respectively, p <0.001). Similarly, the effect of hae on sleep duration was significantly inhibited by flumazenil (79 3 minutes and 30 2 minutes for hae and hae plus diazepam, respectively, p <0.001). The animals were treated with saline (control) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract (hae) before injection of pentobarbital (30 mg / kg ip). Flumazenil (1 mg / kg) was administrated 30 minutes before diazepam or hae . * * * p <0.001 vs. control; ###p <0.001 vs. hae; p values are presented as mean sem (n = 8). As shown in figure 3, all three fractions of hae exhibited sleep - prolonging activity . Duration of sleep in groups receiving 50 mg / kg of wf, eaf, and nbf was 40 4.5 minutes (p <0.001 vs. control), 30 2 minutes (p <0.001 vs. vehicle) and 42 2 minutes (p <0.001 vs. vehicle), respectively . Among the fractions, nbf induced the maximum prolongation of sleep . The animals were treated with 10% dmso (vehicle) or 50 mg / kg of water fraction (wf), ethyl acetate fraction (eaf) or n - butanol fraction (nbf) before administration of pentobarbital (30 mg / kg, ip). * * * p <0.001 vs. control and vehicle; #p <0.05 vs. eaf group; values are presented as mean sem (n = 8). Figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . When compared to the control (6.7 0.4 minutes), diazepam significantly decreased sleep latency to 3.5 0.4 minutes (p <0.001). The latency time in animals receiving 25, 50, and 100 mg / kg of hae was 4.2 0.2 minutes (p <0.001), 4.5 0.5 minutes (p <0.01), and 4.4 0.4 minutes (p <0.01), respectively . The effect of diazepam was not statistically different from that observed with 25, 50, or 100 mg / kg of hae . The animals were treated with saline (control) and diazepam (3 mg / kg) or hydro - alcoholic extract (hae). * * p <0.01 vs. control; * * * p <0.001 vs. control; b, mice were treated with 10% dmso (vehicle) or 50 mg / kg of water fraction (wf), ethyl acetate fraction (eaf) or n - butanol fraction (nbf). * * * p <0.01 vs. control; ###p <0.01 vs. wf and nbf . Values are presented as mean sem (n = 8). Among the different fractions of hae, however, nbf significantly decreased the latency time to 4.7 0.3 minutes (p <0.001). Although wf could decrease sleep latency from the control level (6.7 0.4 minutes) to 5.2 0.2 minutes, this effect was not statistically significant . The highest dose, which did not kill any mice, and the lowest dose, which led to the death of one mouse, were 1.6 and 3.2 g / kg, respectively . The mean of these two doses (2.4 g / kg) was considered as ld50 . The possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells (figure 5). It was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . In the presence of 50, 100, 200, 400, and 800 g / ml of the extract, cell viability was 100 3, 102 2, 103 2, 110 3, and 113 3%, respectively, as compared to the vehicle (100 2.5%). Regarding l929 cells, the viability was 106 1.5, 108 2, 112 2, 113 1, and 123 2.5% in the presence of 50, 100, 200, 400, and 800 mg / ml, respectively . Again, there was no significant difference in the cell viability when compared to the vehicle (100 2%). The cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum . The present study showed that o. basilicum further enhances sleep behavior, confirming that this plant has a hypnotic action as claimed in traditional medicine . To our knowledge, this is the first pharmacological study showing the effects of the macerated extract of this plant on sleep duration and sleep latency . Also, this is the first work to determine the ld50 value for hae of o. basilicum and to assess possible cytotoxicity of this extract on neuronal cells . Yet, results of this study are preliminary and need to be confirmed by further clinical trials . The hypnotic assessment method was based on prolongation of sleep induced by pentobarbital, which is the most commonly used method for screening sedative - hypnotic agents (11 - 13). In agreement with the previously published works and as expected, diazepam significantly increased pentobarbital - induced sleeping time, indicating that our study method was optimized (17, 18). The effect of hae of o. basilicum on sleep latency was lower than that of diazepam . However, the sleep - prolonging effect of hae was comparable to that of diazepam, and, even at a dose of 50 mg / kg, hae had a greater effect . Also, the hypnotic effect of hae of o. basilicum is greater when compared with the effect of essential oil of o. basilicum reported by ismail (4-fold and 2-fold increase in sleep duration, respectively) (7). According to our results, the hypnotic effect of hae was not dose - dependent in the range of given doses, and the maximum effect occurred with a dose of 50 mg / kg . Therefore, we used this dose to investigate the effects of different fractions of hae . The fractions of hae were prepared to obtain better insight into the nature of compounds responsible for the hypnotic effect of o. basilicum: 1, wf which extracts water - soluble constituents (e.g., glycosides, quaternary alkaloids, tannins); 2, eaf which extracts compounds of intermediate polarity (e.g., some flavonoids); and 3, nbf which extracts low polar agents (e.g., sterols, alkanes, and some terpenoids) (19, 20). Although wf, eaf, and nbf at a dose of 50 mg / kg were all able to increase sleep duration, not one of them could enhance sleep duration to the level induced by 50 mg / kg of hae . Because hae contains the active constituents of all the above - mentioned fractions, it can be concluded that an additive effect was caused by the interaction between these constituents when hae was administrated . Therefore, both polar and non - polar constituents in o. basilicum extract are involved in the sleep - prolonging effect of this plant . Among wf, eaf, and nbf fractions, nbf not only induced the maximum prolongation of sleep duration, but also was the only fraction to induce a significant decrease in sleep latency . On the other hand therefore, nbf contains a higher concentration of constituents responsible for the hypnotic effect of o. basilicum . These include terpenoids (e.g., linalool, eugenol), flavonoids (e.g., quercetrin, luteolin), alkaloids (e.g., rosmarinic acid), steroids (e.g., beta - sitisterol), and saponins (21, 22). It has been shown that o. basilicum contains high amounts of linalool, eugenol, and rosmarinic acid (7). Linck and coworkers demonstrated that linalool increases barbiturate - induced sleeping time in mice (23). Similarly, the sleep - prolonging effect of eugenol was reported by sharma et al . Rosmarinic acid is found in methanolic extract of o. basilicum and can be isolated from the nbf of plant extracts (24, 25). It plays a major role in the sedative - hypnotic actions of some medicinal plants (26). Neurons located in the anterior hypothalamus release gamma - aminobutyric acid (gaba) to inhibit wake - promoting areas in the hypothalamus and brainstem (27, 28). Barbiturates such as pentobarbital act on the gaba receptor s ionophore complex and favor the binding of gaba . Benzodiazepines such as diazepam increase the affinity of gaba for its receptor and thereby enhance pentobarbital - induced sleeping time (29). Consistent with this, we observed that pretreatment of mice with flumazenil decreases the sleep - prolonging effect of diazepam . In agreement with our finding, awad et al . Reported that rosmarinic acid, which is found in the extract of o. basilicum, can act as a gaba transaminase inhibitor and therefore increases the brain level of gaba (30). Therefore, it is rational to suggest that the sleep - prolonging effect of o. basilicum is mediated, at least in part, through the potentiating of the gabaergic system . It is now accepted that some natural compounds interact with the gabaergic system to increase sleep behavior (31, 32). The toxicity assay showed that the ld50 value for hae of o. basilicum is 2.4 g / kg . This dose is far from its hypnotic doses (25 - 100 mg / kg). Also, hae, even at high concentrations, did not decrease the viability of neuronal and fibroblast cells . Therefore, it seems that the hypnotic effect of o. basilicum is not accompanied by neurotoxicity . In conclusion, present data demonstrated that the macerated extract of o. basilicum potentiates sleeping behaviors without any cytotoxicity.
Timber harvesting and logging is one of the vital activities carried out in the forestry sector in malawi . This is because it creates a room for establishment of new stand in the process of removing the old age . This also reduces the impact which may follow if the stands are left unharvested past their harvesting age . Timber harvesting activities cause some degree of soil disturbance on soil physical, chemical, and biological properties; this also reduces site productivity . Ground - based logging systems can cause serious disturbance to the physical properties of forest soil due to soil compaction . Bulk density, soil porosity, and temperature are amongst the seven physical properties of soil; hence harvesting and logging has an impact on them . The bulk density of the soil is defined as the mass per unit volume of the soil and represents the ratio of the mass of solids to the total or bulk volume of the soil . Soil porosity is that part of the bulk volume not occupied by either mineral or organic matter but it is an open space occupied by air or water while soil temperature is the thermal diffusivity or conductivity of soil at different weather conditions . For instance, soil bulk density reflects the soil's ability to function for structural support, water and solute movement, and soil aeration . In addition, soil temperature plays an important role in many processes, which take place in the soil such as chemical reactions and biological interactions . The metabolic activity of soil microorganisms, seed fermentation, and plant roots is directly influenced by soil temperature in terms of water movement and soil freezing . Pinus kesiya royle ex gordon occurs naturally in himalaya region (asian): burma, china, india, laos, philippines, thailand, tibet, and vietnam . This species particularly grows well at altitudes from 600 to 1800 m above sea level . The trees can reach heights of 3035 or 45 m with straight, cylindrical trunk . Pinus kesiya is a major exotic plantation species in malawi and other southern african countries . Its success as an exotic is due to its fast growth rate and wide adaptability . Current studies have shown a large degree of site specificity both in soil and tree growth responses to soil compaction [12, 13]. These research results have encouraged new intuitions on the effects of soil compaction on forest productivity in different soil types and proved the importance of having site - specific soil quality assessments in forests . Moreover, there is still limited information on the relationships between soil compaction / disturbances and tree growth . Generalization about negative effects of harvest - related soil disturbance on tree growth may be in error because these impacts depend on their type and severity and on soil properties and climatic conditions [12, 14]. In addition many studies have assessed tree growth and soil response in logged sites using retrospective approach, which may not allow ascertaining the original type, degree, and extent of disturbance . Therefore, the present study was undertaken to determine the impacts of soil disturbance and compaction on soil physical properties and tree growth and the effectiveness of tillage in maintaining or enhancing site productivity for intensively managed pinus kesiya sites in the study area using prospective approach . The study was conducted in malawi located in southern africa in the tropical savannah region at chongoni plantation (14 19 s, 34 16 e, and 1650 m above sea level). The rain season starts from around mid - november to late april and the rainfall pattern is bimodal with peak periods in january and february . Chongoni plantation receives 1200 mm to 1800 mm rainfall per annum, with annual temperature ranging from 7c to 25c . The dry season runs from may to october . It is situated about 85 km southeast of lilongwe the capital . In addition, the experimental site has homogenous soil and topographic conditions . The site is on gentle slope (<10%); the soils are deep, well - drained, mostly stone - free, and high in ferralsols, acrisols, and nitosols, with site index of 20 m at 25 years for pinus kesiya . The old - growth stand previously occupying the site was planted in 1980 with pinus kesiya and was harvested from may to august 2010 . Treatments plots are 70 m by 70 m (0.49 ha). The treatments were as follows: t1:stem - only removal with soil compaction;t2:stem - only removal with soil compaction plus tillage;t3:stem - only removal with no soil compaction (control). Stem - only removal with soil compaction; stem - only removal with soil compaction plus tillage; stem - only removal with no soil compaction (control). The stem - only removal harvest followed merchantability standards of 5.5 m log length and 8 to 13 cm small end top diameter (i.e., cut - to - length (ctl) operation). The harvested trees were directionally hand - felled between june and august 2010 so that all tree tops remained within the plot . In t1 and t2 logs were pulled by a tractor from the stump area to the road side before being transported to the sawmill . In t2 further thorough soil tillage to the depth of 60 cm was accomplished by using a plough . In t3 sulkies were used to remove the logs from the stump area to the road side . Ten equally spaced traffic lanes were identified in each plot to be trafficked to make soil disturbance comparable across blocks . Every other lane was trafficked twice to more closely mimic traffic patterns that operationally occur when a tractor or a sulky is used during harvesting . In december 2010, the plots were planted with pinus kesiya seedlings on a 2.75 m by 2.75 m spacing (1320 trees ha) using hole planting . All treatments including spot weeding in the three growing seasons after planting were carried out in order to eliminate the confounding effects that soil disturbance and compaction can have on vegetation communities and competition pressure . Twenty (20) soil samples from each treatment were collected to a depth of 010 cm and 1020 cm in september 2010 using a 31.2 mm diameter punch tube volumetric sampler . According to walworth, soil samples of 15 to 25 collected from randomly selected locations in a field are a good representative of the entire field . Samples with significant amounts of sound or decayed wood material were discarded and replaced with new samples . In compacted and compacted and tilled plots, sampling was restricted to traffic lanes . Soil samples collected were then oven - dried at 105c for determination of bulk density and soil gravimetric and volumetric water content . The measurements were taken at the point where each tree was planted and the rut depth from the original soil level was recorded . Furthermore, around every other measurement tree, the percentage of each disturbance class in a tree centred 2.75 by 2.75 m square area was recorded . Then the percentage of the ground area and the tree frequency in each soil disturbance class was calculated . Soil strength was measured in september 2012 in twenty locations per plot using a cone penetrometer at 5 cm intervals to the 60 cm depth on each treatment . High outlier values created when roots or buried wood was hit by the penetrometer were noted on the cards in the field and those measurements were discarded from the dataset . Soil cores were randomly collected in all the treatments using 75 cm cylinder rings centred at 5 cm and 15 cm depths to characterize the 010 cm and 1020 cm depths, respectively . Soil water retention curves and particle density were determined at science laboratory of malawi college of forestry and wildlife . Total porosity was determined by both the gravimetric method with water saturation and the particle density method . Volumetric water contents were determined after equilibrating the soil with water at tensions of 10, 20, 200, 400, and 1500 kpa . Pore size distribution and water retention were then determined by the procedure as outlined by ares et al . . Measurements of soil temperature were taken monthly between 08.00 hours and 10.00 hours from january 2011 to december 2013 at 20 cm depth in all the treatment plots . Trees in all treatments were measured immediately after planting and yearly in the three consecutive growing seasons . The parameters measured were total height (h) using a telescopic pole, stem basal diameter (bd) measured at a permanently marked location 15 cm above the ground level in growing seasons one through three, and stem diameter (dbh) at 1.3 m above ground in the growing seasons two and three . Stem diameters were measured using a diameter tape . A stem volume index (svi) data obtained were subjected to kolmogorov - smirnov d and normal probability plot tests using statistical analysis of systems software version 9.1.3 . . Then, harvesting and logging effects on soil strength and bulk density were analysed as a mixed model of repeated measures data with soil depth and soil disturbance as fixed effects and block as a random effect . The model was fitted using the mixed procedure in sas, which estimates variance components using restricted maximum likelihood methods . Differences between treatment means were separated using fischer's least significant difference (lsd) at the 0.05 level . A repeated measures analysis was appropriate because measurements of bulk density and soil strength were done at different depths at the same sampling point, and, therefore, sampling errors were not independent . Measurements taken at adjacent depths are expected to be more correlated than measurements taken some distance apart . The covariance structures associated with the within subject factor were selected by choosing those with lowest value for the bayesian criterion and akaike's information criterion . A mixed model approach was also used to test for soil disturbance effects on tree growth in years 0 to 3 . The result for ground area affected by the soil disturbance is presented in figure 1 . The results indicate that about fifty - two percent of the area of compacted plots was affected by the vehicular traffic . Seventy percent of the trees within the measurement plots of the compacted treatment were located on some degree of soil disturbance . Rut depths were shallow to moderate with averages of 2.8 cm for t2 and 13.1 cm for t3, respectively . Soil bulk density as related to soil depth and soil disturbance in the study site is presented in table 1 . The results show that there was a significant (p <0.001) difference for soil bulk density between soil depths . The depth of 1020 cm had higher soil bulk density than the 010 cm depth . Similarly, there was a significant difference for soil bulk density among treatments, with t3 having a higher soil bulk density than t1 and t2 . Relative to t1, soil bulk density at the depth of 010 cm increased 28.9% for t2 and 48.9% for t3 . At the depth of 1020 cm, the increase in soil bulk density was 22.2% and 38.9% for t2 and t3, respectively, in relation to t1 . Soil bulk density significantly (p <0.001) increased with both soil disturbance and depth . The results indicate that soil strength significantly (p <0.001) increased with soil disturbance and depth, with t1 producing a lower soil strength than class t2 and t3 . However, there was no significant (p> 0.05) difference for soil strength between t2 and t3 with increase in depth . Total soil porosity for all the treatments related to soil depth in the study sites is given in table 2 . The results indicate that there was no significant (p <0.05) difference for soil porosity between soil depth, even though the depth of 010 cm produced higher soil porosity than 1020 cm depth . There was a significant (p <0.001) difference for soil porosity among the different treatments with noncompacted soil producing higher soil porosity than the compacted and compacted plus tillage treatments in both soil depths of 010 cm and 1020 cm, respectively . However, there was no significant (p <0.05) difference between noncompacted and compacted plus tillage treatments in soil porosity in both soil depths of 010 cm and 1020 cm, respectively . On compacted soil, total porosity at 010 cm depth decreased by 13.8% compared with noncompacted soil and by 16.1% at the 1020 cm depth . The results indicate that there was a significant (p <0.001) difference in water retention by different treatments with compacted soil consistently retaining more water at 20 to 400 kpa water potential than noncompacted or compacted plus tillage soil at both 010 cm and 1020 cm depth . Soil temperature, measured once a month at 020 cm depth, was not significantly (p> 0.05) different between compacted and noncompacted areas (data not shown). Tree mortality and growth for pinus kesiya throughout the three growing seasons are presented in table 3 . The results indicate that there were no significant (p> 0.05) differences in mean height, stem basal diameter, diameter at breast height, stem volume index, and mortality for trees growing on compacted, compacted plus tillage, and noncompacted treatments at years one to three . This study illustrates that the amount of soil disturbance in cut - to - length (ctl) operation by a tractor is about forty - eight percent . Tepp reported a 38% soil disturbance using a ctl harvester and forwarder, while geist and cochran found that tractor harvesting using ctl produced 36% soil disturbance . However, the present study results are higher than those reported by [25, 26]. Armlovich reported that the amount of soil disturbance in a cut - to - length (ctl) operation had an average disturbance of 23.2% while in a study by gingras average disturbance was 25% . This difference may arise because in the present study, equipment traffic was applied when soil water contents were at somewhat drier than field water capacity, which is considered to correspond to a given soil water potential [12, 27]. According to heninger et al . And mcnabb and boersma, the degree of soil disturbance depends on the intensity of site treatment, type of equipment used, time of year when harvesting occurs, frequency of entry, amount of slash or litter on site, soil moisture, and soil type . Soil bulk density significantly (p <0.001) increased with both soil disturbance and depth . The results are in agreement with those reported by [12, 30]. According to froese, in the upper soil, biological activity (roots, animals, etc .) Can act to reduce resistance and soil bulk density while at lower depths soil texture, gravel content, and structure may increase soil resistance and soil bulk density; hence soil bulky density increases with depth . Soil strength in this study also increased after vehicle trafficking to about 60 cm depth but it was not detrimental for tree root growth of 2000 to 3000 kpa depending on tree species and soil conditions [12, 31, 33]. The mean soil strength by depth increment in the study site never exceeded 1200 kpa . The compacted soils decreased the total soil porosity at the study site by 13.8% and 16.1% in the depths of 010 cm and 1020 cm, respectively, compared to the noncompacted soils . These results are within the range of those reported by [12, 34]. According to kim, the decrease of soil porosity in t2 as compared to t1 is because of soil compaction . In t2 soil is highly compacted due to soil texture and structure by determining the size, number, and interconnection of pores . Coarse - textured soils have many large (macro)pores because of the loose arrangement of larger particles with one another . Because fine - textured soils have both macro- and micropores, they generally have a greater total porosity, or sum of all pores, than coarse - textured soils . This is in agreement with the results reported by [12, 38]. According to ares et al . Compacted soil retained more water than noncompacted soil because compression converted larger pores to smaller ones, especially in the upper part of the soil profile . The study reveals that mean height, stem basal diameter, diameter at breast height, stem volume index, and mortality for pinus kesiya trees species were not reduced by soil disturbance or compaction despite the fact that the soil physical properties were affected . Values for these growth indicators were even greater for trees on disturbed soil than those on nondisturbed soil in years 2 and 3 . Reported that douglas - fir growth was not reduced by soil disturbance and compaction even though the soil physical properties were affected from ground - based harvest . According to ares et al ., since the growth was not reduced by the soil disturbance or compaction, this is an indication that bulky density, soil strength, and macroporosity did not reach levels in compacted areas that can reduce tree growth . The observed bulk density in the compacted area in this study ranged from 0.45 to 0.66 mg m, which is within the 0.50 to 1.11 mg m range for pinus kesiya seedlings growth [40, 41]. Furthermore, the soil strength observed in this study did not exceed 1200 kpa; that is, it remained well below the critical threshold for tree root growth considered to be around 2000 to 3000 kpa depending on tree species and soil conditions . Soil physical properties particularly soil bulk density, soil strength, and total soil porosity were greatly affected by compaction which was brought by harvesting and logging . However, the extent of disturbance was not detrimental for early growth of planted pinus kesiya . The high organic matter content, low bulk density, and low compressibility of the soil contributed to buffering the harvest impacts and made this soil conducive for intensive forest management and ground based harvesting systems . Soil compaction could also be considered to be beneficial to early tree growth at three years . Increased water available in the 20 to 400 kpa range on compacted traffic lanes may explain this increased growth.
Physical activity during preschool age has significant effects on physical, social and psychological health of children . Fundamental movement skills (fms) including locomotor (e.g. Run, gallop, hop, leap, horizontal jump and slid), object control (e.g. Catch, kick, overhand throw and dribble) and body management (e.g. Balance, climb and forward roll) provide a base for more advanced physical skills . Fms develop during early childhood and are essential for complex activities at adulthood . Some studies have shown that the level of locomotor skills of children is positively correlated with the levels of participation in physical activities at adulthood[34]. D'hondt et al reported that the levels of gross motor skills in obese children are lower than the levels of these skills in normal - weight children . Therefore it seems likely that more proficient children in fms are more physically active, have more self confidence, are less obese and would be healthier in their adulthood . Fms would not be developed naturally as a result of growth and maturation in all children . It has been suggested that the optimal ages for fms learning are between 2 to 7 years[2, 6]. Brian w et al have recommended that physical activity promotional programs for preschool children should be on base of children's natural activities such as being spontaneous and intermittent . Also preschool children activities should be enjoyable and contain gross motor plays and locomotor activities . Therefore, it seems that an effective physical activity program for preschool children needs to be developmentally appropriate for children in the listed age range . Furthermore, physical, emotional and psychosocial needs of children should be considered in development of appropriate physical activity program for these children . Ideally physical activity programs in nursery schools also a well developed training course for these instructors is essential in efficient teaching of movement skills to children in nursery schools[1, 9]. In addition to the role of fms in long - term health, fms play a cardinal role in physical development of preschool children . To our knowledge there is no national curriculum for preschool children physical activity education in iranian nursery schools . The objectives of this study were a) to develop a physical activity program for nursery schools in iran, and b) to evaluate the effects of this program on improvement of fundamental movement skills of preschool children in selected nursery schools in iran . This quasi - experimental study evaluated the effect of a 10 weeks physical activity program on fundamental movement skills levels of nursery children in iran . The participants were 147 children with age range between 4 to 6 years that were selected by convenience sampling . Children selected form five available nursery schools in five different cities in iran . A manual for nursery physical activity instructors was developed by a panel of 20 experts in different related fields in iran including sports medicine specialist, pediatrician, expert nursery teachers, psychologist, nutritionist, physical education specialist, phd in sports physiology, and phd in movement and behavior . This manual contained two parts; a) instructions for nursery physical activity instructors about goals and structure of the program, and materials on children's growth and development, children's nutrition, physical education, psychology of children, and health and safety, b) twenty four sections which cover different lessens containing physical activity and games aimed at developing locomotor and object control skills of children aged 4 to 6 years . Some sections of this package were developed based on the iranian traditional games and plays that were appropriate for preschool ages . Five nursery schools were selected in five different cities (tehran, isfahan, shahrood, neishaboor and gorgan) and volunteer nursery teachers of these nursery schools were enrolled in an educational course . This one week course was designed to train nursery physical activity instructors to be able to physically train children in nursery based on our designed physical activity program . The aim of this short - term educational program was to ensure that all physical activity instructors are alert to their duties about implementing of the developed physical activity manual . Also physical activity instructors were educated to test children using the test of gross motor development - edition 2 (tgmd-2) before and after intervention . Persian version of this test has been employed by akbari h, et al . And bakhtiari s, et al . In iran[9, the levels of gross motor development of all subjects were measured before intervention and after 10 weeks physical activity program employing tgmd-2 . Tgmd-2 is a valid and reliable (test - retest reliability = 0.88 - 0.96) criterion - referenced instrument designed to assess gross motor development among children . Each subtest consists of 6 locomotor items (run, hop, gallop, leap, horizontal jump, and slide), and 6 object control items (throw, catch, kick, strike, dribble, and roll). Subtest raw score was defined by sum of 6 items of each subtests of locomotor or object control (scored between 0 - 48). Standard scores of each subtest (scored between 0 - 20) and also gross motor quotient were calculated in accordance with tgmd-2 manual . (gmq) for each subject was reported as very superior (gmq> 130), superior (gmq = 121130), above average (gmq = 111120), average (gmq = 90110), below average (gmq = 8089), poor (gmq = 7079), and very poor (gmq <70) according to the suggestions of the tgmd-2 manual . According to developed physical activity manual, physical activity program was conducted 5 days a week for 10 weeks by trained nursery physical activity instructors . Paired - samples t test was used to determine whether there were significant differences between the levels of locomotor and object control of participants before and after intervention . Independent samples t - test was used to compare differences between boys and girls on variables . In this quasi - experimental study 147 children with mean (sd) age of 4.95 (0.8) (range 3 - 6) year from five nursery schools in 5 cities in iran were included . Of all subjects 49% (72) were girls and 51% (75) were boys . The base line and post intervention tgmd-2 scores of subjects are presented in tables 1 and 2 . In the baseline, there were no statistically significant differences between the locomotor and object control raw scores of boys and girls (p=0.49 and p=0.9) respectively . Base line and post intervention tgmd-2 scores of all subjects sd: standard deviation means (sd) of base line and after intervention tgmd-2 raw scores of all subjects by age and gender sd: standard deviation after intervention, differences between the locomotor raw scores of boys and girls were not statistically significant (p=0.5). However, there was a statistically significant difference between boys and girls in the object control raw scores (p=0.048). The gmq of all subjects which is the most reliable score of tgmd-2 was statistically significantly increased after 10 weeks of intervention . Both subtests of tgmd-2 including locomotor raw score and object control raw score in both genders were statistically significantly increased after 10 weeks of intervention (table 1). Descriptive rating of gmq for subjects before and after intervention is shown in table 3 . Before intervention only 11.5% of all subjects were rated superior / very superior in gmq scores (i.e. Gmq> 120), however this rate increased to 49.7% of all subjects after 10 weeks of intervention . Before the intervention, 26.6% of all subjects were rated as poor / very poor, this rate decreased to 2% of subjects after 10 weeks intervention . Age equivalents of subjects on the locomotor and object control subtests before and after intervention are shown in table 4 . Age equivalents or developmental age defined as developmental level or age that corresponds to a raw score made by an individual . Descriptive rating of the gross motor quotient of all subjects before and after intervention age equivalents for locomotor and object control raw scores according to age groups of all subjects before and after intervention the main finding of this study was that the developed physical activity intervention program that was focused on gross motor skills development had a significant positive effect on proficiency in fundamental movement skills in preschool children from selected nursery schools in five cities in iran . Differences in tgmd-2 results before and after the intervention were significant in locomotor, object control, sum of standard cores and gross motor quotient in all subjects . To our knowledge there has been no published data on the levels of physical activity of iranian preschool children . However, base line tgmd-2 subtests scores of our subjects in this study were in the range of reported data from the united states (table 5). Comparison of tgmd-2 standard scores of subjects in this study with reported data from the united states, mean (sd) sd: standard deviation our data suggested that a supervised physical activity program could increase these scores to be better than normative reported data . There was no significant difference in the base line mean of the locomotor and object control raw scores between girls and boys (30 vs 29.5) and (24.8 vs 26), respectively . However after the intervention the mean of both object control raw scores (p=0.048) and object control standard scores (p=0.02) in girls were higher than these scores in boys . Cliff et al have reported that locomotor raw score in preschool children was higher in girls compared with boys but there was no significant difference between girls and boys in the object control raw score . In this study, base line means of standard scores of the locomotor (9.3 vs 8.9), object control (9.1 vs 8.1) and gmq (95.5 vs 91.2) were not significantly different between girls and boys . In contrast cliff et al have reported significant differences between girls and boys in the mean of locomotor standard scores (9.9 vs 7.9), object control standard scores (10.1 vs 8.6) and gmq (99.7 vs 88.2). One study has reported that locomotor skills proficiency is higher in girls and in contrast boys are more proficient in object control skills . Some studies have suggested that the levels of moderate and vigorous physical activities in children with better motor performance are significantly higher than the levels of these activities in children with less developed skills[4, 19]. Therefore, conduction of a physical activity program such as the program used in this study may help children to improve their fms which may help to have a higher physical activity in their future . First, same person conducted both training sessions and outcomes measurement in each nursery school . Furthermore, to our knowledge there was no normative data of tgmd-2 for iranian children to be used for comparison with our data . Further studies are needed to evaluate the long - term effects of physical activity intervention on fms in iran . It seems that our developed physical activity program conducted by trained nursery physical activity instructors could be an effective and practical way of improving gross motor skills of preschool children in short term in iran . Conduction of this program in nursery schools could indirectly help with increasing health levels and levels of physical activities in the society . We recommend using of this kind of physical activity programs in all nursery schools in iran and similar counties.
Glomerular visceral epithelial cells (gecs; podocytes) play a key role in the maintenance of glomerular permselectivity [14]. Under normal conditions, podocytes are in contact with extracellular matrix, and there appears to be little turnover of podocytes, as these cells are highly differentiated (terminally differentiated). Gecs form a tight network of interdigitating foot processes, which are bridged by filtration - slit diaphragms, and permselectivity of the glomerular capillary wall is dependent on the maintenance of appropriate structure of podocytes and of their foot processes . The complex cellular architecture of the podocyte is maintained by an organization of actin filaments in the cytoplasm . Proteins including nephrin, neph1, fat, podocin, cd2ap and zo-1 are found within or near the slit diaphragm, and nephrin, neph family proteins, and cadherins form complexes with scaffolding proteins, cd2ap and zo-1 [2, 4]. These connect the slit - diaphragm protein complex with actin filaments, which are joined by -actinin-4 proteins . Acquired gec injury is frequently associated with effacement of the foot processes, disruption of the filtration - slit diaphragms, and proteinuria [1, 2]. Moreover, heritable mutations in several gec structural proteins alter podocyte ultrastructure and cause heritable proteinuria, implying that impairment of the slit diaphragm or cytoskeletal structure underlies proteinuria [4, 5]. Focal segmental glomerulosclerosis (fsgs) is an important cause of proteinuria and nephrotic syndrome in humans . In recent years, evidence has accumulated to support the view that the early pathogenesis of fsgs is associated with gec injury, leading to apoptosis, detachment of gecs from extracellular matrix, and podocytopenia, which is followed by glomerulosclerosis [13, 68]. Acquired gec injury may be triggered by immunological factors (e.g., t cell or other factors affecting permeability), oxidants, human immunodeficiency virus, toxins, and other substances . In addition, fsgs may be associated with heritable mutations in several distinct proteins that play key roles in maintaining gec ultrastructure, including podocin, -actinin-4, transient receptor potential cation channel, subfamily c, member 6 (trpc6), inverted formin 2 and others . In this paper, we focus on mutations in -actinin-4, which are associated with an autosomal dominant late - onset fsgs in humans . We discuss the structure and function of -actinin-4, and we review mechanisms by which mutations in -actinin-4 may lead to podocyte injury and fsgs . There are four -actinin genes (actn1 - 4) that encode highly homologous proteins (-actinin-1, 2, 3 and 4), which form ~100 kda head - to - tail homodimers . -actinins-2 and -3 are ca - insensitive muscle -actinins expressed in z - discs of striated muscle cells, while -actinins-1 and -4 are widely distributed, nonmuscle isoforms . All family members are actin crosslinking proteins that share a number of structural features and regulatory regions . These include a central rod with four spectrin - like repeats, two n - terminal calponin - homology domains (ch1 and ch2), which contain three actin - binding sites (abs1 - 3), a c - terminal calmodulin - like domain, containing two putative ef hands, and a phosphoinositide - binding plextrin homology domain . The spectrin - like repeats facilitate homodimerization and may confer a degree of flexibility to the -actinin molecule, allowing it to resist mechanical strain . For its interaction with actin filaments, the ch1 and ch2 domains adopt a closed conformation, where abs 2 and 3 are exposed, while abs1 remains buried and inaccessible to f - actin . This association with actin is highly dynamic and can be controlled by the regulatory regions along the -actinin sequence . All -actinin isoforms contain two putative ef - hand motifs at the c - terminus of each monomer, but only isoforms 1 and 4 demonstrate sensitivity to ca [15, 16]. For these nonmuscle isoforms, binding of ca is believed to reduce the affinity of -actinin for actin, providing a mechanism for -actinin - dependent cytoskeletal remodeling . Additionally, extensive biochemical studies on the effect of phosphoinositide binding to -actinin-1 have been performed . Binding of phosphatidylinositol (3,4,5)-trisphosphate (pip3), and to a lesser extent phosphatidylinositol (4,5)-bisphosphate (pip2), decreases the association between -actinin-1 and f - actin, suggesting that phosphoinositides mediate cytoskeletal remodeling [17, 18]. For -actinin-4, in contrast to findings for -actinin-1, our studies showed that phosphoinositides increase the interaction of -actinin-4 with f - actin . Tyrosine phosphorylation at position 12 was found to be dependent on focal adhesion kinase (fak), supporting a role for -actinin in cell adhesion . Phosphorylation of -actinin-1 reduced its association with f - actin in vitro, suggesting that -actinin mediates fak - dependent cytoskeletal remodeling . The amino acid sequence of -actinin-4 reveals an analogous tyrosine residue at position 32, suggesting that phosphorylation may also regulate the binding of -actinin-4 to f - actin . However, we were unable to demonstrate fak - dependent tyrosine phosphorylation of -actinin-4 (unpublished observations). Expression of these isoforms at the cytoplasmic face of several types of cellular interaction sites, including focal adhesions, adherens junctions, and hemidesmosomes suggests a degree of versatility . The first -actinin binding partners identified were the subunits of integrins and intercellular adhesion molecule-1 . Since -actinins also bind to various regulatory molecules, -actinin may also serve as a scaffolding protein to integrate signaling molecules at various adhesion sites . -actinin-1 may link membrane proteins such as talin, vinculin, and -integrins with the cortical actin cytoskeleton [10, 2225]. Similarly, the tight junction protein, magi-1, interacts with the c - terminus of -actinin-4, providing a physical link from the cell periphery to the cytoskeleton . The list of interacting proteins is likely to grow and will provide a better understanding of the diverse functions of -actinins in nonmuscle tissues . However, the best defined function of -actinin-4 is to crosslink / bundle actin filaments, and it may enhance cell motility and elicit tumor - suppressor activity . Intriguingly, -actinin-4 is reported to shuttle between the cytoplasm and the nucleus . Cytoplasmic localization was associated with an infiltrative phenotype and correlated significantly with a poorer prognosis in cases of breast cancer . In this context, the relevance of nuclear -actinin-4 remains unknown, although it has been suggested that nuclear translocation may attenuate metastatic potential . The nuclear localization of -actinin-4 suggests a role for -actinin-4 in gene expression . -actinins-1 and -4 were identified as histone deacetylase 7 (hdac7)-interacting proteins, and the interaction domain was mapped to the c - terminus of -actinin 4 . Hdac7 can participate in multiple cellular processes, including the regulation of myocyte enhancer factor-2- (mef2-) mediated transcription . A point mutation in hdac7 disrupted its association with -actinin-4 and mef2, implying that -actinin-4 and mef2 binding sites overlap . Overexpression of -actinin-4 also potentiated estrogen receptor -mediated transcription by counteracting a negative regulatory effect of hdac7, while knockdown of -actinin-4 decreased expression of estrogen receptor target genes and affected proliferation of cultured breast cancer cells . Another study demonstrated that -actinin-4 colocalized along actin stress fibers and in membrane lamellae with nuclear factor-b (nf-b). Incubation of cells with epidermal growth factor or tumor necrosis factor- induced translocation of -actinin-4 and the p65 subunit of nf-b into the nucleus . As nf-b regulates transcription of a large number of genes in response to diverse stimuli, the study further supports a regulatory role for -actinin-4 in transcription . Human gecs express only -actinin-4 (although mouse gecs also express -actinin-1) [10, 30]. Immunoelectron microscopy studies showed that -actinin localizes to contractile microfilaments within podocyte foot processes [31, 32]. In cultured mouse gecs, -actinin-4 was found along actin stress fibers, focal adhesions, and within the cortical actin network at the cell periphery (figure 1). This specific subcellular localization of -actinin-4 facilitates its regulation of adhesion and cytoskeletal dynamics [19, 33, 36]. Interestingly, -actinin-4 was also present in the nucleus of some rat gecs [34, 35], in keeping with earlier reports in other cell lines . Normally, -actinin-4 may be required for integrin - dependent adhesion of gecs . Indeed, knockout of -actinin-4 in mice resulted in reduced glomerular podocyte number, accompanied by the appearance of urinary wilm's tumor-1 (wt-1), a podocyte biomarker . Such podocyte loss is consistent with defective glomerular basement membrane adhesion, as tunel assays failed to detect any apoptotic cells in -actinin-4 null glomeruli . The severity of the resulting phenotype seen in -actinin-4 null mice (i.e., glomerular disease, renal failure, and early death) indicates that -actinin-4-mediated podocyte adhesion is critical for filtration barrier function [30, 37, 38]. On the other hand, transgenic podocyte overexpression of wild - type -actinin-4 in mice these findings suggest that podocytes tolerate a wide range of -actinin-4 expression, but that a minimum threshold level is essential for normal gec adhesion to the glomerular basement membrane through its interaction with integrins . Deficiency of -actinin-4 leading to human disease is not described, but several point mutations or single amino acid deletions in -actinin-4 are found in heritable forms of human fsgs . Most mutations in actn4-associated fsgs families congregate at the ch1-ch2 interface [9, 40]. An example is the autosomal dominant k255e mutation (k256e is the mouse mutant corresponding to human k255e). Such mutants show increased binding to actin filaments, compared with the wild - type protein [30, 41, 42]. Interestingly, -actinin-4 k256e is insensitive to regulation by ca and phosphoinositides (pip2 and pip3), suggesting that its gain of affinity for f - actin blunts its responsiveness to regulatory factors . Expression of an -actinin-4 k256e transgene in mouse podocytes in vivo (under the control of the nephrin promoter) resulted in development of proteinuric fsgs . In addition, homozygous (but not heterozygous) knock - in of the mutant -actinin-4 allele into the actn4 locus in mice induced proteinuria, confirming the pathogenic potential of -actinin-4 mutations . The underlying biochemical mechanism, whereby fsgs - associated actn4 mutations enhance affinity for actin, was uncovered by pollak's group . They showed that disease - causing mutations disable an important intramolecular hinge that normally holds ch1 and ch2 in a closed conformation . The mutant protein adopts an open conformation forcing an interaction of all three actin - binding sites with the actin filament, thereby increasing the binding affinity by lowering its rate of dissociation from actin . Although -actinin-4 is widely expressed, human podocytes appear to be selectively vulnerable to mutations in -actinin-4 (including k256e and other analogous mutants), perhaps because podocytes are terminally differentiated cells with limited regenerative capacity . The downstream cellular mechanism(s) by which mutant -actinin-4 leads to podocyte injury and fsgs is poorly understood . One possibility is that injury is secondary to alterations in the mechanical properties of the podocyte via increased affinity of mutant -actinin-4 for f - actin (figure 2). Gecs that express -actinin-4 k256e show defective spreading and motility (figure 1). Moreover, exposure of -actinin-4 k256e expressing gecs to cyclical equibiaxial stretch, an in vitro mimic of the mechanical forces due to glomerular capillary pressure, significantly reduced cell surface area and caused retraction of cellular processes . Lastly, the enhanced association with f - actin alters the subcellular localization of -actinin-4, restricting its presence at the cell periphery (figure 1), and potentially altering its capacity to interact with various binding partners [19, 33]. Such abnormalities could, at least in part, be attributable to cytoskeletal disruption, as well as loss of integrin engagement, effectively yielding a phenotype similar to that of the -actinin-4 null mouse [36, 43]. Since the actin cytoskeleton is a key component contributing to the unique morphological characteristics of the podocyte foot processes, as well as being connected to both slit diaphragm and adhesion complexes, altered -actinin-4 biology must have important implications for podocyte health . An alternate and/or parallel mechanism, which we also consider in this paper, is that mutant -actinin-4 induces proteotoxicity in podocytes (i.e., an impairment of podocyte function caused by misfolding of a protein), ultimately leading to apoptosis (figure 2). Consistent with this notion, -actinin-4 k256e forms actin - associated aggregates in cultured gecs and in podocytes of both homozygous k256e knock - in mice and humans with actn4-associated fsgs [30, 33, 35]. Such misfolded / aggregated protein may be associated with the activation of stress pathways in podocytes (see below). Prior to discussing the proteotoxic potential of mutant -actinin-4, this section provides a brief overview of the ubiquitin - proteasome system and er stress . The ubiquitin - proteasome system plays a key role in regulating the half - life of short - lived cellular proteins, and in selective degradation of damaged or abnormal proteins [45, 46]. The proteasome degrades aberrant cytoplasmic or cytoskeletal proteins, and misfolded er proteins, which are retrotranslocated selectively to the cytoplasm . The latter process is known as er - associated degradation (erad) [47, 48]. Ubiquitin - proteasome pathway - mediated protein degradation involves tagging of the substrate by covalent attachment of ubiquitin molecules via a three - step reaction, and degradation of the tagged protein by the 26s proteasome complex . Ubiquitinated proteins typically undergo efficient degradation by the proteasome, but sometimes, large amounts of misfolded proteins are not degraded effectively, and may form covalently - linked aggregates . Such misfolded proteins and/or aggregates may impair the function of the proteasome and lead to the activation of stress pathways and cytotoxicity . Secretory, luminal, and membrane proteins normally attain their correctly folded conformation in the er via er - resident chaperones . To rescue misfolded proteins, the er has in place quality control machinery, including the unfolded protein response (upr) [4953], and erad [47, 48]. In the upr, accumulation of misfolded proteins in the er results in the activation of three er sensors . Activating transcription factor-6 moves from the er to the golgi, where it is cleaved by proteases . The cleaved cytosolic fragment translocates to the nucleus to activate transcription of er chaperones, for example, the glucose - regulated proteins (grp), grp94 and bip (grp78), and others . In parallel, inositol requiring-1 activates its endoribonuclease activity, cleaving x - box binding protein-1 mrna and changing the reading frame to yield a potent transcriptional activator . Normally, er chaperones assist in posttranslational processing of proteins and in their exit from the er, and may complex with defective proteins to target them for degradation . During stress, induction of er chaperones may enhance the protein folding capacity, and limit accumulation of abnormal proteins . Misfolded proteins in the er also activate perk (pkr - like er kinase), which then phosphorylates the eukaryotic translation initiation factor-2 subunit (eif2). This process reduces initiation aug codon recognition, and the general rate of translation is blunted (which decreases the protein load on a damaged er). The upr allows cells to recover from stress, and may be protective to additional insults, but substantial / prolonged er stress may lead to apoptosis . For example, certain mrnas may be translated preferentially after eif2 is phosphorylated . Among these is activating transcription factor-4, which induces expression of several genes, including chop (c / ebp homologous protein-10; also known as gadd153), a gene closely associated with apoptosis and/or growth arrest [49, 51]. Apoptosis may also result from activation of caspase-12 or protein kinases [49, 51]. Impairment of the ubiquitin - proteasome system can be associated with exacerbation of er stress [49, 54], perhaps by interference with erad . The potential for mutant -actinin-4 to impair podocyte function is suggested by the characteristics of this mutant protein to form microaggregates, undergo ubiquitination, impair the ubiquitin - proteasome system, enhance er stress, and enhance apoptosis (figure 2). Density - gradient centrifugation of the k255e mutant -actinin-4 showed abnormal sedimentation, suggesting that the mutant protein forms high molecular mass aggregates . In keeping with this result, expression of -actinin-4 k255e (and other fsgs - inducing mutants), but not wild - type -actinin-4 in cultured mouse gecs resulted in formation of aggregates, as visualized by fluorescence microscopy [30, 33, 42, 43]. Analogous results were obtained in rat gecs stably transfected with -actinin-4 k256e, where in some cells, the mutant (but not the wild type) protein formed aggregates . Unlike the wild - type protein, the mutant was not present in the nucleus of rat gecs [34, 35]. In addition, -actinin-4 k255e formed aggregates in podocytes of homozygous k255e knock - in mice and humans with actn4-associated fsgs . Misfolded / aggregated proteins may result in the activation of stress pathways . In pulse - chase metabolic labeling experiments, mutant -actinin-4 was degraded more rapidly, compared with the wild - type protein, with the mutant showing a half - life of ~15 h, and the wild type of over 30 h . Consistent with this result, after stable transfection in rat gecs or transient transfection in cos cells (a monkey kidney cell line, which allows for high levels of protein expression), the level of -actinin-4 k256e protein was considerably lower, compared with the wild - type protein (figure 3) [34, 35]. Together, these results suggested that -actinin-4 k256e may undergo ubiquitination prior to degradation via the ubiquitin - proteasome pathway . Indeed, in transiently transfected cos cells, mutant -actinin-4 was polyubiquitinated, whereas the wild - type protein was not (figure 3). Treatment of gecs that express -actinin-4 k256e with proteasome inhibitors enhanced expression of -actinin-4 k256e, in keeping with ubiquitination and degradation of the mutant protein by the proteasome [30, 35]. -actinin-4 k256e and wild - type proteins were transiently overexpressed in cos cells to study their effects on the ubiquitin - proteasome system . The function of the ubiquitin - proteasome system was monitored in viable cells by use of a reporter consisting of a short degron, cl1, fused to the c - terminus of green fluorescent protein (gfp). This gfp proteasome reporter is rapidly degraded when ubiquitin - proteasome function is normal, whereas impairment of the ubiquitin - proteasome system, for example, via enhanced flux of a mutant / aggregated protein will reduce degradation of gfp, resulting in an increased level of gfp expression . In cos cells transfected with wild - type -actinin-4, expression of gfp declined significantly, in keeping with proteasomal degradation of gfp (figure 3). In contrast, expression of -actinin-4 k256e retarded the degradation of gfp, implying that the ubiquitin - proteasome system was impaired in the presence of mutant -actinin-4 . -actinin-4 is a cytosolic / cytoskeletal protein that normally would not enter the er to undergo posttranslational modification . Nonetheless, compared with -actinin-4 wild type, transient transfection of -actinin-4 k256e in cos cells enhanced the upr, as evidenced by increased expression of the er chaperone, grp94, and the proapoptotic gene, chop (figure 3). In gecs, expression of the k256e mutant or wild type -actinin-4 (by stable transfection) did not increase expression of the er chaperone, bip, or chop, compared with parental (untransfected) cells . This difference between cos cells and gecs may be due to lower expression of stably transfected proteins in the gecs . However, after incubation of gecs with tunicamycin (a drug that blocks n - linked glycosylation and causes an accumulation of misfolded proteins in the er), the gecs stably transfected with -actinin-4 k256e showed increases in bip and chop that significantly exceeded the increases seen in cells expressing the wild - type protein, as well as the increases in parental gecs . The effect of -actinin-4 k256e on the induction of the upr by tunicamycin was particularly robust, given that expression of the mutant was substantially lower than the wild - type protein . Thus, although stable expression of -actinin-4 k256e in gecs did not induce the upr directly, the mutant appeared to adversely affect the integrity of the er, which may render these cells more susceptible to additional stress and induction of proapoptotic genes . Together, these results are in keeping with the view that exacerbation of er stress may be secondary to impairment of the ubiquitin - proteasome system [49, 54]. Stable expression of -actinin-4 k256e in gecs led to a reduction in cell number, as well as increased apoptosis and caspase-3 activity, compared with gecs that stably express -actinin-4 wild type, or parental (untransfected) gecs . These changes in cell number and apoptosis in the presence of -actinin-4 k256e occurred despite the significantly lower expression level of the mutant, compared with the wild - type -actinin-4, highlighting the potential detrimental consequences of this mutation . In cos cells, -actinin-4 k256e had only a slight effect on apoptosis and cell number, suggesting that this cell type may be more resistant to cytotoxicity . Nevertheless, -actinin-4 k256e markedly exacerbated apoptosis and reduced cell number in the context of mild proteasome inhibition . This result supports the view that apoptosis induced by mutant -actinin-4 may be associated with an impairment in proteasome function . The effects of -actinin-4 k256e on the upr (discussed above) are based on studies in cultured cell lines, but importantly, analogous changes also occurred in vivo . As stated above, transgenic mice that express an -actinin-4 k256e transgene in podocytes develop proteinuria and fsgs . Expression of the er chaperones, bip and grp94, eif2 phosphorylation, as well as expression of the proapoptotic protein, chop, were increased in glomeruli of transgenic mice, compared with control . Based on these results, it is reasonable to propose that in the -actinin-4 k256e model of fsgs, there is pronounced er stress, which may be contributing, at least in part, to gec apoptosis . The maintenance of a highly dynamic actin - based cytoskeleton is critically important to podocyte morphology and function . Microfilaments in the foot processes tether the actin cytoskeleton to the slit diaphragm and adhesion complexes, while forming the architectural infrastructure for the foot processes . -actinin-4 provides structural support to these microfilaments via its crosslinking and bundling activities, while linking them to components of the slit diaphragm and sites of adhesion . The gain affinity mutations in fsgs - associated -actinin-4 substantially alter the properties of the actin cytoskeleton, rendering it more rigid and less dynamic . Therefore, the underlying pathogenesis of actn4-associated podocyte injury, glomerular filtration barrier dysfunction and the appearance of fsgs lesions are at least partly attributable to an aberrantly high interaction of -actinin-4 with f - actin and its impact upon the cytoskeleton . In addition, the enhanced actin--actinin-4 interaction generates misfolded protein / aggregates, which could provide a parallel mechanism of podocyte dysfunction . As discussed above, misfolded proteins may induce dysfunction of the ubiquitin - proteasome system, that is, the misfolded proteins choke or this process may enhance proapoptotic stress in cellular compartments, including the er . In addition, since ubiquitination regulates many essential cellular processes, including normal protein degradation, cell cycle, transcription, dna repair, and protein trafficking, a disrupted ubiquitin - proteasome system may have broader adverse consequences for cell function . Thus, the pathogenesis of fsgs associated with -actinin-4 k256e may resemble processes in certain age - related or neurodegenerative diseases, where signs of er stress, ups dysfunction, and protein misfolding are observed [30, 45, 54, 5658]. For example, in huntington's disease, the expansion of a glutamine stretch within the n - terminal region of huntingtin gene generates a protein with severe neurotoxic properties . Expression of mutant huntingtin leads to a pronounced defect in erad, and upr activation was noted in postmortem huntington's disease brains . Familial amyotrophic lateral sclerosis has been linked to mutations in the gene encoding superoxide dismutase-1, and these mutations induce misfolding and aggregation of this protein, which is believed to contribute to neuronal dysfunction and death . Activation of the upr is observed in mutant superoxide dismutase-1 transgenic mice, and increased levels of er stress markers, as well as wild - type superoxide dismutase-1 aggregates have been reported in spinal cord tissue of sporadic amyotrophic lateral sclerosis . Modulation of er stress pathways protected mice with experimental amyotrophic lateral sclerosis from disease progression . Other examples of neurodegenerative diseases associated with protein misfolding / aggregation and er stress are alzheimer's, parkinson's, and prion diseases . Protein misfolding and ups dysfunction also appears to play a role in desmin - related cardiomyopathies, which result in congestive heart failure . By analogy to experimental neurodegenerative disease models, one must, however, be cautious in extrapolating the cell culture events, which are based on overexpression and a relatively brief experimental time frame, to a disease process that in humans takes many years to become established . Finally, the shuttling of the wild type, but not mutant -actinin-4 between the cytoplasm and the nucleus, as well as the potential for disruption of gene regulation in the presence of mutant -actinin-4, will require additional consideration as a potential mechanism of podocyte injury in fsgs.
Through the last twenty years, the knowledge on thyroid disease during pregnancy has rapidly expanded . It is well documented that women with overt hypothyroidism during pregnancy have an increased risk of pregnancy loss and adverse pregnancy outcome [13], but the consequences of subclinical hypothyroidism and the significance of concomitant thyroid peroxidase antibodies (tpoab) are debated [46]. Subclinical hypothyroidism is a condition in which a slightly raised thyroid stimulating hormone (tsh) signal is representing an early, mild thyroid failure . The persistency of mild (subclinical) hypothyroidism may differ according to ethnicity and age; however, tsh increase with age as well as the prevalence of tpoab positivity is increasing with age . Further the presence of tpoab does seem important, as resolution to euthyroidism is reported much higher in tpoab negative subjects compared to tpoab positive . Women with asymptomatic autoimmune thyroid disorders, who are euthyroid in early pregnancy, carry a significant risk of developing hypothyroidism progressively during gestation . The aim of this study was to estimate the significance of tsh, thyroid peroxidase antibody (tpoab), and mild (subclinical) hypothyroidism in women from the danish general suburban population study (gesus) on the number of children born, the number of pregnancies, and the number of spontaneous abortions . The danish general suburban population study (gesus) was initiated in january 2010 and finished in october 2013 . Gesus is a cross sectional study of the adult danish suburban general population in naestved municipality (70 km south of copenhagen) selected on the basis of the danish central population register code . The gesus study was designed to invite only 25% of younger women of 2029 years age influencing the distribution of age . The health examination included a comprehensive physical examination (body mass index (bmi)), biochemical tests and a self - administrated questionnaire (prevalent disease (diabetes mellitus, hypothyroidism, and hyperthyroidism), antihypertensive and cholesterol lowering medication, thyroid and antithyroid medication, contraception, smoking, income, education, employment, the number of pregnancies, the number of children born, age of first child, and the number of spontaneous abortions). Gesus had an overall participation rate of women of 45% (n = 11565). In this study we included participants of european origin (99% danish) (n = 11387) and excluded participants with missing values of tsh, ft4, and tt3 (n = 50) and missing values of the number of pregnancies, the number of children born, and the number of spontaneous abortions (n = 31). Prevalent hypothyroidism was defined as history of hypothyroidism or intake of t4/t3 medication, and prevalent hyperthyroidism was defined as history of hyperthyroidism or intake of antithyroid medication . Thyroid hormones within the reference interval were defined as ft4 = 10.024.0 pmol / l, tt3 = 1.202.90 nmol / l, and tsh = 0.43.7 mu / l . Mild (subclinical) hypothyroidism was defined as tsh> 3.7 mu / l and ft4 and tt3 within the reference interval, no history of thyroid disease, and no intake of t4/t3 or antithyroid medication . Controls were defined as having 0.4 <tsh 3.7 mu / l and ft4 and tt3 within the reference interval, no history of thyroid disease, and no intake of t4/t3 or antithyroid medication [13, 14]. Thyroid peroxidase antibody (tpoab) positivity was defined by the cut - off value of tpoab> 60 u / ml . All biochemical tests were performed at the same laboratory at naestved hospital . Measurements of tsh and of thyroid hormones - free thyroxin (ft4) and total triiodothyronine (tt3) were performed using an electrochemiluminescent immunoassay (roche cobas 6000, basel, switzerland). L (cv <5%), and tt3 reference range 1.202.90 nmol / l (cv <4%). Tpoab was measured by kryptor antitpon (brahms, henigsdorf, germany), with detection limit of 10 all statistical analyses were performed using stata version 13.0 for windows (statacorp, college station, tx, usa). A value of p <0.05 was considered statistically significant . Tsh and tpoab were not normally distributed and therefore log - transformed (logtsh and logtpoab). We performed the following statistical models: for the full cohort of women: linear regression analyses of tsh and tpoab of children born and the number of pregnancies, logistic regression for spontaneous abortion;for women with prevalent hypothyroidism versus controls: logistic regression analyses for 1 children born, 1 pregnancies, and spontaneous abortion;for women with subclinical hypothyroidism versus controls: logistic regression analyses for 1 children born, 1 pregnancies, and spontaneous abortion.models designed were either age - adjusted or multifactorially adjusted using age, bmi, diabetes, contraception, education, income, employment, smoking, antihypertensive medication, cholesterol lowering medication, and menopause as listed in table 1 . For the full cohort of women: linear regression analyses of tsh and tpoab of children born and the number of pregnancies, logistic regression for spontaneous abortion; linear regression analyses of tsh and tpoab of children born and the number of pregnancies, logistic regression for spontaneous abortion; for women with prevalent hypothyroidism versus controls: logistic regression analyses for 1 children born, 1 pregnancies, and spontaneous abortion; logistic regression analyses for 1 children born, 1 pregnancies, and spontaneous abortion; for women with subclinical hypothyroidism versus controls: logistic regression analyses for 1 children born, 1 pregnancies, and spontaneous abortion . Logistic regression analyses for 1 children born, 1 pregnancies, and spontaneous abortion . The study was approved by the regional ethics committee of zealand, denmark (reg . Number rvk sj-177, sj-113, and sj-114), registered with clinicaltrial.gov (nct01335802), and reported to the danish data protection agency . Table 1 shows characteristics of women . In total, 758 (6.7%) had mild (subclinical) hypothyroidism, 9.4% prevalent hypothyroidism, and 4.2% prevalent hyperthyroidism . In women with mild hypothyroidism tpoab logtsh and logtpoab were negatively linearly associated with the number of children born and number of pregnancies in the full cohort in age - adjusted and multiadjusted models (table 2). Mild (subclinical) hypothyroidism was associated with a risk of not having children and a risk of not getting pregnant in age - adjusted and multiadjusted models . Prevalent hypothyroidism was not associated with the number of children born, the number of pregnancies, or spontaneous abortions (table 3). In the present study, we showed that with higher tsh levels the less number of children born and the less number of pregnancies . Coefficients for logtsh were higher than for logtpoab; thus, the effect of tsh seems higher than for tpoab . Mild hypothyroidism was also associated with a higher age of first child born and risk of not having children and not getting pregnant . Tpoab was not a significant confounder in the models, and there was no interaction with age . This analysis is cross sectional and in a way retrospective for those women who are now menopausal . Also, for women who are premenopausal we may not have the life - time full number of children born, the number of pregnancies, and the number of spontaneous abortions . We cannot adjust for age of the mother at the birth of her 1st child as this only applies for those who have had children; thus, those with no children have missing values in that category . We have included the information in table 1, and it appears that there is a significant difference between those with mild hypothyroidism and controls . Furthermore, were we not able to perform analyses of pregnancy and child birth history restricted to only within few years of the tsh examination . This may be caused by the fact that, during fertile life, early miscarriage may have gone unnoticed or been forgotten . Had recently reported that women with a combination of subclinical hypothyroidism and thyroid autoimmunity were found to have the highest risk of miscarriage before 20 weeks of gestation compared to only tpoab positivity or subclinical hypothyroidism . In contrast, previous prospective studies have confirmed an association between mild hypothyroidism during pregnancy and pregnancy loss . Allan et al . Showed that stillbirth was significantly more frequent in women having tsh higher than 6.0 reported that the risk of child loss was increased with higher levels of maternal tsh, whereas concentrations of maternal ft4 and child loss were not associated . Observed that the evolution of pregnancy did not depend on whether the hypothyroidism was overt or mild but depended on adequate treatment during pregnancy . These findings correspond to our results, since the odds ratios for 1 pregnancy and child birth versus none were not significant comparing prevalent hypothyroidism versus controls reflecting a well - treated condition . Furthermore that mild hypothyroidism is representing nonadequate treatment and mild thyroid failure was associated with a risk of not having children and a risk of not getting pregnant . Accordingly, in our study we observed that elevated tsh above the upper reference limit in mild hypothyroidism correlated with the number of pregnancies and child births . Several studies have reported a possible association between thyroid dysfunction during pregnancy and specific adverse pregnancy outcome like preeclampsia [19, 20], placental abruption, or preterm delivery [21, 22]. It has been suggested that higher tsh and tpoab positivity were independently associated with lower likelihood of reversion to euthyroidism . In a 20-year follow - up study, an increased serum tsh level was predictive of progression to overt hypothyroidism, and the annual rate of progression to overt hypothyroidism was more than 4% in women with both raised serum tsh and antithyroid antibodies . Showed a trend towards slightly higher serum tsh levels in women with thyroid autoimmunity in the first trimester of pregnancy compared to women without thyroid autoimmunity and that women with asymptomatic autoimmune thyroid disease who were euthyroid in early pregnancy carried a significant risk of developing hypothyroidism progressively during gestation, despite a marked reduction in antibody titers . This retrospective cross sectional study showed that among women without any history of thyroid disease or antihyperthyroid / antihypothyroid medication 6.7% had mild (subclinical) hypothyroidism . The prevalence of mild hypothyroidism was comparable to previous studies like the colorado study in which 8.5% had subclinical hypothyroidism increasing with age explained by the chosen value of tsh cut - off defining mild hypothyroidism . The presence of mild hypothyroidism influenced the level of ft4 within the reference range as ft4 was decreased in women with mild hypothyroidism . This could be explained by an increased intracellular deiodination of t4 to t3 in women with mild hypothyroidism to avoid decreased levels of active intracellular thyroid hormone concentrations . We are not able to determine the status during pregnancy as the present study is retrospective, but it seems unlikely that tpoab would have an effect directly on metabolism and peripheral thyroid hormone regulated cellular function . However, we cannot exclude the possibility that the women presenting with mild hypothyroidism have had a previous normalized tsh level . This population study was performed in naestved municipality eastern denmark representing a mild iodine deficiency . In denmark, the iodine intake was stable at a low level for many years and in 2000, the mandatory iodine fortification of bread salt and household salt began . Follow - up studies reported an increase in prevalence of hypothyroidism and tpoab positivity especially among younger women . The present study is limited by the fact that all clinical observations are self - reported questionnaire data . Furthermore, we did not have any information about the numbers of induced abortions which could influence the data of child births although it is rarely recommended to induce an abortion only due to high levels of tsh during first trimester of pregnancy . In addition, individuals were classified based on a single blood test; thus, we cannot distinguish between transient and permanent tsh elevation; however, somwaru et al . Showed that subclinical hypothyroidism was persistent in 56% after 4-year follow - up . Finally, due to financial reasons the gesus study was designed to invite only 25% of younger women aged 2030 years old influencing the distribution of age . The strength of our study was the high number of participants with blood samples of tsh, ft4, tt3, and tpoab in 11254 women . Furthermore, for the analyses of mild (subclinical) hypothyroidism we excluded women with any self - reported thyroid disease or use of t4/t3 or antithyroid medication and only compared euthyroid and mild hypothyroid women . Taken together, we observed that with higher tsh levels the less number of children born and the less number of pregnancies . Mild hypothyroidism was also associated with a higher age of first child born and risk of not having children and not getting pregnant . In conclusion, we observed that impaired fertility is associated with tsh, tpoab, and mild (subclinical) hypothyroidism in a danish population of women.
In many instances, oral implants provide ideal replacement of missing teeth . Although the overall implant outcome is favourable, implant - related mechanical and biological complications are still frequently encountered . One of the contributing factors that influence the longevity of implant prosthesis is dental occlusion . Uncontrolled occlusal forces can lead to implant overloading and detrimental stresses development.1 strains beyond the physiological limit of bone can evoke bone loss around implants and eventual failure of the implant.2 mechanically, excessive strains on implants can cause different failure types involving the implant, its components, or the prosthesis.13 as a result, it is reasonable to propose an occlusion scheme that will minimize the stresses applied on the implant, without compromising the function and the esthetics of the prosthesis.123 in the literature, several occlusion schemes were described for natural teeth.45678 although they are similar in maximal intercuspation (mi), they vary significantly during excursion . The most commonly described occlusion schemes are canine - guided (cg) occlusion, group function (gf) occlusion, and balanced occlusion.4 cg occlusion occurs when the overlap between the maxillary and mandibular canine teeth disengages the posterior teeth during excursive movements.9 it relies on the anatomy and proprioceptive abilities of canine teeth.10 gf occlusion is described as multiple lateral occlusal contacts on the working side.9 the simultaneous contacts are thought to distribute occlusal forces.56 balanced occlusion is distinguished with the presence of simultaneous contacts on working and non - working sides during excursion.9 however, due to the expected risks associated with balanced occlusion, there has been a recommendation to avoid this scheme.11 another occlusion scheme described in the literature and has been observed naturally is the long centric (lc) occlusion, where many teeth maintain the contact in the initial stages of excursion, followed by cg occlusion at the later stages of excursion.12 these schemes are relevant as they have been commonly used to restore teeth and exist in the natural dentitions.13 biomechanically, key differences exist between natural teeth and osseointegrated implants . The natural tooth root is retained within the alveolar bone by periodontal ligament (pdl), which absorbs and distributes forces applied on the tooth . Furthermore, the pdl maintains inherent mobility of the natural tooth.1 in addition, the pdl provides proprioception feature, which allows for protection against occlusal overload . On the other hand, pdl is missing between alveolar bone and implant, and the implant is directly anchored within the bone with a movement of 20 times less than natural tooth movement.1 due to the lack of pdl, the proprioception around the implant is reduced.1 therefore, due to the inherent lack of implant mobility and reduced proprioception, the implant and its prosthesis are prone to overloading . During normal mastication, teeth become compressed in their sockets, leaving the implant prosthesis in hyperocclusion.13 as a consequence of overloading, the implant may suffer from alveolar bone loss14 and mechanical complications, such as ceramic chipping, screw loosening, or components fracture.21516 to overcome the inherent limitations of implant prosthesis, occlusal considerations were discussed in the literature for the purpose of reducing the implant overloading.1718192021 an occlusion scheme specific to implant prosthesis is termed implant - protected (ip) occlusion.123 in light occlusion, the prosthesis should not be touching the opposing tooth . However, under heavy biting forces, the prosthesis should be in light contact as the natural teeth are intruded.1 in the excursions, there should be no contact on the implant prosthesis laterally . Instead however, in order to provide objective recommendation, it is beneficial to validate the impact of different occlusion schemes . Therefore, the aim of this study is to investigate the effect of four different occlusion schemes and different excursive positions on peri - implant strains of a maxillary canine implant . The hypotheses are (1) there is an effect of occlusion scheme on peri - implant strains, and (2) there is an effect of the excursive position on peri - implant strains . The right canine of the maxillary model was trimmed . The maxillary and mandibular models were replicated by laboratory silicone (pinkysil addition cured silicone; barnes, bankstown, nsw). Methylmethacrylate resin (vertex self - curing resin; henry schein, waterloo, nsw) was poured in the silicone moulds and heat flasked according to the manufacturer's instructions . Methylmethacrylate resin was selected because it's young's modulus is relatively similar to human bone . The generated maxillary and mandibular resin models were articulated on a hanau modular articulator (014503 - 000; whip mix, louisville, kentucky) using average values . The occlusion was rechecked for maximum intercuspation and lateral gf occlusion was achieved on both sides . Initial gf occlusion was selected as it allowed alteration of the occlusion scheme by modifying the canine palatal anatomy . Implant drills were used to create an ideal vertical hole in the area of the maxillary right canine, which was slightly larger than the implant diameter . The hole was filled with freshly mixed methylmethacrylate resin and an implant (5.0 mm diameter, 13 mm long branemark mk iii external hex implant, nobel biocare) was inserted in the hole simultaneously . The model was returned to the heat flask to complete the curing of the resin . Four different crowns were fabricated for the purpose of achieving four different lateral occlusion schemes on the right side . A wide platform prefabricated titanium abutment (branemark snappy abutment, nobel biocare, macquarie park, nsw, australia) was attached on the implant and torqued to a recommended value of 35 ncm . A cross - pin design was used to avoid screw access in the area of loading and to allow ease of changing the canine crowns while maintaining the same loading set - up (fig . The abutment was scanned by 3shape scanner (3shape d-640; wieland - imes, pforzheim, germany) and each crown was digitally designed to represent a defined lateral occlusion scheme . The applied lateral occlusion schemes were: (1) cg occlusion, where the overlap between the maxillary and mandibular canines evokes immediate disclusion of the rest of the dentition upon excursion; (2) gf occlusion, where all the teeth, including the canines, are in contact during excursion; (3) ip occlusion, where the contact between the maxillary and mandibular canines exists only at maximal interscuspation, but not in excursion; and (4) lc occlusion, where the occlusion is similar to gf occlusion for the first 1 mm of excursion, after which it changes to cg occlusion . The digital fabrication method was followed to ensure the differences between the crowns were restricted on the palatal guiding surface . A resin pattern for each crown was 3d printed, fitted on the abutment, and verified on the articulator . The most apical part of the crowns was broadened to allow incorporation of the cross - pin . The first point was on the cingulum and corresponded to the maximal intercuspation (mi) position . The other points were 1 mm and 2 mm buccal from the mi, which corresponded to the loading after 1 mm and 2 mm excursion, respectively . The three points were selected to allow simulation of occlusal contacts that occurred during functional and parafunctional range of movement.2223 one rectangular stacked 45-degree rosette strain gauge (vishay precision group, raleigh, nc, usa) was attached with cyanoacrylate resin (m bond 200 adhesive vishay micro - measurements, raleigh, nc, usa) to the mid - buccal crestal area overlaying the implant body . The gauge was composed of three strain foils: horizontal, vertical, and diagonal foils . The model was not moved until the completion of the loading . A linear variable differential transformer (ldvt) horizontal arm (solartron, qc systems pty ltd ., vic, australia) was placed against the most convex area on the buccal surface of the crown to measure lateral movement under loading . The purpose of the ldvt was to detect any displacement of the crowns during loading . The ldvt was attached to a signal conditioning units (solartron metrology pty, vic, australia). Eventually, the data recorded from each measuring tool were monitored by computer software (lab view 7 express, national instruments corp ., austin, tx, usa). For each crown, a computer - controlled precision universal testing machine (mts 810 materials test system; mts systems corp ., eden prairie, mn, usa) was used to apply repeated vertical static load of 140 n at the three marked points (fig . A 140 n load was chosen as it fits within the physiological load range during mastication.1424 each crown was cycled 10 times to maximum load at each loading site . Therefore, a total of 30 readings were generated for every lateral occlusion scheme . For each loading condition, the strain generated from each gauge foil (vertical, horizontal, and diagonal) was recorded . Subsequently, maximum and minimum principal strain values were calculated for each loading according to the following equations: maximum principal strain=1+32 + 12(1-2)2+(2-3)2 minimum principal strain=1+32 - 12(1-2)2+(2-3)2 where 1 is the horizontal gauge strain, 2 is the vertical gauge strain, and 3 is the diagonal gauge strain . The shear strain was subsequently measured by calculating the difference between the maximum and minimum principal strain values: shear strain = maximum principal strain - minimum principal strain the average shear strain and standard deviation were calculated for the 10 readings of each crown and loading site . The effects of crown morphology (cg, gf, lc, ip) and loading site (mi, 1 mm, 2 mm) were evaluated and the interactions between them were investigated by the two - way analysis of variance test followed by tukey post hoc test at a significance level of 5% . The statistical analysis was performed via spss software package (ibm spss statistics, version 22, spss inc ., chicago, il, usa). The shear strain values for each crown were plotted in a box and whisker diagram . For all the crowns and excursive positions, the lateral displacement did not exceed 70 m . There was no significant influence of the different occlusion schemes and excursive positions on the lateral displacement . Therefore, major crown displacement that could influence the strains measurements was not observed in any loading condition . The shear strain data indicate that the occlusion scheme and the excursive position had effects on localized peri - implant strain magnitude . However, greater impact was detected from altering the excursive position than from altering the occlusion scheme . For all the occlusion schemes, it is clear that as the excursive position moves buccally, the peri - implant strains increases (fig ., there was a significant difference among the occlusion schemes (p <.005). Gf was associated with the least strains, followed by ip, cg, and lc . At 1 mm, this was followed by ip, cg, and lc (p <.005). However, the difference was not significant between gf and ip (p = .65) and between cg and lc (p = .32). At 2 mm, the difference was significant between them (p <.005). A significant difference existed within the different schemes, except between the ip and lc occlusions (p = .16). Overall, the outcome reflected that at the earlier stages of excursion, gf and ip were associated with the least strain values . However, as the excursion increased, the strains generated from ip became considerably high . Regardless of the occlusion scheme, as the excursion increased, the peri - implant strains increased linearly . For all the schemes, the difference in peri - implant strains within the different positions was significant (p <.005). The 2-way analysis of variance test revealed that there was a significant interaction between the occlusion scheme and the site of loading . The schemes that had less strain values at the mi position tended to have less strain values for the rest of the positions . This study confirms that variation in occlusion scheme and excursive position influences the peri - implant strains . Thus, the hypotheses that the peri - implant strain magnitudes would be influenced by the occlusion scheme and the excursive location were accepted . Axial forces are preferred as they place the peri - implant bone, implant components, and prosthesis under compression . In addition, the forces are evenly distributed within the peri - implant bone.131516 on the contrary, the non - axial forces are associated with localized stress concentration 1516 and can eventually evoke greater peri - implant strains.14 much of the peri - implant bone strains will be in the crestal region, which is more susceptible to resorption.1516 in this investigation, the occlusion scheme was altered by modifying the canine palatal contour . Furthermore, varying the site of load application simulated implant crown loading in maximal intercuspation and excursive positions . It is very clear from this study that the palatal morphology and the excursive position can influence the peri - implant strains, which are most likely related to altering the proportion of axial and non - axial forces on the implant . Applying forces on inclined morphology was proven to increase the non - axial forces and the bending moment applied on the implant and the crown,219 as opposed to applying forces on flat surface . As the inclination increases, the lever arm between the point of load application and the fulcrum point at centre of implant increases, which will eventually accentuate the torque . In addition, the non - axial forces will increase by lateral loading of the implant crown . As the occlusal forces move laterally from the implant centre, the implant and the relevant components will be subjected to additional flexion and torqueing.20 tooth morphology plays a considerable role in occlusion . This is particularly evident for the canines due to their location in the corner of the arch, which allows them to control the eccentric movements and the pattern of lateral contacts.410 as an effect of altering the canine morphology, the lateral occlusion scheme can range from cg occlusion to gf or ip occlusions . Cg and lc occlusions are associated with greater overlap between the maxillary and mandibular canines to allow for posterior teeth disclusion.17 this has been established in this study by increasing the incline of the canine palatal contour . As a result, these occlusion schemes were associated with greater peri - implant strains at mi and the early stage of excursion . On the contrary, shallow canine palatal morphology, such as in gf or ip occlusions, tends to have less strains at mi and the early stage of excursion (1 mm)., who found that premolar implant crowns with steeper cusp inclination had greater peri - implant strains than crowns with shallower cusp inclination.19 however, as the excursion extends to 2 mm buccally, a different pattern was observed, and the ip occlusion had experienced a considerable localized increase of peri - implant strains . Although ip occlusion is thought to evoke less peri - implant strains, this was not consistently observed through the whole excursion path . This reflects the complexity associated with dental occlusion and palatal contour that can cause variation in the steepness along the excursion path . The crowns were constructed to test the effect of bucco - lingual morphology, but the different crowns exhibited different mesio - distal morphology, which might have had effects on the results . Differences in the mesio - distal morphology can cause the load pointer to shift down the ridge mesially or distally . While it may appear minimal, it will lead to strain fluctuation in experiments due to additional torsional strains being superimposed . In addition, although the differences between the occlusion schemes are statistically significant, it is very difficult to speculate the clinical impact of such difference . The high statistical difference can be due to repeating the load application on single crowns . As a result, a low standard deviation was observed . Additional experimentation should evaluate multiple crowns in each scheme . In order to simulate the natural pattern of occlusal contacts, each crown was loaded in mi and two excursive positions . These were adopted because it was thought to be clinically relevant and reflect the functional loading on the crown, as several reports pointed that functional occlusal contacts occurred within a range of few millimetres.2223 functional movements, such as chewing and physiological grinding, occur naturally within 0.5 mm from the mi position.78 the excursive range from 0.5 mm to edge - to - edge position is used during parafunctional activities . Therefore, the experiment covers the possible occlusal force occurrence during functional and parafunctional range of activities . It is very clear that regardless of occlusion scheme, as the excursion increases, the peri - implant strains increase as well . At mi, the force is directed parallel to the implant which will render the force of compressive nature . However, when the excursion occurs, the force direction will shift buccally, producing a coupling force to the centre of the implant and resulting in a larger bending moment1521 . The increase of peri - implant strains in the excursive positions illustrates the negative consequences of localized stresses during parafucntional activities . It is likely that the implemented occlusion scheme is a less important for implant overloading than the presence of parafunctional activities . This is in accordance with a clinical study that reported a direct relationship between bruxism and increased implant prostheses complications.18 contrary to the occlusion scheme, the excursive position has a greater impact on peri - implant strains . This indicates that the location of the load is much more significant in strain development than the palatal contour of the crowns . Therefore, altering the occlusion scheme for the purpose of changing the lateral contact pattern is more important than modifying the crown morphology and cuspal inclination . While this study had loaded all the crowns in all the excursive locations, the load might be different in clinical situations . From the clinical perspective, the canines in cg occlusion and lc occlusions are be the only teeth subjected to lateral forces in excursive positions.9 on the other hand, in gf occlusion, the lateral forces are distributed between the canine and the rest of the posterior teeth on the working side.9 an earlier study had reported that implant prostheses with gf was more comfortable than cg over a period of few months,17 which may be attributed to less restricted mandibular movement.5 while it is attractive to assume that gf is a safer occlusion, other investigators have reported increased chance to grind patients' teeth in eccentric positions . This has been observed by elevated emg readings of masticatory muscles and increased condylar displacement with gf occlusion.56 cg occlusion exhibits a protective mechanism from the proprioceptive feature of canine teeth . However, whether the proprioceptive mechanism will offset the negative effect of palatal steepness is yet to be determined . Nevertheless, ip occlusion will be safer for the implant due to the absence of lateral occlusal contacts . For implant prostheses, several authors suggested using the natural teeth for occlusion guidance and alleviation of occlusal contacts on the implant prosthesis during excursive movement.120 this is envisioned to minimize the nonaxial forces on implant components and reduce the risk of mechanical complications through micromovement or flexure.20 although ip is likely to be beneficial in eliminating lateral contacts on implant restoration, utilizing this scheme for anterior implant restoration may result in shallow morphology and shortened clinical crown that can hinder the dental esthetics . A recent systematic review did not confirm the superiority of any occlusion scheme for teeth or implant prostheses.11 in a retrospective study, kinsel and lin evaluated the effect of lateral occlusion on implant - supported prosthesis complications . Patients with gf occlusion had significantly greater chance of ceramic chipping than those with cg . However, the investigators did not relate the ceramic chipping solely to the occlusion scheme . In addition, they have reported more critical factors, such as bruxism and opposing implant prosthesis . On the other hand, several authors have reported there is no specific occlusion scheme that should be implemented for implant restorations . Instead, the lateral occlusion scheme for implant prostheses can mimic the the occlusion scheme on tooth supported prostheses.113 as a result, no specific occlusion scheme will be selected for all implant prostheses . While this study has the advantage of simulating a clinical scenario, it has some limitations . For example, a true representation of natural cortical bone and trabecular bone is impossible in a laboratory study . This experiment assumes isotropic mechanical behaviour of bone . However, bone is inhomogeneous due to the variations in cortical bone thickness and trabecular bone porosity . As a result, variation in bone quality may react differently to occlusal forces.14 in addition, laboratory experimentations assume complete implant integration, which does not represent actual implant osseointegration . Within the limitations of this preliminary study, it can be concluded that the occlusion scheme and the excursive position influence the peri - implant strains . According to the present experimental set - up, it appears that the impact of eccentric position is greater than that of the occlusion scheme . Implementing an occlusion concept that reduces the occurrence of occlusal contacts in excursive positions is preferable to reduce the strains within the peri - implant region.
Hemangiomas are categorized as racemose, capillary, cavernous, and venous, according to the size of their vascular spaces . Cavernous and venous hemangiomas have low blood flow, because they lack arterial or capillary components . Histopathologically, venous hemangiomas contain dilated vessels with thick, fibrous walls, whereas cavernous hemangiomas have capillary - sized vessels lined by flat endothelial cells . We report the first case of a venous hemangioma of the pps and discuss its typical radiographic findings . A 49-year - old female patient was referred for evaluation of swelling in the right submandibular region . She had swelling for the first time 3 weeks earlier, and it had not improved after medical treatment . Clinical examination revealed a soft mass in the right submandibular region and a bulge at the right lateral pharyngeal wall . A contrast - enhanced computed tomography (ct) scan revealed a cystic lesion, 4.63.0 cm, with contrast non - enhancement in the parapharyngeal space (pps). Mri showed a well - circumscribed non - enhancing mass with high signal intensity on t2-weighted images (fig . The cystic lesion of the pps was surgically removed using a transcervical approach . During surgery the lesion bled profusely, but the mass was bluntly dissected from the surrounding structures . The surgical specimen, measuring 4.34.52 cm, showed a pale - to - dark brown soft cut surface with a blood - filled spongy vascular lesion . Microscopic examination revealed blood - filled sinusoidal spaces with large irregular lumens and thick walls lined by endothelial cells (fig . Postoperative recovery was uneventful, and there has been no evidence of cranial nerve palsy or tumor recurrence after one year . Pps has a complex anatomy and close proximity to vital anatomical structures, with which it may become involved by various pathological processes . Presenting symptoms of pps tumors may be attributed to the size of the mass and compression of neighboring structures (1). We had difficulty with the preoperative diagnosis, due to the absence of typical symptoms or signs of a vascular lesion in the pps, such as pulsation or bruit . Fine - needle aspiration cytology (fnac) was also not helpful in reaching a diagnosis . Although an exact tissue characterization of hemangioma in the pps could not be made on fnac, contrast - enhanced ct and magnetic resonance imaging (mri) should be included in differentiating these lesions from other tumors of the pps . In general, mr imaging is superior to ct imaging in its ability to ascertain the soft tissue characteristics of pps tumors (2). Changes in the blood flow dynamics within a hemangioma result in thrombus formation and phleboliths (3). Phleboliths are calcified nodules that can be regarded as a characteristic property of venous or cavernous hemangiomas . Ct with contrast is an excellent imaging technique for revealing phleboliths . Although mri is not sensitive for the detection of small amounts of calcification, large amounts of calcification show discrete low signal intensity on all pulse sequences mri can produce high signal intensities, representing the blood, as well as focal heterogeneities, representing areas of thrombosis, fibrosis, or calcification (4). Although mri is very useful for the detection of vascular lesions, the detectability of phleboliths on ct images is superior to that by mri . Some reports have shown that plain x - ray films may also reveal calcified lesions . When imaging shows calcification in the pps, the differential diagnosis may include pleomorphic adenoma of the deep lobe of the parotid gland and metastatic thyroid carcinoma of the pps . Pleomorphic adenoma with foci of chondroid or osteoid stroma can demonstrate opacities on imaging, but usually shows minute, scattered flecks . Additionally, in cases of metastatic thyroid carcinoma of the pps, large flocculent calcification is seen on ct images (5). Venous hemangioma cannot be clinically or radiologically differentiated from cavernous hemangioma, because of their similar characteristics . However, perfusion and blood pool scintigraphy has been demonstrated to have high sensitivity for detecting head and neck hemangioma, and can also differentiate between cavernous and venous hemangiomas (6). They tend to be larger and less well circumscribed, and show no tendency to regress (7). Attention needs to be given to the tumor location, extent, growth rate, and accessibility as well as the patient's age and esthetic concerns . The excision of large tumors in the pps can be challenging, given the risk for severe hemorrhage and nerve injury . A surgical approach should be chosen according to the tumor size and location, its relationship to the great vessels, and any suspicion of malignancy . Generally, hemangiomas in the pps, including venous hemangioma, can be resected by using a transcervical approach . Profuse bleeding often occurs during surgery, but after all of the pathology has been removed, the bleeding will cease . When surgery is impossible, the use of corticosteroids, cryotherapy, feeding vessel ligation, embolization, and fibrosing agents can be considered as alternatives (8). In summary, we present the first reported case of venous hemangioma occurring in the parapharyngeal space . Considering that a presumptive diagnosis of most pps tumors can be made based on imaging studies, preoperative imaging findings are very important in approaching these lesions . It may be that multiple, spotty, calcific nodules, 13 mm in size, within a cystic lesion on ct images indicate the pathognomonic finding of hemangioma in the pps.
The tetracycline class of antibacterial agents has seen widespread clinical use for over 50 years due to its broad spectrum antibacterial activity. (1) tetracyclines inhibit bacterial growth by preventing protein biosynthesis through binding to the 30s ribosome, thereby blocking the binding of aminoacyl - trna to the acceptor site. (2) this class includes a number of naturally occurring compounds, such as chlorotetracycline (1)(3) and tetracycline (2),(4) as well as several semisynthetic analogues, such as doxycycline (3)(5) and minocycline (4) (figure 1). (6) in addition to their use as antibiotics for serious infections, tetracyclines have also been prescribed for use in acne and rosacea. (7) as with many classes of antibiotics, widespread use of tetracyclines has led to resistance and has limited their use in more complicated bacterial infections . Examples of naturally occurring (1 and 2) and semisynthetic (36) tetracyclines . Two major bacterial resistance mechanisms have been identified for tetracyclines: (1) active transport from bacterial cells via efflux pumps(10) and (2) ribosomal protection. (11) the efflux pumps are comprised of inner membrane tetracycline efflux proteins that were first identified in gram - negative organisms (tet(ae)) and were later found in gram - positive bacteria (tet(kl)). (10c) the ribosomal protection mechanism (tet(mo)) involves the expression of proteins that bind to the ribosome and allow protein synthesis to proceed even in the presence of tetracyclines . Since these early discoveries, the number of efflux and ribosomal protection mechanisms identified has increased. (12) the presence of the strongly activating 10-hydroxy group readily enables chemical modification of the tetracycline d - ring at the 7- and 9-positions . This strategy led to the introduction of the 7-dimethylamino group in compounds 46, all of which have demonstrated improved activity in bacterial strains bearing efflux pump resistance mechanisms . More recently, the 9-substituted glycylcyclines, such as tigecycline (5),(13) and the aminomethylcyclines, such as amadacycline (6),(14) have shown improved activity for resistant organisms bearing the ribosomal protection mechanism . In 2005, myers and co - workers published a fully synthetic route to the tetracyclines, the key step of which involves the michaeldieckmann condensation of the ab precursor 8 with structurally diverse d - ring precursors . This route provides potential access to a broad range of tetracyclines that are inaccessible via traditional semisynthesis. (17) one example of this is the introduction of a nitrogen atom into the d - ring, resulting in azatetracyclines . One 7-azatetracycline and one 9-azatetracycline were disclosed in myers original work, resulting in compounds with only modest antibacterial activity . Herein we demonstrate this powerful approach using pyridyl d - ring precursors 7 to generate novel 8-azatetracyclines, 10 (scheme 1). To explore 7- and 9-substituted-8-azatetracyclines, we required intermediates that could be readily modified either prior to or after construction of the protected compound 9 . Thus, the halopyridines 15 and 16 were prepared according to schemes 2 and 3 . 3,5-dichloroisonicotinic acid was treated with sodium benzylate to give compound 12, which was esterified to the phenyl ester 13 by conversion to the acid chloride, followed by treatment with phenol and 4-(dimethylamino)pyridine (dmap). Suzuki coupling(18) using methylboronic acid and pdcl2cy2 catalyst(19) gave the parent d - ring precursor 14 in good yield . N - oxide formation with hydrogen peroxide in acetic acid followed by treatment with either phosphorousoxychloride or phosphorousoxybromide gave 15a and 15b, respectively . For 15a, the reaction gave a 4.5:1.0 ratio of the desired regioisomer, while a 25:1 ratio was observed for 15b . The regiochemistry was assigned by conversion to the dimethylamino compound 15c through a palladium - mediated amination. (20)h nmr studies indicated an noe between the methyl groups of the amine and the adjacent ring methyl group . The methoxy derivative 15d was prepared from 15b by treatment with sodium methoxide in 2-methylpyrrolidinone (nmp) at 120 c . This gave the acid directly, which was re - esterified to the phenyl ester . Reagents and conditions: (i) nah (2 equiv), bnoh, nmp, 80 c; (ii) (cocl)2, cat . Dmf, ch2cl2; (iii) phoh, et3n, dmap, ch2cl2; (iv) ch3b(oh)2, pdcl2cy2, k3po4, toluene, water, 100 c; (v) h2o2, acoh, 80 c; (vi) pocl3, toluene, 100 c; (vii) pobr3, toluene, 100 c; (viii) dimethylamine in thf, pd2dba3, xantphos, k3po4, 1,4-dioxane, 100 c (sealed); reagents and conditions: (i) for 15a, mcpba, ch2cl2; for 15b, h2o2, acoh, 80 c; (ii) pobr3, toluene, 100 c; (iii) bocnh2, pd2dba3, xantphos, cs2co3, 1,4-dioxane, 80 c; (iv) hcl, 1,4-dioxane; (v) hf - pyridine, nano2, pyridine; (vi) dimethylamine in thf, pd2dba3, xantphos, cs2co3, 1,4-dioxane, 60 c (sealed). The dihalo compounds 16a and 16b were prepared by n - oxidation of 15a and 15b followed by treatment with pobr3 (scheme 3). Yields were typically moderate (4550%), with reduction of the n - oxide to give the starting pyridines 15 observed as major side reactions in both cases . Palladium - mediated coupling of 16b with t - butylcarbamate gave a 2.5:1 mixture of regioisomers, which were readily separated after conversion to the free amine 16c upon treatment with hcl . Treatment of 16c with sodium nitrite in the presence of hf - pyridine gave the fluoro derivative 16d in good yield. (21) similarly, a 2.4:1.0 ratio of regioisomers was observed in the palladium - mediated reaction of 16b with dimethylamine to give 16e and 16f . Interestingly, the regioselectivity could be reversed by heating 16b in the presence of dimethylamine without catalyst to give 16f as the major isomer in a 3:1 ratio . The 9-amino d - ring precursors 18a and 18b were prepared according to scheme 4 . Once again, palladium - mediated couplings using t - butylcarbamate proved to be highly effective for introduction of the amino group . Further protection of the mono - boc intermediates 17 with boc2o and dmap in dmf gave the di - boc precursors 18 . Reagents and conditions: (i) bocnh2, pd2dba3, xantphos, cs2co3, 1,4-dioxane, 80 c; (ii) boc2o, dmap, dmf . Most of the d - ring precursors were lithiated with lithium diisopropylamide (lda) at 78 c, followed by treatment with the enone 8 . Warming to 20 c yielded the fully protected tetracycline precursors 19 . For d - ring precursors 16a, 16d, and 18b, a one - pot reaction was performed in which lithium bis(trimethylsilyl)amide (lhmds) was added to a 78 c solution of the precursor and the enone 8 followed by warming to 20 c . Yields varied from 20 to 84% depending on substrate, with larger reaction scales generally providing better results . The lhmds procedure also typically gave higher yields but was only possible on the more highly electron deficient d - ring precursors . The 7-bromo intermediate 19c was further derivatized at this stage via suzuki couplings with methylboronic acid and phenylboronic acid to give 19d and 19e, respectively . Similarly, palladium - mediated amination with 19h and t - butylcarbamate provided the 7-chloro-9-amino compound 19j . Deprotection of the intermediates 19 with hf followed by hydrogenation in the presence of palladium on carbon gave the final 8-azatetracyclines 20ak . Reagents and conditions: (i) for 14, 15bd, 16e, and 18a, lda, tmeda, 78 c, thf then 8, 78 c to 20 c; (ii) for 16a, 16d, and 18b, lhmds, 8, thf, 78 c to 20 c; (iii) ch3b(oh)2 or phb(oh)2, pdcl2dppf2-ch2cl2, k3po4, toluene, 1,4-dioxane, water, 100 c; (iv) bocnh2, pd2dba3, xantphos, k3po4, 1,4-dioxane, 100 c; (v) aqueous hf, ch3cn or 1,4-dioxane; (vi) 10% pd / c, h2, hcl, meoh, 1,4-dioxane . To compare the 8-azatetracyclines directly to tigecycline, the 9-glycylamido-8-azatetracyclines 21ac were prepared (scheme 6). Treatment with bromoacetylchloride in thf followed by excess t - butylamine gave the acylated intermediates, which were deprotected under standard conditions to give the final analogues 21ac . Reagents and conditions: (i) hcl, 1,4-dioxane; (ii) bromoacetylchloride, thf then t - bunh2, 50 c; (iii) aqueous hf, ch3cn or 1,4-dioxane; (iv) 10% pd / c, h2, hcl, meoh, 1,4-dioxane . The bromopyridine intermediates proved to be useful for the introduction of alkylamines via palladium - mediated aminations and alkyl groups via suzuki couplings, ultimately providing the 9-alkylamino-8-azatetracyclines 24 and 9-alkyl-8-azatetracyclines 26 (scheme 7). Thus, 19h was treated with various amines in the presence of pd2dba3 and 9,9-dimethyl-4,5-bis-(diphenylphosphino)xanthenes (xantphos) in 1,4-dioxane at 100 c . Potassium phosphate was found to be the most effective base for this transformation, with cesium carbonate and sodium t - butoxide leading to rapid decomposition . Low yields were generally observed due to decomposition of both the starting material and products upon heating . Reaction times of 24 h were eventually settled on as a balance between product formation and decomposition . Copper - catalyzed aminations were also explored, but rapid decomposition of the starting material was observed . Hindered amines generally gave low yields as did reactions with the 7-dimethylamino compound 19 m . Alternatively, the alkylamines could be introduced prior to the michaeldieckmann reaction using either palladium or copper - mediated aminations(22) with 16a, 16d, and 16e . Protection of the resulting aminopyridines upon treatment with lhmds followed by boc2o or cbzcl gave the d - ring precursors 22 . Suzuki reactions with 19h or 19 m and various boronic acids were also carried out in moderate yields, providing the 9-alkyl intermediates 25 . Reagents and conditions: (i) r2r3nh2, pd2dba3, xantphos, k3po4 or cs2co3, 1,4-dioxane, 100 c or n - propylamine, cui, 2-acetylcyclohexanone, k3po4, dmf, 100 c; (ii) lhmds, thf then boc2o or cbzcl; (iii) lda, tmeda, 78 c, thf then 8, 78 c to 20 c or lhmds, 8, thf, 78 c to 20 c; (iv) aqueous hf, ch3cn, or 1,4-dioxane; (v) 10% pd / c, h2, hcl, meoh, 1,4-dioxane; (vi) r2b(oh)2, (ph3p)4pd, k3po4, toluene, 1,4-dioxane, h2o, 90 c . The in vitro antibacterial activities of all analogues were determined for a panel of gram - positive (e.g., staphylococcus aureus and streptococcus pneumoniae) and gram - negative (e.g., escherichia coli and klebsiella pneumoniae) bacteria . The activities of the 7-substituted-8-azatetracycline as well as several control tetracyclines for a representative set of these organisms are found in table 1 . The parent 8-azatetracycline 20a showed comparable activity to tetracycline, with reasonable minimum inhibitory concentrations (mics) of 0.251 g / ml for the tetracycline - susceptible strains . Lacking a substituent at either c-7 or c-9, 20a demonstrated poor activity for the strains bearing tet(m), tet(k), and tet(a). Overall, the 7-position appears to be very tolerant of substitution, allowing for bulky (20e), electron withdrawing (20b and 20i), and electron donating (20f and 20 g) groups while maintaining activity against the tetracycline - susceptible strains . As with minocycline, introduction of the 7-dimethylamino group in 20f led to improved activities for most strains tested . Modest (12 dilution) improvements in mics were observed for the tet(m) strains, while a more significant improvement was seen for the strain bearing the tet(k) resistance mechanism . With the exception of the 7-chloro analogue, all of the 7-substituted-8-aza compounds exhibited improved mics for the tet(k) strain, correlating well with literature reports on 7-substituted tetracyclines . Unfortunately, all of the 8-azatetracyclines appear to be substrates for the tet(a) efflux pump, exhibiting no antibacterial activity at the highest concentration tested (32 g / ml). In general, substitution at the 7-position appears to be inefficient at overcoming the ribosomal protection resistance mechanism of the tet(m) strains . The only compound with two dilution improvements for both tet(m) strains was compound 20i, the 7-fluoro analogue . Obtained from marilyn roberts lab at the university of washington . To make a direct comparison to tigecycline, the 9-glycylamido-8-azatetracyclines were prepared (table 2). Whereas tigecycline is a very potent antibacterial agent against all of the organisms in our panel, including the four strains bearing tetracycline resistance mechanisms, the corresponding 8-azatetracycline analogues 21ac were found to have poor overall activity . The aniline precursors 20j and 20k, however, showed significant improvements in mics for both tet(m) strains relative to the corresponding 9-unsubstituted compounds 20b and 20i . For the 7-cl analogue 20j, antibacterial activity also improved for the tet(k) and wild type strains . These compounds were also assessed for their ability to inhibit protein synthesis in a cell - free transcription / translation assay (table 3). In this assay, compounds 20j and 20k were about 2-fold more potent than tetracycline, in agreement with their relative mics for e. coli . Compounds 21ac, on the other hand, inhibited protein synthesis with ic50s between 0.42 and 0.90 g / ml, similar to tigecycline, despite having only modest antibacterial activity in the whole - cell assays . These data suggest that the compounds are active at the target, but that compounds 21ac are not capable of getting to the target in the whole - cell assay, possibly due to poor cell permeability . Atcc 25922 is a tetracycline - susceptible strain . On the basis of the results of compounds 20j and 20k, compounds bearing a small, unbranched alkylamino group at the 9-position (24ac) proved to be very potent antibacterial agents against the two tetracycline - susceptible gram - positive strains, with mics between 0.016 and 0.25 g / ml . A 4-fold improvement in activity against the tet(m) s. aureus strain was observed for all of these compounds relative to 20j, while mics for the s. pneumoniae tet(m) strain were largely unchanged . For the tet(k) s. aureus strain, antibacterial activity improved with increasing size of the alkyl group . Among the tetracycline - susceptible gram - negative strains, however, there was a clear loss in potency as the size of the alkyl group increased, a trend that continued for the larger branched alkyl compounds 24df . This is not surprising, as it is known that gram - negative antibacterial agents tend to be smaller and more polar. (23) these larger side chains also adversely affected the antibacterial activity for s. pneumoniae while having less effect on the mics for the s. aureus strains . The 7-fluoro-9-n - propylamino (24n) and 7-dimethylamino-9-n - propylamino (24o) analogues were prepared as comparisons to 24c . While the 7-fluoro compound had mics that tracked fairly closely to the 7-chloro analogue, the 7-dimethylamino compound exhibited no antibacterial activity for any of the strains at the highest concentration tested (32 g / ml) with the exception of the tetracycline - susceptible s. pneumoniae strain . All three of the compounds had similar, albeit weak, activity in the transcription / translation assay, indicating the differences in mics might be due to poor permeability of the 7-dimethylamino analogue . Introduction of a more polar heteroatom into the side chain (24gi, k) resulted in improved antibacterial activity against the s. pneumoniae and gram - negative strains but had minimal effect on activity toward either resistant s. aureus organisms . Interestingly, the oxygen bearing side chains (24 g and 24h) yielded significantly improved antibacterial activity toward tetracycline - susceptible gram - positive strains relative to the all carbon side chains (24e and 24f), while the nitrogen analogues (24k and 24i) were less potent against s. aureus and showed more modest improvements against s. pneumoniae . The combination of a branched side chain with a distal amino group gave the most balanced antibacterial compounds in this series (24l, 24 m, and 24j). These compounds combine good antibacterial activity toward both gram - positive and gram - negative organisms, with modest activity against tet(a) e. coli . Both compounds 24l and 24 m are less potent than tigecycline but compare favorably to amadacycline . The antibacterial activity of the 9-alkyl and phenyl substituted compounds were generally modest (table 5). Here again, a larger substituent at c-9 yielded improved activity for tet(m) and tet(k) strains but resulted in loss of activity for wild type s. pneumoniae and gram - negative strains . Introduction of a 7-cl or 7-dimethylamino group also gave improved activity for most of the strains in the panel . When comparing the 7-cl-9-alkyl compounds (26b) to the 7-cl-9-aminoalkyl compounds (24c), it is clear that the nitrogen confers significant improvements in antibacterial activity . In the 7-dimethylamino case, however, the 7-dimethylamino-9-alkyl compound 26a is a balanced gram - positive antibacterial agent while the 9-aminoalkyl compound 24o has no significant activity . Were selected for further profiling . Both compounds inhibited protein synthesis in the transcription / translation assay with ic50s comparable to tigecycline . Susceptibility testing was performed, and mic50 and mic90 values were calculated for recent clinical isolates of s. aureus, s. pneumoniae, haemophilus influenzae, and e. coli (table 6). The panels contained both tetracycline - susceptible and resistant strains, including 12 methicillin - resistant s. aureus (mrsa) strains . As expected from the initial mic data, compound 20f had very potent antibacterial activity for the tetracycline - susceptible strains but performed poorly against the resistant organisms . The compound did have mic90s that were significantly lower than tetracycline . Despite having higher mics in the primary screen relative to tigecycline, compound 24l performed similarly in the s. aureus, s. pneumoniae, and h. influenzae panels, exhibiting clinically relevant mic90s in each case . These organisms are particularly relevant, as they are responsible for infections in a large portion of community - acquired bacterial pneumonia cases . The pharmacokinetic profiles of the two 8-azatetracyclines were determined in spraguedawley rats (table 7). Compound 20f had very similar auc, clearance, and volume of distribution profiles to both tetracycline and tigecycline, although the half - life was somewhat shorter . Low oral bioavailability (% f) of 12.7% was observed but was comparable to tetracycline (12.1%). In humans, tetracycline is generally reported to have oral bioavailability of 5070%. (24) we and others have found the oral bioavailability of several tetracycline derivatives to be significantly lower in rat than reported data in humans . Compound 24l had a 4-fold higher auc and 5-fold lower clearance than the other three compounds as well as a significantly lower volume of distribution . Compounds were dosed at 1 mg / kg iv and 10 mg / kg po with sterile water as vehicle . The compounds were also investigated in mouse s. aureus and e. coli septicemia models (table 8). Both 8-azatetracyclines performed well in the s. aureus model following iv administration, with pd50 values comparable to the two marketed control tetracyclines . 20f, the most potent of the four compounds, also provided the best protection, with 100% survival at the lowest dose at which it was administered (0.30 mg / kg). Despite having an 8-fold higher mic, compound 24l provided equivalent protection relative to tigecycline, likely due to its higher auc and lower clearance. (25) the e. coli model proved to be more challenging, requiring higher doses for protection . Compound 20f had a pd50 of 4.3 mg / kg compared to 2.1 mg / kg for tigecycline . Compound 24l was 4-fold less potent than 20f and was comparable to tetracycline in this model . S. aureus atcc 13709 (smith) or e. coli atcc 25922 was mixed with 5% mucin and inoculated by intraperitoneal injection at 2.1 10 cfu / mouse (s. aureus) or 2.0 10 cfu / mouse (e. coli). One hour postchallenge, mice received iv treatment (vehicle = sterile water) with the test article at concentrations ranging from 0.0510 mg / kg (s. aureus) or 0.3030 mg / kg (e. coli). The pd50 in mg / kg was calculated as survival after 48 h. lowest dose was 0.30 mg / kg with 100% survival at all doses . A novel, fully synthetic series of 8-azatetracycline analogues was prepared and optimized . Through modification of the 7- and 9-positions, compounds were found that overcome both tet(k) efflux and tet(m)-mediated ribosomal protection . The 7-chloro-9-alkylamino-8-azatetracycline 24l exhibits clinically relevant mic90 profiles against both gram - positive and gram - negative organisms that include these resistance mechanisms . This compound also demonstrated in vivo efficacy in murine models of infection that is equivalent to currently marketed tetracycline antibacterial agents . The 7-dimethylamino-8-azatetracycline 20f was efficacious when administered intravenously and had oral bioavailability in rat equivalent to tetracycline . The current work demonstrates the ability of this chemistry platform to generate antibacterial compounds that are inaccessible through traditional semisynthesis, providing the medicinal chemist a broader array of tools through which essential antibacterial properties can be modulated . Importantly, this includes the ability to overcome ever evolving resistance mechanisms and to identify structureactivity relationships to deliver compounds with desired pharmacokinetic properties . Air and moisture sensitive liquids and reagents were transferred via syringe or cannula and were introduced into flame - dried glassware under a positive pressure of dry nitrogen through rubber septa . Analytical thin - layer chromatography was performed on em science precoated glass - backed silica gel 60 f-254 250 m plates . Visualization of the plates was effected by ultraviolet illumination and/or immersion of the plate in a basic solution of potassium permanganate in water followed by heating . Column chromatography was performed on a flashmaster personal system using isolute flash si ii silica gel prepacked cartridges (available from biotage). Preparative reversed - phase hplc chromatography (hplc) was accomplished using a waters autopurification system with mass - directed fraction collection . All intermediate compounds were purified with a waters sunfire prep c18 obd column (5 m, 19 mm 50 mm; flow rate = 20 ml / min) using a mobile phase mixture of h2o (a) and ch3cn (b) containing 0.1% hco2h . The final 8-azatetracycline compounds were purified using a phenomenex polymerx 10 rp 100a column (10 m, 30 mm 21.2 mm; flow rate = 20 ml / min) using a mobile phase mixture of 0.05n hcl in h2o (a) and ch3cn (b). All final 8-azatetracycline compounds were isolated as mono-, di-, or trihydrochloride salts following freeze - drying . H nmr spectra were recorded on a jeol ecx-400 (400 mhz) spectrometer and are reported in ppm using residual solvent as the internal standard (cdcl3 at 7.24 ppm, dmso - d6 at 2.50 ppm, or cd3od at 3.31 ppm). High performance liquid chromatographyelectrospray mass spectra (lc - ms) were obtained using a waters alliance hplc system equipped with a binary pump, a diode array detector, a waters sunfire c18 (5 m, 4.6 mm i.d . 50 mm) column, and a waters 3100 series mass spectrometer with electrospray ionization . The eluent was a mixture of h2o (a) and ch3cn (b) containing 0.1% hco2h at a flow rate of 1 ml / min . Purity of the final compounds was assessed by the following method: (a) time = 0, 100% a; (b) time = 0.5 min, 100% a; (c) time = 3.5 min, 100% b; (d) time = 5 min, 100% b (e) time = 6 min, 100% a; (f) time = 7 min, 100% a. all final products were 95% purity as assessed by this method . Sodium hydride (60% dispersion in mineral oil, 4.37 g, 109 mmol) was added portionwise to a solution of 3,5-dichloroisonicotinic acid (10.2 g, 53.3 mmol) in nmp (100 ml). After gas evolution ceased, benzyl alcohol (5.52 ml, 53.3 mmol) was added dropwise . After 1 h, the reaction was complete and was allowed to cool to room temperature and stand overnight . The reaction mixture was diluted with water (300 ml) and was washed with et2o (2 100 ml, discarded). The aqueous layer was brought to ph 2 with conc hcl, causing a precipitate to form . The mixture was diluted with brine (100 ml) and was allowed to stand for 30 min . The solid was collected by filtration, was washed with water (3), and was dried in a 45 c vacuum oven overnight . This gave 9.36 g (67%) of the title compound as a white solid . H nmr (400 mhz, dmso - d6) 14.3014.10 (bs, 1 h), 8.52 (s, 1h), 8.34 (s, 1 h), 7.507.30 (m, 5 h), 5.33 (s, 2 h). Oxalyl chloride (4.9 ml, 56 mmol) was added to a suspension of 12 (3.71 g, 14.1 mmol) in ch2cl2 (50 ml) over 2 min . Dmf was added dropwise in 20 l portions every 5 min until complete solution was achieved . After stirring for an additional 30 min, the resulting solid was dissolved in ch2cl2 (50 ml), and phenol (2.65 g, 28.1 mmol), dmap (0.172 g, 1.41 mmol), and et3n (9.76 ml, 70.4 mmol) were added . After 1 h, the reaction mixture was diluted with ch2cl2 (50 ml) and was washed with water (2 100 ml) and nahco3 (saturated, aqueous, 100 ml). The material was purified by column chromatography (biotage 50 g column, 025% etoac in hexanes gradient), yielding 4.20 g (88%) of the product as an off - white solid . H nmr (400 mhz, dmso - d6) 8.70 (s, 1h), 8.48 (s, 1 h), 7.527.30 (m, 8 h), 7.157.08 (m, 2 h), 5.44 (s, 2 h). Compound 13 (2.01 g, 5.92 mmol), methyl boronic acid (1.06 g, 17.7 mmol), dichlorobis(tricyclohexylphosphine)palladium(ii) (102 mg, 0.296 mmol), and k3po4 (3.76 g, 17.7 mmol) were heated to 100 c in toluene (20 ml) and water (2 ml). After 16 h, the reaction mixture was allowed to cool to room temperature and was diluted with etoac (20 ml). This was washed with water (20 ml) and brine (20 ml), dried over na2so4, filtered, and concentrated under reduced pressure . The material was purified by column chromatography (biotage 50 g column, 040% etoac in hexanes gradient), yielding 1.66 g (88%) of the product as a white solid . H nmr (400 mhz, dmso - d6) 8.50 (s, 1h), 8.26 (s, 1 h), 7.527.30 (m, 8 h), 7.207.10 (m, 2 h), 5.37 (s, 2 h), 2.38 (s, 3 h). Ms (esi) m / z 320.27 (m + h). Compound 14 (9.66 g, 30.3 mmol) was heated to 80 c in acetic acid (50 ml) and hydrogen peroxide (30% aqueous solution, 17 ml). After 6 h, lc / ms indicated that the starting material was consumed and the n - oxide present . Most of the acetic acid and water were removed under reduced pressure . The material was then diluted with etoac (200 ml), and the mixture was washed with nahco3 (saturated aqueous solution, 3 50 ml). The organics were dried over na2so4, filtered, and concentrated under reduced pressure, yielding 9.37 g (92%) of the crude n - oxide . The material was dissolved in toluene (60 ml), phosphorousoxychloride (7.69 g, 50.1 mmol) was added, and the reaction mixture was heated to 100 c . After 2 h, lc / ms indicated that the reaction was complete . Upon cooling to room temperature, the reaction mixture was poured into a solution of k2co3 (40 g) in water (200 ml). This mixture was extracted with etoac (3 150 ml), and the combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was redissolved in etoac (30 ml) with heating and was then allowed to cool to room temperature and stand overnight . Another 1.92 g (19%) of the white crystalline solid was obtained from the mother liquor after standing in a refrigerator overnight . H nmr (400 mhz, cdcl3) 8.05 (s, 1h), 7.437.30 (m, 7 h), 7.297.20 (m, 1 h), 7.07 (d, j = 8.2 hz, 2 h), 5.21 (s, 2 h), 2.46 (s, 3 h). Ms (esi) m / z 398.22, 400.22 (m + h). Compound 14 (25.9 g, 81.1 mmol) was heated to 80 c in acetic acid (160 ml) and hydrogen peroxide (30% aqueous solution, 40 ml). After stirring overnight, lc / ms indicated that the starting material was consumed and the n - oxide present . The reaction mixture was concentrated under reduced pressure, was diluted with etoac, and was washed with nahco3 (saturated aqueous solution, 3). The organics were dried over na2so4, filtered, and concentrated under reduced pressure, yielding 25.4 g (93% crude) of the n - oxide as a tan solid . The material was suspended in toluene (200 ml) and was heated to 80 c, at which point most of the solid had dissolved . Upon cooling to room temperature, the reaction mixture was poured into a solution of k2co3 (83 g) in water (300 ml). The combined extracts were dried over mgso4, filtered through celite, and concentrated under reduced pressure . The material was recrystallized from etoac / hexanes (1:1), yielding 20.6 g (64%) of the product as an off - white solid . H nmr (400 mhz, cdcl3) 8.05 (s, 1h), 7.437.30 (m, 7 h), 7.297.20 (m, 1 h), 7.07 (d, j = 8.2 hz, 2 h), 5.21 (s, 2 h), 2.46 (s, 3 h). Ms (esi) m / z 398.22, 400.22 (m + h). Compound 15b (1.06 g, 2.66 mmol), dimethylamine (2.0 m solution in thf, 4.0 ml, 8.0 mmol), k3po4 (1.7 g, 8.0 mmol), tris(dibenzylideneacetone)dipalladium (120 mg, 0.13 mmol), and xantphos (220 mg, 0.40 mmol) were heated to 100 c in 1,4-dioxane (10 ml) in a sealed pressure vessel . After heating overnight, the reaction mixture was cooled to room temperature, was diluted with etoac (100 ml), and was washed with water (3 50 ml) and brine (50 ml). The material was purified by column chromatography (biotage 50 g column, 015% etoac in hexanes gradient), yielding 388 mg (40%) of the title compound as a white solid . H nmr (400 mhz, cdcl3) 7.96 (s, 1h), 7.457.20 (m, 8 h), 7.08 (d, j = 8.2 hz, 2 h), 5.16 (s, 2 h), 2.76 (s, 6 h), 2.37 (s, 3 h). Ms (esi) m / z 363.45 (m + h). Sodium methoxide (38 mg, 0.70 mmol) was added to a solution of compound 15b (118 mg, 0.351 mmol) in nmp (2 ml), and the reaction mixture was heated to 100 c . After heating overnight, additional sodium methoxide (57 mg, 1.1 mmol) was added and heating was continued at 100 c . After an additional 1 h, the reaction mixture was cooled to room temperature, water (20 ml) was added, and the ph was adjusted to 2 with 6 n aqueous hcl . This was extracted with etoac (3 20 ml), and the combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The crude acid intermediate was dissolved in ch2cl2 (10 ml), and oxalyl chloride (0.123 ml, 1.40 mmol) was added . The material was dissolved in ch2cl2 (10 ml), and phenol (66 mg, 0.70 mmol), dmap (4 mg, 0.04 mmol), and et3n (0.245 ml, 1.76 mmol) were added . After 1 h, the reaction mixture was diluted with etoac (50 ml) and was washed with water (2 30 ml) and brine (30 ml). The material was purified by column chromatography (biotage 10 g column, 012% etoac in hexanes gradient), yielding 20.3 mg (17%) of clean product . H nmr (400 mhz, cdcl3) 7.75 (s, 1h), 7.457.24 (m, 9 h), 7.10 (d, j = 7.3 hz, 2 h), 5.13 (s, 2 h), 3.91 (s, 3 h), 2.67 (s, 3 h). Ms (esi) m / z 336.1, 338.1 (m + h). Compound 15a (4.99 g, 14.1 mmol) was dissolved in ch2cl2 (30 ml), and 3-chloroperbenzoic acid (77%, 6.33 g, 28.2 mmol) was added in one portion . After 6 h, an additional portion of 3-chloroperbenzoic acid (3.15 g, 14.1 mmol) was added, and the reaction was stirred at room temperature overnight . The reaction mixture was diluted with ch2cl2 (200 ml) and was washed with na2co3 (saturated aqueous solution, 100 ml) and brine . The material was purified by column chromatography (biotage 70 g column, 550% etoac in hexanes gradient), yielding 3.90 g (75%) of the pure n - oxide as an off - white solid (rf = 0.33 in 66% etoac / hexanes). The n - oxide was dissolved in toluene (30 ml), phosphorousoxybromide (6.36 g, 22.1 mmol) was added, and the reaction mixture was heated to 80 c . After 1 h, the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 (10 g) in water (50 ml). This mixture was extracted with etoac (3 100 ml), and the combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was purified by column chromatography (biotage 50 g column, 010% etoac in hexanes gradient), yielding 2.06 g (45%) of the desired product 16a as an oil which slowly solidified on standing overnight . H nmr (400 mhz, cdcl3) 8.05 (s, 1h), 7.437.30 (m, 7 h), 7.297.20 (m, 1 h), 7.07 (d, j = 8.2 hz, 2 h), 5.21 (s, 2 h), 2.46 (s, 3 h). Ms (esi) m / z 398.22, 400.22 (m + h). Compound 15b (8.54 g, 21.4 mmol) was heated to 80 c in acetic acid (120 ml) and hydrogen peroxide (30% aqueous solution, 30 ml). After stirring overnight, lc / ms indicated that the starting material was mostly consumed and the n - oxide present . The reaction mixture was cooled to room temperature and was diluted with brine (150 ml). The resulting precipitate was collected by filtration and was washed with water / brine (1:1, 2). The solid was dissolved in ch2cl2 and was dried over na2so4, filtered, and concentrated under reduced pressure . This gave 7.83 g (88% crude) of the n - oxide as a tan solid . The material was suspended in toluene (200 ml) and was heated to 100 c, at which point most of the solid had dissolved . A solution of phosphorousoxybromide (10.8 g, 37.8 mmol) in toluene (10 ml) was added rapidly . After 45 min, the reaction mixture was allowed to cool to room temperature and was poured into a solution of k2co3 (40 g) in water (150 ml). After stirring for 30 min at room temperature, the combined extracts were dried over mgso4, filtered, and concentrated under reduced pressure . The material was purified by column chromatography (biotage 50 g column, 08% etoac in hexanes gradient to isolate the product then 15% etoac in hexanes to recover 15b), yielding 5.02 g (49%) of the product as an off - white solid . H nmr (400 mhz, dmso - d6) 7.497.32 (m, 8 h), 7.10 (d, j = 7.3 hz, 2 h), 5.15 (s, 2 h), 2.41 (s, 3 h). Ms (esi) m / z 476.18, 478.16, 480.16 (m + h). Compound 16b (2.15 g, 4.50 mmol), t - butylcarbamate (633 mg, 5.40 mmol), cs2co3 (2.93 g, 9.00 mmol), tris(dibenzylideneacetone)dipalladium (206 mg, 0.225 mmol), and xantphos (380 mg, 0.673 mmol) were weighed into a flask . This was evacuated and backflushed with nitrogen (3), and 1,4-dioxane (15 ml) was added . The reaction mixture was cooled to room temperature, was diluted with etoac (100 ml), and was washed with water (2 50 ml). The material was purified by column chromatography (biotage 50 g column, 012% etoac in hexanes gradient), yielding 1.43 g (62%) of a 4:1 mixture of the two regioisomeric compounds . H nmr (400 mhz, cdcl3) 7.517.46 (m, 2 h), 7.427.32 (m, 7 h), 7.307.24 (m, 1 h), 7.12 (d, j = 8.2 hz, 0.5 h), 7.03 (d, j = 8.2 hz, 2 h), 6.88 (s, 0.25 h), 6.71 (s, 1 h), 5.12 (s, 2 h), 5.02 (s, 0.5 h), 2.43 (s, 0.75 h), 2.34 (s, 3 h), 1.50 (s, 9 h), 1.47 (s, 2.25 h). Ms (esi) m / z 535.10, 537.10 (m + na). The above regioisomeric mixture (1.43 g, 2.78 mmol) was stirred in 4 m hcl in 1,4-dioxane (20 ml) and 1,4-dioxane (5 ml) overnight . The reaction mixture was diluted with etoac (100 ml) and was washed with nahco3 (saturated aqueous solution, 2 100 ml). The material was purified by column chromatography (biotage 20 g column, 030% etoac in hexanes gradient), yielding 805 mg (70%) of the major regioisomer 16c and 270 mg (24%) of the minor regioisomer . H nmr (400 mhz, cdcl3) 7.507.42 (m, 2 h), 7.407.30 (m, 5 h), 7.307.24 (m, 1 h), 7.04 (d, j = 8.2 hz, 2 h), 5.06 (s, 2 h), 4.56 (s, 2 h), 2.16 (s, 3 h). Ms (esi) m / z 413.11, 415.01 (m + h). Nmr (400 mhz, cdcl3) 7.427.34 (m, 8 h), 7.137.10 (m, 2 h), 5.01 (s, 2 h), 4.64 (s, 2 h), 2.35 (s, 3 h); ms (esi) m / z 413.09, 415.09 (m + h). Hf - pyridine solution (70% hf, 2 ml) was added to a 0 c solution of compound 16c (884 mg, 2.14 mmol) in pyridine (1 ml). Sodium nitrite (177 mg, 2.57 mmol) was added (bubbling observed), and the reaction mixture was allowed to slowly warm to room temperature . After 3 days, the reaction mixture was poured into na2co3 (saturated aqueous solution, 30 ml) and was extracted with etoac (3 30 ml). The combined extracts were washed with hcl (1 n aqueous solution, 2 50 ml) and were dried over na2so4, filtered, and concentrated under reduced pressure . The material was purified by column chromatography (biotage 20 g column, 06% etoac in hexanes gradient), yielding 687 mg (77%) of the product as a colorless oil that slowly solidified on standing . H nmr (400 mhz, cdcl3) 7.507.42 (m, 2 h), 7.407.30 (m, 5 h), 7.307.24 (m, 1 h), 7.087.02 (m, 2 h), 5.14 (s, 2 h), 2.33 (s, 3 h). Ms (esi) m / z 416.17, 418.15 (m + h). Compound 16b (1.51 g, 3.16 mmol), cs2co3 (3.1 g, 9.5 mmol), tris(dibenzylideneacetone)dipalladium (145 mg, 0.158 mmol), and xantphos (267 mg, 0.474 mmol) were weighed into a vial equipped with a septum . This was evacuated and flushed with nitrogen (3), and 1,4-dioxane (7 ml) and dimethylamine (2.0 m solution in thf, 4.75 ml, 9.50 mmol) were added . The reaction mixture was heated to 60 c and was stirred for 4 h. the reaction mixture was allowed to cool to room temperature, was filtered through celite, and was concentrated under reduced pressure . The material was purified by column chromatography (biotage 50 g column, 010% etoac in hexanes gradient), yielding 868 mg (58%) of the major regioisomer 16e and 354 mg (24%) of the minor regioisomer 16f . Data for 16e: h nmr (400 mhz, cdcl3) 7.507.46 (m, 2 h), 7.417.30 (m, 5 h), 7.307.24 (m, 1 h), 7.087.02 (m, 2 h), 5.08 (s, 2 h), 2.85 (s, 6 h), 2.32 (s, 3 h); ms (esi) m / z 441.19, 443.18 (m + h); rf = 0.29 in 10% etoac / hexanes . The regiochemistry was confirmed by an noe between the methyl protons and the n, n - dimethylamino protons (0.61%). Data for 16f: h nmr (400 mhz, cdcl3) 7.417.31 (m, 7 h), 7.307.20 (m, 1 h), 7.01 (d, j = 7.3 hz, 2 h), 4.94 (s, 2 h), 3.04 (s, 6 h), 2.33 (s, 3 h); ms (esi) m / z 441.19, 443.18 (m + h); rf = 0.40 in 10% etoac / hexanes . The regiochemistry was confirmed by noe between the benzylic protons of the o - benzyl group and the n, n - dimethylamino protons (0.44%). Compound 16d (538 mg, 1.29 mmol), t - butylcarbamate (302 mg, 2.58 mmol), cs2co3 (840 mg, 2.58 mmol), tris(dibenzylideneacetone)dipalladium (59 mg, 0.065 mmol), and xantphos (109 mg, 0.190 mmol) were weighed into a flask . This was evacuated and backflushed with nitrogen (3), and 1,4-dioxane (3 ml) was added . After 4 h, the reaction mixture was cooled to room temperature, was diluted with etoac (50 ml), and was washed with water (2 25 ml). The material was purified by column chromatography (biotage 20 g column, 012% etoac in hexanes gradient), yielding 451 mg (77%) of the product . Rf = 0.35 in 20% etoac / hexanes . H nmr (400 mhz, cdcl3) 7.427.24 (m, 8 h), 7.046.99 (m, 2 h), 4.99 (s, 2 h), 2.38 (s, 3 h), 1.40 (s, 18 h). Compound 17a (451 mg, 1.00 mmol) was treated with di - t - butyldicarbonate (1.09 g, 5.00 mmol) and dmap (24 mg, 0.20 mmol) in dmf (15 ml). After 30 min, the reaction mixture was diluted with etoac (100 ml) and was washed with water (3 50 ml) and brine (50 ml). The material was purified by column chromatography (biotage 10 g column, 08% etoac in hexanes gradient), yielding 481 mg (87%) of the title compound . H nmr (400 mhz, cdcl3) 7.427.24 (m, 8 h), 7.046.99 (m, 2 h), 4.99 (s, 2 h), 2.38 (s, 3 h), 1.40 (s, 18 h). Compound 16e (467 mg, 1.06 mmol), t - butylcarbamate (372 mg, 3.17 mmol), cs2co3 (1.04 g, 3.17 mmol), tris(dibenzylideneacetone)dipalladium (48 mg, 0.053 mmol), and xantphos (89.3 mg, 0.159 mmol) were weighed into a vial . This was evacuated and backflushed with nitrogen (3), and 1,4-dioxane (3 ml) was added . After 4 h, the reaction mixture was cooled to room temperature and was filtered through celite . The filtrate was concentrated under reduced pressure, and the material was purified by column chromatography (biotage 20 g column, 016% etoac in hexanes gradient), yielding 493 mg (98%) of the product containing 20% of an unidentified impurity . H nmr (400 mhz, cdcl3) 7.437.32 (m, 8 h), 7.307.24 (m, 1 h), 7.157.10 (m, 2 h), 6.80 (s, 1 h), 4.94 (s, 2 h), 2.85 (s, 6 h), 2.31 (s, 3 h), 1.48 (s, 9 h). Ms (esi) m / z 478.27 (m + h). Compound 17b (490 mg, 1.03 mmol) was treated with di - t - butyldicarbonate (672 mg, 3.08 mmol) and dmap (12.5 mg, 0.103 mmol) in dmf (5 ml). After stirring overnight, the reaction mixture was diluted with etoac (25 ml) and was washed with water (3 20 ml) and brine (20 ml). The material was purified by column chromatography (biotage 10 g column, 010% etoac in hexanes gradient), yielding 424 mg (72%) of the product . H nmr (400 mhz, cdcl3) 7.427.24 (m, 8 h), 7.067.00 (m, 2 h), 4.92 (s, 2 h), 2.79 (s, 6 h), 2.36 (s, 3 h), 1.39 (s, 18 h). Ms (esi) m / z 578.33 (m + h). Lda was prepared at 78 c from n - butyllithium (1.6 m solution in hexane, 0.495 ml, 0.792 mmol) and diisopropylamine (0.112 ml, 0.792 mmol) in thf (5 ml). Tmeda (0.318 ml, 2.11 mmol) was added, followed by dropwise addition of a solution of compound 14 (169 mg, 0.529 mmol) in thf (2 ml). After 5 min, a solution of 8 (128 mg, 0.264 mmol) in thf (2 ml) was added . After complete addition, the reaction mixture was allowed to warm to 20 c over 1 h. the reaction was quenched by the addition of ammonium chloride (saturated, aqueous solution, 20 ml) and was extracted with etoac (2 20 ml). The combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was purified by preparative hplc (sunfire prep c18 column, 80100% b gradient), yielding 65.5 mg (35%) of the title compound as a yellow solid . H nmr (400 mhz, cdcl3) 15.77 (s, 1 h), 8.36 (s, 1 h), 8.16 (s, 1 h), 7.557.24 (m, 10 h), 5.405.25 (m, 4 h), 3.93 (d, j = 11.0 hz, 1 h), 3.163.04 (m, 1 h), 2.982.90 (m, 1 h), 2.762.64 (m, 1 h), 2.602.40 (m, 8 h), 2.12 (d, j = 14 hz, 1 h), 0.82 (s, 9 h), 0.26 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 708.72 (m + h). The following compounds were synthesized by methods similar to 19a . Prepared from 15a in 38% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.6 (s, 1 h), 8.12 (s, 1 h), 7.567.49 (m, 4 h), 7.427.32 (m, 6 h), 5.37 (s, 2 h), 5.07 (s, 2 h), 3.91 (d, j = 11.0 hz, 1 h), 3.133.02 (m, 1 h), 2.97 (dd, j = 15.6 4.9 hz, 1 h), 2.652.45 (m, 3 h), 2.50 (s, 6 h), 2.14 (d, j = 15.6 hz, 1 h), 0.82 (s, 9 h), 0.27 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 742.56 (m + h). Prepared from 15b in 71% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.55 (s, 1 h), 8.12 (s, 1 h), 7.557.24 (m, 10 h), 5.405.22 (m, 4 h), 3.90 (d, j = 11.0 hz, 1 h), 3.25 (dd, j = 16.5 hz, j = 4.88 hz, 1 h), 3.123.02 (m, 1 h), 2.622.42 (m, 9 h), 2.14 (d, j = 14.0 hz, 1 h), 0.82 (s, 9 h), 0.26 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 786.63, 788.63 (m + h). Prepared from 15c in 50% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.65 (s, 1 h), 8.01 (s, 1 h), 7.527.22 (m, 10 h), 5.36 (s, 2 h), 5.17 (q, j = 11.0 hz, 2 h), 4.02 (d, j = 11.0 hz, 1 h), 3.05 (dd, j = 17.2 hz, j = 11.6 hz, 1 h), 2.982.86 (m, 1 h), 2.73 (s, 6 h), 2.622.38 (m, 9 h), 2.12 (d, j = 13.4 hz, 1 h), 0.81 (s, 9 h), 0.26 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 751.79 (m + h). Prepared from 15d in 27% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.70 (s, 1 h), 7.83 (s, 1 h), 7.557.24 (m, 10 h), 5.36 (s, 2 h), 5.17 (q, j = 13.4 hz, 2 h), 3.98 (d, j = 11.0 hz, 1 h), 3.72 (s, 3 h), 3.18 (d, j = 16.5 hz, 1 h), 3.04 2.96 (m, 1 h), 2.602.30 (m, 9 h), 2.15 (d, j = 14 hz, 1 h), 0.82 (s, 9 h), 0.26 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 738.66 (m + h). H nmr (400 mhz, cdcl3) 15.59 (s, 1 h), 7.567.24 (m, 10 h), 5.36 (s, 2 h), 4.92 (q, j = 67.8 hz, j = 9.16 hz, 2 h), 3.91 (d, j = 11.0 hz, 1 h), 3.203.02 (m, 2 h), 2.622.45 (m, 9 h), 2.192.12 (m, 1 h), 1.37, (s, 18 h), 0.82 (s, 9 h), 0.26 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 941.59 (m + h). Prepared from 16e in 54% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.65 (s, 1 h), 7.527.22 (m, 10 h), 5.36 (s, 2 h), 5.17 (q, j = 11.0 hz, 2 h), 4.01 (d, j = 11.0 hz, 1 h), 3.05 (dd, j = 17.2 hz, j = 11.6 hz, 1 h), 2.982.86 (m, 1 h), 2.73 (s, 6 h), 2.622.38 (m, 9 h), 2.12 (d, j = 13.4 hz, 1 h), 0.82 (s, 9 h), 0.26 (s, 3 h), 0.13 (s, 3 h). Ms (esi) m / z 829.49, 831.48 (m + h). Compound 16a (793 mg, 1.84 mmol) and compound 8 (885 mg, 1.84 mmol) were dissolved in thf (16 ml), and the solution was cooled to 78 c . Lhmds (1.0 m solution in thf, 5.5 ml, 5.5 mmol) was added slowly via syringe . After 10 min, the reaction mixture was allowed to slowly warm to 0 c over 1 h. a phosphate buffer solution (ph 7.0, 20 ml) was added, followed by the addition of ammonium chloride (saturated, aqueous solution, 50 ml). The resulting mixture was extracted with ch2cl2 (3 50 ml), and the combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The resulting brown solid was washed with cold methanol (3 5 ml) to afford 1.11 g (74%) of the title compound as a yellowbrown powder . The organics were concentrated under reduced pressure and purified by column chromatography (biotage 20 g column, 520% etoac in hexanes gradient), yielding an additional 150 mg (10%) of the product . H nmr (400 mhz, cdcl3) 15.45 (br, 1 h), 7.547.48 (m, 4 h), 7.407.30 (m, 6 h), 5.36 (s, 2 h), 5.03 (abq, j = 10.4 hz, 2 h), 3.87 (d, j = 11.0 hz, 1 h), 3.273.23 (m, 1 h), 3.103.00 (m, 1 h), 2.652.57 (m, 1 h), 2.572.43 (m, 8 h), 2.16 (d, j = 11.0 hz, 1 h), 0.81 (s, 9 h), 0.26 (s, 3 h), 0.12 (s, 3 h). Ms (esi) m / z 820.37, 822.37 (m + h). The following compounds were prepared by methods similar to 19h . Prepared from 16d in 45% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.55 (s, 1 h), 7.567.26 (m, 10 h), 5.36 (s, 2 h), 5.02 (s, 2 h), 3.88 (d, j = 10.4 hz, 1 h), 3.183.04 (m, 2 h), 2.622.58 (m, 1 h), 2.582.40 (m, 8 h), 2.17 (d, j = 14.6 hz, 1 h), 0.81 (s, 9 h), 0.25 (s, 3 h), 0.12 (s, 3 h). Ms (esi) m / z 804.34, 806.34 (m + h). Prepared from 18b in 53% yield, yellow solid . H nmr (400 mhz, cdcl3) 15.50 (s, 1 h), 7.527.24 (m, 10 h), 5.36 (s, 2 h), 4.85 (q, j = 86.1 hz, j = 9.76 hz, 2 h), 4.02 (d, j = 10.4 hz, 1 h), 3.083.00 (m, 1 h), 3.002.82 (m, 1 h), 2.77 (s, 6 h), 2.622.38 (m, 9 h), 2.202.12 (m, 1 h), 1.35, (br s, 18 h), 0.79 (s, 9 h), 0.26 (s, 3 h), 0.12 (s, 3 h). Ms (esi) m / z 966.59 (m + h). A solution of compound 19c (52 mg, 0.067 mmol), methylboronic acid (40 mg, 0.67 mmol), dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii) dichloromethane adduct (3 mg, 0.003 mmol), and k3po4 (142 mg, 0.667 mmol) in toluene (1 ml), 1,4-dioxane (1 ml), and water (0.2 ml) was heated to 70 c . After 2 h, the reaction mixture was heated to 100 c . After an additional 2 h, the reaction mixture was diluted with etoac (20 ml) and was washed with water (15 ml) and brine (15 ml). The material was purified by preparative hplc (sunfire prep c18 column, 80100% b gradient), yielding 18.3 mg (38%) of the title compound as a yellow solid . H nmr (400 mhz, cdcl3) 15.71 (s, 1 h), 8.24 (s, 1 h), 7.557.24 (m, 10 h), 5.36 (s, 2 h), 5.305.20 (m, 2 h), 3.95 (d, j = 10.4 hz, 1 h), 3.102.92 (m, 2 h), 2.622.42 (m, 12 h), 2.12 (d, j = 14.0 hz, 1 h), 0.82 (s, 9 h), 0.26 (s, 3 h), 0.14 (s, 3 h). Ms (esi) m / z 722.72 (m + h). A solution of compound 19c (31 mg, 0.040 mmol), phenylboronic acid (24.5 mg, 0.201 mmol), dichloro[1,1-bis(diphenylphosphino)ferrocene]palladium(ii) dichloromethane adduct (1.6 mg, 0.002 mmol), and sodium carbonate (21.3 mg, 0.201 mmol) in toluene (1 ml), 1,4-dioxane (1 ml), and water (0.2 ml) was heated to 100 c via microwave reactor for 10 min . The reaction mixture was diluted with etoac (10 ml) and was washed with water (5 ml) and brine (5 ml). The material was purified by preparative hplc (sunfire prep c18 column, 80100% b gradient), yielding 16.9 mg (54%) of the title compound as a yellow solid . H nmr (400 mhz, cdcl3) 15.66 (s, 1 h), 8.48 (s, 1 h), 7.527.24 (m, 15 h), 5.425.30 (m, 4 h), 3.97 (d, j = 10.4 hz, 1 h), 3.002.86 (m, 2 h), 2.782.62 (m, 1 h), 2.582.30 (m, 8 h), 2.00 (d, j = 14.0 hz, 1 h), 0.80 (s, 9 h), 0.25 (s, 3 h), 0.14 (s, 3 h). Ms (esi) m / z 784.75 (m + h). Compound 19h (100 mg, 0.122 mmol), t - butylcarbamate (42.9 mg, 0.366 mmol), k3po4 (77.7 mg, 0.366 mmol), tris(dibenzylideneacetone)dipalladium (5.6 mg, 0.006 mmol), and xantphos (10.3 mg, 0.018 mmol) were weighed into a vial equipped with a septum . The vial was evacuated and flushed with nitrogen (3), and 1,4-dioxane (0.5 ml) was added . The reaction mixture was heated to 100 c and stirred for 2 h. the reaction mixture was allowed to cool to room temperature, was filtered through celite, and was concentrated under reduced pressure . The material was purified by preparative hplc (sunfire prep c18 column, 80100% b gradient), yielding 41.7 mg (40%) of the title compound as a yellow solid . H nmr (400 mhz, cdcl3) 15.6515.45 (br s, 1 h), 7.567.50 (m, 2 h), 7.437.34 (m, 7 h), 7.297.20 (m, 1 h), 5.38 (s, 2 h), 4.94 (dd, j = 29.3 hz, j = 11.0 hz, 2 h), 3.90 (d, j = 11.0 hz, 1 h), 3.27 (dd, j = 16.4 hz, j = 11.6 hz, 1 h), 3.123.03 (m, 1 h), 2.682.60 (m, 1 h), 2.602.45 (m, 8 h), 2.18 (d, j = 14.6 hz, 1 h), 1.47 (s, 9 h), 0.85 (s, 9 h), 0.29 (s, 3 h), 0.15 (s, 3 h). Ms (esi) m / z 857.67 (m + h). Aqueous hf (48%, 0.4 ml) was added to a solution of 19a (65.5 mg, 0.093 mmol) in ch3cn (0.6 ml) in a plastic vial . After 18 h, the reaction mixture was poured into a solution of k2hpo4 (4.8 g) in water (20 ml). The combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was dissolved in meoh (1 ml) and 1,4-dioxane (1 ml), and palladium on carbon (degussa, 10 wt%, 5 mg) was added . An atmosphere of hydrogen was introduced, and the reaction mixture was stirred for 2 h. the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure . The material was purified by preparative hplc (phenomenex polymerx column, 0100% b gradient). Fractions with the desired mw were collected and freeze - dried to yield 18.4 mg (41%, 2 steps) of the title compound as a yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 8.55 (s, 1 h), 8.36 (s, 1 h), 4.19 (s, 1 h), 3.262.98 (m, 9 h), 2.71 (t, j = 14.2 hz, 1 h), 2.392.32 (m, 1 h), 1.751.63 (m, 1 h). The following compounds were synthesized by similar methods to 20a . Prepared from 19b in 24% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od) 8.08 (d, j = 6.0 hz, 1 h), 4.09 (s, 1 h), 3.082.92 (m, 9 h), 2.302.15 (m, 2 h), 1.70 1.58 (m, 1 h). Ms (esi) m / z 450.18 (m + h). Prepared from 19d in 34% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 8.35 (s, 1 h), 4.19 (s, 1 h), 3.252.95 (m, 9 h), 2.802.25 (m, 5 h), 1.801.63 (m, 1 h). Ms (esi) m / z 430.46 (m + h). Prepared from 19e in 24% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 8.53 (s, 1 h), 7.62 (s, 5 h), 4.18 (s, 1 h), 3.142.82 (m, 9 h), 2.812.66 (m, 1 h), 2.242.14 (m, 1 h), 1.661.52 (m, 1 h). Ms (esi) m / z 492.48 (m + h). Prepared from 19f in 96% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 8.22 (s, 1 h), 4.19 (s, 1 h), 3.383.30 (m, 2 h), 3.24 (s, 6 h), 3.122.95 (m, 7 h), 2.60 (t, j = 14.2 hz, 1 h), 2.422.34 (m, 1 h), 1.751.63 (m, 1 h). Ms (esi) m / z 459.50 (m + h). Prepared from 19 g in 51% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 7.80 (s, 1 h), 4.15 (s, 1 h), 3.97 (s, 3 h), 3.402.98 (m, 9 h), 2.322.20 (m, 2 h), 1.721.56 (m, 1 h). Ms (esi) m / z 446.39 (m + h). Prepared from 19i in 68% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 7.81 (s, 1 h), 4.17 (s, 1 h), 3.262.96 (m, 9 h), 2.422.25 (m, 2 h), 1.701.58 (m, 1 h). Ms (esi) m / z 434.27 (m + h). Prepared from 19j in 68% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.19 (s, 1 h), 3.202.98 (m, 8 h), 2.352.22 (m, 2 h), 1.681.56 (m, 1 h). Ms (esi) m / z 465.32 (m + h). Prepared from 19k in 49% yield, 2 steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.20 (s, 1 h), 3.402.90 (m, 9 h), 2.422.22 (m, 2 h), 1.701.54 (m, 1 h). Compound 19j (68.3 mg, 0.080 mmol) was stirred in 4 m hcl in 1,4-dioxane for 2 h. the reaction mixture was concentrated under reduced pressure . The material was dissolved in thf (2 ml), and bromoacetylchloride (12.5 mg, 0.080 mmol) was added . After stirring overnight, an additional portion of bromoacetylchloride (0.020 ml) was added . T - butylamine (0.063 ml, 0.60 mmol) was added to a solution of the intermediate (35 mg, 0.040 mmol) in thf (1 ml), and the reaction mixture was heated to 50 c . After stirring overnight, the reaction mixture was concentrated under reduced pressure and was purified by preparative hplc (sunfire prep c18 column, 50100% b gradient). This gave 16.8 mg (49%) of the title compound as a yellow solid . H nmr (400 mhz, cdcl3) 8.20 (br s, 1 h), 7.527.46 (m, 2 h), 7.437.30 (m, 8 h), 5.36 (s, 2 h), 4.95 (dd, j = 40.6 hz, j = 11.0 hz, 2 h), 3.88 (d, j = 11.0 hz, 1 h), 3.323.24 (m, 1 h), 3.123.03 (m, 1 h), 2.662.59 (m, 1 h), 2.582.42 (m, 8 h), 2.18 (d, j = 14.6 hz, 1 h), 1.301.10 (br s, 9 h), 0.82 (s, 9 h), 0.27 (s, 3 h), 0.12 (s, 3 h). Ms (esi) m / z 870.60 (m + h). Aqueous hf (48%, 0.4 ml) was added to a solution of the preceding intermediate (16.8 mg, 0.019 mmol) in ch3cn (0.6 ml) in a plastic vial . After 18 h, the reaction mixture was poured into a solution of k2hpo4 (7.8 g) in water (15 ml). Sodium chloride (10 g) was added to the aqueous layer, and this was extracted with etoac (2). The combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was dissolved in meoh (1 ml) and 1,4-dioxane (1 ml), and palladium on carbon (degussa, 10 wt%, 5 mg) was added . An atmosphere of hydrogen was introduced, and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure . The material was purified by preparative hplc (phenomenex polymerx 10 rp 100a column, 0100% b gradient). Fractions with the desired mw were collected and freeze - dried to yield 1.2 mg (10%, 2 steps) of the title compound as a yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.304.20 (s, 2 h), 4.19 (s, 1 h), 3.252.96 (m, 9 h), 2.482.28 (m, 2 h), 1.721.58 (m, 1 h), 1.43 (s, 9 h). The following compounds were prepared according to methods similar to 21a: prepared from 19k in 68% yield for the acylation / amine displacement and 58% yield for the deprotections, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.244.65 (m, 3 h), 3.262.97 (m, 9 h), 2.382.28 (m, 2 h), 1.691.56 (m, 1 h), 1.44 (s, 9 h). Ms (esi) m / z 562.37 (m + h). Prepared from 19l in 43% yield for the acylation / amine displacement and 55% yield for the deprotections, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.304.40 (m, 3 h), 3.403.25 (m, 8 h), 3.123.04 (m, 4 h), 2.99 (s, 3 h), 2.622.52 (m, 1 h), 2.412.35 (m, 1 h), 1.731.60 (m, 1 h), 1.45 (s, 9 h). Compound 19h (50 mg, 0.061 mmol), k3po4 (39 mg, 0.18 mmol), tris(dibenzylideneacetone)dipalladium (2.8 mg, 0.003 mmol), and xantphos (5.1 mg, 0.009 mmol) were added to a small vial equipped with a septum . After the flask was evacuated and flushed with nitrogen (3), 1,4-dioxane (0.5 ml) and ethylamine (2.0 m solution in thf, 0.091 ml, 0.18 mmol) were added . The reaction mixture was heated to 100 c and stirred for 3 h. the reaction mixture was allowed to cool to room temperature and was filtered through celite . The material was purified by preparative hplc (sunfire prep c18 column, 90100% b gradient). Fractions with the desired mw were collected and freeze - dried to yield 11 mg (23%) of the title compound as a yellow solid . H nmr (400 mhz, cdcl3) 15.6 (br, 1 h), 7.567.49 (m, 4 h), 7.427.30 (m, 6 h), 5.37 (s, 2 h), 5.03 (s, 2 h), 3.89 (d, j = 11.0 hz, 1 h), 3.243.04 (m, 3 h), 2.97 (dd, j = 15.6, 4.9 hz, 1 h), 2.642.43 (m, 3 h), 2.49 (s, 6 h), 2.14 (d, j = 15.6 hz, 1 h), 1.25 (t, j = 6.8 hz, 3 h), 0.81 (s, 9 h), 0.25 (s, 3 h), 0.11 (s, 3 h). Ms (esi) m / z 785.57 (m + h). Aqueous hf (48%, 0.3 ml) was added to a solution of the preceding intermediate (11 mg, 0.014 mmol) in ch3cn (7 ml) in a plastic vial . After 18 h, the reaction mixture was poured into a solution of k2hpo4 (2 g) in water (10 ml). The material was dissolved in meoh (2 ml) and 1,4-dioxane (2 ml), and palladium on carbon (degussa, 10 wt%, 5.6 mg) was added . An atmosphere of hydrogen was introduced, and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure . The material was purified by preparative hplc (phenomenex polymerx 10 rp 100a column, 0100% b gradient). Fractions with the desired mw were collected and freeze - dried to yield 3.2 mg (46%, 2 steps) of the title compound as a yellow solid . H nmr (400 mhz, cd3od) 4.08 (s, 1 h), 3.43 (q, j = 7.4 hz, 2 h), 3.082.92 (m, 9 h), 2.302.15 (m, 2 h), 1.671.55 (m, 1 h), 1.22 (t, j = 7.4 hz, 3h). Ms (esi) m / z 493.24 (m + h). The following compounds were prepared by similar methods to 24a: prepared from 19h and methylamine (1.0 m solution in thf) in 21% yield for the amination step, 19% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od) 4.08 (s, 1 h), 3.41 (s, 3 h), 3.082.92 (m, 9 h), 2.302.15 (m, 2 h), 1.671.55 (m, 1 h). Ms (esi) m / z 479.22 (m + h). Prepared from 19h and n - propylamine in 24% yield for the amination step, 40% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.16 (s, 1h), 3.47 (t, j = 7.3 hz, 2 h), 3.152.96 (m, 9 h), 2.322.20 (m, 2 h), 1.781.55 (m, 3 h), 1.01 (t, j = 7.4 hz, 3 h). Ms (esi) m / z 507.29 (m + h). Prepared from 19h and 2-propylamine in 11% yield for the amination step, 16% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od) 4.214.13 (m, 1 h), 4.08 (s, 1 h), 3.142.92 (m, 9 h), 2.312.15 (m, 2 h), 1.671.56 (m, 1 h), 1.23 (dd, j = 6.4, 1.8 hz, 6 h). Ms (esi) m / z 507.24 (m + h). Prepared from 19h and 2-methylpropan-1-amine in 15% yield for the amination step, 57% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.18 (s, 1h), 3.38 (d, j = 7.3 hz, 2 h), 3.202.96 (m, 9 h), 2.342.20 (m, 2 h), 2.101.98 (m, 1h), 1.681.54 (m, 1 h), 1.00 (d, j = 6.4 hz, 6 h). Ms (esi) m / z 521.40 (m + h). Prepared from 19h and 3-methyl - butan-1-amine in 25% yield for the amination step, 66% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od) 4.09 (s, 1 h), 3.453.40 (m, 2 h), 3.20 (s, 1 h), 3.082.93 (m, 9 h), 2.302.16 (m, 2 h), 1.721.58 (m, 2 h), 1.551.48 (m, 2 h), 0.96 (d, j = 6.4 hz, 6 h). Ms (esi) m / z 535.30 (m + h). Prepared from 19h and 2-methoxyethylamine in 19% yield for the amination step, 44% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.13 (s, 1h), 3.723.60 (m, 4h), 3.40 (s, 3 h), 3.122.95 (m, 9 h), 2.322.21 (m, 2 h), 1.691.56 (m, 1 h). Ms (esi) m / z 523.32 (m + h). Prepared from 19h and 2-(propan-2-yloxy)ethan-1-amine in 9% yield for the amination step, 30% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.19 (s, 1h), 3.823.69 (m, 5h), 3.202.98 (m, 9 h), 2.352.22 (m, 2 h), 1.681.56 (m, 1 h), 1.18 (d, j = 6.4 hz, 6h). Ms (esi) m / z 551.41 (m + h). Prepared from 19h and (3-aminopropyl)dimethylamine in 9% yield for the amination step, 21% for the 2 deprotection steps, yellow solid . H nmr (400 mhz, cd3od) 4.09 (s, 1 h), 3.703.46 (m, 4 h), 3.102.90 (m, 15 h), 2.312.17 (m, 2 h), 2.102.00 (m, 2 h), 1.681.56 (m, 1 h). Compound 16d (100 mg, 0.24 mmol), cs2co3 (235 mg, 0.720 mmol), tris(dibenzylideneacetone)dipalladium (11 mg, 0.012 mmol), and xantphos (20 mg, 0.036 mmol) were weighed into a flask . This was evacuated and backflushed with nitrogen (3), and 1,4-dioxane (0.5 ml) and n, n - dimethylneopentanediamine (0.057 ml, 0.36 mmol) were added . After 1.5 h, the reaction mixture was cooled to room temperature and was filtered through celite . The filtrate was concentrated under reduced pressure and was purified by column chromatography (biotage 10 g column, 03% meoh in ch2cl2 gradient), yielding 76.9 mg (69%) of the intermediate . Lhmds (1.0 m in thf, 0.22 ml, 0.22 mmol) was added dropwise to a 78 c solution of the above intermediate (93 mg, 0.20 mmol) in thf (5 ml). After 5 min, benzyl chloroformate (0.084 ml, 0.60 mmol) after 15 min, the reaction mixture was quenched by the addition of nh4cl (saturated, aqueous solution, 15 ml) and was extracted with etoac . The material was purified by column chromatography (biotage 10 g column, 07% meoh in ch2cl2 gradient), yielding 111 mg (93%) of the product . Nmr (400 mhz, cdcl3) 7.457.20 (m, 13 h), 7.066.98 (m, 2 h), 5.16 (s, 2 h), 4.87 (s, 2 h), 3.78 (br s, 2 h), 2.38 (s, 3 h), 2.18 (br s, 6 h), 1.62 (br s, 2 h), 0.90 (br s, 6 h). Ms (esi) m / z 600.28 (m + h). A solution of compound 22a (54 mg, 0.090 mmol) in thf (0.5 ml) was added dropwise to a 78 c solution of lda (2.0 m solution in thf, 0.112 ml, 0.224 mmol) and tmeda (0.081 ml, 0.54 mmol) in thf (2 ml), resulting in an orange - colored solution . After 10 min, a solution of 8 (43 mg, 0.090 mmol) in thf (0.5 ml) was added dropwise over 30 s. after complete addition, the reaction mixture was allowed to warm to 10 c over 1 h. the reaction was quenched by the addition of ammonium chloride (saturated, aqueous solution), was diluted with water, and was extracted with etoac (2). The combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was purified by preparative hplc (sunfire prep c18 column, 20100% b gradient), yielding 22.9 mg (26%) of the desired product as a yellow solid . H nmr (400 mhz, cdcl3) 15.55 (br s, 1 h), 7.527.20 (m, 15 h), 5.36 (s, 2 h), 5.205.00 (m, 2 h), 4.80 (s, 2 h), 3.90 (d, j = 11.0 hz, 1 h), 3.193.05 (m, 2 h), 2.622.57 (m, 1 h), 2.562.12 (m, 19 h), 0.940.74 (m, 15 h), 0.26 (s, 3 h), 0.12 (s, 3 h). Ms (esi) m / z 988.59 (m + h). Aqueous hf (48%, 0.4 ml) was added to a solution of 23a (22.9 mg, 0.0232 mmol) in ch3cn (0.8 ml) in a plastic vial . After 18 h, the reaction mixture was poured into a solution of k2hpo4 (4.8 g) in water . The mixture was extracted with etoac, and the combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was dissolved in meoh (1 ml), 0.5 m hcl in meoh (0.2 ml), and 1,4-dioxane (1 ml), and palladium on carbon (degussa, 10 wt%, 2 mg) was added . An atmosphere of hydrogen was introduced, and the reaction mixture was stirred for 3 h. the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure . The material was purified by preparative hplc (phenomenex polymerx column, 0100% b gradient). Fractions with the desired mw were collected and freeze - dried to yield 9.9 mg (67%, 2 steps) of the desired product as a yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.16 (s, 1 h), 3.43 (s, 2 h), 3.152.90 (m, 17 h), 2.302.22 (m, 1 h), 2.222.12 (m, 1 h), 1.651.54 (m, 1 h), 1.17, (s, 6 h). The following compounds were prepared according to methods similar to 24j: prepared from 16a and n, n - dimethylethylenediamine . H nmr (400 mhz, cd3od) 4.13 (s, 1 h), 3.843.77 (m, 2 h), 3.443.38 (m, 2 h), 3.102.92 (m, 15 h), 2.322.20 (m, 2 h), 1.681.55 (m, 1 h). Ms (esi) m / z 536.25 (m + h). Prepared from 16a and n, n - dimethylneopentanediamine . H nmr (400 mhz, cd3od) 4.10 (s, 1 h), 3.513.36 (m, 4 h), 3.102.92 (m, 15 h), 2.332.18 (m, 2 h), 1.681.57 (m, 1 h), 1.174 (s, 3 h), 1.169 (s, 3 h). Ms (esi) m / z 578.35 (m + h). Prepared from 16a and 2,2-dimethyl-3-(pyrrolidin-1-yl)propan-1-amine . H nmr (400 mhz, cd3od) 4.09 (s, 1 h), 3.883.78 (m, 2 h), 3.503.39 (m, 2 h), 3.18 2.92 (m, 9 h), 2.322.08 (m, 6 h), 1.671.56 (m, 1 h), 1.15 (s, 6 h). Ms (esi) m / z 604.29 (m + h). Prepared from 16d and n - propylamine . H nmr (400 mhz, cd3od) 4.09 (s, 1 h), 3.33 (t, j = 6.9 hz, 2 h) 3.423.38 (m, 2 h), 3.20 (s, 1 h), 3.072.94 (m, 8 h), 2.91 (dd, j = 14.6, 4.6 hz, 1 h), 2.232.12 (m, 2 h), 1.681.55 (m, 3 h), 0.96 (t, j = 7.3 hz, 3 h). Copper(i) iodide (4.7 mg, 0.025 mmol) and k3po4 (209 mg, 0.984 mmol) were added to a vial equipped with a septum . After the vial was evacuated and flushed with nitrogen (3), a solution of compound 16e (217 mg, 0.492 mmol) in dmf (2 ml), 1-propylamine (0.162 ml, 1.97 mmol), and 2-acetylcyclohexanone (0.013 ml, 0.098 mmol) were added . The reaction mixture was heated to 100 c for 3 h. the reaction mixture was allowed to cool to room temperature, was diluted with etoac (50 ml), and was washed with water (3 40 ml). The material was purified by column chromatography (biotage 10 g column, 012% etoac in hexanes gradient), yielding 74.6 mg (36%) of the title compound as a thick oil . H nmr (400 mhz, cdcl3) 7.507.46 (m, 2 h), 7.417.30 (m, 5 h), 7.307.24 (m, 1 h), 7.087.02 (m, 2 h), 5.08 (s, 2 h), 2.85 (s, 6 h), 2.32 (s, 3 h). Ms (esi) m / z 420.34 (m + h). Lhmds (1.0 m solution in thf, 0.167 ml, 0.167 mmol) was added to a solution of phenyl 5-benzyloxy-2-(dimethylamino)-3-methyl-6-(propylamino)isonicotinate (63.5 mg, 0.152 mmol) in thf (2 ml), resulting in a bright - yellow solution . A solution of di - t - butyldicarbonate (99.5 mg, 0.456 mmol) in thf (1 ml) was added . After 10 min, the reaction was quenched by the addition of ammonium chloride (saturated, aqueous solution) and was extracted with etoac (2). The material was purified by column chromatography (biotage 10 g column, 012% etoac in hexanes gradient), yielding 13.6 mg (17%) of the product . H nmr (400 mhz, cdcl3) 7.407.28 (m, 7 h), 7.287.22 (m, 1 h), 7.057.00 (m, 2 h), 4.90 (s, 2 h), 2.79 (s, 6 h), 2.34 (s, 3 h), 1.761.66 (m, 2 h), 1.561.50 (m, 2 h), 0.91 (t, j = 7.56 hz, 3 h). Ms (esi) m / z 520.38 (m + h). A solution of 22b (24 mg, 0.047 mmol) in thf (0.5 ml) was added dropwise to a 78 c solution of lda (0.5 m solution in thf, 0.102 ml, 0.051 mmol) and tmeda (0.041 ml, 0.27 mmol) in thf (2 ml), resulting in a yellowishorange colored solution . After 10 min, a solution of 8 (16.4 mg, 0.034 mmol) in thf (0.5 ml) was added dropwise . After complete addition, the reaction mixture was allowed to warm to 20 c over 45 min . The reaction was quenched by the addition of ammonium chloride (saturated, aqueous solution) and was extracted with etoac (2). The combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was purified by preparative hplc (sunfire prep c18 column, 90100% b gradient), yielding 6.1 mg (20%) of the desired product as a yellow solid . H nmr (400 mhz, cdcl3) 15.56 (s, 1 h), 7.527.24 (m, 10 h), 5.36 (s, 2 h), 4.994.64 (m, 2 h), 4.02 (d, j = 11.0 hz, 1 h), 3.102.81 (m, 2 h), 2.78 (s, 6 h), 2.622.40 (m, 10 h), 2.202.14 (m, 1 h), 1.801.45 (m, 2 h), 1.441.15 (m, 11 h), 1.000.70 (m, 12 h), 0.25 (s, 3 h), 0.12 (s, 3 h). Ms (esi) m / z 908.65 (m + h). Aqueous hf (48%, 0.4 ml) was added to a solution of 20o (6.1 mg, 0.007 mmol) in ch3cn (0.6 ml) in a plastic vial . After 18 h, the reaction mixture was poured into a solution of k2hpo4 (4.8 g) in water (15 ml). The combined etoac extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was dissolved in meoh (1 ml) and 1,4-dioxane (1 ml), and palladium on carbon (degussa, 10 wt%, 2 mg) was added . An atmosphere of hydrogen was introduced, and the reaction mixture was stirred for 4 h. the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure . . Fractions with the desired mw were collected and freeze - dried to yield 2.8 mg (70%, 2 steps) of the desired product as a yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.19 (s, 1 h), 3.44 (t, j = 7.6 hz, 2 h), 3.243.12 (m, 7 h), 3.122.97 (m, 8 h), 2.392.26 (m, 2 h), 1.731.56 (m, 3 h), 0.98 (t, j = 8.4 hz, 3 h); ms (esi) m / z 516.42 (m + h). Compound 19 m (105 mg, 0.126 mmol), tetrakis(triphenylphosphine)palladium(0) (15 mg, 0.013 mmol), and k3po4 (80 mg, 0.38 mmol) were weighed into an 8 ml vial . This was sealed with a septum and was evacuated and backflushed with nitrogen (3). Toluene (1 ml), 1,4-dioxane (1 ml), and water (0.5 ml) were added, followed by allylboronic acid pinacol ester (63.7 mg, 0.379 mmol). After 2.5 h, the reaction mixture was cooled to room temperature and was concentrated under reduced pressure . The material was purified by preparative hplc (sunfire prep c18 column, 90100% b gradient), yielding 28.6 mg (29%) of the title compound as a yellow solid . Aqueous hf (48%, 0.4 ml) was added to a solution of 25a (28.6 mg, 0.036 mmol) in 1,4-dioxane (1 ml) in a plastic vial . After 18 h, the reaction mixture was poured into a solution of k2hpo4 (4.8 g) in water . The mixture was extracted with etoac, and the combined extracts were dried over na2so4, filtered, and concentrated under reduced pressure . The material was dissolved in meoh (1 ml), 0.5 m hcl in meoh (0.5 ml), and 1,4-dioxane (1 ml), and palladium on carbon (degussa, 10 wt%, 5 mg) was added . An atmosphere of hydrogen was introduced, and the reaction mixture was stirred for 1 h. the reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure . The material was purified by preparative hplc (phenomenex polymerx column, 070% b gradient). Fractions with the desired mw were collected and freeze - dried to yield 14.3 mg (69%) of the title compound as a yellow solid . H nmr (400 mhz, cd3od with 1 drop dcl) 4.20 (s, 1 h), 3.383.15 (m, 7 h), 3.142.94 (m, 8 h), 2.912.82 (m, 2 h), 2.56 (t, j = 13.8 hz, 1 h), 2.402.32 (m, 1 h), 1.861.76 (m, 2 h), 1.741.62 (m, 1 h), 1.01 (t, j = 7.34 hz, 3 h). The following compounds were prepared according to methods similar to 26a: prepared from 19h and allylboronic acid pinacol ester, yellow solid . H nmr (400 mhz, cd3od) 4.10 (s, 1 h), 3.222.92 (m, 9 h), 2.812.74 (m, 2 h), 2.492.38 (m, 1 h), 2.262.19 (m, 1 h), 1.77 1.60 (m, 3 h), 0.97 (t, j = 7.3 hz, 3 h). Ms (esi) m / z 492.27 (m + h). Prepared from 19h and methylboronic acid, yellow solid . H nmr (400 mhz, cd3od) 4.09 (s, 1 h), 3.222.92 (m, 9 h), 2.85 (s, 3 h), 2.492.38 (m, 1 h), 2.262.19 (m, 1 h), 1.77 1.60 (m, 1h). Ms (esi) m / z 464.30 (m + h). Prepared from 19h as an over - reduced side product in the synthesis of 26c . H nmr (400 mhz, cd3od) 7.78 (s, 1 h), 4.12 (s, 1 h), 3.222.91 (m, 9 h), 2.78 (s, 3 h), 2.492.21 (m, 1 h), 1.771.62 (m, 1h). Ms (esi) m / z 430.46 (m + h). Prepared from 19h and phenylboronic acid . H nmr (400 mhz, cd3od) 8.047.98 (m, 2 h), 7.477.39 (m, 3 h), 4.08 (s, 1 h), 3.142.92 (m, 9 h), 2.572.47 (m, 1 h), 2.282.07 (m, 1 h), 1.731.65 (m, 1h). Ms (esi) m / z 526.26 (m + h). Prepared from 19h as an over - reduced side product in the synthesis of 26e . H nmr (400 mhz, cd3od) 8.047.98 (m, 2 h), 7.477.39 (m, 3 h), 4.08 (s, 1 h), 3.142.92 (m, 9 h), 2.572.47 (m, 1 h), 2.282.07 (m, 1 h), 1.731.65 (m, 1h). Tetracycline - susceptible isolates sa100 (s. aureus atcc 13709, smith), sa101 (s. aureus atcc 29213), sp106 (s. pneumoniae atcc 49619), ef103 (e. faecalis atcc 29212), ec107 (e. coli atcc 25922), kp109 (k. pneumoniae atcc 13883), ab110 (acinetobacter baumannii atcc 19606), ec108 (enterobacter cloacae atcc 13047), and pa111 (pseudomonas aeruginosa atcc 27853) were obtained from the american type culture collection (atcc, manassas, va). Tetracycline - resistant isolates s. aureus sa158 (tet(k)), s. pneumoniae sp160 (tet(m)), e. faecalis ef159 (tet(m)), e. coli ec155 (tet(a)), k. pneumoniae kp153 (tet(a)) were obtained from marilyn roberts lab at the university of washington . S. aureus sa161 (tet(m)) was obtained from micromyx (kalamazoo, mi). Minimal inhibitory concentration (mic) determinations were performed in liquid medium in 96-well microtiter plates according to the methods described by the clinical and laboratory standards institute (clsi). (26) cation - adjusted mueller hinton broth was obtained from bbl (cat . 212322, becton dickinson, sparks, md), prepared fresh and kept at 4 c prior to testing . A0432, pml microbiologicals, wilsonville, or) was used to supplement medium, as appropriate . All test methods met acceptable standards based on recommended quality control ranges for all comparator antibiotics and the appropriate atcc quality control strains . Compound stocks prepared and diluted in sterile deionized water were assayed for inhibition of coupled in vitro transcription / translation using an e. coli s30 extract system with a firefly luciferase readout from promega (cat . Briefly, compounds were diluted into water and added to reaction mix aliquoted to black - walled 96-well microtiter plates (cat . An appropriate three - point titration was used for each compound, and reactions were run in duplicate . Plates were incubated at 37 c for one hour and then placed on ice for 5 min to arrest transcription / translation . E1500) was added to each well and luminescence was detected on a bmg labtech lumistar - optima instrument . Positive assay control values, from reactions without inhibitor, were averaged per plate to determine percent inhibition of luciferase production . Results were plotted using microsoft excel and 50% inhibition values (ic50) were determined.
Humans are infected naturally by 3 species of hookworms; necator americanus, ancylostoma duodenale, and ancylostoma ceylanicum . Individual people can be infected with 1 species, 2 species, or all 3 species of hookworms [1 - 5]. Although a. ceylanicum is often considered to be a zoonotic pathogen, since it is also found in dogs and cats, its importance as a significant soil - transmitted helminth of humans is increasingly being recognized . Two haplotypes of a. ceylanicum have been identified; one largely found in humans and another that infects humans, dogs, and cats . A. ceylanicum is considered to be of emerging importance as a helminth of humans in the asia pacific region . A fourth hookworm also reported from humans, ancylostoma caninum represents a canine species with zoonotic potential . This species has mainly been recorded as causing eosinophilic enteritis in people in tropical australia, but never establishes a patent infection and hence humans are regarded as accidental hosts . Human hookworm is common in the solomon islands, with an estimated 192,000 people (36% of the population) infected, but the different species involved are yet to be determined [9 - 11]. The report to the rockefeller hookworm campaign in 1928 described only n. americanus as being present on these islands . The only previous report of a. ceylanicum from the solomon islands was a 5-year - old boy who imported the species in 1938 from the shortland island group of the solomon islands to australia . The solomon islands is a small pacific nation to the north east of australia, with a predominantly melanesian population . The regional assistance mission to solomon islands (ramsi) occurred between 2003 and 2013, at the request of the solomon island s government, to assist in stabilizing internal troubles occurring within the nation . Ramsi consisted of 2,200 police and soldiers from many pacific island nations and was led by australia . This case report describes the finding of a. ceylanicum in an australian soldier who developed symptomatic disease after returning from a deployment in solomon islands with ramsi . The patient was a 26-year - old male living in townsville, north queensland, australia who presented in march 2004 with central poorly defined abdominal pain of 4 weeks duration and increased frequency of defecation . A peripheral eosinophilia had been noted on blood tests in february prior to symptom development, and in march, when eosinophils were 6.2010/l whilst red blood cell indices were normal (hemoglobin 146 g / l, mcv 93 fl, hematocrit 0.44, red cell count 4.710/l). The patient had served with ramsi in the solomon islands from late july to early december 2003 . His duties involved travel to various remote areas and provided multiple opportunities to become infected with hookworms . Routine treatment with albendazole had been given by the australian defense force on return to townsville in december, 2 months prior to development of symptoms . Initially, in march 2004 no parasite eggs were detected in feces by formalin - ethyl acetate concentration, but infective larvae of hookworms were found on harada - mori culture . Infective larvae were sheathed, had inconspicuous buccal spears, the intestine was narrower than the esophageal bulb, and inconspicuous transverse striations were noted on the sheath in the tail region . The infective larvae, measured after preservation in hot formalin, had the following dimensions, meansd (range): length 657.19.6 m (633 - 669 m), width 21.40.9 m (20.2 - 22.2 m), esophagus 156.73.5 m (151 - 162 m), tail 77.93.8 m (69 - 81 m), length of sheath 763.78.6 m (779 - 746 m); esophagus / length 23.80.5%, and tail / length 11.90.6% . Due to the failure of single dose post - deployment albendazole therapy to eradicate this infection, the patient was treated with 100 mg of mebendazole twice daily for 3 days . Twelve adult hookworms (6 females and 6 males) were recovered by dissection of stools passed during the 48 hr immediately following treatment . The en face view revealed a robust ventral cutting plate bilaterally with a prominent point on the dorsal end and less obvious point on the ventral end (fig . The width of the bursa in lateral view was greater than its length, and the mediolateral and posteriolateral bursal rays were parallel (fig . The morphology of the adult hookworms was consistent with a. ceylanicum with key features being: the large ventral cutting plates terminating at the dorsal end in a large single tooth; and in the male the parallel mediolateral and posteriolateral bursal rays . Although the morphology and dimensions of the infective larvae are more consistent with a. ceylanicum than n. americanus, differentiating them from infective larvae of a. duodenale is unreliable . Although molecular taxonomy is valuable in identifying hookworm species, it was not available in this case and, as the specimen was subsequently accidentally disposed of, molecular taxonomic studies cannot be performed now . However, the morphological features were sufficient to identify this species as a. ceylanicum . This represents only the second report of human a. ceylanicum infection having been acquired in this nation, with the first being made almost 80 years ago . This finding not only expands the known range of human a. ceylanicum infection but also raises the question of whether a. ceylanicum is in fact a common cause of human hookworm infection in some regions of the solomon islands, as it has recently been described as being in many parts of asia . The only previous reported description of hookworm species in the country was almost 80 years ago and was anecdotal, not providing descriptions of number of people tested or how the species identifications were arrived at . A. ceylanicum infection has recently been reported from two people in western australia . Prior to this, a. ceylanicum had been reported from dogs and cats in northern australia, including those from townsville . Hence, the infection could have been acquired in north queensland or while deployed with ramsi . The likelihood of infection with ancylostoma from humans in the solomon islands is high, but negligible in north queensland since human feces are disposed of safely . However, since a. ceylanicum is present in dogs and cats in north queensland, there is a possibility of a zoonotic australian source even though this infection has not been seen in humans in this region . Many ramsi personnel were infected with hookworms during their mission to the solomon islands, and an isolated case of a. ceylanicum infection had been reported from this area early in the 20th century . This adds to the likelihood that the case presented here was acquired in the solomon islands and not elsewhere . The clinical symptoms displayed by our patient were consistent with the phase of hookworm infection during which the host attempts to eliminate the parasite using an allergic response, manifesting morphologically as eosinophilic enteritis . For n. americanus this response begins about 10 days after infection and persists for up to a month . A. caninum and a. duodenale have a more complicated life cycle in which some infective larvae can enter a state of hypobiosis (arrested development) and can orchestrate entry to the intestine to coincide with the wet season and physiological events [23 - 26]. Whether a. ceylanicum has the failure of a routine post - deployment albendazole treatment to eliminate the infection in our patient supports a hypothesis that the current infection was due to hypobiotic larvae activated after the anthelmintic was given . Acute enteritis has been described previously in soldiers with a. ceylanicum acquired in west papua (formerly dutch new guinea) and in a traveller returning from myanmar to france . Treatment with albendazole in the latter patient cleared the gut infection but symptoms and parasites recurred after 3 months adding evidence for hypobiosis in a. ceylanicum . Our patient was treated after diagnosis with mebendazole due to its far greater tissue penetration and therefore greater capacity to kill hypobiotic larvae in the tissue . The key points from this case report are: a. ceylanicum should be considered as a cause of acute eosinophilic enteritis in returned travellers, workers, or defense force personnel who have lived or visited tropical and subtropical countries; laboratory confirmation may require persistence to make the diagnosis of hookworms and will require special tests (morphology of adults after treatment; molecular taxonomy on stools or infective larvae) to identify the species; and human infection with a. ceylanicum is probably endemic in the solomon islands and further study of the prevalence of different hookworm species present in this country is warranted.
Verruciform xanthoma (vx), which has almost similar histologic features, is also a rare lesion usually found on the oral mucosa or the genital area . It is presumably associated with the inflammatory response to mucosal damage.1 however, xanthoma and vx of the esophagus are extremely rare . Since the first report by remmele and engelsing2 only 13 cases of esophageal xanthoma have been reported,1,2,3,4,5,6,7,8,9 and since the report by herrera - goepfert et al.,10 only four cases of vx of the esophagus have been reported.10,11,12,13 the etiologies of both lesions are not understood . We describe herein a new case, including a review of all reported cases of xanthoma and vx of the esophagus . A 70-year - old man with an unremarkable medical history was hospitalized with a complaint of epigastric pain . Serum total cholesterol, triglyceride, high density lipoprotein cholesterol, and low density lipoprotein cholesterol levels were 151, 215, 33, and 102 mg / dl, respectively . Multiple shallow gastric ulcers and a duodenal ulcer were detected and suspected to be the cause of the pain . Besides the ulcers, in the upper esophagus 20 cm from the incisors, a 3-mm yellowish granular elevated mucosal lesion was found and a biopsy was performed (fig . 1). Microscopically, large round cells were aggregated in the lamina propria immediately beneath the squamous epithelium . The etiologies are different, as xanthoma is caused by hyperlipidemia and vx arises presumably as a result of an inflammatory response to continuous mucosal damage.1 however, the etiologies of the two lesions arising in the esophagus are not understood . The characteristics of all reported cases of xanthoma and vx of the esophagus are summarized in table 1 . Fourteen cases of xanthoma and four cases of vx of the esophagus have been reported . However, some reports loosely stratified vx into esophageal xanthoma, whereas others have excluded it.6,8 in terms of clinical data, both diseases were found predominantly in men than in women: 9 versus 3 in xanthoma and 3 versus 1 in vx . The median age was 59 years (range, 37 to 74) in xanthoma and . The predominant location was the lower esophagus for xanthoma (lower, 7; middle, 2; upper, 3), whereas vx was not reported in the lower esophagus (upper, 2; middle, 2). The median size was not different: 3 mm (range, 2 to 10) for xanthoma and 4 mm (range, 3 to 20) for vx . The associated medical conditions were diverse; however, two patients with malignant tumors were included in each group: hepatocellular carcinoma and ileocecal lymphoma in xanthoma, and gastric cancer and multifocal cancer (cancer of the glottis, liver, and trachea) in vx, although there was no definite association . Vx is characterized by its histologic features, including papillomatosis, acanthosis, and hyperparakeratosis.11 also, the external morphology is verrucoid . Nevertheless, findings of large round foam cells in the lamina propria under the squamous epithelium are the same as those in xanthoma . It is difficult to differentiate between the two lesions on the basis of gross examination when they arise on the esophagus . Exophytic and verrucoid features seen in vx of the skin were not observed in the esophagus because most of the reported cases were small in size.10,12 considering that xanthoma and vx are nonneoplastic lesions, differentiating between them could be a waste of effort . However, these lesions have to be grossly distinguished from ectopic sebaceous glands and small subepithelial tumors such as carcinoid and granular cell tumor because most of the reported esophageal xanthomas are yellowish or white mucosal elevated lesions . In terms of microscopic findings, signet ring cell carcinoma, which contains round cells with abundant cytoplasm, while signet ring cell carcinoma has an eccentrically located nucleus because of the intracellularly abundant mucin, xanthoma has a centrally located and small nucleus . Positive immunohistochemical staining for cd68, which indicates a histiocytic origin, is another characteristic finding of xanthoma.5 moreover, esophageal cancer and ectopic sebaceous glands do not commonly stain with lugol's solution; thus, endoscopists need to be aware of these lesions for the differential diagnosis.6,14 with more case reports of esophageal xanthoma and vx of the esophagus, the characteristics of both lesions will be more clearly elucidated.
The sizing of pda for device implantation is based on the automatic calculations of dimension by the inbuilt sizing software in the cardiac catheterisation laboratory and many experienced interventionists believe their eyeball measurements more . The estimations can be erroneous if the calibrations are not proper, leading to catastrophic embolisation of the device, needing emergency surgical removal as was needed in our patient . Calibration by comparing with the fluoroscopic images of measured metallic sizing devices placed outside body can be misleading because of magnification errors.1 in order to circumvent these problems we suggest some novel ways which take care of magnification errors and avoid the need for aortic root angiography . An eight year child with echocardiographically documented 6 mm patent ductus arteriosus (pda) was planned for device closure . Femoral venous access was obtained using 8f sidearm sheath and the artery cannulated with a 6f sidearm sheath . A 6f pigtail catheter was advanced through the femoral arterial sheath into the descending aorta at the mouth of the pda and an angiogram obtained in a lateral projection delineating the ductus (figure 1). The minimum diameter at the aortic end was 6 mm and a 8 - 10 mm pda device was selected for deployment . A 7f swan - ganz catheter was advanced into the the pulmonary artery through venous route and a 0.35 " terumo wire advanced through the pda into the descending aorta .the terumo wire was exchanged with an 0.35 " extra stiff amplatzer wire and the swan - ganz catheter with the pda delivery system . An 8 - 10 mm size pda device was released across the ductus and after confirming the proper position by repeat angiogram which showed a small residual shunt flow which may be expected . Angiogram in lateral plane showing a small residual left to right shunt with pda device in place before unscrewing of the stylet after a short while, the device suddenly embolised into the left pulmonary artery (figure 2). The patient had a brief episode of mild hypotension which improved spontaneously and his oxygen saturations remained static . An attempt was made to retrieve the device percutaneously by a snare and later by a bioptome but the trial was given up as the device tended to go more distally, in which case even surgical removal would be difficult . The patient was discussed with the cardiac surgeon who shifted the patient for emergency surgery . After considerable debate surgeon decided for a left thoracotomy hoping to avoid cardiopulmonary bypass and closed the ductus . Later the left pulmonary artery was separated from the surrounding tissues and umbilical tape passed around it proximally for control of bleeding . A 1.5 cm longitudinal incision was made distal to the umbilical tape which was kept under traction and a long curved forceps passed toward the pulmonary hilum and the device retrieved . Cine image in lateral plane showing device embolized into left pulmonary artery soon after its release from stylet . Showing the pda device being retrieved from left pulmonary artery via left thoracotomy without cardiopulmonary bypass . In order to prevent such eventualities we tried to devise some alternative ways of sizing a pda .the first involves measuring a fully inflated tyshak pulmonary balloon outside the human body with a caliper and then inflating the balloon to the same pressure in the right ventricular outflow tract passed over a 0.35 " extra - stiff wire, and repeating the measurements, and using the same calibration factor for sizing of pda visualized by aortic arch angiogram . Previously, some radio opaque sizing devices have been used placing them outside human body which can have magnification errors if not placed exactly at the level of heart.2 the second method involves advancing a tyshak balloon 2 - 4 mm bigger than the echocardiographic dimension across the pda and inflating the balloon with dilute dye under low pressure without dilating the ductus so that it forms an impression of the ductus which is recorded in a cine mode in multiple planes, and the plane delineating the exact anatomy of the ductus is used for measurements after proper calibrations as mentioned above without the need for aortic arch angiogram (figures 4 and 5). Showing measurement of diameter of inflated tyshak balloon by a caliper after inflating it with fixed quantity of contrast . Angiogram in lateral plane showing an inflated tyshak balloon across the pda making an impression of the ductus . Technological advances have made nonsurgical closure of the pda a simple and a routine percutaneous intervention . The use of the amplatzer device occluder (ado) has further simplified the method and improved the results with minimal complications . However there are situations where complications are encountered and surgical help is required to ameliorate them . Faella and colleagues reported 15 procedure related complications in 316 patients which included hemolysis, left pulmonary artery artery stenosis, device protrusion into aorta causing coarctation, device misplacement and one death following device embolisation.2 late embolisation of the device to the left pulmonary artery has been reported with impaired left pulmonary perfusion six months after implantation requiring surgical removal . M vavuranakis et al reported severe hemolysis with jaundice, anemia and hemogloginura on the second day following deployment of smaller sized coil due to improper sizing which needed removal and replacement by an ado after repeat sizing using balloon tipped catheter.3 the complications can be reduced by proper expertise and optimal sizing of the ductus . Sizing of atrial septal defect (asd) by inflating a balloon across the asd till a circumferential waist is created and measuring the waist (stretched balloon diameter) and also inflating the balloon outside by same amount of dye across sized rings, is a standard method.45 using a similar method for the pda is not routine since angiographic visualization is usually adequate . However making an impression of the ductus by inflating a balloon across it can give a detailed anatomy about the length and breadth of the ductus in multiple planes without the need for multiple dye injection and may help in the selection of proper sized device.
Extrahepatic portal venous obstruction (ehpvo) is accompanied by replacement of the extrahepatic portal vein by a cavernoma with or without thrombosis of the intrahepatic portal, splenic, or superior mesenteric veins . In developing countries, ehpvo has been reported to be the most common cause of upper gastrointestinal bleeding (ugib) in children (70% in some reports) and is also a common cause of variceal bleeding in adults . In western countries, ehpvo is second only to cirrhosis as a cause of portal hypertension, but its relative incidence is much lower compared with that in the developing countries . Its aetiology is still not clear but has been attributed to umbilical sepsis after birth with thrombosis extending to the portal system via the patent umbilical vein or portal pyaemia following intra - abdominal sepsis . However, notwithstanding a lack of knowledge about its cause, most children and adults with ehpvo are generally from the so - called lower economic strata . However, the outcome after a bleed is better compared to bleeding in cirrhotics (if adequate blood replacement facilities are at hand), because patients with ehpvo have normal liver function (and histology) which helps them to sustain bleeding episodes without decompensation . However mortality rates of between 5 and 30% have been reported for a single bleeding episode because of the large volumes of blood lost in patients who do not have access to sophisticated medical facilities including blood transfusion . Till the middle of the 20th century, surgery was the only treatment available for these patients . However, with the advent of endoscopic therapy, this soon became the predominant treatment modality for the control of acute bleeding and also an important method for the prevention of a repeated bleeding episode . The main disadvantages of endotherapy are that it requires multiple sessions and a long - term followup with a recurrence rate of up to 40% in some studies . Because the prevalence of ehpvo is the highest in developing countries and the condition affects mainly the poor [2, 19], most of whom do not have access to blood transfusion facilities and are not treatment compliant, the benefits of using a less invasive procedure like endoscopic therapy must be weighed against surgery which, in the best centres carries an operative mortality of 1%, is a onetime treatment, is not associated with encephalopathy and followed by rebleeding rates of less than 10% . Moreover, operations like a splenectomy and proximal lienorenal shunt eliminate a large painful spleen and hypersplenism and restore a normal growth pattern in children . Thus, any new treatment for ehpvo must be compared with the results of shunt surgery which have stood the test of time especially if it has been performed by an experienced surgeon . A history of major upper gastrointestinal bleeding in a child who has oesophagogastric varices in endoscopy and normal liver function tests should raise the suspicion of extrahepatic portal venous obstruction . Although a similar picture can also be present in well compensated child a, cirrhosis ehpvo is much more common in children especially in the developing world . Investigations to confirm the diagnosis are usually simple and readily available at most centres . Massive splenomegaly is present in 90% of patients, and a complete blood count may reveal a low haemoglobin level and decreased total leukocyte and platelet counts due to hypersplenism in 50% . Liver function tests are nearly always normal, unlike in cirrhotics, but in the long - term the prothrombin time and albumin levels may be deranged due to the prolonged decreased portal blood flow and hence decreased synthetic function . Ultrasonic doppler (usg doppler) examination of the upper abdomen should be the first radiological investigation performed to confirm the diagnosis as it has a sensitivity of 7090% and a specificity of 99% in diagnosing ehpvo . The characteristic findings are the replacement of portal vein by multiple tortuous vessels, also known as cavernous transformation, with hepatopetal blood flow in the collaterals (figure 1). Upper gastrointestinal endoscopy (ugie) is done to confirm the presence of varices and to grade their size . Splenoportovenography by splenic puncture or selective coeliac / superior mesenteric angiography provides excellent imaging of the portal venous system but is invasive and has largely been replaced with computed tomography and magnetic resonance angiography . Ct arterial portography is highly accurate, not operator dependent, and useful in circumstances when bowel gas obscures the findings on ultrasound examination . However, its high cost, exposure to radiation, and the systemic toxicity of the contrast agents used are its main disadvantages (figure 2). Mr angiography is noninvasive and has a diagnostic accuracy that is similar to ct scans . However, it has limited availability and also has a high cost . With newer mr techniques, a clear portal venogram can be obtained and the direction and velocity of blood flow in the portal system can be determined . It should be done only if a patient presents with abnormal lfts or has hepatic dysfunction due possibly to coincident hepatitis following repeated blood transfusions for previous bleeding episodes . In spite of many years of familiarity with the disease, the causal factors have not been established . Umbilical sepsis at birth, portal pyaemia, and thrombosis, and so forth are rare clinical features . Larroche in his landmark paper in 1970 described portal vein thrombosis occurring after umbilical vein catheterization, but most of these resolved within short period of time and did not progress to ehpvo . However, there seems to be a strong association with levels of hygiene and poverty as the disease seems to affect the socially disadvantaged and has all but disappeared in most western countries and japan where it was more common in the late nineteenth century . There have been occasional reports of tests for dysmorphic megakaryocytes and endogenous erythroid colony assessment as being sensitive in distinguishing these patients from a normal population, but these are not readily available and therefore not commonly used . Tests for venous thromboembolism such as the presence of factor v leiden, protein c, s and antithrombin iii levels, and prothrombin gene mutation may also be positive in certain adult patients [23, 24] but have not had a high yield in the indian scenario . Variceal bleeding being the most common and most serious presentation of ehpvo, it remains the most common indication for treatment which should be tailored depending on the patient's social circumstances and his or her access to medical facilities . Before the 1980s, conservative medical management was recommended [2629] which included bed rest and blood transfusions as it was thought that these children would eventually grow out of their disease possibly by forming more collaterals to decompress the raised portal venous pressure . However, when it was realized that a single episode of variceal bleeding could carry a mortality rate of up to 30% in western countries, most physicians now follow a more active approach . In india, basu followed 25 patients with ehpvo for five years who had not had any intervention and reported that all had died . The main treatment options available today, as with other forms of variceal bleeding, are pharmacotherapy with beta blockers, endoscopy, and surgery . Prevention of a first episode of variceal bleeding with drugs has been well studied in adults with cirrhosis . In children with child a cirrhosis and portal hypertension, ozsoylu et al one of the most probable reasons may be that these patients usually present with bleeding as their first symptom (rather than splenomegaly), and hence primary prevention is difficult to study . Further studies are required to assess the role of drugs for primary prevention of variceal bleeding in ehpvo . Secondary prophylaxis with beta blockers has been shown to be effective in reducing presinusoidal portal hypertension in animals and humans, but their efficacy in preventing variceal bleeding in ehpvo has not been proved . Excellent results have been achieved using endoscopic therapy in the control of acute bleeding, and this has now become the therapeutic modality of choice in this situation with injection sclerotherapy and elastic band ligation being effective in 90% of cases . In those few patients who continue to bleed or are bleeding so massively that endoscopic control is not a possible surgical treatment in the form of portosystemic shunts or oesophagogastric devascularisation, it has been shown to be effective [3436] but carries mortality rates ranging from 1030% . Both cirrhotic and noncirrhotic children with portal hypertension found that 42% of patients with oesophageal varices who had no intervention presented with bleeding as compared to 24% patients in whom sclerotherapy was used for primary prevention . However, there was no difference in survival and patients with sclerotherapy had more bleeding episodes from ectopic sites after the procedure . But since few ehpvo patients in the developing world present before a bleeding episode, endoscopic therapy for primary prophylaxis has not become an established mode of treatment . Complete eradication of varices with endoscopic sclerotherapy (est) and variceal band ligation (evl) occurs in 8090% of patients with ehpvo . At present, endoscopic variceal eradication therapy is mainly indicated if it is used as a primary treatment modality, the vessels are too small for anastomosis, extensive thrombosis of the portal venous system which means there are no veins available for shunting, and in those patients who cannot tolerate the surgical procedure due to underlying comorbidities . It is used as a primary treatment modality, the vessels are too small for anastomosis, extensive thrombosis of the portal venous system which means there are no veins available for shunting, and in those patients who cannot tolerate the surgical procedure due to underlying comorbidities . However it must be remembered that endotherapy does not relieve the underlying portal hypertension and may result in an increased incidence of gastric and ectopic varices . Est is associated with higher rates of ulcer and stricture formation than evl which has the additional advantages of eradicating varices in fewer sessions . A randomized study by zargar et al . Has shown superiority of evl over est, but others have found that evl alone may be associated with increased risk of recurrence of varices (40% in one study). Poddar et al . In 2005 showed that evl + est compared to est alone was a better method in treatment of oesophageal varices in children with ehpvo because of fewer treatment sessions and fewer complications . Variceal recurrence rates were low in both the groups over a followup of 27 months (6.6% in evl + est group as compared to 10% in the est group). Further studies are, however, needed to establish the superior efficacy of evl + est over est or evl alone . However, est is still favoured over evl in many places due to its low cost . Zargar et al . Have shown 88% success with injection sclerotherapy in a followup of 15 yrs . Most of their patients had recurrent bleeding in the first 4 yrs after variceal eradication which was also managed by endoscopic therapy, and therefore the authors recommended annual endoscopy for the first 4 yrs after variceal eradication . In contrast, a similar study from king's college hospital in london, uk, had previously shown that after a mean followup of 8.7 yrs, recurrent bleeding occurred in 31% . We believe that it should be recommended as the treatment of choice for secondary prophylaxis in developing countries where the disease is more common and the accessibility to health care resources is poor . Surgical intervention in variceal bleeding in ehpvo is indicated if there isfailure of endoscopic management in acute variceal bleeding, bleeding not amenable to endoscopic treatment such as portal hypertensive gastropathy and ectopic varices, as a onetime treatment for secondary prophylaxis in those who have difficult access to specialized centresfor associated complications like portal biliopathy, growth retardation, hypersplenism, and massive splenomegaly leading to poor quality of life . Failure of endoscopic management in acute variceal bleeding, bleeding not amenable to endoscopic treatment such as portal hypertensive gastropathy and ectopic varices, as a onetime treatment for secondary prophylaxis in those who have difficult access to specialized centres for associated complications like portal biliopathy, growth retardation, hypersplenism, and massive splenomegaly leading to poor quality of life . These procedures divert blood flow from the high pressure portal circulation to low pressure systemic circulation by creation of an anastomosis between a tributary of the portal vein (splenic, superior mesenteric, and left gastric, left gastroepiploic) and a systemic vein (renal, inferior vena cava, and adrenal). The shunts may be selective (i.e., only decompressing the varices) or nonselective (decompressing the entire portal venous system). The main requirement for shunt procedure is the presence of a vessel free of thrombus and of sufficient size . For shunt to be effective and remain patent, it should be at least 10 mm in diameter although bismuth et al . And prasad et al . Have had patency rates ranging from 84 to 96% after anastomosing veins of down to 4 mm in diameter . These are the most commonly performed procedures and include proximal splenorenal (psrs), mesocaval, and portacaval shunts . The initial results with nonselective shunts were not promising since shunt thrombosis and rebleed and encephalopathy occurred in a large proportion of patients [29, 45]. But recent series have shown rebleed rates of 211%, a mortality rate of <2%, and no postshunt encephalopathy [9, 10]. Orloff et al . In 1994 found similar results with proximal side - side splenorenal shunts with or without splenectomy and end - side cavomesenteric shunts . They showed survival rates of> 96% after 10 years and shunt thrombosis rates of <2% over a 15 yr followup . It is advantageous in that, along with diversion of blood flow to decrease portal pressure and control bleeding, it also relieves the patient from symptomatic enlarged spleen and the effects of hypersplenism . Psrs has shown good long - term results . In a study by prasad et al ., the 15 yr survival was 95% in 160 patients of ehpvo treated with psrs with a rebleeding rate of 11% . The long - term outcomes with shunts are shown below (table 2). In a recent prospective randomized study by wani et al . In which the authors compared endoscopic sclerotherapy and shunt surgery revealed that rebleeding rates were significantly lower in the shunt surgery group (3.3% versus 22.6%). Treatment failure rates were also much less in the surgery group (6.7% versus 19.4%). A study by krishna et al . Evaluating qol after endoscopic or surgical treatment of ehpvo showed that endoscopic variceal eradication had no significant effect on qol, but the postsurgery group had improvement in physical, psychosocial, and total qol scores . The risks of overwhelming postsplenectomy infection have been described, but many studies including those from india and mexico have shown low rates of postsplenectomy sepsis [9, 48, 49]. Other disadvantage is rebleeding due to shunt thrombosis . Rates of shunt thrombosis vary from 416% [811], and rebleeding in these patients is usually easily controlled by endoscopic therapy . Surgical shunts in cirrhotics have been shown to be associated with neurological disturbances . In ehpvo, however this complication is rare, but there have been occasional reports of abnormal findings on electroencephalography, late cns side effects, and emotional disorders even after 20 yrs . Minimum hepatic encephalopathy (mhe) rates were higher in surgically shunted patients as compared to nonshunted children (but the difference was not significant). Shunt procedures, however, are not popular in the emergency setting because they are (i) time consuming, (ii) need technical expertise and associated with (iii) high rates of shunt thrombosis, and encephalopathy . However, the rate of shunt thrombosis depends on the experience of surgeon . A study by orloff et al . Showed shunt thrombosis rates of 0.5% in emergencies . Selective shunts aim to decompress only the gastrosplenic circulation leaving the blood flow to the liver intact, therefore, theoretically at least decreasing the rebleeding risk from oesophagogastric varices whilst preserving hepatopetal flow . The distal splenorenal shunt (warren shunt) is the most commonly performed selective shunt although other shunts also have been described like the left gastroepiploic to left renal vein and left gastric to left renal vein shunts . The patency rates were 92%, rebleeding rates of 12%, shunt dysfunction occurred in 25%, and encephalopathy in none of their patients . Such shunts cannot be performed in patients with thrombosis of the splenic vein or those who have a history of splenectomy . Superina et al . In a study of 34 patients showed that mesenterico left portal vein bypass (mlpvb) or rex shunt was successful in 91%, and they concluded that it was a more physiological shunt for ehpvo . But it requires the presence of a patent superior mesenteric vein, intrahepatic left portal vein, and internal jugular vein . These procedures are indicated if (i) performed as salvage therapy in variceal bleeding not controlled with endoscopic measures, (ii) a suitable size vein is not available for a shunt procedure, (iii) surgical expertise for a shunt procedure is not available . Splenectomy alone is not recommended since it does not decompress the portal circulation and also leads to thrombosis of splenic vein which thereafter cannot be used for shunting later . Mathur et al . Evaluated the role of oesophagogastric devascularisation with or without gastro - oesophageal stapling in acute variceal bleeding . In their study, 20 patients had ehpvo and the operative mortality was 5%; recurrent varices occurred in 5% with rebleeding in 11% . None of their patients had encephalopathy . In a retrospective study of 24 patients with ehpvo undergoing salvage surgery for variceal bleeding (13 had devascularisation procedures; 11 had proximal splenorenal shunts), they achieved control of bleeding in 96% . Retrospectively reported a four - year followup of 22 patients with noncirrhotic portal hypertension in whom the bleeding was not controlled with endoscopic therapy . In their study, the rebleeding rate was 10% and overall survival was 95% . Both endoscopy and shunt surgeries have shown good long - term results in secondary prophylaxis . However, treatment should take into account the socioeconomic status of the patient and facilities available locally . Splenectomy and proximal lienorenal shunt being a one - time procedure with, if performed by experienced surgeons, low mortality and occlusion rates and an absence of postprocedure encephalopathy should be considered as the main treatment in patients with difficult access to sophisticated medical facilities . The role of shunt surgery has expanded since it is also effective in correcting portal biliopathy, hypersplenism, and growth retardation and may improve the quality of life.
Cancer genomics has gone through a dramatic period of progress due to the availability of genome - wide technologies . (tcga, https://cancergenome.nih.gov/) and the international cancer genome consortium (icgc, http://icgc.org/), involve thousands of patients and have generated petabytes of data . One of the major assets of such projects is the public availability of the data allowing their integration with data from other initiatives . In that way, data from these initiatives can push a more focused and deeper analysis either in a specific gene set or in a specific cohort of patients / samples . The human surfaceome, the collection of cell surface proteins in human cells, has been defined and studied by us previously . By using bioinformatics pipeline and an experimental approach based either on real - time pcr or on other gene expression technologies, we were able to identify potential new biomarkers for few tumor types and have characterized new cell surface putative cancer - testis (ct) antigens [1, 2]. Relevant roles of surface proteins include nutrient and ion transport, adhesion to substrates, signaling, and intercellular interaction . Due to these roles and their subcellular localization, easily accessible to therapeutic agents, surface proteins are important targets for cancer intervention . Since our original publication few reports have further explored the human surfaceome [26], mostly in the context of a mass - spectrometry - based characterization of the cell surface of tumor cells . Data from tcga / icgc allow the development of new metrics that evaluate the frequency of gene alterations in different cancer types . Recently, we developed a new scoring system for the identification and prioritization of cancer genes . The s - score method integrates information derived from different omics technologies to generate a gene - specific score that indicates whether that specific gene is a tumor suppressor (negative s - score) or an oncogene (positive s - score). The numerical value indicates the frequency in which that gene is altered in the cohort of samples used in the calculation . We have used the s - score metric to identify cancer genes in a set of human homologs of yeast genes characterized as suppressors of genome instability in yeast . The availability of the s - score system provides a quantitative way to identify and prioritize cancer genes in a particular set of samples . Here, we capitalize on the availability of data from the tcga project to further and deeper investigate the status of the human surfaceome in 15 tumor types, including gbm and colorectal and breast tumors, all analyzed in our previous publications [1, 2]. This generated a pan - cancer landscape of the human surfaceome with the identification of shared and tumor - specific markers . Furthermore, the use of the s - score system allowed us to identify gene signatures associated with overall survival in breast cancer patients . These signatures can be ultimately used in the development of new and more efficient diagnostic and therapeutic protocols . The protein - coding sequences of all human genes were obtained from the ncbi refseq (release 64). The illumina human bodymap 2.0 dataset was obtained from embl - ebi expression atlas (https://www.ebi.ac.uk/gxa/experiments/e-mtab-513). Tcga data was retrieved from both, the tcga web site (https://cancergenome.nih.gov/) and the cbio cancer genomics portal (http://www.cbioportal.org/). To predict plasma membrane subcellular localization, the ncbi reference sequence dataset was submitted to tmhmm version 2.0 (http://www.cbs.dtu.dk/services/tmhmm/). All sequences containing at least one tm domain were selected . To avoid false positives, sequences containing only one tm domain in the first 50 residues, which could be a signal peptide, were excluded and classified as secreted protein . Furthermore, sequences were also filtered based on the identification of signal peptide cleavage sites by signalp, release 4.1 (http://www.cbs.dtu.dk/services/signalp/). Since tm domains are not exclusive to cell surface proteins, the sequences were grouped according to subcellular localization as defined by gene ontology . We excluded sequences that were exclusively located at the following cellular compartments: lysosome, endoplasmic reticulum, mitochondria, cytoskeleton, endosome, liposome, nucleolus, nucleus, and ribosome . This step was conducted using in - house perl scripts . To validate the obtained list of surfaceome genes, we classified these genes as belonging to the following classes: g - protein - coupled receptors (gpcrs), solute carrier (slc) proteins, and cluster of differentiation (cd) antigens . The gpcr genes were obtained from gpcrdb (http://gpcrdb.org), while cd and slc genes were collected from hgnc (http://www.genenames.org/genefamilies/a-z#r). S - scores were calculated for the human surfaceome and for the 15 tumor types as previously defined . The distribution of s - scores was used to calculate z - scores for all genes using r statistical package . The go enrichment analyses of the surfaceome gene cluster were conducted using clusterprofiler, implemented in r, with p values <0.01 as a cutoff . Genes with extreme s - score (s - score <2 and> 2 for breast tumors) were selected to test any putative association with overall survival in breast cancer samples derived from tcga (without subtype distinction). The altered set comprised samples within which the respective genes were differentially expressed (z - score> 2 or z - score <2, as reported by tcga), amplified or deleted, or presenting deleterious mutations (nonsense, frameshift, and splice - site). After that, for each gene, the survival analysis was performed using the kaplan - meier method and the difference in survival curves was tested for statistical significance using the log rank test p value . We then selected a nonredundant set of 20 genes with the lowest p value (cutoff of 0.05) and tested all possible groups of three genes . First of all, we decided to reannotate the set of human genes coding for putative cell surface proteins . This was required due to (i) an improvement in the annotation of gene and protein databases regarding a protein's subcellular localization and (ii) the inclusion of new human genes in the reference sequence collection . The same approach used by da cunha et al . (2009) generated now a set of 3,758 human genes, composing the human surfaceome (figure 1). The complete list of human genes coding for cell surface proteins is provided as supplementary table s1, in supplementary material available online at http://dx.doi.org/10.1155/2016/8346198 . As expected, the great majority (85%) of surfaceome genes present in our dataset in 2009 remained classified as such in 2016 . New genes were added (585), mostly due to their inclusion in the reference sequence collection and some other genes (529) were excluded due mainly to new functional annotation that classified their protein products as belonging to other subcellular compartments . To assess the robustness of our approach, we performed the same analysis reported by us in our original 2009 paper checking the representation of three known families of cell surface proteins (g - protein - coupled receptors (gpcrs), solute carrier (slc) proteins and cluster of differentiation (cd) antigens). Since these are large and well - studied families of cell surface proteins, we envisaged that they would be appropriate for a benchmark analysis . For gpcrs, 98% of their known members were represented in our dataset . For slc proteins and cd antigens we found 77% and 88% represented in the surfaceome set, respectively . Overall, 90% of members of these three families were represented in our present surfaceome set, compared to 83% in our previous analysis . This improvement is expected due to a better annotation of the sequences in public databases . Capitalizing on the availability of surfaceome sets derived from mass - spectrometry analysis, we decided to compare our dataset to the dataset from bausch - fluck et al . . Although this type of comparison is problematic for different reasons, including (i) the nonexhaustive nature of the wet - based approach (due to the method itself and the samples screened) and (ii) the different premises of both methods (the requirement of at least one tm domain per protein in our pipeline and the lack of such requirement in which allowed the authors to characterize gpi - anchored proteins, e.g. ), the analysis may be illuminating in the sense that it can highlight important differences in both methodologies . We found that 66.6% (664 out of 996) of the proteins classified by bausch - fluck et al . Were present in our dataset while only 17.6% (664 out of 3758) of our proteins were present in their dataset . This was expected due to the issues raised above . To illustrate the complex nature of this comparison, 23.8% of all cell surface proteins found in have no tm domain, as identified by tmhmm . Next, the s - score method was used to identify cancer genes within the surfaceome set . S - score threshold was defined for each tumor type as the s - score representing the average s - score plus / minus three standard deviations (z - score 3 or 3). The list of all cancer genes coding for cell surface proteins in all 15 tumors types is shown in supplementary table s1 . Using the above z - score threshold, we found 248 surfaceome genes classified as a cancer gene in at least one tumor type . In the heatmap representation of the surfaceome cancer genes (figure 2(a)) we can clearly identify three distinct clusters based on the s - score values for all 15 tumor types . Although all three groups have a variety of oncogenes and suppressors, some features deserve further comments . For example, the first group is mainly composed of suppressors, especially in melanoma and colorectal and lung adenocarcinoma and uterine corpus endometrial carcinoma . Genes in this group include several members of the cadherin superfamily (pcdhgb3, pcdha2, pcdha7, pcdh15, pcdhgb5, pcdh11x, pcdhac1, fat1, fat2, and fat4). There is a set of oncogenes in group 2 shared by almost all tumors and involving 30 genes, including ephb1 and ephb3 . There is no clear pattern in group 3 and oncogenes and suppressors seem to be distributed evenly across all tumors . To better understand the pattern presented in figure 2(a), we performed a gene ontology (go) enrichment analysis (using the biological process ontology) for the three different clusters . As expected, all three groups shared go categories associated with the cell surface such as transmembrane transport and cell surface receptor signaling pathway . More interestingly, however, is the fact that specific go categories were enriched in individual groups (figure 2(b)). Go categories exclusively found in group 1 were clearly associated with nervous system including neuromuscular process; memory; and neuronal action potential . The same pattern was observed for group 2 although the go categories represented different aspects of nervous system including the following: sensory perception of pain and other categories related to axonogenesis . Regarding group 3, go analysis lent further support for the current concept of ion transport associated with cancer, including manganese ion transport . Additionally, this group presented genes related to antigen processing and presentation, highlighting that the interplay between immune and tumor cells is complex . Several of the identified surfaceome cancer genes are known for their involvement in different aspects of cancer biology, especially the ones classified in group 2 . Abcc5, a cell surface transporter, was involved in resistance to anticancer drugs and overexpression of atp11b has been linked to drug resistance in ovarian cancer . Both genes were regarded as oncogenic by our work especially in lung squamous cell carcinoma and ovarian cancer . On the other hand, ephb3 has already been suggested as a candidate target gene for both lung small cell carcinoma and colorectal cancer . Finally, a transferrin receptor (tfrc) has shown an increased expression in many malignant tumors and was also found to be highly oncogenic in this work . As previously discussed by us, the s - score method allows the prioritization of cancer genes based on clinical parameters . For example, we have identified genes associated with both short- and long - term survival in ovarian cancer . To test whether we could identify genes in the surfaceome set associated with clinical parameters, we decided to look at overall survival in breast tumors, since this type of tumor is the one with the largest cohort in tcga . For this specific analysis a more relaxed threshold (z - score <2 or> 2) was used to classify a gene as a cancer gene in breast tumor to increase the number of genes under test without compromising the quality of the classification (a heatmap, similar to figure 2(a) and generated using the dataset with a more relaxed threshold, is presented in supplementary figure 1). For each surfaceome gene classified as oncogene or suppressor in breast tumor, we split the breast cancer samples into two groups: altered (genes with differential expression, genes amplified / deleted, or genes mutated) and unaltered . For each gene, the two groups were then compared by a kaplan - meier analysis to evaluate whether they had significantly different overall survival . Twenty - three genes, seven oncogenes and 16 suppressors, were significantly (p - value <0.05) associated with differences in overall survival in breast cancer patients (supplementary table 2). These genes are involved in cell adhesion and ion transport, two of the main categories enriched in our gene ontology analysis . Next, all possible combinations of these genes were similarly tested for differences in overall survival . Although we found several combinations with statistically significant differences in overall survival, we have focused on wnt5a, cnga2, and igsf9b due to statistical significance (it is the most significant three - gene set in supplementary table 2) and novelty . Patients in which one of the three genes was altered had a significantly shorter survival (p value = 1.82e)compared to patients where these three genes were unaltered (figure 3). The wnt5a, cnga2, and igsf9b genes have negative s - scores in breast cancer (2.59, 3.39, and 2.56, resp . ), demonstrating a tumor suppressor profile ., the loss of wnt5a has been associated with poor prognosis, in agreement with the suppressor status defined by the respective s - score . On the other hand, wnt5a was recently reported to promote cancer cell lines invasion and proliferation, a feature typical of oncogenes . Wnt5a is present in pathways where wnt signaling is involved through interaction with frizzleds (fzd10, e.g.) And dishevelled . Cnga2, a homotetrameric channel in olfactory sensory neurons, has not been reported in association with cancer . However, cnga2 represents the alpha subunit of a cyclic nucleotide - gated olfactory channel possessing a role in calcium signaling pathway acting through calmodulin - like 6 and calcium / calmodulin - dependent protein kinase iv directly involved with protein kinase a (pka), a biological target in cancer therapy . Igsf9b was only recently identified as an inhibitory synaptic adhesion molecule and no link with cancer was found in the literature . We have updated the set of human genes coding for cell surface proteins, the human surfaceome . Using tcga data for 15 tumor types and a new method of cancer genes classification that integrates information from different omics technologies and allows a ranking based on clinical parameters (s - score), we identified several potential therapeutic targets within the surfaceome set . Furthermore, we present evidence that a three - gene set wnt5a, cnga2 and igsf9b was associated with shorter survival in breast cancer patients . Our results clearly show the importance of large - scale genomics datasets from cancer patient cohorts, like the one provided by tcga . We envisage that the data we provide here will be extremely useful to researchers who aim to characterize cell surface targets for a variety of tumor types.
A recent national cross - sectional survey showed that the overall prevalence of diabetes was estimated to be 11.6% in the chinese adult population . Diabetes is a progressive disease, due in part to the loss of -cell function, with the reduction in function probably commencing 10 to 12 years prior to diagnosis and aggravated by increasing fasting plasma glucose (fpg) levels . Furthermore, east asian patients with type 2 diabetes have a higher risk of developing renal complications than europeans, and, with regard to cardiovascular complications, a predisposition for developing strokes . These results denote that more studies are needed to explain these interethnic differences, and, most importantly, effective strategies are required to facilitate earlier identification and prevention to combat these growing disease burdens, particularly in china . Methylenetetrahydrofolate reductase (mthfr) is the main regulatory enzyme for folate / homocysteine metabolism . Mthfr converts 5,10-methylene - tetrahydrofolate (thf) into 5-methyl - thf, the dominant circulating form of folate . The 5-methyl - thf product donates a methyl group to homocysteine in the generation of s - adenosylmethionine, a major source of methyl groups used for dna methylation . Polymorphism of mthfr 677ct leads to a reduction in enzyme activity, resulting in increased concentrations of plasma homocysteine and lower levels of serum folate, and thereby confers a higher risk for stroke, particularly in those with low folate intake . A recent meta - analysis of case control studies found that the homocysteine concentration in individuals with type 2 diabetes was significantly higher than that in control subjects (0.94 mol / l, 95% confidence interval [ci] 0.401.48), and the odds ratio (or) associated with type 2 diabetes for mthfr 677tt relative to 677cc was 1.38 (95% ci 1.181.62). Our recent study also showed that participants with mthfr 677tt genotype had a higher prevalence of diabetes than those with cc genotype . Furthermore, plasma homocysteine levels were significantly inversely associated with estimated glomerular filtration rate (egfr), and mthfr 677tt genotype was associated with a higher risk for decreased kidney function independent of homocysteine levels . However, no prospective studies investigating the association between mthfr 677ct polymorphism, homocysteine, renal function, and new - onset diabetes (nod) have been conducted . Furthermore, the frequency of the mthfr 677 tt genotype, which showed marked ethnic variations, was more common in china than in most of the european countries . The chinese also had higher homocysteine levels, lower folate concentrations, and did not have a policy of mandatory folic acid fortification in food . These characteristics make the chinese population particularly suited for testing the possible genotype (mthfr 677ct polymorphism) and phenotype (homocysteine and egfr levels)-disease association using the same analytical framework . Therefore, in the current study, we aimed to evaluate the effect of mthfr 677ct polymorphism, homocysteine, and egfr levels on the risk of nod in a rural chinese cohort, and to identify the possible effect modifiers . Our findings may possibly provide insights into the ethnic differences in diabetic complications seen between east asian patients and europeans . This report followed the strobe (strengthening the reporting of observational studies in epidemiology) statement for cohort studies . Study participants were from an epidemiological study of metabolic syndrome conducted during 2003 to 2005 in rural communities (dongzhi and wangjiang) in anqing, anhui province of china . Detailed protocol and the details regarding study population and data collection have been previously described . Briefly, 6301 of the study subjects from dongzhi community who received baseline screening examination were invited for a follow - up visit in 2011, and 2901 (46%) of them responded . The nonresponders did not differ from the responders substantially with respect to baseline characteristics (data not shown). This study was approved by the institutional review boards from the nanfang hospital in guangzhou and the institute of biomedicine in the anhui medical university . Written informed consent was obtained from each study participant . Fpg, total cholesterol (tc), triglyceride (tg), high - density lipoprotein - cholesterol (hdl - c), and homocysteine were measured on the hitachi 7020 automatic analyzer (hitachi, tokyo, japan). Serum creatinine concentrations were determined using an enzymatic method (sarcosine oxidase - peroxidase anti - peroxidase). Plasma insulin was measured using an enhanced chemiluminescence method on an elecsys 2010 system (roche, basel, switzerland). Mthfr 677ct genotype was determined by taqman assay designed and manufactured by applied biosystems (foster city, ca). The homeostasis model assessment of insulin resistance (homa - ir) was calculated from the fasting concentrations of insulin and glucose using the following formula: fasting insulin (u / ml) fasting glucose (mmol / l)/22.5 . We excluded participants with self - reported physician diagnosis of diabetes or fpg 7.0 mmol / l at baseline in the final analysis . Nod was defined as fpg 7.0 mmol / l or self - reported physician diagnosis of diabetes at follow - up year . Current alcohol drinking was defined as drinking alcohol at least once per week in the last year . The question about insomnia was phrased as follows: do you frequently suffer from insomnia?, and a choice of 3 responses (frequent: almost every week, medium: 13 times per month, and seldom) was provided . Baseline characteristics are presented as mean or percentage, except for fasting insulin and homocysteine, which are presented as median (q1q3) because of the skewed distribution . Between - group differences in baseline characteristics were tested using the student t test, the signed rank test, or the test, accordingly . The effects of mthfr 677ct polymorphism (cc, ct, and tt), homocysteine (<10, 1016, and 16 mol / l), and egfr (120, 90120, <90 ml / min/1.73 m) on the risks of nod in males and females were estimated using logistic regression models with adjustment for baseline covariates including age (year), baseline fpg (5.6 vs <5.6 mmol / l), body mass index (bmi, 23 vs <23 kg / m), blood pressure (<130/85, 130/85140/90, 140/90 mm hg), tg (1.7 vs mmol / l), hdl - c (<1.3 [females]/1.0 [males] vs 1.3/1.0 mmol / l), current cigarette smoking, current alcohol drinking, and insomnia (frequent, medium, and seldom). R software, version 2.15.1 (http://www.r-project.org) was used to perform all statistical analyses . Overall, 2901 subjects were revisited . In this report, study participants with self - reported physician diagnosis of cardiovascular disease (cvd, n = 28), diabetes (n = 10), hypertension (n = 124), or with any missing data (n = 186) regarding baseline values for age, cigarette smoking status, alcohol drinking status, fasting glucose, homocysteine, mthfr 677ct polymorphism or insomnia, or with any missing data (n = 61) for fpg or self - reported physician diagnosis of diabetes at follow up, or who had an fpg value of 7.0 mmol / l at baseline(n = 70) were excluded . The prevalence of mthfr 677 cc, 677 ct, and 677 tt genotypes was 36.8%, 47.4%, and 15.8%, respectively . This population had no significant deviations in genotype distributions from expected hardy weinberg equilibrium . Those with mthfr 677 tt genotype had significantly higher homocysteine levels (median, 12.0 in males and 10.1 mol / l in females) than those with ct (10.4 and 9.1 mol / l) or cc (10.4 and 8.6 mol / l) genotype (p <0.05 for either of these genotypes in males or females). Furthermore, there was an inverse association between homocysteine and egfr levels in males (r = 0.43, p <0.001) and females (r = 0.63, p <0.001). The follow - up time ranged from 5.81 to 7.57 years, with a mean of 7.02 (sd 0.31) years . There were no significant differences in the follow - up time between subjects with and without nod in males (6.97 [0.31] vs 7.02 [0.31], p = 0.137) and females (7.05 [0.34] vs 7.03 [0.32], p = 0.506). The baseline characteristics of the study subjects stratified by nod status and sex are summarized in table 1 . Nod patients had significantly higher tg, bmi, insulin levels, and homa - ir compared with those without in both males and females . However, nod patients had significantly higher mthfr 677 tt genotype rates in females only (table 1). Baseline characteristics according to nod status by sex the incidence rates of nod in males and females were 8.0% and 7.0%, respectively . Compared with subjects with mthfr 677 cc genotype, those with tt genotype had a higher risk of nod in females (or 2.78, 95% ci 1.395.56) but not in males (0.80, 0.401.61, p for interaction = 0.008). <10 mol / l) was not associated with nod in males (0.88, 0.421.85) or females (1.52, 0.653.57). However, mildly decreased egfr (<90 vs 90120 ml / min/1.73 m) was associated with nod mainly in males (1.96, 1.013.78; females, 0.74, 0.321.72, p for interaction = 0.134) (table 2). Relationship of mthfr 677ct polymorphism, baseline homocysteine levels, and egfr with the risk of nod in the full models, we observed that higher tg, bmi, fpg levels, and insomnia, in addition to mthfr c677 t polymorphism in females, and higher fpg levels and insomnia in addition to lower egfr levels in males remained independent risk factors for the risk of nod (data not shown). In the stratified analyses, the mthfr 677ct polymorphism (cc, ct, and tt genotypes) was more strongly associated with the risk of nod among females with higher bmi (23 vs <23 kg / m, p for interaction = 0.009) or lower hdl - c levels (<1.3 vs 1.3 mmol / l, p for interaction = 0.015). However, the association of mthfr 677ct polymorphism with increased risks of nod in females appeared to be similar among subgroups classified according to baseline homocysteine (10 vs <10 mol / l, p for interaction = 0.061), egfr (<110 vs 110 ml / min/1.73 m, p for interaction = 0.229), fpg (5.6 vs <5.6 mmol / l, p for interaction = 0.635), tg levels (1.7 vs <1.7 mmol / l, p for interaction = 0.189), or insomnia (frequent vs medium or seldom, p for interaction = 0.831). Furthermore, similar trends for egfr category and increased risk of nod in males were observed among subgroups stratified by insomnia (p for interaction = 0.946), baseline fpg (p for interaction = 0.977), homocysteine levels (p for interaction = 0.573), or mthfr 677ct polymorphism (p for interaction = 0.880) (table 3). Stratified analysis of multivariate ors for nod among 1172 women according to mthfr 677ct polymorphism further adjustment for homa - ir (n = 2144) did not substantially change the relationship of mthfr 677ct polymorphism (tt vs cc, 2.20; 1.024.73) in females or egfr levels (<90 vs 90120 ml / min/1.73 m, 1.85; 0.923.71) in males with the risk of nod . Previous studies have shown conflicting results regarding the homocysteine levels in patients with diabetes . A recent meta - analysis of 14 case control studies found that the mean homocysteine concentration was greater in patients with type 2 diabetes than in control subjects . However, the 3 studies that contributed most to the overall estimate included patients with type 2 diabetes accompanied by varying degrees of kidney disorders . These results suggest that it may be renal dysfunction but not diabetes that mostly explains the difference in homocysteine levels between type 2 diabetes patients and control subjects in this meta - analysis . In fact, in our present prospective study, we did not find significant associations between homocysteine levels and nod in males or females . However, consistent with this meta - analysis, female subjects with tt genotype in the current study had a higher risk of nod (tt vs cc genotype, 2.78; 1.395.56). To explain the significant relationship of mthfr gene 677ct polymorphism (but not homocysteine levels) with the risk of nod, we speculate that, first, the homocysteine levels (median 10.7 mol / l in females) in the present study was possibly not high enough to cause obvious organ damage . Second, plasma homocysteine may just serve as a reliable functional marker of folate status, and folate may have other actions, including antioxidant actions, effects on cofactor availability, or direct interactions with the enzyme endothelial no synthase, in addition to homocysteine lowering that influence health status . A previous report showed that folic acid given to patients with coronary heart disease resulted in improvement in endothelial function without any change in plasma homocysteine concentration . Unfortunately, we were not able to directly examine the association between mthfr 677ct polymorphism, folate, and the risk of nod in the current study due to the lack of baseline folate data . Additional studies are required to further address this topic and to evaluate the role of folic acid supplementation in reducing the risk of diabetes, particularly in populations without folic acid fortification . Meanwhile, we did not have enough information to explain the sex - specific effect of mthfr 677ct polymorphism, which may relate to the possible role of hormones in folate / homocysteine metabolism . Our current study detected a detrimentally interactive effect between the mthfr 677ct polymorphism and higher bmi (23 vs <23 kg / m), or lower hdl - c (<1.3 vs 1.3 obese subjects without diabetes have been shown to exhibit an enhanced rate of glucose production . We hypothesize that a higher bmi state may augment the mthfr 677ct polymorphism - mediated risk of nod . Furthermore, mthfr 677ct polymorphism appeared to modify the efficacy of the drug pravastatin in reducing risk of cardiovascular events . A significantly protective effect against coronary heart disease (hazard ratio [hr] 0.71, 95% ci 0.580.87) was shown in subjects with cc genotype but not in subjects with ct (hr 1.25, 95% ci 0.971.61) or tt genotype (hr 0.80, 95% ci 0.501.28, p for interaction = 0.004). Consistently, we also observed an interaction between the mthfr 677ct polymorphism and hdl - c levels on the risk of nod . These results suggest that an investigation of the possible modifying effect of mthfr 677ct polymorphism on cvd associated with hdl - c increasing therapy maybe provide some clues regarding the conflicting results gathered from these kinds of trials . Overall, our results indicate that the mthfr 677tt genotype, along with homocysteine, bmi, and hdl - c levels, may help to identify apparently healthy females at increased risk for diabetes . Menon et al reported that hyperhomocysteinemia did not appear to be a risk factor for all - cause or cvd mortality, and prior studies demonstrating an association between homocysteine and cvd risk may have inadequately adjusted for the confounding effects of kidney function . We also observed that mildly decreased egfr (<90 ml / min/1.73 m), but not homocysteine, was associated with the risk of new - onset disease . Nevertheless, more studies are needed to verify if our findings can be generalized to other populations or ethnicities, particularly individuals with lower egfr levels or obvious chronic kidney disease . The strengths of our study include the 7-year prospective follow - up of middle - aged rural chinese men and women, and the comprehensive adjustments for the major traditional risk factors for nod, including baseline fpg and ir . First, we did not measure glycosylated hemoglobin levels or perform glucose - tolerance tests . Second, we did not have information regarding family history of diabetes . However, further adjustment for family history of diabetes did not change the significant association between mthfr 677ct polymorphism and prevalence of diabetes in our previous study . Lastly, previous studies have shown that nonalcoholic fatty liver disease (nafld) was strongly associated with ir and diabetes . Unfortunately, we were not able to examine this effect in the current study due to the lack of data on nafld ., we found that individuals with mthfr 677tt genotype had a significantly higher risk of nod among females, particularly in those with higher bmi or lower hdl - c levels . The higher risk of nod associated with mildly decreased egfr (<90 ml / min/1.73 m) also warrants more investigation . Our study findings, if further confirmed, will provide new strategies to identify apparently healthy population at increased risk for diabetes . Furthermore, considering the higher frequency of mthfr 677tt genotype in china and the higher stroke risk associated with tt genotype, our results also provide some explanations for the ethnic differences in diabetic complications seen between east asian patients and europeans.
Soft tissue coverage for wounds remains a difficult management problem for patients sustaining traumatic injury and burns . There are several methods to achieve wound coverage secondary healing, primary suturing, skin grafting, and flap surgeries as described in reconstruction ladder . A skin graft is the most commonly used modality for coverage of wounds in reconstructive plastic surgery . The skin graft needs to undergo various stages of healing for a good take on the recipient bed . There are different methods employed to secure the graft to recipient bed for a few days with a basic idea to ensure that the graft is not elevated off the bed by formation of haematoma / seroma under it . Repeated tie over dressings are required in situations where the dressing needs to be changed more frequently as in cases of infected raw area, bleeding tendency, patients on anticoagulant drugs, and in convex areas of body such as buttocks, breast, and the scalp, where the dressing is difficult to secure . In this novel method, a sterile sample container was cut at its upper part [figures 1 and 2]. The skin graft was applied on the raw area and fixed with skin staplers and tie over sutures . Once paraffin gauze and adequate padding is applied on the raw area, the tie over threads were passed from inside out of the container [figures 3 and 4] and pulled at the appropriate tension to keep the dressing in place . The lid of the container was tightened to complete the dressing ensuring that the graft was maintained in close approximation with the wound surface . Sterile plastic container upper part of the sterile container is cut tie over threads being passed from inside out the lid is tightened over the dressing the dressing can be changed repeatedly with sterile precautions depending on the requirement, as an outpatient procedure by unscrewing the lid [figures 5 - 8] which can then be easily reapplied . The procedure can be used on wounds of any size by changing the size of the sterile container chosen . Post - toilet mastectomy raw area covered with the skin graft the tie over dressing applied in ot tie over dressing repeated in an outpatient department we have used this method in eight cases of the post toilet mastectomy raw areas with very good results [figures 9 and 10]. Post - toilet mastectomy raw area skin graft once applied has to be covered with petrolatum gauze to avoid its separation from the wound bed at the time of change of dressing . An ideal method of graft fixation should be simple, rapid, repeatable, able to be performed in the outpatient department, prevent hematoma or seroma formation, soak the exudates well, and allow the graft bed to be inspected easily . There are multiple methods of securing dressings over the skin graft, some of them can be applied only once and some can be repeated . The dressings that can be applied only once are like foam, hydro cellular dressing (highly absorbent and can be easily changed), negative pressure therapy dressing (stabilises the graft, increases the vascularity of bed, takes away toxic chemicals), and gas bag (transparent, can see graft and monitor any haematoma). These traditional methods can stabilize the graft till the first dressing post operatively . In some contaminated wounds, the dressing needs to be removed earlier, especially if there is drainage or foul smell . This approach may also be proper for graft, used to cover defects of some anatomical regions with increases risk of contamination, such as perineal, axillary, and genital or it can be used in areas where base of wound is difficult to immobilize like breast / pectoral region . Repeated tie over dressings can be done by keeping interrupted sutures long to be used as tie over dressings . These ties over dressing can be made of sutures or rubber bands . When taking a tie over stitch, both the threads can be left long and only one thread is tied at a time, the other thread is left long for next time . These techniques are difficult for small dressings especially the bra hooks; the silk loops method is very cumbersome and takes long time to do . The novel method being discussed has a very small learning curve and is very fast . It hardly takes 5 min in the hands of a plastic surgeon to complete the dressing . This dressing technique maintains the advantage of conventional tie over dressing with rapidity and repeativity . Good graft take can be expected whether split - thickness or full - thickness with appropriate methods of stabilisation . We recommend a novel, low cost, simple, rapid method of graft fixation that can be used repeatedly and can be applied to a wound of any size.
The stomach is a sensitive digestive organ that is susceptible to exogenous pathogens to which it is exposed by the diet . In response to such pathogens, the stomach induces oxidative stress, which might be related to the development of both gastric organic disorders such as gastritis, gastric ulcers, and gastric cancer and functional disorders such as functional dyspepsia [2, 3]. One of the most important possible causes of gastric ulcers is oxidative stress, which is involved in the pathogenesis of gastric inflammation and ulcerogenesis, but also in lifestyle related diseases including atherosclerosis, hypertension, diabetes mellitus, ischemic heart diseases, and malignancies [4, 5]. Nowadays, researchers and companies are inclined to investigate herbal medicines, more and many plants with anti - ulcerogenic properties have been found by different groups . Herbs, medicinal plants, spices, vegetables, and crude drug substances are considered to be potential candidates for use in the treatment of gastric ulcer . Among them, ganoderma lucidum () has been found to have anti - tumor, anti- cancer, immunomodulatory and immunotherapeutic effects, to inhibit platelet aggregation, and to lower blood pressure, cholesterol and blood sugar . Recentiy, pharmacopuncture treatment has been widely used and different types of herbs have been found to be effective for treating various diseases . This study was accomplished to investigate the effect of ganoderma lucidum pharmacopuncture (glp) in treating chronic gastric ulcer induced in rats by using ethanol (etoh). Ganoderma lucidum caps, 500 g grown in south korea were washed thoroughly with distilled water, cut into pieces, and submerged in 4 l of 25% alcohol for 10 hours at room temperature to be extracted . The alcohol extract was condensed to 500 ml, by using a rotary evaporator, and impurities were removed with a 0.22 m filter, the extracts was then, sterilized and stored at a temperature of 20. the extract was dissolved in ethanol before administrations, and was diluted with 5% dw (dextrose water, jw pharmacoceutical) to keep the final concentration of ganoderma lucidum at 10% . Adult male, sprague - dawley (sd) rats (weighing 220 240 g, 15 weeks old and housed five rats per cage) were purchased from sam - taco co. they were provided with standard food and water ad libitum and were maintained in an animal house at a controlled temperature (22 2) with a 12 hours light / dark cycle . The rats were divided randomly into 4 groups of 8 rats each: the normal, the control, the normal saline (np) and the glp groups . The study was approved by the ethics committee for animal experimentation, dong - eui university . In this study, the np and the glp groups were treated with injection of saline and glp respectively . Two local acupoints cv12 () and st36 () were used . A pharmacopuncture needle (29 gauge: 1 ml disposal insulin injection syringe from hwajin co. busan, korea) was used . All laboratory rats were administered treatment for 15 days . On last day, the rats were sacrificed and their stomachs were immediately excised . The reactivities, activities and deaths of rats in each group were observed during the experiment . The animals were killed by cervical dislocation after administration of an overdose of ether after the two week experiment . The whole stomach was cut and removed 1.5 cm away from the cardia and the pylorus . For general specimen observation then, the specimen was placed in formaldehyde and glutaraldehyde, and was stored in a liquid nitrogen solution for later observation . The length and the width of the injured gastric mucosa region were measured with a vernier caliper . The gastric mucosa ulcer index (ui) was determined according to the guth standard: spot erosions were recorded as 1 point, erosion lengths <1 mm were recorded as 2 points, erosion lengths from 1 to 2 mm were recorded as 3 points, those from 2 to 3 mm were recorded as 4 points, and those> 3 mm were recorded as 5 points; the score was doubled when the erosion width was> 1 mm . Ulcers of the gastric mucosa appear as elongated bands of hemorrhagic lesions parallel to the long axis of the stomach . The inhibition percentage (%) was calculated by using the following formula: to investigate whether glp had affeced the activity of radical scavenging enzymes, we measured the activity of superoxidase dismutase (sod) in the gastric mucosa according to the method of mccord and fridovich . The standard assay was performed in 3 ml of 50 mm potassium phosphate buffer at ph 7.8 containing 0.1 mm ethylene diamine triacetic acid (edta) in a cuvette thermostated at 25. the reaction mixture contained 0.1 mm ferricytochrome c, 0.1 mm xanthine, and sufficient xanthine oxidase to produce a reduction rate of ferricytochrome c at 550 nm of 0.025 absorbance unit per min . A kinetic spectrophotometric analysis was started, with the addition of xanthine oxidase at 550 nm . Under these conditions, the amount of sod required to inhibit the reduction rate of ferricytochrome c by 50% was defined as 1 unit of activity . After the opened stomachs had been cut into small pieces, they were preserved in 10% buffered formalin overnight . Next, the biopsies were embedded in paraffin wax, sectioned into 5 m thicknesses by using a microtome and then stained with haematoxylin and eosin (h&e). The tissue sections were assessed for histopathological changes such as congestion, edema, hemorrhage and necrosis by using a light microscope . Tissue section slides were heated at 60 for approximately 25 minutes in a hot oven . Immunohistochemical staining was conducted according to the manufacturer s protocol (dakocytomation, usa). Briefly, endogenous peroxidase was blocked by using a peroxidase block (0.03% hydrogen peroxide containing sodium azide) for 5 minutes . Tissue sections were washed gently with wash buffer and then incubated with bcl-2-associated x protein (bax) (1: 200), b - cell lymphoma 2 (bcl-2) (1: 200), and transforming growth factor - beta 1 (tgf-1) (1: 100) biotinylated primary antibodies for 15 minutes . Then, the tissue sections were rinsed gently in wash buffer and placed in a buffer bath . Following washing and counterstaining with hematoxylin for 5 seconds, we added diaminobenzidine substrate chromagen to the tissue sections and incubated them further for 5 minutes . The total numbers of immune positive cells per field (10 m) were counted in at least 15 randomly chosen fields at 100 magnification . The statistical significances of the differences among groups were assessed with a one way analysis of variance (anova, post hoc analysis). A value of p <0.05 was considered significant . Scale bar = 1 cm; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . P <0.05 vs. control; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . Scale bar = 200 m and magnification = 40; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . P <0.05 vs. control; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . Scale bar = 100 m and magnification = 100; bax, bcl-2-associated x; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . Scale bar = 100 m and magnification = 100; bcl-2, b - cell lymphoma 2; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . Bax, bcl-2-associated x; bcl-2, b - cell lymphoma 2; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . Scale bar = 100 m and magnification = 100; tgf-1, transforming growth factor - beta 1; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . P <0.05 vs. the control; tgf-1, transforming growth factor - beta 1; np, injection of saline; glp, injection of ganoderma lucidum pharmacopuncture . The areas of gastric ulcer formation were reduced in the np and the glp groups compared with the control group (fig . The formation of the ulcers was significantly suppressed in the glp group compared to the control group (p <0.05). However in the control group, scattered mucosal damage and local congestion were observed . In the np and the glp groups, the gastric mucosal injuries were obviously slightly lwss severe than they were in the control group (fig . The treatment with glp caused a significant increase (p <0.05) in the enzyme activity of sod (fig . An increased level of bax protein was observed in the control group, whereas, the expressions of bax protein in the np group was decreased compared with those in the control group and those in the glp group were more significantly decreased compared with those in the control group (p <0.01) (figs . Bcl-2 protein expression was hardly observed in the control group and the expressions of bcl-2 protein in the np group were increased significantly compared with those in the control group (p <0.05) (figs . Bcl-2 protein expressions in the glp group were more significantly decreased compared those in the control group (p <0.01) (figs . The increase in the glp group was more significant (p <0.01) than it was in the control group (figs . The gastric mucosa is one of the most important tissue in an organism, because of its function, structure, and the pathological processes that can take place in this tissue . The integrity of the gastric mucosa depends on the protection provided by the gastric mucosal barrier, and the gastric mucosa can be damaged by a variety of internal or external factors with the production of a number of inflammatory mediators and cytokines, resulting in secondary mucosal damage . Ethanol is an ulcerogenic agent that is known to produce erosions, ulcerative lesions, and petechial bleeding in the mucosa of the stomach . Ethanol rapidly penetrates the gastric mucosa, causing membrane damage, exfoliation of cells, erosion, and ulcer formation . In the present study, using the chronic injury model, gastric mucosal lesions in rats were induced by using etoh . Administration of an ethanol solution for 15 days induced hemorrhagic lesions in the gastric mucosa in the control group . In the control group, the np and the glp groups had reduced areas of gastric ulcer formation when compared with the control group (fig . 1). In the glp, compared with the np group, the formation of ulcers was significantly suppressed (fig . 2). Gastric ulcers were induced on the anterior walls of the stomachs in rats by using an etoh solution . The rats of the control group showed loss of mucosa, but compared with control group, the mucosal losses were less in the np and the glp groups (fig . Ethanol administration reduced sod activity in the control group, compared with the normal group (fig . This might have resulted from the utilization for the decomposition of superoxide anion generated by lipid peroxidation . However, the activities in the glp group were significantly compared to the control group (fig . 4). Apoptosis, programmed death of cells through dna fragmentation, cell shrinkage, and dilation of endoplasmic reticulum is normally followed by cell degeneration and the formation of membrane vesicles, called apoptosis bodies . Ethanol by itself was revealed to be able to induce apoptosis in the gastric epithelium . The up regulation of the pro apoptotic factor, bax protein, and the down regulation of antiapoptotic bcl-2 are two main indicators for apoptosis . The level of bax protein was increased in the control group, but in the np and the glp groups, the level of bax protein were decreased compared with the control group . Especially in the glp group, on the other hand, bcl-2 immunoreactivites in the np group were up regulated compared with the control group . Glp not only up regulated the levels of bcl-2 but also down regulated the levels of bax compared with the control group (fig . Therefore, we assume that glp suppressed apoptosis by regulating the mitochondrial damage mediated endogenous pathway, which could be one of the important mechanisms for preventing gastric ulcer disease . Tgf-1 is well known to be a multi functional cytokine that regulates many biological processes such as cell proliferation, cell differentiation, adhesion, inter cell signaling, and the also production and the degradation of extracellular matrix proteins, thus playing an essential role during wound healing and tissue repair . This indicates that tgf-1 expression is part of the normal healing response of gastric tissue . In this study, the level of tgf-1 was significantly increased in the glp group (figs . The present study indicated the protective efficacy of glp against etoh induced gastric ulcer in sd rats . This conclusion was based on gross appearance, histology, and immunohistochemistry staining for bax, bcl-2 and tgf-1.
Improper nutritional knowledge is one of the most important causes of nutritional problems, which can affect practice and cause more complications . It has been recommended that food choices and dietary behaviors can be impressed by knowledge about diet (1). To effectively improve healthy eating, it is necessary to understand the nutritional attitudes and beliefs of the general community (2). Age, education level, gender and marital status can influence nutritional knowledge, attitude and practice (kap) (3). The association of socioeconomic status (ses) with nutrition knowledge and beliefs has been confirmed (4). Many studies have found that nutrient intakes and dietary patterns of people in low ses groups threaten the general health and raise the risk of nutritional disease (5,6). People in low ses group, due to lack of access to health care, improper living conditions, less knowledge and greater psychological stress, may be at a higher risk of poorer health conditions more than others (7 - 10). With respect to the association of ses index with health behaviors (11) and undesirable health consequences, it is of prime importance to evaluate the effect of ses index on health - related behaviors . In this study, the association between nutritional kap with ses was assessed among the iranians in urban and rural regions of 31 provinces of iran . The study population consisted of 14,136 iranian households who lived in urban and rural regions of 31 provinces, selected by multi - stage cluster sampling . Non - iranian households were not included in the survey, and the households who were absent for tree times at the time of the interview were also excluded . The method of study has been published in a previous survey (12). Mothers or any over 15-year old member of the households, who were in charge of cooking for the entire family, were considered as statistical units of the study . A structured questionnaire and interview with a qualified person in families nutritional knowledge questions inquired about main food groups, causes of consuming food, role of main food groups, sources of protein intake, and role of dietary fiber as well as nutritional attitude toward health - related behaviors and food choices . To assess the practice of the households, the household members were asked about the frequencies of different consumed foods . Ses was an index that included household assets (house ownership, number of rooms in the house, having such equipment as tv, cell phone, car, freezer, washing machine, dish washing machine, phone, microwave, access to internet), occupation and education level of the heads of the families and the respondents and number of family members . Data were analyzed using the stata version 11.0 (stata corp, college station, tex .) (survey analysis). Data were analyzed using the stata version 11.0 (stata corp, college station, tex .) (survey analysis). The percentage of nutritional knowledge was significantly higher in families with good ses and it linearly increased with family ses . Most people consumed food to prevent disease and be healthy (54.7%, 95% ci: 53.5, 56.0). More than half percent of households were aware of grain, meat and legumes, and dairy group (60.5%, 69.7%, and 52.5%, respectively). The percentage of knowledge about identification of fruit, vegetable and fat groups was less than half percent . About 73.1% of the households (95% ci: 71.9, 74.3) were familiar with the role of dairy group (growing and strengthen teeth and bones), although less than 10% of them were aware of the role of fruit groups in providing dietary fiber (8.6%, 95% ci: 7.1, 9.2). The percent of participants knowledge about legumes and soy, another source of protein, was 45.5% (95% ci: 44.2, 46.8) and 40.1% (95% ci: 38.8, 41.5), respectively . Only 10.1% of the participants were familiar with the concept of dietary fiber; among them, the most percentage of knowledge belonged to bowel movement . * (% (95% ci)), p<0.05, ses; socioeconomic status the percentage of attitude is shown in table 2 . More than 97% of the participants had a favorable attitude towards the importance of nutrition and diet in health . Respectively, 90.3%, and 70.4% of the participants had a favorable attitude toward importance of nutritional requirements of children rather than adults and the necessity of equal food intake in both genders when there is few food . The percentage of a favorable attitude of the family with good, moderate and weak ses about preferring fruit consumption than bread at time of hunger was 39.8%, 50.8% and 58.5%, respectively . The percentage of participants who disagreed with consuming steam cooked rice was significantly higher in families with weak ses . About 80.5% of the participants had a favorable attitude toward preferring consumption of tuna fish and the lowest percentage was related to weak ses families . Households with good ses had a more favorable attitude about the necessity of daily consumption of vegetables or salad and the need for milk consumption in any age besides childhood period (95.3%, and 93.1%, respectively). The families with weak ses had the highest favorable attitude about preferring whole meal bread on other kinds of breads . The percentage of favorable attitude toward drinking water in the middle of eating food and the necessity of keeping body fitness in girls at puberty was 66.5% and 56.1%, respectively . When the participants were asked about the difference between nutrition fact of meat and mushrooms, only 25.4% agreed . * (% (95% ci)), p <0.05, ses; socioeconomic status table 3 demonstrates the practice of households based on ses . Most households consumed fruit, vegetable, milk, yoghurt, cheese and sugar daily . Consumption of foods such as rice, red meat, butter, cream, egg, legumes, dough, chicken and poultry was weekly in most participants . The other items such as viscera, tuna, nuts and synthetic juice were rarely or never consumed . Families with good ses significantly consumed more fruit, vegetable, dairy group, red meat, chicken and poultry, fish and egg, while sugar consumption was significantly higher in families with weak ses . * (% (95% ci)), p<0.05, ses; socioeconomic status the aim of this study was to assess the association between nutritional kap with ses among iranian households . The best knowledge in all items was seen in families with good ses index and it linearly increased with family ses . Households with weak ses had the best favorable attitude toward the difference between mushroom and meat nutrition fact, preferring whole meal bread on other kinds of breads and preferring fruit consumption than bread at time of hunger . The consumption of food items such as red meat, chicken and poultry, fish, egg, dairy group, fruit, vegetable and nuts was significantly higher in households with good ses index while the other items including rice, tuna, legumes and sugar were consumed the most in weak families . These findings are supported by other surveys that have shown that more intake of fruit and vegetable are related to more diet costs, and diet rich in fat and sugar is contributed to lower costs (13,14). Findings from a survey on 4,356 us adults suggested that better ses index independently promotes the possibility of adequate fruits and vegetables intake and overall diet quality . They also reported that nutritional knowledge and belief can affect the positive association between ses and diet quality indicators(4). As it is confirmed in other studies, the socio - demographic variation in intake can be associated with nutritional knowledge as a partial mediator in improving diet . The result of this study also revealed that healthy eating was significantly associated with knowledge and possibility of meeting current recommendations for fruit, vegetable and fat intake (15). The association of ses and dietary knowledge or income and diet is supported by other studies (16 - 19). Another way that ses can influence diet is related to food purchasing differences . A study on australians in 2000 showed that food purchasing differences due to household income is related to diet via food - cost concern (20). Food purchase decisions due to a person s attitude toward food price can influence diet quality . Based on this survey, people who care about food price were more likely to live in low - income, food - insecure households, they had low education, were tenants and did not own homes, and were service workers . They were more susceptible to diseases such as overweight, high blood pressure, heart disease and diabetes than the others (21). Our results showed higher consumption of food items such as sugar, tuna and lower consumption of nuts and protein sources such as meat, fish, egg and dairy product in families with weak ses, which can be associated with an increased rate of some diseases . Thus, implementing measures to guide people in the line of healthier nutrition is necessary and it can help decrease the rate of diet - related diseases, especially in low ses households . With respect to the increasing nutritional disease and the important role of dietary behaviors, increasing nutritional kap may be a way to change life style and health related behaviors, but it is not enough . Therefore, targeted policies should be coupled with efforts to promote diet and nutritional kap for those people with unfavorable socio - economic status . Some cost - effective strategies should be presented for the low - income groups in the society to neutralize the negative effect of income on food purchasing patterns and health related life style.

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