Hypertension and proteinuria are medical complications associated with the multisystemic effects of long-term hypercortisolism in dogs with hyperadrenocorticism (HAC).This study investigated the relationships among adrenocorticotropic hormone (ACTH)-stimulation test results, systemic blood pressure, and microalbuminuria in clinically-healthy dogs (n = 100), in dogs affected with naturally occurring pituitary-dependent , or adrenal-dependent hyperadrenocorticism .p = 0.803). However the incidence of hypertension was highest in dogs with ADH (p = 0.017), followed by dogs with PDH, with the lowest levels in clinically healthy dogs (p = 0.019). Presence of microalbuminuria and albuminuria in clinically healthy dogs and dogs affected with HAC was significantly different (p < 0.001); incidences of albuminuria followed the same pattern of hypertension; highest incidence in dogs with ADH, and lowest level in clinically healthy dogs; but microalbuminuria showed a different pattern: clinically healthy dogs had highest incidences and dogs with ADH had lowest incidence. The presence of albuminuria was not associated with blood pressure values, regardless of whether dogs were clinically healthy or affected with ADH or PDH (p = 0.306).Mean systemic blood pressure was similar between clinically healthy dogs and dogs with HAC (Higher incidence of hypertension and albuminuria, not microalbuminuria was seen in dogs affected with HAC compared to clinically healthy dogs; incidence of hypertension and albuminuria was significantly higher in dogs affected with ADH compared to PDH. However, presence of albuminuria was not correlated with systemic blood pressure. Hyperadrenocorticism  is a common endocrine disorder in dogs and is characterized by chronically elevated circulating concentrations of the steroid hormones produced by the adrenal cortex (e.g. cortisol) . The mulApart from hypertension, many complications associated with Cushing's syndrome in humans are also linked with increased urinary albumin excretion rate. These include dyslepidaemia, reduced insulin sensitivity, and hyper-coagulative status. These complications are also associated with canine HAC ,16,18,19Microalbuminuria is defined as the presence of a small amount of albumin (1 to 30 mg/dL) in the urine . MicroalThe purposes of this study were to report the relationship between ACTH-stimulation tests, systemic blood pressure, microalbuminuria and albuminuria in dogs affected with naturally occurring pituitary-dependent (PDH) and adrenal-dependent hyperadrenocorticism (ADH).Informed consent was obtained from all dog owners prior to the study, and number of animals of each breed in each group are shown in Table One hundred clinically healthy client-owned dogs visiting the National Taiwan University Veterinary Hospital from 2005 to 2009 for routine health checkups and/or annual vaccination were included in this study. Of the clinically healthy dogs, 29 were intact females, 19 were spayed females, 45 were intact males, and 7 were castrated males. The average age was 5.1 ± 3.5 years (range 0.9 to 15 years), and average body weight was 11.6 ± 9.1 kg (range 1.8 to 35.0 kg). Thorough physical examination, blood pressure measurements and routine blood work  were performed to exclude heart and renal disorders that might affect blood pressure or cause microalbuminuria or albuminuria.Medical records of 278 dogs in the National Taiwan University Veterinary Hospital during 2005-2009 with clinical signs of HAC and results of ACTH stimulation tests consistent with hyperadrenocorticism were screened. Only 70 of these records included conclusive results from an ACTH stimulation test, ultrasonographic evidence of adrenal glands, blood pressure measurements and microalbuminuria analysis. Therefore, only 70 dogs were included in the study.Inclusion criteria of HAC were clinical signs consistent with a diagnosis of HAC , results of routine serum biochemical analyses that were consistent with a diagnosis of HAC , and affirmative results of adrenocorticotropic hormone (ACTH) stimulation tests (post-ACTH cortisol concentration >20 μg/dL) . Dogs wiAll included dogs were required to have had an ACTH stimulation test with intramuscular injection of 0.25 mg synthetic ACTH . Informed consent was obtained from all owners prior to diagnostic testing. Blood samples for serum cortisol measurements were collected via cephalic venipuncture immediately before, and 1 hour after synthetic ACTH was injected. Serum cortisol concentrations were measured by a validated radioimmunoassay  .In this study, 70 dogs were affected with HAC, with the mean age of 11.7 ± 2.7 years and ranged from 4.5 to 18 years. Dogs had a mean body weight of 9.0 ± 7.6 kg ranging from 2.1 to 43.4 kg. Twenty-seven were spayed females, 21 were intact males, and 11 each of castrated males and intact females. Among these 70 cases, 40 dogs affected with PDH and 30 dogs affected with ADH . The mean age of the 40 dogs affected with PDH was 10.7 ± 2.7 years, ranging from 4.5 to 16 years with a mean body weight of 9.9 ± 8.6 kg, ranging from 2.1 to 43.4 kg. Sixteen were spayed females, 6 were intact females, 12 were intact males and 6 were castrated males. The mean age of the dogs affected with ADH was 12.9 ± 2.2 years, ranging from 8 to 18 years. This group had a mean body weight of 7.9 ± 5.6 kg, ranging from 3.0 to 24.5 kg. Eleven were spayed females, 5 were intact females, 9 were intact males, and 5 were castrated males. Breeds of 70 dogs affected with HAC are shown in Table Systemic blood pressure values of 100 clinically healthy dogs and 70 dogs affected with (pre-treated) HAC were evaluated by the same protocol--Doppler sphygmomanometry. A Doppler Ultrasonic Flow Detector  with an inflatable cuff width of 1.9, 2.5, 3, 4, or 5 cm (depending on the circumference of the antebrachium) was used. The cuff was wrapped around the middle part of the antebrachium, and a Doppler probe coated with ultrasonic transmission gel was positioned over the palmar area to detect blood flow from the arteria digitalis palmaris communis. The cuff was then inflated and deflated to obtain a systemic blood pressure reading via an aneroid pressure gauge. During systemic blood pressure measurement, the antebrachium was maintained at the level of the heart. A series of 5 readings (with 10 to 20 seconds between consecutive measurements) was obtained for each dog, and all measurements were completed within 6 minutes. To minimize procedural stress, all dogs were allowed to assume a comfortable position with only gentle restraint by their owners. The dogs remained in the same position throughout systemic blood pressure measurement. The final systemic blood pressure value was calculated as the mean of 5 readings . The heaFreshly voided urine samples from 100 clinically healthy dogs and 70 dogs affected with (pre-treated) HAC were collected. Urine albumin analysis was carried out within four hours after the freshly voided urine samples were collected. Urine samples with hematuria (≥10 RBC/high power of field), pyuria (≥5 WBC/hpf), or bacteriuria was excluded after a light-microscopic examination of urine sediment. The urine albumin and creatinine concentrations were semi-quantitated  and urinary albumin:creatinine ratio (UACR) was calculated accordingly. In this study, no microalbuminuria, microalbuminuria and albuminuria were defined as UACR < 30 mg/g (0.03), 30-300 mg/g (0.03-0.3), and >300 mg/g (0.3), respectively.p value < 0.05.Comparison of systemic blood pressure between clinically healthy dogs and dogs affected with either PDH or ADH were evaluated by means of ANOVA. Relationship between post-ACTH cortisol concentrations and systemic blood pressure was analyzed by use of linear regression. Association between systemic blood pressure, microalbuminuria status  and health status  were evaluated by Pearson's chi-square test. All statistical analyses were performed using commercially available software . Continuous data are presented as mean ± SD. Statistical significance was determined by a The mean systemic pressure of 100 clinically healthy dogs was 152.8 ± 27.3 mmHg. One  was hypotensive, 87% (87/100) were normotensive, and 12% (12/100) were hypertensive. Among the 100 clinically healthy dogs, 53% (53/100) did not have microalbuminuria, 45% (45/100) had microalbuminuria, and 2% (2/100) had albuminuria  were normotensive, and 31.4% (22/70) were hypertensive. In 70 dogs with HAC, 12.9% (9/70) did not have microalbuminuria, 38.6% (27/70) had microalbuminuria, and 48.6% (34/70) had albuminuria  were normotensive, and 20% (8/40) were hypertensive. Among the 40 dogs affected with PDH, 15% (6/40) did not have albuminuria, 52.5% (21/40) had microalbuminuria, and 32.5% (13/40) had albuminuria. Pre- and post-ACTH cortisol concentrations in the dogs affected with PDH were 4.6 ± 2.9 (ranging from 1.9 to 14.1) and 25.5 ± 11.6 (ranging from 21.6-63.1) μg/dL, respectively  were normotensive, and 46.7% (14/30) were hypertensive. Among the 30 dogs with ADH, 10% (3/30) did not have microalbuminuria, 20% (6/30) had microalbuminuria, and 70% had albuminuria. Pre- and post-ACTH cortisol concentrations in dogs affected with ADH were 8.5 ± 6.0 (ranging from 2.9 to 28.7) and 33.2 ± 23.7 (ranging from 23.6-110.2) μg/dL, respectively (Table p = 0.803). However, incidence of hypertension between clinically healthy dogs and dogs affected with HAC was significantly different (p = 0.019). Presence of microalbuminuria and albuminuria in clinically healthy dogs and dogs affected with HAC was significantly different (p < 0.001); significantly higher incidence of microalbuminuria, but significantly lower incidence of albuminuria in clinically healthy dogs compared to dogs affected with HAC. Overall, presence of microalbuminuria or albuminuria was not associated with the status of blood pressure in either clinically healthy dogs or dogs with HAC (p = 0.141).The mean systemic blood pressure was not different between clinically healthy dogs and dogs with HAC (p = 0.004). Incidence of hypertension in dogs affected with PDH and ADH was significantly different (p = 0.017). Presence of microalbuminuria and albuminuria in dogs affected with PDH and ADH was significantly different (p = 0.007). However, albuminuria was not associated with the status of blood pressure (p = 0.306).The mean systemic blood pressure was significantly different between dogs affected with PDH and ADH (p < 0.001). No correlation between systemic blood pressure in either pre-ACTH  or post-ACTH cortisol concentrations  was found.Both pre- and post-ACTH cortisol concentrations between dogs affected with PDH and ADH were significantly different (p = 0.019); incidences of albuminuria followed the same pattern: highest incidence in dogs with ADH, and lowest level in clinically healthy dogs (p < 0.001); but microalbuminuria showed a different pattern: clinically healthy dogs had highest incidence and dogs with ADH had lowest incidence.Overall, the incidence of hypertension was highest in dogs with ADH, followed by dogs with PDH, with the lowest levels in clinically healthy dogs (The incidence of hypertension in dogs affected with HAC has been reported to range from 52.6 (n = 10/16) to 86.1% n = 31/36) [1/36 3,44. In thiIn this study, higher systemic blood pressure and higher incidence of hypertension was associated with ADH compared to PDH. This finding has not been reported in dogs affected with hyperadrenocorticism. However, no correlation between the cortisol concentrations and systemic blood pressure was found. Similar results were also reported in human patients with Cushing's syndrome ,18. The Incidence of microalbuminuria in clinically healthy dogs in this study (45%) was similar to previous studies, ranging from 19 to 52% ,32. SurpIn this study, incidence of hypertension and albuminuria was significantly higher in dogs affected with ADH compared to PDH. This finding has not been reported in dogs affected with HAC. The correlation between systemic blood pressure and albuminuria would be anticipated in dogs with HAC, however, this was not supported in this study. Hypertension is not the only factor associated with presence of albuminuria. Increased urinary albumin excretion rate is also linked to dyslepidaemia, reduced insulin sensitivity, impaired endothelial function and hyper-coagulative status . These cThe cause of albuminuria in dogs affected with HAC can not be answered in this study. Nevertheless, long-term monitoring of albuminuria in terms of treatment and survival time is needed to clarify the importance of albuminuria and HAC in dogs.Incidences of hypertension and albuminuria, but not microalbuminuria, were highest in dogs affected with ADH, followed by dogs with PDH, with the lowest levels observed in clinically healthy dogs. However, the presence of albuminuria was not correlated with systemic blood pressure.The authors declare that they have no competing interests.YHL participated in the designs of the study and carried out the recruitment of cases of hyperadrenocorticism. She also drafted the manuscript. TYH performed measurement of systemic blood pressure, carried out the analysis of urinary albumin and creatinine, and the recruitment of clinically healthy dogs as control in this study. HPH participated in the designs of the study, carried out the recruitment of cases of hyperadrenocorticism and performed the statistical analysis and the manuscript writing. All authors read and approved the final manuscript.


Gastroesophageal reflux is one of the most common causes of chronic cough in the general population. Reflux occurs frequently in patients with cystic fibrosis (CF). We undertook laparoscopic Nissen fundoplication in adult CF patients with a clinical diagnosis of reflux cough who had failed conventional medical therapies.We determined the response to the surgical route in the treatment of intractable reflux cough in CF.Patients with refractory cough were assessed by 24 h pH monitoring and oesophageal manometry. Pre-and post-operation cough, lung function and exacerbation frequency were compared. Cough was assessed by the Leicester Cough Questionnaire (LCQ), lung function by spirometry and exacerbation frequency was defined by comparing the postoperative epoch with a similar preoperatively.P = 0.04), and FVC improved from 2.62 to 2.87 (P = 0.05). Fundoplication lead to a marked fall in cough with the total LCQ score increasing from 11.9 to 18.3 (P = 0.01). Exacerbation events were reduced by 50% post operatively.Significant abnormalities of oesophageal function were seen in all patients studied. 6 patients , with the mean age of 34.5 years consented to surgery. Their mean number of reflux episodes was 144.4, mean DeMeester score was 39.2, and mean lower oesophageal sphincter pressure 12.4 mmHg. There was a small change in the FEV1 from 1.03 L to 1.17 (Whilst there is an obvious attention to respiratory causes of cough in CF, reflux is also a common cause. Fundoplication is highly effective in the control of reflux cough in CF. Significant reduction in exacerbation frequency may indicate that reflux with possible aspiration is a major unrecognised contributor to airway disease. Cystic fibrosis (CF) is a multisystem disease which impacts the digestive system, sweat glands, and the reproductive tract, but progressive pulmonary insufficiency continues to be major cause of morbidity and mortality. The mainGastroesophageal reflux, which is increasingly recognised as one of the most common causes of chronic cough in general population, occurs frequently in patients with CF. About onThe medical treatment of oesophageal reflux causing cough is challenging, particularly in the presence of non-acid reflux. In contrast to the treatment of gastroesophageal reflux disease (GORD) causing heart burn, where acid is a vital component, patients with chronic cough frequently fail to respond to full acid suppression with proton pump inhibitors (PPI) and H2 blockers. In the treatment of resistant chronic cough, Nissen fundoplication has been shown to be an effective tool.We hypothesised that in CF patients with reflux cough not responding to maximal medical therapy, laparoscopic Nissen fundoplication (LNF) could be an alternative approach. Maximal medical treatment was defined as therapeutic trials of the medication listed in table 30 adult patients with CF diagnosed on the basis of clinical presentations and confirmed by sweat tests or genetic investigation, are under the care of the Hull adult CF unit. 18 patients were considered to have symptoms of reflux cough based on a semi-structured questionnaire, (Appendix 1)  and presth of June 2008)For this study ethical approval was waived by the ethics committee of Hull and East Riding, UK.  upper border of the lower oesophageal sphincter (LOS), and presLaparoscopic fundoplication was performed in a standard fashion under general anaesthesia with full muscle relaxation using a five port technique. In every case the oesophageal hiatus was fully dissected and the oesophagus mobilised. At least one non absorbable suture was placed to approximate the crura posterior to the oesophagus and to minimise the risk of post-operative herniation. In cases where there was a large pre-existing hiatal defect, additional posterior crural sutures were placed as required. In most cases there was no obvious hiatus hernia demonstrated at surgery. Calibration of the oesophageal hiatus was clinical but in cases of doubt a 56 Fr endoluminal bougie was available to calibrate the appropriate size.Our primary outcome measure was the change in cough as assessed by self-administered Leicester Cough Questionnaire (LCQ). Pre- and post-operative changes in spirometry and exacerbation rate as defined by an event requiring antibiotics , were secondary endpoints. Results are expressed as means and standard deviations (SD), and the comparisons pre and post- operatively were made by paired t-test with 95% confidence interval (CI), using SPSS software version 13.Of 18 patients with clinically diagnosed reflux cough, four men and two women with a mean age of 34.5 ± 14.7 years, failed to show any improvement with the standard medical therapy of reflux cough. In the 5 patients consenting to oesophageal studies, the mean number of reflux episodes was 144.4 (range from 97 to 178), and pre-operative mean DeMeester score was 39.2 (± 24). The mean resting pressure of lower oesophageal sphincter was 12.4. Reported cough was highly associated with reflux episodes. (Table P = 0.01 (CI: 2.2 to 10.4).Patients were asked to report the severity of their symptom using LCQ pre- and post-operatively. LCQ results were calculated by the sum of scores from each of its three domains. Total LCQ score improved significantly from 11.9 (± 3.2) to 18.3 (± 1.2), P = 0.002 (CI: 1.5 to 3.9). Mean scores from social domain increased from 4 (± 1.4) to 6.5 (± 0.6), P = 0.01 (CI: 0.8 to 4.2). In the psychological domain, scores increased from 4.5 (± 0.9) to 5.7(± 0.5), P = 0.06 (CI of -0.1 to 2.4).  to 6(± 0.4), .002 CI: .5 to 3.9P = 0.04 (95%CI = 0 to 0.25), and forced vital capacity (FVC) improved from a mean of 2.62L (± 0.88) to 2.87L (± 0.96) and P = 0.05.Forced expiratory volume in one second (FEV1) increased from a mean of 1.03L (± 0.32) to 1.17L (± 0.41), P = 0.01) Figure Post fundoplication epoch was compared to the same pre-operation era for the number of exacerbations; which were defined as the need for oral or IV antibiotic therapy. The mean number of exacerbations halved from 8 (± 7.1) to 3.1 ± 2.3). In other words, patients had one exacerbation per two months before the operation, while it was one in 4 months after fundoplication (.3. In otCough is the commonest symptom of medical importance. Chronic cough is a frequent symptom in general population with the incidence of 12%. Whilst aCough is one of the characteristic symptoms of CF, initially presenting as pulmonary exacerbations. In a study of objective cough frequency in 14 CF children with exacerbations, cough rates per hour were reported as 18.2 during the day and 5.8 during the night. In some patients with CF, cough becomes a persistent chronic symptom. Patients will describe two sorts of cough. One productive and associated with exacerbations and a second dry cough usually characterised by irritation in the throat. We sought the clinical history of reflux cough in our patients with chronic cough and were struck by the high incidence of reflux associated features. As a result, we invited them to undergo oesophageal function studies.In the normal western population, excessive acid reflux is a common finding with the prevalence of between 10 to 20%. It is weSuggested mechanisms of excessive reflux included abnormalities of pancreatic and duodenal function leading to increased enteroglucagon levels, and hence delayed gastric emptying. Gastric acid secretion may be excessive. Reflux mIn all of our studied patients acid reflux was confirmed. The recommended strategy in treatment of reflux cough is therapeutic trials; startingFundoplication has been reported as extremely effective in the treatment and management of reflux symptoms. It has bWhilst the effect of fundoplication in CF has not previously been studied, experiences in the context of lung transplantation have shown improvement in episodes of acute and chronic rejection and the serial lung function.,31 TheseTechnically, fundoplication is subtly more difficult in CF patients than in normal subjects with reflux. The tissues are generally more friable and edematous perhaps as a result of previous use of systemic steroids or as part of the systemic disease. In our patients, there were no operative or post-operative complications. Weight loss and dysphagia, recognised operative complications were trivial and short lived.Our experience in the diagnosis and treatment of chronic cough, led us to the realisation that many patients with CF complain of a similar symptom profile indicative of reflux. In common with other reports, these patients have excessive acid reflux which we found to be unresponsive, in terms of cough, to medical anti-reflux therapy. Fundoplication produced a dramatic symptomatic improvement associated with a halving in the rate of pulmonary exacerbations. If this experience is replicated in larger studies, then reflux and aspiration may be demonstrated to be an important previously unrecognised mechanism of disease progression in CF.The authors declare that they have no competing interests.HF carried out data collection, literature review, statistical analysis, and drafted the manuscript. TM carried out data collection on lung functions and looked after the patients during exacerbations. JD looked after patients' diet. WJ carried out the 24 hour pH studies and manometries. PS carried out the operations and post op care. AHM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Using one patient’s data (20 scans) as an example surrogate for a spherical tumor, it was calculated that a 13% change in volume (4.2% change in diameter) could be determined with statistical significance at P=0.05. For this case, cursor positioning error in linear measurement of object size, by even 1 pixel on the CT image, corresponded to a significant change in volume (P=0.05). The precision of volume determination was dependent on total volume. Precision improved with increasing object size (r2 =0.367). Given the small dimensions of the adrenal glands, the ~18% COV is likely to be a high estimate compared to larger tumors. Modern CT scanners working with thinner sections (i.e. <1 mm) are likely to produce better measurement precision. The use of volume measurement to quantify changing tumor size is supported as a more precise metric than linear measurement.This study aims to determine the precision (reproducibility) of volume assessment in routine clinical computed tomography (CT) using adrenal glands as surrogate tumors. Seven patients at our institution were identified retrospectively as having received numerous abdominal CT scans . The adrenal glands were used as surrogate tumors, assuming no actual volume change. Left and right adrenal gland volumes were assessed by hand segmentation for each patient scan. Over 1240 regions of interest were outlined in total. The reproducibility, expressed as the coefficient of variation (COV), was used to characterize measurement precision. The average volumes were 5.9 and 4.5 cm Tumor volume change after therapy is thought to be predictive of therapeutic response for some cancers and cancer treatments.–3 The as8et al reported 12% to 13% measurement variability in pharyngeal cancer imaged on MR./mean) was used to assess inter-observer variability.2), was used to compare the variation in the measured volumes of paired organs over time. All statistics were calculated with standard spreadsheet software . Comparative analyses were made using the t-test with statistical significance assumed at P < 0.05 in all cases.The coefficient of variation (COV), defined as the standard deviation divided by the mean, was used to evaluate the precision in adrenal volume measurement across a series of CT scans. The coefficient of determination, also known as the square of the correlation coefficient  between observers for the three LA and three RA volume assessments was 24.7% and 19.1%, respectively.3, a 20 mm diameter sphere has a volume of 4.2 cm3, and a 30 mm diameter sphere has a volume of 14.1 cm3. The mean volumes of the LA and RA were 5.9 ± 1.6 [standard deviation] cm3 and 4.5 ± 2.1 cm3, respectively, which correspond to equivalent spherical lesions with diameters of approximately 22.5 mm and 20.5 mm. The mean volumes of the LA and RA are provided in 3 (Patient 2) and the largest was 8.0 cm3 (Patient 3).The volumes of the LA and RA are plotted for three patients against the CT scan number in Figures 2=0.367) with COV decreasing as volume increased.The COV of LA and RA volume measurement for each patient is tabulated in 3, SD = 0.64 cm3) from Patient 5 were evaluated in a hypothetical exercise of the statistically detectable change in volume. Using the two-sided paired t-test, it was found that a 13.0% increase in true volume results in a statistical difference at P=0.05, and a 17.8% increase in true volume results in a statistical difference at P=0.01. Assuming isotropic growth of the tumor, these volume changes of 13.0% and 17.8% correspond to linear changes in the dimension of the tumor of 4.2% and 5.6%, respectively.As an example, the 20 RA measurements (mean volume = 3.4 cm2) between LA and RA volume measurements over time, as represented by the series of CT scans performed, was calculated for all patients [The coefficient of determination . If the correlation were high between LA and RA organ volumes, this would suggest that factors other than straightforward measurement precision may be causing temporal changes in measured volumes.As seen in Figures 3). What the results of this work demonstrate is that for objects (e.g. tumors) in the size range of the adrenal glands (2.5 to 9 cm3), a subtle change in volume would be easily observable given the level of measurement precision documented here.The RECIST criterion defines 3, SD = 0.64 cm3) from Patient 5 as an example, a statistically “true” change in volume would be detectable at a 13.0% increase in volume (P=0.05) and at a 17.8% increase in volume (P=0.01). Assuming isotropic growth of the tumor, these volume changes of 13.0% and 17.8% correspond to linear changes in the dimension of the tumor of 4.2% and 5.6%, respectively. A completely spherical tumor with a volume of 3.4 cm3 has a diameter of 12.8 mm. An abdominal CT scan produces pixel dimensions of about 0.68 mm, depending on the field of view (350 mm field of view / 512 pixels). A positioning error of even 1 pixel on the CT image therefore produces a 5.3% change in linear dimension, which is greater than the linear change (4.2%) associated with a spherical lesion growing by a statistically detectable amount (P=0.05), as described above. In other words, when using a linear measurement to measure the change in size of an object with volume close to that of the adrenal glands, small positioning errors, even by a single pixel, in that linear measurement are enough to incorrectly identify a statistically significant change in volume. As a result, this suggests that volume measurements will have more precision than linear measurements of tumor dimension, suffering less from such small errors in outlining fidelity. These findings propose that volume measurement is a more precise measure of volume change than linear measurement and thus support a paradigm shift toward the use of volumetric measurement as a preferred tool for tracking tumor growth.As described in the results, using the 20 RA measurements (mean volume = 3.4 cm3), up to an 18% variability in volume measurement can be expected that is otherwise not attributable to true change in the object’s size. The adrenal glands were used as surrogate tumors, assuming no actual volume change; however, reports on adrenal gland volume as a function of age are conflicting, with some studies reporting no significant volume change over time[3). For Patient 7, whose imaging studies spanned the longest period of time (5.25 years), this model would predict a 3% change in adrenal gland volume over that period, which is much smaller than the 18% volume measurement variability identified in our study.The volume assessment reproducibility of approximately 18% found in this study is consistent with the findings of others who have studied volume assessment of tumors. Van de Langenberg reported 19.7% inter-observer variability in volume assessment of vestibular schwannomas imaged using MR. Studies 1112over time15 and otover time which coThis study also verifies that volume assessment precision improves with increasing object size, as one might expect . This is3 (range: 2.7 cm3 to 8.4 cm3). This result can be interpreted as the expected precision in repeated volumetric measurement of adrenal-sized objects, in a real-world clinical imaging setting. Since volume measurement is less susceptible to cursor positioning fidelity than linear measurement, it more precisely quantifies volume change. These results support the use of volumetric measurement rather than linear measurement in monitoring tumor growth, as in disease progression, or in monitoring response to therapy. This study also showed that precision improves with increasing object size. Clinically, volume measurement should be used to evaluate changes in tumor size, where 18% volume change serves as a threshold for adrenal-sized tumors, defining the measurement precision. Below the threshold, the observed volume change may be due to lack of measurement precision; above the threshold, the measurement may be a true volume change. Such a threshold would be smaller for increasing tumor size. Future studies exploring the dependence of volume measurement precision on scan protocol parameters, such as CT section thickness, the use of contrast agents, window/level settings, and segmentation algorithms, may build upon the findings of this study to better define the precision for specifically defined scan protocols.In this study, a series of 92 CT scans performed serially in seven patients (13 glands) was used to evaluate CT-based volume measurement precision, using the left and right adrenal glands as surrogates for tumors. It was assumed that the adrenal glands did not have true volume changes over the measurement interval for each patient. It was found that the relative precision for human observers hand-outlining lesions, expressed as the coefficient of variation, averaged about 18%  for organs with typical volume of 5.2 cm


To examine the hypothesis that colorectal carcinomas with and without TP53 mutations may be characterised by aetiological heterogeneity, we analysed a group of 107 patients with primary Dukes' C colorectal cancer seen at the Memorial Sloan-Kettering Cancer Center (MSKCC) from 1986 to 1990. We assessed p53 overexpression using the monoclonal antibody PAb 1801, and identified 42 (39%) patients displaying p53-positive phenotype, defined as > or = 25% of positive cells. Patients with two or more first-degree relatives with cancer had an odds ratio (OR) of 2.9 (95% CI 1.0-8.3) for p53 overexpression in comparison with those without a family history of cancer . A possible association between body weight and p53 overexpression was observed. The ORs were 1.9 for the second quartile, 1.9 for the third quartile and 3.4 for the highest quartile in comparison with the lowest quartile . No association between occupational physical activity, smoking, drinking, parity and p53 overexpression was identified. The results suggest that p53 overexpression may be related to genetic predisposition to colorectal cancer, and p53-positive and p53-negative colorectal cancers may be controlled by different aetiological pathways.


To describe the changes in HIV services provided and the patient population utilizing voluntary counseling and testing (VCT) services at private testing laboratories in Chennai, India in 2001 and 2006.In 2001, a cross-sectional descriptive survey was conducted to assess the services provided and client population of 1,031 private laboratories. A subset of labs (9%) that had been surveyed in 2001 were also studied in 2006.In 2001, significantly more high volume labs (>10 HIV tests per month) offered HIV diagnostic tests than low volume labs (<10 HIV test per month) (p<0.001). More high volume labs (20.0%) provided pre-test counseling as part of HIV testing than low volume labs (11.1%) (p = 0.003). Between 2001 and 2006, the number of labs that provided HIV diagnostic tests significantly increased, including ELISA (87.8% vs. 40.0%), Western Blot (84.4% vs. 13.3%), and Tridot (98.9% vs. 72.2%) (p<0.001). Also the number of labs that reported greater than 10 women seeking HIV testing per month significantly increased from 14.5% to 79.0% (p = 0.006). More labs provided pre-test counseling in 2006 (34.4%) than in 2001 (21.1%) (p = 0.046).Though HIV diagnostic testing services have increasingly become available, counseling services have not expanded commensurately. Further outreach and education is necessary to expand comprehensive HIV VCT services in both urban and rural India. It is estimated that 2–3 million Indians are currently living with HIV and the overall HIV prevalence continues to rise Voluntary Counseling and Testing (VCT) is recognized as an integral element of any effective HIV public health primary prevention and care program In India, VCT has been available in certain locations since 1986 Recent studies in India have demonstrated that ongoing counseling and testing can lead to reduced risk taking behaviors and increased condom useThe study was conducted by YRG Centre for AIDS Research and Education, a large tertiary HIV care facility in South India In 2001, a cross-sectional descriptive survey was conducted to assess the services provided and client population of 1,031 private laboratories in Chennai, the capital of Tamil Nadu state in southeastern India with a population of over 7 million. The Chennai metropolitan area was divided into eight zones in which field workers identified private laboratories street by street. Hospitals, nursing homes, and other clinical facilities that may also provide laboratory testing services were also identified within each zone. All consenting laboratory testing facilities were then administered the survey. Labs that performed greater than 10 HIV tests per month in 2001 were defined as high-volume facilities (N = 140), and those with less than 10 HIV tests per month in 2001 were defined as low-volume facilities (N = 891). A subset of labs (9%) that had been surveyed in 2001 were also studied in 2006. This study was conducted among privately operated labs because the primary source of healthcare for Indians has been private facilities.The survey assessed the current availability of basic clinical testing services, STD testing, HIV testing, counseling services, and willingness to build skills in offering VCT services. The survey was administered by outreach staff to consenting lab personnel who provided written consent to take part in the survey. This study received institutional review board (IRB) approval at both the free-standing YRGCARE IRB and the Brown University-affiliated Miriam Hospital IRB.2) statistics and independent t-tests were used. A p value of less than 0.05 was considered statistically significant. Data entry, database management, and statistical analyses were performed with SPSS software .Descriptive statistics were used to calculate the frequency, mean, median, and standard deviation (SD). Diagnostic tests were performed, and the mean+/−SD was used for variables that were normally distributed; and the median and interquartile range (IQR) were calculated for variables that were influenced by extreme variables. To compare proportions, chi-square (χIn 2001 (N = 1031), 64.7% of survey respondents were lab technicians, 20.4% were owners/directors, and 9.2% were physicians; the breakdown of respondents was similar in 2006. The median number of years of operation was 6 years (IQR: 3–12) for low volume labs and 10 years (IQR: 5–17) for high volume labs. The median number of employees was 2 personnel (IQR: 1–3) for low volume labs and 5 personnel (IQR: 2–10) for high volume labs. The mean length of time counselors had been with the lab was 0 years for low volume labs and 1.4 years for high volume labs. In 2001, labs offered the following basic testing services: 68.6% liver function testing, 95.5% hemoglobin, 81.1% complete heamogram, 93.8% renal function testing, 92.1% cholesterol, and 81.0% triglycerides; there were modest increases in these basic testing services in 2006 (N = 90).In 2001, significantly more high volume labs offered HIV diagnostic tests than low volume labs, including ELISA (59.3% vs. 20.0%), Tridot (79.3% vs. 49.6%), and Western Blot (15.0% vs. 2.5%) (p<0.001) See . SignifiBetween 2001 and 2006, the number of labs that provided HIV diagnostic testing significantly increased, including ELISA (87.8% vs. 40.0%), Western Blot (84.4% vs. 13.3%), and Tridot (98.9% vs. 72.2%) (p<0.001) See . In 2006The number of labs receiving greater than 10 physician referrals for HIV tests significantly increased between 2001 (30.0%) and 2006 (78.9%) (p<0.001); and the number of labs receiving greater than 10 walk-ins for HIV testing also significantly increased between 2001 (5.5%) and 2006 (14.4%) (p = 0.034). From 2001 to 2006, labs increasingly received referrals from physicians not in the same area as the lab facility (5.6% to 37.8%) (p<0.001). In 2006, significantly fewer labs (40.0%) referred clients testing positive for HIV to a physician or another institution than in 2001 (57.7%) (p = 0.025). A small minority of labs (<5%) referred clients testing positive for HIV to alternative medical providers both in 2001 and in 2006. More labs provided pre-test counseling in 2006 (34.4%) than in 2001 (21.1%) (p = 0.046).Between 2001 and 2006, more labs reported that the reason clients sought HIV testing was due to migration (8.9% vs. 27.8%), and fewer labs reported job related reasons (91.1% vs. 70.0%) (p = 0.001). In 2001, more high volume labs (60.7%) expressed interest in having a staff member trained in counseling than low volume labs (38.3); however among the 90 labs assessed in both 2001 and 2006, fewer labs (6.7%) in 2006 were interested in having a staff member trained in counseling than in 2001 (58.9%).In addition to an increase in performing HIV testing, assays for other common pathogens significantly increased from 2001 to 2006: 87.8% to 100% offered screening tests for syphilis (RPR/VDRL); 32.2% to 73.3% syphilis confirmatory testing, 31.1% to 81.1% Trichomonas wet mount, 74.4% to 97.8% Hepatitis B Surface Antigen (HBsAg), and 73.3% to 86.7% acid fast bacilli (AFB) smear (p<0.005).This study demonstrates that a considerable number of private laboratories in Chennai currently have the infrastructure to provide comprehensive STD and HIV testing services with a high volume of clients. Though in India public awareness and knowledge of HIV and AIDS remains relatively low Increasing efforts in India have been underway to encourage general physicians to order HIV testing along with other routine lab tests Over three fourths of labs reported over ten women seeking HIV testing in a month in 2006 compared to 2001 when less than a fourth of these same labs reported over ten women seeking HIV testing in a month. In contrast, only close to a third of labs in 2006 reported more than ten men seeking HIV testing in a month. It is possible that increasingly Indian women are seeking HIV testing services at private laboratories as part of antenatal screening. Also, most Indian men may be diagnosed with HIV after presenting to clinical care with an opportunistic infection, and their asymptomatic wives may then be consequently tested for HIVThe labs surveyed in 2001 and 2006 reported that the reasons clients sought HIV testing was due to migratory and job-related reasons. Though the number of labs reporting migration significantly increased from 2001 to 2006 relative to job-related reasons, it is very likely that job-related reasons are the same as migratory reasons, as both reasons provide documentation necessary for Indians to seek work overseas. Currently, there is no mandatory HIV testing for domestic employment neither in the Indian public nor private sectors. However, the Gulf States, which have a large number of South Asian nationals working as guest workers, have required mandatory HIV testing of all foreign nationals since the 1990s Fewer labs in 2006 referred their clients testing positive for HIV to external physicians or institutions than in 2001. It is likely that more private labs provide in-house HIV care and treatment at affiliated institutions, however studies have shown that private Indian institutions may lack adequate training in providing HIV services Though HIV testing services at the most experienced private laboratories have considerably expanded from 2001 to 2006, there is still a substantial underutilization of counseling services as part of HIV testing. As most of the assessed labs are structured primarily as testing centers that receive clients via physician referral, these facilities may not have experience in providing client-centered counseling. Health counseling itself is a relatively new concept in India where openly discussing sexual lifestyles is often taboo and patients may not be proactive in seeking health care services In the context of a lack of adequate universal HIV/AIDS treatment, there are potentially many perceived deterrents to being tested for HIV which can outweigh the benefits of knowing one's status. However it is possible with the increasing availability of ART, more individuals may seek VCT services As the current study documents, VCT services have now become available in urban settings, but increasingly as HIV spreads to rural India, VCT services must also geographically expand. As developing countries, including India, are increasingly able to provide antiretroviral treatment, VCT will become an important tool for linking people to care. The current study showed an increased use of HIV rapid testing with same-day results, which may allow more individuals to receive their test results. It has been suggested that the cost-effectiveness of VCT services can be achieved through supporting VCT services in high-prevalence areas, by linking VCT services to reach high-prevalence populations, and by targeting individuals who are or were sexual contacts of known HIV-infected individuals


Six out of 120 rats fed dioxane in drinking water at levels of 0.75, 1.0, 1.4 or 1.8% developed carcinomas in the nasal cavity. Spontaneous tumors at this tissue localization have not been reported to occur in laboratory animals. The carcinomas were pre-eminently of epidermoid type with few adenocarcinomatous areas and epithelial papillomas. Four rats with carcinoma of the nasal cavity had hepatocellular carcinoma in addition.


Millet protein concentrate was easily digested by trypsin in vitro. Essential amino acids were above the amounts recommended by the Food Agricultural Organization/World Health Organization (FAO/WHO/UNU) for humans. Yellow millet protein concentrate (YMPC) possessed the highest differential scanning calorimetry result , white millet protein concentrate (WMPC) had the lowest . The millet protein concentrates had molecular sizes around 14.4 and 97.4 kDa. They have U-shape solubility curves. Water-binding capacity was in the range of 5.0 and 7.0 g/g, while oil absorption capacity ranged between 4.8 and 5.9 g/g. WMPC had higher bulk density (0.22 g/mL) and emulsifying capacity than YMPC and Soy Protein Concentrate (SPC). Foam capacity and foam stability ranged from 137 to 73 g/mL for WMPC, from 124 to 61 g/mL SPC and from 124 to 46 g/mL for YMPC. Millet protein concentrates are potential functional food ingredients.Two varieties of foxtail millet protein concentrates (white and yellow) were characterized for  The functional properties of plant proteins have been exploited in a multitude of applications  and had resulted into an ever increasing demand for plant protein ingredients with improved processing and functional characteristics. This potential usefulness, however, will also depend on their functional properties, which affect the sensory characteristics of food and play an important role in the physical behavior of food or its ingredients during preparation, processing, and storage. Functional properties include emulsification, foam formation, viscosity, improvement of appearance, texture, and water holding and oil holding capacities. On the basis of these properties, the specific protein selected to be used in a certain food will depend on its required function in the final product ,2. FunctSetaria italica L.) is also known as Italian millet and is one of the world’s oldest cultivated crops. In the northern area of China it has been widely used as a nourishing gruel or soup for pregnant and nursing women, and has been applied in food therapy [Millets typically contain higher quantities of essential amino acids and are higher in fat content than maize, rice, and sorghum . Millet  therapy . Foxtailin vitro digestibility, amino acid composition, differential scanning calorimetry (DSC), protein solubility, molecular size, foaming, emulsifying, bulk density water and oil holding capacity of the two varieties of defatted foxtail millet protein concentrates compared to commercial soy protein concentrate.The digestibility of the nutrients must be known in order to evaluate fully the significance of nutrient concentration. Protein functional properties are determined to a large extent by a protein’s physicochemical and structural properties . Protein2.2.1.The two varieties of foxtail millet (white and yellow) were purchased from a local market in Wuxi, China. All chemicals used in the experiments were of analytical grade. The foxtail millet (1 kg) was defatted twice with hexane for 8 hours at room temperature. The defatted white millet flour (DWMF) and the defatted yellow millet flour (DYMF) were air-dried for 24 hours under a vacuum drier. The defatted flour was milled using a laboratory-scale hammer miller and the resulting flour was sieved through a 60 mesh screen and stored at 5 °C in sealed glass jars until used. The commercial soy protein concentrate was purchased from a supermarket in China, produced by Fuxin Flour Mill Co. Ltd .2.2.The proximate composition of white millet flour (WMF), yellow millet flour (YMF), DWMF and DYMF was determined according to the method described by Ceirwyn . The moi2.3.2SO4 was added. The mixture was heated under reflux for 1 hour with the heating mantle. The hot mixture was filtered through a fibre sieve cloth. The filterate obtained was discarded and the residue was returned to the fibre flask to which 100 mL of (0.31 N NaOH) was added and heated under reflex for another 1 hour. The mixture was filtered through a fibre sieve cloth and 10 mL of acetone added to dissolve any organic constituents. The residue was washed twice with about 50 mL hot water on the sieve cloth before it was finally transferred into a crucible. The crucible and the residue were oven-dried at 105 °C overnight to drive off moisture. The oven dried crucible containing the residue was cooled in a dessicator and later weighed to obtain the weight W1. The crucible with weight W1 was transferred to the muffle furnace for ashing at 550 °C for 4 hours. The crucible containing white or grey ash  was cooled in the dessicator and weighed to obtain W2. The difference W1–W2 gives the weight of fibre . The percentage fibre was obtained by the formula:The sample (2 g) was accurately weighed into the fibre flask and 100 mL of 0.25 N H2.4.Defatted millet flour protein concentrate was prepared according to the procedure described by Olayide , with so2.5.® ninhydrin reagent was added with post column instrument . The light absorbance of amino acids was detected with an UV Visible detector (Pickering Laboratories Inc.) at 570 nm wavelength and the amino acids were quantified by comparing with standard amino acid profiles. Methionine and cysteine were determined separately by oxidation products according to the performic acid procedure of Moore [o-phthalaldehyde derivatization followed by fluorescence detection by Ravindran and Bryden [For the determination of the amino acids, samples of protein concentrate (100 mg) for all the samples and 5 mL 6 M HCl were added to a 50 mL stopper bottle and sealed. The air was removed by keeping the sample in a vacuum chamber. The sealed samples were placed in an oven at 120 °C for 16 hours to hydrolyze. After hydrolysis, 5 mL of 2 mM norleucine internal standard was added and the solution was filtered in a 0.2 μL Gelman membrane filter. 1 mL of stock sample was pipetted into a 50 mL borosilicate glass serum bottle and dried in a freeze-drier. 1 mL of sodium diluent buffer (pH 2.2) was added to the freeze-dried residue and transferred to a 1.5 mL micro-centrifuge tube for HPLC analysis. The prepared samples were injected as 2.5 μL volumes and run on a Waters HPLC  at a flow rate of 0.4 mL/min with a Pickering sodium ion-exchange column of 4 × 150 mm  and sodium eluent (pH 3.15 and 7.40). TRIONEof Moore  before hd Bryden . Amino a2.6.SDS-PAGE was done according to the method described by Laemmli , with 122.7.In vitro digestibility was carried out according to the method described by Elkhalil et al. [l et al. , with slWhere: A: total protein content (mg) in the sample.B: total protein content (mg) in TCA precipitate.2.8.Protein solubility was determined according to the procedure of Bera and Mukherjee , with slWhere: PS: Amount of protein in supernatant.PIS: Protein in initial sample.2.9.et al. [Foaming capacity was evaluated by the method of Bernardi Don et al. , with mi2.10.et al. [The emulsifying capacity was determined by the method of Yasumatsu et al. . The sam2.11.Sample (0.5 g) was taken and mixed with 3 mL of distilled water or refined groundnut oil. The slurry was centrifuged at 750 × g for 15 min. The pellet was drained for 30 min and the gain in weight per unit weight was reported as water or oil absorption capacity (g/g), respectively.2.12.A known weight of the protein concentrate was added to a graduated measuring cylinder. The cylinder was gently tapped and volume occupied by the sample was determined. Bulk density was reported as weight per unit volume (g/mL).2.13.Thermal properties of protein concentrates were evaluated using differential scanning calorimetry . 70 mg of the various samples were dissolved in 1 mL of 0.05 M phosphate buffer (pH 7.0) containing 0.1 M NaCl. The protein solutions (45 μL) were transferred and hermetically sealed in a stainless steel pan. The samples were heated by scanning from 25 to 135 °C at a rate of 10 °C per min against a reference containing 45 μL buffers without protein in a differential scanning calorimeter (Perkin-Elmer Corp.) The denaturation peak temperature and enthalpy were calculated by a thermal analysis data software program.2.14.Data were analyzed by analysis of variance (ANOVA) using SAS statistical software package . Each value was determined by at least three replicates. Results were given as mean ± standard deviation.3.3.1.The results of the proximate chemical composition of WMF, YMF, DWMF and DYMF are shown in 3.2.in vitro trypsin digestibility, amino acid composition, protein solubility, thermal properties, molecular size, emulsifying, foaming, water and oil holding and bulk density properties.The protein content of the white and yellow millet protein concentrates obtained have a protein content of 80.04 and 75.69% respectively. The millet protein concentrates were used for the investigation of 3.3.The essential amino acid composition of foxtail millet flour, millet protein concentrates and soy protein concentrate are shown in 3.4.WMPC and YMPC revealed polypeptides of a wide range of molecular weights. WMPC and YMPC showed slight variation in the banding patterns. WMPC contain about six polypeptides with estimated molecular weight ranged from 14.4 to less than 66.2 kDa . However3.5.In vitro trypsin digestibility of WMPC and YMPC were determined and compared to that of SPC (P < 0.01) t of SPC . WMPC wa3.6.et al. [The pH-protein solubility profiles of WMPC, YMPC and SPC had very similar solubility profiles, exhibiting a U-shaped curve in which the WMPC had highest solubility values at alkaline pH. In acidic condition, all proteins had solubility (above 40%) at pH 2.0, but SPC has lower solubility than WMPC and YMPC at pH 4.0 and pH 5.0 (less than 20%). At pH 6.0 or above, all proteins dissolved between (50% and 80%), with WMPC having slight higher solubility than SPC and YMPC . These tet al. . The max3.7.P < 0.01) than that of SPC. The foam capacity WMPC, SPC and YMPC, ranged from 137, 124 and 124 g/mL, respectively  were observed between WMPC, YMPC and SPC. Petruccelli et al. [et al. [SPC have significantly lower emulsifying activity than WMPC and YMPC . Emulsifi et al.  reportedi et al. . The hig [et al. , which c3.9.P < 0.01). SPC possessed the lowest water binding capacities (5 g/g), followed by YMPC (6 g/g) and WMPC had the highest (7 g/g). Chandi and Sogi reported much lower values of 3.87 g/g [The water absorption capacity (WAC) of the WMPC, YMPC and SPC ranged from 7, 6 and 5 g/g, respectively , but wer3.87 g/g . Interac3.87 g/g . In foodP < 0.01). WMPC possessed oil absorption capacity similar to SPC   . WMPC ha3.11.et al. [DSC is a rapid, easy, and capable technique for supplying both thermodynamic  and kinetic data (reaction rate and activation energy) on protein denaturation, and has been used extensively in various food systems. The information on protein thermal properties is useful for food-processing strategies and heat-processing design . Data fret al.  reportedet al. .4.Defatted foxtail millet protein concentrates from both varieties (white and yellow) exhibited substantial amounts of protein content. Furthermore, the results of the protein characterization show that millet protein concentrate is a potential functional food ingredient. SPC was more digestible than WMPC and YMPC. The essential amino acid pattern of foxtail millet protein concentrates suggests their possible use as a supplementary protein source to most cereals because this protein is rich in lysine, which is the first limiting amino acid in most cereals. The two concentrates varied in their denaturation temperatures and revealed a wide range of molecular weights polypeptides. The three samples showed lower solubility at pH 4.0 and 5.0. WMPC exhibited the highest solubility. WMPC and YMPC have higher emulsifying capacity, foam capacity and stability than SPC. Millet protein concentrates thus have excellent applications for future product development by virtue of their functional properties.


The 22q11.2 microdeletion syndrome is a common condition that is associated with cardiac as well as extra-cardiac manifestations. Its prevalence and manifestations from north India has not been reported. This study was designed to determine the prevalence and ability of clinical criteria to predict 22q11.2 microdeletion.et al (1999).A total of 146 cases of cardiac malformation requiring tertiary care at a teaching hospital were prospectively screened for 22q11.2 microdeletion using fluorescence in situ hybridization test. Detailed clinical information was obtained as per guidelines of Tobias, Nine out of 146 patients (6.16%) was found to have 22q11.2 microdeletion. All the positive patients showed the presence of extra-cardiac features of 22q11.2 microdeletion syndrome. None of the cases with isolated cardiac defect were positive for microdeletion.It seems that 22q11.2 microdeletion syndrome is over-suspected in children with isolated congenital heart defects. Screening for 22q11.2 microdeletion should be considered in those cardiac malformation cases which have extra-cardiac manifestations in the form of facial dysmorphism and hypocalcaemia. The 22q11.2 microdeletion syndrome is characterized by hemizygous microdeletion of 22q11.2 region of chromosome 22. It occurs at a frequency of 1 in 4,000 to 6,000 live births . The 22qSeveral studies have tried to ascertain the incidence of this condition in general population ,5,6; andFrom August 2006 to March 2010, 146 cases of structural heart defects, with or without extra-cardiac anomalies including dysmorphic features, that required tertiary care management at the All India Institute of Medical Sciences prospectively enrolled into the study. They were referred from various States of north India for tertiary care management. All the patients have undergone cardiac catheterization or operative procedure as required for their condition. A few older patients were recruited from the outpatients. The study was approved by the Institutional Human Ethics Committee.et al (1999) [All patients underwent cardiac, as well as clinical genetics evaluation including echocardiography and CT/catheter angiography. Clinical genetics evaluation was carried out as per guidelines of Tobias, l 1999)  . Interphase cell suspension was prepared by standard method . Blood nhttp://www.biologia.uniba.it/rmc/; courtesy Professor Mariano Rocchi) for the study. The clones were received as bacterial LB agar stab culture, which were sub-cultured on LB agar plate before growing in large amount in LB medium. Probe DNA was extracted using a commercial BAC extraction kit . All probes were labelled using nick translation method with FITC-12-dUTP (Roche) or TRITC-12-dUTP (Roche) or Cy3 . Working concentration of probe DNA was between 100-200 ng/μl.Microdeletion status was determined by FISH using non-commercial DNA probes. Bacterial artificial chromosome (BAC)/Phage artificial chromosome (PAC) clones RP5-882J5 (22q11.2), RP11-22M5 (22q11.22) & CTA-154H4 (22q11.21) spanning approximately 2 mega base (Mb) in length from 22q11.21 through 22q11.23  were obtained from European Resource Centre for Molecular Cytogenetics, University of Bari, Italy Slide was washed in acetic acid for 2 min, and dehydrated in 70, 90, 100% ethanol, 3 min each. Nuclei on the slide were digested with pepsin (100 mg/ml) in 0.01 N HCl for 20 min at 37°C, rinsed in double distilled water and followed by PBS, and fixed in 1 % paraformaldehyde in PBS for 10 min at 4°C. Slides were then rinsed in PBS, twice in double distilled water and then dehydrated through ethanol series as before. The hybridization buffer  containing labelled probe was applied to the slides under a circular cover slip (11 mm in diameter). The probes and nuclear DNA were denatured together at 76°C for 6-7 min. Hybridization was performed in a dark moist chamber at 37°C for overnight. After hybridization, cover slips were removed and slides were washed with NP40 (0.03% solution) at 72°C for 2-3 min, followed by NP40 (0.01% solution) for 2 min at room temperature. Then slides were dehydrated in ethanol series, as before and mounted in antifade  with 1 μg/ml 4,6 diaminidino-2- phenylindol . The slides were screened under Olympus BX 51 fluorescent microscope with 100 watt mercury bulb using 100× plan-apochromatic objective and single band pass filter for DAPI, FITC and TRITC (Olympus Japan). FISH image was captured through spectral imaging system. A total of at least 1000 interphase nuclei and at least 10 metaphase nuclei were scored from each case. Presence of two signals in 100% metaphases and 90% interphase cells was considered as normal whereas demonstration of one signal in 100% metaphases or 85-90% interphase cells was considered as microdeletion positive. When presence of both one and two signals in interphase (15% deleted) as well as in metaphase (10% deleted) cells was observed, the case was considered to have mosaicism.A total of 146 consecutive cases with congenital structural heart defect requiring surgical treatment were studied by FISH. The mean age of the patients was 7.7 years, range 5 days to 29 years. There were 97 males and 49 females. Among 146 cases seventy six patients had conotruncal heart defect and 70 had non-conotruncal heart defect . All microdeletion positive cases had characteristic extra-cardiac manifestations besides cardiac defect as tetralogy of Fallot Table . This frThis syndrome is inherited from parents in 5-10% of cases with 50% risk for transmission. Hence, it is recommended  to consider FISH analysis for possible 22q11.2 microdeletion to parents of all affected child. It seems also reasonable to offer prenatal diagnosis where required.It is concluded that 22q11.2 microdeletion syndrome is over-suspected in children with isolated congenital heart defects. We recommend routine FISH testing in cases with conotruncal congenital heart disease associated with extra cardiac anomalies .Written informed consent was obtained from the parents of patients for publication of cases and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.AH formulated activity plan, checked & interpreted results, besides being the Principal investigator of the project funded by Indian Council of Medical Research, India. He also reviewed clinical findings, prepared manuscript and responded to the queries of reviewers. He is the guarantor of the manuscript. MJ carried out all FISH related activity under guidance of AH, besides working as Research fellow for the project under AH. IC also carried out FISH related activity under guidance of AH. MK was involved in clinical management of the cases. All authors read and approved final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/101/prepub


Lung cancer accounts for 4% of all newly diagnosed cancers in Saudi Arabia. The pattern of presentation is unknown. The objectives of this study were to assess the clinical, radiological, pathological, biochemical and bronchoscopic abnormalities in lung cancer patients and to compare our findings with those reported in the literature.A total of 114 patients with proven lung cancer were selected for the study. A questionnaire concerning patients' demographic data was obtained; the abnormalities and the cell types of lung cancer were recorded prospectively in each subject.P ≤ 0.001)A total of 114 patients with lung cancer were studied. Mean age ± SD was (59.8 ± 10.8) years, and (71.1%) were smokers and 95.1% of them were male, (90.1%) smoked >20 pack/yr (96.2%) for 20 years or more. Cough (76.3%) and clubbing (40.4%) were the most common symptom and physical abnormality respectively. The right lung (64.9%) was more commonly affected than the left (37.7%). Metastases were present in (49.1%) at presentation. The right and left upper bronchi (24% vs. 16%) were the mostly affected. Hypercalcemia was more common in squamous cell, while hyponatremia was more common in adenocarcinoma, and small cell. Squamous cell carcinoma was the most common cell type (51.8%) and significantly associated with smoking (Squamous cell carcinoma was the most common cell type, and significantly associated with smoking. The incidence of metastasis was high at presentation. The right lung and right upper bronchus were often affected. Hypercalcemia and hyponatremia were the most common biochemical abnormalities. Though tThis is a four year prospective study that was performed from October 2004 to September 2008 at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia. Ethical approval of the study was granted from the Internal Review Board at KAUH. All participants gave written informed consent. A total of 140 consecutive patients with highly suspicious primary lung cancer detected clinically and radiologically were included for the study. The initial work up to diagnose lung cancer was either through performing flexible fiberoptic bronchoscopy (FFB), and/or computed tomography (CT)-guided biopsy. FFB was used primarily to diagnose central lung lesions while CT-guided biopsy was performed mainly to diagnose peripheral ones. Open lung biopsy was performed if bronchoscopy and/or CT guided biopsy had failed to obtain a diagnostic tissue. Mediastinoscopy and or pleural biopsy were performed when indicated to reach diagnosis. Total body scan of the bones and CT scan of the brain were used to assess the presence of metastasis. After the initial work up, a total of 26 patients were excluded from the study, 24 due to the benign nature of their illness and two withdrew from the study. A total of 114 patients with proven histopathology of primary lung cancer were interviewed, and clinically examined by the principle investigator (P.I.). A questionnaire concerning patient's demographic data, and smoking status as in 23P value <0.05 throughout analysis.Data management was carried out using statistical package for social science SPSS 10 . DescriptP = 0.051, while adenocarcinoma showed a higher frequency among females (48.0%) as compared to males (21.3%), [P = 0.051. Clearly Squamous cell carcinoma was significantly associated with smoking as compared with adenocarcinoma, P ≤ 0.001 [P = 0.312 [P = 0.239 [A total of 114 lung cancer patients were studied, 78.1% were males. Mean age ± SD was (59.8±10.84) with a minimum age of 42 years and maximum of 80 years. A great proportion (71.1%) of patients were cigarette smokers, (95.1%) were male and more than half of them were heavy smokers >36 pack/yr. Less than 10% of the patients were shisha smokers (hubbly bubbly) . Table 2(21.3%), , P = 0.0 ≤ 0.001 . Table 8 = 0.312 . In cont = 0.239 .vs. 19%) and clubbing was more common in nonsmall cell lung cancer than small cell lung cancer (35% vs. 4%).[vs. 11%); however, we report similar findings of clubbing being more common in non-small cell lung cancer than small cell lung cancer (87% vs. 17).[26vs. 38%) respectively. Furthermore, the right and left upper lobes had the highest incidence of lung cancer (60% vs. 74%) radiologically as compared with middle  and lower lobes  respectively. It was not clear why the upper lobes were more susceptible to lung cancer than other lobes. However, it is well known that upper lobes are less vascular, and more aerated than the lower ones, and more affected by smoking, therefore, we question whether some or all of these factors may have an effect on the distribution of the lung cancer among affected patients. In this study, lung mass, atelectasis/consolidation and pleural effusion were the most common abnormalities found as detected by chest radiographs  result that rose to  by CT scan of the chest respectively. Furthermore, CT scan of the chest is more sensitive than chest radiographs in detecting early stage of lung cancer and in assessing the pattern of distribution of affected lymph nodes in patients with lung cancer. Mediastinal lymph nodes have the highest incidence of involvement in lung cancer (51%) followed by the hilar (30%) abnormalities that can't be assessed clearly in chest radiographs.[2626vs. 16%) followed by right middle bronchus and lingula (12% for each) while the least affected sites were the right and left lower bronchi (9% vs. 6%). The reported pattern of distribution of lung cancer in which upper lobes were affected more than middle ones and the middle ones were affected more than lower ones was a very interesting observation not previously reported. CT guided transthoracic needle aspiration (TTNA) is the procedure of choice for sampling peripheral lung lesion with a diagnostic accuracy of 80–95%.[372426724444629The main findings were as follow: i) The most common presenting symptoms of lung cancer was cough 76.3%) followed by dyspnea (67.5%). ii) The most common physical signs were: clubbing of the fingers 40.4%) and pallor. iii) The right lung (64.9%) was affected more often than the left 37.7%), and lung mass (69.3%) and pleural effusion (35.1%) were the most common radiological abnormalities. iv) Almost 50% of the patients had distant metastasis at the time of diagnosis. V) Squamous cell carcinoma was the most common (51.8%) cell type followed by adenocarcinoma 27.2%). The clinical presentations of lung cancer result from the effects of local growth of the tumor, regional growth or spread through the lymphatic system, hematogenous distant metastatic spread, and remote paraneoplastic effects from tumor products or immune cross-reaction with tumor antigens.–27 In th.2%. The 262626262626 vs. 4%).31 In ours. 17).26 Chest ras. 17).26–34 In oubes 27%, % respectgraphs.26 In our pbes 27%, % respectaphs.2626 Almost 3.7%, and aphs.262640 The ov.4% and paphs.262640 In our80–95%.37242644 In5%.37242644 In KSA6.3% foll%.3724267244445 sq


The rising prevalence of obesity has had a profound impact on female reproductive health. Increased body mass index (BMI) is associated with ovulatory subfertility and anovulatory infertility. Overweight and obese women have poorer outcomes following fertility treatment. They respond poorly to clomiphene induction of ovulation and require higher doses of gonadotrophins for ovulation induction and superovulation. Ovarian stimulation for assisted reproduction produces fewer follicles resulting in the harvest of fewer oocytes. Fertilization rates are poorer and the embryo quality is impaired in younger women who are obese. Pregnancy rate in some studies is lower and there is an increased risk of early pregnancy loss. Weight loss regularizes menstrual cycles and increases the chance of spontaneous ovulation and conception in anovulatory overweight and obese women. Gradual sustained weight loss is beneficial whereas crash dieting is detrimental. These figures are expected to rise to 2.3 billion and 700 million, respectively, by 2015.. These women also face a higher risk of cycle cancellation [OR 1.86 (95% CI: 1.13–3.06)] and are less likely to ovulate [OR 0.44 (95% CI: 0.31–0.61)]. However,2, the odds ratio of cycle fecundity for women with BMI ≥25<30kg/m2 was 0.70 (95%CI 0.35-1.40) and those for women with BMI>30 kg/m2 was 0.87 (95% CI. 0.35-2.18). in comparison with those with BMI of <25 kg/m2. Gonadotrophin requirements were higher  in obese women (BMI>30 kg/m2) when compared to non obese women.. The combined odds of pregnancy were 1.47  for a woman with a BMI 20-30 kg/m2 as compared to women with a BMI of >30 kg/m2.  37] and 2 were 1.33(95% CI: 1.06-1.638). The odds of miscarriage were 1.53(95% CI: 1.27-1.84)[2 when compared to women of BMI< 30 kg/m2. However, the authors of this meta-analysis were unable to adjust for confounders such as age and co-morbidities such as PCOS as individual patient data were not available to them.A systematic review of literature showed that when compared with women of BMI<25, the odds of miscarriage in women with BMI of ≥ 25 kg/m2  following spontaneously conceived pregnancies as well as following ovulation induction .[Another recent meta-analysis of the available evidence suggested that there was a significant increase in the odds of miscarriage in women with a BMI of ≥ 25 kg/m6–14.83). However,6–14.83). to calcu2 had an ongoing pregnancy rate per cycle of 45.5%, compared with 38.3% for those with BMI> 25 kg/m2.[The reasons behind an increased risk of miscarriage amid overweight or obese women have been debated. It has been suggested that this is due to impaired folliculogenesis and poor oocyte quality in obese women. The proponents of this view cite a cohort study which concluded that uterine receptivity was unimpaired in women with increased BMI when hormonal support and embryo quality were standardized. The alte25 kg/m2. In anoth25 kg/m2.Most authors agree that there is an increased risk of miscarriage in overweight and obese women after spontaneous conception; however, any further increase in risk after IVF or ICSI is debatable. The reasons of an increased risk of miscarriage could possibly be the higher prevalence of PCOS among overweight and obese women.2 to 35 kg/m2.[2, although in those younger than 37 years with normal FSH, weight reduction to BMI less than 30 kg/m2 is preferred.[Due to the adverse impact of BMI on treatment outcomes, there have been calls for restricting publically funded fertility treatment to women with high BMI. The threshold BMI used as a cut off value for restricting access to publically funded fertility treatment varies around the world. Across the UK, there is no uniformity of practice with cut-off values for access to IVF varying from 30 kg/m35 kg/m2. The NICE35 kg/m2. British referred. A 5-10% Available data suggest that as little as 5%–10% weight loss can improve fertility outcomes. Other stWeight loss can be achieved by lifestyle modification, dietary restriction, physical activity and pharmacotherapy with varied results. Life style modification programs  have been shown to be associated with poor levels of compliance and are In a large randomized controlled trial pharmacological measures like metformin, at a dose of 850 mg twice daily, have not been shown to affect menstrual frequency, body weight or insulin sensitivity, despite a fall in total testosterone and waist circumference. There haDecision making around postponement of fertility treatment to allow weight loss to take place needs to accommodate the effect of further increase in age in older women. A recent study demonstrated that the effect of BMI on IVF success was strongly influenced by age. With increasing age, the effect of BMI alone was attenuated such that, it became less influential than age, in women aged 36 years or older.Obesity in women has impacts on fertility and fertility treatment. Increase in BMI reduces the chance of conception in ovulatory women and affects the outcome of ovulation induction treatment.Obese women undergoing IVF require higher doses of gonadotrophins, respond poorly to ovarian stimulation and have fewer oocytes harvested. Obesity is associated with lower fertilization rates, poor quality embryos and higher miscarriage rates. Weight loss in these women improves their reproductive outcomes; however, in order for this to be effective it has to be gradual and sustained.


In pain processing, long term synaptic changes play an important role, especially during chronic pain. The immediate early gene Arc/Arg3.1 has been widely implicated in mediating long-term plasticity in telencephalic regions, such as the hippocampus and cortex. Accordingly, Arc/Arg3.1 knockout (KO) mice show a deficit in long-term memory consolidation. Here, we identify expression of Arc/Arg3.1 in the rat spinal cord using immunohistochemistry and in situ hybridization following pain stimuli.de novo expressed in dorsal horn neurons after nociceptive stimulation. Expression of Arc/Arg3.1 was induced in an intensity dependent manner in neurons that were located in laminae I (14%) and II (85%) of the spinal dorsal horn. Intrathecal injection of brain derived neurotrophic factor (BDNF) also induced expression of Arc/Arg3.1. Furthermore, 90% of Arc/Arg3.1 expressing neurons also contained the activity marker c-Fos, which was expressed more abundantly. Preproenkephalin mRNA was found in the majority (68%) of the Arc/Arg3.1 expressing neurons, while NK-1 was found in only 19% and GAD67 mRNA in 3.6%. Finally, pain behavior in Arc/Arg3.1 KO mice was not significantly different from their wild type littermates after application of formalin or after induction of chronic inflammatory pain.We found that Arc/Arg3.1 is not present in naïve or vehicle treated animals, and is We conclude that Arc/Arg3.1 is preferentially expressed in spinal enkephalinergic neurons after nociceptive stimulation. Therefore, our data suggest that Arc/Arg3.1 dependent long term synaptic changes in spinal pain transmission are a feature of anti-nociceptive, i.e. enkephalinergic, rather than pro-nociceptive neurons. The experience of pain is usually initiated by the activation of nociceptors, which are the peripheral terminations of nociceptive ganglion neurons. The central projections of these neurons enter the dorsal horn of the spinal cord to terminate on second order neurons . After sde novo expression of Arc/Arg3.1 in a limited number of neurons in the superficial dorsal horn, depending on the type of stimulus. Further, Arc/Arg3.1 is predominantly expressed in spinal interneurons located in lamina II and many of these neurons also contain the opioid neurotransmitter enkephalin. Finally, we found that the pain behavior in Arc/Arg3.1 knockout (KO) mice after nociceptive stimuli was not significantly different from their wild type (WT) littermates.Our findings show that Arc/Arg3.1 is not expressed at detectable levels in naïve spinal cord. However, after peripheral nociceptive stimulation we found In the spinal cord of naïve rats and mice there was no detectable expression of Arc/Arg3.1 mRNA or protein when using in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. However, after application of a peripheral nociceptive stimulus to the hind paw, Arc/Arg3.1 was expressed in a limited number of cells in the superficial layers of the lumbar dorsal horn. ISH using the alkaline phosphatase (AP) reaction produced a bluish/brownish reaction product in the cytoplasm and in some occasions in the nucleus and primary dendrites of Arc/Arg3.1 mRNA expressing neurons Fig. . Arc/ArgFor both ISH and IHC, we observed that the intensity of the labeled neurons varied in a single section. We did not observe any labeling indicative of localization of Arc/Arg3.1 in intermediate or distal dendrites. Although labeling patterns obtained with ISH and IHC were identical, labeling efficiency was higher for ISH than for IHC. Therefore, ISH was used for the quantification of neurons expressing Arc/Arg3.1. The specificity of our detection techniques was assessed by omitting the probes/primary antibodies in the ISH and IHC procedures, respectively, and by applying ISH and IHC on spinal tissue of Arc/Arg3.1 KO mice. These experiments did not show any labeling in the spinal cord. ISH performed on cortex of naïve rats showed Arc/Arg3.1 mRNA labeling in the cortex and the hippocampus as previously reported .Several types of nociceptive stimuli applied to the hind paw induced Arc/Arg3.1 mRNA expressing neurons on the ipsilateral side Fig.  but not The number of neurons expressing the neuronal activation marker c-Fos or Arc/Arg3.1 mRNA was counted in separate sections treated with IHC or ISH, respectively. c-Fos labeled neurons outnumbered Arc/Arg3.1 mRNA labeled neurons Fig. , except p>0.05, unpaired t-test)  Fig. .In this experiment, the colocalization of Arc/Arg3.1 with various neuronal markers was investigated Fig. . We founIn order to identify Arc/Arg3.1 in inhibitory neurons, FISH for GAD67 mRNA, the specific marker for GABAergic neurons, and fluorescent IHC for Arc/Arg3.1 protein were combined. Very few of the Arc/Arg3.1 labeled neurons were GABAergic  . c-Fos expression after BDNF + NBQX injection was also not significantly different from BDNF injection alone (p= 0.08 for lamina II)  induced Arc/Arg3.1 mRNA expression in the superficial dorsal horn neurons . We found that 45% ± 8 of Arc/Arg3.1 mRNA labeled neurons were located in lamina I and 55% ± 8 in lamina II. 93.6% ± 2.5 of Arc/Arg3.1 mRNA labeled neurons expressed NeuN, 55.6% ± 9.1 expressed c-Fos, and 16.8% ± 6.4% expressed NK-1. Since it has been shown  that admII) Fig. . IntrathFreely moving Arc/Arg3.1 knockout (KO) mice did not display any overt behavioral abnormalities in comparison with their wild type (WT) littermates, as reported previously . With rep>0.05) was found between the WT and Arc/Arg3.1 KO mice in licking or fluttering behavior  nor the total numbers of flutters (WT 100 ± 21 (SEM); KO 128 ± 32 (SEM); p>0.05, unpaired t-test) were significantly different. In addition, c-Fos expression due to the formalin injection did not appear different from the c-Fos expression in the WT mice.Subcutaneous injection of formalin in the hind paw induced a two-phased pain behavior in both WT and Arc/Arg3.1 KO mice, consisting of licking and fluttering of the injected paw. In both groups, the first phase was apparent in the first 10 minutes after injection, and the second phase began 25 minutes after injection with licking as the prominent behavior. No significant difference  and immunohistochemistry (IHC) to show that nociceptive stimulation induced Arc/Arg3.1 mRNA and protein in the superficial dorsal horn of the spinal cord. Both techniques specifically identified Arc/Arg3.1 since standard controls, most notably nociceptively stimulated spinal cord of Arc/Arg3.1 knockout (KO) mice, did not show any specific labeling. In naïve or vehicle treated animals expression of Arc/Arg3.1 mRNA and protein was absent in the spinal cord, in agreement with a study using RT-PCR . This stpocampus  and cortpocampus .Arc/Arg3.1 mRNA and protein were induced in the superficial dorsal horn in the acute phases of all pain models that we tested, i.e. after nociceptive stimulation with capsaicin, CFA, formalin and mustard oil. Injection of CFA induces an inflammatory process  that leaNeurons expressing Arc/Arg3.1 in the spinal cord are most likely driven by direct input from afferent nociceptive fibers that use glutamate as their main neurotransmitter . Apart fFollowing nociceptive stimulation, Arc/Arg3.1 was often expressed in activated neurons as identified by c-Fos. Especially after nociceptive stimulation with capsaicin, and after chronic inflammatory pain, the number of neurons expressing Arc/Arg3.1 is low as compared to those showing c-Fos expression. This finding may be interpreted to indicate that Arc/Arg3.1 is only expressed in activated neurons that received the strongest input from nociceptive fibers. This assumption is in line with our finding that Arc/Arg3.1 expression is intensity dependent. On the other hand, there may be specific subpopulations of spinal nociceptive neurons that are capable of producing Arc/Arg3.1, while others are not. In search of such a neuronal subpopulation that specifically expressed Arc/Arg3.1, we focused on neurons that were characterized by the expression of the neurokinin-1 (NK-1) receptor, Protein Kinase C gamma (PKC-γ), calbindin, GAD67 or preproenkephalin. We found a high percentage of Arc/Arg3.1 expressing neurons (68%) to contain preproenkephalin, while percentages of colocalization with other markers were less prominent . NK-1 expressing neurons project to supraspinal sites  and are In order to understand the functional role of Arc/Arg3.1 in enkephalinergic neurons at the behavioral level, we employed Arc/Arg3.1 KO mice and their WT littermates. The only significant difference between these mice was that in the hotplate test the thermal threshold of naïve Arc/Arg3.1 KO mice was significantly higher as compared to naïve WT mice. This finding is difficult to interpret since naïve WT mice, like their KO littermates, do not show Arc/Arg3.1 expression in the spinal cord. One explanation may be that there is a very low basal expression of Arc/Arg3.1 that we and others  were notSeveral studies have shown that in hippocampus knockdown of Arc/Arg3.1 leads to enhanced LTP in the early phase but impaired consolidation of LTP and long term depression (LTD) in the late phase . In the The low number of spinal projection neurons that express Arc/Arg3.1 may be explained by the fact that, in contrast to other areas of the brain, structural long-term changes in the excitability of these spinal neurons are counterproductive if they persist after the healing process has been completed. Our finding that Arc/Arg3.1 is expressed predominantly in enkephalinergic neurons may suggest that in these neurons long term changes are actually consolidated. However, Arc/Arg3.1 KO mice that lack consolidation of long term changes show normal pain behavior. This would not exclude that enkephalinergic neurons, which have an inhibitory effect on pain transmission, may serve as an anti-nociceptive mechanism against strong nociceptive inputs that may occur in the future.Our data show that Arc/Arg3.1, which is critically involved in consolidating long term structural changes in the forebrain, is preferentially induced in spinal enkephalinergic neurons after nociceptive stimulation. This finding suggests that Arc/Arg3.1 dependent memory formation in spinal pain transmission is a predominant feature of neurons, which are anti-nociceptive rather than pro-nociceptive.In this study we used 99 male Wistar rats and 16 Arc/Arg3.1 KO mice and their wild type littermates.2/70%N2O. In experiments applying mustard oil (MO)  the animals were kept under anesthesia during entire survival time and subsequent perfusion. For 25% MO application  the left paw was shaved and wrapped in a gauze soaked with MO and then covered with foil. For application of 10%  and 50%  MO, the left paw was shaved and MO was applied once at the beginning of the experiment using a brush. For the experiments using intrathecal injections, the same protocol was used as described in [f]quinoxaline-7-sulfonamide  was injected concomitantly with BDNF . For control intrathecal experiments, 25 nmol N-Methyl-d-asparate , or only vehicle  or only NBQX  was injected intrathecally. After the injections, the rats were placed back in their cages. For induction of neuropathic pain, the spared nerve injury (SNI) model and a control operation were used [50 μl of 0.3% capsaicin (Sigma-Aldrich) solution consisting of 80% saline, 10% Tween-80, and 10% ethanol 100%  or 100 μl of Complete Freund's Adjuvant  was injected in a hind paw under anesthesia with 2% isofluorane in 30%Oribed in . Brain-dere used . In shorAll mice were habituated for 5 days to the experimenter, the experiment room, and the transparent cage that was used for the Von Frey measurements. Thereafter, prior to each experiment the mice were habituated for 30 minutes to the room in which the behavioral experiments took place.before each Von Frey measurement, the mice were allowed to habituate to a transparent cage (15 cm × 15 cm with a gridded floor) for 10 minutes. We used calibrated von Frey filaments, which were applied for 2 seconds at 5 seconds interval, and the threshold was set at 3 evoked responses in a maximum of 5 applications.the thermal thresholds were assessed by measuring the time a mouse spent on the hotplate (51°C) before showing a response like fluttering or licking of the hind paw, or jumping. Immediately after a response or after maximally 45 seconds, the mouse was taken off the hotplate.the mice were restrained by the experimenter and 15 μl of formalin, i.e. a freshly made solution of 4% paraformaldehyde (PFA) in phosphate buffer (PB), was injected subcutaneously in the left hind paw. The number of flutters and the time spent licking of the injected paw were measured during 55 minutes post-injection. After 90 minutes the mice were perfused and the tissue was processed as described below. n = 4 for Arc/Arg3.1 KO mice; n = 4 for WT littermates.25 μl of CFA was injected in a hind paw of restrained mice and thereafter the mechanical and thermal thresholds were assessed at 1.5 h, 4 hrs, 1 d, 3 d, 4 d, 8 d, 14 d, 21 d, 28 d, 34 d, and 42 d post-injection. n = 4 for Arc/Arg3.1 KO mice; n = 4 for WT littermates.t-test or a repeated measures ANOVA was performed, p < 0.05 was taken as significant.An unpaired After experiments the mice were sacrificed and further processed for immunohistochemistry (IHC) or in situ hybridization (ISH). Histological examination of Arc/Arg3.1 KO mice spinal cord did not reveal any morphological abnormalities in comparison with their WT littermates.At the end of the survival times the animals received an overdose of sodium pentobarbital and were transcardially perfused with 100 ml saline (rats) or 10 ml (mice) followed by 750 ml of 4% PFA (rats) or 50 ml (mice) dissolved in 0.12 M phosphate buffer (PB), pH 7.4. The spinal cord was dissected and left overnight in a solution of 4% PFA and 30% sucrose at 4°C. Subsequently, sections were cut (30 μm) on a freezing microtome and collected in RNAse-free PB. Serial sections were cut and collected in 9 separate jars, and therefore sections in one jar were at least 270 μm apart. The sections were kept in a solution of 40% glycerol, 40% ethyleenglycol and 20% RNAse-free PB for long-term storage at -20°C.2O2 and a self made FITC tyramide according to protocol described in [The partial cDNA templates encoding the following mRNAs were used: Arc/Arg3.1 , GAD67 , preproenkephalin . The riboprobes were obtained by linearizing the recombinant plasmids with the appropriate restriction enzymes and RNA polymerases. The transcription was performed in the presence of digoxigenin (DIG)- or fluorescein-labeled 11-UTP (Roche). ISH based on alkalic phosphatase (AP) reaction was performed following the protocol described previously . For fluribed in . ThereafAnalysis was carried out on L4 and L5 segments of the spinal cord, except for the BDNF experiments, in which also S1 and S2 segments were included in the analysis. Slides were systematically examined starting from the first section in the first row for the appropriate segmental level. The first 5 to 6 sections that were encountered and were not damaged during the procedure were included in the analysis . For illt-test was performed, and p < 0.05 considered significant.For double labeling based on FISH combined with fluorescent IHC, confocal images were analyzed using the Zeis LSM image browser. For each section, the dorsal horn showing Arc/Arg3.1 labeled neurons was analyzed in a vertical plane consisting of 9 slices with an optical thickness of 2.46-2.76 μm. Every fifth section was analyzed for double labeled neurons. For markers that label the cytoplasm, the criterion was that the diameter of a profile was at least 10 μm to be counted as a neuron. For statistical analysis, an unpaired The authors declare that they have no competing interestsMH performed or contributed to all experiments, analyzed data and drafted the paper. JLMJ contributed to experiments and analysis. KB contributed to experiments. DK provided KO mice and gave advice. JCH conceived and supervised the project and edited the manuscript. All authors read and approved the final manuscript.


Procalcitonin (PCT) and C reactive protein (CRP) have been used as infection parameters. PCT increase correlates with the infection's severity, course, and mortality. Post-cardiocirculatory arrest syndrome may be related to an early systemic inflammatory response, and may possibly be associated with an endotoxin tolerance. Our objective was to report the time profile of PCT and CRP levels after paediatric cardiac arrest and to assess if they could be use as markers of immediate survival.A retrospective observational study set in an eight-bed PICU of a university hospital was performed during a period of two years. Eleven children younger than 14 years were admitted in the PICU after a cardiac arrest. PCT and CRP plasma concentrations were measured within the first 12 and 24 hours of admission.In survivors, PCT values increased 12 hours after cardiac arrest without further increase between 12 and 24 hours. In non survivors, PCT values increased 12 hours after cardiac arrest with further increase between 12 and 24 hours. Median PCT values (range) at 24 hours after cardiac arrest were 22.7 ng/mL (0.2 – 41.0) in survivors vs. 205.5 ng/mL (116.6 – 600.0) in non survivors (p < 0.05). CRP levels were elevated in all patients, survivors and non-survivors, at 12 and 24 hours without differences between both groups.Measurement of PCT during the first 24 hours after paediatric cardiac arrest could serve as marker of mortality. Procalcitonin (PCT) and C reactive protein (CRP) have been used as infection parameters. PCT concentration increases because of several microbial infections and this increase correlates with the infection's severity, course, and mortality -5. OtherPaediatric cardiac arrest is associated with high in-hospital mortality . Only onA recent study has shown that PCT serum levels were significantly higher in cardiac arrest patients who died of refractory shock than in those who died of neurological failure or survive. There aA retrospective observational study set in an eight-bed PICU of a university hospital was performed. During a period of two years, eleven children younger than 14 years were admitted in our PICU after a cardiac arrest. PCT and CRP plasma concentrations were measured within the first 12 and 24 hours of admission. Plasma CRP was measured on a Vitros 5.1 Fusion Chemistry System . For the determination of PCT, time-resolved amplified cryptate emission (TRACE) technology was used on a Kryptor analyzer . Limits of detection were 0.3 mg/dl and 0.02 ng/ml for CRP and PCT methods, respectively. The study received ethics approval by the Hospital Ethics Research Committee.U-test was used to compare two independent samples. A p-value ≤ 0.05 was considered significant.We compared CRP and PCT plasma levels among survivor and non survivor patients. Mann-Whitney Eleven children, six males successfully resuscitated after cardiac arrest were admitted in our PICU. Seven patients survived and four did not survive at hospital discharge. Age, diagnosis, place of arrest, cardiac arrest duration, outcome and cause of death for each individual patient are shown in Table Median PCT values (range) at 12 hours after cardiac arrest were 23.6 ng/mL (3.1 – 78.2) and 79.1 ng/mL (32.8 – 300.8) in survivors and non survivors, respectively (p NS), whereas at 24 hours were 22.7 ng/mL (0.2 – 41.0) and 205.5 ng/mL (116.6 – 600.0) in survivors and non survivors, respectively (p < 0.05). Median CRP values at 12 hours were 2.1 mg/dL (0.4 – 8.5) and 1.3 mg/dL (0.1 – 3.0) in survivors and non survivors, respectively (p NS) whereas at 24 hours were 9.1 mg/dL (2.0 – 24.4) and 4.5 mg/dL (4.0 – 10.5) in survivors and non survivors, respectively (p NS).Figures In spite of the reduced number of patients, it is interesting to point out that time of cardiac arrest and time to initiation of CPR were correlated with PCT values at 24 h Figure . This reWe found in a small group of paediatric patients that PCT levels higher than 100 ng/mL 24 hours after cardiac arrest may identify high risk of mortality. Several studies have demonstrated PCT utility for the diagnosis of severe bacterial infections,18. Its Another important finding is that PCT elevation is not directly related with infection in these cases. According with the literature, cut-off PCT values for diagnosis of bacterial infections are between 0.5 and 5.0 ng/mL whereas cut-off values for diagnosis of shock are much higher,5,18. SpThe main limitation of our study is the small number of patients. Because cardiac arrest is uncommon in paediatric age a multicenter study could be necessary to have a bigger sample.Measurement of PCT during the first 24 hours after paediatric cardiac arrest can predict non survival of patients. PCT values higher than 100 ng/mL 24 hours after cardiac arrest could serve as marker of mortality. CRP did not show this quality.PCT: procalcitonin; CRP: C-reactive protein; CRA: cardiac arrest; PICU: paediatric intensive care unit; CPR: cardiopulmonar resuscitation.CR has received remuneration for holding lectures on the topic of prognosis markers of sepsis by BRAHMS, Germany. The authors declare that they have no further competing interests.MLA participated in the design of the study, analyzed data and drafted the manuscript. CR conceived the study, participated in the design and drafted the manuscript. AC and AM participated in the design, and reviewed the manuscript. BP was responsible for biochemical determinations and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: