Split (1)

train · 133k rows

text stringlengths 0 868k
A biloma is a rare abnormal accumulation of intrahepatic or extrahepatic bile caused by traumatic or spontaneous rupture of the biliary tree1, 2). It is most commonly caused by surgery, percutaneous transhepatic cholangiography (ptc), percutaneous transhepatic biliary drainage (ptbd), and abdominal trauma11, 12). We report here a patient who complained of chills, fever and right upper quadrant abdominal discomfort . A 78-year - old woman presented to the emergency room with her guardian, with acute fever and chills that developed the day before admission . The patient was diagnosed with diabetes a few years previously, but this was not being treated . On admission to the emergency room, the vital signs were as follows: blood pressure 70/40 mmhg, heart rate 118/min, respiratory rate 30/min and temperature 40.3. the patient appeared to be in acute distress; she was lethargic, the sclerae were slightly yellow and the conjunctivae were pale . The abdominal examination revealed slight tenderness in the right upper quadrant, but there was no palpable mass . The laboratory studies were as follows: wbc 52,400/mm, hb 12.3 g / dl, platelets 90,000/mm, ast 153 iu / l, alt 135 iu / l, alkaline phosphatase 255 iu / l, total bilirubin 2.07 mg / dl, total protein 5.9 g / dl, albumin 3.0 g / dl, bun / cr 25/1.9 mg / dl, esr 25 mm / hr, and crp 20.4 mg / dl . The serum electrolyte levels were normal except for an elevated potassium 7.0 mmol / l . The abga data were as follows: ph 7.24, pao271 mmhg, paco2 19 mmhg, hco3 15.1 the hepatitis viral marker tests, including hbsag, anti - hbs ab and anti - hcv, were all negative . Standard x - rays on the day of admission revealed lung infiltrates and no specific findings in the abdomen . The abdominal computed tomography (ct) revealed a 9.58.5 cm subcapsular cyst with a clear boundary in the left hepatic lobe and enlargement of the intrahepatic bile duct around the cyst . Although the left hepatic lobe had some atrophic findings, there were no significant signs of an enlarged bile duct or abnormal gallbladder (figure 1). On the day of admission after the computed tomography, the abga revealed a ph 7.05, pco2 64.8 mmhg, po2 73.3 mmhg, hco3 the patient was intubated and put on a respirator; she was treated with antibiotics and vasopressors . On the second day of admission, percutaneous drainage with an 8.5 fr pigtail catheter, under the guidance of an abdominal ultrasound was performed for diagnosis and treatment . We removed 565 ml of fluid that was dark brown in color . For a diagnosis, biochemical tests were carried out on the bile, and these tests revealed a total bilirubin of 22.3 mg / dl and a direct bilirubin of 18.9 mg / dl . The leukocyte count was 4,560/mm, and gram staining showed a gram negative bacillus . On the third day of admission, her vital signs were as follows: blood pressure 100/65 mmhg, heart rate 110/min and temperature 37.2. there was 200 ml of drained bile, the s - fibrinogen was 894 mg / dl (200 - 415), the d - dimer was 9,040 ng / ml (0 - 255), and the fdp was elevated up to 97 ug / ml (normal range: 0 - 5). On the eighth day of admission, the patient's mental status improved, the vital signs were stable and voluntary respirations recovered . On the thirteenth day of admission, pancreatico - biliary magnetic resonance imaging (mri) revealed a biloma that was connected to the intrahepatic bile duct (figure 2). On the sixteenth day of admission, the biloma that was connected to the intrahepatic duct and common bile duct was noted again via a tubogram performed with a catheter . Over time, the amount of bile drainage decreased . On the twentieth day of admission, 64 ml of bile was drained, the leukocyte count was 340/mm and there were no signs of bacteria on culture or gram staining of the bile . The follow - up ct showed that the size of the cyst had decreased to 5.44.6 cm and the density around the cyst had increased . The amount of drainage did not decrease afterward, and the connection between the biloma and the bile duct was still noted . On the 25th day of admission, the drainage tube was blocked and follow - up of the bilirubin in blood and the size of the biloma were assessed . On the 29th day of admission, follow - up ct showed a marked decrease in the size of the cyst (figure 3), and the serum bilirubin was normal (0.22 mg / dl). On the 34th day of admission, the drainage tube was removed after evaluating the size and connection of the biloma via a catheter tubogram (figure 4). They reported a case with extrahepatic bile leakage after trauma to the upper right quadrant of the abdomen; the bile did not cause peritonitis, but it accumulated in an encapsulated form . This concept was later applied to all cases with biliary tract lacerations caused by bile leaks that formed a capsule inside or outside of the liver1). The mechanism of encapsulation is explained by a large amount of bile leaking at a fast pace that causes biliary peritonitis, or a small amount of bile leaking at a slow pace that causes mild inflammation . This leaked fluid is simultaneously trapped by the greater omentum and mesentery, where it becomes encapsulated2). Although the amount of bile leaked may be minor, the occurrence of an inflammatory reaction contributes to the appearance of a growth in size of the biloma3). As diagnostic techniques continue to develop, more cases of biloma are being reported, but cases of spontaneous biloma and biloma accompanied by other diseases have rarely been reported to date . From 1979 to 1997, 25 cases of spontaneous biloma were analyzed by fujiwara; the underlying causes included choledocholithiasis (16 cases), cholangiocarcinoma (2 cases), acute cholecystitis (1 case), liver infarction (1 case), hepatic abscess (1 case), nephrotic syndrome (1 case), obstructive jaundice (1 case), sickle cell anemia (1 case), and tuberculosis (1 case)4). In korea, there have been two cases of choledocholithiasis accompanied by a biloma, and they were treated by nonsurgical methods5, 6). There is one report of a case of biloma after trauma7), and 1 case of a biloma after endoscopic retrograde cholangiopancreatography (ercp)8). One of the patients with a choledocholithiasis who suffered a nontraumatic intrahepatic rupture of the bile duct, had a surgical repair9). The causes of biloma include trauma to the liver, which is the most common cause, abdominal surgery, endoscopic surgery and percutaneous catheter drainage10 - 13). Currently, the term biloma is generally used to describe intrahepatic or intraperitoneal focal biliary stasis14). The clinical symptoms of a biloma are nonspecific, and they can range from no symptoms to abdominal pain and distention, jaundice and fever; leukocytosis can also be present15). In the case there were complaints of sudden chills, fever, upper abdominal discomfort and sensations of abdominal distention . Physical examinations showed tenderness in the upper right area of the abdomen, slight jaundice, hypotension, leukocytosis and fever over 40. the size and location of the biloma are determined by the cause of the rupture of the bile duct, the location and speed of bile leakage and the rate the bile is reabsorbed by the peritoneum2). Fujiwara reported that the locations for bile leakage included the left hepatobiliary duct (8 cases), the gallbladder (5 cases), the right hepatobiliary tract (2 cases), the common bile duct (1 case), and unknown (9 cases); the locations where the bilomas formed were the left lobe (11 cases), the right lobe (10 cases), and the upper abdomen (4 cases). Vazquez analyzed 21 cases of biloma and found that 16 cases occurred in the upper abdominal area near the liver and five in the left upper abdominal area4). The diagnosis of a biloma can be assisted by surgery, a history of trauma with abdominal ultrasonography, abdominal ct and mri . A definite diagnosis can be made by a 99mtc - disida scan, percutaneous aspiration or by ercp1, 2). Abdominal ultrasonography shows a mass of low echogenicity with a clear border in the liver, and the follow up tests show changes in size16). The abdominal ct will give a more accurate view of the location of the biloma and show its relationship to the surrounding organs . A standard ct view will show a low - density mass in the liver at a ct number lower than 20 . Ultrasonography and abdominal ct are sometimes unable to differentiate bilomas from seromas, lymphoceles and angiomas . Mris or hepatobiliary scans can be useful when continuous bile leakage is present, but they are not diagnostic when continuous leakage is not present7, 17). Radiological image - guided aspiration tests, when testing for bilirubin, can be diagnostic18). When the aspirated substance is a clear yellow liquid, then microbiological tests must be done to rule out an infection . Ercp can be used to determine the location and severity of an active bile leak . However, the presence of small biliary cysts or bilomas, located in the lower areas of the liver that can be hidden by gastrointestinal shadows, can be difficult to diagnosis19). We were able to identify a cystic lesion by abdominal ct; a tubogram and magnetic resonance cholangiopancreatography (mrcp) were used to confirm a bile leak from the common bile duct . The fluid obtained by the transabdominal ultrasonography assisted percutaneous cyst drainage containing 22.3 mg / dl of total bilirubin and 18.9 mg / dl of direct bilirubin; these findings confirmed the diagnosis of a biloma . Treatment for bilomas that have a diameter of only a few centimeters is not always necessary; these lesions can be watched . However, most bilomas require treatment . In the past, surgery was the main approach to treatment . Currently, there are a much wider variety of options such as percutaneous catheter drainage, endoscopic sphincterotomy (est), endoscopic nasobiliary drainage (enbd) and endoscopic drainage4, 6, 15, 19, 20). Surgery is now performed only in cases with a persistent bile leak or for treatment of underlying disease . In this case, a spontaneous biloma with bacterial infection was found after the patient presented in septic shock . After percutaneous drainage was performed, the amount of bile excreted did not decrease to lower than 64 ml per day . As there was no difficulty in flow through the common bile duct, the biochemical tests performed after removal of the drainage catheter showed no increase in total bilirubin or any increase in the size of the biloma, thus demonstrating a rare case of recovery.
The construct of unintended pregnancies (ups) is multifactorial and broadly encompasses pregnancies that are either unwanted or mistimed.1 in many instances, ups are likely to end by induced abortion; worldwide estimates suggest that 50% will be voluntarily terminated.2 in 2008, it was estimated that 43.8 million abortions occurred worldwide, of which 86% occurred in low-/middle - income countries.3 furthermore, between 2003 and 2008, the number of induced abortions was found to have decreased in high - income countries but to have increased in low-/middle - income countries . During this same period of time, the proportion of unsafe abortions increased and they were believed to account for 13% of maternal deaths, with the majority of these concentrated in countries with restrictive laws on abortion.4 elective abortion in brazil is considered non - legal, unless the pregnancy resulted from a rape, would cause a life - threatening condition to the mother, or the fetus has anencephaly or any other malformation that is incompatible with the extrauterine life . As a result, data on prevalence and associated costs of abortion are limited and remain a challenge to collect . However, it is recognized worldwide that direct health costs of up and resulting abortions can significantly impact local health services and the families affected.5 health system and societal costs can also arise from ups that do not end in abortion and are carried to parturition . In the united states, the public health costs attributed to unintended births in a single year cost taxpayers $11.1 billion (2006).6 furthermore, previous studies have reported that ups are more likely to result in preterm births and low - birth - weight babies, which would increase health costs for neonatal care and costs associated with long - term disabilities.7 this is particularly relevant for ups in adolescence, which are more likely to result in low - birth - weight births, which can increase health costs.8 over the past few decades, brazil, like many fast - emerging economies, has experienced a fertility transition noted by a dramatic reduction in the total fertility rate, which currently sits at 1.8 births per woman.9,10 furthermore, during the fertility transition, the rate of contraception has also increased.11 despite increased contraception use, the cumulative rate of spontaneous and induced abortion changed very little between 1996 and 2006, for which population survey estimates are available.12,13 furthermore, a survey of more than 1,000 women aged 1024 years in a single postnatal unit in brazil reported that more than 50% of all births were unintended, suggesting that possibly significant unmet contraception need still exists.8 similarly, for the whole country, the data from the last demographic and health survey (dhs) performed in 2006 indicates that 55% of all births were unintended.14 prevention of up using publicly funded programs in high - income countries has proven to generate significant cost savings for health services and public services.15 previous economic studies evaluating the consequences of up have mostly been performed in high - income countries where publicly funded fertility services and abortion legislation are different from those in low-/middle - income countries.15 the aim of this study was to evaluate the cost consequences of up in brazil, an emerging economy with vastly different fertility planning services than most advanced economies and with restrictive abortion laws . To the best of our knowledge, this is the first published study that describes the economics of up, and it is believed that this work can aid decision makers regarding access to contraceptives . A model was developed to evaluate the humanistic burden and financial impact of ups in brazil . The analysis factored in costs and outcomes for 1 year post - delivery of ups . Possible birth outcomes of up included induced abortion, miscarriage, and ongoing pregnancy resulting in birth . All birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus, the data system for the ministry of health in brazil . Available data was further separated into vaginal (61.0%) and cesarean section (c - section) deliveries (38.4%).16,17 outcomes and resulting costs for infants were also followed . These included stillbirths and infant survival and complications from term and preterm deliveries . Complications included admission to a neonatal intensive care unit (icu), hospitalization during the first year, and cerebral palsy . Data from datasus were used when possible to populate the model to reflect the reality of the public health care system . However, for parameters not identified in datasus, other relevant sources were used . A decision tree model was constructed in microsoft excel (microsoft corporation, redmond, wa, usa) from the perspective of the brazilian public health system (figure 1). The total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9%, respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% . The up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20, ten, and 30 deaths per 100,000 births were used for miscarriage, vaginal delivery, and c - section delivery, respectively.2123 in addition, a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods, it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births . Differential infant mortality rates were used for term and preterm births, with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications, including admissions to neonatal icu, hospitalization during the first year, and cerebral palsy . All preterm birth infants were assumed to require neonatal intensive care, compared to 7.6% of term birth infants.27 for preterm infants, the average number of hospitalizations during the first year was 1.7, mostly from respiratory issues.28 for term birth infants, hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births, respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . In brazil the public national health system (sus) pays for around 70%75% of all reproductive procedures . However, in this model, it is assumed that the costs are those from the public system, although, in approximately one - quarter of ups, the costs are probably higher when related to women s care covered by insurance or privately . Costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system . Costs of miscarriage were related to the following procedures: pelvic exam, blood test, ultrasound, and dilation and curettage . The cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission, while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 . A model was developed to evaluate the humanistic burden and financial impact of ups in brazil . The analysis factored in costs and outcomes for 1 year post - delivery of ups . Possible birth outcomes of up included induced abortion, miscarriage, and ongoing pregnancy resulting in birth . All birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus, the data system for the ministry of health in brazil . Available data was further separated into vaginal (61.0%) and cesarean section (c - section) deliveries (38.4%).16,17 outcomes and resulting costs for infants were also followed . These included stillbirths and infant survival and complications from term and preterm deliveries . Complications included admission to a neonatal intensive care unit (icu), hospitalization during the first year, and cerebral palsy . Data from datasus were used when possible to populate the model to reflect the reality of the public health care system . However, for parameters not identified in datasus, other relevant sources were used . A decision tree model was constructed in microsoft excel (microsoft corporation, redmond, wa, usa) from the perspective of the brazilian public health system (figure 1). The total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9%, respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% . The up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20, ten, and 30 deaths per 100,000 births were used for miscarriage, vaginal delivery, and c - section delivery, respectively.2123 in addition, a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods, it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births . Differential infant mortality rates were used for term and preterm births, with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications, including admissions to neonatal icu, hospitalization during the first year, and cerebral palsy . All preterm birth infants were assumed to require neonatal intensive care, compared to 7.6% of term birth infants.27 for preterm infants, the average number of hospitalizations during the first year was 1.7, mostly from respiratory issues.28 for term birth infants, hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births, respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . In brazil the public national health system (sus) pays for around 70%75% of all reproductive procedures . However, in this model, it is assumed that the costs are those from the public system, although, in approximately one - quarter of ups, the costs are probably higher when related to women s care covered by insurance or privately . Costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system . Costs of miscarriage were related to the following procedures: pelvic exam, blood test, ultrasound, and dilation and curettage . The cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission, while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 . Based on the modeled parameters we estimate 1.79 million annual ups and 1.47 million annual planned pregnancies . We estimate annual maternal deaths of 351, of which 49 (14%) are attributed to abortions and 302 to complications from miscarriages and deliveries . The number of infant deaths within the 12 months following birth was estimated at 32,864 . The number of estimated neonatal admissions for the year 2010 associated with up was 224,631 which included all preterm deliveries and 7.6% of all term deliveries . The disaggregated values for different pregnancy outcomes attributed to up are described in table 2 . Costs of abortions are not reimbursed in the public health system and are not included in the analysis . The total costs attributed to up are estimated to be r$4.1 billion annually, of which approximately r$32.9 million (0.8%) was attributed to miscarriage and r$4.07 billion (99.2%) to births and resulting complications . From the direct birth - related costs, antenatal care accounted for approximately r$233 million (5.7%) of birth costs, with labor and delivery costs responsible for r$988 million (24.3%). The remainder of birth costs were attributed to infant complications, estimated to be r$2.8 billion (70.0%), with neonatal costs accountable for approximately r$628 million (15.4%) and hospital readmission in the first year costs of r$2.1 billion (52.9%) of all birth costs annually . Based on the national cost estimates and the number of annual ups our analysis estimates the costs and outcomes associated with unintended pregnancies to derive a cost per up of r$2,293 . The cost per up factors in a range of health - related costs attributed to those resulting in abortion and resulting live births for those carried to parturition . This figure is broadly aligned with previous analyses in the united states of $1,609 per up after factoring in the purchasing power parity between the two countries.35 it is also worth noting that the estimates provided here are likely an underestimate of all costs . For example, the analysis described here focused on the costs that occur within the public health systems . Consequently, costs of elective abortions paid for by individuals are not represented in this analysis . Furthermore, because elective abortion is illegal in brazil except in extreme and rare cases, hidden costs from elective abortions may filter into the public health system . For instance, costs associated with post - abortive care are likely to be classified as miscarriages when reported to national authorities . Additionally, the long - term societal costs that arise from up and reduced educational attainment and lost productivity of young mothers has not been accounted for in our analysis.3639 our analysis highlights that considerable cost savings can be achieved by reducing up, which are thought to represent 55% of all pregnancies in brazil.20 the analysis accounted for both untimed and unwanted births to estimate the up rate and associated costs, in which live births resulting from unwanted pregnancies represented the largest share of health costs, representing 99% of all costs . This is also attributed to the fact that abortion is a relatively inexpensive procedure that is paid for outside of the health service . Because a substantial proportion of pregnancies are attributed to mistimed pregnancies, ie, pregnancies that would have occurred at some point in the future, but occurred sooner due to mistimed pregnancy, the actual cost savings to be realized by reducing up is less than described here and would mostly be associated with averting those pregnancies considered to be unwanted . To put the potential cost savings into perspective, an analysis conducted for the state of california, usa estimated that preventing many up in a single year will offer savings of $1.1 billion up to 2 years of age of the child, and that every dollar spent on averting up offers $2.76 of saving in 2 years and $5.33 at 5 years post - delivery.15 a national - level analysis that focused on the federally financed medicaid program in the united states noted that taxpayer savings of $4.7$6.2 billion could be achieved by reducing unintended pregnancies.40 based on the cost savings attributed to up, it is anticipated that future health researchers can build on the research described here to better understand the cost savings that may be achieved through improved contraceptive use . The analysis sought to incorporate a broad range of costs associated with up to illustrate that costs extend beyond the point of delivery . For instance, ups often occur in adolescent and young adults and can be predictive of low birth weight, preterm birth, and neonatal admission.8,4143 the precise mechanism by which up influences preterm birth is likely to be multifactorial . However, some studies have observed that women with ups are less likely to receive adequate prenatal care44 and are also more likely to smoke and drink alcohol during pregnancy, which contributes to adverse outcomes.44,45 it is important to highlight these costs because it suggests that ups place unnecessary demand on health services that are often stretched to capacity . Since up, for the most part, can be avoided, this illustrates the health service benefits and health service capacity that could be released by reducing up . Much of the humanistic burden attributed to up relates to maternal mortality and morbidity of women who pursue unsafe abortions . In our analysis, we estimate 49 annual deaths from abortions representing 13% of the estimated maternal deaths . It has been suggested that access to improved contraceptive services could prevent maternal mortality by 25%35%.46 furthermore, estimates from the world health organization reported that unsafe abortion disables approximately 5 million women each year, suggesting reductions in morbidity could also be achieved through improved contraception use.4 empirical evidence and clinical guidelines agree that the most effective approach to preventing up is through education and contraceptive use, of which long - acting contraceptive (larc) methods are the most effective intervention.4751 in particular, among adolescents, prevailing evidence suggests that education and contraception are the main interventions for reducing up.49 in advanced economies, the percentage of women using larc methods has been steadily increasing . A recent study noted that the proportion of women using larc methods has increased from 2.4% of women aged 1544 years since 2002 to 8.5% of women in 2009.52 in the united states, the american college of obstetricians and gynecologists (acog) advocates use of intrauterine contraceptives (both copper - intrauterine device and the levonorgestrel - releasing intrauterine system) and implants as first - line therapy for adolescent women because they contribute to reduce the number of ups.48 larc has also proven to be cost - saving compared with combined oral contraceptives in the united kingdom . Investigators reported that this finding was influenced almost entirely by the lower failure rates associated with larc.50,53 an analysis conducted in the united states also noted that cost savings could be achieved by switching women from oral contraceptives to larcs, and approximately 50% of costs of up were attributed to contraceptive adherence.54 despite the positive benefits of larc, use in brazil lags behind that of other countries . A survey conducted in 2008 in northern brazil reported that only 1% of women attending a post - abortion clinic elected for an intrauterine device, despite 93% of women having knowledge of the product.55 several barriers have been identified, which often limit uptake of larc methods.56 these include health care provider lack of knowledge of risks, myths, misconceptions, and lack of training for insertion of the various devices . Also, misinformation and fear of pain at insertion among women can limit demand for larc methods . Furthermore, larc can often involve considerable up - front expense, although evidence does suggest that these methods are cost - effective compared with alternative contraceptive methods . Because of the expense, reimbursement can influence the rate of uptake . Even recognizing the importance of abortion, and especially induced and unsafe abortion, for maternal mortality and morbidity in brazil, there are no reliable and available data confirming a pandemic of such conditions as commonly reported by media and gray literature . Data extracted from the 2006 brazilian dhs indicated that only 1.5% self - reported induced abortions among all pregnancies for the whole period of the survey.14,19 a probable contribution for this scenario is the high prevalence of and easy access to contraceptive use among women in reproductive age in the country during the last 2 decades . Although all induced abortions are technically classified as unsafe, this is not the impression of the majority of obstetricians working in the field . There is a general belief that complications due to unsafe induced abortions are less and less frequent and that there was recently a marked drop in their occurrence . With no direct information to confirm this, one indirect point of evidence is the decrease in the proportional contribution of abortion as cause of maternal mortality in the country . In fact, official brazilian data confirm that it was 16.4% in 1990 (representing the third cause of maternal mortality), while only 8.9% in 2000 and 9.0% in 2010 (the fifth cause, behind even the indirect obstetric causes).20 in the meantime, there was a spread of the use of misoprostol as an agent for inducing abortion, not only in health facilities for situations where abortion is allowed, but mainly for situations not legally permitted, wherein the women obtain misoprostol in the black market and self - administer it vaginally . This is believed to be the current most common way of inducing medical abortion and also to be associated with the low rates of severe complications due to abortion, especially infection and hemorrhage, in brazil . There are also at least two possible limitations for the external validity of the current cost estimates . The first refers to the assumption that all miscarriages are really diagnosed and treated with dilation and curettage . However, it is possible that a proportion of individuals will never reach health facilities and receive no intervention . Currently, in brazil, the majority of medical abortions are performed by the woman through self - administration of misoprostol acquired off - label or in the black market . With these procedures, the rate of complications due to medical abortions has dropped significantly during the last decade, with some of these cases arriving at hospitals already as complete abortions that need no complementary procedures . Although insufficient data are available to support this scenario, this would probably impact strongly on the estimates of post - abortion care, taking into account the correspondent reduced morbidity and mortality . The cost consequences of live births attributed to up in adolescents can extend beyond the observed costs of increased prenatal care, preterm births, and abortions . Studies have noted that adolescent women are more likely to dropout from school following pregnancy . Furthermore, others have suggested that future generations will follow the reproductive pattern of their parents, which could perpetuate the likelihood of up in the offspring of these mothers.57 as reported, education has been shown to break the association of adolescent fertility across generations, suggesting the longer - term benefits to be achieved by reducing up . Our analysis illustrates the cost consequences associated with up in brazil . In the past 2 decades, brazil has made considerable progress toward reducing abortion rates, but, despite these efforts, the humanistic and direct economic costs associated with up remain high.58 to achieve economic savings from reducing the up rate will require education, training of providers, and improved access to effective contraceptive measures . It is believed that the research described herein can inform the burden of up and the benefits of improved fertility planning . The cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report . It is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries . The cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report . It is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries.
Pmds is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically, as well as genotypically, normal man . It is characterized by the persistence of the uterus, fallopian tubes and upper vagina in otherwise normally virilized boys . Despite the normal male genotype (46 xy) and the subsequent normal development of fetal testes, mllerian structures do not regress either due to absence of mllerian inhibiting substance (mis) or lack of response to it . The persistence of a large uterus - like paramesonephric duct in a man is in itself clinically unusual, but when it forms a part of the contents of a hernial sac, it must be considered a rarity . We report the case of a 70 year old man with unilateral cryptorchidism on the right side and left obstructed inguinal hernia containing uterus and fallopian tube (that is, hernia uteri inguinalis; type i male form of pmds) coincidentally detected during an operation for an obstructed left inguinal hernia with right cryptorchidism . A 70 year old man presented to our hospital with a painful left - sided inguinal swelling of one day duration . The patient gave history of asymptomatic left inguinal swelling from past 20 years and absence of the right testis since birth . He had been married for fifty years and was having 4 children, the youngest one being 38 year old female . General physical examination revealed a man of sub - average built with well developed secondary sexual characters . His urethra and penis were fully developed with a poorly developed right hemi - scrotum and no palpable right testis in the scrotum or inguinal canal . There was a non - reducible, tender swelling measuring approximately 10 8 cm in the left inguinal region with absent cough impulse . Exploration of the inguinal canal revealed an indirect inguinal hernia containing a globular structure resembling uterus, fallopian tubes with an atrophic right testis embedded in the broad ligament and attached to pelvis with a thick fibrous band [figs . 1 and 2]. Total excision of the uterus with fallopian tubes and atrophic right testis was performed and the operation was completed with left inguinal hernioplasty [figs . 3 and 4]. Grossly, the specimens removed were identified as a uterus with patent endometrial and endocervical linings and two fallopian tubes . The right testis measuring 2 1 1 cm, was atrophic and embedded in the right broad ligament . The specimen was sent for histopathological examination which revealed uterine muscular tissue with its cavity lined by endometrial tissue and congested fallopian tubes . Male pseudo - hermaphroditism is a condition in which the gonads are testes but the internal genitalia are not completely virilized . Pmds is a rare form of internal male pseudo - hermaphroditism in which mullerian duct derivatives are seen in men . The exact cause of pmds is not known, however it is thought to result from a defect of the synthesis or release of mif, or from defects in the mif receptor . Defects in the mif gene lead to the persistence of a uterus and fallopian tube in males . It is likely that remnant mullerian structures lead to cryptorchidism by hindering the normal testicular descent mechanism . Patients with pmds usually have normal development of external genitalia and secondary sexual characteristics . The typical patient with pmds has unilateral or bilateral cryptorchidism and is assigned to the male sex at birth without hesitation, as they have normal male genotypes and phenotypes . The male form is encountered in 8090% of cases, characterized by unilateral cryptorchidism with contralateral inguinal hernia, and can be one of the two types: the first type is hernia uteri inguinalis, which is characterized by one descended testis and herniation of the ipsilateral corner of uterus and fallopian tube into the inguinal canal . The second type is crossed testicular ectopia, which is characterized by herniation of both testes and the entire uterus with both fallopian tubes . Clinically, the persistence of a uterus and fallopian tubes leads to either cryptorchidism or inguinal hernia depending on whether or not mullerian derivatives can be mobilized during testicular descent . If the uterus and fallopian tube are mobile, they may descend into the inguinal canal during testicular descent . Pmds is usually coincidently detected during surgical operation, as in our patient's case . However pre - operative ultrasonography, computerized tomography and mri allow possible pre - operative diagnosis . An element of deciding on intervention is largely based on reducing risk of malignancy, while maintaining maximum reproductive function . In pmds patients with undescended testes, the rate of testicular cancer is about 12% which is comparable to the rate seen in undescended testes not associated with pmds . Several authors have asserted that the mullerian structures should not be removed as there is minimal risk associated with their retention, and excision of the mullerian structures risks damaging primary blood supply to the pmds testis via the internal spermatic and deferential arteries . However most recent reports have demonstrated rare malignancies including adenocarcinomas of the mullerian duct associated with retained mullerian structures . Pmds is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically, as well as genotypically, normal man . Since patients are phenotypically male, the diagnosis is usually not suspected until surgery is performed for cryptorchidism or hernia repair . Hernia uteri inguinalis is type i of the male form of pmds, characterized by one descended testis and the herniation of the ipsilateral corner of the uterus and fallopian tube into the inguinal canal . In order to prevent further complications such as infertility and malignant change, the surgeon should be aware of pmds while dealing with patients who present with unilateral or bilateral cryptorchidism . In summary, in cases of unilateral or bilateral cryptorchidism associated with hernia, as in our patient's case, a written informed consent was obtained from the patient for publication of the case report and various images which may be used with the same . The patient being a major gave a written consent of the same after assuring him that name and hospital mrd no of the patient will not appear in the case report . All the authors have contributed either as being part of surgical and anaesthesia team or helping in the photography, paper writing, submission etc . Afak yusuf sherwani, abdul qayoom shah, abdul majeed wani, farooq ahmad sofi and ashfaq amin wani were in the surgical team . Bashir a chalkoo, bashir a khan and ab hamid sherwani in the anaesthesia team . Wasim lone, mehmood a sheikh and raj reshi sharma did photography and paper writing.
Spider nevus (also known as spider angioma or vascular spider) is a common benign vascular anomaly that may appear as solitary or multiple lesions . Spider nevi are represented by telangiectases that consist of a large arteriole from which radiate numerous small vessels that resemble spider's legs . They are found in the distribution of the superior vena cava, that is, on the face, neck, arms, and upper trunk . Spider nevi are usually <2 cm in diameter and rarely grow to assume large size more than 2 cm . We hereby report a case of very large spider nevus in a patient with hepatitis c virus (hcv) related liver cirrhosis . A 48-year - old - female presented with a history of abdominal distension and jaundice of 3-month duration . Physical examination revealed pallor, icterus, pedal edema, hepatospleenomegaly and tense ascites . She had multiple classical spider nevi over the chest . Also, she had a large spider nevi situated on the forehead, measuring 8 cm in largest dimension with palpable dilated vessels and venous hum [figure 1]. Deep compression on the central area of the lesion produced complete disappearance with blanching and when released refilling of vessels occurred . Laboratory tests revealed a hemoglobin level of 9.8 g / dl, a white - cell count of 4240/mm, and a platelet count of 77,000/mm . Anti - hcv antibody for hcv was positive with hcv rna of 1.6 10 iu / ml . An ultrasound abdomen was suggestive of liver cirrhosis with portal hypertension (portal vein diameter of 14 mm), ascites and splenomegaly . Thus, a diagnosis of cirrhosis liver with portal hypertension was made (ctp-9, class b, and meld 11). She was put on diuretics for the control of ascites but developed hepatic encephalopathy and renal dysfunction . Diuretics were stopped and in view of refractory tense ascites, she was taken up for a transjugular intrahepatic portosystemic shunt . However, during the procedure she had massive intrahepatic bleed and in spite of intensive resuscitative measures she died 48 hours later . Spider nevi can be seen in pregnancy, in patients with thyrotoxicosis, rheumatoid arthritis, oral contraceptive use and most commonly, liver cirrhosis . These frequently appear in alcoholic cirrhotic or when liver function deteriorates in patients of the chronic liver disease . Found elevated levels of vascular endothelial growth factor and basic fibroblast growth factor in patients with liver cirrhosis, especially in those with spider nevi . In patients with nonalcoholic cirrhosis, the levels of substance p are elevated which may play an important role in the pathogenesis of spider nevi by causing vasodilatation . Most remain small in size and rarely do they assume the size as seen in our patient . The present case is unique because of its very large size, rare presentation over the forehead and palpable mass like vascular lesion in a patient with nonalcoholic cirrhosis . Electrodesiccation and laser treatments under local anesthesia are effective therapeutic procedures for facial spider nevi . Spider nevi, however, usually do not require any treatment in cirrhotic patients due to a high risk of bleeding from these vascular lesions.
Primary hyperhidrosis is a common disease that is characterized by excessive sweating of face, palms, or axilla, occurring in 0.6 - 3% of a population (1, 2). Since kux performed endoscopic thoracic sympathetic surgery (3), it has been popular during the past few decades as the surgical treatment of choice for facial, palmar, and axillary hyperhidrosis (4 - 6). Although endoscopic thoracic sympathetic surgery offers permanent cure for hyperhidrosis, it is often accompanied by serious complications, such as compensatory hyperhidrosis . In order to decrease compensatory hyperhidrosis, endoscopic thoracic sympathetic surgery has attempted to reduce the extent of resection of the sympathetic nerve, but these procedures did not significantly decrease the occurrence of compensatory hyperhidrosis (7). We performed sympathetic nerve reconstruction surgery using intercostal nerve in 19 patients with severe compensatory hyperhidrosis after endoscopic thoracic sympathetic surgery and evaluated the results of the procedure . From february 2004 to august 2007, we performed endoscopic thoracic sympathetic surgery in 184 patients with primary hyperhidrosis . Among theses patients, reconstruction surgery of sympathetic nerve using intercostal nerve was performed in 19 patients with severe compensatory hyperhidrosis after endoscopic thoracic sympathetic surgery . Fourteen patients were male and 5 were female (median age: 28 yr, range: 19 - 61). This study was approved by the institutional review board of our hospital (irb no: 3 - 20080021). All procedures of reconstruction surgery were performed under general anesthesia with a single lumen endotracheal tube . The patients were in a semi - fowler position with their arms extended . In 18 patients except for one patient with severe pleural adhesion, starting on the left side, two separate skin incisions were made along the previous thoracoscopic scars and thoracoscopic ports were placed . After co2 gas insufflation into the thoracic cavity with less than 10 mmhg of pressure to deflate the lung, the pleural space was inspected with 5-mm thoracoscope . The intercostal neurovascular bundle was dissected in lengths of 5 - 7 cm and the distal part was resected . After the proximal and distal part of previously operated sympathetic nerve was exposed, the nerve sheaths of proximal and distal part of exposed sympathetic nerve and harvested intercostal nerve end were removed with an electro - surgical tip cleaner (surgisite, ethicon, gargrave, skipton, uk). The intercostal nerve end was placed between the proximal and distal part of the exposed sympathetic nerve and the fibrin glue was applied to contact surface of the sympathetic and intercostal nerve (fig . The same procedure was repeated on the right side . In cases of sympathetic nerve clipping, this procedure was performed after clip removal . In one patient with severe pleural adhesion we reviewed the clinical charts of all patients who underwent sympathetic nerve reconstruction surgery using the intercostal nerve . Patients were followed by telephone questionnaire on the effects of the surgery and postoperative complications . The degree of improvement of compensatory hyperhidrosis was graded as " definite ", " mild ", or " absent " . " Definite " means that patients felt fully satisfied after the reconstruction surgery, " mild " means that patients felt satisfied to a certain extent, and " absent " means that patients felt no improvement . Median interval between the sympathetic nerve reconstruction surgery and questionnaire was 22 (range: 1 - 45) months . In one patient (patient number 17 in table 1), digital infrared thermographic imaging was performed preoperatively and postoperatively . Primary hyperhidrosis patients were composed of 9 facial, 8 palmar, and 1 axillary, and one patient had both facial and palmar hyperhidrosis . Initial endoscopic thoracic sympathetic surgery for primary hyperhidrosis was t3 sympathicotomy in 5 patients; t2 sympathicotomy in 8; t2, 3 sympathicotomy in 1; t2, 3, 4 sympathicotomy in 1; t2 clipping in 2; t2, 3 clipping in 1; and t2 clipping with t3 sympathicotomy in 1 patient . The median interval between the first endoscopic thoracic sympathetic surgery for primary hyperhidrosis and sympathetic nerve reconstruction surgery was 47 (range, 4 - 111) months . R3 intercostal nerve was used for sympathetic nerve reconstruction surgery in 15 patients, r4 intercostal nerve in 2, r2 intercostal nerve in 1, and r3 with r5 intercostal nerve in 1 patient . Three patients replied that the effects of reconstruction were " definite ", 6 responded with " mild ", and 8 said " absent " . Postoperative complications were numbness of the chest wall in 2 patients, chest wall pain in 2 patients, and temporary ptosis in 1 patient in whom ptosis spontaneously resolved after 3 months . Preoperative and postoperative digital infrared thermographic imagings performed in one patient showed the thermal change in chest and back . Since kux advocated thoracoscopic sympathetic surgery (3), recent developments in thoracoscopy and specialized instruments have facilitated the procedure . And, it became the treatment of choice for hyperhidrosis because of its low morbidity, short hospital stay, and excellent cosmetic results (6). Although it has many advantages, some patients suffer from compensatory hyperhidrosis, which is by far the most common and disagreeable complication after endoscopic thoracic sympathetic surgery . According to the previous reports, compensatory hyperhidrosis occurs in 59.8 - 90% of patients after sympathetic surgery (8 - 10). The mechanism of compensatory hyperhidrosis is not clear, but it seems to be associated with compensation for thermoregulatory function (11, 12). Because the incidence and degree of compensatory hyperhidrosis appear to be related to the extent of resection of the sympathetic chain, some clinicians have suggested that the extent of resection should be limited (13). For these reasons, many treatment methods, such as different level sympathectomy or sympathicotomy, ramicotomy, and clipping, have been attempted to reduce the extent of resection (14 - 18), although the effects of these methods remain controversial . In mild compensatory symptoms, antiperspirants including aluminum - based compounds, iontophoresis, and systemic or topical anticholinergic drugs can be used . However, if the symptoms are severe, the management is more difficult and results are unsatisfactory . Since philipeaux and vulpian reported the first experimental nerve graft in 1870, many successful nerve grafts were reported in the field of orthopedic surgery . In the field of thoracic surgery, schoeller et al . (19) reported successful phrenic nerve reconstruction using sural nerve in patient with mediastinal tumor resection . Telaranta reported that reconstruction of the sympathetic chain using sural nerve graft diminished compensatory sweating in a male patient who underwent sympathicotomy for palmar hyperhidrosis (20). Miura et al . (21) reported that sympathetic nerve reconstruction surgery using the intercostal nerve was useful after resection of the sympathetic nerve involved by tumor . Although the sural nerve is the most commonly used for nerve graft, the intercostal nerve has several advantages over the sural nerve . First, the intercostal nerve has more sympathetic nerve fibers than the sural nerve, hence the intercostal nerve is more appropriate for sympathetic nerve reconstruction surgery . Second, the sural nerve can be used only as free graft but the intercostal nerve can be used as pedicled graft and harvested as a neurovascular bundle . Therefore, a sufficient blood supply in the graft can be maintained . So, an additional incision is not needed and donor site morbidity decreases . In our experience, thoracoscopic intercostal nerve could be harvested in all patients except 1 patient with severe pleural adhesion, leading us to believe that the intercostal nerve is a useful graft for sympathetic nerve reconstruction surgery . Epineural and fascicular sutures are the most used for nerve anastomosis, but the foreign body reaction caused by suture material to nerve is another possible problem (22, 23). Some reports demonstrated the successful nerve anastomosis using fibrin sealant without suture technique (24, 25). If microscopic suture technique is used in sympathetic nerve reconstruction surgery, it inevitably needs thoracotomy . The type of anastomosis performed in this procedure is the modification of end - to - side neurorrhaphy where axonal sprouting occurs (26). The nerve is generally anastomosed in the original direction, but the free graft should be prepared to be anastomosed in the original direction in this operation . Some reports revealed the anastomosis in reverse orientation did not influence the nerve conduction (27, 28). In the questionnaire on the effects of sympathetic nerve reconstruction surgery, nine patients replied " definite " or " mild " . In 3 patients who replied that the results were " definite ", compensatory sweating decreased and anhidrosis of the affected areas after endoscopic thoracic sympathetic surgery improved . Patients, who replied mild improvement, complained still uncomfortable compensatory hyperhidrosis, although amount and frequency of perspiration decreased . These results of the reconstruction surgery were not different between variables such as age, sex, affected area, and interval from endoscopic thoracic sympathetic surgery due to the small number of cases in this study . The digital infrared thermographic imaging is a useful tool in evaluation of body temperature distribution . In the image, before and after sympathetic nerve reconstruction, we performed the digital infrared thermographic imagings in 22 yr - old man complaining of compensatory hyperhidrosis in chest after t3 sympathicotomy . After the reconstruction surgery, trunk temperature has increased, and this result corresponded with patient's symptom . Even though the number of patients is not sufficient for data analysis, outcome of the reconstruction surgery did not correlate with the interval between the sympathetic surgery and reconstructive surgery . To evaluate the exact effects of sympathetic nerve reconstruction, more cases and longer postoperative complications of the reconstruction surgery were seen in 5 patients; prolonged chest wall pain which was tolerable in 2 patients, and numbness of chest wall in 2 patients . In addition, because compensatory hyperhidrosis depends on climate and season, the timing of a questionnaire survey is important . In this study, the questionnaire was performed on october when it is relatively cool and dry in korea ., our results suggest that sympathetic nerve reconstruction with intercostal nerve may be one of the useful surgical methods in severe compensatory hyperhidrosis patients . The reconstruction surgery must be decided very carefully in highly selected patients with severe compensatory hyperhidrosis.
Endometrial cancer (ec) is one of the most common gynecologic malignancies, occupying the sixth place in malignant tumor throughout the world with an increasing trend . The initial treatment of ec is comprehensive staging surgery, which consists of total hysterectomy, bilateral salpingo - oophorectomy, pelvic and para - aortic lymphadenectomy and peritoneal cytology . Laparotomy is the traditional approach, but laparoscopic surgery is widely proposed recent years since it was first reported using for the treatment of ec in 1992 by childers and surwit . In lots of retrospective and prospective studies, laparoscopy has been showed with many advantages compared to laparotomy, such as smaller incision, less pain, and faster recovery . It seems that laparoscopic surgery for ec also has similar recurrence rate and survival outcome with laparotomy procedure . However, most of these studies have focused on patients with early stage or low - risk disease and few specifically on patients with advanced stage or high risk . What's more, data of long - term survival outcomes about laparoscopic surgery remain limited . Willis et al have published an article about laparoscopic hysterectomy in a high - risk series of patients with ec, showed that laparoscopy provide all the benefits inherent in minimal access surgery with no compromise in terms of outcomes, but there was no laparotomy data as control . Therefore, to compare the long - term safety and efficacy between laparoscopic surgery and laparotomy in patients with high - risk ec, we conducted a retrospective analysis based on our decade of clinical data using propensity score matching (psm) in this research . A total of about 400 medical case notes of patients with histologically confirmed ec who has been received surgical treatment in beijing chao - yang hospital affiliated capital medical university from 2000 to april 2014 were reviewed . We selected subjects according to the following criteria: high - risk (grade 2, histologic nonendometrioid, myometrial invasion 1/2, positive pelvic or para - aortic nodes or stages ii iv) patients who had undergone comprehensive surgical staging in the form of peritoneal cytology, total hysterectomy, bilateral salpingo - oophorectomy, pelvic lymphadenectomy and para - aortic lymphadenectomy or sampling, + / cytoreductive surgery by the same experienced surgical team via laparotomy (total abdominal hysterectomy, tah) or laparoscopy (total laparoscopic hysterectomy, tlh). Para - aortic lymphadenectomy was performed when para - aortic lymph nodes sampled positive and nonendometrioid carcinomas . Patients who were enrolled (excepted staged i ag2 with histologic endometrioid) received postoperative adjuvant therapy including 3 to 6 cycles of chemotherapy (paclitaxel, 175 mg / m + carboplatin, area under the concentration time curve [auc] 5 or adriamycin, 45 mg / m + cisplatin, 50 mg / m + paclitaxel, 160 mg / m) and vaginal cuff brachytherapy or pelvic irradiation (45 gy over 5 weeks). Patients had antibiotic prophylaxis (cephalosporins or quinolones) half an hour before operation and venous thrombosis prophylaxis after operation by using molecular weight enoxaparin (0.4 ml every day for 45 days) and lower extremity sequential compression device or graduated compression stocking . Patients associated with severe cardiopulmonary disease (myocardial infarction, heart failure, or cerebral infarction), other malignancies or a bulky uterus 12 weeks of gestation were excluded . Because our data source was case notes and the missing data were few, we deleted the individuals with missing data . The collected data of patients included age, body mass index (bmi), the scope of operation, operating time, blood loss, intraoperative and postoperative complications, first flatus time, postoperative hospital stay, surgical stage, histologic type and grade, number of lymph nodes dissection, and clinical outcome . The staging of patients was done according to the international federation of gynecology and obstetrics (figo) 2009 staging classification . Intraoperative complications included injuries to bladder, ureter, intestine or large vessels, bleeding requiring blood transfusion, conversion from laparoscopy to laparotomy and transferred to intensive care unit (icu) for any unintended reasons . Postoperative complications included fever (> 38c) after 24 hours postoperatively, wound infection or dehiscence, deep - vein thrombosis (dvt), and pulmonary embolism (pe) within 30 days after operation . The overall survival (os) was defined as the period from the date of surgery to death or the date of last contact for living people . Follow - up is completed by telephone, e - mail, or outpatient service . All procedures were approved by the ethics committee for human experiments of the capital medical university . Because parts of the baseline characteristics were statistical different between patients received laparoscopic surgery and laparotomy eight covariates entered in the propensity model, including age, bmi, the scope of operation, myometrial invasion, pathologic type and grade, lymph node metastasis, and surgical stage . Propensity scores were calculated using a nonparsimonious multivariable logistic regression model to estimate the conditional probability of a patient receiving a surgery approach . Then a 1:1 match between the laparoscopy and laparotomy groups was performed by using the nearest available neighbor matching . After matching, data of patient baseline characteristics, operation outcome and incidence of intraoperative and postoperative complication were compared using t test, chi - square test, or fisher exact test . Survival data were estimated using kaplan meier curves and compared between groups by log - rank statistics . The 5- and 10-year survival rate and os were reported . All of the statistical analysis was completed by spss 19.0 (spss version 19.0; spss, inc ., chicago, il). Statistical significance was defined as p <0.05 . Three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . Among these 225 patients, 31 individuals with severe cardiopulmonary disease, other malignancies or uterus 12 weeks of gestation were excluded; 4 individuals with missing surgical or pathological data were deleted . Finally, a total of 220 patients with high - risk ec met the study criteria, 107 were treated by laparotomy and 113 by laparoscopy . Table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . Before matching, significant differences were not found in age, myometrial invasion, pathological type, and grade and lymph node metastasis between 2 groups but in bmi, scope of operation, and figo stage which could be the confounding factors . After matching, 81 pairs of patients were matched successfully . In the matched cohorts baseline patient characteristics pre- and postpropensity score matching the mean operative time was similar between 2 groups, 253 minutes in laparoscopy group and 258 minutes in laparotomy group . The mean blood loss were significant less in laparoscopy group (107 ml) than laparotomy group (414 ml) (p <0.01). The laparoscopy cohort also has significant shorter first flatus time (2.0 days vs. 2.4 days, p <0.01) and postoperative hospital stay (14.7 days vs. 17.7 days, p = 0.02) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy . The mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference (p <0.01). However, the mean numbers of para - aortic lymph nodes were comparable between 2 groups (p = 0.11) (table 2). Comparison of operation outcome intraoperative complications were significant different between the 2 groups (6.2% for laparoscopy and 25.9% for laparotomy, p <0.01). The patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy (p <0.01). For the incidence of transferred to icu on account of poor cardiopulmonary resuscitation, it was also statistically higher in patients treated by laparotomy than by laparoscopy . Moreover, no injury to bladder, ureter, or large vessels occurred in both 2 groups, only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair . There was no significant difference in postoperative complications between the 2 groups (19.8% for laparoscopy and 29.6% for laparotomy, p = 0.14). Dvt occurred more often in laparotomy patients than laparoscopy patients, 14.8% versus 3.7% (p = 0.01). Six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference (table 3). Comparison of intraoperative and postoperative complications clinical outcome of 2 groups was shown in table 4 . The median follow - up was 45 months (rang 5176). During the follow - up time, there were 16 (9.9%) recurrences which were similar between the 2 treatment cohorts, 7 (8.6%) were observed in laparotomy group at peritoneal, liver, lung, vaginal, and abdominal incision versus 9 (11.1%) in laparoscopy group at peritoneal, liver, lung, and vaginal . Twenty patients died, 11 in the laparotomy group and 9 in the laparoscopy group . The kaplan meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2%, 85.3% and at 10 years was 75.8%, 85.3%, respectively . No statistically significant difference was found between the 2 groups when os was compared (p = 0.97) (figure 1). It showed that no significant difference between laparotomy and laparoscopy cohorts (75.8% vs. 85.3%, p = 0.97). Three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . Among these 225 patients, 31 individuals with severe cardiopulmonary disease, other malignancies or uterus 12 weeks of gestation were excluded; 4 individuals with missing surgical or pathological data were deleted . Finally, a total of 220 patients with high - risk ec met the study criteria, 107 were treated by laparotomy and 113 by laparoscopy . Table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . Before matching, significant differences were not found in age, myometrial invasion, pathological type, and grade and lymph node metastasis between 2 groups but in bmi, scope of operation, and figo stage which could be the confounding factors . After matching, 81 pairs of patients were matched successfully . In the matched cohorts the mean operative time was similar between 2 groups, 253 minutes in laparoscopy group and 258 minutes in laparotomy group . The mean blood loss were significant less in laparoscopy group (107 ml) than laparotomy group (414 ml) (p <0.01). The laparoscopy cohort also has significant shorter first flatus time (2.0 days vs. 2.4 days, p <0.01) and postoperative hospital stay (14.7 days vs. 17.7 days, p = 0.02) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy . The mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference (p <0.01). However, the mean numbers of para - aortic lymph nodes were comparable between 2 groups (p = 0.11) (table 2). Intraoperative complications were significant different between the 2 groups (6.2% for laparoscopy and 25.9% for laparotomy, p <0.01). None of the laparoscopic cases was converted into open procedures . The patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy (p <0.01). For the incidence of transferred to icu on account of poor cardiopulmonary resuscitation, it was also statistically higher in patients treated by laparotomy than by laparoscopy . Moreover, no injury to bladder, ureter, or large vessels occurred in both 2 groups, only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair . There was no significant difference in postoperative complications between the 2 groups (19.8% for laparoscopy and 29.6% for laparotomy, p = 0.14). Dvt occurred more often in laparotomy patients than laparoscopy patients, 14.8% versus 3.7% (p = 0.01). Six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference (table 3). The median follow - up was 45 months (rang 5176). During the follow - up time, there were 16 (9.9%) recurrences which were similar between the 2 treatment cohorts, 7 (8.6%) were observed in laparotomy group at peritoneal, liver, lung, vaginal, and abdominal incision versus 9 (11.1%) in laparoscopy group at peritoneal, liver, lung, and vaginal . Twenty patients died, 11 in the laparotomy group and 9 in the laparoscopy group . The kaplan meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2%, 85.3% and at 10 years was 75.8%, 85.3%, respectively . No statistically significant difference was found between the 2 groups when os was compared (p = 0.97) (figure 1). It showed that no significant difference between laparotomy and laparoscopy cohorts (75.8% vs. 85.3%, p = 0.97). For accurately estimating the effect of intervention, randomized controlled trial (rct) is considered the optimal approach but often limited by ethic and other problems . Psm can eliminate the imbalance between groups and reduce the effects of confounding to achieve a random effect in observational studies . In the present study, the patient baseline characteristics of laparotomy and laparoscopy group were adequately balanced after matching, and then, the surgery outcomes achieved comparability . This retrospective study demonstrated that laparoscopic technique is effective and even safer compared with laparotomy for patients with high - risk ec . In 2009, gynecologic oncology group (gog) compared laparoscopy and laparotomy for comprehensive surgical staging of ec in a randomized multicenter trial which involved 2616 patients and reported that laparoscopic surgery resulted in fewer complications and shorter hospital stay but longer operation time . In another large randomized trial published in 2010, laparoscopic surgery was associated with a shorter hospital stay and less amount of blood loss, but no beneficial effect was observed in terms of major complications and operation time . However, our data showed evidence of less blood loss, lower proportion of major complications and shorter hospital stay with laparoscopy versus laparotomy and a similar operation time . Operation time is much related to the experience of the surgical team and learning curve . As the improvement of surgical technique, it is a trend that laparoscopic operation time becomes shorter . Moreover, our study indicated that the first flatus time was shorter in laparoscopy group than in laparotomy . Patients treated with laparoscopic surgery had smaller incision and less pain compared to those treated with laparotomy . Laparoscopy can also bring out an earlier resumption of bowel function and shorter hospital stays . We found that the postoperative hospital stays in our data were longer than that reported in most of the previously studies and just similar to kong's study in korea . Kong reported a mean hospitalization of 16.4 days for laparoscopy and 23.3 days for laparotomy . But in gog study, the median hospital stays was 3 days for laparoscopy and 4 days for laparotomy . Patients in china are accustomed to a longer hospitalization, which is mainly decided by the social factors . In chinese traditional opinion, patients are willing to discharge from hospital only when they have complete recovery (after removing drainage tube and urinary catheter or taking out stitches), what's more, they need not worry about the medical expense because of the coverage of insurance . So, some patients even have been in hospital until the end of the first chemotherapy . In the present study, intraoperative complications were significantly lower in the laparoscopy group and no injuries to bladder, ureter, or large vessels occurred . The incidence of major complications can be highly reduced when the procedure was performed by experience surgeon . However, it seems that laparoscopy had no benefit in the incidence of postoperative complications . But we found dvt occurred significantly often in patients treated by laparotomy than those treated by laparoscopy . In gog study, there was no significant difference in incidence of venous thromboembolism (vte) and pe between different approaches . In contrast, fader's study showed that the laparoscopy cohort had significantly fewer episodes of vte . Lots of studies indicate that the combination of underlying malignancy and pelvic surgery is the risk for developing a vte . In a japanese large research about risk factors for vte, the age (50 or 55 years), a diagnosis of cancer, operating time (4 hours), blood loss (1000 ml), and blood transfusion (2000 ml) were reported significantly related to postoperative vte . It may account for the difference of dvt incidence between the 2 groups that laparotomy had longer operating time and more blood loss in our study . What's more, trendelenburg position during laparoscopic surgery may play a role in preventing vte . Most of the previous studies demonstrate that the pelvic or para - aotic lymph nodes count removed by laparoscopy approach were similar to those dissected by laparotomy . Laparoscopy has been proven to be even more efficient in lymphadenectomy because of its clearer vision . However, obermair and kalogiannidis reported lower number of lymph node dissections in the laparoscopy group . In the present study, the mean number of dissected para - aotic lymph nodes was comparable between the 2 groups, but the pelvic lymph nodes were significant fewer in laparoscopy cohort than laparotomy . Two large rcts reported by benedett and kitchener showed no evidence that lymphadenectomy could decrease the risk of recurrence or mortality rate in patients with early ec . Conversely, another retrospective research gave a result that lymphadenectomy improved the survival of patients with high - risk ec and stage i b to stage iv . Moreover, an article published in lancet in 2010 reported that para - aotic had a significantly benefit in terms of os for patients with ec at mediate or high risk . It seems that the difference of pelvic lymph nodes count between the laparoscopy and laparotomy groups had not influenced the os in our study . Lots of previous studies have proven that laparoscopy surgery was similar to laparotomy in short - term survival . A meta - analysis reported in 2012 based on 8 rcts showed that there was no significant difference in disease - free survival, cancer - related survival, or os . Cho et al reported a similar 5-year os and progression - free survival between laparoscopy and laparotomy groups based on 10-year experience . In 2012, the gog published the survival data of their large rct in 2009 that the 5-year os was almost identical in both laparoscopy and laparotomy cohorts at 89.8% . Between 2000 and 2010, we conducted a randomized trial and reported that the 5-year os rates were 91% of laparotomy and 96% of laparoscopy with no significantly difference . In another retrospective study focus on high - grade ec in 2012, progression - free and os were not significantly different between the surgical cohorts at 44-month median follow - up . In the present study, parts of patients were follow - up more than 10 years . With a median follow - up time of 45 months, the 5- and 10-year survival rate for the laparotomy were 89.2% and 75.8% compared with 85.3% and 85.3% for the laparoscopy . There was no significant difference in os between 2 cohorts and also the recurrence rate . Although the patients enrolled were not assigned to different approaches randomly which was a limitation of our study, we performed a psm and successfully to control a certain confounding factors yet . When randomized trial was limited by the objective conditions, psm might be a feasible alternative to reduce selection bias . In conclusion, it seems that laparoscopic surgery is as effective as laparotomy in the long term and can be safely carried out in patients with high - risk ec for surgery treatment . Furthermore, other long - term follow - up data on high - risk ec of randomized trials is still needed to confirm these results.
Syringomyelia is the development of a fluid - filled cavity within the spinal cord . Despite different etiologies that cause syringomyelia, it is largely agreed that the alteration of cerebrospinal fluid (csf) flow dynamics plays an important role in the development of a syrinx . Known causes of syringomyelia include chiari 1 malformation and basal arachnoiditis [postinfectious, inflammatory, postirradiation, blood in subarachnoid space (sas) amongst others]. Primary spinal syringomyelia refers to syrinx formation due to a spinal pathology, e.g., spinal cord tumor, spinal infection, trauma, and surgery . The term idiopathic syringomyelia has been used to describe syrinx formation in cases where causative mechanism remains undetermined after clinical and routine radiological work - up . The term does not imply the absence of a cause but just the absence of a cause that can be detected using standard diagnostic methods . There are very few reports in the literature describing arachnoid bands / webs as causes of idiopathic syringomyelia . We present our experience in the management of cases of syringomyelia due to dorsal spinal arachnoid bands and review the current available evidence of the diagnostic work - up and management of this etiology . A 43-year - old male was referred with a 2-year history of progressive dissociative sensory loss in his lower limbs with myoclonic twitches . This was associated with neck and low back pain in addition to left - sided radiating leg pain . There was no history of previous trauma, central nervous system (cns) infection, or spinal surgery . On examination, there was non - dermatomal numbness in left lower limb but normal power in all four limbs . He had been investigated previously in another neurosurgical center and the magnetic resonance imaging (mri) scan revealed a cervicothoracic syrinx but in the absence of any apparent cause; the patient was managed conservatively for 2 years till he was eventually referred to our center for a second opinion . Mri scan [figure 1] showed twin syrinx cavities in the cervical and upper thoracic regions with an unusual distribution and no apparent etiology . The patient underwent a computed tomography (ct) myelogram [figure 2] that showed csf block at t1/t2 suggestive of arachnoid pathology . During the ct myelogram, a small air bubble was inadvertently introduced that ascended superiorly and was trapped at the level of the csf block . He was now experiencing increasing dysesthetic pain down both arms along with left flank numbness and gait disturbance . In view of progressive deterioration, he underwent a t1/t2 thoracic laminectomy for intradural exploration that revealed a thickened dorsal band of arachnoid . Preoperative (top row) and postoperative (bottom row) mri for case 1 showing cervical significant syrinx prior to surgery and collapsed down following surgery . The postoperative scan was done 6 months after the surgery preoperative ct myelogram and axial mri for case 1 . The abrupt change in cord caliber / displacement demonstrated by the myelogram was not evident on mri studies . Note inadvertent bubble of air that ascended superiorly to the level of arachnoid web, which was useful in confirming the presence and level of the web postoperatively, the patient recovered well from the operation and there was gradual improvement in his symptoms with normal gait and return of normal function of hands . Six months after the surgery, the patient reported complete symptom resolution and follow - up mri scan [figure 1] showed significant reduction in the size of the syrinx . A 45-year - old male was referred following an episode of legs giving way that was followed by paraparesis for a few hours that recovered fully . On examination mri scan [figure 3] revealed a lower thoracic syrinx with some signal change in spinal cord . Follow - up imaging after 18 months showed no change . However, 4 months later the patient reported increasing discomfort and numbness in both the arms with subjective lower limb weakness and gait disturbance . A ct myelogram [figure 4] was performed that revealed displacement of the spinal cord at t3 to t5 and compression to the left likely secondary to either arachnoid band or cyst . Pre- (top row) and postoperative (bottom row) mri for case 2 showing significant syrinx prior to surgery and collapsed down following surgery . The postoperative scan was done 12 months after the surgery preoperative ct myelogram for case 2 demonstrating the cord displacement and change in caliber by now, the patient had developed right lower limb spastic weakness along with left radiating leg pain . On examination he had brisk reflexes, positive hoffmann's sign bilaterally, positive ankle clonus, reduced sensation in the right leg, and upgoing plantars . Intradurally, we found a dorsal arachnoid band that was excised and histology confirmed arachnoid tissue . Postoperatively, the patient recovered well from the operation with improvement in right leg strength and the left leg pain . At 6-month follow - up, he reported complete symptomatic resolution, and neurological examination was normal . His follow - up mri [figure 3] at 6 months postoperatively revealed significant reduction in the syrinx size . A 43-year - old male was referred with a 2-year history of progressive dissociative sensory loss in his lower limbs with myoclonic twitches . This was associated with neck and low back pain in addition to left - sided radiating leg pain . There was no history of previous trauma, central nervous system (cns) infection, or spinal surgery . On examination, there was non - dermatomal numbness in left lower limb but normal power in all four limbs . He had been investigated previously in another neurosurgical center and the magnetic resonance imaging (mri) scan revealed a cervicothoracic syrinx but in the absence of any apparent cause; the patient was managed conservatively for 2 years till he was eventually referred to our center for a second opinion . Mri scan [figure 1] showed twin syrinx cavities in the cervical and upper thoracic regions with an unusual distribution and no apparent etiology . The patient underwent a computed tomography (ct) myelogram [figure 2] that showed csf block at t1/t2 suggestive of arachnoid pathology . During the ct myelogram, a small air bubble was inadvertently introduced that ascended superiorly and was trapped at the level of the csf block . He was now experiencing increasing dysesthetic pain down both arms along with left flank numbness and gait disturbance . In view of progressive deterioration, he underwent a t1/t2 thoracic laminectomy for intradural exploration that revealed a thickened dorsal band of arachnoid . Preoperative (top row) and postoperative (bottom row) mri for case 1 showing cervical significant syrinx prior to surgery and collapsed down following surgery . The postoperative scan was done 6 months after the surgery preoperative ct myelogram and axial mri for case 1 . The abrupt change in cord caliber / displacement demonstrated by the myelogram was not evident on mri studies . Note inadvertent bubble of air that ascended superiorly to the level of arachnoid web, which was useful in confirming the presence and level of the web postoperatively, the patient recovered well from the operation and there was gradual improvement in his symptoms with normal gait and return of normal function of hands . Six months after the surgery, the patient reported complete symptom resolution and follow - up mri scan [figure 1] showed significant reduction in the size of the syrinx . A 45-year - old male was referred following an episode of legs giving way that was followed by paraparesis for a few hours that recovered fully . On examination mri scan [figure 3] revealed a lower thoracic syrinx with some signal change in spinal cord . Follow - up imaging after 18 months showed no change . However, 4 months later the patient reported increasing discomfort and numbness in both the arms with subjective lower limb weakness and gait disturbance . A ct myelogram [figure 4] was performed that revealed displacement of the spinal cord at t3 to t5 and compression to the left likely secondary to either arachnoid band or cyst . Pre- (top row) and postoperative (bottom row) mri for case 2 showing significant syrinx prior to surgery and collapsed down following surgery . The postoperative scan was done 12 months after the surgery preoperative ct myelogram for case 2 demonstrating the cord displacement and change in caliber by now, the patient had developed right lower limb spastic weakness along with left radiating leg pain . On examination he had brisk reflexes, positive hoffmann's sign bilaterally, positive ankle clonus, reduced sensation in the right leg, and upgoing plantars . Intradurally, we found a dorsal arachnoid band that was excised and histology confirmed arachnoid tissue . Postoperatively, the patient recovered well from the operation with improvement in right leg strength and the left leg pain . At 6-month follow - up, he reported complete symptomatic resolution, and neurological examination was normal . His follow - up mri [figure 3] at 6 months postoperatively revealed significant reduction in the syrinx size . We report a very uncommon and hence under - recognized cause of symptomatic syringomyelia . Whilst arachnoid bands / webs have previously been reported as the etiologic factor for idiopathic syringomyelia first reported the importance of recognizing occult arachnoid webs, pouches, and cysts in idiopathic syringomyelia . In a large series of 28 patients with thoracic arachnoid pathology, 6 cases of ventral or dorsal arachnoid bands were reported . However, there is uncertainty not only regarding the pathophysiology of syrinx formation with arachnoid webs but also regarding the ideal radiological investigations . Arachnoid adhesions due to trauma / infection / previous surgery are well - recognized causes of syringomyelia . Idiopathic arachnoid bands have been hypothesized to develop from the septum posticum, the membrane dividing the midline dorsal arachnoid space of the cervicothoracic spinal canal . It is however equally possible that they represent localized arachnoiditis due to factors that are not always elicited in the history . Frequently cited theories in syrinx formation are those of gardner, williams, and oldfield . Greitz proposed that increased intramedullary pulse pressure in the spinal cord relative to the nearby sas is the distending force in the production of syringomyelia and that syrinx consists of extracellular fluid accumulated in the distended spinal cord . Directly addressed the pathophysiology of primary spinal syringomyelia in their prospective study of 36 patients . Their findings were consistent with their theory that a spinal subarachnoid block increases subarachnoid pulse pressure above the block, producing a pressure differential across the obstructed segment of the sas, which results in syrinx formation and progression . They reported that their findings were similar to that of their previous studies, which examined the pathophysiology of syringomyelia associated with obstruction of sas at the foramen magnum, inferring a common mechanism for both . Three - dimensional computational models of the spinal sas have been used to study csf flow either in unobstructed sas or with sas obstructed by a porous region simulating dorsal and circumferential arachnoiditis . The findings suggest that syrinx formation may be related to a change of temporal csf pulse pressure dynamics . Consequently, it would appear that restoring csf flow across the arachnoid webs / bands should result in syrinx resolution . Alternatively, syrinx formation may occur due to resonance in a mechanical system with a pathology - induced nodal point . However, the recognition of a secondary imaging finding the scalpel sign can suggest the presence of an arachnoid web . This is a focal indentation of the thoracic spinal cord, resembling a scalpel on sagittal mri images with the scalpel blade pointing posteriorly . However, none of our patients had any clear evidence suggesting an arachnoid web on conventional mri . In all the early reported series of arachnoid webs, ct myelography was the investigation of choice . Indeed, it has been reported that cardiac gated mri techniques were less successful in studying csf flow in the spine than at the craniovertebral junction . However, in a large series of 125 patients with idiopathic syringomyelia, mauer et al . Performed cardiac gated phase contrast mri of csf flow in the median sagittal plane in the spine and reported that these were more reliable compared to myelography . Cardiac gated phase contrast cine - mode mri in multiple axial planes has also been used to quantitatively analyze csf flow in those planes for the diagnosis of arachnoid webs . Whilst in these papers, arachnoid webs were indirectly diagnosed using flow studies; direct visualization of arachnoid membranes has been reported with high resolution using retrospectively cardiac gated cine steady - state free precession mri . However, all these mri techniques are susceptible to movement artifacts and also require a 3tesla mri that may not always be available . Arachnoid webs may be missed even with mri csf flow sequences, hence ct myelography remains useful . It is worth noting that the inadvertent air bubble introduced during the ct myelogram of our first patient provided an excellent clue about the location of the csf block . The location of the syrinx is not a reliable clue regarding the level of arachnoid web / csf block . The arachnoid web was just caudal to the syrinx in our first patient and at the cranial end of the syrinx in the second patient . We recognize that myelography is an invasive procedure with potential morbidity and with recent significant advancements in mr technology, it may be prudent to investigate symptomatic idiopathic syringomyelia with mri csf flow studies in the first instance and failing that, to proceed to myelography . We believe that restoring csf flow by excision of the arachnoid band and thereby improving sas compliance offers the best possible outcome in these symptomatic cases . In the earliest reported series, all patients treated by web excision improved whereas both patients treated with syringo - pleural shunts worsened . Mixed result were obtained with shunting in other series but excision of the webs was almost always associated with successful outcome . Shunting syrinxes is associated with high rates of recurrence, poor long - term outcomes, and sometimes worsening of neurological status . In fact, most patients with any short segment arachnoid pathology causing primary spinal syringomyelia, generally have good outcomes after surgical decompression / detethering procedures . It is the patient group with long segment pathology that often requires multiple surgeries, frequently for csf diversion . This was corroborated in a recent systematic review, which suggested that arachnolysis rather than shunting was the only initial surgical treatment that is likely to have a statistically significant effect on lowering recurrence rates . This once again highlights the importance of exhaustively looking for a rectifiable surgical targets, such as arachnoid webs and cysts in the presumed idiopathic syrinxes, which would otherwise have be treated with shunts . Arachnoid webs / bands are a rare and hence often underdiagnosed cause of idiopathic syringomyelia, leading to delayed diagnosis and treatment . In symptomatic / deteriorating patients with idiopathic syringomyelia, imaging studies should be closely inspected for the presence of a transverse arachnoid web . Conventional mri should always be augmented with mri csf flow studies in the sagittal plane and if needed in multiple axial planes to aid in the diagnosis . Ct myelography should be performed if these studies do not yield answers or also if the surgeon feels further information is needed prior to definitive treatment, especially as myelography provides more robust localization of the level of block . Focussed surgical decompression and excision of the offending arachnoid tissue usually leads to good clinical and radiological outcomes.
Approximately 30% of women have sustained at least 1 vertebral fracture by the age of 75 . These fractures are an important and common cause of morbidity in osteoporotic patients; moreover, fractures evidenced both clinically and at radiographic examination are associated with an increased mortality rate . Back pain owing to vertebral fractures has a significant affect on osteoporotic patients [46], and the number and severity of these fractures also increases the risk of developing chronic back pain . This has a marked negative impact on the quality of life (qol) and functional impairment of the affected patients . Conventional treatments for osteoporosis including bisphosphonates, selective estrogen receptor modulators (serms), and estrogen have been shown to reduce the rate of bone resorption and to preserve bone mass . Another therapeutic option includes rhpth, an agent that has been shown to increase both bone mass and bone strength . When injected, teriparatide (rdna origin), the amino - terminal fragment of human pth (rhpth 134), is a potent bone formation agent for the treatment of severe osteoporosis . It increases osteoblast production / growth and prevents osteoblast apoptosis; at the same time, it enhances absorption of calcium from the intestine, renal reabsorption of calcium, while decreasing the excretion of phosphates in the kidney [1114]. When administered once daily by subcutaneous injection, rhpth increases bone density and improves trabecular architecture, cortical geometry, and strength . Not only does rhpth increase trabecular bone density by stimulating bone formation, but it also stimulates osteoclastic bone resorption [1719]. In several studies, rhpth has been shown to increase bone mineral density in postmenopausal osteoporosis, in senile osteoporosis in men, and in glucocorticoid - induced osteoporosis . Teriparatide acts via the pth-1 receptor on osteoblasts and bone marrow stromal cells to induce osteoblastic bone formation, that is, osteoid synthesis and accelerated mineralization . This, in turn, results in reductions in skeletal fractures to levels equivalent to, or over, those obtained by using antiresorptive agents . The increase in bone mineral density induced by rhpth is substantial, ranging 10% to 15% over 2 to 3 years in most studies [10,2024]. Moreover, rhpth can cause demonstrable increases in bone mineral density and changes in markers of bone turnover within 3 months since the start of treatment . Quality of life (qol) can be measured to compare the effect of different treatments for osteoporosis . Measuring pain scores only for these patients would be inadequate, because apart from acute and chronic back pain, patients with vertebral fractures also experience anxiety, constant fear of falling, or suffering another fracture while their daily living activities are impaired . Most information has been collected thanks to the efforts made by some researchers to develop specific instruments to test the physical and emotional disability generated by the disease . Generic tools available for measuring qol are useful to evaluate general health but they lack disease specificity . More recently, some specific instruments have been developed to measure the qol in osteoporosis more accurately . The most widely used are the osteoporosis quality of life questionnaire (oqlq), the osteoporosis assessment questionnaire (opaq), the osteoporosis - targeted quality of life questionnaire (optqol), and the quality of life questionnaire of the european foundation for osteoporosis (qualeffo-41). One of the first ones was qualeffo-41, which has been translated and validated in different languages including italian . This questionnaire has proved to be repeatable, coherent, and able to discriminate between patients and controls . In the last few years, other specific questionnaires have been developed, but not all of them have been as extensively used and validated in different countries as qualeffo-41 . The goal of this study was firstly to assess the validity of rhpth treatment in a cohort of postmenopausal women with severe osteoporosis; secondly, to evaluate the improvement in qol and pain symptoms after several months of rhpth therapy . A follow - up qualeffo-41 questionnaire was used to quantify the patient s pain and the affect on qol after rhpth therapy . The study was a 18 months, randomized prospective cohort study conducted at department of molecular and clinical endocrinology and oncology, university of naples federico ii, naples, italy . Inclusion criteria for this study consisted of back pain, postmenopausal osteoporosis (t - score 2.5 at lumbar spine or femoral neck), the presence of 2 osteoporotic vertebral fractures, previous treatment for osteoporosis . The exclusion criteria were: an increased risk of osteosarcoma (ie, patients with paget disease bone, previous skeletal exposure to external beam radiotherapy, or previous malignant neoplasm involving the skeleton), hypercalcemia, malignant neoplasms, impaired renal function, liver disease, history of diseases other than postmenopausal osteoporosis that affect bone metabolism, nephrolithiasis, alcohol or drug abuse . Secondary osteoporosis was excluded in order to avoid the interference of the primitive disease with the patient s quality of life . Informed consent was obtained from all subjects and the study protocol was approved by the hospital / science s ethical committee . Eighty - one postmenopausal women were enrolled and divided in two groups with no statistically significant differences in any of the considered variables: group a forty - two women (mean age 659 yrs; mean body mass index bmi 24.52.6 kg / m), with severe postmenopausal osteoporosis (mean lumbar bmd 3.880.70, mean femoral neck bmd 3.070.60 and with 2 vertebral atraumatic fractures), persistent back pain and previous treatment with biphosphonates for osteoporosis; group b thirty - nine women matched for age (6014.4 yrs), bmi (22.88.8 kg / m), menopausal status, affected by back pain, severe postmenopausal osteoporosis (lumbar spine bmd 3.900.73, mean femoral neck bmd 3.020.61 and with 2 vertebral atraumatic fractures) previously treated for osteoporosis with biphosphonates . Bmi, age at menopause, lifestyle habits (i.e., smoking, drinking, nutrition style), nutrition anamnesis (calcium intake), history of diseases other than osteoporosis, family history of osteoporosis were considered . Groups were randomized to daily treatment with 20 g s.c . Of recombinant human parathyroid hormone (rhpth 134), self - administered injections (group a) or 70 mg per os of alendronate every week (group b). All women received 1,000 mg elemental calcium daily and 800 iu of vitamin d daily for 18 months . Biochemical markers of bone turnover were dosed in all the selected female patients: alkaline phosphatase (alp: 35104 u / l), n - terminal propeptide of type i procollagen (pinp: 19102 g / l), n - telopeptide crosslinks (ntx: 565 nmol / mmol crea) were assessed at baseline, 3, 12 and 18 months (t0, t3, t12, t18). The bmd of the lumbar spine and the proximal femur was measured by dual - energy x - ray absorptiometry (dual - energy x - ray absorptiometry qdr 1000; hologic, waltham, ma, usa) at baseline and at 18 months (t0, t18). All women underwent anteroposterior and lateral radiography of thoracic and lumbar spine at t0 and t18 . The qol questionnaire of the european foundation for osteoporosis (qualeffo) was administered at baseline and at the end of the study to evaluate the impact of rhpth on health - related qol . Originally, the questionnaire consisted of 48 questions and 6 visual analogue scales . In the qualeffo validation study, this resulted in the qualeffo-41, which consisted of 41 questions in 5 domains: pain, physical function, social function, general health perception, and mental function . All answers were recorded so that all items range from 1 to 5, and all answers were standardized so that 1 represents the best and 5 the worst qol, with the exception of questions e23 - 25 (questions with 3 answer options), questions e24 - 26 - 27 - 28 (4 answer options), and questions g33 - 34 - 35 - 37 - 39 - 40 (answer with reverse scores: 1 is the worst while 5 is the best). Domain scores are calculated by averaging the answers of 1 domain and transforming the scores to a score from 0 to 100 . Data are expressed as mean sd or percentage; moreover, to assess the affect of teriparatide treatment on markers of bone turnover, bone mineral density, and on health - related qol, pearson correlation coefficients were computed in the 2 study groups . Therefore, percentage changes of biomarkers, bone mineral density, and qualeffo results were calculated . There was no significant difference in these characteristics between the 2 groups . In the group a, 39 (93%) out of the 42 recruited women completed the study: 2 patients discontinued the drug therapy because of lack of compliance with the study treatment; 1 patient discontinued because of a new vertebral atraumatic fracture . The aim of this study was to evaluate percentage changes from baseline in biochemical markers of bone turnover, values of bone mineral density (measured at lumbar spine and proximal femur), and measurements of qol . Follow - up checks, where we also evaluated clinical conditions, adverse events, compliance to treatment and use of non - steroid anti - inflammatory drugs, were carried out at 3, 12 and 18 months since the beginning of treatment . In group a serum levels of pinp increased of 90%, 145% and 127% at t3, t12, t18, respectively; bone alp levels increased of 57%, 79% and 65%; ntx levels increased of 53% at t3, of 100% at t12, of 110% at t18 . In group b percentage changes from baseline of serum levels of pinp were 50%, 70% and 74% at t3, t12, t18, respectively; bone alp levels decreased of 30%, 48% and 41%; ntx levels were reduced by 55% at t3, of 69% at t12, of 72% at t18 . Mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 . Mean pinp values were 426 g / l at t0, 8012 g / l at t3, 10334 g / l at t12, 9526 g / l at t18 in group a (t0 vs t3 r: 0.81, p<0.001; t0 vs t12 r: 0.88, p<0.001; t0 vs t18 r: 0.86, p<0.001) and 7816 g / l, 395 g / l, 2313 g / l, 208 g / l at t0, t3, t12, t18 respectively (t0 vs t3 r: 0.67, p<0.001; t0 vs t12 r: 0.76, p<0.001; t0 vs t18 r: 0.82, p<0.001). In group u / l, 10720 u / l at t3, 12245 u / l at t12 and 11235 u / l at t18 (t0 vs t3 r: 0.72, p<0.001; t0 vs t12 r: 0.46, p<0.01; t0 vs t18 r: 0.85, p<0.001); instead, in group b mean alp was 72 15 u / l at baseline, 5015 after 3 months, 3717 u / l at t12 and 427 u / l at the end of the study (t0 vs t3 r: 0.91, p<0.001; t0 vs t12 r: 0.53, p<0.001; t0 vs t18 r: 0.65, p<0.001). Ntx mean values in group a were 317 nmol / mmol crea, 4721 nmol / mmol crea, 6211 nmol / mmol crea, 6523 nmol / mmol crea at t0, t3, t12 and t18 respectively (t0 vs t3 r: 0.22, p>0.1; t0 vs t12 r: 0.57, p<0.001; t0 vs t18 r: 0.49, p<0.01) while in group b ntx mean values were 534 nmol / mmol crea at t0, 29 11 nmol / mmol crea at t3, 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 (t0 vs t3 r: 0.74, p<0.001; t0 vs t12 r: 0.58, p<0.001; t0 vs t18 r: 0.69, p<0.001). By the end of the study period, the bmd values expressed in terms of t - score in our total pool displayed important changes (figure 3). At month 18, lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% . Specifically, in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 (r: 0.88; p<0.001); instead, in group b, mean t - score at t0 was 3.900.73 and mean t - score at t18: 3.750.72 (r: 0.98; p<0.001). The bmd in the femur increased from baseline at month 18, in group a, by 5.2% and by 1.99% in group b. in group a, mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study (r: 0.87; p<0.001); in group b, mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 (r: 0.99; p<0.001). At t0, patients treated with teriparatide were more protected against new fractures, compared with patients treated with bisphosphonates; in fact, only 1 new vertebral fracture occurred in group a (2.4%) at study endpoint (t18) vs 6 new vertebral fractures that occurred in group b (15.7%) at t18 . The most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women; nausea, reported by 10%; headache and dizziness that were reported by only 1 and 2 women . The reported adverse effects were abdominal pain in 9 patients, arthralgia in 4 patients, and dyspepsia in 1 patient . We evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 (figure 4). First domain (domain a) result indicated a serious reduction in pain (22%) after treatment with rhpth: mean scores measured was 729.2 at start and 5614 after 18 months (r: 0.61; p<.001) compared with group b (9.7%; 718.7 at t0 and 6511 at t18; r: 0.53; p<.001). For everyday activities (domain b), an average of 5513.6 was measured at t0 and of 4022.9 at t18 (r: 0.44; p<.01) in group a which had a total improvement of 27.3%, while the improvement was of 11% in group b (61.118 at t0 and 54.315.7 at t18; r: 0.68; p<.001). The performed domestic job domain (domain c) showed an improvement of 29% in group a (6010.7 at t0; 42.613.3 at t18; r: 0.68; p<.001) and of 2.9% in group b (62.113.3 at t0; 60.313.7 at t18; r: 0.88; p<.001). The mean score of domain d (locomotor function) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% (46.88.6 at t0; 41.410.05 at t18; r: 0.69; p<.001). The quality of free time and of the social activities (domain e) at the 2 time points they were 36.28.6 and 25.98.6, indicating a percentage change of 28.4% in group a (r: 0.50; p<.01); the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only (r: 0.87; p<.001). Patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health (domain f) (5925 at t0; 3916.7 at t18; r: 0.84; p<.001) versus 12.8% improvement of group b (63.228.2 at t0; 55.124.8 at t18; r: 0.90; p<.001). In the mood domain (domain g), the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a (r: 0.71; p<.001). These data demonstrated a considerable improvement (29.7%) compared with group b (1.8%; 32.59.8 at t0; 33.19.9 at t18; r: 0.18; p>.1). As a consequence of pain relief, the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a, while it did not decrease in group b. in group a serum levels of pinp increased of 90%, 145% and 127% at t3, t12, t18, respectively; bone alp levels increased of 57%, 79% and 65%; ntx levels increased of 53% at t3, of 100% at t12, of 110% at t18 . In group b percentage changes from baseline of serum levels of pinp were 50%, 70% and 74% at t3, t12, t18, respectively; bone alp levels decreased of 30%, 48% and 41%; ntx levels were reduced by 55% at t3, of 69% at t12, of 72% at t18 . Mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 . Mean pinp values were 426 g / l at t0, 8012 g / l at t3, 10334 g / l at t12, 9526 g / l at t18 in group a (t0 vs t3 r: 0.81, p<0.001; t0 vs t12 r: 0.88, p<0.001; t0 vs t18 r: 0.86, p<0.001) and 7816 g / l, 395 g / l, 2313 g / l, 208 g / l at t0, t3, t12, t18 respectively (t0 vs t3 r: 0.67, p<0.001; t0 vs t12 r: 0.76, p<0.001; t0 vs t18 r: 0.82, p<0.001). U / l, 10720 u / l at t3, 12245 u / l at t12 and 11235 u / l at t18 (t0 vs t3 r: 0.72, p<0.001; t0 vs t12 r: 0.46, p<0.01; t0 vs t18 r: 0.85, p<0.001); instead, in group b mean alp was 72 15 u / l at baseline, 5015 after 3 months, 3717 u / l at t12 and 427 u / l at the end of the study (t0 vs t3 r: 0.91, p<0.001; t0 vs t12 r: 0.53, p<0.001; t0 vs t18 r: 0.65, p<0.001). Ntx mean values in group a were 317 nmol / mmol crea, 4721 nmol / mmol crea, 6211 nmol / mmol crea, 6523 nmol / mmol crea at t0, t3, t12 and t18 respectively (t0 vs t3 r: 0.22, p>0.1; t0 vs t12 r: 0.57, p<0.001; t0 vs t18 r: 0.49, p<0.01) while in group b ntx mean values were 534 nmol / mmol crea at t0, 29 11 nmol / mmol crea at t3, 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 (t0 vs t3 r: 0.74, p<0.001; t0 vs t12 r: 0.58, p<0.001; t0 vs t18 r: 0.69, p<0.001). By the end of the study period, the bmd values expressed in terms of t - score in our total pool displayed important changes (figure 3). At month 18, lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% . Specifically, in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 (r: 0.88; p<0.001); instead, in group b, mean t - score at t0 was 3.900.73 and mean t - score at t18: 3.750.72 (r: 0.98; p<0.001). The bmd in the femur increased from baseline at month 18, in group a, by 5.2% and by 1.99% in group b. in group a, mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study (r: 0.87; p<0.001); in group b, mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 (r: 0.99; p<0.001). At t0, all patients of groups a and b showed baseline vertebral fractures . Patients treated with teriparatide were more protected against new fractures, compared with patients treated with bisphosphonates; in fact, only 1 new vertebral fracture occurred in group a (2.4%) at study endpoint (t18) vs 6 new vertebral fractures that occurred in group b (15.7%) at t18 . The most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women; nausea, reported by 10%; headache and dizziness that were reported by only 1 and 2 women . The reported adverse effects were abdominal pain in 9 patients, arthralgia in 4 patients, and dyspepsia in 1 patient . We evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 (figure 4). First domain (domain a) result indicated a serious reduction in pain (22%) after treatment with rhpth: mean scores measured was 729.2 at start and 5614 after 18 months (r: 0.61; p<.001) compared with group b (9.7%; 718.7 at t0 and 6511 at t18; r: 0.53; p<.001). For everyday activities (domain b), an average of 5513.6 was measured at t0 and of 4022.9 at t18 (r: 0.44; p<.01) in group a which had a total improvement of 27.3%, while the improvement was of 11% in group b (61.118 at t0 and 54.315.7 at t18; r: 0.68; p<.001). The performed domestic job domain (domain c) showed an improvement of 29% in group a (6010.7 at t0; 42.613.3 at t18; r: 0.68; p<.001) and of 2.9% in group b (62.113.3 at t0; 60.313.7 at t18; r: 0.88; p<.001). The mean score of domain d (locomotor function) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% (46.88.6 at t0; 41.410.05 at t18; r: 0.69; p<.001). The quality of free time and of the social activities (domain e) at the 2 time points they were 36.28.6 and 25.98.6, indicating a percentage change of 28.4% in group a (r: 0.50; p<.01); the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only (r: 0.87; p<.001). Patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health (domain f) (5925 at t0; 3916.7 at t18; r: 0.84; p<.001) versus 12.8% improvement of group b (63.228.2 at t0; 55.124.8 at t18; r: 0.90; p<.001). In the mood domain (domain g), the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a (r: 0.71; p<.001). These data demonstrated a considerable improvement (29.7%) compared with group b (1.8%; 32.59.8 at t0; 33.19.9 at t18; r: 0.18; p>.1). As a consequence of pain relief, the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a, while it did not decrease in group b. our results demonstrate that daily injections of rhpth for 18 months are an efficacious and generally well - tolerated therapy in postmenopausal women with severe osteoporosis . More importantly, in our experience rhpth therapy results in decreased fractures and pain symptoms in patients with osteoporosis, and teriparatide is a bone - forming agent and its effect is demonstrated by increases in biochemical markers of bone turnover over the study period: bone formation markers showed more rapid and higher increases than resorption ones, suggesting an early imbalance of bone turnover in favor of formation; these data are in agreement with previous studies . Treatment with teriparatide resulted in a greater increase in bone mineral density (bmd). After 18 months of therapy with rhpth, bone mineral density in the lumbar spine and of the proximal femur increased by 12.4% and 5.2%; these reported percentage increases are consistent with results of other studies . At month 18 in patients treated with bisphosphonates, instead, lumbar spine increased only by 3.85% and the bone mineral density in the femur increased by 1.99% . The differences in the percentage increases between the 2 groups of patients can be explained with substantial differences between antiresorptive and anabolic therapeutic effects on bone mass and architecture as well as on bone mineral density . Moreover, rhpth increases trabecular connectivity, whereas the majority of bone mineral density increases observed with bisphosphonate treatment are a result of increased mineralization of existing bone matrix . Teriparatide has been referred to be an efficacious treatment against new fractures, making a significant change in the course of severe osteoporosis, which can lead rapidly not only to varying degrees of disability, the loss of self - sufficiency, and to institutionalization, but also to death [3436]. In this study, we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms . In addition, use of teriparatide caused a considerable decrease of pain, accompanied by a consequent reduction of the need for nonsteroidal anti - inflammatory drugs by the patients; this was the main factor that assured an absolute compliance of the patients . In severe osteoporosis vertebral fractures are an important cause of back pain owing to muscle weakness and altered posture, resulting in serious acute and chronic pain that contributes to further disabilities; in fact, vertebral fractures are the top health condition accounting for length of hospitalization, and added significantly to the length of hospitalization in patients admitted for other medical problems . Apart from the physical disabilities had by these patients in fact, patients with vertebral fractures often experience impaired physical functions, limited activities of daily living, limited leisure and recreational activities, and significant emotional distress with loss of self - esteem and depression [3436]. In this study, we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms . To analyze all these variables and to measure the values of qol at baseline and at the end of the study, we used the qualeffo - test, developed by lips and associates in 1997 . Specifically, we used qualeffo-41, that represents a qol questionnaire which is brief, easy to administer, and with adequate preliminary psychometric properties . We preferred this test to the generic ones (such as nhp, sip, sf-36, eq - sd) because of its specificity in the evaluation of qol in people affected by osteoporosis with vertebral deformities; in fact, disadvantages of generic instruments are that they usually include irrelevant questions and that these questionnaires are less able to investigate those aspects that mostly influence qol of osteoporotic patients [2628]. This makes the qualeffo-41 potentially useful during routine clinical practice and research for the treatment and the follow - up of postmenopausal women with severe osteoporosis . By using this test, our study evidences that the use of rhpth influences all considered domains of qol; in fact, pain, physical and social functions, mood significantly improved in nearly all patients treated with rhpth at the end of the study . Thus, the reduction of back pain observed from baseline of active treatment through posttreatment follow - up is consistent with the reduction of new vertebral painful fractures, that means improved qol for this kind of patients . Our data clearly demonstrate that rhpth is more effective than bisphosphonates in acting on back pain and all the domains of qol . Adverse events (back pain, headache, nausea, dizziness) in our patients receiving rhpth were mild and transient, with a percentage lower than in previous studies . The adverse effects attributed to teriparatide did not stop the patients from continuing with the treatment . Our results demonstrate that rhpth increases bone mineral density considerably, reduces the occurrence of new fractures, and the need for analgesic therapy . Therefore, this study evidences that this anabolic agent represents a valid therapeutic option in severe postmenopausal osteoporosis; moreover, patients after this treatment experience, improvements not only in pain relief, but also in certain aspects of emotional functioning, activities of daily living, and leisure activities, improve qol considerably.
A 22 years old female patient was admitted to our outpatient clinic with a complaint of dyspnea for the previous three years . A right bundle branch block was detected on her electrocardiogram . In the transthorasic echocardiography (tte), a 22 mm secundum type atrial septal defect (asd), right heart dilatation and pulmonary arterial hypertension (pap 45 mmhg) were detected . Transesophageal echocardiography (tee) showed that the defect was 25 mm, the superior rim was 16 mm and the aortic rim was 8 mm in size . The defect was closed through a right femoral route with a 30 mm asd occluder after balloon sizing with tte . In the cineangiography and tte early after the procedure, the device was observed to be in the correct position (fig . The tte revealed that the device was not in the defect and it had embolized to the pulmonary artery . The patient was referred to the cardiovascular surgery unit and underwent surgery promptly . Under general anaesthesia the pulmonary artery was incised along its vertical axis and the occluder device was removed (fig . The right atrium was opened and the defect was then closed with a pericardial patch . Secundum asd is the most common type, which accounts for 50% to 70% of all asds and located in the fossa ovalis . Sinus venosus type asd is located at the upper portion of the interatrial septum where the vena cava superior opens . A shunt between atria leads to chronic right ventricular volume overload, which in turn causes lower filling rates of left ventricle . Surgical or percutaneous closure in asymptomatic patients is indicated in the case of right ventricular volume overload and if the pulmonary to systemic flow ratio increases beyond 1.5 . In adult patients, the defect should be repaired as soon as possible after it is detected because patients who undergo repair after 25 years of age have a shorter life span than a control population . The most important factor for success in percutaneous closure is the correct patient selection . In patients with secundum asd, percutaneous closure can be performed safely with careful evaluation of anatomical factors like defect size and sufficiency of the rims . In a previous report, it was shown that atrial defects up to 43 mm in diameter can be closed percutaneously . A sufficient anterior rim is not necessary to bring the device into a stable position . Conversely, deploying the device in patients with deficient cranial rims can be difficult and may lead to an unstable position of the device and increased risk of device embolization . The most common type of complications in percutaneous closure is device embolization, with a rate of 0.5% to 1% . In a study with 450 patients, seven cases of device embolization into pulmonary artery in the first 12 hours one hour after intervention we detected device embolization into the pulmonary artery after performing echocardiography since the patient had dyspnea . After that, emergency surgery was performed within one hour with a median sternotomy and under cardiopulmonary bypass ., snares can be used to take out the device, but most of the time, surgical extraction of the device and asd patch closure is needed . Percutaneous asd closure is a widely used and has been performed successfully throughout the world in recent years . However, it should be kept in mind that improper patient and device selection may result in deadly complications like device embolization, cardiac perforation and tamponade.
Extracellular vesicles (evs) are a heterogeneous group of vesicles that can be subdivided based on their size, biogenesis, and molecular composition . Using the biogenesis as a classification tool, evs can be divided into three major groups, namely, exosomes, microvesicles (mvs), and apoptotic bodies [13]. Even though the molecular composition of these three subsets of evs is different, several markers overlap . So far, identification of a specific marker that with certainty can distinguish or identify the particular ev subset still awaits . It can be expected that the different ev subsets may cover different biological roles, but the function of evs has also been described to depend both on the cellular source and on the recipient tissue / cell . Nevertheless, it is now recognized that evs are involved in numerous physiological processes, including intercellular communication and delivery of proteins, lipids, and genetic material to recipient cells [2, 3, 57]. In addition, evs have been associated with the development and progression of different pathological conditions, including cancer and infectious diseases [815]. The immunological effects of evs comprise a broad range of mechanisms, including immune activation, immune suppression, and modulation of immune surveillance . Cells from both the innate and the adaptive immune system have been shown to release evs, such as t and b cells, dendritic cells (dcs), macrophages, mast cells, and natural killer (nk) cells [1624]. The effect of evs is directly related to their molecular composition and several studies have ascribed an immunostimulatory effect of evs to the presence of a very specific molecular content [20, 23, 2531]; for instance, cd56-positive and perforin - containing evs from nk cells can mediate ev - induced cytotoxicity . Several immunosuppressive effects of evs have also been reported [21, 32, 33]; for instance, fas ligand (fas - l) evs released from regulatory t cells are able to inhibit dc - induced cytotoxic t - lymphocyte (ctl) responses . Furthermore, inhibitory roles of evs derived from immature dcs have been observed in relation to transplant tolerance [3436]. Thus, identification of a specific molecular signature of evs released by immune cells can provide knowledge that can lead to the use of evs in a therapeutic setting . Numerous studies have investigated the effects of evs released from different leukocytes, leading to an incipient understanding of the physiological functions of these evs . Nevertheless, several basic questions concerning specific characteristics, like the protein composition of the different types of leukocyte - derived evs, remain unclear . The present study investigated the expression of selected immunological lineage - specific markers and selected vesicle - related markers on five major leukocyte subpopulations, namely, cd4 t cells, cd8 t cells, nk cells, b cells, and monocytes . The expression was determined for leukocytes present in freshly isolated whole blood and on cultured isolated leukocyte subpopulations and compared to the presentation of these markers on the corresponding leukocyte - derived evs . The ev array, used for phenotyping of evs, is optimized for detection of small evs with a size below 150 nm that present cd9, cd63, and/or cd81, such as exosomes and exosome - like vesicles . However, as these markers may be present on several types of evs and as the intracellular origin of the characterized evs was not determined, the ev subset investigated in the current study was denoted by small evs (sevs). Blood samples were obtained from ten healthy volunteers at the department of clinical immunology, aalborg university hospital . From each donor two blood samples were collected, one tube containing edta (k3edta, vacuette, greiner bio - one, austria) for immediate analysis of noncultured leukocytes and one tube containing cpda (vacuette, greiner bio - one, austria) for the vesicle analysis (plasma). Plasma was isolated by centrifugation at 1800 g for 6 min at room temperature (rt), after which the plasma supernatant was aliquoted and stored at 40c until analysis . Buffy coats were obtained from three healthy donors at the department of clinical immunology, aalborg university hospital, and used for isolation of peripheral blood mononuclear cells (pbmcs). Buffy coats were diluted (1: 4) in sterile pbs (spbs) and pbmcs were isolated by density gradient centrifugation using lymphoprep (axis - shield, oslo, norway). Pbmcs were subsequently washed twice in growth medium (rpmi1640 (gibco, life technologies, carlsbad, ca, usa), 10% heat - inactivated fetal calf serum (fsc) (gibco), 100 u / ml penicillin, and 10 g / ml streptomycin (amplicon, odense, denmark)) and counted in trypan blue and resuspended in isolation buffer (ca- and mg - free pbs, 2 mm edta, and 0.1% bovine serum albumin (bsa)). Magnetic dynabeads were used for isolation of human cd4 and cd8 t cells according to the manufacturer's instructions (dynabeads cd4 positive isolation kit and dynabeads cd8 positive isolation kit, life technologies). Briefly, 1 10 cells / ml were mixed with washed beads and incubated for 20 min at 4c with gentle rotation . The cell suspension was subsequently placed in a magnet for 3 - 4 min . The supernatant was removed and the bead - bound cells were incubated with detachabeads for 45 min at rt with gentle rotation . The cell suspension was placed in a magnet for 3 - 4 min and the supernatant containing the detached cells was collected . The detached cells were washed once in growth medium and adjusted to 3 10 cells / ml . From the cd4 depleted pbmcs, b cells were isolated using the dynabeads untouched human b cells kit (life technologies) according to the manufacturer's instructions . From the cd8 depleted pbmcs, monocytes were isolated using the dynabeads untouched human monocytes kit (life technologies) according to the manufacturer's instructions . Human nk cells were isolated from pbmcs by negative selection according to the manufacturer's instructions (dynabeads untouched human nk cells kit, life technologies). Briefly, 1 10 cells / ml were mixed with the antibody cocktail and incubated for 20 min at 4c followed by washing and incubation with dynabeads for 15 min at 4c while rotating . The cell suspension was subsequently placed in a magnet for 3 - 4 min . The supernatant, containing the cells of interest, was removed and centrifuged at 500 g, for 5 min at rt . The pellet was washed once in growth medium and adjusted to 3 10 cells / ml . For each isolation, the purity of the cells was determined by flow cytometry . Isolated leukocytes were adjusted to 3 10 cells / ml and cultured in either 12-well (3 10 cells / well, 1.5 ml / well) or 24-well plates (1.5 10 cells / well, 1 ml / well) (nunc, thermo scientific, carlsbad, ca, usa) for 4448 hours at 37c and 5% co2 . Following incubation, the plates were centrifuged at 600 g for 10 min at rt and the supernatants containing the cell - derived evs were harvested from the plates . Complete protease inhibitor, edta - free (roche, de, usa), was added to the ev - rich supernatants, which were subsequently upconcentrated using amicon ultra 100k spin columns according to the manufacturer's instructions . Briefly, pbs was added to the harvested supernatants (total volume of 5 ml) and the supernatants were subsequently centrifuged at 600 g for 7 min at rt . The supernatants were transferred to 50 ml spin columns and centrifuged at 3200 g for 10 min at rt . Prior to adding the supernatants, the spin columns were washed once in 5 ml pbs (3200 g, 10 min, rt). Following the first centrifugation of the supernatant, 5 ml of pbs was added to the retention volume and centrifuged again . The retention volume, containing the upconcentrated supernatants, was harvested and the filter unit was washed in 50100 l pbs . Due to varying cell counts between the subpopulations and across the experiments, every supernatant was adjusted to a volume corresponding to 5.5 10 cells / ml . No further purification of the evs was performed and the upconcentrated supernatants were aliquoted and stored at 40c until analysis by the ev array . The cells were harvested in pbs, washed, and resuspended in pbs with 0.5% bsa and 0.09% nan3, followed by a subsequent surface marker staining . For the ev array, the following antibodies were used for capturing: cd11a (hi111) from ab biotech (san diego, ca, usa); flotillin-1 and tsg101 (5b7) from abcam (cambridge, ma, usa); cd3 (hit3a), cd14 (m5e2), cd16 (3g8), cd28 (l293), cd49d (l25), and cd56 (3g8) from bd biosciences (mountain view, ca, usa); cd41 (hip8), cd63 (mem-259), hla - abc (w6/32), and alix (3a9) from biolegend (san diego, ca, usa); icam-1 (r6.5) from ebiosciences (san diego, ca, usa); cd9, cd42a, cd81, and ctla-4 (anc152.2/8h5) from lifespan biosciences (seattle, wa, usa); annexin v (af399), cd4 (34930), cd8 (37006), cd19 (4g7 - 2e3), cd37 (424925), cd45 (2d1), cd80 (37711), cd82 (423524), cd83 (hb15e), mic a / b (159207), tnf ri, and tnf rii from r&d systems (minneapolis, mn, usa); tlr3 (3.7) from santa cruz biotechnologies (dallas, tx, usa); hla - dr / dp / dq (hb-145) from loke diagnostics (aarhus, denmark); fas ligand (10f2) from serotec (oxford, uk); and pd - l1 from sino biological (beijing, china). The following antibodies were used for detection (biotinylated): cd9, cd63, and cd81 from lifespan biosciences . For flow cytometry the following antibodies were used: cd45 apc (t29/33), cd45 fitc (t29/33), and cd3 fitc (ucht1) from dakocytomation (glostrup, denmark); cd3 apc (ucht1), cd4 fitc (rpa - t4), cd4 apc - h7 (sk3), cd8 apc - h7 (sk1), cd9 percp - cy5.5 (m - l13), cd14 fitc (m5e2), cd16 fitc (3g8), cd16 pe - cy7 (3g8), cd19 apc (hib19), cd56 apc (b159), and cd56 pe - cy7 (b159) from bd biosciences (mountain view, ca, usa); cd63 pe - cy7 (h5c6) from ebiosciences; and cd81 pe from lifespan biosciences . In addition, isotype- and fluorophore - matched control antibodies were included . For analysis of noncultured leukocytes, 100 l of freshly drawn whole blood was labeled with fluorescence - conjugated antibodies for 30 min at rt, red blood cells were lysed, and the remaining leukocytes were washed and resuspended in bd facsflow sheath fluid (bd biosciences) with 1% paraformaldehyde . The cultured leukocytes were labeled with fluorescence - conjugated antibodies for 30 min at rt . The leukocytes were washed twice in pbs with 0.5% bsa and 0.09% nan3 and resuspended in bd facsflow sheath fluid (bd biosciences) with 1% paraformaldehyde . Cells were analyzed on a facscanto ii flow cytometer (bd biosciences) using the bd facsdiva software version 6.1.3 (bd biosciences). The acquired data files were analyzed by flowjo vx.0.7 (treestar, ashland, usa), first by adding a leukocyte - gate based on morphologic characteristics (fsc / ssc) and subsequently by the use of the lineage - specific markers; cd3 and cd4 for cd4 t cells; cd3 and cd8 for cd8 t cells; cd16 and cd56 for nk cells; cd19 for b cells; and cd14 for monocytes . For the production of the protein microarray, microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin (arrayit, sunnyvale, ca, usa). Epoxy coated slides (75.6 mm 25.0 mm; schott nexterion, jena, germany) were used as microarray basis . The antibodies listed above were diluted in pbs containing 5% glycerol and printed at a concentration of 180200 g / ml . As a positive control, 100 g / ml of biotinylated human igg in pbs with 5% glycerol was printed . As a negative control, for catching, visualization, and data analysis, the procedures were performed as previously described . In short, the slides were incubated with 100 l plasma, prediluted 1: 10, or 100 l upconcentrated cell culture supernatant, prediluted 1: 2 in wash buffer (pbs with 0.05% tween-20). After overnight sample incubation and a subsequent wash, the slides were incubated with a cocktail of biotinylated detection antibodies (anti - human cd9, cd63, and cd81), diluted 1: 1500 in wash buffer . After incubation, cy5-labeled streptavidin (invitrogen, frederick, md, usa) diluted 1: 1500 was used for detection . Prior to scanning, the slides were washed and dried using a microarray high - speed centrifuge (arrayit). Briefly, the intensity of the antibody signal was calculated by subtracting the mean of the background (without sample / blank) from the mean of the triplicate antibody spots . This signal was then divided by the signal from the mean of the triplicate negative spots (without capture antibody, with sample). Leukocytes can be subdivided based on morphological characteristics upon staining into polymorphonuclear cells (the granulocytes) and mononuclear cells (the lymphocytes and the monocytes). The present study investigated the sevs produced by the different subpopulations of mononuclear cells found in peripheral blood and compared the vesicular phenotype to the cellular phenotype . In addition, sevs present in plasma were phenotyped for the same panel of markers . The purity of the different subpopulations throughout the three individual experiments was 9799.5% for the cd4 and the cd8 t cells, 8295% for the b cells, 6894% for the monocytes, and 8597% for the nk cells . The isolated subpopulations of leukocytes were cultured for 4448 hours and the supernatants, containing the leukocyte - produced evs, were investigated for the presence of a panel of selected immunological and ev - related markers using the ev array (table 1). It was investigated whether sevs from isolated leukocyte subpopulations presented the lineage - specific markers that define the parent cells . The results demonstrated that not all lineage - specific markers could be observed on the corresponding sev subpopulations (figure 1(a)). For instance, cd19, which is a well - defined lineage marker for b cells, could not be detected on sevs in supernatant from cultured b cells . Likewise, cd14, cd16, cd56, and cd3 could not be detected on sevs from cultured monocytes, nk cells, and t cells, respectively (figure 1(a)). In contrast, cd4 and cd8, which are lineage markers for two different populations of t cells, were detected on sevs from the supernatants of these two t cell subpopulations . In addition, sevs from cultured pbmcs presented cd8, but none of the other markers . Aside from the parental lineage - specific markers, likewise, cd45 and cd16 could be detected in one of the experiments with sevs from cd4 t cells . A panel of ev - related markers was assayed on the different subsets of the leukocyte - derived evs (figure 1(b)). Cd9 was detected on all sev subsets, though at very low levels on sevs from t cells and nk cells . Similarly, cd81 was detected on all sevs but at very low levels on sevs from b cells and nk cells . Cd82 was detected at high levels on sevs from monocytes and pbmcs and at very low levels on sevs from b cells and t cells but was absent on sevs from nk cells . Furthermore, alix was detected at very low levels on sevs from the cultured cd8 t cells . In contrast, annexin v, tsg101, and flotillin-1 could not be detected on the leukocyte - derived sevs . Furthermore, the presence of more general immunological markers was investigated on the different sev populations . A total of 18 markers were chosen based on their relevance for the function of leukocytes (table 1). The majority of markers could not be detected on the sev subpopulations using the ev array (figure 1(c)). However, cd49d was found on sevs from all the different leukocyte subpopulations and cd41 was detected on sevs from all subpopulations besides sevs from nk cells . Additionally, sevs from cultured cd4 and cd8 t cells presented several of the immunological markers, including cd11a, tlr3, cd28, ctla-4, and the fas - l . The data presented in the heat maps were from three individual experiments and illustrated a degree of variation between the individuals . The presence of a natural variation in the phenotype of sevs between healthy individuals was also observed upon phenotyping of the sevs present in plasma from 10 healthy individuals (figure 2). Plasma sevs presented several of the markers that were detected on the leukocyte - derived sevs . In addition, plasma sevs presented some lineage - specific markers, including cd3, cd14, and cd19 that were not detected on sevs from cultured leukocytes . Furthermore, tnf ri, tlr3, cd42a, cd80, and cd83 as well as annexin v, flotillin-1, and alix were detected on the majority of the plasma sevs . The cellular expression pattern of cd9, cd63, and cd81 was determined for all the leukocyte subpopulations by flow cytometry (figure 3). The expression of these vesicle - related markers was investigated for leukocytes present in freshly drawn whole blood (n = 10) as well as for isolated subpopulations of leukocytes following two days of culture (n = 3). Figures 3(a) and 3(b) demonstrate the expression levels in histograms from two representative donors . None of the leukocyte subsets from whole blood presented cd9 on their surface (figure 3(a)). In contrast, both cd63 and cd81 were expressed on all leukocytes, but the expression pattern varied between the different subsets . The lymphocytes, including the cd4 t cells, the cd8 t cells, the b cells, and the nk cells, expressed cd81 at high levels, while they all expressed cd63 at low - to - intermediate levels . The opposite situation was observed for the monocytes that presented high levels of cd63 on their surface, but only low - to - intermediate levels of cd81 . Regarding the cultured leukocytes, each subpopulation was isolated from three different donors and cultured for 4448 hours (figure 3(b)). The results showed that, in contrast to the freshly isolated leukocytes, the cultured leukocytes presented cd9 on their surface . For the lymphocyte populations, the expression of cd9 was low, while the monocyte population expressed intermediate levels of cd9 . All cultured lymphocyte subpopulations expressed cd63 and cd81 at low - to - intermediate levels, while the monocytes expressed high levels of both markers . Even though the levels varied, the expression patterns of cd63 and cd81 on the cultured leukocyte subpopulations were similar to the patterns observed on leukocytes present in freshly isolated blood . Based on the obtained minimum and maximum mfi values, it was clear that the expression level of the three markers varied between the individuals illustrating a natural variation . In order to investigate the phenotypic homogeneity between the sevs and the parent cells, the cellular expression patterns of selected lineage - specific and vesicle - related markers were compared to the vesicular presentation of these markers (figure 4). In relation to the selected lineage - specific markers, cd3, cd14, cd16, and cd19 could not be detected on sevs from cultured t cells, monocytes, nk cells, and b cells, respectively, indicating that not all cell surface molecules are transferred to the sev surface . For cd9, cd63, and cd81, the majority of the subpopulations presented vesicular levels that overall correlated with the cellular expression . Exceptions were observed with cd63, in which case the cellular expression on monocytes was high, but the vesicular presentation was low . Similarly, the cellular expression of cd63 was intermediate or low for both nk cells and b cells, but no cd63 could be detected on the corresponding sevs . Several studies have reported that evs are released from different leukocyte subsets; however, a thorough simultaneous investigation of the major leukocyte subsets with focus on lineage - specific markers has not yet been described . In the current study the phenotype of five different subsets of leukocyte - derived sevs was determined and related to both the phenotype of sevs present in plasma and the cellular phenotype of the leukocyte subpopulations . The ev array, used for the investigation of the phenotype of sevs, is a protein microarray technique that provides the opportunity to detect and characterize sevs for up to 60 markers in a high - throughput manner and with high sensitivity [37, 38]. One major advantage of the ev array is the ability to phenotype sevs from unpurified material without a requirement for preanalytical purification . Isolation of specific ev subsets is in many cases highly warranted, but for phenotyping of evs using the ev array, crude plasma or cell - free supernatants are applicable . In order to investigate whether lineage - specific proteins present on leukocytes could be detected on the corresponding sevs, leukocytes were phenotyped for selected lineage - specific markers using flow cytometry, while the sevs were phenotyped using the ev array . The results showed that none of the included lineage - specific markers could be detected on the sevs released from b cells, nk cells, and monocytes in monocultures (cd19, cd56, and cd14, resp . ). In contrast, sevs from monocultures of cd4 and cd8 t cells did present cd4 and cd45 or cd8 and cd45, respectively . None of the t cell - derived sevs presented cd3 . A study by kornek et al . Investigated evs from jurkat t cells activated with phytohemagglutinin (pha) and observed that the fraction of large evs (sedimented at 10.000 g) only presented low levels of cd3, while the fraction of sevs (sedimented at 100.000 g) presented high levels of cd3 . Regarding cd19, a study by admyre and coworkers from 2007 showed that evs from a b cell line presented cd19, which differs from the observations obtained in the study at hand . Similarly, another study has described that evs from resting nk cells present typical nk cell markers like cd56 and fas - l, but not cd16 . In terms of cd14, a study by aharon et al . Observed this marker on large evs (mvs) released from a monocytic cell line . The discrepancy between some of the results may be due to investigations of different preparations of evs and/or may be explained by the fact that the ev - producing cells were different, for example, cells isolated from peripheral blood versus cell lines . In addition, differences in the activation state of the ev - producing cells are of importance for the outcome . Upon stimulation, cells change their phenotype and this phenotypic change depends on the specific stimulus given to the cells [28, 42]. In the present study, leukocyte subpopulations were isolated and cultured without any activation signal, while several of the other studies investigated the phenotype of the evs released upon adding a stimulus to the cells . The presented results obtained with sevs released from cultured pbmcs support this phenomenon as these sevs apparently displayed fewer markers than sevs from monocultures . Overall, these data indicate that the phenotype of the vesicle subset is context - dependent . However, during the current conditions, several of the leukocyte - derived sevs did not present the lineage - specific markers found on the parent cell type (figure 1(a)). Variations in the molecular content between cells and evs were also observed in a study by hunter et al . That reported significant differences in the presence of mirnas between plasma evs and pbmcs . For comparison, the presentation of lineage - specific markers on plasma sevs was determined . In contrast to the leukocyte - derived sevs, plasma sevs presented several lineage - specific markers, including cd3, cd14, and cd19 . Compared to the evs present in cell supernatants, evs in plasma are a mixture of evs produced by many different cell types . Thus, plasma can act as a liquid biopsy and will present a very heterogeneous ev phenotype that provides information about numerous cellular processes . The detection of cd3, cd14, and cd19 on plasma sevs, but not on sevs from cultured leukocyte subpopulations, emphasizes that the biological context has a great impact on the phenotype of the released evs . Interestingly, some of the sev subsets presented other markers, for example, sevs from cd8 t cells presenting cd16 . Several studies have described the expression of cd16 on a small portion of different types of t cells and even shown a functional role of cd16 on t cells [44, 45]. Thus, the observations of cd16 on sevs released from cultured t cells combined with the fact that functional molecules, such as perforin and lytic granules, can be stored inside evs may indicate a specific functionality of these evs, for example, surrogates or extensions of the effector cell in antibody - dependent, cell - mediated cytotoxicity . Apart from the selected lineage - specific markers, the sevs were phenotyped for the presence of 26 different immunological or vesicle - related markers . Regarding the leukocyte - derived sevs, cd49d was the only marker detected on all sev subsets (figure 1(c)). Cd49d is an 4 integrin that is expressed as a heterodimer on t, b, and nk cells and monocytes as well as on other immune cells . Together with cd29, cd49d forms the very late antigen-4 (vla-4) that is involved in leukocyte trafficking, adhesion, and extravasation [46, 47]. So far, two studies have reported on the presence of cd49d / vla-4 on evs from b cells [48, 49], and these results can be confirmed by the present study . In terms of plasma sevs, surprisingly, cd41 was observed on sevs from t cells, b cells, monocytes, and pbmcs as well as on plasma sevs from half of the healthy donors (figure 1(c)). However, according to the protein atlas, cd41 is expressed by pbmcs and even though the function is unclear, the presence of cd41 on sevs released from these cells, either in monocultures or as pbmcs, is possible . In addition, sevs from cultured t cells presented an array of other immunological markers, ranging from more general markers, like cd45, to specific markers like cd28, and the tlr3 . Moreover, sevs from cd8 t cells presented ctla-4 and the fas - l . Even though the mechanisms are quite different, both of these molecules play a role in downregulation of an immune response, either by transmitting an inhibitory signal in activated t cells or by inducing activation - induced cell death, respectively . The presence of such regulatory molecules on evs suggests that t cell - derived evs can be involved in immune regulation . The presence of fas - l on evs from t cells has also been observed in other studies [5254], but to the best of our knowledge, studies observing ctla-4 on evs from leukocytes have not previously been published . Upon evaluating the presence of markers on evs, it is important to consider the natural variation in the molecular composition of the ev pool that exists between healthy individuals as visualized by the ten plasma samples . Thus, the present results are a snapshot providing insight about the current vesicular presentation of selected markers . The sevs investigated in this study were identified by the presence of cd9, cd63, and/or cd81 . Thus, in order to investigate the similarity of the leukocytes and the corresponding sevs, the cellular expression patterns of cd9, cd63, and cd81 were determined . The expression pattern was determined for both leukocytes present in whole blood and cultured leukocyte subpopulations . The results demonstrated a lack of cd9 on the surface of leukocytes present in freshly drawn whole blood (figure 3(a)). In contrast, cd9 was present on every leukocyte subpopulation following two days of culture . In accordance with the present results, a recently published study also observed cd9 on the surface of isolated populations of cd3, cd14, and cd19 cells . The expression patterns of cd63 and cd81 were quite similar between the two cell preparations but with minor differences in the expressions levels . Overall, these findings are in agreement with previously described results [5661]. When looking at both the cellular and the vesicular presentation of these markers, it is clear that, for the majority of the cultured subpopulations, the presentation of these markers was comparable . However, for the nk cells, the b cells, and the monocytes, the cellular expression of cd63 was very different from the presentation observed on sevs . A comparison between the presentation of cd9, cd63, and cd81 on whole blood leukocytes and plasma evs is a more complex procedure that needs to take into account that plasma evs are a very heterogeneous group of evs that emanate from multiple cell types . The tetraspanin signals observed on the plasma sevs represent all tetraspanin - positive sevs irrespective of their origin . Thus, the presentation of tetraspanins on plasma sevs cannot be expected to correlate with the expression of tetraspanin on leukocytes . Nevertheless, it is clear that the cd63 signals in plasma generally were low, while the cellular expression of cd63 for several of the leukocyte subpopulations is medium to high, indicating that the presentation of cd63 is different from sevs to cells . Overall, the vesicular signal intensities for cd63 were much lower than the intensities observed for cd9 and cd81, suggesting that cd63 might be a poor marker for sevs in general, which has also been observed in other studies [38, 6264]. The results underline the importance of detecting evs with a cocktail of antibodies against tetraspanins, as detection with a single marker may overlook some subsets of evs . Surface molecules on evs are responsible for the biodistribution and the ligation to target cells . Thus, the protein composition is related to the functionality of evs, why phenotyping of evs can be used to gain knowledge about the functionality . In summary, the presented data regarding the lineage - specific markers and the tetraspanins support the accumulating observations suggesting that the transfer of molecular cargo into evs is tightly controlled . A tightly regulated sampling of molecules to evs would match with the fact that evs play an important role as systemic regulators, traveling through tissues providing key intercellular communication as well as transfer of biologically active components.
In march 2014, an unprecedented outbreak of ebola virus disease (evd) began in western africa that as of july 2015 is ongoing and has claimed more than 11000 lives . The current outbreak is caused by viruses belonging to the species of the zaire ebolavirus, a member of the filoviridae family . Filoviridae are enveloped, filamentous viruses with lengths that may reach> 1000 nm . Ebola virus can be excreted in bodily fluids, including vomit, stool, blood, saliva, semen, and breast milk . Ebola virus loads of up to 10 genome copies ml have been reported in blood, 10 genome copies ml in stool, and 10 genome copies ml in urine; however, the conversion between genome copies and infectious units is unknown . Once infected, individuals may produce up to 9 l of liquid waste per day, primarily watery diarrhea . Ebola virus is considered a potential bioterrorism agent . In response to the evd epidemic, both the world health organization (who) and the u.s . Centers for disease control and prevention advised direct disposal of ebola - contaminated liquid waste into sewage systems (wastewater collection and treatment systems) and latrines without disinfection . Initial recommendations were made on the basis of an expected limited persistence of ebola virus in the environment, as ebola virus is an enveloped virus, and a lack of strong evidence for a waterborne transmission route . As stated by a who guidance document, ebola virus is likely to inactivate significantly faster in the environment than enteric viruses with known waterborne transmission (e.g., norovirus, hepatitis a virus). However, as has been noted in a recent review, the persistence of enveloped viruses in the water environment varies by> 2 orders of magnitude . Recommendations for ebola virus - contaminated wastewater disposal were met with debate (e.g., refs (1618)) because of uncertainty about ebola virus persistence within wastewater matrices and the lack of a risk - based analysis for waste handling . Wastewater handling recommendations have since been revised to recognize uncertainty in this area and to recommend disinfection of latrines and holding of wastewater prior to handling to allow ebola virus inactivation . Additionally, some facilities have opted to provide additional disinfection prior to disposal of liquid waste into sewer systems . Recent research found both ethanol and hypochlorite to be effective disinfectants for ebola virus dried on surfaces; however, the disinfection kinetics of ebola virus within liquid matrices remains unknown . Various wastewater disinfection approaches have been recently suggested for pathogen control in an outbreak setting . Currently, no data on ebola virus persistence in wastewater exist, hindering risk estimation and examination of potential environmental exposure routes . The necessity of evaluating ebola virus persistence in wastewater matrices has previously been highlighted, as wastewater in ebola virus outbreak settings may be temporarily held in open containers or disposed of in open sewers . Historically, the transmission of ebola virus via environmental routes (droplets, aerosols, or fomites) has been thought to be unlikely due to epidemiological evidence and environmental sampling . The primary ebola virus transmission route is via direct contact with bodily fluids . Transmission has previously occurred without known direct contact with infected individuals, providing supporting evidence that ebola virus transmission may be possible via large droplets . The potential for transmission of ebola virus via wastewater is currently unknown . To address uncertainties regarding ebola virus persistence in wastewater, we have conducted an initial evaluation of ebola virus persistence within wastewater to address uncertainty and inform ongoing risk assessments . A current ebola virus outbreak strain from guinea (makona - wpgc07) was spiked to two end concentrations (10 and 10 tcid50 ml) into a domestic wastewater (untreated sewage) sample . The ebola virus - containing wastewater was sampled for 8 days, and the viability of ebola virus was determined in these samples . Study results are presented, and study limitations and implications are discussed, as well as recommendations for an ongoing research agenda . Untreated wastewater was collected from an anonymous regional wastewater treatment facility in western pennsylvania that receives wastewater from seven communities (combined population of approximately 60000). The total raw wastewater flow to the treatment facility is <10 mgd (million gallons per day). The wastewater was frozen at 80 c to minimize compositional changes prior to analysis . Wastewater characteristics (table 1) were determined at an epa - certified analysis facility (microbac laboratories, marietta, oh). The region from which the sample was collected uses a combined sewer system that experiences significant infiltration, and the determined composition is typical for the region . Ebola virus cultivation experiments were conducted at rocky mountain laboratories under bsl4 conditions . Wastewater samples were shipped to rocky mountain laboratories overnight on ice . Upon receipt, samples were sterilized with 5 mrad of -irradiation . Sterilization was performed to limit cell culture death due to wastewater microbial activity leading to a false positive . Stock virus (ebola virus guinea makona - wpgc07, 10 tcid50 ml) was handled as described previously . Ebola virus was diluted in wastewater to achieve two separate virus titers (10 and 10 tcid50 ml), and both experiments were completed in triplicate . The ebola virus concentration in sewage has not been previously measured or estimated; thus, two separate concentrations were utilized to cover possible concentration scenarios . Tcid50 is an end point dilution series that is used to determine at what dilution 50% of the infected wells produce cell death . An approximation of focus - forming units (ffus) can be made from a poisson distribution utilizing the formula tcid50 0.69, assuming each ffu is formed from a single virus . Spiked wastewater was then distributed into three labeled vials for each concentration, and samples were taken daily for 8 days . Tests were conducted at 20 c and 40% relative humidity . At each time point, including the time zero measurement, 50 l of wastewater from the bulk wastewater vial was added into 450 l of dulbecco s modified eagle s medium (dmem, sigma) supplemented with heat - inactivated fetal bovine serum (fbs, gibco) to a final concentration of 2%, pen / strep (gibco) to a final concentration of 50 units / ml penicillin and 50 g / ml streptomycin, and l - glutamine (gibco) to a final concentration of 2 mm in an appropriately labeled 2 ml screw top vial and frozen at 80 c . Negative controls were 50 l of nonspiked wastewater in 450 l of dmem . To perform the titrations, a 96-well 1.1 ml well dilution plate (axygen, corning, ny) was prepared . The thawed sample (400 l) was placed in the top row of the plate and a 10-fold dilution conducted by passing 40 l of sample into 360 l of dmem . Next, 100 l from each well of the dilution plate was transferred to a 96-well cell culture plate seeded with vero cells . The cells were incubated with virus dilutions for 1 h; then the medium was removed from the two highest concentrations and rinsed two times with pbs, and 200 l of fresh culture medium was added . Fresh culture medium (100 l) was also added to the remaining wells in the plate . The plates were incubated at 37 c for 7 days, inspected for the cytopathic effect (cpe), and scored . The natural logarithm of c / co tcid50 was plotted and fit with a linear trendline for estimation of the inactivation constant (k). A literature review was then performed to compare observed inactivation to other environmental matrices . Ebola virus was spiked into wastewater at two concentrations and assayed for 8 days to determine persistence in a wastewater matrix . The time zero time point was measured immediately following addition of ebola virus to the wastewater . No viable ebola virus was recovered from samples spiked with 10 ebola virus tcid50 ml after the initial time zero sampling . Virus viabilities from the initial 10 ebola virus tcid50 ml concentration are shown in figure 1 and detailed in table s1 . Ebola virus titer was rapidly reduced (approximately 99%) within the first day of the test, consistent with an inability to identify infectious ebola virus from the initial sample with 10 ebola virus tcid50 ml on day 1 . Persistence of an initial ebola virus concentration of 10 tcid50 ml in domestic wastewater (untreated sewage) (a) including the time zero time point and (b) excluding the time zero time point to mitigate potential aggregation effects . Fit inactivation constants (k) were determined to be 1.08 when including time zero and 0.35 when excluding time zero . Error bars are 1 standard deviation . There was a rapid decrease (approximately 99%) in ebola virus titer within the first day of the test . In addition to inactivation, viral particle aggregation or adsorption to wastewater particles may play a role in the apparent rapid viral decrease and enhanced viral persistence . In this case first, aggregated particles would not be detected as multiple infectious units, resulting in an apparent rapid decrease . Second, it has been previously recognized that aggregation increases viral persistence and resistance to inactivation stressors . Similarly, viral association with particles has been recognized to provide protection from inactivation, including association of viral particles with wastewater solids . Mechanistically, particle association is believed to protect viral particles from inactivation by shielding them from environmental stressors and is dependent on the organism and particle type . Utilizing the current assay, it cannot be determined if the initial rapid decrease in viral titer was due to viral inactivation or aggregation . Aggregation would result in an apparent viral titer decrease as each viral aggregate would function as an infectious unit in the cell culture assay . To address this uncertainty, we plotted two inactivation curves, both including (figure 1a) and excluding the measured time zero point (figure 1b). A linear trendline, as has been previously suggested for viral and ebola virus persistence, showed a lower fit (r = 0.59 including the time zero time point, and r = 0.67 excluding the time zero time point) than that previously observed for ebola virus in deionized water (r> 0.91), but a fit better than that previously observed for ebola virus in human blood (r <0.29). The inactivation constant (k) was determined to be 1.08 when including the time zero time point and 0.35 when excluding the time zero time point . On the basis of the model fit, the t90 (time for 90% inactivation) would be 2.1 days including the time zero time point and 6.6 days excluding the time zero time point . The observed ebola virus inactivation in wastewater was slower than that observed for deionized water, which required 1.8 days for 90% inactivation at 21 c . The observed ebola virus inactivation in wastewater was more rapid than that reported for human blood, which required 20 days for a 90% inactivation, and results are consistent with recent studies that identified viable ebola virus to persist in infected macaque blood for> 8 days . In general, ebola virus was found to be less persistent in wastewater than model enteric viruses . While the t90 for ebola virus in wastewater was found to be <1 day, the t90 for hepatitis a is greater than 17 days and the t90 for enteric adenovirus is 33 days; however, the t90 for poliovirus is 5 days, which is between the observed t90 values including or excluding the time zero time point . The results demonstrate a more rapid initial viral titer decrease but overall enhanced persistence of ebola virus in wastewater compared to the proposed enveloped surrogate bacteriophage phi6 . This study has two primary limitations that may alter the persistence of ebola virus compared to what may be observed in the field . First, the tested wastewater was more dilute than would be expected in typical latrine waste . In general, interaction with constituents within the wastewater (e.g., ammonia) would be expected to contribute to more rapid inactivation of viruses; however, the true effect of these constituents on ebola virus persistence is unknown . Second, the wastewater was frozen to minimize compositional changes and disinfected (-irradiation) prior to utilization to limit microbial activity resulting in false positive viral cell culture . Microbial activity within wastewater matrices would be expected to contribute to more rapid inactivation of infectious viral particles; however, the true effect of microbial activity on ebola virus persistence is unknown . Microbial activity reduces viral persistence through both the production of metabolites detrimental to viral persistence and direct usage of the viral particles as a nutrient source . Additionally, the influence of other environmental characteristics (e.g., temperature, ph, and mixing) on ebola virus persistence is unknown and may contribute to altered environmental behavior . As such, we believe these results to be an upper bound for ebola virus persistence in wastewater matrices . Subsequently, we caution extrapolation of these results without a holistic assessment of all factors, including wastewater composition, dilution, and potential exposure routes . The results of this study suggest a potential exposure route to infectious ebola virus via wastewater; however, any assessment of potential exposure routes must consider the effects of wastewater composition, dilution of contaminated wastewater, and inactivation of ebola virus during treatment and holding . Additionally, the possibility for ebola virus transmission via wastewater and subsequent infection remains unknown . The who updated guidelines in january 2015 to recommend holding of latrine waste for 1 week prior to further handling or transport . The objective of this holding period is to allow ebola virus die - off . On the basis of these results, it would be reasonable to approximate a three - log (i.e., 99.9%) removal of ebola virus due to this holding period . The resulting risk would ultimately depend upon the initial ebola virus concentration in wastewater, potential for exposure, and susceptibility to infection via wastewater exposure . The wastewater travel times for wastewater via a sewer system to a centralized sewage treatment works would typically be <1 day, depending on system dynamics . Further assessment is necessary to determine ebola inactivation and dilution within this period and potential human exposure routes, including workers within the sewer system and ebola virus persistence within wastewater sludges . The greatest exposure risk would be expected for persons in contact with contaminated wastewater prior to significant dilution, treatment, or holding . These results demonstrate a persistence of ebola virus in wastewater greater than what has previously been suggested and the potential of a wastewater exposure route to infectious ebola virus . While ebola virus was found to be generally less persistent than enteric viruses in wastewater, the identified survival period suggests value in a nuanced evaluation of wastewater exposure risks during an epidemic response . Specifically, these findings highlight the value of a precautionary approach to wastewater handling within an outbreak scenario, in response to both ebola virus and other emerging viruses.
Pulmonary nodules and lung tumours move substantially during respiration with significant differences according to the tumour localization . Respiratory tumour motion is a big challenge for radiotherapy, especially for high precision techniques . An additional target dose escalation which is deemed necessary for a curative approach of lung cancer is limited as so far as tumour motion is not sufficiently quantified and integrated into modern treatment planning . Thus, the risk of side effects within the surrounding normal tissue is unacceptably high . Radiation pneumonitis and the development of lung fibrosis are the physiological reactions of the lung which are not acceptable in severely ill patients . Thus modern techniques of adaptive radiotherapy will greatly benefit from devices which take lung and tumour motion into account in radiotherapy planning . The final objective is an additional increase in the radiation dose within the tumour volume together with maximum protection of the adjacent normal lung parenchyma . A basic requirement is the accurate measurement and quantification of the mobility of pulmonary tumours . But also for follow - up examinations of patients with pulmonary tumours, a confident registration of the respiratory level is important in order to achieve easy identification and valid measurements . Integration of the knowledge about lung and lung tumour motion is essential for planning of high precision radiotherapy . Four - dimensional imaging using computed tomography (4d - ct) is under development for optimization of radiotherapy by integration of motion [24]. These developments also include online imaging with a linac - integrated cone beam ct . The particular advantages of magnetic resonance imaging (mri) are the high soft - tissue contrast, differentiation of tumour and atelectasis, the possibility to choose the optimal plane for motion quantification and the possibility to integrate further dynamic parameters, e.g. Lung perfusion or o 2 quantification, into one imaging session . The temporal resolution is already reasonably high for monitoring lung tumour motion in real - time . Novel mri techniques allow for non - invasive acquisition of the motion of lung and pulmonary tumours during the respiratory cycle with high spatial and temporal resolution [8, 9]. Continuous improvements and new developments in mr scanner technology, such as high performance gradient systems and pulse sequences, have made this possible and address the inherent challenges of mri of the chest, such as low proton density with an unfavourable signal to noise ratio and short t2 relaxation times with substantial susceptibility artefacts . Parallel imaging techniques make use of the different spatial sensitivities for the receiver coils in order to employ them for the simultaneous acquisition of image data, significantly reduce the phase encoding steps and achieve a marked improvement in spatial and temporal resolution . Thus, important requirements were met to measure the respiratory movement of the lung quantitatively . Fast - low - angle - shot (flash) sequences with a temporal resolution of one image per second demonstrate chest wall motion per se and postoperative changes of respiratory motion, i.e. Changes of the cranio - caudal distance before and after lung volume reduction surgery . Modified true - fast - imaging - with - steady - state - precession (truefisp) sequences, widely used in cardiac mri, were successfully applied in volunteers (fig . 1). By adding a mathematical model, a continuous quantitative measurement of lung volume was achieved, which again showed a high correlation with spirometry . After successful tests in volunteers, dynamic mri was transferred to patients with solitary pulmonary tumours in order to visualise lung tumour motion by mri in two dimensions (fig . Quantification was performed by measuring the cranio - caudal distance and the diaphragmatic length over time . Again, the results showed good correlations with the forced expiratory capacity in 1 s . The diaphragmatic length was particularly useful in the assessment of respiratory mechanics and the respective effects of pulmonary tumours . In such studies the truefisp sequence was superior to a flash sequence due to its higher t2 signal . The respiratory motion of patients with a pulmonary tumour showed a significant difference between the tumour bearing and the non - tumour bearing hemithorax . In addition, the location of the nodule had a marked effect on the regional mobility of the lung . Thus, mri is capable of providing important complementary quantitative regional information, which might be especially useful during follow - up . Depending on size and location of the tumour, differences in motion were observed in the three axes (x-, y - and z - direction). For this assessment pulmonary nodules were grouped as cranial, middle and caudal according to giraud et al . Especially, the cranio - caudal motion (y -direction) showed a significant dependence on tumour size and location . Small tumours in the caudal lung area can move up to 5 cm during maximum respiration . But also during shallow breathing, nodules in the lower lung zone exhibited a mean dislocation of 9.5 mm in the cranio - caudal direction and 6.6 mm in the anterior posterior direction (fig . Theoretical calculations for 20 patients with pulmonary nodules demonstrated that this information for treatment planning might already lead to a marked increase of the target dose . A sufficient safety margin might not be larger than 3.4 mm for the upper, 4.5 mm for the middle, and 7.2 mm for the lower lung zone . This is substantially smaller than the conventional safety margin of 5 or 10 mm, respectively, and might allow for an additional dose escalation especially in the upper lung zone . But it has to be mentioned that an individual quantification of the tumour motion is recommended as the interindividual differences in tumour motion are high . Simultaneous application of markers on the external chest wall demonstrated correlation and influencing factors between internal and external motion . Further developments in parallel imaging technology, especially view sharing techniques, have resulted in a further improvement in temporal resolution . This advantage can be applied to establish a three - dimensional flash sequence for a continuous volumetric visualisation of respiratory motion of lung and tumours . In fig . 4, the 3d - flash sequence is compared to the 2d - truefisp sequence . A 3d - flash data set contains 52 slices with isotropic 3 mm voxels and a temporal resolution of one data set per second . Motion of pulmonary nodules can be visualised and quantified in all three axes in order to be transferred in potential treatment concepts . Segmentation of such data sets, however, is still demanding and time - consuming and cannot be recommended for clinical use, yet . Besides the mere information about tumour motion, three - dimensional techniques also allow for quantification of changes in tumour volume during the respiratory cycle . Changes in tumour volume are associated with deformations and changes of the main rotation axes of the nodules . Although this is not real rotation, the rotation of the axes can add up to 30 and impressively reflects the degree of deformation . However, this information seems to be highly important, e.g. For further improvements in position and adaptation of collimators in radiotherapy . In addition, the knowledge about deformation of nodules and changes in volume during the respiratory cycle has substantial impact on the accuracy of volume measurement during the follow - up of pulmonary nodules and attempts to calculate nodule doubling times . Radiotherapy very quickly leads to a significant, often temporary reduction of motion of the treated hemithorax when compared to the situation prior to radiotherapy . This ipsilateral reduction is compensated for by an increased motion range of the contralateral lung . Obviously, such changes between right and left lung cannot be detected by global lung function tests . Further studies are warranted to demonstrate which patients might benefit from preventive treatment, e.g. To prevent clinical aggravation . Additional split lung analysis is feasible revealing important insights into differences between right and left lungs undetectable by global pulmonary function tests . Motion of pulmonary tumours can be measured in two or three dimensions, effects of respiration on volume and shape can be calculated . However, presently the evaluation of such data sets is extremely time - consuming . Substantial portions of this paper and the figures were originally published in plathow c, meinzer hp, kauczor h - u . Respiratory cycle from maximum expiration to maximum inspiration within 1 s. top row shows a healthy volunteer with synchronous movement of both lungs . Bottom row shows a patient with lung cancer on the left and subsequently reduced motion range of the left lung . Lung motion is reflected by measuring the distance from the apex to the top of the diaphragm (l). Cranio - caudal lung tumour motion is measured with regard to the intervertebral space t 6/7 (1); antero - posterior motion with regard central anterior margin of the spine (2); and medio - lateral motion with regard to the centre line of the spinal processus (3). Maximum lung tumour motion (mm) during shallow breathing in cranio - caudal (cc), medio - lateral (ml) and antero - posterior (ap) direction depending on tumour localization in the upper, middle or lower lung zone . Top row, 2d - truefisp sequence; middle row, 3d - flash sequence with a temporal resolution of one volume data set per second; bottom row, volume segmentation of lung and tumour from the 3d - flash data set.
A 21-month - old male patient, 75 cm in height and 10 kg in weight, experienced tricuspid valve regurgitation (tvr) and was scheduled to undergo tricuspid valve plasty . The tvr was discovered in a follow - up test 1 year after he had undergone ventricular septal defect repair surgery . Before tricuspid valve plasty, a tee was performed, which showed a level 3 - 4 tvr and severe right ventricular hypertrophy . Atropine, thiopental sodium, and vecuronium were administered to induce anesthesia and muscle relaxation . A catheter was placed at the left radial artery to measure the constant blood pressure and analyze the abga (table 1). There were complications due to the adhesion of the svc and ivc near the surgical site . Therefore, a 20 fr and 22 fr cannula was placed on them, respectively . 2 u of packed red blood cells (prc), albumin, and mannitol was used as the priming solution . Immediately after activating the cardiopulmonary bypass, the measured hematocrit level was 27.5% . The acid - base balance management mid - surgery was performed with an -stat . During extracorporeal circulation, the body temperature was aimed at a moderately low degree of 25. the perfusion rate was kept at 0.5 - 1.0 l / min . After tricuspid valve surgery, the patient was weaned from the cardiopulmonary bypass by reducing the perfusion rate of the machine and starting mechanical ventilation . The total anesthesia, surgery and extracorporeal circulation time was 510 minutes, 420 minutes and 180 minutes, respectively . The amount of blood loss was estimated to be 800 ml . During surgery, 600 ml of a crystalloid solution, 3 u prc and 2 u platelets were administered . However the patient's eyes could not focus, and his upper and lower limbs experienced intermittent convulsions . Brain computed tomography revealed findings indicative of a cerebral infarction in the occipital lobe (fig . Eeg was performed, which showed no abnormal findings of alpha rhythm loss, which is customary in cortical blindness . Phenobarbital was administered until 19 days after surgery under the suspicion of a connection between the ocular deviation and the convulsions . The convulsions did not return, but the reaction to light or objects in front of the eyes did not trigger an avoidance response from the patient . There were no hopeful clinical signs of a visual recovery but fundoscopy showed normal findings . On the postoperative day 28, when the flash visual evoked potential (fvep) and mri had been planned, the patient's eyes refocused . He was able to recognize the objects in front of his eyes, could walk and avoid things in his path, and was able to recognize his guardians . Post surgery visual loss or reduction can be caused by a variety of factors, and occurs 0.1% to 1% of cases . Visual loss is caused by damage to any connecting striate area between the cornea and occipital cortex . Direct pressure on the eyeball, indirect increase in ocular tension due to improper positioning and blood vessel damage can cause central retinal artery occlusion or ischemic optic nerve damage, which can lead to visual loss . In addition, a severe decrease in blood pressure, emboli, and thrombus mid- or post - surgery can cause an infarction in the occipital cortex, which can lead to cortical blindness . Visual loss after a cpb might be caused primarily due to emboli, severe low blood pressure, acute anemia, hypoxia or a combination of these factors . In the present case twenty seven cases were cortical blindness, of which 22 cases occurred after cardiac surgery (81%). Aldrich et al . Reported a retrospective study of 25 patients, where the causes of cortical blindness were natural ischemic stroke in 8 patients (32%), cardiac surgery in 5 patients (20%), cerebral angiography in 3 patients (12%), non - surgery in 4 patients (16%), seizure in 2 patients (8%), and other factors, such as damage to the head and peritoneal dialysis, in 3 patients (12%). There were also cases of temporary cortical blindness reported after post - cardiac surgery angiography . In the case of children, the early indicators of brain damage after surgery are age, the complexity of surgery, metabolic acidosis, increase in lactate, and artery acidosis . Diagnosing cortical blindness is simple when the patient makes a complaint but as in our case, a pediatric patient has limited ability to communicate . Therefore, conducting an eeg, fvep, angiography, and funduscopy can assist in making a diagnosis . In this case, the eeg did not show the abnormal finding of a loss in alpha rhythm . However, computed laminography revealed an infarction in the occipital cortex we checked for visual loss with light and objects to test the patient's avoidance response but he did not respond properly . However, microemboli caused by extracorporeal circulation can obstruct the blood flow in the cerebral vessels, particularly in the watershed area . Braekken et al . Reported that cardiac surgery using extracorporeal circulation causes microemboli in most cases . In addition, in patients with neural damage, there are more cases of transcranial doppler detection of microembolic signals in the right middle cerebral artery than in patients without . Microemboli mid - surgery occurs most often when blocking the aorta (18%) and unblocking the aorta (13%). Rodriguez and belway stated that in pediatric cardiac surgery, the likelihood of microemboli can increase with increasing time of extracorporeal circulation depending on the oxygenator, circuit, adnexa parts, and components . The surgery in this case was not complicated, but may have increased the likelihood of microembolization due to the long extracorporeal circulation time . However, we should have been more careful in the selecting the -stat and ph - stat method . Nevertheless, the strength of the ph - stat should have been considered regarding neural sequelae . The cpb priming solution in pediatric patients, unlike adults, can cause blood thinning, which can lead to possible ischemic organ failure . Aldrich et al . Reported that conditions, such as the age below 40, no diabetes or high blood pressure, and no impairment in cognitive skills, language skills or memory, indicate a good prognosis for visual recovery . In addition, eeg tests can assist in a diagnosis but are not helpful in accurately predicting the prognosis . In this study, two weeks after the surgery, he recovered his speech and cognitive skills, so we did expected a good prognosis . The patient's vision recovered rapidly from postoperative day 28 and he could be discharged . Other tests such as a fvep were not performed, so the precise extent of the recovery rate is unknown . Considerable effort is required to reduce neural sequelae after pediatric cardiac surgery, such as visual loss . The cpb machine's circuit components and type choice should be made on what can best prevent the likelihood of emboli . A pediatric transesophageal scan and cerebral oxymeter can help in the early identification of neural side - effects . A transesophageal scan plays an important role in eliminating emboli, so it should be prepared for all suitable pediatric patients . The pathological difference between an adult and pediatric cardiopulmonary bypass should be recognized, and proper measures should be followed . When cortical blindness is suspected, the ophthalmologist and neurologist should perform a combined examination, being cautious of permanent sequelae . In conclusion, with the reported visual loss in our pediatric patient, surgeons should consider the possibility of visual loss and cerebrovascular injury after a cardiac surgery using cardiopulmonary bypass.
Neonatal period (the first 28 days after birth), which is the period of various physiological adaptations to the extrauterine life, is a vulnerable time . More than 10 million children die before the age of 5 years, 8 million of whom die before the age of 1 year and more than half of these deaths occur in the first 4 weeks after birth . About 98% of the neonatal deaths occur in developing countries . According to the world health organization (who), the rate of deaths is 30 times more in the countries with the highest rate compared to the countries with the lowest rate . According to the reports of ministry of health and medical education, the index of neonatal death in iran is 16 - 20 deaths out of 1000 survived births, which is higher than that in developed countries . Who has urged iran to halve its neonatal death rate by 2015 . Risk factors in studying neonatal death are categorized into three groups: prenatal, intrapartum, and postnatal factors . All over the world, infection, preterm delivery, and birth asphyxia contribute to about 87% of neonatal deaths . In most societies, congenital malformations and premature birth babies with low birth weight (lbw) and extremely lbw account for 6 - 7% and 1% of babies death, respectively . These causes, however, are the second and third reasons, respectively, which are responsible for one half of the neonatal deaths . Prematurity is responsible for about 60 - 80% of neonatal deaths all over the world in babies without congenital anomalies . In many cases, neonatal diseases are caused by respiratory problems, of which hyaline membrane disease (hmd) can be mentioned as the foremost . About 1.3% of all neonatal deaths are caused by hmd or its complications in premature newborns . Have mentioned five main causes of neonatal death, including prematurity, internal bleeding, septicemia, respiratory distress syndrome, and congenital anomalies, while gheibi et al . Have reported prematurity (68%), hmds (51%), asphyxia (13%), sepsis (13%), and congenital cardiac malformations (8%) as the most important causes of neonatal death . The baby's gender has also been considered as an important factor in his or her death . One of the most important issues in babies death is suitable management and timely referral of newborn babies with high - risk levels and being hospitalized in neonatal intensive care unit (nicu). Nicu is a critical section in a hospital, dealing with infections (e.g. Septicemia, pneumonia, surgical infections, and other infections) that are difficult to control . In the usa, management, appropriate care, and supportive cares are the important factors in preventing babies death . Rostami et al . Have reported that the rescue chances of the babies who have received preventive nasal continuous positive airflow pressure (ncpap) are three times higher than the other babies . Considering that an analytical and comprehensive study has not been done about the role of neonatal risk factors and the therapy given for hospitalized neonatal death in west azerbaijan, the study is case control study, wherein the case group included 250 died babies who were hospitalized in the nicu during 2007 - 2009 . The control group also included 250 newborn babies who were hospitalized in the same unit and were discharged alive and healthy after they were given appropriate treatment . Only the newborn babies from shahid motahhari therapy - training center were selected in order to homogenize the sample and eliminate some threatening factors (factors involved in referral, death, etc . ). Data collection was carried out through a researcher - designed questionnaire which was designed based on other studies and scholars points of view . Inclusion criteria included the babies complete profiles, and the criteria for exclusion included their incomplete profiles . The questionnaire contained six sections (mother's and baby's demographic information, causes of hospitalization, during - treatment complications, therapeutic actions, and causes of death) for alive babies and five sections for dead ones . Chi - square test, odds ratio (or), and logistic regression were applied to analyze how the variables were related . Based on single - variable analysis, neonatal risk factors affecting the newborn babies hospitalized in the nicu are as follows: 51.6% of case babies and 18.3% of control babies had first - minute apgar score of less than 6, which proved to have a significant correlation with babies death (p <0.001). About 9.6% of babies of case group and 2.4% of babies in the control group had during - birth asphyxia which proved to have a significant correlation with neonatal death (p = 0.001) [table 1]. The distribution of independent variables by independent variables; p value based on chi - square test in this study, one of the factors causing death of babies hospitalized in the nicu was hmd; 77.6% of case babies and 44.8% of control babies suffered from this disease (p <0.001). Fourteen percent of case babies and 2% of control babies had sepsis which showed a significant relation with babies death (p <0.001). Moreover, 58.8% of case babies and 71.5% of control babies were born through cesarean section which also proved to have a significant effect (p <0.001) [table 1]. Congenital anomalies have been recognized as one of the significant risk factors of death of babies hospitalized in the nicu . Eight percent of case babies and 2.8% of control babies were reported to have congenital anomalies (p <0.001) [table 1]. Regarding therapeutic actions, the results of the study also showed that having ncpap played a significant role in decreasing death of babies hospitalized in the nicu (p <0.001). There was also a significant relation between supportive cares and neonatal death (p <0.001). Increased number of pregnancies also had a significant effect on neonatal death (p <0.001) [table 1]. Variables like lbw, baby's gender, blood group, history of preterm labor, and consanguinity had no significant role in the death of neonates hospitalized in the nicu . In order to gauge the key effects of the mentioned factors, the adjusted or calculated from advanced logistic regression model was measured through advance selection method . The results showed that the or of neonatal death was 4.02 times greater for neonates with first - minute apgar score of less than 6 compared to those with first - minute apgar score of 6 and more, for asphyxia, the or was 6.16, for hmd it was 4.08, and for sepsis it was 6.42 . Applying ncpap and multiparity led to a decrease in the death of neonates hospitalized in the nicu [table 1]. In the present study, the most important neonatal risk factors affecting neonatal death were found to be sepsis, birth asphyxia, hmd, first - minute apgar score of less than 6, congenital anomalies, and delivery method . In our study, the most common risk factor for neonatal deaths was sepsis which raised the risk of death by infection to 6.42 times more than the other causes ., sepsis and its complications were reported to be the first cause of death, but in the investigations of nayeri et al . And amin et al ., sepsis had higher ratings . This may be due to the factor that premature babies are prone to infection, but it should be more investigated because of the high number of deaths resulting from it . This spread of sepsis has been reported to be in 1 - 10 cases out of 1000 live births, but 10 - 50% of deaths occur because of it . The second risk factor in the present study was at birth asphyxia, which is in line with tariq and kundi's study which mentioned sepsis and neonatal asphyxia as the main causes of death, but in the studies carried out by sereshtedari et al ., it was the cause of the 16% deaths in 18 provinces of the country, in bangladesh approximately 26% of neonatal deaths are caused by asphyxia . The other serious clinical problem is respiratory difficulty, and in this study, it manifested as respiratory distress and hmd . In our study, it was the third risk factor of infant death . The study of abdul in malaysia showed that this problem with sepsis is the most important cause of infants death . There is also a second leading cause of infant death in tariq and kundi's study, and respiratory distress syndrome which were studied by kumar et al . In india, jehan et al . In pakistan, naghavi in 18 provinces of iran, javanmardi et al . In isfahan, amani et al . In ardebil, and in the studies of nayeri et al . In tehran and sereshtedari one of the causes of infant death, low apgar score at birth, is increasingly mentioned in many studies to be a risk factor . Low apgar score at birth could be correlated to factors such as neonatal infections, birth asphyxia, meconium aspiration, respiratory distress syndrome, and other causes which may result in infant mortality . In our study, ncpap was applied in the control group two times more than in the case group, which resulted in a decrease of babies death by 0.43 . In their study, rostami et al . Also reported that applying preventive ncpap to newborn babies will increase their survival chances by three times . Congenital anomalies was found to be in the second position in the studies of sharifi, hematyar and yarjo, javanmardi et al . In esfahan, and naghavi's study in 18 provinces . It was mentioned as the second and first cause in the studies of sareshtedati et al . And's study, congenital anomalies and infection were mentioned as the leading factors of infant death . The rate of abnormalities in our study compared with other studies, which shows that it is a less important factor in the incidence of neonatal death . This may be due to high morbidity and mortality caused by other factors such as immaturity, lack of attention and careful examination at the time of birth, lack of screening for neonatal abnormalities, and not performing autopsy in dead neonates . Death rate in babies with lbw is about 40 times higher than in babies with normal weight . Unlike these studies this finding is in correlation with that of nayeri et al . In the present study, normal vaginal delivery was higher in the case group than that in the control group . This finding shows that cesarean section can be better when there is an emergency case or prematurity . Although emergency cesarean section is performed to save the lives of the mother and baby, the selection is not always uncomplicated . In a study, it has been reported that two - third of babies who died were born through cesarean section . In our study, despite the high level of infant mortality in males, there was no significant relation between gender and the cause of death . An important risk factor that was investigated in this study was the number of pregnancies . The results of this investigation showed that death rate decreased as the number of pregnancies increased . Number of births higher than 5, pregnancy number above 3, and high rate of birth were mentioned as effective factors causing babies death in studies conducted by shirvani and khosravi, chaman et al ., and titaley et al ., respectively . Appropriately carrying out care and supportive cares can be effective factors in preventing babies death . In our study, 51.6% of case babies and 2.3% of control babies had received supportive care from birth . Higher death rate in case babies can be due to bad physical conditions and other threatening factors . The results of the study show that incorrect methods of transfer can increase the death rate . In this study, special attention was devoted to increasing the care standards in order to decrease the death rate . According to the results of the study that took into consideration factors such as age, occupation, consanguinity, blood group, preterm delivery background, and sex in two groups with equal distribution, it can be concluded that factors related to accompanying diseases and care during delivery play a significant role in neonatal death . Therefore, regarding the fourth goal of millennium development, decreasing babies death to two - fourths by 2015 in countries with high rate of mortality, it seems to be better to spot pregnancies with high risk of babies death through pre - delivery care and to transfer mothers who are likely to bear premature or sick babies to centers that have nicus . In cases where it is impossible to specify whether a delivery is risky before birth, the subsequent hospitalization problems can be prevented by providing appropriate conditions of transfer and therapeutic actions in intensive care units . The major risk factors in this study (sepsis, asphyxia during birth, hmd, congenital anomaly, and apgar score less than 6 in the first minute of birth) are considered as the major complications of preterm labor and preterm infants . So, strict preventive programs should be undertaken for premature preterm labor and preterm infants; taking care of the infants in four stages (before conception, during pregnancy, during delivery, and thereafter) can be highly effective as well.
Lung cancer remains the leading type of cancer worldwide and in latin america [1, 2]. The disease burden is significantly high, with around 2.5 million new cases per year and 1.5 million deaths worldwide . The two main histological subtypes of lung cancer are small - cell lung cancer (sclc), which comprises 15% of cases, and non - small - cell lung cancer (nsclc) accounting for 85% of cases which include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma . Among all newly diagnosed nsclc cases, adenocarcinomas are the most frequent subgroup following by squamous cell carcinomas [6, 7]. Cigarette smoking is the major risk factor for lung cancer but around 1020% of cases are found in never smokers; also wood - smoke is a major risk factor in countries like mexico [811]. Surgery is the selected treatment for early stage nsclc with the greatest probability of long - term survival in such patients . In advanced nsclc, conventional therapies are based on chemotherapy and radiotherapy but with low efficacy . Over the last decade, there have been advances in the study of molecular pathways underlying tumor development leading to the development of targeted therapies such as tyrosine kinase inhibitors (tkis) and antibodies directed against the two main actionable genes in nsclc up to now: mutations in the epidermal growth factor receptor (egfr) gene targeted by tkis like gefitinib [13, 14], erlotinib [9, 15, 16], and afatinib [1719] and translocations involving the anaplastic lymphoma kinase (alk) gene treated with the tki crizotinib, alectinib, and ceritinib . Benefits have been shown in a subset of 1520% of patients harboring egfr mutations which correlate with definite clinical characteristics: adenocarcinoma histology, female sex, asian ethnicity, and nonsmokers [2325]. Despite these improvements in therapeutic strategies, early diagnosis is very difficult; most cases are diagnosed at an advanced stage and cancer metastasis is very frequent; therefore, there is still an exceedingly low 5-year survival rate of 1124% [2628]. The immunotherapy approach has opened new therapeutic options in advanced nsclc with the advent of antibodies against immune checkpoints [29, 30]. Recently, the anti - programmed death-1 (pd-1) antibodies nivolumab and pembrolizumab have been approved in the treatment of advanced metastatic nsclc based on results from clinical trials after prior chemotherapy [31, 32]. Both antibodies block signaling through pd-1 and may restore antitumor immunity with benefits in overall survival [33, 34]. For example, nivolumab, a fully human monoclonal antibody, has recently shown greater overall survival than docetaxel . These antibodies exhibit a reasonable toxicity profile but they should be administered in selected patient populations based on biomarkers such as pd - l1 expression to avoid serious immune - mediated adverse effects . Although these checkpoint inhibitors have proven efficacy in patients, their mechanism of action implies side effects as the onset of autoimmune diseases and a series of endocrine disorders [37, 38]. This is the rationale for further research into other molecular and cellular factors of the immune system that could be effectively targeted to develop novel therapeutic strategies for the management of advanced nsclc . Recent findings indicate that inflammation plays a key role in tumor progression and survival across several cancer types . Cancer related inflammation affects many aspects of malignancy including proliferation, survival, angiogenesis, and tumor metastasis . Inflammatory components in the development of the neoplasm include diverse leukocytes populations, like macrophages and neutrophils, which respond immediately to inflammatory stimulus . Immunoregulatory cytokines secreted in a proinflammatory environment also contribute to tumor growth and metastases and identify patient subsets in advanced nsclc with differential prognosis . Both macrophages and neutrophils are increased in patients with lung cancer; this is associated with poor clinical outcomes, suggesting that these cells might have important tumor - promoting activities [43, 44]. Tumors escape phagocytosis and immune response through overexpressing cd47 that interacts with the signal regulatory protein alpha (sirp) preventing engulfment . Macrophages within the tumor microenvironment are called tumor - associated macrophages (tams). Tams have a complex relationship with tumor cells; at an early stage they attack tumor cells avoiding tumor spread; however, over time they begin producing reciprocal growth factors and establish a symbiotic relationship with tumor cells . Macrophages are polarized into two functionally distinct forms m1 and m2, mirroring the th1 and th2 nomenclature of t cells . Differentiation of the m1 macrophages is induced by interferon-, lipopolysaccharides, tumor necrosis factor (tnf), and granulocyte - monocyte colony - stimulating factor . The m1 macrophages produce high levels of interleukin- (il-) 12, il-23, tnf, il-1, il-6, cxc ligand 10 (cxcl10), inducible nitric oxide synthase (inos), human leukocyte antigen- (hla-) dr, and reactive oxygen and nitrogen intermediates [47, 48]. Differentiation of the m2 macrophages is induced by il-4, il-10, il-13, il-21, activin a, immune complexes, and glucocorticoid . The m2 macrophages express high levels of il-10, il-1 receptor antagonist, cc ligand 22 (ccl22), scavenger, mannose receptor, galactose receptor, arginase i, and cd163 antigen, reduce the expression of inos, and inhibit antigen presentation and t cell proliferation [47, 49]. Factors that shift tams towards a m2 phenotype include the location of tams within the tumor microenvironment, tumor stage, and type of cancer . Nevertheless, it is still not fully defined whether the diversity within the tam population is due to the maturation of unique monocytic precursors or due to various factors within the local tumor microenvironment . The m2 macrophages have been found to encourage the growth of various tumour cells in vitro and to increase tumor cell survival [51, 52]. Studies suggest that in solid tumors established and progressively growing tams are reprogrammed to induce immune suppression in situ in the host through cytokines, prostanoids, and other humoral mediators [54, 55]. Il-1 and il-6 expression in tams differs in ovarian cancer compared to peripheral blood monocytes . Tams in the ovary produce low levels of il1 and increase the release of il-6, which contributes to elevated acute phase proteins and increased malignancy . There is an association between the number of macrophages and prognosis in a variety of human tumors . Tam infiltration increased in carcinomas of breast, cervix, and bladder and correlates with a poor prognosis . However, in prostate, lung, and brain, increasing tams is associated with regression of tumors . Tams can regulate the development of new blood vessels within tumors . In hypoxic sites, they stimulate the production of enzymes and extracellular matrix molecules that regulate endothelial cell activity by stimulating factors such as vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf), tumor necrosis factor- (tnf-), transforming growth factors- and (tgf-,), interferons, thrombospondin, il-8, and epidermal growth factor (egf). Innate immunity in lung involves alveolar macrophages (ams) which act as a barrier avoiding penetration of pathogens . Conversely, macrophages contribute in part to the pathogenesis of lung disease due to toxic particles ingestion, releasing lysosomal enzymes that can kill the macrophage itself, or contribute to the recruitment of new macrophages inducing chronic inflammation . Clinical evidence has indicated that the activation of alveolar macrophages by sio2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis . Exposure to cigarette smoke activates nf - e2-related factor 2 (nrf2) in macrophages and reduces neutrophil recruitment, reduces ams phagocytic ability and expression of several important recognition molecules, and impairs clearance of apoptotic cells through oxidant - dependent activation of rhoa [60, 61]. In current smokers, the exposure to cigarette smoke affects several important recognition molecules on ams and downregulates cd31, cd91, cd44, and cd71 on these cells . Ams with defective phagocytosis lead to chronic inflammation and significantly increase the likelihood of developing chronic obstructive pulmonary disease, lung injury, and cancer (figure 1). The infiltration of alveolar macrophages promotes the death of tumor cells in those sites of primary tumor growth and/or metastasis in lung . The antitumor activity of alveolar macrophages from lung cancer patients decreases with increased metastasis, tumor size, and development of pleural invasion . The onset of malignant disease triggers the immune response recruiting tams into the tumor site . High numbers of intratumor tams have been linked with invasion, angiogenesis, hypoxia, and early occurrence of metastasis in different tumor types including lung cancer [48, 50] (figure 2). In patients with nsclc, the m1 macrophage phenotype has been associated with the expression of il-1, il-12, tumor necrosis factor- (tnf-), and inos and also has been correlated with extended survival time . In a study, m1 tams were identified using cd68 and inos markers in tumor compared to nontumor tissue in nsclc patients . Results indicate that inos expression is lower in tissues from patients with adenocarcinoma and squamous cell carcinoma compared to nontumor tissues but surprisingly this was not the case in large cell lung carcinomas . The classically activated m1 macrophages produce effector molecules such as reactive oxygen intermediates, reactive nitrogen intermediates, and tnf, to limit tumor growth . Overall there is an association of m1 tams with better lung cancer prognosis . At the other end are the alternatively activated m2 macrophages which have been correlated with tumor initiation, progression, metastases, by secretion of matrix - degrading enzymes, angiogenic factors, and immunosuppressive cytokines chemokines, inhibiting inflammation [65, 67, 68]. M2 macrophages polarized by cigarette smoke lead to proliferation, migration, and invasion of alveolar basal epithelial cells, and exposition to these cigarette smoke - induced m2 macrophages also significantly increased the cell population in g2/m phase causing proliferation in lung cancer cells . In nsclc, the concentration of macrophages m2 was 70% in comparison with 30% m1 . Density of macrophages m1 in the tumor islets, stroma, or islets and stroma was positively associated with patient's survival time . Also, neutrophils are also polarized into n1/n2 subgroups, n1 being proinflammatory, while n2 is anti - inflammatory . N1 and n2 represent a dichotomy in neutrophil subpopulations present in patients and animal models with cancer where they play distinctive roles in the pathogenesis of disease . They displayed an activated phenotype that included chemokine receptors as ccr5, ccr7, cxcr3, and cxcr4 . Also, tans produced proinflammatory factors mcp-1, il-8, mip-1, and il-6, as well as the anti - inflammatory il-1r antagonist . Also, tans exhibit high activated phenotype compared with peripheral neutrophils . In cancer patients, tans could drive antitumoral immunity through regulating cytotoxic t lymphocytes . In early stages of lung cancer disease, tans increased t cell ifn- production and activation and increase t cell proliferation . The blockage of tgf- is able to polarize n2 tans to n1 tans in murine models of mesothelioma and lung cancer . If apoptotic neutrophils within the tissues are not removed in an efficient and timely manner, they will become necrotic and release cytotoxic granule proteins that may perpetuate host tissue damage . Thus, neutrophils apoptosis and clearance is a critical limiting factor for the successful resolution of inflammation . In colon adenocarcinoma cell line, so far, the possible mechanisms by which neutrophils are increased in nsclc patients have not been described; despite this, these cells are dysfunctional; increased levels of il-8 could explain this accumulation; however, the mechanisms by which this occurs are not known . Neutrophils are recruited to tumor sites through transendothelial migration involving the cd47:sirp recognition (signal regulatory protein alpha) creating an inflammatory environment . Malignant cells escape phagocytosis displaying high levels of cd47 on their surface which binds to sirp in macrophages and dendritic cells . After binding to sirp, cd47 induces a dephosphorylation cascade preventing phagocytosis through impaired synaptic myosin accumulation . In this way, cd47 can regulate the function of cells in the monocyte / macrophage lineage [8082]. Cd47 is a ubiquitous cell - surface molecule from the immunoglobulin (ig) superfamily that interacts with sirp, thrombospondins, and integrins . Cd47 was first isolated in association with integrin in neutrophil granulocytes and was later shown to regulate integrin function [84, 85]. It plays a role in cellular processes like proliferation, apoptosis, adhesion, and migration and in immunological processes such as inflammatory response, immune response, and tumor immunity [87, 88]. This receptor is recognized as a marker of self highly expressed by circulating hematopoietic stem cells, red blood cells, macrophages, macrophages neutrophils, and many cancer types . Cd47 has also been identified as a tumor marker, and its dysregulation contributes to cancer progression and evasion of antitumor immunity [9194]. Cd47 is expressed ubiquitously whereas its counter - receptor sirp is more abundant in myeloid - lineage cells such as macrophages, neutrophils, and dendritic cells . Several processes are regulated through the cd47:sirp signaling system of macrophages, including phagocytosis mature red blood cells (rbcs) in the spleen, phagocytosis of senescent cells and apoptotic bodies, rejection of transplants of hematopoietic stem cells (hscs), and immunosurveillance thereby preserving tissue integrity and function [9699]. Remarkably, there are many factors positively regulating phagocytosis while sirp-cd47 is the only negative regulator preventing self - phagocytosis . Cd47 is critical for transepithelial and transendothelial migration of neutrophils or polymorphonuclear leukocytes (pmn) facilitating diapedesis through endothelial cells while targeted cd47 deletion decreases neutrophil extravasation [100, 101]. The sirp-cd47 interaction initially recruits pmns to tumor sites or sites of injury but later negatively regulates these cells to end the inflammatory response . However, in a postacute stage of inflammation, neutrophils experience cleavage of the cytoplasmic signaling domains of sirp, correlating with increased recruitment and neutrophil - associated damage . Truncated sirp acts like a decoy, able to bind cd47 but not signaling intracellularly therefore maintaining the inflammatory microenvironment and being a caveat for cd47 targeted therapies [102105]. Additionally, sirp binding to cd47 in vitro downregulates cd18 as marker of neutrophil activation thus playing a role in the inflammatory activation state of pmns [106, 107]. The dual role of cd47 in promoting inflammation through neutrophil migration and recognition of self through blocking phagocytosis in macrophages plays a role in the development of cancer and later in tumor immune evasion . Loss of cd47 induces phagocytosis by macrophages in vitro and blocks tumor development and metastasis in vivo . This receptor is strongly overexpressed in several cancer types including both hematological and solid tumors [80, 91, 109, 110]. A high cd47 expression has been a poor prognostic factor for patients with these diseases [80, 111, 112]. Cd47 is also highly expressed in tumor initiating cells (tics) or cancer stem cells (csc) where it is a marker of more aggressive tumor cells, with higher metastatic potential, and less sensitive to engulfment by macrophages, thereby escaping from immune surveillance while increasing cell proliferation through activation of the pi3k / akt pathway [92, 113116]. Therefore, cd47 becomes an attractive target for therapeutic approaches with both antitumor and anti - inflammatory properties and anti - cd47 antibodies are being tested with positive results in preclinical and clinical settings [80, 111, 112, 117]. In lung cancer and in several types of cancers including breast, bladder, colon, pancreatic, and hematological cancers, blocking cd47 in tumor cells the cd47:sirp interaction is involved in the pathogenesis of lung cancer and other cancer types when tumors release cytokines promoting tumor growth and stimulating the conversion of macrophages from m1 to m2 phenotype . Systemic administration of nanoparticles with anti - cd47 sirna showed efficient inhibition of lung metastasis to about 30% of controls . In patients with lung metastasis, the number of circulating tumor cells (ctc) with the phenotype epcam(+)cd44(+)cd47(+)met(+) were associated with poor overall survival and increased metastasis and cd47 was a marker associated with the fraction of metastasis - initiating cells within the pool of ctcs . Antisense suppression of cd47 in squamous lung tumors prior to irradiation showed benefit obtaining a 71% tumor size reduction . This protection could possibly be exerted through thrombospondin-1 signaling to recover from radiation stress, revealing a strategy to protect normal tissues from radiation damage using anti - cd47 antibodies which could be useful in the application of combined radiation with targeted therapies in lung cancer . There is a close relationship between macrophage, neutrophil infiltration, and upregulation or cd47 with poor prognosis and lack response to treatment . Nowadays, therapies are developed to block the interaction of tumor cells with macrophages through cd47, thereby offering an opportunity to turn tams against nsclc cells by allowing the phagocytic behavior of resident macrophages . Also, anti - cd47 could regulate the recruitment of neutrophils into tumor and diminish the chronic inflammation figure 3 . Inhibition of csf-1 receptor, which is essential for macrophage differentiation significantly increased survival and suppressed established tumors, accompanied by decreased m2-like tam . Treatment with metformin is able to reduce the metastases in vivo, through blocked matrix metalloproteinase-9 and expression of mmp-2, maintaining the components of the extracellular matrix, avoiding the separation of tumor cells, inhibiting the growth and metastasis of tumors . Also, metformin prevented m2-polarization of macrophages regulated ampk1 and, besides, inhibited il-1 induced release of the proinflammatory cytokines il-6 and il-8 in macrophages [124, 125]. Glycodelin (gene name paep) is a proliferation suppressor and apoptosis inducer of t cells, monocytes, b cells, nk, and regulated pulmonary immune response in asthmatic inflammation . However, atypical expression is observed in squamous cell carcinomas and adenocarcinomas of nsclc . In vitro, silencing by sirna - transfection of paep in two nsclc cell lines resulted in significant upregulation of immune system modulatory factors such as pdl1, cxcl5, cxcl16, mica / b, and cd83 as well as proliferation stimulators edn1 and hbegf . This kind of therapy provides a mechanism to overcome tumor immunosurveillance . As mentioned above, currently the only fda - approved immunotherapies for the treatment of nsclc are nivolumab and pembrolizumab . These antibodies inhibit checkpoint molecules such as ctl-4 and pdl-1, improving the survival and response to treatment . Ctla-4 is thought to regulate t cell proliferation early in an immune response, primarily in lymph nodes, whereas pd-1 is upregulated in current smokers and suppresses t cells . These antibodies switch on immune system cells mediated by t cells, increasing their ability to recognize and destroy cancer cells [128, 130]. Monoclonal antibodies specific for tumor cell antigens, coupled with appropriate cytokines, may provide rational basis for designing trials to employ the neutrophil cytotoxic potential as adjuvant therapy in cancer patients . Chronic inflammation seems to play a major role in the onset and development of cancer . Understanding the interaction between the cellular and molecular factors that mediate inflammation in nsclc, including the rather unexplored components of innate immunity such as macrophages and neutrophils, can elucidate novel targets affecting key oncogenic pathways in this malignancy and allow preventing cancer cell proliferation, angiogenesis, and metastasis . Inhibiting cd47 as promoter of neutrophil extravasation and migration may reduce inflammation thereby preventing cancer, and blocking the antiphagocytic signal of cd47 on the surface of tumor cells can overcome immune suppression, harnessing the immune system to target malignant cells more effectively . On the other hand, the potential side effects should be addressed by careful selection of patient populations based on biomarkers such as tumor cd47 overexpression.
Gastroenteropancreatic neuroendocrine tumors (gepnets) are rare malignant neoplasm with an incidence ranging from two to five cases/100,000/yr . Because it originates from the enterochromaffin serotonin - producing cell, it has a unique feature of hormone secretion and expression of distinctive differentiation markers [1 - 6]. Some patients remain asymptomatic for several years, or complain of episodic flushing, abdominal pain, nausea, vomiting, and diarrhea . In most cases, due to the vagueness of symptoms, a diagnosis is delayed (3 - 10 years on average), with an increased risk of developing metastases . The ability of the imaging method to localize primary or metastatic site of gep - net also has some limitations . Most gep - nets are highly vascular, thus could easily be detected by a contrast enhanced computed tomography (ct); however, approximately 6% to 20% are hypovascular and often difficult to be evaluated by a ct scan or other imaging methods . Gep - nets originating from jejunum and ileum are also often difficult to identify on an image due to their small size . Therefore, non - invasive parameters, indicating gep - pet, are needed for diagnosis, following - ups and prognosis . Chromogranin a (cga), a glycoprotein contained in secretion granules of neuroendocrine cells, is the most abundant granin in gep - nets and widely used as a circulating tumor marker, but only few studies have been published on the role of cga in patients with gep - pet, and the range of sensitivity were variously reported [8 - 10]. We investigated to evaluate whether biochemical response using serial plasma cga is reliable in concordance with the tumor response based on response evaluation criteria in solid tumors (recist) criteria in gep - nets irrespective of chemotherapeutic agents . A total of 27 cases in 18 patients, who were pathologically diagnosed in gep - nets, were analysed between march 2011 and september 2013 . For all cases, non - functioning was defined to the absence of clinical syndromes of hormonal hypersecretion, such as hypoglycemia, peptic ulcer, diarrhea, steatorrhea, acromegaly, cushing s syndrome, and gallstone . The following clinicopathological characteristics of all 18 patients were collected: age, sex, primary site, tumor grade in accordance to the 2010 world health organization (who) classification, liver metastasis, number of metastatic site, site of metastasis, and information of chemotherapy . Systemic chemotherapies for gep - net included octreotide, vip (vincristine, ifosfamide, cisplatin), xelox (capecitabine, oxaliplatin), ep (etoposide, cisplatin), pazopanib, sunitinib, everolimus, and xeliri (capecitabine, irinotecan). Biochemical efficacy was estimated according to the criteria proposed by the italian trials in medical oncology (itmo) group for evaluating markers (biochemical response). Partial response (pr) was defined as 50% decrease in plasma cga compared to the baseline cga; stable disease (sd) was defined as a decrease <50% or as an increase <25%; and progressive disease (pd) was defined as an increase 25% . The level of cga was determined from venous blood samples drawn into edta - containing tube after overnight fasting and collected before systemic treatment . The plasma cga level was measured by cga - ria (chromoa assay, cis bio international, saclay, france) with a normal range of 27 - 94 ng / ml . Chromoa is based on sandwich enzyme - linked immunosorbent assay, using two monoclonal antibodies (the same antibodies as cga - riact) directed against the central domain of the molecule (145 - 245), which is less sensitive to proteolysis . The tumor size was measured by using a ct or magnetic resonance imaging by recist criteria ver . 1.1 . Group comparisons were performed using a nonparametric test of mann - whitney or kruskal - wallis, followed by a dunn multiple comparison test, as appropriate . Comparisons of paired values were performed using a nonparametric test of wilcoxon . The chi - square test and the fisher exact test (for value less than 5) were employed to compare the sensitivity and rate of concordance in different groups . In all statistical tests, progression - free survival (pfs) was measured as the time from the date of chemotherapy to the date of first documented disease progression or death . The pfs were estimated using the kaplan - meier method with log - rank analysis . A total of 27 cases in 18 patients, who were pathologically diagnosed in gep - nets, were analysed between march 2011 and september 2013 . For all cases, non - functioning was defined to the absence of clinical syndromes of hormonal hypersecretion, such as hypoglycemia, peptic ulcer, diarrhea, steatorrhea, acromegaly, cushing s syndrome, and gallstone . The following clinicopathological characteristics of all 18 patients were collected: age, sex, primary site, tumor grade in accordance to the 2010 world health organization (who) classification, liver metastasis, number of metastatic site, site of metastasis, and information of chemotherapy . Systemic chemotherapies for gep - net included octreotide, vip (vincristine, ifosfamide, cisplatin), xelox (capecitabine, oxaliplatin), ep (etoposide, cisplatin), pazopanib, sunitinib, everolimus, and xeliri (capecitabine, irinotecan). Biochemical efficacy was estimated according to the criteria proposed by the italian trials in medical oncology (itmo) group for evaluating markers (biochemical response). Partial response (pr) was defined as 50% decrease in plasma cga compared to the baseline cga; stable disease (sd) was defined as a decrease <50% or as an increase <25%; and progressive disease (pd) was defined as an increase 25% . The level of cga was determined from venous blood samples drawn into edta - containing tube after overnight fasting and collected before systemic treatment . The plasma cga level was measured by cga - ria (chromoa assay, cis bio international, saclay, france) with a normal range of 27 - 94 ng / ml . Chromoa is based on sandwich enzyme - linked immunosorbent assay, using two monoclonal antibodies (the same antibodies as cga - riact) directed against the central domain of the molecule (145 - 245), which is less sensitive to proteolysis . The tumor size was measured by using a ct or magnetic resonance imaging by recist criteria ver . Group comparisons were performed using a nonparametric test of mann - whitney or kruskal - wallis, followed by a dunn multiple comparison test, as appropriate . Comparisons of paired values were performed using a nonparametric test of wilcoxon . The chi - square test and the fisher exact test (for value less than 5) were employed to compare the sensitivity and rate of concordance in different groups . In all statistical tests, progression - free survival (pfs) was measured as the time from the date of chemotherapy to the date of first documented disease progression or death . The pfs were estimated using the kaplan - meier method with log - rank analysis . The rectum was the second most common site of gep - net (22.2%) in this study . According to who classification, ten of the 27 cases (37%) were grade 3 neuroendocrine carcinoma . Grades 1 and 2 nets were nine (33.3%) and eight (29.6%), respectively . Measurable lesions were found on ct images in 18 out of the 27 cases . Among the 27 cases included in this study, no difference in the basal cga level was observed in terms of gender, primary tumor site, tumor grade (who classification), liver metastasis, number of metastatic site, and line of chemotherapy with serial cga monitoring . The plasma cga level ranged from 33.18 to 895 ng / ml (table 1). The overall response rate (rr) by recist criteria was six out of the 27 cases (22.2%) and eight out of the 27 cases (29.6%) for biochemical rr . The concordance of response between recist criteria and biochemical criteria were 74% (table 3). Compared with the baseline values, a decrease of 50% in the cga level were observed in three out of six cases (50%) with pr by recist criteria (figs . 1a, 2). In only grades 1 and 2 gep - nets cases (n=17), the concordance of disease control between recist criteria and biochemical criteria were 94.1% (fig . There was a significant difference for pfs between responders and non - responders (35.73 months vs. 5.93 months, p=0.05) based on the biochemical criteria (fig . 3). A subgroup analysis of pfs between responders and non - responders, in accordance to recist response, tumor grade, and primary site, were not statistically significant, but showed longer pfs in the biochemical responder group (fig . The rectum was the second most common site of gep - net (22.2%) in this study . According to who classification, ten of the 27 cases (37%) were grade 3 neuroendocrine carcinoma . Grades 1 and 2 nets were nine (33.3%) and eight (29.6%), respectively . Among the 27 cases included in this study, no difference in the basal cga level was observed in terms of gender, primary tumor site, tumor grade (who classification), liver metastasis, number of metastatic site, and line of chemotherapy with serial cga monitoring . The plasma cga level ranged from 33.18 to 895 ng / ml (table 1). The overall response rate (rr) by recist criteria was six out of the 27 cases (22.2%) and eight out of the 27 cases (29.6%) for biochemical rr . The concordance of response between recist criteria and biochemical criteria were 74% (table 3). Compared with the baseline values, a decrease of 50% in the cga level were observed in three out of six cases (50%) with pr by recist criteria (figs . 1a, 2). In only grades 1 and 2 gep - nets cases (n=17), the concordance of disease control between recist criteria and biochemical criteria were 94.1% (fig . There was a significant difference for pfs between responders and non - responders (35.73 months vs. 5.93 months, p=0.05) based on the biochemical criteria (fig . 3). A subgroup analysis of pfs between responders and non - responders, in accordance to recist response, tumor grade, and primary site, were not statistically significant, but showed longer pfs in the biochemical responder group (fig . Cga is the most abundant granin in gep - nets and represents the best general marker in the tissue and blood . Cga expression generally correlates with the number of dense core granules in the neuroendocrine cells . Thus, cga monitoring may be helpful in assessing the response to the different therapeutic options . In some studies, however, it is still controversial whether serial cga changes reflect tumor response for treatment . This study showed that the change of the cga level was correlated with tumor response in nonfunctioning gepnets . Additionally, biochemical response based on serial cga may be a predictive marker for pfs in gep - nets . Changes in circulating cga have been reported to represent tumor burden and treatment response . To our knowledge, there were no definite measuring criteria of plasma cga for tumor response and conflicting result of sensitivity and specificity in accordance to treatment response were reported in the literature . Previous studies [2 - 10,18] showed acceptable sensitivity (54%-78%) and specificity (60%-86%) for regression and sd; jensen et al . Also reported that plasma cga concentration is important to disclose tumor progression with specificity and sensitivity, 86% and 86%, respectively . In our study, the concordance of response between recist criteria and biochemical criteria was 74% . Compared with the baseline values, a decrease of 25% in the cga level was observed in four out of six patients (66.7%) with pr based on recist criteria and an increased cga levels was shown in all 4 patients with pd (100%). Among 11 patients available for serial cga levels, all five patients with sd or partial remission had a more than 20% decrease in the cga levels compared to the baseline value . All six patients with pd showed a less than 20% decrease or increase in the cga levels . Several other studies also showed the possibility of cga as a biochemical marker of treatment response (table 4). High - baseline cga value has been considered to be an independent poor prognostic marker for gep - nets in previous studies [21 - 25]. However, whether changes of the cga levels compared to the baseline values could predict the prognosis has not been established . In this study, there was a significant difference for pfs between the responders and non - responders for biochemical criteria (p=0.05). It suggested that a decrease in the cga levels from the baseline levels may be an important predictive marker for survival in gep - nets . This study was a retrospective analysis with small sample size, and heterogeneous patient population . A recognized international standard for cga assay is not available and variations in assay types may influence results . Nevertheless, this analysis identified the usefulness of serial cga monitoring as a biomarker that predicts treatment response and survival . This study revealed that the changes of the plasma cga levels were associated with tumour response . Additionally, biochemical response based on serial cga may be a predictive marker for pfs in gep - nets.
Subjective cognitive complaints (scc) are quite prevalent among older adults, with some estimates suggesting that between 25% and 50% of all older adults have self - perceived memory impairment [1, 2]. In clinical practice, it is often difficult to assess the veracity and severity of subjective cognitive complaints, primarily because such complaints vary widely from individual to individual . As a result, clinicians and caregivers perhaps do not consider subjective complaints to have the same weight as objective findings . However, studies have shown that subjective complaints may be valid indicators of current and future cognitive impairment . A recent study by amariglio and colleagues showed that certain subjective complaints, such as i have trouble finding my way around familiar streets, are correlated with impairment in delayed recall, naming, and semantic fluency . A review conducted by jonker and colleagues showed that memory complaints may be predictive of dementia or alzheimer's disease onset within two to four years, especially in individuals with a diagnosis of mild cognitive impairment (mci). Subjective cognitive complaints also have clinical implications that are outside the realm of cognitive function . A review by mol and colleagues found that scc correlated with depression, anxiety [4, 5], and low level of well - being, even in the absence of objective cognitive impairment . Scc and objective cognitive function in older patients with and without major depression and found no changes in objective cognitive function between the two groups, but significantly more scc in the depressed group . Scc have also been correlated with decreased functional ability, even when depression is controlled for . Although the direction of causality between many of these variables remains unclear, scc should be considered in a clinical setting because of their associations with objective impairment, dementia, depression, low quality of life, and functional ability . Previous research in the area of scc has largely focused on memory complaints [13, 7, 8]. In the current study, we have broadened the scope of scc in order to examine other areas of cognition that have shown impairment following complaints . The most common method appears to be a single, dichotomous, yes or no assessment of complaints (e.g., do you find that you have trouble with your memory?) [713], or a combination of similar yes or no questions [3, 14, 15]. Furthermore, most of the existing literature on scc examines community - dwelling, healthy, and older populations [3, 7, 9, 12, 16]. Subjective cognitive complaints, in addition to predicting the onset of a neurodegenerative process, may yield important information about areas of impairment, specifically in patients with mild disease . Two such disorders are mild cognitive impairment (mci) and its vascular equivalent, vascular mild cognitive impairment (vamci). Differences in functional impairment between individuals with mci and those with vamci, which may be overlooked by objective cognitive testing, may be predicted by an analysis of significance of scc . In the current study, we compared the profile of scc in individuals with a diagnosis of mci to those with vamci using the neuropsychological impairment scale (nis). The nis is a comprehensive scale - based questionnaire, which may elicit differences in presence and severity of scc between the two groups . We hypothesized that the severity of subjective cognitive complaints would not differ between the two groups, because objective cognitive functioning is similar . However, we hypothesized that the specific types of scc might differ between mci and vamci, due to the differing pathologies of these two disorders . Twenty patients with mci and twenty patients with vamci matched on demographic characteristics (age, education) were recruited from the memory disorders clinic at st . Patients are referred to the clinic by their primary or secondary care physicians, for assessment and diagnosis of cognitive impairment . Patients typically undergo a standardized history and a standardized cognitive exam as part of the workup . In addition, routine blood work including tsh, b12, and rbc folate is done to rule out reversible causes of dementia . As well, most patients receive structural imaging (either computed tomography (ct) or magnetic resonance imaging (mri)) and in some cases functional imaging (single photon emission computed tomography (spect)). For this study, only patients clinically diagnosed with either mci or vamci were recruited . The diagnostic criteria for mci describe cognitive impairment which is more severe than normal aging but less severe than dementia and typically not associated with serious impairment in everyday functions . Vamci is described as the vascular equivalent of mci, thereby indicating a similar level of objective cognitive performance despite possible differences in mechanism and etiology . Currently, there are no specific neuroimaging or vascular criteria that are necessary for a diagnosis of vamci . Some patients underwent mri scans instead of ct scans as part of their clinical visit . As we used different imaging modalities we could not compare degree of white matter disease across study subjects in a quantifiable manner . Rather, patients were assigned to the vamci group if scans showed evidence of white matter lesions, lacunar infarcts, and/or moderate to severe microangiopathic change . Cognitive testing and data intake occurred during the patients' initial visits to the clinic . Follow - up visits to the memory disorders clinic were not considered for this cross - sectional analysis . Subjective cognitive complaints were assessed using the neuropsychological impairment scale (nis) [20, 21]. The nis is a questionnaire consisting of 95 complaints, such as i am forgetful and i am easily distracted . Subjects rated these statements on a five - point likert scale according to applicability and intensity . When scored, the nis divides complaints into seven domains: critical items (e.g., head injury, stroke, and dizziness), cognitive efficiency (e.g., confusion, mental slowness), and attention, memory, frustration tolerance, verbal learning, and academic skills (e.g., counting change, learning new tasks). The nis also includes three scores that give a general, comprehensive picture of complaints: the global measure of impairment (gmi) score is a simple sum of all domain subscores; the total items circled (tic) score indicates how many complaints are reported; and the symptom intensity measure (sim) score indicates the average intensity of complaints . The nis also includes three validity checks: defensiveness, inconsistency, and affective disturbance . Thus, the nis can detect if subjects are overly defensive or inconsistent; and the affective disturbance validity check controls for levels of depression and other emotional disturbances . The mini - mental state examination (mmse) was used to measure cognitive performance from a general perspective . The bna consists of five subcategories: memory, attention, naming, visuospatial function, and executive function . Functional ability was objectively assessed using the older american resources and services (oars) activities of daily living questionnaire . This questionnaire assesses fourteen activities of daily living (adls): seven basic adls (eating, dressing and undressing, grooming, walking, getting in and out of bed, bathing, and going to the bathroom) and seven instrumental adls (using the telephone, traveling, shopping, preparing meals, doing housework, taking medications, and handling money). Subjects were asked about their own ability to perform the above adls; where possible, informants who knew the subjects well were asked to supplement and verify responses . Mean scores for each group (mci and vamci) were compared via an independent samples t - test . Age, education, and other demographic factors were also compared between the two groups . The mci and vamci groups were matched on demographic measures such as age, years of education, and hollingshead two - factor index of social position (table 1). Functional ability, as measured by the oars questionnaire, was also similar between the two groups (p = 0.919) (table 2). Objective cognition function was measured by the mmse and the bna (table 2). Mmse scores were similar between the two groups (p = 0.330), which implies similar levels of general cognitive function . Overall bna scores were also similar between the mci and vamci groups (p = 0.177). Scores from subcategories of the bna were also compared between groups to look for specific areas of cognitive impairment (table 2). Performance on all subcategories of the bna (attention, memory, naming, visuospatial function, and executive function) was similar between mci and vamci groups . Performance on the individual tasks which comprise the above five subcategories was also similar between groups, with the exception of the explaining proverbs task (p = 0.034). On this task, which falls into the subcategory of executive function, mci subjects averaged a better score than vamci subjects (3.15 compared to 2.30). Despite similar performance on objective functional and cognitive measures (oars, mmse, and bna), the mci and vamci groups achieved markedly different results when subjective cognitive complaints were assessed by the nis . In general, vamci subjects had significantly higher scores on the nis than mci subjects (table 3); higher scores indicate greater scc . The nis global measure of impairment (gmi) score gives a broad, comprehensive view of complaints . The vamci group averaged a gmi score of 117.0, compared to just 79.3 for the mci group (p = 0.050). A qualitative analysis of the total items circled (tic) scores shows that vamci subjects had a greater number of complaints than mci subjects (52.8 compared to 44.2); however this difference was not statistically significant (p = 0.136). The symptom intensity measure (sim) scores reveal that vamci subjects' complaints were greater in severity than those of mci subjects (2.0 compared to 1.7); however, again, this difference was not statistically significant (p = 0.065). Thus, we cannot attribute the difference in gmi scores to just one cause . Rather, it is likely that difference in gmi scores stems from differences in both complaint quantity and complaint severity . Quantitatively, the vamci group had higher scores in all seven domains of the nis (table 3). Four of these domains yielded differences in scores that were statistically significant: critical items (p = 0.017), cognitive efficiency (p = 0.043), memory (p = 0.046), and verbal learning (p = 0.027). The nis also includes three validity checks to test for defensiveness, affective disturbance, and inconsistency . In our sample of subjects, there was no statistical difference in any of the above variables between the mci and vamci groups (table 3), confirming that the complaints were valid . Although vamci subjects had qualitatively higher scores on the three validity measures, their scores were not significantly different from those of the mci group . Mci and vamci are clinical conditions that precede the development of neurodegenerative disorders including alzheimer's disease and vascular dementia, respectively . Apart from differences on neuroimaging there has been very little study of how these two clinical conditions differ in terms of their subjective and objective clinical profiles . Taken altogether, our findings from the mmse, bna, oars, and nis show that despite similar levels of functional ability and objective cognitive function, vamci subjects have significantly more scc than mci subjects . Our findings support the existing view of mci and vamci as disorders with similar presentations with regard to objective function, but ultimately different etiologies . As previously discussed, mci and vamci subjects were matched on age, education, and social status and achieved statistically similar scores on the mmse and bna . The one component of the bna that differed between the two groups was the explaining proverbs task; however, this seeming disparity may have resulted from differences in cultural background . In our sample of patients from the diverse city of toronto, 17 out of 40 subjects (unfamiliarity with english proverbs certainly confounded this result and may explain this difference observed on the bna . Leaving aside this single anomaly, our findings show that mci and vamci subjects had comparable levels of objective cognitive function . However, our findings from the nis questionnaire show that vamci subjects had more subjective cognitive complaints than their mci counterparts . This discrepancy suggests that vamci patients are exhibiting particular cognitive deficits, which are not identified by the bna, or that vamci patients are more sensitive to and aware of mild deficits in these particular areas . The nature of vamci - specific deficits may be determined from the domains in which vamci patients report greater complaints: critical items, cognitive efficiency, memory, and verbal learning . The critical items domain references events in a patient's history that are evident predictors of cognitive difficulty: dizziness, concussion, head trauma, stroke, and so forth . Vamci subjects reported greater complaints in this domain than mci subjects; this finding may be explained by simply considering the diagnostic criteria of vamci compared to those of mci . As mentioned above, history of clinical stroke can differentiate vamci from mci . A vamci patient is more likely than an mci patient to have experienced stroke or stroke - like symptoms, so he / she is more likely to have complaints in the critical items domain . Vamci subjects' greater complaints in the memory, verbal learning, and cognitive efficiency domains may be explained by examining the pathology of vamci, which differs from that of mci upon neuroimaging . A study by vannorsdall et al . Found that white matter hyperintensities in both periventricular and subcortical regions correlated with poorer working memory . Found that deep white matter hyperintensities correlated with decreased performance on verbal fluency tasks, while periventricular hyperintensities were not associated with any particular domain . White matter lesions have also been correlated with slowed information processing [25, 27]. Recent research has demonstrated that this decrease in processing speed may be specific to white matter lesions in the anterior thalamic radiation . Our analysis may be hampered by the limitations of our cognitive testing methods . In this study, the mmse and bna may have suffered from ceiling effects, which would have masked any differences in cognitive function between the mci and vamci groups . However, the advantages of the mmse and bna are numerous and informed our decision to use these tests . In addition, more sensitive cognitive testing would not have been possible at the study facility, given the amount of time allotted for a clinical visit . The differences we have found in scc between mci and vamci groups may suggest a need for alternate cognitive testing methods, which, unlike the mmse or bna, would elicit different results between the two groups and would not suffer from ceiling effects . Our study has demonstrated that vamci patients may be exhibiting particular cognitive deficits in memory, verbal learning, and cognitive efficiency, separate from those exhibited by mci patients . These deficits may be explained by the greater white matter burden in vamci patients, as compared to age - matched mci patients . Further research should be done to elaborate on the nature of vamci - specific deficits, particularly in the domains we have identified in this study . As well, vamci - specific deficits should be related to neuroimaging findings, in order to associate deficits with particular locations and severities of white matter disease . Finally, tools for more sensitive cognitive testing must be developed, so that we may identify vamci - specific cognitive deficits.
The 42 participating spanish hospitals were selected according to earss criteria (1,7). The total catchment population was 9 million people, or 22.5% of the spanish population . The first blood e. coli isolates obtained from each patient between 2003 and 2006 were included . Each laboratory identified the strains and tested their susceptibilities according to standard microbiologic procedures; all used commercial microdilution systems . Susceptibility data were interpreted according to clinical laboratory standards institute criteria (8). For epidemiologic purposes, intermediate susceptibility to amc was considered as resistance . Multidrug resistance was defined as resistance to> 3 of the following antimicrobial agents: ciprofloxacin, gentamicin, cotrimoxazole, and cefotaxime . To assess the comparability of susceptibility test results, an external quality assurance exercise (uk national external quality assessment scheme) was performed yearly . Hospital - acquired infections were defined as infections acquired at least 48 hours after hospital admission . Outpatient consumption of penicillin/-lactamase inhibitors (world health organization code j01cr02) for the period 20022006 was assessed from the especialidades consumo de medicamentos database, which showed retail pharmacy sales of all medicines acquired with national health system prescriptions and covered nearly 100% of the spanish population (5). The information was tabulated, and the number of units was converted into defined daily doses (ddd) of active drug ingredients according to who methodology (9). The number of ddd per 1,000 inhabitants per day (dids) was calculated for each active drug ingredient . Differences in the antimicrobial resistance prevalence between different groups were assessed by fisher exact test . Association was determined by calculation of the odds ratio (or) with 95% confidence intervals (ci). Statistical analyses were performed by using graphpad prism version 3.02 software (graphpad software, inc . Participating hospitals reported data on 9,090 cases of e. coli bacteremia during the study period, corresponding to the same number of patients; 4,526 (49.8%) were male patients and 4,564 (50.2%) were female patients . A total of 1,531 cases (16.8%) were diagnosed in 2003; 2,526 (27.8%) in 2004; 2,438 (26.8%) in 2005; and 2,597 (28.6%) in 2006 . Of the total number of isolates, 328 (3.6%) were obtained from children <14 years of age; 2,857 (31.4%) were obtained from patients> 15 and <64 years of age; and 5,909 (65%) were obtained from patients> 64 years of age . There were 3,384 (37.9%) isolates implicated in hospital - acquired infections and 5,540 (62.1%) in community - acquired infections; information was missing for 166 cases . Of the 9,090 e. coli isolates tested, 1,136 (12.5%) were nonsusceptible to amc, 5.1% were resistant, and 7.4% were intermediate . The prevalence of amoxicillin / clavulanic acid nonsusceptibility in relation to gender, age, infection origin, and resistance to other antibimicrobial drugs is detailed in the table . * the most prevalent phenotypes included multidrug resistance to ciprofloxacin, cotrimoxazole, and gentamicin, which was detected in 73 nonsusceptible amc isolates (36.9% of multiresistant isolates and 6.4% of isolates overall), and resistance to ciprofloxacin, cotrimoxazole, and cefotaxime was detected in 55 isolates (27.8% of multiresistant isolates; 4.9% of isolates overall). Multidrug resistance was more prevalent in nosocomial (23.5%) than in community - acquired isolates (15.1%; or 1.99, 95% ci 1.462.72; p<0.0001). Among nonsusceptible amc isolates, susceptibility to other antimicrobial drugs, including ciprofloxacin, gentamicin, cefotaxime, and cotrimoxazole, was more frequent in community- (28.7%) than in hospital - acquired isolates (13.3%; or 1.68, 95% ci 1.272.22; p = 0.0003). The overall rate of invasive e. coli nonsusceptibility to amc increased from 9.3% (2003) to 15.4% (2006) (test for trend 36.51; p<0.0001) (figure 1); this increase was observed in 64.3% of the participant hospitals . This increase was also detected in both intermediate and resistant isolates, with annual distributions of 5.6% and 3.8%, respectively, in 2003; 6.8% and 4.8% in 2004; 7.5% and 5.4% in 2005; and 9.4% and 6% in 2006 . Evolution of amoxicillin - clavulanic acid nonsusceptibility of escherichia coli from blood isolates, spain, 20032006 . Amc nonsusceptibility according to age groups increased over the study period as follows: children <14 years of age (10.6% in 2003, 14.6% in 2004, 14.3% in 2005, and 16.3% in 2006); patients> 15 and <64 years 9.6% in 2003, 11.2% in 2004, 11.7% in 2005, and 13.3% in 2006); patients> 64 years (8.8% in 2002, 11.3% in 2004, 15.9% in 2005, and 16.3% in 2006). The prevalence of amc nonsusceptibility in community - acquired infections increased from 8.9% (2003) to 15.6% (2006) (test for trend 29.43; p<0.0001). Amc nonsusceptibility in nosocomial infections increased from 9.2% (2003) to 15.2% (2006) (test for trend 11.94; p = 0.0006). In the final 2 years of the study period (20052006) this increase was due to community - acquired e. coli isolates only; the nonsusceptible proportion varied from 11.5% (2005) to 15.6% (2006) (or 1.42, 95% ci 1.161.74; p = 0.0009) in community - acquired isolates compared with 15.4% (2005) to 15.2% (2006) in hospital - acquired isolates (figure 1). Community - acquired infection probably included healthcare - associated infections, a recently described epidemiologic category distinct from both community - acquired and nosocomial status . In this study, the number of blood isolates of e. coli producing extended - spectrum -lactamase (esbl) was 614 (6.7%); 188 of them (30.6%) were nonsusceptible to amc . When esbl - producing e. coli isolates were excluded from analysis, amc nonsusceptibility increased from 8.4% (2003) to 14.3% (2006) (test for trend 34.39; p<0.0001) in total isolates; from 8.2% (2003) to 14.5% (2006) (test or trend 25.23; p<0.0001) in community - acquired isolates; and from 8.9% (2003) to 13.3% (2006) (test for trend 6.35; p = 0.012) in hospital - acquired isolates (figure 1). The proportion of isolates highly susceptible to amc (mic <4 mg / l) steadily decreased over the study period as follows: 70.2% (2003), 70% (2004), 64.8% (2005), and 57.4% (2006) (test for trend 99.36; p<0.0001). Community consumption of penicillin/-lactamase inhibitors, predominantly amc, increased 34.7% from 2000 to 2006 (figure 2), whereas total antimicrobial drug consumption remained relatively constant (19.6 dids in 2000 compared with 19.1 dids in 2006). After amc, the most used -lactam antimicrobial agents in the community in spain were amoxicillin, cefuroxime, and cefixime; their consumption did not vary or slightly decreased from 2002 through 2005 (6). Evolution of consumption of outpatient penicillin/-lactamase inhibitors (world health organization code j01cr02), spain, 20002006 . The increased amc resistance of e. coli isolates from blood observed in this study is of serious concern from clinical and epidemiologic standpoints because amc is the first - choice antimicrobial treatment for many invasive e. coli infections . Increased amc resistance coincided with growing amc consumption at the community level . In urinary infections, previous treatment with amc is a risk factor for the development of amc resistance (10). Amc resistance mechanisms (-lactamase overproduction, ampc cephalosporinase hyperproduction, and inhibitor - resistant penicillinases) (11) might be favored by strong amc consumption.
A 51-year - old caucasian female was referred to our institution because of an abnormal nodule detected in her right lung . This had at first been detected four months earlier when she underwent an urgent hartmann procedure due to refractory adenocarcinoma of the sigmoid colon (pt4n1 stage). Due to the urgency of the surgery, the patient was not staged prior to the surgery but was initially staged after the operation . The only pathologic finding in the computed tomography (ct) of the lung was a nodule in the lower right lobe of 2.5 cm at its longest dimension (fig . She received 4 cycles of chemotherapy for the sigmoid adenocarcinoma before she was referred to us for excision of what was believed to be a single metastatic nodule . Although the radiologic findings supported a benign tumor, neither positron emission tomography - ct nor any other procedure to attempt to diagnose the nodule was performed due to the patient's willingness to undergo complete removal of the mass even if it were benign . The patient underwent video - assisted thoracoscopic (vats) wedge excision of the tumor . The specimen of the resected lung contained a firm intrapulmonary white - colored well - defined mass with dimensions of 2.52.32.2 cm . Microscopically, the tumor had a " patternless pattern, " with proliferation of bland spindle cells in alternating hypocellular and hypercellular areas, accompanied by a collagenous stroma (fig . The tumor had no evidence of increased mitotic activity (0 to 1 mitoses per 10 high power fields using an olympus bh-2 microscope, with a 40 field, 0.5 mm diameter, and area of 0.196 mm), any significant atypia, or necrosis . Immunohistocemically, the tumor cells were positive for vimentin, cd34 (fig . 2c), and bcl-2 . The index of cellular proliferation with the antibody ki-67 is low and estimated at <1% of the cellular population . Immunohistochemistry staining of the tumor cells was negative for alfa - smooth muscle actin (sma), muscle specific actin (hhf-35), h - caldesmon, cd117 (c - kit), s-100 protein, epithelial antigens (wide spectrum cytokeratins [ae1/ae3] and epithelial membrane antigen), and thyroid transcription factor-1 . Solitary fibrous tumors (sfts) were first pathologically described by klemperer and rabin in 1931 . To the present, sft is the preferred term for an uncommon, but histomorphologically distinctive spindle cell neoplasm, that was identified in the past as fibrous mesothelioma, localized fibrous mesothelioma, localized fibrous tumor, localized mesothelioma, pleural fibroma, solitary fibrous mesothelioma, or submesothelial fibroma . There are reports of sfts arising either from mediastinal, diaphragmatic, or parietal pleura, or from within a lung fissure and in various extrapleural locations, such as the retroperitoneum, mediastinum, thyroid gland, nasal cavities, meninges, or parietal surfaces of the intra - abdominal viscera . Less than 20 cases of intraparenchymal sfts have been reported in the english literature [1 - 5]. To prove and establish the fact that these lesions are indeed of pulmonary origin, it is important to consider the clinical, radiologic, and pathologic findings to demonstrate the lack of continuity with the visceral pleura and to exclude an endophytic growth in a pleural - based lesion . Hypoglycemia and seizures can be encountered in some cases due to secretion of insulin - like growth factor ii by the tumor [1 - 3,5]. Hemoptysis due to sft as well as hypertrophic osteoarthropathy secondary to sft of the lung has also been reported . There is no association with smoking or asbestos exposure . Due to its non - specific symptoms, it can potentially mimic solitary pulmonary metastasis, as was the case with our patient . On gross examination, histologically, it is associated with variable patterns, the most common being the " patternless pattern . " The cytologic features consist of cellular areas composed of spindled to ovoid cells with scanty cytoplasm, occurring either solely or in groups . Immunohistochemistry plays a substantial role in supporting the diagnosis made by morphologic features and is also helpful in differentiating these tumors from mesothelioma, neurofibroma, and other spindle - cell lesions . A differential diagnosis of intrapulmonary sft includes, among other lesions, pulmonary adenofibroma, benign neural neoplasms, leiomyoma and leiomyosarcoma, synovial sarcoma, spindle cell thymoma, spindle cell carcinoid tumor, nerve sheath tumor, fibrosarcoma, sarcomatoid carcinoma, and sarcomatoid mesothelioma . The morphologic features and immunohistochemical profile of sft are sufficiently distinct to allow separation from such conditions in the majority of the cases . Several studies have reported positivity of tumor cells with cd34 antibody in almost 100% of cases, and with cd99 antibody in 70% of cases, whereas bcl-2, sma, and epithelial membrane antigen are positive in 20% to 35% of the cases . The histologic criteria for classifying the malignant variants of sft of the lung and pleura were described by england et al . In 1989 and vallat - decouvelaere et al . In 1998 . They established the following features suggestive of malignancy: 1) more than 4 mitoses per 10 high - power fields, 2) presence of necrosis, 3) hemorrhage, 4) hypercellularity as detected by nuclear crowding and overlapping, 5) nuclear atypia, 6) pleomorphism, 7) stromal or vascular invasion, and 8) size exceeding 10 cm . . The absence of these characteristics does not exclude malignant behavior . On the other hand, encapsulation, pedunculated and resectability with free surgical margins are considered to be favorable prognostic factors even in histologically malignant variants . Positive margins are associated with an aggressive clinical course and high rates of local recurrence and metastasis . Resection with free margins is considered to be the treatment for sft located either in the lung or on the pleura . Wedge resection may be accomplished by vats or standard thoracotomy, according to the anatomic position and size of the lesion and the experience of the surgeon . Adjuvant therapy may have a place in recurrent or systemic disease, but its benefit is undefined . In general, sfts have an unpredictable course, depending on their potential for malignancy . In large series, the recurrence rate of benign sfts is reported to be low (1.4%), while the recurrence rate of malignant variants is reported to be higher (range, 9% to 19%). A staging system based on pedunculated versus sessile attachment and malignant versus benign histology that predicts recurrence has been proposed by perrot et al . (table 1). Due to the possibility of local recurrence and/or distal metastasis after surgical removal of the primary sft, long - term follow - up is recommended . Local recurrence detected early is amenable to reoperation and resection, with good long - term results . The long - term survival rate for both benign and malignant variants is reported to be more than 90% [1 - 3,6]. In conclusion, sfts are neoplasms that usually arise from the pleura . An intraparenchymal or endobronchial location is a rare occurrence . Resection with clear margins is curative and final diagnosis and prognosis can be defined only after surgical resection . The prognosis of patients with rare sfts of the lung depends on the completeness of the tumor resection . Variants of sfts of the lung that are malignant or suspected to be malignant should be managed as lung cancer with regard to the surgical resection of the tumor and follow - up strategy.
Synthesis of metal nanoparticles with the help of plant extracts is an emerging field of nanotechnology, due to their novel properties, terrific applications in biomedicine and its eco - friendly nature . In recent past great efforts were made for synthesis of environment benign and eco - friendly nanoparticle from plants such as iron oxide nanoparticles from medicago sativa, copper nanoparticles from magnolia kobus, calcium nanoparticles from boswellia ovalifoliolata, gold nanoparticles from avena sativa, zinc oxide nanoparticles from catharanthus roseus, and silver nanoparticles (agnps) from syzygium alternifolium . Silver is one among the metal nanoparticles focused much interest due to its wide variety of applications . It has different biological activities such as antimicrobial, anthelmintic, antilarvicidic, antioxidant, anticancer, anti - inflammatory, hepatoprotective, and wound healing activity . Conventional methods for synthesizing agnps are mainly by chemical, physical, and microbe - mediated synthesis . In these chemical and physical methods, usage of hazardous chemicals, high energy requirements, difficult and wasteful materials generate potential and biological hazards to the environment . Whereas in the case of microbe - mediated synthesis . Therefore biological synthesis of agnps by using plant materials is easy, efficient, and eco - friendliness in comparison to chemical mediated or microbe - mediated synthesis and they possess secondary metabolites having the redox capacity to reduce metal nanoparticles in an easiest way . Adansonia digitata l. belongs to the family malvaceae is a large tree indigenous to africa and found in many countries also . Traditionally, the fruit pulp dissolved in water or milk is used as a drink or sauce for food in africa . Fruit pulp and powdered seeds are used for the treatment of diarrhea and dysentery in india . The high content of vitamin c in fruit pulp is recommended to pregnant women s for daily intake . In recent times, different scientists prove different activities of pulp such as hepatoprotective, antimicrobial, antiviral, antioxidant, antidiarrheal, hypoglycemic, anti - inflammatory, and antioxidant activities . Due to high medicinal values and mythological significance of this plant is known as kalpavriksha (a tree which fulfill all desires) in india . In our previous studies, synthesis of agnps from stem bark and leaf extract of a. digitata acts as excellent reducing agents and show potential antimicrobial activity against different microorganisms . However, the potentiality of fruit pulp mediated synthesis of agnps is not carried out so far . Hence, the present study is aimed to synthesize agnps from a. digitata fruit pulp extract, characterize and to know the potentiality of agnps against different microbial pathogens . 2 to 4 kg weight mature fruits possess a great amount of pulp is collected from acharya ranga agricultural university, tirupati and cross checked by herbarium (voucher no: svu362) deposited in department of botany, sri venkateswara university, tirupati . Cut open the fruit and separate the seeds adhesive to pulp . Grounded the collected pulp with the help of mortar and pestle, sieved the content for the synthesis of nanoparticles . For synthesis, 25 g of fruit pulp is extracted with 100 ml of distilled water on boiling water bath for 30 min, filter the content with whatman no . 1 filter paper and stored at room temperature . From this, 5 ml of the extract is taken into 250 ml of erlenmeyer conical flask and titrated against with the solution of 1 mm agno3 at a 60 - 80c temperature . The contents are cooled and centrifuged at 10,000 rpm for 20 min to avoid the presence of any biological impurities, and it is used for further characterization and antimicrobial studies . Confirmation of synthesized nanoparticles is agnps by ultraviolet (uv)-vis spectrophotometry absorption spectra using a spectro uv 2080 double beam, between the wavelength scan range of 190 - 700 nm, 1200 l / mm spectrophotometer, analytical technologies, india . To know the possible bio - molecules responsible for reduction and stabilization of agnps by fourier transform infra - red (ftir) spectra in the scan range of 4,000 to 500 cm transmittance with an alpha interferometer, bruker, ettlingen, karlsruhe, germany by kbr pellet method . X - ray diffraction of synthesized nanoparticles is examined by an x - ray diffractometer (xrd) (shimadzu, xrd-6000) equipped with cu ka radiation source using ni as a filter at a setting of 30 kv/30 ma to know the crystalline nature of agnps . Purity of agnps was analyzed by fei quanta 200 feg high resolution (hr)-scanning electron microscopy (sem) machine equipped with edax instrument . To know the size, shape, agglomeration pattern, and dispersed nature of the nanoparticles are done by atomic force microscopy (afm) by nova nt - mdt solver next, russia . Transmission electron microscopy (tem) using hf-3300 advanced 300 kv tem / stem from hitachi . The antimicrobial activity of biologically synthesized nanoparticles are analyzed on seven pathogenic bacteria such as bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538 (gram - positive), escherichia coli atcc 25922, klebsiella pneumonia atcc 43816, proteus vulgaris atcc 13315, pseudomonas aeruginosa atcc 15442, salmonella typhimurium atcc 14028 (gram - negative), and five fungal pathogens such as alternaria solani atcc 32904, aspergillus flavus atcc 9643, aspergillus niger atcc 16404, penicillium chrysogenum atcc 11709, and trichoderma harzianum atcc 20476 procured from department of microbiology, sri venkateswara university, tirupati . Disc diffusion method was followed for testing antimicrobial activity against biologically synthesized agnps, and comparative studies were made with plant pulp extract, 1 mm agno3 as negative controls and streptomycin or fluconazole as a standard for bacteria and fungi, respectively . 1 filter paper and 20 l of plant extract, 1 mm agno3 solutions, and 10 g / disc streptomycin / fluconazole are loaded on separate discs and allowed to air dry for 1 h at sterile conditions . Apart from these, a 5, 10, 20, 40, 60, 80 g / ml concentrations of synthesized agnps are tested separately to know the minimum inhibitory concentration . 20 and 40 g / ml concentration of agnps show minimum inhibitory effect and 80 g / ml concentrations of agnps show maximum inhibitory concentration . Due to this, we prefer 80 g / ml concentrations of agnps to check the antimicrobial activity on different microbial organisms . Freshly prepared nutrient agar media for bacteria and potato dextrose agar media for fungi are poured into sterile petri plates, allowed to 30 min for solidification . The plates are swabbed with 100 l of microbial cultures and placed the previously prepared discs; the experiment is carried out in triplicates . The plates are incubated at 37c for 24 - 48 h, and then the zone of inhibition is measured with the help of a scale and tabulated the results . 2 to 4 kg weight mature fruits possess a great amount of pulp is collected from acharya ranga agricultural university, tirupati and cross checked by herbarium (voucher no: svu362) deposited in department of botany, sri venkateswara university, tirupati . Cut open the fruit and separate the seeds adhesive to pulp . Grounded the collected pulp with the help of mortar and pestle, sieved the content for the synthesis of nanoparticles for synthesis, 25 g of fruit pulp is extracted with 100 ml of distilled water on boiling water bath for 30 min, filter the content with whatman no . 1 filter paper and stored at room temperature . From this, 5 ml of the extract is taken into 250 ml of erlenmeyer conical flask and titrated against with the solution of 1 mm agno3 at a 60 - 80c temperature . The contents are cooled and centrifuged at 10,000 rpm for 20 min to avoid the presence of any biological impurities, and it is used for further characterization and antimicrobial studies . Confirmation of synthesized nanoparticles is agnps by ultraviolet (uv)-vis spectrophotometry absorption spectra using a spectro uv 2080 double beam, between the wavelength scan range of 190 - 700 nm, 1200 l / mm spectrophotometer, analytical technologies, india . To know the possible bio - molecules responsible for reduction and stabilization of agnps by fourier transform infra - red (ftir) spectra in the scan range of 4,000 to 500 cm transmittance with an alpha interferometer, bruker, ettlingen, karlsruhe, germany by kbr pellet method . X - ray diffraction of synthesized nanoparticles is examined by an x - ray diffractometer (xrd) (shimadzu, xrd-6000) equipped with cu ka radiation source using ni as a filter at a setting of 30 kv/30 ma to know the crystalline nature of agnps . Purity of agnps was analyzed by fei quanta 200 feg high resolution (hr)-scanning electron microscopy (sem) machine equipped with edax instrument . To know the size, shape, agglomeration pattern, and dispersed nature of the nanoparticles are done by atomic force microscopy (afm) by nova nt - mdt solver next, russia . Transmission electron microscopy (tem) using hf-3300 advanced 300 kv tem / stem from hitachi . The antimicrobial activity of biologically synthesized nanoparticles are analyzed on seven pathogenic bacteria such as bacillus subtilis atcc 6633, staphylococcus aureus atcc 6538 (gram - positive), escherichia coli atcc 25922, klebsiella pneumonia atcc 43816, proteus vulgaris atcc 13315, pseudomonas aeruginosa atcc 15442, salmonella typhimurium atcc 14028 (gram - negative), and five fungal pathogens such as alternaria solani atcc 32904, aspergillus flavus atcc 9643, aspergillus niger atcc 16404, penicillium chrysogenum atcc 11709, and trichoderma harzianum atcc 20476 procured from department of microbiology, sri venkateswara university, tirupati . Disc diffusion method was followed for testing antimicrobial activity against biologically synthesized agnps, and comparative studies were made with plant pulp extract, 1 mm agno3 as negative controls and streptomycin or fluconazole as a standard for bacteria and fungi, respectively . 7 mm sterile discs are prepared from whatman no . 1 filter paper and 20 l of plant extract, 1 mm agno3 solutions, and 10 g / disc streptomycin / fluconazole are loaded on separate discs and allowed to air dry for 1 h at sterile conditions . Apart from these, a 5, 10, 20, 40, 60, 80 g / ml concentrations of synthesized agnps are tested separately to know the minimum inhibitory concentration . 20 and 40 g / ml concentration of agnps show minimum inhibitory effect and 80 g / ml concentrations of agnps show maximum inhibitory concentration . Due to this, we prefer 80 g / ml concentrations of agnps to check the antimicrobial activity on different microbial organisms . Freshly prepared nutrient agar media for bacteria and potato dextrose agar media for fungi the plates are swabbed with 100 l of microbial cultures and placed the previously prepared discs; the experiment is carried out in triplicates . The plates are incubated at 37c for 24 - 48 h, and then the zone of inhibition is measured with the help of a scale and tabulated the results . Reduction of ag into ag nanoparticles was observed visually by means of a color change pattern of the reaction medium . A. digitata fruit pulp having thick cream color, upon synthesis the color change from creamy to light yellow indicates the formation of nanoparticles . This color changed nanoparticles solution was analyzed by uv - vis spectrophotometry shows a broad absorption peak at 434 nm further confirms the formation of nanoparticles are agnps [figure 1]. Ftir spectroscopic studies of these synthesized nanoparticles show broad transmittance peaks at 3322 cm assigned for an o - h bond of phenols and 1636 cm assigned for an n - h bond of primary amines [figure 2]. Xrd spectrum of synthesized agnps shows the crystalline nature of agnps and gives four intensive peaks at 38.1, 46.2, 64.5, and 77.3 of 2 degrees of x - axis corresponds to 111, 200, 220, and 311 bragg reflections of y - axis [figure 3]. The mean particle diameter of synthesized agnps is 44 nm, calculated according to debye - scherrer equation (d = k/ cos) and it coincides with powder diffraction file of joint committee on powder diffraction standards file no . The full width at half maximum values, i.e. K = 0.44 was derived from 38, 46, 64, and 77 bragg reflections of the x - axis . 2 m resolution studies of biologically synthesized agnps with afm reveal the particles are polydispersed, spherical in shape, having the size range from 25 to 57 nm, and there is no agglomeration observed between the particles [figure 4a]. Raw data obtained from this afm microscope is treated with a specially designed image processing software (nova - tx) to further exploit the three - dimensional (3d) image of nanoparticles [figure 4b]. 500 nm resolution studies with 20 kv electron energy passing through the nanoparticles coated thin films on a clean glass slide reveals the nanoparticles are spherical in shape, polydispersed and having the size range from 18 to 32 nm [figure 5a]. The same sample was analyzed with the help of edax instrument shows 33.28% presence of ag in the sample [figure 5b] along with 05.29% of carbon, 03.28% of nitrogen, 31.80% of oxygen, 08.37% of sodium, 02.57% of magnesium, 01.01% of aluminum, 00.78% of silicon, 07.87% of aurum, and 05.74% of calcium [table 1]. Crystalline nature of synthesized agnps was analyzed using selected area electron diffraction (saed) by directing the electron beam perpendicular to nanoparticles . The saed pattern of synthesized agnps shows the spots had been corresponding to 111, 200, 220, and 311 of the crystallographic nature of face - centered cubic structures [figure 6a]. 20 nm resolution studies with 300 kv electron energy passing through nanoparticles coated thin films on copper grid show the nanoparticles are polydispersed, spherical in shape and size range from 3 to 7 nm [figure 6b]. To know the antimicrobial potency of biologically synthesized agnps from fruit pulp of a. digitata was analyzed against two gram - positive, five gram - negative, and five fungal pathogens by disc diffusion method . Among the bacterial pathogens, the highest inhibition zones were observed in p. vulgaris followed by k. pneumoniae, p. aeruginosa, s. typhimurium, e. coli, b. subtilis, and s. aureus . Whereas in the case of fungi, the highest inhibition zones were observed in t. harzianum, followed by a. niger, a. flavus, p. chrysogenum, and a. solani . Ultraviolet - vis spectrum of synthesized silver nanoparticles shows broad peak at 434, 280, and 247 nm of narrow peaks is due interference of phytoconstituents in the medium . Inset figure shows color change pattern from cream to light yellow fourier transform infra - red spectrum of synthesized silver nanoparticles shows broad peaks at 3322 cm and 1636 cm x - ray diffractometer pattern of synthesized silver nanoparticles shows four intensive peaks atomic force microscopy micrograph of synthesized silver nanoparticles (agnps), (a) 2 m resolution studies 25 - 57 nm size, spherical shaped, polydispersed particles, (b) three - dimensional image of agnps analyzed by nova - tx software scanning electron microscopy micrograph of synthesized silver nanoparticles (agnps) (a) 500 nm resolution studies shows 18 - 32 nm size, spherical shaped particles, (b) edax spectrum of synthesized agnps shows 33.28 weight percent of ag metal in the sample metal analysis by edax spectrum shows different weight percentages of sample transmission electron microscopy micrograph of synthesized silver nanoparticles (agnps), (a) selected area electron diffraction pattern shows characteristic crystal spots of elemental silver, (b) 20 nm resolution studies of agnps shows 3 - 7 nm size, spherical shaped nanoparticles antimicrobial studies of biologically synthesized silver nanoparticles (agnps) from adansonia digitata pulp extract, (a) bacillus subtilis, (b) staphylococcus aureus, (c) escherichia coli, (d) klebsiella pneumoniae, (e) proteus vulgaris, (f) pseudomonas aeruginosa, (g) salmonella typhimurium, (h) alternaria solani, (i) aspergillus flavus, (j) aspergillus niger, (k) penicillium chrysogenum, (l) trichoderma harzianum, (1) plant extract, (2) agno3, (3) agnps, (4) streptomycin / fluconazole zone of inhibition (mm) of agnps on microbial pathogens and comparative studies with different controls when the addition of agno3 solution to the plant extract the color of the plant extract is changed gradually according to the quantitative addition of agno3 solution . This color change is due to the reduction of nanoparticles with the help of electrons present in the fruit pulp extract coming from nad and ascorbic acid as electron donors present in the plant extracts . Significantly higher amount of ascorbic acid, i.e.,> 300 mg/100 g was reported in fruit pulp of a. digitata . This solution was subjected to uv - vis spectrophotometry between the scan ranges of 190 - 750 nm, a broad peak is obtained at 434 nm . The same type of results was observed in leaf extract mediated synthesis of agnps from couroupita guianensis . The color change pattern and broad peak obtained in uv - vis spectrophotometry are due to surface plasmon resonance nature of agnps present in the medium . These nanoparticles absorb light at different wavelengths and excited due to charge density at the interface between conductor and insulator to give a respective peak on uv - vis spectrophotometry . Ft - ir is a sensitive tool to analyze functional groups present in the biological samples . It relies on the light absorbance between 4000 cm to 500 cm of the electromagnetic, infrared region . In our case, the same type of results was observed in myristica fragrans seed extract mediated synthesis of agnps . From this ftir study, clearly reveals hydroxyl groups of phenols and amide groups of proteins from plant extract forming a layer to the nanoparticles and acting as a capping agent to prevent agglomeration and providing stability in the medium . Based on these ftir studies, we suggest that the bio - molecules present in plant extracts play dual role in formation and stabilization to agnps . Afm is a primary tool for analyzing size, shape, agglomeration pattern and also offers visualizations of 3d views of the nanoparticles, unlike the electron microscopes . It has an advantage over combination of hr, samples do not have to be conductive and does not require the high - pressure vacuum conditions . Better resolution and percentage presence of nanoparticles were demonstrated by sem with edax instrument provides reliable characterization and morphology of the particles when compare to afm . The four intensive bragg reflections are appear in xrd pattern [figure 3] is correlated with saed pattern [figure 6a] of agnps, clearly indicates the nanoparticles are crystalline in nature . Higher resolution studies with tem analysis shows size, shape, and agglomeration pattern of nanoparticles . It achieves better resolution than sem due to electron energies higher than 20 kv used in sem . Finally, all the microscopic studies reveal the biologically synthesized agnps are spherical in shape, having the size range from 3 to 57 nm polydispersed and no agglomerations were found between the particles . To test the inhibitory effect of biologically synthesized nanoparticles on different microorganisms bacterial species show the highest inhibitory activity when compare to fungi because the cell walls of fungi are made up of chitin is more rigid when compare to bacterial cell walls containing peptidoglycans . Among the bacteria, gram - negative species show the highest inhibitory zones when to compare to gram - positive species, because the gram - negative species containing a less amount of peptidoglycans . Silver is a precious metal used as an effective antimicrobial agent before the advent of agnps . Due to overuse of silver products, decreased the efficiency of silver agents as antibiotic . In recent times with the advancement of nanotechnology, the interest in the use of agnps as antibacterial agents had been rekindled . Small sized nanoparticles have higher antimicrobial activity than larger particles because they have large surface area to interact bacteria efficiently . In recent times, the scientists produce 30 to 40 nm size, spherical shaped agnps from fruit extract of vitis vinifera shows excellent antimicrobial activity against b. subtilis and k. planticola . 10 to 70 nm size, spherical shaped agnps synthesized from emblica officinalis fruit extract shows potential antimicrobial activity against s. aureus, b. subtilis, e. coli, and k. pneumoniae . Euphorbia hirta leaf mediated synthesis of agnps having a spherical shape with 40 - 45 nm size shows good antifungal efficacy . In our studies also 3 - 57 nm size, spherical shaped agnps produced from a. digitata pulp extract confirmed by hr microscopic studies with afm, sem, and tem proved these agnps are eco - friendly antimicrobial agents against different microorganisms . In the present study, we report an eco - friendly, non - toxic, cost - effective method for synthesis of agnps from a. digitata pulp extract as reducing agent . In this method, naturally occurring materials are acts as reducing agents such as bio - molecules such as phenols and proteins present in plant extract as a simple and alternative to complex physical or chemical synthetic procedures . 3 to 57 nm size, spherical shaped, polydispersed nanoparticles are produced from a. digitata pulp extract confirmed by afm, sem, and tem prove this plant extract as an effective reducing agent for the production of narrow range of nanoparticles by industrial scale . Further antimicrobial studies reveal that small size, spherical shaped particles have immense activity against different microbial pathogens and acts as eco - friendly antimicrobial agents.
Malakoplakia is a rare chronic inflammatory disease that occurs most commonly in the genitourinary tract, especially the urinary bladder . Most patients have associated conditions characterized by some degree of immunosuppression, as seen in solid - organ transplants, autoimmune diseases requiring steroid use, chemotherapy, chronic systemic diseases, alcohol abuse and poorly controlled diabetes.1) despite some characteristic histological features including the presence of large histiocytes with clusters of michaelis - gutmann (mg) bodies, malakoplakia of the kidney is difficult to diagnose because of its rarity and varying histological pictures.2) to our knowledge, there have been no case reports of malakoplakia of the kidney in a secondary adrenal insufficiency from korea . Here, we report an unusual case of renal malakoplakia involving the perirenal space and extending to the descending colon in a 65-year - old korean woman with secondary adrenal insufficiency and diabetes mellitus . In march 2010, a 65-year - old woman was transferred from the national police hospital (nph) to our facility, shinchon severance hospital (seoul, korea) via the emergency room (er), presenting with a decreased level of consciousness . According to her medical history, she had hypertension, diabetes mellitus for 10 years, recurrent cystitis, and degenerative arthritis . She had been taking dexamethasone (5 mg, once a day) for the degenerative arthritis for approximately five years up to the time of admission . Three weeks before the admission, she had been hospitalized at nph due to drowsy mentality for 3 or 4 hours . On the day of admission at nph, she presented with a fever (38.8) and dyspnea . The patient was given antibiotics (ceftriaxone and azithromycin) for 14 days, on suspicion of community - acquired pneumonia . Thereafter, her presenting symptoms were improved, but she complained of consistent pain in the left lower quadrant of the abdomen . Abdomen - pelvis computed tomography (ct) scan was performed, which showed the mass - like lesion in the left renal area suspecting abscess . While being treated with ciprofloxacin, she developed a decreased level of consciousness . Upon physical examination on the day of the admission to our hospital, her initial vital signs were as follows: blood pressure 134/71 mm hg, pulse rate 95 bits per minute, body temperature 36.4, and respiration rate 18 breaths per minute . Her breathing sound was clear, and her heart sound was normal without any murmurs . Laboratory studies had the following results (reference values): low sodium (na) levels of 112 mmol / l (135.0 - 145.0), potassium (k) 3.4 mmol / l (3.5 - 5.5), chloride (cl) 77.0 mmol / l (98 - 110), total co2 21.0 mmol / l (24 - 30), low serum osmolality 240.0 mosm (289.0 - 308.0), and normal urine osmolality 315.0 (50.0 - 1200). Initial urinalysis results showed 3 - 5 white blood cells / high - power field and yeast - like organisms . In the radiological examination, there was no acute ischemic lesion in the brain magnetic re sonance imaging and angiography with neck angiography (3d), and no specific finding on the chest x - ray . Sodium replacement therapy for hypotonic euvolemic hyponatremia had been started from the day of admission . At that stage, secondary adrenal insufficiency due to exogenous steroid was suspected . A low dose adrenocorticotropic hormone (1 g cosynthropin) stimulation test was conducted to diagnose the secondary adrenal insufficiency . The stimulation test showed that there was adrenal insufficiency; basal level of cortisol 8.56 g / dl, peak level during the hour - long test 13.94 g / dl (table 2). Steroid hormone (hydrocortisone sodium succinate; solucortef) was replaced . On the third day after admission, her general condition including clear consciousness was improved, with a normal level of serum sodium (136 mmol / l), and her steroid hormone dosage had been gradually tapered down and was maintained at 7.5 mg (5 mg, 2.5 mg two times a day) of prednisolone acetate . However, the patient complained of myalgia, which was accompanied with leukocytosis (white blood cell count 13,650 g / l [neutrophil 84.9%]). Radiological reading of the abdomen - pelvic ct conducted on the second day after admission showed that there was an ill - defined inflammatory lesion with multifocal abscess pockets in the left perirenal space, which was suspected of actinomycosis of the descending colon, extended to the left kidney (figure 1a). Compared to the abdomen - pelvic ct finding performed outside, the lesion seemed to have decreased slightly in size . Urinalysis was repeated and showed many white blood cells per high - power field and yeast - like organisms, which was confirmed as yeast in the urine culture . Considering the laboratory and radiologic findings, intravenous antibiotic treatment was started (ubacillin 1,500 mg a day) for suspected actinomycosis . No specific lesion was found at the colonoscopy performed to confirm the diagnosis of actinomycosis . On the 12th day after admission the histology of the lesion showed a collection of foamy histiocytes also known as von hansemann cells with von - kossa stain positive spheres (michelis - gutmann body) (figure 2). The patient presented dysuria and urgency, and antibiotic treatment was changed to oral ciprofloxacin (500 mg a day). On the 15th day after admission, she was discharged, as her presenting symptoms and laboratory findings were improved . About 4 months after discharge, the patient was readmitted presenting with general weakness for one month . The findings of the abdomen - pelvic ct conducted upon readmission (figure 1b) showed that the extent of the malakoplakia in the perirenal space had decreased since 9 week prior, as she had been taking the oral antibiotics . She has had follow - up in our outpatient clinic ever since, taking oral trimethoprim - sulfamethoxazole . Malakoplakia is an uncommon but distinctive type of chronic granulomatous inflammation . Upon microscopic examination, malakoplakia is characterized by the presence of sheets of histiocytes with granular cytoplasm (von hansemann cells) and the formation of intracellular bacterial inclusions called michaelis - gutmann bodies.3 - 7) the pathogenesis is poorly understood, but it is thought to result from acquired bactericidal defects in macrophages occurring mostly in patients with chronic debility or immunosuppression, a kidney transplant requiring steroids, or azathioprine.8,9) these observations suggest that an altered immune response may play a role in the pathogenesis of the disease . Individuals with diabetes mellitus have a greater frequency and severity of infection . From the earliest reports on studies of adrenocortical function, there has been impressive evidence that adrenocortical hormone, and more specifically cortisol, is essential for normal immunity . Otherwise, prednisone, triamcinolone, dexamethasone and similar commercially popular steroids have the dangerous side - effect of lowering resistance to infection, which is common to all known glucocorticoids.10) according to previous studies, gram - negative bacteria, particularly escherichia coli, have been isolated from involved sites of genitourinary malakoplakia in more than two - thirds of patients.11) bacteria other than yeast were not isolated in the urine culture in the present case with cystitis because the patient was on antibiotic therapy before admission to our hospital . Although we did not carry out a detailed investigation of the patient's immunity, we believe that unilateral renal malakoplakia extending to the perirenal space and the descending colon, as observed in the present case, may result from an interaction between various factors such as bacterial infection, impaired host immune response and diminished leukocyte function under the influence of a systemic illness such as secondary adrenal insufficiency and diabetes mellitus . The common presenting features of malakoplakia of the kidney are high fever, loin tenderness, and a palpable mass with a history of urinary tract infection.8) based on the reported studies, the degree of decreased kidney function varies up to acute renal failure.12,13) ultrasound and ct features are variable and only briefly mentioned in many reviews . Features include enlarged kidneys,14) focal hypoechoic renal masses that simulate abscesses15) and heterogeneous masses that calcify, show central areas of necrosis and are mistaken for tumors.16) the imaging appearance of malakoplakia is therefore non - specific, and malignant renal tumor is a common misdiagnosis . The final diagnosis of malakoplakia is usually attained only upon histopathologic examination of the excised specimen . There is no established treatment for patients with malakoplakia . However, there are treatment options for malakoplakia that include antibiotics, surgical excision, or a combination of both, depending on the site and extent of involvement . Some researchers have suggested that such antibiotics as trimethoprim - sulfamethoxazole and ciprofloxacin were beneficial because of their ability to penetrate intracellularly.8,17 - 20) we started the treatment with ciprofloxacin . During follow - up in the outpatient clinic, we changed the antibiotic treatment to trimethoprim - sulfamethoxazole due to the patient's continued complaint of cystitis symptoms . In conclusion, renal malakoplakia should be taken into account in the differential diagnosis of a patient who presents with urinary tract infection, fever, abdominal pain, and abscess - like lesion in ultrasonography or ct, especially if the patient concerned has diabetes mellitus and a long history of use of exogenous steroids . It is considered that family physicians' early diagnosis would enable prompt treatment and regular monitoring, leading to avoidance of long - term morbidity and mortality.
Ghrelin, a 28-amino - acide peptide, is a potent stimulator of growth hormone release that has been implicated in the control of food intake and energy homeostasis in human begins and rodents [15]. Ghrelin is not secreted into the gastrointestinal tract like digestive enzymes but into blood vessels to circulate throughout the body . Ghrelin causes weight gain by increasing food intake and reducing fat use [7, 8]. Ghrelin has effects on nutrient intake and growth hormone (gh) release, subsequently on physical development and growth . Tumor necrosis factor (tnf-) and interleukin-6 (il-6) are pleiotropic cytokines with numerous immunologic and metabolic actions [10, 11]. Il-6 is generally considered to be an important cytokine in the network of cytokines that regulate immune reactions and acute phase responses . The relationship between congenital heart disease (chd), malnutrition, and growth retardation is well documented . Infants with congenital heart disease are prone to malnutrition for several reasons including decreased energy intake, increased energy requirements, or both . Different types of cardiac malformations can affect nutrition and growth to varying degrees . Although nutritional and growth status were investigated in children with cyanotic and acyanotic heart disease, serum ghrelin levels have not been established . The objective of this study is to investigate and compare the functional role of ghrelin on the regulation of energy balance in children with cyanotic and acyanotic congenital heart disease and the association of ghrelin with tnf-, il-6, that were not entirely confirmed in literature by now . The study was conducted on 47 children with acyanotic chd, 21 children with cyanotic chd, and 25 healthy children . All patients' cardiac diagnoses were made on the basis of clinical and laboratory examinations . Body mass index (bmi) was calculated as the ratio of body weight (kg) and squared height (m). All blood samples were drawn at 08 - 09 am and stored 20c until the procedure . Serum ghrelin, tnf-, and il-6 levels were analyzed with elisa kits (tnf-, il-6 kit was purchased from bio - source international inc . (camarillo, calif, usa); ghrelin kit from phoenix international, inc, usa). The given data were compared between groups using one - way anova, followed by post - hoc; bonferroni test . In 47 acyanotic patients, mean age was 30.518.4 months, in 21 cyanotic patients was 28.415.6 months and in 25 control subjects was 31.115.1 months . Age and anthropometric data of the patients and the control subjects are shown in table 1 . There was no significant difference between groups (the acyanotic patients, the cyanotic patients) in terms of mean age, weight, height, bmi . Serum ghrelin levels were significantly higher than in acyanotic and cyanotic groups compared to in the control group (p<.0001) (table 3). Serum ghrelin levels in the acyanotic patients were significantly higher than in the cyanotic patients (p<.0001). Tnf- levels were significantly higher than in cyanotic and acyanotic patients with chd compared to in the control groups (p<.001, p<.0001, resp . ). Serum tnf- values were higher in the acyanotic patients compared to the cyanotic patients with chd (p<.001). Serum il-6 levels were higher than in cyanotic and acyanotic patients with chd compared to in the control groups (p<.0001, p<.001, resp . ). In both acyanotic and cyanotic groups, serum ghrelin levels were negatively correlated with bmi (r=.549, p<.05 and r=.688, p<.01, resp .) Tnf- levels were not related to bmi in the acyanotic and cyanotic patients with chd . Ghrelin levels were also correlated with tnf- in the acyanotic and cyanotic groups (r=.485, p<.05 and r=.573, p<.01, resp .) Ghrelin levels were not related to il-6 in the acyanotic and cyanotic patients with chd (r=.263, p>.05 and r=.398, p>.05, resp . ). Inadequate caloric intake, malabsorption, and increased energy requirements caused by increased metabolism may all contribute . However, inadequate caloric intake appears to be the most important cause of growth failure in chd [13, 15, 16]. Patients with acyanotic heart disease had a greater growth deficit in weight, and those with cyanotic heart disease had a greater growth deficit in stature as demonstrated by both decreased height and weight . Although growth impairment is most pronounced in infants with cyanotic chd, growth failure does not correlate well with the degree of hypoxia . In this study, the cyanotic patients had a more pronounced retardation in both height and weight than in the acyanotic patients [13, 17]. Ghrelin is accepted as a good marker of the nutritional state, mainly in situations of malnutrition, like anorexia nervosa, owing its fast recovery after weight gain . The inverse correlation between ghrelin levels and bmi we observed the mentioned correlation, both in children with cyanotic heart disease and in children with acyanotic heart disease . Although the cyanotic patients had a more pronounced retardation in both height and weight than in the acyanotic patients, we found that serum ghrelin levels significantly elevated in the acyanotic patients than in the cyanotic patients (p<.0001). Growth failure in cyanotic children has not been shown to be proportional to the severity of cyanosis, suggesting that multiple factors are involved in the pathogenesis of their growth disturbance . Alteration of endocrine mediators of growth has been implicated as a possible mechanism of growth failure in cyanotic patients . Cyanotic newborn lambs have decreased levels of serum insulin - like growth factor i without a corresponding decrease in growth hormone or hepatic growth factor receptors . Weintraub et al . Reported that while insulin - like growth factor i levels were linearly related to height and weight in patients with cyanotic lesions, no such correlation was found in their cyanotic patients . These studies suggest that chronic tissue hypoxia may have independent role in growth failure . We found that serum tnf- significantly increased in the cyanotic patients and in the acyanotic patients . Similarly, serum il-6 was increased in both groups but the change was more distinctive in the cyanotic patients . Tnf- and il-6 appear to be important cachectic process mediators, although this association is not completely established [23, 24]. Cachexia results in decreased muscle strength and function and compromised immune function [25, 26]. This syndrome is likely to occur in children who have chronic congestive heart failure, chronic shunt hypoxemia . In addition to inadequate calorie and protein intake, there is evidence that this syndrome may be caused by circulating tumor necrosis factor, which stimulates catabolism . In the present study, ghrelin correlated to positively with tnf-, in acyanotic patients and cyanotic patients with chd . The relation of ghrelin with tnf- raises the possibility of the direct effect of tnf- upon ghrelin or the impact of heart failure severity upon both ghrelin and tnf-. Nagaya et al . Have shown that plasma ghrelin level is increased in cachectic patients with congestive heart failure as a compensatory mechanism in response to anabolic - catabolic imbalance . In conclusion, serum ghrelin level is elevated in cyanotic and acyanotic patients with chd . Increased ghrelin levels additionally, the relation of ghrelin with cytokines may be explained by the possible effect of chronic congestive heart failure and chronic shunt hypoxemia.
Enteric viruses are major etiologic agents of acute gastroenteritis among infants and young children worldwide . Rotavirus, norovirus, adenovirus, and astrovirus are the recognized viral causes of pediatric gastroenteritis . The world health organization (who) data showed that each child practically has viral diarrhea irrespective of race and socioeconomic status within the first 5 years of life and this has great economic burden for the system of public health services and all society [2, 3]. Viral intestinal infections are the most common cause of acute infectious diarrhea in the pediatric group and accounted for approximately 70% of episodes of acute infectious diarrhea in children . There is paucity of information as regards viral enteropathogens of diarrhea in many developing countries including nigeria . The reason(s) attributed to this may be as a result of the poor health care system in the country where important health issues are taken for granted such as the aforementioned case . Thus, as a result of the nonperformance of viral tests for diarrheal patients, information on viral enteropathogens is lost and thus measures to implement control strategies become difficult . Against this background, this study was carried out to ascertain the prevalence of rotavirus, adenovirus, and norovirus infection in young children with diarrhea in two primary health centers in edo state, nigeria . It also aimed at assessing the coinfection rates of the viral agents, age, seasonal distribution of infection, and the association between clinical symptoms and viral diarrhea . This study was carried out in two primary health centers located in ikpoba - okha local government area of edo state, nigeria . The health centers attend to the primary health needs of the people within and around the locality . Cases attended to include malaria, diarrhea, immunization of infants and children, antenatal and postnatal cases, and other minor health issues within the scope of the health personnel . The two primary health centers are aduwawa and evbomodu primary health centers and they are neighboring communities with a fast growing population made up of indigenes with new residents from the main city alongside other inhabitants from other parts of the country . A total of two hundred and eighty - two (282) stool specimens comprising 223 diarrhea and 59 nondiarrhea stool specimens were collected from children aged between 0 and 36 months attending two primary health centers . As regards children with diarrhea, males were 121 while females were 102 . Verbal informed consent was obtained from patients or guardian of the children prior to sample collection . The samples were collected from patients at the time of clinic visit as well as other times when the child defecates . Sterile wide mouth specimen containers were used for specimen collection and they were processed within 6 hours of collection . Rotavirus, adenovirus, and norovirus were detected by the immunochromatographic technique (ict). Rotavirus and adenovirus were detected using vikia rota - adeno rapid test device manufactured by biomerieux, france . Briefly, 2 drops of liquid stool specimen was added to the specimen dilution buffer and shaken vigorously to homogenize . Two drops of the diluted sample was transferred to the sample well of the test device (cassette) and was timed for 10 minutes . Similarly, norovirus was detected using rida quick norovirus (ni403) test device manufactured by r - biopharm ag, germany . Briefly, 1 ml of sample dilution buffer (diluent) was placed in a separate labeled test tube and 100 ml of liquid stool was added to it and shaken vigorously to homogenize . This was allowed to settle for 2 minutes and 250 l of the supernatant was placed in a clean labeled test tube . Six drops of conjugate 1 was added to the test tube and was shaken vigorously to homogenize . The mixture was emptied into the sample well of the test device and incubated for 10 minutes at room temperature . Four drops of conjugate 2 was added to the reaction window of the test device and incubated for 1 minute at room temperature . 10 drops of wash buffer was added to the reaction window and was allowed to stay until the buffer was completely absorbed . Six drops of substrate was added to the reaction window and timed for 3 minutes . Statistical analysis was carried out using odd ratio and chi - square () tests . A total of 223 children with diarrhea were tested for three viral agents (rotavirus, adenovirus, and norovirus). A total of 95 (42.6%) were positive for at least one viral agent while none of the 59 children without diarrhea was positive for any viral agent . The overall result showed that rotavirus had a prevalence of 63 (28.3%) (table 1). The sex distribution of enteric viruses showed that males had 54 (44.6%) positive cases while females had 41 (40.2%), and this was not statistically significant (p = 0.60). Age group distribution of infection showed that 712 months had the highest infection rate with 48 (58.5%) and was closely followed by 06 months which had 32 (50.8%). There was a statistical significance between age group and infection (p <0.0001) (table 2). The pattern of coinfecting viruses showed rotavirus - adenovirus mixed infection as the most prevalent with 12 (5.4%) (table 3). The seasonal pattern of enteric viruses showed that rainy season had 60 (46.9%) while dry season had 35 (36.8%), and this was not statistically significant (p = 0.17). The distribution of enteric viruses according to health centers was not statistically significant (p = 0.89) (table 4). The distribution of viral agents with respect to clinical symptoms is showed in table 5 . The prevalence of rotavirus, adenovirus, and norovirus infections was investigated among children with diarrhea . The results of this study when compared to other studies carried out in nigeria and other parts of the world showed that the incidence of viral agents varied from one locality to another . The 28.3% of rotavirus in this study is lower than 55.9% in ilorin, nigeria, 35% in jos, nigeria, 33.3% in two districts in nigeria, and 39% in ghana . It is higher than 27% in zaria, nigeria, 28.1% in edo state, nigeria, 9.2% in botswana, and 16.9% in korea . The 19.3% of adenovirus in this study is lower than 22.3% in northwestern nigeria and 23.0% in tanzania . It is higher than 6.7% in nigeria, 3.0% in south africa, and 7.8% in botswana . The 3.6% of norovirus in this study is lower than 37.3% in nigeria, 16.4% in ghana, 39% in brazil, 11.6% in korea, and 15% in ghana . The differences among studies reporting viral infections in different countries might be explained by the different age group, seasonal variations, and viral detection methods used . There was a statistical significance between infection and age group (p <0.0001). Age group of 712 months had the highest prevalence of viral diarrhea with 48 (58.8%). This is consistent with the report of moyo et al ., who also found 712 months as the group with the highest infection . The reason for this may be due to the fact that it is the period of activities for many children . Crawling and walking stages are at this period and in the process, children could pick harmful materials into their mouth especially in unhygienic conditions . It was observed in this study that, within the first year of life, viral infection rate was 84.2% and in the second year, it was 97.9% . This is in accordance with other studies where infection was most prevalent in children within the first 2 years of life [11, 16, 19, 20]. The relatively low prevalence of viruses among older children could be partly due to immunity acquired through previous exposures . In this study, coinfection rate was 7.6%, and rotavirus - adenovirus coinfection was the highest with 12 (5.4%). This, however, did not agree with the reports of chung et al . And koh et al ., who had rotavirus - norovirus mixed infection as the most prevalent . The seasonal pattern of viral infection was not statistically significant (p = 0.17), though infection was more during the rainy season (46.9%) than in the dry season (36.8%). Reports from nigeria have showed that viral diarrhea occurs throughout the year but with variations with respect to seasons [8, 13, 16]. Viral diarrhea with respect to the two primary health centers did not show statistical significance (p = 0.89), as infection rates for evbomodu and aduwawa health centers were 43.0% and 42.1%, respectively . The most commonly associated clinical symptom observed in this study with rotavirus, adenovirus, and norovirus positive cases was vomiting with 55.6%, 55.8%, and 75.0%, respectively . This finding is similar to other studies [2325] where vomiting was the most commonly associated symptom with viral diarrhea . Rotavirus positive patients with respect to clinical symptoms such as fever, dehydration, and abdominal pain were low in this study compared to other studies [23, 26, 27] which reported high percentage of clinical symptoms . Similarly, for adenovirus positive patients, fever, dehydration, and abdominal pain were low compared to other studies with moderate symptoms [13, 24, 28, 29]. However, as regards norovirus positive patients, fever, dehydration, and abdominal pain were also low, but other studies with norovirus showed moderate to high clinical symptoms [25, 30, 31]. Thus, the differences in clinical symptoms with viral diarrhea as seen from the different studies may be attributed to seasonal variations, geographical location, nutritional status of the patients, and type of viral pathogens causing the infection . Coinfection of viral agents was found to be 7.6% and the most common clinical symptom was vomiting . This is worrisome considering the burden of these viruses on the young children in this locality . The fact that viral diagnostic tests are not routinely done or rarely done in any of the hospitals in this locality means that such vital information on viral diarrhea is missed out due to the poor attention given to health care in the country . Thus, there is need to test stool specimens of clinically confirmed diarrheal patients for enteric viruses as this will go a long way in reducing the wasteful use of antibiotics which are used as blind treatment for persistent diarrhea that may be of viral origin . Finally, the primary health care centers should be provided with all the necessary diagnostic test materials to address cases of viral diarrhea.
Alpha ()2-adrenoceptor agonists have been used as adjuvant to anesthetic agents in peri - operative period for its several beneficial actions . These drugs improve hemodynamic stability during endotracheal intubation and surgical stress by its central sympatholytic action, and thus reduce anesthetic and opioids requirements . Dexmedetomidine, the pharmacologically active d - isomer of medetomidine, is highly selective and specific 2-adrenoceptor agonist . The analgesic effect of dexmedetomidine is qualitatively different as compared to opioids and can be used as an alternative to opioids in general anesthesia . The anesthetic sparing effect of dexmedetomidine demonstrated in earlier studies is confounded by the use of opioids . Opioids, when used as analgesic in general anesthesia, are known to decrease mac value of sevoflurane . Inhalation agents were titrated based on either hemodynamic criteria and/or bispectral index values in studies done earlier, and none of them demonstrated the effect of dexmedetomidine alone on requirement of sevoflurane with the use of entropy as a measure of depth of anesthesia . The primary objective of our study was to evaluate the effect of continuous infusion of dexmedetomidine alone, without use of opioids, on sevoflurane requirement during general anesthesia with continuous monitoring of depth of anesthesia by entropy analysis . The study protocol was approved by ethical committee of the institute, and written informed consent was obtained from all patients included in the study . The study population comprised of 60 patients, aged 18 - 55 years, with american society of anesthesiologists (asa) status of 1 and 2, undergoing major elective surgical procedures . We excluded patients older than 55 years, those with a history of psychiatric / neurological illness, cardiovascular disease, hypertensive patients, morbid obese patients, pregnant and nursing women, with known allergic reaction to any of the study medication, recent use of sedatives or analgesics, and with significant laboratory abnormalities . The consenting patients were randomly allocated into one of the two anesthetic groups based on the simple randomization table generated: group a (control group): sevoflurane - fentanyl and group b (test group): sevoflurane - dexmedetomidine . After arrival in the operating room, glycopyrrolate 0.004 mg / kg and ondansetron 0.15 mg / kg intravenous (iv) were given as pre - medication in both the groups . The patients in group b received dexmedetomidine(2 ml diluted in 48 ml of saline) iv in a dose of 1 mcg / kg over10 minutes through infusion pump prior to induction . Monitoring consisted of continuous electrokardiogram (ekg), non - invasive blood pressure (nibp), pulse oximetry (spo2), co2 expired fraction (etco2), sevoflurane inspired fraction (fisevo), sevoflurane expired fraction (etsevo), and electroencephalograph (eeg) analysis by entropy (response and state entropy). After the start of drug infusion, heart rate (hr), blood pressure (bp), respiratory rate (rr), spo2, and entropy were measured at 5 and 10 minute . Patients in group a received fentanyl 2 g / kg intravenous slowly, 2 minutes prior to induction . After pre - oxygenation for 10 minutes, general anesthesia was induced with thiopentone sodium 5 mg / kg iv slowly ., dexmedetomidine infusion was continued between 0.2 and 0.8 mcg / kg / h depending on hr, bp, and entropy value changes . Anesthesia was maintained with sevoflurane to a maximum of 2.5% end tidal to maintain heart rate and blood pressure within 20% of baseline value and entropy value between 40 and 60 . The anesthesiologist was permitted to treat hemodynamic events, defined as heart rate and blood pressure more than 20% of baseline, in spite of increasing sevoflurane concentration to 2.5% and dexmedetomidine infusion to 0.8 mg / kg with supplemental analgesia in the form of fentanyl 1 mcg / kg . If rise in heart rate and blood pressure persisted further after supplementation with fentanyl, anesthesiologist was permitted to administer incremental doses of metoprolol 2 - 5 mg . Ephedrine 5 mg iv was given for fall in heart rate and blood pressure more than 20% of baseline . In the group b, dexmedetomidine was stopped approximately 15 - 20 minutes before completion of surgery, diclofenac sodium aqueous 1 mg / kg was given iv in both groups at the time of skin closure . Reversal of neuromuscular blockade was achieved with neostigmine 0.05 mg / kg and glycopyrrolate 0.008 mg / kg iv slowly . Tracheal extubation was done when respiration was satisfactory and adequate muscle tone was achieved . Hr and bp were recorded before induction, at the time of induction, intubation, and then at 5, 10, 15, 30, 45, and 60 minute after intubation and at extubation.depth of anesthesia was evaluated by entropy (state entropy, response entropy) analysis (datex - ohmeda s/5 avance workstation, ge healthcare, helsinki, finland) at 5, 10, 15, 30, 45, and 60 minute after intubation and at extubation.end tidal concentration of sevoflurane was assessed with anesthesia gas assessment module e - caiovx at same interval as entropy analysis . Hr and bp were recorded before induction, at the time of induction, intubation, and then at 5, 10, 15, 30, 45, and 60 minute after intubation and at extubation . Depth of anesthesia was evaluated by entropy (state entropy, response entropy) analysis (datex - ohmeda s/5 avance workstation, ge healthcare, helsinki, finland) at 5, 10, 15, 30, 45, and 60 minute after intubation and at extubation . End tidal concentration of sevoflurane was assessed with anesthesia gas assessment module e - caiovx at same interval as entropy analysis . Statistical analysis was conducted with epi info software (version 3.5.3, 2011, centers for disease control and prevention, atlanta, ga, usa) for windows statistical package using unpaired t - test for continuous variables with normal distribution . The non - parametric kruskal - wallis test was used for variables not normally distributed . For categorical variables, chi - square test was used . The two groups were similar regarding age, sex, weight, and asa physical status [table 1]. Pre - induction heart rate, systolic and diastolic blood pressures were similar between two groups (p> 0.05). The depth of anesthesia as assessed by response entropy (re) and state entropy (se) was comparable between two groups at all time points during maintenance period (p> 0.05). The re and se was maintained between 40 and 60 during the period of observation . The fisevo concentration was significantly less in group b as compared to the group a at all time points (p <0.05) [figure 1]. The average etsevo concentration during anesthetic maintenance was 1.35% and 1.72% in the group b and a, respectively . A significant decrease of 13% to 33% of etsevo concentration was seen with group b from 5 min to 60 minutes post - intubation (p <0.05) during surgery [figure 2]. Fraction of inspired sevoflurane at different time interval (pi - post - intubation, fi - fraction of inspired) end tidal sevoflurane at different time intervals (pi - post - intubation) during anesthesia maintenance, group b showed a statistical significant decrease in heart rate, systolic and diastolic blood pressure from baseline at all time points as compared to the group a (p <0.05). An average 6.5% fall in heart rate from baseline in group b as compared to 3.7% rise in the group a, 8% fall in systolic blood pressure from baseline as compared to 3.6% rise in the group a, and 8.16% fall in diastolic blood pressure from baseline as compared to 3.3% rise in the group a was observed [figures 3 and 4]. Changes in sbp and dbp at various time points post - intubation (pi - post - intubation, sbp - systolic blood pressure, dbp - diastolic blood pressure) changes in hr at various time points post - intubation (pi - post intubation, hr - heart rate) in our study, none of the patients in either group required supplemental analgesia or anti - hypertensive drugs intra - operatively . Bradycardia was observed in 2 patients in group b within 10 mins post - extubation, which promptly responded to atropine 0.6 mg iv . The major observation in our study was that dexmedetomidine infusion as an adjuvant in general anesthesia causes decreased requirement of sevoflurane without compromising adequate depth of anesthesia, thus it has anesthetic - sparing property . A study done on patients undergoing hysterectomy showed a 30% reduction of maintenance concentration of isoflurane . Similarly, a reduction in 35% to 50% in isoflurane concentration with low or high dose of dexmedetomidine was found in a study on healthy human volunteers . This larger reduction in isoflurane requirement seen as compared to our study might be due to difference in study population (healthy human volunteers vs. patients posted for elective surgery) and the type of stimulus (tetanus nerve stimulus vs. surgical stimulus). The results of our study results are consistent with that of the study done by fragen, et al . In elderly patients, which showed a 17% reduction . Dexmedetomidine by its sympatholytic action decreases heart rate and blood pressure, thus assessing the depth of anesthesia by hemodynamic parameters would be unreliable in evaluating its effect on requirement of inhalational agent . Several electroencephalogram - dependent indices such as bispectral index and entropy have been used to measure the depth of anesthesia . Entropy is a useful monitor for measuring the electroencephalographic effects of increasing and decreasing sevoflurane concentration and assessing the depth of anesthesia . Analogous to the bispectral index, entropy displays a high degree of specificity and sensitivity in assessing consciousness during anesthesia . Using bispectral index to assess the depth of anesthesia, magalhes et al . Showed decreased requirement of sevoflurane with continuous infusion of dexmedetomidine during general anesthesia . In our study, we used entropy to measure the depth of anesthesia, thereby eliminating the bias of evaluation by hemodynamic parameters as in earlier studies . Use of dexmedetomidine produces intra - operative and post - operative opioids - sparing effect . Dexmedetomidine by its sympatholytic action attenuates symapthoadrenal response to tracheal intubation . In patients undergoing laparoscopic tubal ligation, a 33% decrease in morphine use post - operatively was observed when dexmedetomidine was used at a dose of 0.4 mcg / kg . Dexmedetomidine when administered as infusion at a dose of 0.5 mcg / kg / h has specific analgesic effect and provides visceral pain relief . In morbidly obese, dexmedetomidine produces a greater decrease in sympathovagal balance intra - operatively than fentanyl along with better post - operative analgesia . Dexmedetomidine, when used as sole substitute for remifentanil in ambulatory gynecologic laparoscopic surgery, provides better peri - operative hemodynamic stability and post - operative analgesia . Dexmedetomidine provides similar intra - operative hemodynamic response and better post - operative analgesia compared to remifentanil in patients undergoing supratentorial craniotomy . Use of opioids along with dexmedetomidine would confound its effect on requirement of inhalation agent . Hence, in our study, fentanyl was not administered in dexmedetomidine group b, and we found a 21.5% decrease in the etsevo, in contrast to 33.12% decrease in study done by magalhes e et al . Where fentanyl was used . One limitation of our study was that we included both laparoscopic as well as open surgeries . To conclude, the continuous infusion of dexmedetomidine, as adjuvant in general anesthesia, significantly decreases the requirement of sevoflurane for maintaining adequate depth of anesthesia . Studies measuring plasma concentration of dexmedetomidine should be undertaken to establish the precise correlation between its dose and inhalational agent's requirements.
Rkshv.219 virus is latently infected in islk.rkshv219 cell line, derived from islk cell line, as previously described by . Rkshv.219 cells were seeded at 5 10 cells into 150-mm dish (one day prior to induction). For induction of kshv reactivation, the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2 g / ml doxycycline . Kshv reactivation was induced at 0, 6, 12, 24, and 48 h. the 0 h sample was treated identically to other timepoints, however doxycycline was not added . Two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay . Following doxycycline induction of kshv reactivation, islk.rkshv219 cells were harvested at 0, 6, 12, 24, and 48 h at 2.5 10 cells . Media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10 min at room temperature, to cross - link the chromatin structure . 125 mm glycine was then added to stop the formaldehyde cross - linking reaction . Nucleus isolation buffer, 0.3 m sucrose, 2 mm mgoac2, 1 mm cacl2, 1% nonidet p-40, 10 mm hepes, and ph 7.8 were then added to islk.rkshv219 cross - linked cells . The cells were scraped and placed within a 50 ml conical tube, and the nuclei were isolated by centrifugation at 1000 g for 5 min at 4 c . Micrococcal nuclease, mnase, is a internucleosomal cleavage reagent, which does not cleave the dna protected by a nucleosome . Islk.rkshv219 nuclei were digested with a titration of mnase: 4 u / ml, 2 u / ml, 1 u / ml, and 0.5 u / ml (worthington biochemical corp .) In mnase cleavage buffer (5 mm kcl, 4 mm mgcl2, 1 mm cacl2, 12.5% glycerol, and 50 mm tris cl (ph 7.4)). A titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented . The nuclei, following mnase digestion, were treated with 0.2 g/l proteinase k and 1% sodium dodecyl sulfate . Mnase digested dna for all samples was resolved on a 2% agarose gel, and mononucleosomally protected dna, ~ 150 bp, was isolated by gel excision . Following gel excision, the mononucleosomally protected dna was purified by electroelution, and for each sample's mnase titration the mononucleosomally protected dna was combined . The mononucleosomally protected dna was further purified with phenol chloroform extraction and precipitated with alcohol . Islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55 c overnight for bare genomic dna isolation . We utilized a high - resolution dna microarray, which we customized to contain 1 kb upstream and downstream of the transcription start sites of 462 immunity - related genes . Nimblegen's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array . Sequences were repeat - masked around the tsss, and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray . Oligonucleotide probes, sized at 60 bp, were tiled on each subarray with a mean spacing of 47 bp overlap . Approximately 180 probes of spacing of 12.5 bp were printed on the microarray for each locus . The samples were fluorescently labeled: nucleosomally protected dna with cy3 and bare genomic dna (reference) with cy5 . Samples were hybridized to the dna microarray according to nimblegen's instructions (http://www.nimblegen.com/technology/index.html). The resulting .tiff files were used for microarray data feature extraction, primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow . Specifically, loess spatial correction was applied to the raw data; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal . The ratio between nucleosomal (cy3) and bare genomic cy5 signals was then calculated and stored as a general feature format, .gff subsequent analysis was performed using a software developed in our laboratory dennislab.r, which runs in the r environment for statistical computing . Dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome . Finally, we analyzed the nucleosome distribution changes at the 24 hour timepoint on a per chromosome basis, by calculating the t - statistic and p - value (assuming unequal variance) for the pairwise comparison of the range of values between all chromosomes . In our previous work we demonstrated that nucleosome redistributions are widespread, transient, and dna - directed, . We next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner . We first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint (fig . These results comport with our previous observation that the greatest positioning occurs at the 24 hour timepoint, however do not indicate chromosome specificity with respect to increased positioning . We next wanted to investigate if the nucleosome redistribution changes at the 24 hour timepoint reflected increased or decreased nucleosomal occupancy . We calculated the median nucleosome occupancy values on each chromosome for all of the loci measured . We discovered that nucleosome occupancy values change in a chromosome specific manner (fig . Chromosomes 18, 19, and 22 show the greatest median nucleosome occupancy values at the 24 hour timepoint . Conversely, chromosomes 1, 2, 9, 10, 12, 15, and 17 show the lower median nucleosome occupancy values at the 24 hour timepoint . We were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24 hour timepoint . Most of the ranges of nucleosome occupancy values are not statistically significantly different from one another (fig . 1c, yellow highlights). These two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . At the other end of the scale, chromosomes these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint, nucleosomal occupancy changes in a chromosome specific manner . Rkshv.219 virus is latently infected in islk.rkshv219 cell line, derived from islk cell line, as previously described by . Rkshv.219 cells were seeded at 5 10 cells into 150-mm dish (one day prior to induction). For induction of kshv reactivation, the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2 g / ml doxycycline . Kshv reactivation was induced at 0, 6, 12, 24, and 48 h. the 0 h sample was treated identically to other timepoints, however doxycycline was not added . Two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay . Following doxycycline induction of kshv reactivation, islk.rkshv219 cells were harvested at 0, 6, 12, 24, and 48 h at 2.5 10 cells . Media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10 min at room temperature, to cross - link the chromatin structure . 125 mm glycine was then added to stop the formaldehyde cross - linking reaction . Nucleus isolation buffer, 0.3 m sucrose, 2 mm mgoac2, 1 mm cacl2, 1% nonidet p-40, 10 mm hepes, and ph 7.8 were then added to islk.rkshv219 cross - linked cells . The cells were scraped and placed within a 50 ml conical tube, and the nuclei were isolated by centrifugation at 1000 g for 5 min at 4 c . Micrococcal nuclease, mnase, is a internucleosomal cleavage reagent, which does not cleave the dna protected by a nucleosome . Islk.rkshv219 nuclei were digested with a titration of mnase: 4 u / ml, 2 u / ml, 1 u / ml, and 0.5 u / ml (worthington biochemical corp .) In mnase cleavage buffer (5 mm kcl, 4 mm mgcl2, 1 mm cacl2, 12.5% glycerol, and 50 mm tris cl (ph 7.4)). A titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented . The nuclei, following mnase digestion, were treated with 0.2 g/l proteinase k and 1% sodium dodecyl sulfate . Mnase digested dna for all samples was resolved on a 2% agarose gel, and mononucleosomally protected dna, ~ 150 bp, was isolated by gel excision . Following gel excision, the mononucleosomally protected dna was purified by electroelution, and for each sample's mnase titration the mononucleosomally protected dna was combined . The mononucleosomally protected dna was further purified with phenol chloroform extraction and precipitated with alcohol . Islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55 c overnight for bare genomic dna isolation . We utilized a high - resolution dna microarray, which we customized to contain 1 kb upstream and downstream of the transcription start sites of 462 immunity - related genes . Nimblegen's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array . Sequences were repeat - masked around the tsss, and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray . Oligonucleotide probes, sized at 60 bp, were tiled on each subarray with a mean spacing of 47 bp overlap . Approximately 180 probes of spacing of 12.5 bp were printed on the microarray for each locus . The samples were fluorescently labeled: nucleosomally protected dna with cy3 and bare genomic dna (reference) with cy5 . Samples were hybridized to the dna microarray according to nimblegen's instructions (http://www.nimblegen.com/technology/index.html). The resulting .tiff files were used for microarray data feature extraction, primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow . Specifically, loess spatial correction was applied to the raw data; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal . The ratio between nucleosomal (cy3) and bare genomic cy5 signals was then calculated and stored as a general feature format, .gff subsequent analysis was performed using a software developed in our laboratory dennislab.r, which runs in the r environment for statistical computing . Dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome . Finally, we analyzed the nucleosome distribution changes at the 24 hour timepoint on a per chromosome basis, by calculating the t - statistic and p - value (assuming unequal variance) for the pairwise comparison of the range of values between all chromosomes . In our previous work we demonstrated that nucleosome redistributions are widespread, transient, and dna - directed, . We next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner . We first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint (fig . These results comport with our previous observation that the greatest positioning occurs at the 24 hour timepoint, however do not indicate chromosome specificity with respect to increased positioning . We next wanted to investigate if the nucleosome redistribution changes at the 24 hour timepoint reflected increased or decreased nucleosomal occupancy . We calculated the median nucleosome occupancy values on each chromosome for all of the loci measured . We discovered that nucleosome occupancy values change in a chromosome specific manner (fig . Chromosomes 18, 19, and 22 show the greatest median nucleosome occupancy values at the 24 hour timepoint . Conversely, chromosomes 1, 2, 9, 10, 12, 15, and 17 show the lower median nucleosome occupancy values at the 24 hour timepoint . We were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24 hour timepoint . Most of the ranges of nucleosome occupancy values are not statistically significantly different from one another (fig . 1c, yellow highlights). These two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . At the other end of the scale, chromosomes 1 and 12 are statistically indistinguishable, yet different from all other chromosomes . These results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint, nucleosomal occupancy changes in a chromosome specific manner.
Primary pleuropulmonary synovial sarcoma (ppss) is a rare soft tissue malignancy representing only 0.1% - 0.5% of all primary lung malignancies . The frequency of cardiac metastasis from soft tissue sarcomas ranges from 3.5% to 35% with only two cases of metastatic synovial sarcoma of the heart reported: one involving the right ventricle and the other the left ventricle . Both cases had a primary tumor in the extremities rather than in the lungs without focusing on the imaging findings of these rare lesions (2 - 4). We report a case of ppss that eventually metastasized to the heart in a 17-year - old female . A 17-year - old female presented with dull pain in the left upper chest alternating with brief periods of chest discomfort lasting a few months . She had no serious medical or surgical history and grew up in normal residential and social environments . All laboratory data including blood tests, standard biochemical tests, and urinalysis were within normal limits . Chest radiographs (figure 1a and b) revealed a well - defined round mass in the left upper hemithorax . The mass was at an obtuse angle to the chest wall and was approximately 8 cm long . Chest ct (figure 1c - e) confirmed a well - defined oval - shaped mass with heterogeneous enhancement in the left upper hemithorax abutting the pleura . In the pre - contrast image, the lesion showed homogeneous soft tissue attenuation and a lack of calcification, but a small amount of high - attenuation fluid was present in the left pleural space, suggestive of hemothorax . After contrast enhancement, the mass showed heterogeneous enhancement, with attenuation similar to that of the back muscles, and contained subtle low densities suggestive of necrosis or hemorrhage . The mediastinal and hilar lymph nodes were not enlarged . In the scanned portion of the abdomen f-18 fluorodeoxyglucose positron emission tomography - ct (18f fdg pet / ct) revealed increased fdg uptake in the mass, with a maximum standardized uptake value of 31.7 (figure 1f). From these imaging findings, the diagnoses of a localized fibrous tumor of the pleura, malignant mesothelioma, metastatic pleural malignancy, and rare primary pulmonary sarcoma (e.g., pleuropulmonary synovial sarcoma, fibrosarcoma, leimyosarcoma, sarcomatoid carcinoma, malignant nerve sheath tumor, hemangiopericytoma, and malignant fibrous histiocytoma) were considered . These diagnoses were based on the tumor s appearance as a sharply marginated, heterogeneously enhanced mass with low attenuation foci and no involvement of the bone or calcifications . Ct - guided core needle biopsy of the mass yielded two samples of yellowish gelatinous material . Microscopic examination showed relatively uniform spindle - shaped cells with occasional mitoses arranged in tight fascicles . The tumor cells were immunoreactive to vimentin and cd99 and were focally positive for epithelial membrane antigen (ema), suggesting a monophasic synovial sarcoma . Medical and surgical oncologists agreed to treat the tumor with surgical resection and to initiate a combination chemotherapy regimen consisting of five cycles of vincristine, doxorubicin, and cyclophosphamide, alternating with four cycles of ifosfamide and etoposide . A thoracotomy was performed, and the mass was found to arise from the visceral pleura and to adhere to the left first to third intercostal spaces, with invasion into the left first intercostal muscle with adjacent hemothorax . The surgical specimen consisted of an 8.0 6.5 5.5 cm well - circumscribed but unencapsulated tumor . The tumor was whitish - yellow, soft, and fleshy with cystic degenerative changes and hemorrhage . Histologically, the tumor was composed of densely packed, cellular sheets of spindle cells, and some of the tumor cells were arranged in intersecting fascicles in a herringbone pattern, suggesting fibrosarcoma - like changes (figure 1 g - h). The tumor cells were relatively uniform with ovoid nuclei, scant cytoplasm, and occasional mitotic figures . Immunohistochemical staining revealed immunoreactivity of the tumor cells to cytokeratin, ema, and cd99 but not to cd34 or desmin (figure 1i - j). A final diagnosis of a monophasic synovial sarcoma from the visceral pleura was made based on the histological and immunohistochemical findings . She was discharged after surgical resection without complications, after which she underwent adjuvant chemotherapy and radiotherapy . The patient remained stable for 28 months until follow - up contrast - enhanced chest ct revealed a 1.6 cm low attenuation nodule with poor enhancement in the left pericardial area and a small amount of left pericardial and pleural fluid (figure 2a). Subsequent 18f fdg pet / ct showed slightly increased fdg uptake in the developing nodule with suspected metastasis . Five months after discovery, follow up contrast - enhanced chest ct demonstrated a well - circumscribed polycyclic marginated mass with heterogeneous enhancement in the left pericardial region that had enlarged from 1.6 cm to 5.1 cm (figure 2b). Although mri is the most accurate technique for demonstrating cardiac mass, oncologists found the following mri might be ineffective for choosing further treatment for the pericardial mass due to high cost . They had no choice but to do surgery for the mass whether to perform mri or not . Therefore, they agreed to operate surgical resection to treat pericardial mass without obtaining cardiac mri . The tumor specimen was a 6.0 5.0 2.0 cm well - circumscribed, whitish - yellowish, fleshy to rubbery mass with zones of necrosis and hemorrhage . The tumor was lateral to the left ventricular wall with invasion into the pericardium, myoendocardium, and endocardium of the left ventricle . Microscopically, the tumor contained tight clusters and fascicles of spindle cells with scant cytoplasm, identical to the ppss, confirming a metastatic synovial sarcoma with left pericardial, myoendocardial, and endocardial involvement . After metastatectomy, the patient received adjuvant chemotherapy and radiotherapy . The patient s postoperative period was complicated by recurrent pneumonia . Ppss is a rare mesenchymal spindle cell malignancy representing a type of pulmonary sarcoma that comprises 0.1% - 0.5% of all primary lung malignancies . Typically, it arises from the chest wall, lung parenchyma, pleura, or mediastinum, but rarely at the level of the bronchial tree, and during the fifth decade of the patient s life (1). The 5-year survival rate varies from 36% to 76% (6). A small but increasing number of cases of ppss have been reported over the past decades (5), but very few of these cases have involved cardiac metastasis . Cardiac metastasis from soft tissue sarcomas varies from 3.5% to 35%, with only two cases of metastatic synovial sarcoma of the heart reported: one involving the right ventricle and the other the left ventricle . Both of those cases had a primary tumor in the extremities but not in the lung without focusing on the imaging findings of these rare lesions (2 - 4). Additionally, a literature review of soft tissue sarcomas included only 105 cases of cardiac metastasis . In that review, the predominant histological type was leiomyosarcoma (22%) followed by rhabdomyosarcoma (19%), and liposarcoma (9%); 27% of the cases were unspecified . That report suggested that the clinical manifestation of cardiac metastasis can be subtle, as one - third presented with congestive cardiac failure, which led to other complications such as embolism, tumor thrombus, cardiac tamponade, and arrhythmia (2). In our case, the patient was initially asymptomatic except for dull chest pains with only pericardial effusion on follow - up chest ct . Symptoms frequently associated with these tumors are chest pain, dyspnea, and cough; up to 40% of patients are asymptomatic (7). The radiological features of ppss are summarized as follows: chest radiographs typically demonstrate a well - defined pleural - based or parenchymal mass - like lesion occasionally associated with ipsilateral pleural effusion . Generally, the contralateral lung is not involved . Ct and magnetic resonance imaging (mri) are relatively useful tools for evaluating ppss, although the findings are nonspecific . The most common ct finding of ppss is a well - defined, heterogeneously enhancing mass often with combined ipsilateral pleural effusion . The inner low - density area often contains necrosis, hemorrhage, or fibrous components . Associated lymphadenopathy as well as calcifications, cortical bone destruction, or tumor infiltration of the chest wall and musculature are infrequent . Ppss often exhibits heterogeneous high signal intensity on t2-weighted images and iso- or low signal intensity on t1-weighted images . After administering gadolinium differential diagnoses for ppss include metastasis from the extrapulmonary synovial sarcoma, a primary tumor such as a pleural fibroma or mesothelioma, or other primary sarcomatous or sarcomatoid neoplasms . The typical clinical and radiologic features can help narrow down the differential diagnosis of ppss . A history of asbestos exposure and the presence of pleural plaques are indicative of malignant mesothelioma . Metastatic disease rarely manifests as a large solitary pulmonary lesion, although many other rare primary sarcomatous or sarcomatoid neoplasms cannot be distinguished reliably from ppss based on clinical and radiologic features alone (5). Monophasic synovial sarcomas are much more common, representing 94% of ppss, and are composed of uniform spindle cells . The surrounding plump spindle cells have hyperchromatic nuclei with scant cytoplasm resembling the tumor cells of monophasic synovial sarcoma . Immunohistochemically, most synovial sarcomas show focal expression of cytokeratin and ema . Furthermore, 30% are protein s-100 positive, 60% - 70% cd99 positive, and 75% - 100% bcl-2 positive (8). The prognosis of ppss remains uncertain but is usually poor . The 5-year survival rate varies from 36% to 76% depending on the patient s age, tumor size, and tumor resectability (6). There is no recommended treatment for ppss, but the mainstay of treatment for all soft tissue sarcomas is surgical resection . Aggressive surgery can reduce the tumor size before or after chemotherapy, and complete surgical excision seems to improve the survival of patients with ppss . Combination chemotherapy consisting of doxorubicin and ifosfamide has been associated with higher rates of survival in patients with synovial sarcoma (6). In summary, ppss is a very rare, aggressive malignancy that must be considered in the differential diagnosis of lung and pleural malignancies . The imaging findings of ppss are nonspecific, and ppss is difficult to confirm based on imaging; detailed immunohistochemical staining is required for definitive diagnosis . Ppss with cardiac metastasis is also extremely rare, and only two such cases have been reported . However, cardiac metastasis of ppss can lead to immediate or delayed death; therefore, early detection and appropriate management are very important to extend survival.
The work is self - sponsored by the first author who conceptualized and designed and wrote the manuscript . The second author assisted in the surgery provided some literature review and made necessary correction which lead to the final manuscript . The ethical committee of first choice specialist hospital where the authors carried out the procedure approved the work . He understood that part of his images will be published and also understood that due to the rarity of the case, that it is worthwhile publishing it for education and for health care delivery . The second author assisted during the surgery, provided immediate care, provided some literature review and made necessary correction which lead to the final manuscript.
A number of skin disorders have been described in patients with acquired immunodeficiency syndrome (aids). Among them, secondary infections are common, but their incidence has decreased considerably following the introduction of combined therapies targeted to the human immunodeficiency virus (hiv). Some clinical presentations may be puzzling, particularly in secondary syphilis exhibiting lesions showing a marked tendency to polymorphism . A 29-year - old woman with a three - year history of aids presented with polymorphic papules on the face and abdomen . The skin lesions were asymptomatic but the patient complained of discrete malaise, stiff neck, myalgia headache, and mild fever . A biopsy specimen was taken from a papule on the abdomen . A series of 5-m thick sections were cut from the formalin - fixed paraffin - embedded biopsy . An immunohistochemical assessment was performed using a rabbit polyclonal antibody directed to treponema pallidum (1:200 biocare medical, walnut creek, ca, usa). The envision (dakopatts, glostrup, denmark) polymer - based revelation system and fast red (dakopatts) staining were used . Negative immunohistochemical controls were performed by omitting or substituting the primary and the secondary antibodies in the laboratory procedure . The dermoepidermal junction contained a band - like infiltrate composed mostly of lymphocytes, histiocytes, and plasma cells . A deeper cell infiltrate of similar composition extended along the microvasculature, hair follicles, and sweat glands . The typical spiral, corkscrew, and threadlike spirochetes were highlighted by the red chromogen, and the contrast with the clear background was striking (figure 1a, b, c). Their presence inside the lichenoid infiltrate was associated with a dense superficial and deep perivascular cuff of spirochetes . The latter slender spirochetes were clustered in the dermal stroma (figure 1a) and in rims confined to the perivascular areas (figure 1b, c). In addition, some t. pallidum were evident in the cytoplasm of cells, particularly endothelial cells (figure 1c). Dermal homing of t. pallidum on immunohistochemistry: (a) multiple interstitial clumps of spirochetes (200); (b) vascular trapping of spirochetes (400); (c) prominent accumulation of spirochetes in the microvasculature wall (400). Early syphilis . Dermal homing of t. pallidum on immunohistochemistry: (a) multiple interstitial clumps of spirochetes (200); (b) vascular trapping of spirochetes (400); (c) prominent accumulation of spirochetes in the microvasculature wall (400). In the present case, skin immunohistochemistry shed some light on the diagnosis of syphilis in an aids patient and showed the dermal homing and the microvascular tropism of t. pallidum . It is acknowledged that during a five - year period after inoculation, t. pallidum spreads to every organ . A long time later, it commonly re - emerges as a chronic and deadly illness . At any stage in its evolution, syphilis may mimic a number of other unrelated diseases . During the past decade, a sizable proportion of the population with syphilis corresponded to gay men coinfected with hiv . When the clinical diagnosis of syphilis is not established, a skin biopsy sometimes is submitted to the dermatopathologist without any relevant information . At the conventional histological examination, the diagnostic clues for syphilis are not always obvious because the disease presentation depends on both the host immunological response to the infection and the diverse angioinvasive propensity of the t. pallidum strains indeed, the histochemical silver stain may be difficult to interpret owing to heavy background staining . Immunohistochemistry using an antibody directed to t. pallidum was reported to improve the histological diagnostic accuracy of syphilis . The present finding was assumed to illustrate the migration of t. pallidum toward the microvasculature during early syphilis . In summary, t. pallidum were abundant and heavily clustered in some specific portions of the skin . The peculiar homing of t. pallidum in the skin appears quite specific as it has not been reported for any other infectious microorganism.
Hadrontherapy is an innovative form of radiotherapy, based on high - technology equipment using proton or carbon ions beams to destroy tumours [1, 2]. This treatment method enables significantly higher ballistic precision to be achieved, compared to photons (x - rays) with, as expected therapeutic benefit, an improvement of quality of life and chances of recovery . Carbon ions are also specifically characterised by superior biological efficacy (relative biological effectiveness from 1.5 to 3), overcoming the radiation resistance of certain cancers to photons and even protons . Indeed, carbon ion beams when compared to x - rays represent a distinct advantage for the treatment of highly radiation - resistant tumours . An initial study assessing recruitment potential for proton therapy was conducted in 1998 in italy, showing an incidence of 10 825 cases / year . One year later, a second study was carried out in the context of the medaustron project for the construction of a carbon ions therapy centre in austria . Considering patients living in austria and neighbouring countries, the patient recruitment potential (proton and carbon) was estimated with an incidence of 13 145 cases / year . In france, in 2002, a third study was conducted in the context of the etoile medical project . This study revealed an annual incidence of approximately 5320 cases / year for carbon ions . Concomitantly, a fourth study was conducted, again in the context of the medaustron project, estimating the incidence of cases eligible for hadrontherapy in austria at 2044 cases / year . Finally, again in 2002, in the context of the project for the construction of the cnao centre in italy, an epidemiological study was conducted, estimating the incidence of cases eligible for carbon therapy at 3694 cases / year and proton therapy at 1885 cases / year . . Since 2004, no other epidemiological studies have been published for hadrontherapy . In 2011, however, the japanese team from the national institute for radiological sciences (nirs), pioneer in carbon therapy, published the results of its 1994 to 2011 clinical activity . Patients were included in clinical trials and the number of patients receiving carbon therapy was 6157 . This value is the incidence of treated cases, over 17 years without any notion of recruitment potential and with a rather slow ramp up of the activity . A list of priority indications that can be treated by hadrontherapy (table 1) was drawn up by the gcs etoile medical group, in collaboration with national and international experts, based on published data ranked using an evidence - based medicine this literature review was based on provided or expected medical service criteria (survival and quality of life). According to the classification used by the european survey group of rare diseases, the eligible tumours that can be treated by hadrontherapy conform to the criteria of rare diseases based on the following thresholds of epidemiological indicators: prevalence 50 cases/100 000 persons within a given population and incidence 6 cases/100 000 persons / year . Indeed, for all tumor types identified during the previous hadrontherapy epidemiological studies [58], the calculated and cumulative incidences are all below these thresholds (table 2). Amongst the hadrontherapy indications, 25 are tumours listed in the orphanet international database of rare diseases (cf . The exact incidence of each disease eligible for hadrontherapy is difficult to determine from the available resources . Indeed, the published data [513] pertain to series of patients for which the aim was not to assess the level of demand for treatment . These were rather series of patients who had received the therapy, but whose recruitment had not been accounted for in tumour registries . Considering the scarcity and specific nature of the tumours concerned by hadrontherapy, it is difficult to extrapolate and assess the treatment demand from these results . Hadrontherapy indications are defined according to anatomical location of the tumours, clinical stage, pathology, therapeutic alternatives (e.g., surgical contraindications), and patient characteristics (general condition and comorbidities). French cancer registries record data required to estimate cancer incidence but fail to provide sufficient detail of the tumour stage or of therapeutic data . Moreover, the registries cover only approximately 15% of the french population and mainly for the most frequent cancers . Therefore, these registries do not allow the incidence of these tumours to be estimated . This regional study will provide us with more detailed figures concerning the potential number of new cases eligible for hadrontherapy, allowing us to better adapt the future care offer delivered by the national hadrontherapy centre . This survey should also provide us with information on certain medical indicators to assist in the design of future hadrontherapy clinical trials (detailed incidence data, patient population characteristics, tumour stages, recruitment pools, etc . ). Finally, this epidemiological field study is also eventually intended to be used to organise a network of highly specialised cares . According to the first epidemiological study carried out in france, in association with (i) the results of the literature review that defined the list of hadrontherapy indications and (ii) the indications chosen for proton therapy in france, the number of cases in the rhne - alpes region eligible for hadrontherapy is estimated at 200 per year (the rhne - alpes region is home to 10% of the french population). The main goal is to asses, within the rhne - alpes region, the incidence of cancers with hadrontherapy indication . The secondary goals are (1) to characterise the affected population, (2) to describe the characteristics of the observed cancers, and (3) to describe the characteristics of the treatments implemented when hadrontherapy is unavailable . This is a prospective, multicentre incidence study, lasting 24 months, conducted with the rcp (runion de concertation pluridisciplinaire multidisciplinary tumor board) groups of the rhne - alpes region in france, that offer a therapeutic strategy for cancers for which hadrontherapy is an alternative . This study required the involvement of a part - time consultant epidemiology engineer, who totalled one woman - year of work to provide (1) phone canvassing to identify and involve regional cancer research players; (2) the protocol construction and testing phase, along with the data collection tools; (3) meetings with 3c (cancer coordination centres) coordinators and rcps; (4) investigator centres inclusion meetings . These included year of birth; gender; patient's department of residence; world health organization (who) performance status, patient's therapeutic status: initial stage or recurrence; initial staging (uicc tnm classification: international union against cancer); treatments received before recurrence; time to recurrence; staging at time of current tumour management; icd-10 (international classification of diseases); icdo (international classification of diseases for oncology) histological type; site of surgery and margin quality; postsurgical histopathological stage (ptnm stage); prior radiotherapy; proposed treatment . The criteria included adults and children, no age limit, and patients presenting with hadrontherapy indications listed in table 1, whose medical files have been discussed by experts during an rcp . Compatible general and psychological condition refers to patients not suffering from life - threatening comorbidities (no acute or chronic diseases whose short - term lethal risk is dominant relative to the cancer), capable of adhering to a disease monitoring protocol and of understanding and accepting a complex treatment requiring close cooperation and staying for several days away from home . Tumour inclusion criteria . These include unresectable tumour, belonging to a known radiation - resistant pathological group, mainly in a locoregional development stage, recurrent or local relapse, with low metastatic potential or m+ dissemination (presence of one or more remote metastases at the time of diagnosis) presenting a low threat (slow development and/or metastases that can be readily treated). These inclusion criteria are the results of a consensus synthesis of a systematic literature review performed during 8 years, pathology by pathology with the participation of about one hundred of european medical, surgical, and radiation oncologists, managed by physicians trained in hadrontherapy (mainly protontherapy). The review was carried out by an academic laboratory specialized in clinical trial setup and analysis and was made according to the evidence - based medicine definition of tumour inclusion criteria radiation resistance: tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent (mpde) for surrounding organs necessarily irradiated under applicable technical conditions, assessed by a radiation oncologist (guide des procdures de radiothrapie externe 2007 (guidelines for external radiotherapy procedures); joint effort by the french society for oncology radiotherapy (socit franaise de radiothrapie oncologique sfro), and the french society for medical physics (socit franaise de physique mdicale sfpm), conducted in collaboration with the representatives of the french association of electroradiology paramedical workers (association franaise du personnel paramdical d'electroradiologie afppe)). Unresectable: tumour which local or locoregional expansion stage renders excision either is technically impossible (opinion of an experienced surgical team) or surgically unacceptable due to the irreparable damage that would be necessary (cancer: principles & practice of oncology 2005; devita, v. general articles on treatment strategies in cancer). Locoregional: it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion (t of tnm) and/or by lymphatic diffusion, maintaining a rank of n in tnm staging, in other words, any tumour at stage m0 (latest edition of the tumour tnm classification published by the (uicc) international union against cancer or the (ajc) american joint committee for cancer staging). Low metastatic potential: m0 situations whose medium - term (5-year) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means . This threshold is difficult to determine; a level greater than 50% seems unreasonable . As an example, nonthreatening m+ dissemination or controlled by medical treatments: this means the presence of one or more remote metastases at the time of diagnosis, presenting no immediate threat as displaying slow development and/or accessible to effective treatment (typical case of some low - grade sarcomas lung metastases). These situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy . Local relapse or local recurrence (these two terms are synonymous): it means the redevelopment, at the same site as initially, of a previously effectively treated tumour (dictionary of medical terms, garnier - delamare). Radiation resistance: tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent (mpde) for surrounding organs necessarily irradiated under applicable technical conditions, assessed by a radiation oncologist (guide des procdures de radiothrapie externe 2007 (guidelines for external radiotherapy procedures); joint effort by the french society for oncology radiotherapy (socit franaise de radiothrapie oncologique sfro), and the french society for medical physics (socit franaise de physique mdicale sfpm), conducted in collaboration with the representatives of the french association of electroradiology paramedical workers (association franaise du personnel paramdical d'electroradiologie afppe)). Unresectable: tumour which local or locoregional expansion stage renders excision either is technically impossible (opinion of an experienced surgical team) or surgically unacceptable due to the irreparable damage that would be necessary (cancer: principles & practice of oncology 2005; devita, v. general articles on treatment strategies in cancer). Locoregional: it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion (t of tnm) and/or by lymphatic diffusion, maintaining a rank of n in tnm staging, in other words, any tumour at stage m0 (latest edition of the tumour tnm classification published by the (uicc) international union against cancer or the (ajc) american joint committee for cancer staging). Low metastatic potential: m0 situations whose medium - term (5-year) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means . This threshold is difficult to determine; a level greater than 50% seems unreasonable . As an example, nonthreatening m+ dissemination or controlled by medical treatments: this means the presence of one or more remote metastases at the time of diagnosis, presenting no immediate threat as displaying slow development and/or accessible to effective treatment (typical case of some low - grade sarcomas lung metastases). These situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy . Local relapse or local recurrence (these two terms are synonymous): it means the redevelopment, at the same site as initially, of a previously effectively treated tumour (dictionary of medical terms, garnier - delamare). They are the description of the tumours and population characteristics, along with the treatments proposed and implemented by the rcps . Those were healthcare establishments (private and public) in the rhne - alpes region hosting rcp groups and having radiotherapy departments . They consisted of groups of oncology specialists with expertise in the following diseases: musculoskeletal sarcomas and tumours, head and neck (h and n), gastroenterology, paediatrics, dermatology, endocrine and central nervous system tumours . Case screening is performed continuously by the physicians and clinical research technician in light of the list of indications (table 1), during each rcp . The questionnaire, along with the organisation of the investigation, was assessed beforehand by a sample population of investigators (3c coordinators and cras) via a semiguided telephone interview . For the questionnaire, the evaluation focused on general understanding, the form and length of the questionnaire, time required for completion, amount of data to collect, and the relevance of the selected criteria . The evaluation of the investigation method pertained to the choice of individuals involved in data collection organisation, the data recording method, the organisation for data recording, and the identification source for clinical cases . Variables are encoded in a uniform and standard manner in an investigator's guide book, which contains detailed information concerning the definitions of each indicator to collect . Dual computer data input is performed, along with checks for missing or improbable data (encoding errors, date inconsistencies). Statistical methods . The incidence of cancers is expressed as the number of cases reported from the demographic figures of the rhne - alpes region over the studied period and expressed per 100,000 inhabitants . The study involves 13 clinical research technicians, 50 rcp coordinating physicians and 12 cancer coordination centres (see table 3). The study mobilises 27 rhne - alpes region healthcare centres, of which 8 university hospitals, 1 regional cancer centre, 1 public - private cancer institute, 12 general hospitals, the lyon paediatric oncology hospital institute, and 4 private clinics . This led to a sense of ownership and motivation on the part of the centres . As a whole, the study covered the region's 11 hospital areas and involved 52 groups, out of some sixty listed, of cancer specialists with expertise in the diseases that were to identify . During the study, mean patient age was 43 years [289], and men were more numerous (or = 1.52). The highest number of cases (39.6%) was in the 218 years age group . The number of patients with recurrence was slightly lower (45.3%) than the number of patients receiving initial care (54.7%). Carcinomas, sarcomas (table 4), and paediatric tumours (table 5) were the most frequently identified diseases, and their locations are varied . During the rcp, radiotherapy, chemotherapy, and surgery were prescribed, respectively, in 58%, 57%, and 57% of cases . Previous treatments were chemotherapy, surgery, or conventional radiotherapy in 62.5%, 58.3%, and 58.3% of cases, respectively . The number of cases identified during the study was approximately four times lower than the potential estimate . (i.e., 2.5 cases / rcp), and the rhne department identified 56.6% (i.e., 1.2 cases / rcp). Isre identified twice as many cases as rhne, even though it only has nine expert groups, that is, 2.6 times less than in rhne, which has 24 . The geographic areas not covered by the study correspond to the location of 15 private healthcare centres and to the medical activity of six general and four specialist rcp groups . The first six groups had no recruitment potential according to the statement of their coordinators . This preliminary data collection, conducted over a one - year period, reported 53 cases of hadrontherapy indications, compared to the estimated cap of 200 cases . The estimated incidence of cancers eligible for hadrontherapy in the rhne - alpes region in 2010, regardless of disease, is of 8.5/100,000 inhabitants . The diseases to identify in this study are little known, their distribution has not been studied, and we are unaware of any epidemic outbreaks . Moreover, considering that the cancers to identify are rare, it is likely that the number of cases to identify per investigator centre will be very low (estimated incidence of at the most 1 to 5 cases / year). Finally, the primary goal of the rcp groups is to provide an immediate therapeutic solution . Case identification for an observational study is thus unusual in the context of an rcp . Because of this, sustaining team vigilance and motivation for case identification is difficult to obtain and requires regular refreshing throughout the study . This step also served to circulate hadrontherapy information and to facilitate adhesion, ownership, and mobilisation around the project . The individuals involved in study organisation were in charge of systematically directing each cancer patient to the elective experts . Patients suffering from diseases eligible for hadrontherapy were frequently in relapse condition and rather at the end of their therapeutic options . This is due to the fact that these diseases have usually a slow and long evolution; therefore, this study had more chance to catch them somewhere in an advanced stage than at the beginning . It can also be considered that the lack of satisfactory treatment means as hadrontherapy makes these populations have more chance to show more advanced disease than it will be expected to be seen in the future . The identification and decision to include a patient in the study were made by the rcp groups including these experts . Under these conditions, we have confidence in the case orientation and identification system . Selection criterion is one of the criteria used to identify patients eligible for hadrontherapy . In our current state of knowledge this can be explained in part by the fact that it is easier to create multidisciplinary expert groups in public hospitals and to set up clinical research . Due to their structure, these establishments benefit from human resources with more varied medical specialities than in private radiotherapy practices . In all likelihood, cases eligible for hadrontherapy could have been treated by these private centres during the study and ignored . One should be very cautious when comparing the results of this study with those of published incidence studies . The methodologies employed in these previous studies are heterogeneous, and/or their flow is insufficiently documented . Moreover, their goal was to estimate a recruitment potential based on the extrapolation of the results . The methodologies used were not intended to provide information concerning an exhaustive incidence, as proposed by our study . Finally, the published studies were conducted in attractive institutions, specialising in cancer care and selected for this reason; this may bias the assessment of prevalence . Under these conditions, it is easy to confuse incidence and prevalence . These elements could, in part, explain the observed difference between the preliminary results obtained in our study and the estimate calculated from the epidemiological results published in the literature . Only the publication of results obtained from other exhaustive epidemiological studies conducted in the same areas could support this hypothesis . It is unlikely that the lack of case recording associated with the medical activity of the four groups not involved in this data collection could alone explain the observed difference between the calculated incidence estimates and the preliminary results obtained . For several clinicians involved in the study, the prognosis and risk of metastatic development were difficult to estimate considering the lack of knowledge for these rare diseases . Moreover, the novelty of hadrontherapy, which can potentially modify usual treatment strategies, could have led to the omission of this indication in some cases, in particular conditions unusually treated by radiotherapy as hepatic and biliary tumours . These difficulties, along with the more sustained vigilance effort made by some groups relative to others, could account for the nonhomogeneous data collection and the observed difference between predictions and obtained results, despite the high motivation of the investigators . Furthermore, the reorganisations (case of two major rcp clusters), changes of persons involved and the low frequency of cases to report, lead to a constant erosion of vigilance, requiring continuous action by the organiser . However, some important institutions with their own recruitment targets did not wish to participate, which reduces the scope of our results . About the detailed conditions of the recruited cases, there are a large proportion of recurrent diseases as explained above, and some metastatic diseases have also been included according to defined characteristics: (i) the number of metastasis was limited (1 to 3 metastasis); (ii) their very slow growing speed was not immediately threatening; (iii) the possibility of effective treatment of these pauci - metastatic conditions, essentially by ablative procedures . Actually, the principle to treat, with curative intend, patients in such situation is more and more accepted by expert oncologists . The estimated incidence of cancers, irrespective of type and location, eligible for treatment by hadrontherapy, for the study, that is, 8.5/100,000 individuals, is mildly higher than the reference incidence threshold of 6 cases/100,000 individuals, as defined in the orphanet international database of rare diseases . This figure can be obviously explained by the fact that the incidence was calculated by adding the five major cancer categories and 34 different locations . If one was to calculate the incidence for each of the disease types, their respective incidence would be significantly below the reference threshold of 6 cases/100,000 . Indeed, for our study, the minimum number of cases listed per disease is of 1 case and the maximum is of 7 cases . Table 4 . Considering all of the difficulties and limitations of this preliminary phase of the study, the yield of approximately 25% notification, compared to the expected maximum, appears satisfactory . In the perspective of a (necessary) ramp up of a future hadrontherapy centre, there is thus a sufficient population to initiate operation and to progressively establish recruitment for this centre, without immediately generating an insufficient offer effect . Future epidemiological studies will benefit from focusing on the characterisation of the metastatic development of diseases eligible for hadrontherapy . All cancers have their own development pattern, and, in this respect, the literature provides few elements concerning diseases eligible for hadrontherapy . This information should serve both to improve diagnosis procedures, medical care, and the surveillance of these rare diseases . This study of hadrontherapy healthcare decision networks in the rhne - alpes region, able to identify areas not properly covered by rcps, should enable us to analyse the potential effect of this lack on the outcome of the patients of these areas . They shed no light on posttreatment medical activity: follow - up visits, medical imaging, and other care beyond the irradiated activity per se . One of the most valuable outcomes of this study has been to make the existence of some particularly rare diseases known and to provide information concerning an innovative treatment: hadrontherapy, whose existence will doubtless have a favourable effect on the very knowledge of these diseases . An additional benefit of this approach has been to open the way for a new treatment system in a region of france that is home to approximately 10% of the country's population . At last, these elective indications will have to be validated by health authorities to register them as part of the good professional practices of oncology and give an equal chance to each patient to receive hadrontherapy through the well - established decision process of the rcps.
Type 2 diabetes continues to present a public health burden across the world, and asia is considered to be at the epicenter of this diabetes epidemic, with 60% of the disease burden borne by this region1 . The increase in diabetes prevalence across asia has been rapid, fuelled by increasing obesity, aging populations and urbanization, which in turn have been driven by rapid economic development, a sedentary lifestyle and increased consumption of refined foods3 . These socioeconomic changes have been particularly significant in countries across southeast asia4, and reflected by dramatic rises in type 2 diabetes prevalence over the past decade (figure 1). Indeed, indonesia and the philippines are projected to be among the top 10 countries in the world with the highest number of estimated cases by 20301 . Another issue curtailing the type 2 diabetes rise in asia is the high number of undiagnosed individuals . For instance, in hong kong and taiwan it was reported that almost 53% of diabetic patients remain undiagnosed5 . Furthermore, the prevalence of prediabetes (impaired glucose tolerance) further adds to the already burgeoning burden of overt diabetes and its consequences in this region (figure 1). Comparative prevalence of diabetes in countries across southeast asia in 2011 (calculated by assuming that every country and region has the same age profile). These data are based on figures compiled by the international diabetes federation in the international diabetes atlas, 2011 (available at: http://www.idf.org/atlasmap/atlasmap). It is widely recognized that diabetes is a major source of morbidity, mortality and economic burden6 . Prolonged and poor glucose control increases the risk of developing macrovascular (coronary artery disease, stroke) and microvascular (nephropathy, retinopathy and neuropathy) complications7, which can greatly reduce life expectancy and quality of life8 . In fact, the risk of allcause mortality (cardiovascular and noncardiovascular) is higher in diabetic than nondiabetic patients9 . Consequently, the rapidly growing prevalence of diabetes in asia is likely to result in large increases in diabetesassociated mortality10 . From an economic perspective, the impact of diabetes on healthcare resources and personal expenditure, as well as the financial implications associated with loss in productivity and disability, is enormous11 . In taiwan, the direct healthcare cost associated with diabetes was 11.5% of the total national healthcare cost and was 4.3fold higher than the care of nondiabetic individuals12 . In hong kong, the annual total cost of type 2 diabetes equated to usd 1,725 2,044 per patient . Furthermore, complications increased treatment costs associated with inpatient hospital stay by between 6 and 300% compared with treatment of type 2 diabetes patients without complications14 . Of note is the considerable heterogeneity in healthcare spending for diabetes between countries across southeast asia . The international diabetes federation estimated that, in 2010, treatment of diabetes accounted for 5 and 7% of the total healthcare expenditure in vietnam and indonesia, respectively, compared with 15 and 16% in singapore and malaysia, respectively11 . Taken together, it is clear that early diagnosis and disease control are important healthcare initiatives to improve the lives, outcomes and economies of these atrisk countries . In determining the optimal management approach, recognizing the differences in clinical characteristics and risk of diabetes between asian and caucasian populations is important to understand . For instance in asians, diabetes develops over a shorter time, in younger people and in those with a lower body mass index (bmi)15 . Furthermore, asians have a higher proportion of body fat and abdominal obesity, putting them at a higher risk of developing insulin resistance16 . Reports that asians show early cell dysfunction might further explain their increased susceptibility to type 2 diabetes16 . The thrifty gene and thrifty phenotype hypotheses have been proposed as plausible explanations for the increasing diabetes epidemic seen across asia18 . Although considered protective during periods of famine or food shortage, thrifty genes have rendered individuals highly predisposed to obesity in times of plenty . Alternatively, the thrifty phenotype relates to the impact of an unfavorable intrauterine environment (e.g. Undernutrition) and low birth weight on the subsequent development of diabetes and metabolic syndrome in adults18 . Several diabetesassociated genes have been implicated to account for the heightened predilection in asians, especially asian indians, and include polymorphisms of the peroxisome proliferatoractivated receptor coactivator1 alpha (pgc1 alpha) gene, plasma cell glycoprotein (pc1) gene and insulin receptor substrate (irs2) gene (reviewed in radha and mohan)19 . Given the increased prevalence of diabetes among younger populations in asia and early exposure to hyperglycemia, it has been suggested that they are also at a high risk of endorgan damage and are likely to have to live with complications for longer20 . Indeed, the asia pacific cohort studies collaboration (apcsc), which assessed the impact of diabetes on cardiovascular outcomes and mortality, found a greater risk of fatal coronary heart disease, hemorrhagic and ischemic stroke among younger compared with older individuals21 . Apcsc also showed an association between diabetes and increased risk of death as a result of renal disease, cancer, respiratory infections, and other infective and inflammatory causes21 . Although optimal blood glucose control has been shown to reduce the risk of both macrovascular and microvascular complications6, the marked prevalence of renal dysfunction in asia reflects, in part, the continuing challenge in achieving this goal22 . The aim of the present article was to review the barriers to achieving optimal blood glucose control in asian patients with type 2 diabetes who have declining renal function and to discuss the treatment modalities that might be appropriate for the management of these special patient populations . Of the diabetesassociated complications, the most debilitating is chronic kidney disease (ckd) and its pathogenic progression to endstage renal disease (esrd) that requires dialysis or transplantation23 . Defining and diagnosing ckd in clinical practice has evolved since its first classification by the national foundation kidney disease outcomes quality initiative (nkfkdoqi) in 200224 . In these guidelines, ckd was categorized into five stages according to the severity of disease as defined by decreasing glomerular filtration rates (gfr) alone24 . In the more recent update, the assessment criteria have been modified to encompass both gfr and albuminuria measures as correlates of a patient's prognosis . In addition, a subdivision of the previously described gfr stage 3 has been proposed to help further define the severity of kidney dysfunction in patients with ckd25 . Risk factors for the development of reduced kidney function in southeast asian populations have been identified and include the presence of diabetes, systolic hypertension (> 159 mmhg), hyperuricemia (> 6.29 mg / dl), elevated bmi (> 24.9 kg / m) and hypercholesterolemia (> 248.3 mg / dl)26 . Microalbuminuria is the earliest clinical manifestation of ckd in type 2 diabetes and persistent microalbuminuria represents an early clinical marker for the development of diabetic nephropathy (dn)27 . Although the number of studies exploring the epidemiology of microalbuminuria across asia has been limited, the microalbuminuria prevalence study (maps) of 6,800 hypertensive diabetic patients from 10 countries in the region reported an overall prevalence rate of 39.8% (95% confidence interval [ci] 39.240.5)22 . These data show how a high proportion of hypertensive, type 2 diabetes asian patients are showing early signs of declining renal function, and further support a need for early intervention so as to reduce accelerated progression of cardiorenal complications and the associated healthcare burden28 . With the exception of taiwan (chiang sc et al . Jcma 2011; 74: 310), these data are based on subanalyses of the microalbuminuria prevalence study of 6,800 hypertensive diabetic patients from 10 countries across asia (wu ay et al . 2005; 48: 1726 .) In recognition of the utility of measuring microalbuminuria, guidelines recommend annual screening of this clinical marker for identifying atrisk populations32 . Patients identified with microalbuminuria early in the course of disease might require stricter glycemic and blood pressure control targets . Indeed, several prospective studies have shown the importance of tight glycemic control alone and combined with blood pressure lowering in the prevention of microvascular complications . For instance, the uk prospective diabetes study compared the effects of intensive bloodglucose control with either sulfonylureas or insulin and conventional treatment (including diet) on the risk of microvascular and macrovascular complications in type 2 diabetes patients . Intensive glucose control with sulfonylureas or insulin resulted in a 33% relative risk reduction in the development of microalbuminuria . Furthermore, this reduction in risk was sustained in the 10year followup study during which longterm benefits on cardiovascular disease (cvd) risk also emerged33 . In the kumamoto study, the frequency and severity of diabetic microvascular complications were assessed in japanese type 2 diabetes patients after treatment with either intensive glucose control (multiple insulin injections of three or more) or conventional therapy (insulin injections administered once or twice daily). There was a significant reduction in the progression of nephropathy and other microvascular complications (retinopathy) after intensive insulin therapy (p <0.05)35 . The effect of an intensive, stepwise multifactorial intervention, targeting hyperglycemia, hypertension, dyslipidemia and microalbuminuria, on the initiation and progression of microvascular complications in type 2 diabetes patients with microalbuminuria was assessed in the steno2 study . Type 2 diabetes patients receiving early intensive diabetes management including tight glucose control and angiotensin converting enzyme inhibitors (acei) to reduce blood pressure were significantly less likely to develop nephropathy than patients receiving standard therapy (odds ratio 0.27; 95% ci 0.100.75). This reduction in risk was maintained in an observational follow up of 5.5 years36 . In the most recent analysis of the action in diabetes and vascular disease: preterax and diamicronmodified release controlled evaluation (advance) trial, which included a large number of asian type 2 diabetes patients, further support for the benefits of combining bloodpressure lowering with intensive blood glucose control was provided38 . Patients received either intensive glucose control (target glycated hemoglobin [hba1c] 6.5%) or standard glucose control (defined by local guidelines) with placebo or acei plus indapamide therapy . A combination of intensive glucose control plus acei plus indapamide therapy led to a significant percentage in risk reduction in new or worsening nephropathy (95% ci 1250%; p = 0.005); a 54% reduction in the development of newonset macroalbuminuria (95% ci 3568%; p <0.001), and a 25% reduction in the risk of newonset microalbuminuria (95% ci 1633%; p <0.001). Furthermore, the effects of blood pressure lowering and intensive glucose control were independent of each other, so that on combining these agents, additive benefits were observed, particularly on renal outcomes38 . Indeed, the benefits of early and tight target control strategies on the prevention of dn in chinese type 2 diabetes patients have been observed . In this asian cohort with normoalbuminuria, achievement of american diabetes association (ada)recommended targets of hba1c (<7%), systolic blood pressure (<130 mmhg), diastolic blood pressure (<80 mmhg) and lipid profiles (lowdensity lipoprotein [ldl] <100 mg / dl; triglycerides <150 mg / dl; highdensity lipoprotein> 4050 mg / dl) reduced the risk of developing newonset microalbuminuria compared with those who were unable to attain these targets39 . Interestingly, in a separate study of taiwanese type 2 diabetes patients with microalbuminuria, almost 36% of patients achieved remission to normoalbuminuria following a tight, multifactorial target control approach40 . Worldwide, rates of ckd have been increasing, and have accompanied the increasing prevalence of type 2 diabetes, hypertension and cvd . For instance, in the usa, it is estimated that 30% of patients with type 2 diabetes will develop some form of kidney disease as reflected by protein leakage, microalbuminuria, macroalbuminuria or a decline in the glomerular filtration rate (gfr)41 . Nephropathy is a common complication of asians with type 2 diabetes, as reflected by the accelerating prevalence and incidence rates of observed ckd23 . Indeed, incidence rates of esrd across asia have shown increases over a 6year period (20032008) of 15.8% in hong kong, 22% in malaysia and thailand, 24% in singapore, and up to 31% in the philippines43 . Although incidence rates of esrd appear to have declined in taiwan since 2005, this country has the third highest rate of esrd in the world, with an incidence rate of 384 per million population (pmp), highlighting the disparity between the rate of kidney disease and a country's economic status42 . Furthermore, it has become apparent that a high proportion of esrd cases are as a result of diabetes, ranging from 41% to 62% in individual countries across southeast asia (figure 2)43 . Incidence of endstage renal disease (esrd) as a result of diabetes in selected countries across southeast asia in 2008 . These data are based on figures compiled by the us renal disease system (figure adapted from 2010 us renal disease system annual report chapter 12 international comparisons; volume 2; pages 383396). For singapore, the 2008 data have been extracted from the national registry of diseases office, released 1 march 2011 (inp111). The presence of ckd is also associated with a high cvd risk, which is further exacerbated by the presence of diabetes and hypertension45 . Indeed, cvd remains the main cause of death in patients with ckd rather than kidney failure, and it is estimated that cvdassociated mortality is 1030fold higher in patients with advanced ckd receiving dialysis than the general population46 . Given that the prevalence of hypertension in asia is increasing, and that diabetes and hypertension commonly coexist (type 2 diabetes patients are twice as likely to have elevated blood pressure than nontype 2 diabetes patients), screening these highrisk patients for ckd early might help slow the course of renal function decline and reduce the greater cardiovascular mortality risk observed in such individuals47 . Microalbuminuria is the earliest clinical manifestation of ckd in type 2 diabetes and persistent microalbuminuria represents an early clinical marker for the development of diabetic nephropathy (dn)27 . Although the number of studies exploring the epidemiology of microalbuminuria across asia has been limited, the microalbuminuria prevalence study (maps) of 6,800 hypertensive diabetic patients from 10 countries in the region reported an overall prevalence rate of 39.8% (95% confidence interval [ci] 39.240.5)22 . These data show how a high proportion of hypertensive, type 2 diabetes asian patients are showing early signs of declining renal function, and further support a need for early intervention so as to reduce accelerated progression of cardiorenal complications and the associated healthcare burden28 . With the exception of taiwan (chiang sc et al . Jcma 2011; 74: 310), these data are based on subanalyses of the microalbuminuria prevalence study of 6,800 hypertensive diabetic patients from 10 countries across asia (wu ay et al . Diabetologia . 2005; 48: 1726 .) In recognition of the utility of measuring microalbuminuria, guidelines recommend annual screening of this clinical marker for identifying atrisk populations32 . Patients identified with microalbuminuria early in the course of disease might require stricter glycemic and blood pressure control targets . Indeed, several prospective studies have shown the importance of tight glycemic control alone and combined with blood pressure lowering in the prevention of microvascular complications . For instance, the uk prospective diabetes study compared the effects of intensive bloodglucose control with either sulfonylureas or insulin and conventional treatment (including diet) on the risk of microvascular and macrovascular complications in type 2 diabetes patients . Intensive glucose control with sulfonylureas or insulin resulted in a 33% relative risk reduction in the development of microalbuminuria . Furthermore, this reduction in risk was sustained in the 10year followup study during which longterm benefits on cardiovascular disease (cvd) risk also emerged33 . In the kumamoto study, the frequency and severity of diabetic microvascular complications were assessed in japanese type 2 diabetes patients after treatment with either intensive glucose control (multiple insulin injections of three or more) or conventional therapy (insulin injections administered once or twice daily). There was a significant reduction in the progression of nephropathy and other microvascular complications (retinopathy) after intensive insulin therapy (p <0.05)35 . The effect of an intensive, stepwise multifactorial intervention, targeting hyperglycemia, hypertension, dyslipidemia and microalbuminuria, on the initiation and progression of microvascular complications in type 2 diabetes patients with microalbuminuria was assessed in the steno2 study . Type 2 diabetes patients receiving early intensive diabetes management including tight glucose control and angiotensin converting enzyme inhibitors (acei) to reduce blood pressure were significantly less likely to develop nephropathy than patients receiving standard therapy (odds ratio 0.27; 95% ci 0.100.75). This reduction in risk was maintained in an observational follow up of 5.5 years36 . In the most recent analysis of the action in diabetes and vascular disease: preterax and diamicronmodified release controlled evaluation (advance) trial, which included a large number of asian type 2 diabetes patients, further support for the benefits of combining bloodpressure lowering with intensive blood glucose control was provided38 . Patients received either intensive glucose control (target glycated hemoglobin [hba1c] 6.5%) or standard glucose control (defined by local guidelines) with placebo or acei plus indapamide therapy . A combination of intensive glucose control plus acei plus indapamide therapy led to a significant percentage in risk reduction in new or worsening nephropathy (95% ci 1250%; p = 0.005); a 54% reduction in the development of newonset macroalbuminuria (95% ci 3568%; p <0.001), and a 25% reduction in the risk of newonset microalbuminuria (95% ci 1633%; p <0.001). Furthermore, the effects of blood pressure lowering and intensive glucose control were independent of each other, so that on combining these agents, additive benefits were observed, particularly on renal outcomes38 . Indeed, the benefits of early and tight target control strategies on the prevention of dn in chinese type 2 diabetes patients have been observed . In this asian cohort with normoalbuminuria, achievement of american diabetes association (ada)recommended targets of hba1c (<7%), systolic blood pressure (<130 mmhg), diastolic blood pressure (<80 mmhg) and lipid profiles (lowdensity lipoprotein [ldl] <100 mg / dl; triglycerides <150 mg / dl; highdensity lipoprotein> 4050 mg / dl) reduced the risk of developing newonset microalbuminuria compared with those who were unable to attain these targets39 . Interestingly, in a separate study of taiwanese type 2 diabetes patients with microalbuminuria, almost 36% of patients achieved remission to normoalbuminuria following a tight, multifactorial target control approach40 . Worldwide, rates of ckd have been increasing, and have accompanied the increasing prevalence of type 2 diabetes, hypertension and cvd . For instance, in the usa, it is estimated that 30% of patients with type 2 diabetes will develop some form of kidney disease as reflected by protein leakage, microalbuminuria, macroalbuminuria or a decline in the glomerular filtration rate (gfr)41 . Nephropathy is a common complication of asians with type 2 diabetes, as reflected by the accelerating prevalence and incidence rates of observed ckd23 . Indeed, incidence rates of esrd across asia have shown increases over a 6year period (20032008) of 15.8% in hong kong, 22% in malaysia and thailand, 24% in singapore, and up to 31% in the philippines43 . Although incidence rates of esrd appear to have declined in taiwan since 2005, this country has the third highest rate of esrd in the world, with an incidence rate of 384 per million population (pmp), highlighting the disparity between the rate of kidney disease and a country's economic status42 . Furthermore, it has become apparent that a high proportion of esrd cases are as a result of diabetes, ranging from 41% to 62% in individual countries across southeast asia (figure 2)43 . Incidence of endstage renal disease (esrd) as a result of diabetes in selected countries across southeast asia in 2008 . These data are based on figures compiled by the us renal disease system (figure adapted from 2010 us renal disease system annual report chapter 12 international comparisons; volume 2; pages 383396). For singapore, the 2008 data have been extracted from the national registry of diseases office, released 1 march 2011 (inp111). The presence of ckd is also associated with a high cvd risk, which is further exacerbated by the presence of diabetes and hypertension45 . Indeed, cvd remains the main cause of death in patients with ckd rather than kidney failure, and it is estimated that cvdassociated mortality is 1030fold higher in patients with advanced ckd receiving dialysis than the general population46 . Given that the prevalence of hypertension in asia is increasing, and that diabetes and hypertension commonly coexist (type 2 diabetes patients are twice as likely to have elevated blood pressure than nontype 2 diabetes patients), screening these highrisk patients for ckd early might help slow the course of renal function decline and reduce the greater cardiovascular mortality risk observed in such individuals47 . Despite the advent and availability of a range of antidiabetic and blood pressure lowering agents, many patients in asia remain suboptimally controlled and therefore at risk of developing complications . In the maps study, just 10.6% of type 2 diabetes asian patients with microalbuminuria achieved blood pressure targets below 130/80 mmhg and mean hba1c levels of 7.9%22 . Similarly, in a crosssectional survey in china, the majority of type 2 diabetes patients with nephropathy (66.9%) had a mean hba1c level of> 7.5%, indicating poor glycemic control in patients with complications50 . These data are supported by the recent international diabetes management practice study (idmps) that explored the barriers to achieving optimal glycemic control across asia, latin america and eastern europe51 . In asia, of the 5,372 type 2 diabetes patients assessed, 35.8% had microalbuminuria and just 37.3% achieved the hba1c target of <7.0%; 21.8% achieved the blood pressure target of <130/80 mmhg; and 37% achieved the ldl cholesterol target of <100 mg / dl . Another significant and worrying finding was that just 4.7% of patients in asia attained all three treatment targets51 . Factors that might be contributing to such low control rates have been reported to include: difficult medical access by patients in some developing countries, physician perception of target levels, prescribing habits, as well as knowledge of guidelines an aging population provides additional challenges in managing type 2 diabetes, as aging itself is associated with changes in kidney structure and function, and these agerelated renal changes can be accelerated by the presence of comorbid conditions52 . Furthermore, a study of the hong kong diabetes registry revealed how long disease duration and complexity of treatment regimens might also play a role in suboptimal glycemic control54 . Poor control might also result in type 2 diabetes patients who present and commence treatment when their renal function is normal, but subsequently go on to develop kidney disease . Without appropriate monitoring of gfr levels and diagnosis of kidney disease or its progression, the need for dose adjustments or reevaluation of prescribed therapies might be missed and, consequently, adverse sideeffects might arise55 . Hypoglycemia is a particular challenge in the management of type 2 diabetes, especially in patients with a decline in renal function . For instance, diabetic patients with ckd are twice as likely to experience hypoglycemic events than those without kidney disease56 . This might be a result of reduced insulin clearance, reduced gluconeogenesis by the kidney and increased accumulation of drugs excreted by the kidney57 . The reduction in drug clearance results in prolonged exposure to the drug itself or its metabolites, which can lead to adverse sideeffects . This is a major issue in moderate to severe ckd (stages 35) when renal dysfunction is particularly pronounced55 . The american diabetes association guidelines recommend target hba1c levels of <8% for patients with more advanced microvascular complications and increased risk of hypoglycemia, such as those with more advanced ckd . However, in practice, the use of antidiabetic medications in achieving these glycemic targets is often challenging58 . There are now an array of antihyperglycemic agents available that include both injectable, such as insulin and the incretin mimetics glp1 analogs (exenatide, liraglutide), and oral antidiabetics (oads). Among the oads are biguanides (metformin), second generation sulfonylureas (glimepiride, glipizide, glicazide), meglitinides (nateglinide, repaglinide) glucosidase inhibitors (acarbose), thiazolidinediones (tzd; pioglitazone) and incretinbased therapies that include dipeptidylpeptidase iv (dpp4) inhibitors (linagliptin, sitagliptin, vildagliptin, saxagliptin). In addition, oral sodiumglucose transporter 2 (inhibitors are currently in development (empagliflozin, dapagliflozin). Despite the range of medications available for the treatment of type 2 diabetes and its complications, many of these require dose modification or avoidance and close monitoring55 . This poses a particular challenge in more advanced disease (ckd stages 35), where contraindications or the increased risk of hypoglycemia serve to limit the number of treatment options (table 2)55 . As all these agents differ in their mechanism of action, efficacy, sideeffects and costs, the choice of therapy should be guided by several factors . Patient demographics, presence of comorbid conditions, risk or severity of renal function decline, tolerability, and risk of developing adverse events, such as hypoglycemia, weight gain and edema, should help in the treatment decision process57 . With most of the oads currently available for the treatment of type 2 diabetes, their use in patients with kidney dysfunction is compromised55 . However, sulfonylureas and their metabolites are affected by kidney function by increasing in potency as renal function declines . Furthermore, in type 2 diabetes patients with esrd receiving dialysis, treatment with sulfonylureas might lead to severe and prolonged episodes of hypoglycemia61 . However, secondgeneration sulfonylureas, such as gliclazide and glipizide, which have inactive metabolites, carry less risk of hypoglycemia than the firstgeneration sulfonylureas and might therefore be used in type 2 diabetes patients with declining renal function, but with caution . Similarly, the meglitinide, nateglinide, undergoes hepatic metabolism that gives rise to weakly active metabolites that are excreted predominantly through the kidneys . Furthermore, 15% of the drug is excreted unchanged in urine . Given the reduced clearance in ckd and the likelihood of hypoglycemic episodes, cautious use in these special populations, particularly in more advanced stages of ckd, is warranted . In contrast, repaglinide appears to have a lower risk of hypoglycemia and might be of use in ckd stages 3 and 4, but requires low initial dosing and careful dose titration in these patients62 . The use of metformin, the standard firstline therapy of type 2 diabetes, also poses a risk for diabetic patients with ckd as a result of lactic acidosis, and is therefore contraindicated in patients with advanced renal dysfunction57 . Whether this medication provides an additional risk to the underlying comorbidity or acts directly to bring about lactic acidosis requires further investigation63 . Treatment with glycosidase inhibitors in type 2 diabetes patients with ckd (stage 35) is also not recommended, because of the potential risk of hepatic damage as a result of cumulated dose effects57 . In contrast, a few small, shortterm studies have implicated a renoprotective role for tzd, as it resulted in greater reductions in albuminuria than metformin or sulfonylureas . Whether this translates into direct prevention of renal function decline, however, requires further study64 . Nevertheless, tzd undergo hepatic metabolism and have a low risk of hypoglycemia, making them appropriate for use in ckd . Of note, fluid retention is a known sideeffect of tzd, and in ckd patients this outcome could be exacerbated . Dpp4 inhibitors are the most recent addition to the type 2 diabetes treatment paradigm and have been developed in recognition of the multifactorial nature of type 2 diabetes pathology . This drug class mediates its antihyperglycemic effects by preventing the degradation of the incretin hormones these hormones are released in the intestine after a meal, and target the pancreas by increasing glucosedependent insulin secretion and suppressing glucagon secretion, which subsequently improves both fasting plasma glucose (fpg) and postprandial glucose (ppg) levels . Dpp4 inhibitors are therefore important in regulating glucose homeostasis67 . Furthermore, beneficial effects on pancreatic cells have also been reported with this drug class and therefore it might play a role in preventing or slowing the progression of cell dysfunction68 . There are currently five dpp4 inhibitors approved for type 2 diabetes: sitagliptin, vildagliptin, saxagliptin, alogliptin (japan) and linagliptin; and their efficacy and tolerability have been studied in several clinical trials72 . Although there are limited direct headtohead studies between dpp4 inhibitors that have been carried out to date, observations across studies suggest that all five agents have comparable efficacy as reflected in their levels of hba1c reduction . When compared with other antihyperglycemic agents, headtohead studies showed dpp4 inhibitors to have similar efficacy to metformin, sulfonylureas, tzd and glycosidase inhibitors73 . In terms of tolerability, all five dpp4 inhibitors have been shown to be well tolerated with low rates of adverse effects and, in general, comparable with placebo or comparator72 . Furthermore, the issues of hypoglycemia and weight gain frequently seen with the more traditional antihyperglycemic therapies are not readily observed with dpp4 inhibitors . Although there is little to differentiate between these agents in terms of observed efficacy, each differs in their chemistry and pharmacokinetic profile, which might translate into differences in their clinical applicability79 . Indeed, dpp4 inhibitors differ in their halflives: sitagliptin, alogliptin and linagliptin have longhalf lives, allowing for oncedaily administration; vildagliptin has a short halflife and twicedaily dosing is recommended . Saxagliptin has a short halflife, but given its active metabolite, oncedaily dosing is adequate79 . Furthermore, on examining their metabolism and excretion profiles, differences between the dpp4 inhibitors have emerged . Sitagliptin, vildagliptin, alogliptin and saxagliptin are predominantly excreted through the kidneys72 . Despite the renal excretion profiles of these agents, their favorable tolerability in patients with various severity of renal dysfunction still permits their use in such patients, although dose modifications are recommended in line with increasing kidney dysfunction (table 2). In contrast, linagliptin is excreted through the bile and with approximately 5% being renally excreted80 . Furthermore, recent data suggest that dose modifications of linagliptin are not required irrespective of the severity of renal dysfunction (table 2)81 . Linagliptin might therefore represent an important addition to the currently limited therapeutic armamentarium for achieving glycemic control in type 2 diabetes patients with declining renal function . In recognition of the favorable clinical profile of dpp4 inhibitors, recent international guidelines recommend dpp4 inhibitors as an option in both firstline (when fpg and ppg levels are elevated) and combination therapy82 . Thus, type 2 diabetes patients, particularly special populations like those who are renally impaired, benefit from the emergence and evolution of newer antidiabetic agents, such as the dpp4 inhibitors, that address the limitations of older therapies, and allow for a more tailored and effective approach to disease control . Longterm studies to assess the impact of dpp4 inhibitors on reducing the risk of diabetes complications are awaited . Dpp4 inhibitors represent a good choice of oad for patients with type 2 diabetes . Within this drug class, linagliptin, a novel dpp4 inhibitor, possesses favorable and comparable profiles in terms of both efficacy and adverse events, and is indicated for use in all patients with type 2 diabetes . Furthermore, linagliptin is the only dpp4 inhibitor that is not predominantly dependent on renal metabolism . Many antidiabetic therapies are often of little use in renallyimpaired type 2 diabetes patients because of contraindications or increased risk of hypoglycemia . The favorable pharmacokinetic profile of linagliptin has advantageous implications given the importance of addressing the decline in renal function in asian patients with type 2 diabetes early . In addition, the lack of dose modification requirements and ease of dosing (one dose, once daily) further support the suitability of linagliptin in these difficulttotreat populations.
Pott's disease accounts for 50% of the cases of skeletal tuberculosis (tb), 15% of the cases of extrapulmonary tuberculosis (eptb) and 2% of all cases of tb.1 the diagnosis of pott's disease is mostly based on clinicoradiological observations substantiated by staining and culture methods to detect the causative organism . The typical tubercular lesions of the spine can be diagnosed by radiological methods as their sensitivity is increased by the advent of newer imaging techniques like computed tomography and magnetic resonance imaging (ct and mri). These techniques give sufficient information about lesions in the bone and tissues; although the definite diagnosis is based on tissue / pus examination.2 the conventional bacteriological examination includes ziehl - neelsen (zn) microscopy and culture for acid fast bacilli (afb). Zn microscopy method is a popular technique routinely used in the clinical laboratories worldwide, due to its simplicity, cost effectiveness and rapidity, but it suffers with low sensitivity and requires 10 to 10 bacilli / ml in the clinical specimens to be positive.34 the culture of mycobacterium tuberculosis (m. tuberculosis) is a gold standard method for diagnosis but it also has limitations like that required 6 to 8 weeks due to the slow growth of m. tuberculosis and is often negative, it still needs 10 -10 bacilli / ml (live bacilli) in clinical specimens for culture recovery and also stringent test conditions that is difficult to implement at primary or secondary clinical laboratories.56 moreover, histopathological examination plays a valuable role in the diagnosis of pott's disease but sometime it may be inconclusive and in addition need high expertise and the final reporting also takes more than 1 week.6 recently, the molecular biology technique, polymerase chain reaction (pcr) represents a major advance in the diagnosis of tb.6 with the use of amplification systems, nucleic acid sequences unique to m. tuberculosis can be detected directly in clinical specimens, offering better accuracy than zn microscopy and greater speed than culture . The pcr has shown very promising results for early and rapid diagnosis of the disease due to its detection limit of one to 10 bacilli in various clinical specimens.6 the present study was undertaken to evaluate the efficiency and effectiveness of different laboratory diagnostic modalities along with the role of pcr in the diagnosis of clinicoradiological suspected cases of pott's disease . 62 clinicoradiological suspected cases of pott's disease with neurological complications [table 1] were prospectively enrolled in this study, from 2008 to 2011 in the department of neurosurgery, at a tertiary care hospital, india . The specimens of these patients such as pus and tissue were obtained either during surgery or under ct guidance . Clinicoradiological evidence of suspected pott's disease we included clinicoradiological suspected cases of pott's spine who underwent either open biopsy or ct guided aspiration at our institute . We excluded those subjects who did not give consent for biopsy or ct guided aspiration or biopsy showed neoplastic pathology . The specimens were examined by following methods: histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff (pas) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson (bd, sparks, md, usa) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone (nap) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex (mtbc) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 (123 base pairs [bp]) primer . Histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff (pas) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson (bd, sparks, md, usa) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone (nap) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex (mtbc) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 (123 base pairs [bp]) primer . Dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 . Mb505 bacterial and yeast genomic dna miniprep purification spin kit, hi - media laboratories private limited mumbai, india . The reaction mixture contained 10 l pyrostart fast pcr master mix 2x (dntp, taq polymerase with mgcl2, fermentas, india), 1 l (10 pmole) of each primer, 3 l water (nuclease free) and 5 l of extracted dna . The oligonucleotide primers11 were used forward and reverse sequence: cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively (sbs gene tech co. ltd . ). These primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc . It was performed in a programmable thermal cycler (mj research, ptc-100, gmi, inc . Each cycle comprised denaturation at 94c for 2 min, annealing at 68c for 2 min and primer extension at 72c for 1 min . After the 40 cycles, the additional extension for 10 min at 72c was carried out . The amplified products were separated on 2% agarose gel, visualized on an ultraviolet - transilluminator (bangalore genei, bangalore, india). The presence of 123 bp fragment indicated as a positive test for mtbc [figure 1]. The positive controls included the dna of h37rv strain and negative control included pcr grade water . Polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel . Lane 1 (l1) and l9 ladder 100 base pairs, l2 positive and l3 negative control, l4, 5, 6, 8 showed positive and l7 showed negative specimens a definite treatment either by surgery (46 cases) or by only anti tubercular treatment (att) (9 cases) was offered . We followed the following major indications for surgical intervention in our setup: (1) advanced cases of neurological involvement such as marked sensory or sphincter disturbances, flaccid paralysis or severe flexor spasms . (2) prevertebral cervical abscess with difficulty in respiration and deglutition, seemingly difficult resolution on chemotherapy during conservative treatment . (3) worsening of already present neurological complications during the treatment . (5) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att . A four drug regimen chemotherapy consisting of isoniazid, rifampicin, pyrazinamide and ethambutol (hrze) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol, following or along with surgery . Four - drug regimen for at least 4 - 6 weeks to gauge the response was given (8 - 12 mg / kg / day rifampicin, 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose; and pyrazinamide 15 - 20 mg / kg / day in two divided doses), if not contraindicated the drug therapy was continued for 18 months12 . In cases of no improvements or hepatotoxicity, the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them . The therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity, sensory loss of limbs, autonomic dysfunctions and constitutional symptoms [table 2]. The liver function test was done as per protocol to rule out the subclinical hepatotoxcity . The radiological assessment was done by using followup mri, the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute . Criteria for clinical improvement: (first (pain) is mandatory and any other two improved symptoms / sign) this study was approved by 41 institutional ethics committee (a-04 pgi / imp / ec/41/28/2/2008). The final diagnosis was established by using the collective results of zn microscopy, bactec culture, histopathological findings and response to att . The efficiency of test tools was calculated as (total positive cases / total number of cases) 100 . The positive concordance between traditional tools and pcr were assessed using the kappa coefficient (> 0.75, excellent agreement; <0.4, poor agreement; 0.4 and 0.75, good to a fair agreement)13 and significance difference was analyzed by the chi square (2) test with the help of the spss 15.1 version . The cases were labeled as definite tb if at least one performed test (zn microscopy / bactec culture / or histopathology) was found positive . The specimens were examined by following methods: histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff (pas) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson (bd, sparks, md, usa) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone (nap) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex (mtbc) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 (123 base pairs [bp]) primer . Histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff (pas) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson (bd, sparks, md, usa) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone (nap) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex (mtbc) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 (123 base pairs [bp]) primer . Dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 . Mb505 bacterial and yeast genomic dna miniprep purification spin kit, hi - media laboratories private limited mumbai, india . The reaction mixture contained 10 l pyrostart fast pcr master mix 2x (dntp, taq polymerase with mgcl2, fermentas, india), 1 l (10 pmole) of each primer, 3 l water (nuclease free) and 5 l of extracted dna . The oligonucleotide primers11 were used forward and reverse sequence: cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively (sbs gene tech co. ltd . ). These primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc . It was performed in a programmable thermal cycler (mj research, ptc-100, gmi, inc . Each cycle comprised denaturation at 94c for 2 min, annealing at 68c for 2 min and primer extension at 72c for 1 min . After the 40 cycles, the additional extension for 10 min at 72c was carried out . The amplified products were separated on 2% agarose gel, visualized on an ultraviolet - transilluminator (bangalore genei, bangalore, india). The presence of 123 bp fragment indicated as a positive test for mtbc [figure 1]. The positive controls included the dna of h37rv strain and negative control included pcr grade water . Polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel . Lane 1 (l1) and l9 ladder 100 base pairs, l2 positive and l3 negative control, l4, 5, 6, 8 showed positive and l7 showed negative specimens a definite treatment either by surgery (46 cases) or by only anti tubercular treatment (att) (9 cases) was offered . We followed the following major indications for surgical intervention in our setup: (1) advanced cases of neurological involvement such as marked sensory or sphincter disturbances, flaccid paralysis or severe flexor spasms . (2) prevertebral cervical abscess with difficulty in respiration and deglutition, seemingly difficult resolution on chemotherapy during conservative treatment . (3) worsening of already present neurological complications during the treatment . (5) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att . A four drug regimen chemotherapy consisting of isoniazid, rifampicin, pyrazinamide and ethambutol (hrze) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol, following or along with surgery . Four - drug regimen for at least 4 - 6 weeks to gauge the response was given (8 - 12 mg / kg / day rifampicin, 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose; and pyrazinamide 15 - 20 mg / kg / day in two divided doses), if not contraindicated the drug therapy was continued for 18 months12 . In cases of no improvements or hepatotoxicity, the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them . The therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity, sensory loss of limbs, autonomic dysfunctions and constitutional symptoms [table 2]. The liver function test was done as per protocol to rule out the subclinical hepatotoxcity . The radiological assessment was done by using followup mri, the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute . Criteria for clinical improvement: (first (pain) is mandatory and any other two improved symptoms / sign) this study was approved by 41 institutional ethics committee (a-04 pgi / imp / ec/41/28/2/2008). The final diagnosis was established by using the collective results of zn microscopy, bactec culture, histopathological findings and response to att . The efficiency of test tools was calculated as (total positive cases / total number of cases) 100 . The positive concordance between traditional tools and pcr were assessed using the kappa coefficient (> 0.75, excellent agreement; <0.4, poor agreement; 0.4 and 0.75, good to a fair agreement)13 and significance difference was analyzed by the chi square (2) test with the help of the spss 15.1 version . The cases were labeled as definite tb if at least one performed test (zn microscopy / bactec culture / or histopathology) was found positive . The study group consisted of 62 specimens from suspected pott's spine individuals . On the basis of histopathological examination, 7 cases (5 specimens from open biopsy and 2 from fine needle aspiration [fna]) were excluded from the study as they were diagnosed to be neoplastic . Out of rest 55 cases, 46 specimens were obtained through open biopsy during surgery and 9 were ct guided fna . Further, in all (46) open biopsy specimens, 7 were positive for epithelioid cell granulomas with afb, 27 revealed granulomatous lesions suggestive of tb with or without the presence of the langerhans giant cell . However, the histological diagnosis could not be reached in 12 specimens because of either suspicious granulomatous lesions or on account of inconclusive histology . Five of nine ct guided fna specimens were diagnosed as tubercular and 4 specimens showed no granulomatous / no malignant lesions . The received specimens included 31 (56.4%) pus and 15 (27.3%) tissues through open biopsy along with 6 (11%) pus and 3 (0.54%) tissues were obtained through ct guided fna . The overall collected specimens were consisted 37 pus and 18 tissue specimens (either open biopsy or by ct fna) were analyzed . The zn microscopy was positive in total 20 (36.4%) of 55 cases (18/37 cases in pus and 2/18 cases in tissue specimens). The bactec culture was positive 27 (49%) of 55 cases (22/37 in pus and 5/18 in tissue specimens). The desired amplification of pcr is6110 was positive in 37 (67.5%) of 55 cases (30/37 cases in pus and 7/18 in tissue specimens) [table 3]. The kappa coefficient of pcr with zn microscopy (= 0.37, [p = 0.001]) showed poor agreement and with the bactec culture (= 0.57, [p <0.001]) it showed a good agreement [figure 2]. Result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools . (znm = zn microscopy, bc = bactec culture and hp = histopathology) on histopathological results; 39 (71%) cases were positive and 16 (29%) were negative for tb . Out of 39 positive cases, 31 (56.4%) were positive on pcr also . Thus, the false negative and positive of pcr was found to be 8 (14.4%) and 6 (11%) respectively . Therefore, the kappa coefficient of pcr with histopathology was (= 0.4 [p = 0 . 004]) slightly good [figure 2]. In 55 cases, it was possible to reach a final diagnosis through the combined result of all performed tests (without consideration of pcr results) and with att response . Hence, among all 55 cases, 45 (82%) cases were tb positive test and 10 (18%) were negative by all tests carried out (zn microscopy, bactec culture and histopathology). Although pcr showed 67.3% (37/55) sensitivity at the final diagnosis, while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests . Therefore, the kappa coefficient of pcr with the combined results of all traditional tests (= 0.63 [p <0.001]) ultimately reached to a fair positive agreement [figure 2]. The present study, the combination of (a) zn microscopy with bactec, the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture . (b) zn microscopy with histopathology, the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy . (c) bactec culture with histopathology, the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture, where the histopathology was negative . (d) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology . (e) zn microscopy with pcr, the total positivity was 38 cases, in which 18 more cases were positive on pcr method . (f) bactec culture with pcr, the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . (g) histopathology with pcr, the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr . (h) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [table 4]. The received specimens included 31 (56.4%) pus and 15 (27.3%) tissues through open biopsy along with 6 (11%) pus and 3 (0.54%) tissues were obtained through ct guided fna . The overall collected specimens were consisted 37 pus and 18 tissue specimens (either open biopsy or by ct fna) were analyzed . The zn microscopy was positive in total 20 (36.4%) of 55 cases (18/37 cases in pus and 2/18 cases in tissue specimens). The bactec culture was positive 27 (49%) of 55 cases (22/37 in pus and 5/18 in tissue specimens). The desired amplification of pcr is6110 was positive in 37 (67.5%) of 55 cases (30/37 cases in pus and 7/18 in tissue specimens) [table 3]. The kappa coefficient of pcr with zn microscopy (= 0.37, [p = 0.001]) showed poor agreement and with the bactec culture (= 0.57, [p <0.001]) it showed a good agreement [figure 2]. Result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools . On histopathological results; 39 (71%) cases were positive and 16 (29%) were negative for tb . Out of 39 positive cases, 31 (56.4%) were positive on pcr also . Thus, the false negative and positive of pcr was found to be 8 (14.4%) and 6 (11%) respectively . Therefore, the kappa coefficient of pcr with histopathology was (= 0.4 [p = 0 . 004]) slightly good [figure 2]. In 55 cases, it was possible to reach a final diagnosis through the combined result of all performed tests (without consideration of pcr results) and with att response . Hence, among all 55 cases, 45 (82%) cases were tb positive test and 10 (18%) were negative by all tests carried out (zn microscopy, bactec culture and histopathology). Although pcr showed 67.3% (37/55) sensitivity at the final diagnosis, while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests . Therefore, the kappa coefficient of pcr with the combined results of all traditional tests (= 0.63 [p <0.001]) ultimately reached to a fair positive agreement [figure 2]. The present study, the combination of (a) zn microscopy with bactec, the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture . (b) zn microscopy with histopathology, the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy . (c) bactec culture with histopathology, the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture, where the histopathology was negative . (d) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology . (e) zn microscopy with pcr, the total positivity was 38 cases, in which 18 more cases were positive on pcr method . (f) bactec culture with pcr, the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . (g) histopathology with pcr, the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr . (h) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [table 4]. Pott's disease, accounts for 2% of all tb infections.1 the diagnosis of pott's disease is principally based on classical clinical manifestations of spinal infection supplemented by modern imaging like ct and mri.1415 the ct and mri in particular can detect the subtle changes in the spine, from initial changes in the intensity pattern of the vertebrae to the extreme changes like deformity of the spine including soft tissue shadow, granulation tissue and pus etc.16 however, the various pathologies including neoplastic and other inflammatory conditions simulate with pott's diseases on imaging . The current study excluded 7 of 62 cases; they were diagnosed to be neoplastic by the histopathological examination . It signifies that even strong clinicoradiological diagnosis based on classical or nonclassical radiological and clinical findings can be wrong on histopathological evaluation . The remaining 55 cases, 20 (36.3%) were tested positive with zn microscopy and 27 (49%) with bactec culture . An earlier report by jain et al.17 found 14.8% positivity on zn microscopy and 11.11% positivity on culture method in cases of spinal tb . The zn microscopy and bactec culture reports revealed higher positivity in relation to the earlier published series . Our higher detection rate was because of the fact that we used the liquid culture medium bactec 12b medium . It is the rapid, sensitive and efficient method for the isolation of mycobacterium in a clinical laboratory as compared to solid medium lowenstein jensen.19 moreover, it is established today that all the traditional tests need to have a minimum concentration of bacilli for yielding positive results as mentioned in the introduction . Hence, if the specimens, which are naturally having more concentration of bacilli, like sputum and pus it may become easy to detect m. tuberculosis.20 similarly, in our study the pus specimens had higher recognition of tubercular bacilli on zn microscopy and bactec culture methods [table 3]. The study quoted the detection rate of eptb including pott's spine varying from 53% to 81%.182122 the histopathological diagnosis could be reached in 71% (39 out of 55) of the cases which were either confirmed by the presence of afb in 12.7% or strongly suggestive of tubercular pathology . In our series, we found 16 cases were negative on histology . Among these negative cases, 10 out of 16 cases had been under anti tubercular therapy for more than 6 months and another reason of histology negative might be a possible sampling error during the collection of the representative tissues for processing . The positivity of fna specimens seems to higher than surgical specimens, however, we require a long sample size for definite conclusion . Molecular diagnostic tool, such as pcr has been shown to have higher efficiency in the diagnosis of pulmonary tb as well as eptb.4 it has been studied on clinical specimens of sputum, cerebrospinal fluid, edta - blood, pleural fluid, fluid from fistulae and pus from wounds.2324 in an attempt to increase diagnostic precision, we performed pcr is6110, which was detected in 37/55 (67.3%) cases . Various studies documented the increased positivity by pcr targeting is6110 in specimens of eptb i.e., sekar et al.25 found 63% positivity, negi et al.26 73% and tiwari et al.27 62% positivity among clinical specimens of eptb . The overall positive agreement shows a fair agreement (= 0.63) between pcr and definite tb . The false negative results were observed in 14.5% cases out of 45 cases diagnosed by all performed traditional tests . The reasons of false negative results of pcr may be on account of insufficient specimens, irregular distribution of mycobacterium bacilli in specimens, the presence of extensive necrosis and presence of inhibitors.28 the limitation of our study are a low positive correlation (= 0.4) of pcr with histopathological findings . This can be explained up to some extent by the specimen variation as that is different specimens (such as pus, tissue) from the same subjects . Surgeons prefer to send tissue specimens for histology and pus for zn microscopy, bactec culture and pcr . In this study histology was done on tissue specimens (all 55 cases), while zn microscopy, bactec culture and pcr were done in 37 pus and 18 tissue specimens . In our diagnostic battery, all performed traditional tests were diagnosed 45/55 cases and pcr was alone detected 37/55 cases . Thus, positive results of pcr with the clinicoradiological assumption it would be possible to make a decision in suspected pott's disease to start early treatment to prevent irreversible complications . In the present study, it is observed that no single diagnostic modality is having a good positivity rate . Hence, a combination of diagnostic battery is required for rapid diagnosis and better results . The combination of bactec culture and histopathology has detected a maximum of 45 cases . While the combination of pcr and histopathology has also detected same number of cases table 4 . As culture takes long incubation time for results, the combination of pcr and histopathology can be applied for rapid detection of pott's disease . To conclude, for diagnosis of pott's disease, the combined results of zn microscopy, bactec culture and histopathology are most appropriate . However, for rapid diagnosis the combination of pcr amplification and histopathology offer a better prospect.
Such proteins perform many basic functions including cell signalling, transcription regulation, energy conservation and transformation, and ion exchange . Membrane proteins are difficult to study, since they are inserted into lipid bilayers and expose to the polar outer and inner environments portions of different sizes . It is therefore difficult to purify them in the native, functional form and even more difficult to crystallize them . For such technical reasons, only a small fraction of the protein data bank structures are membrane proteins (<1% of the total number of structures and far less than their estimated abundance in cells) (1). There are a number of computational methods available to predict the topology of membrane proteins, which consists of two basic features: (i) the location of transmembrane domains along the protein chain and (ii) the location of the n- and c - termini with respect to the lipid membrane . Topological models are sufficient in many instances to design simple experiments in order to prove the location of the n- and c - protein termini, that of the inner and outer loops with respect to the membrane plane, and concomitantly the number of transmembrane segments in the chain . However, the best - performing methods are offered at different servers and are endowed with different graphical interfaces . This hampers the direct comparison of predictions, especially for experimentalists interested in comparing their results with computational methods . Currently, two other web servers of which we are aware (2,3) are available and separately comprise two of the predictors that we implement tmhmm2.0 (4) and memsat (5). The novelty of our web server is to include in the same framework ensemble (6) and prodiv (7), two powerful methods that became available only recently . Furthermore the available web servers render only the consensus prediction, without allowing a critical discrimination among different predictions that may be useful, considering that different predictors may highlight different properties, depending on the different implementation . Automatic topology annotation for membrane proteins has been included among the efforts of the biosapiens european network of excellence () with the specific aim of taking into consideration different predictors for annotating membrane proteins in the human genome . The common platform for these efforts is the biosapiens distributed annotation servers (das) (). In this context, the pongo - das server () provides topology annotation for the all - alpha membrane proteins of the human genome . This is done using the das protocol () to answer at das queries that can be seen using specific visualizers such as dasty (8) or ensembl (9). In order to allow users to browse directly the pre - computed transmembrane annotations the annotation is carried out using four selected predictors, namely ensemble1.0 (6) and prodiv (7), in order to allow a direct comparison of the topology prediction for the sequence at hand . The server has also been set up to make it possible to predict the topology of any putative membrane proteins of interest regardless of provenance . The topological models computed by the different predictors can be directly obtained simply by pasting in a box the sequence of interest and looking at the results . Recently developed web technologies (e.g. Ajax) are used to improve the user interface . The website implements the following predictors: memsat is a new version of the memsat predictor of transmembrane helices in proteins (4). Tmhmm2.0 which is a predictor of transmembrane helices in proteins based on hidden markov models (5). It has been shown that it performs quite well taking into account that it uses only single sequence information . Ensemble1.0 is an ensemble of two hidden markov models and one neural network (6). Ensemble takes also advantage of the evolutionary information derived by multiple sequence alignment, both for the neural network and the hidden markov model systems . Prodiv_tmhmm_0.91 is a recent predictor of transmembrane helices in proteins (7) which uses a hidden markov model similar to tmhmm, but exploits the evolutionary information derived by multiple sequence alignment . Spep is a signal peptide predictor based on combination of neural networks (10). This predictor has performance similar to the most widely used signalp (11), and it has been included since it is quite common that signal peptides are mispredicted as transmembrane helices . In the context of the biosapiens project, we downloaded the uniprot dataset (september 22, 2004) which consists of 33 135 human protein sequences, and for future use also the ipi dataset (august 5, 2004) which consists of 46 782 human proteins . The union of the two datasets comprises 50 600 sequences from the human genome . It is worth noticing that these 50 600 sequences do not include the known splice variants, since those variants are not presently included as unique uniprot codes . The choice of annotating uniprot sequences is well justified from the fact that ensembl gene products (in contrast to the uniprot entries) are not stable; for instance, only 60% of the sequences are common (and conserved) between the ensembl releases 34 and 35, respectively . We then use four state - of - the - art predictors already described in the literature in order to identify the most probable integral membrane proteins . In this way the union of the predictions obtained with the four methods gives a set of likely membrane proteins, while the intersection contains those chains on whose annotation as membrane proteins all the predictors agree upon . The pre - computed annotations are filtered with spep before being processed by the transmembrane predictors . This is done since it is quite common that signal peptides are mispredicted as transmembrane helices by all the predictors implemented in our web server . In the case of a positive spep answer, the system cuts the corresponding predicted segment and processes the remaining part of the sequence with the four transmembrane predictors . The website implements the following predictors: memsat is a new version of the memsat predictor of transmembrane helices in proteins (4). Tmhmm2.0 which is a predictor of transmembrane helices in proteins based on hidden markov models (5). It has been shown that it performs quite well taking into account that it uses only single sequence information . Ensemble1.0 is an ensemble of two hidden markov models and one neural network (6). Ensemble takes also advantage of the evolutionary information derived by multiple sequence alignment, both for the neural network and the hidden markov model systems . Prodiv_tmhmm_0.91 is a recent predictor of transmembrane helices in proteins (7) which uses a hidden markov model similar to tmhmm, but exploits the evolutionary information derived by multiple sequence alignment . Spep is a signal peptide predictor based on combination of neural networks (10). This predictor has performance similar to the most widely used signalp (11), and it has been included since it is quite common that signal peptides are mispredicted as transmembrane helices . In the context of the biosapiens project, we downloaded the uniprot dataset (september 22, 2004) which consists of 33 135 human protein sequences, and for future use also the ipi dataset (august 5, 2004) which consists of 46 782 human proteins . The union of the two datasets comprises 50 600 sequences from the human genome . It is worth noticing that these 50 600 sequences do not include the known splice variants, since those variants are not presently included as unique uniprot codes . The choice of annotating uniprot sequences is well justified from the fact that ensembl gene products (in contrast to the uniprot entries) are not stable; for instance, only 60% of the sequences are common (and conserved) between the ensembl releases 34 and 35, respectively . We then use four state - of - the - art predictors already described in the literature in order to identify the most probable integral membrane proteins . In this way the union of the predictions obtained with the four methods gives a set of likely membrane proteins, while the intersection contains those chains on whose annotation as membrane proteins all the predictors agree upon . The pre - computed annotations are filtered with spep before being processed by the transmembrane predictors . This is done since it is quite common that signal peptides are mispredicted as transmembrane helices by all the predictors implemented in our web server . In the case of a positive spep answer, the system cuts the corresponding predicted segment and processes the remaining part of the sequence with the four transmembrane predictors . Pongo has two different usage options: pongo - das accessing the pre - computed annotations using keywords or das queries;pongo - pred that is an enhanced and modern version of a standard through - the - web server application . Pongo - das accessing the pre - computed annotations using keywords or das queries; pongo - pred that is an enhanced and modern version of a standard through - the - web server application . In the case of pongo - das we implemented two types of result visualization: by means of a das, which is a client server system in which a single client integrates information from multiple servers . It allows a single machine to gather up genome annotation information from multiple distant web sites, collate the information, and display it to the user in a single view . Little coordination is needed among the various information providers, anda user - friendly interface that allows the search for a specific prediction.through the das protocol a web client like ensembl can obtain the list of the annotations for each human protein using its uniprot code as requested for the das queries (); in order to check this url the client needs a das client such as the one that will be embedded in ensembl pages at ebi and a uniprot code for the sequence . A user can directly query the database, without using the das infrastructure, using the web interface available at (figure 1) the user can then use a crc64 (the sequence hash code), or the sequence uniprot or ipi code to get the predictions in a graphical view . An example of a sequence filtered with the different predictors is presented together with the detailed sequence annotation (figure 2). A colour code is used to quickly identify the transmembrane segments . By means of a das, which is a client server system in which a single client integrates information from multiple servers . It allows a single machine to gather up genome annotation information from multiple distant web sites, collate the information, and display it to the user in a single view . Little coordination is needed among the various information providers, and a user - friendly interface that allows the search for a specific prediction . Conversely, pongo - pred provides the user with a unique framework for membrane protein topology and signal peptide predictions . Another interesting feature of pongo - pred is the javascript - enabled portlet that in real time refreshes the queue status (without reloading of the submission page and with very lightweight xml http requests). In particular, on the left - side of the page the user can realise whether her / his submission is a new call, whether it is running (in this case the starting date and an absolute link is provided for bookmarking), or whether it has been processed . The pongo homepage . On the left - side it is possible to follow the status of the different queries and the starting of a new action.
The epstein barr virus (ebv) infects b cells . In most cases, ebv - positive patients develop a severe form of infectious mononucleosis which typically manifests with fever, tonsillopharyngitis, lymphadenopathy and hepatosplenomegaly . Deficiencies may be characterized by abnormal activity of lymphocyte function leading to hemophagocytosis and multi - organ failure . Although ebv - associated hlh carries a high mortality rate, quick diagnosis and appropriate treatment can subside the disease . We describe here a patient who developed hepatosplenomegaly and pancytopenia at 2 years of age but achieved automatic recovery ., there was a clinical suspicion of ebv - induced hlh and the patient was given the option of treatment with rituximab . She received antibiotic treatment due to fever and chills . During the hospitalization period, the patient recovered and developed hemophagocytosis with pancytopenia as well as sores on the lip . The patient underwent bma and nothing abnormal was found . In spite of treatment, the disease did not subside and she developed fever, abdominal distention and edema in her lower extremities . Physical examination showed that the patient had decreased deep - tendon reflexes (dtr) and was unable to walk . Clindamycin, ceftazidime, dexamethasone, ivig (intravenous immunoglobulin) and g - csf were administered . Since the patient did not achieve full recovery, she was transferred to our center . Based on clinical symptoms and lab tests (table 1), bma was performed once again and the result raised suspicion of ebv - induced secondary hlh . Rituximab was injected once and the patient developed a blood ebv load of zero within one week . In order to control abnormal stimulation of the immune system, the patient was investigated for mutations in il-2-inducible t cell kinase (itk) and negative test results were received . (the patient was negative for mutations in il-2-inducible t cell kinase (itk)). She is now considered to be an acceptable candidate for transplantation from an hla matched sibling donor . Hlh can be divided into two subgroups: primary or familial hlh and secondary hlh . The primary form is an inflammatory disease which is similar to secondary one on the basis of symptoms . Primary hlh usually arises in young children less than one year old (70% cases); however, in rare cases it can also occur in adults (perforin gene mutations have been identified in patients with primary hlh). For example, type 2 is caused by mutations in genes 21 - 22 and type 5 is due to mutations in the munc18 - 20 that may be accompanied by hypogammaglobulinemia . Hypogammaglobulinemia was not confirmed in the patient because it was not possible to check mutation (the related tests were sent to germany to examine the type 5 disease). Ebv may also be accompanied by hypogammaglobulinemia for a certain period secondary hlh is associated with immunologic stimulation caused by malignancies and bacterial or congenital infections . The most common causes of secondary hlh are viral infections by ebv, cmv, prob19 and hiv . However, it may also be seen in patients with immune system defects ., 4 secondary hlh is a known complication of ebv infection, particularly in patients with x - linked lymphoproliferative disease . The incidence of x - linked lymphoproliferative syndrome is 1 in 1,000,000 male infants . Diagnostic criteria for hlh disease diagnosis of hlh requires the presence of all 5 major criteria . If the patient meets only 4criteria but the clinical suspicion for hlh is high, one should initiate treatment, because delays may be fatal . Alternative criteria 1 or a combination of 2 and 3 may substitute for 1 major criterion . Adapted from henter ji, elinder g, ost a.semin oncol . 1991; 18:29 - 33 . It means that ebv - infected b cells stimulate cytotoxic t lymphocytes leading to hypercytokinemia and stimulation of histolytic cells . More recently, it has been found that chronic stimulation by ebv may cause chronic hlh in the patient . On the other hand, ebv causes stimulation, generation and uncontrolled secretion of t - and nk- cells, as well as generation of il2, infa and il6 . There is another mechanism by which ebv stimulates membrane protein (lmp-1) in cells . Similar to xlp, lmp-1 may cause acquired immune deficiency, leading to hlh accordingly . Treatment follows two goals: first, to suppress immune response using ivig, steroid and hlh protocol . Second, to inhibit undesirable influence of immune response and cytokine - stimulated cell activation using immunochemotherapy . In cases with neurological symptoms the level of viral dna which is measured by quantitative pcr may be useful to demonstrate treatment response predicting mortality . The dna level reached zero in our patient . Using ivig prior to start of protocol 2004 it has also been recommended in the treatment of ebv - induced secondary hlh monthly . Decrease of b cells, which have been contaminated by ebv, actually causes decrease of viruses, as it may be useful for our patient as well . Finally, treatment - refractory patients, transplant candidates, patients with familial type of hlh, or recurrent disease undergo chemotherapy for 8 weeks . Patients who respond favorably to treatment do not undergo transplantation but those who experience relapse and exhibit neurological symptoms are considered for transplantation . Severe infection of ebv is not associated with life - threatening condition, but our case study needs urgent treatment . In patients with long - term ebv infection, pancytopenia, coagulopathy and hepatosplenomegaly, bma will be performed and the serum ferritin level will be measured as well . For patients with secondary hlh, rituximab can be used when hlh is associated with ebv to inhibit the immune response . Thus, the main aim of treatment is to reduce the rate of mortality in patients with hlh.
We used data from the hoorn study, a population - based cohort study on glucose metabolism and other cardiovascular risk factors in a general caucasian population, which has been described in detail previously (12). In brief, men and women aged 5075 years were randomly selected from the population register of the town of hoorn, the netherlands; 2,484 subjects participated (response rate 71%). All subjects had a 75-g oral glucose tolerance test, except those in whom type 2 diabetes had previously been diagnosed . An extensive metabolic and cardiovascular investigation was performed in an age-, sex-, and glucose tolerance stratified, random subsample of 631 participants (89% of those invited), which is used in the present study (12). Participants without representative urine samples available (n = 45) and/or who did not complete at least 7 of the 10 autonomic function tests (n = 31), had a history of neurological disease (n = 5), and were using ace inhibitors (n = 33) and/or drugs known to influence autonomic nerve function (namely antiparkinson drugs, phenytoin, antihistamines, or parasympatholytic, parasympathomimetic, and sympathomimetic drugs) (n = 49) were excluded from the analyses . The present study, therefore, consisted of 490 individuals: 305 with normal glucose metabolism, 71 with igm (including those with impaired fasting glucose and/or impaired glucose tolerance), and 114 with type 2 diabetes . The hoorn study was approved by the ethical review committee of vu university medical centre, amsterdam, the netherlands . Urinary albumin concentration was measured in a first voided sample by rate nephelometry (array protein system, beckman coulter, galway, ireland) with a detection threshold of 6.2 mg / l (intra- and interassay coefficients of variation of 5 and 8%, respectively). Microalbuminuria was present if the albumin - to - creatinine ratio (acr) was in the range of 2.030 mg / mmol (5). The average value of the acr was used if two representative samples (n = 154) were available . Excluding subjects with an acr> 30 mg / mmol (i.e., macroalbuminuria, n = 4) did not materially affect any of the results, which are therefore presented for the entire group . Cardiovascular autonomic function tests were performed as described in detail elsewhere (9). In brief, participants were asked to refrain from smoking and drinking coffee for at least 2 h before the assessments . Tests took place between 8:30 a.m. and 4:00 p.m. in a quiet location with room temperature of 1922c after a resting period of at least 10 min . Ten parameters of cardiac autonomic function were derived from the r - r interval and finger systolic blood pressure (sbp) continuous recordings by a computerized data analyses system developed locally by the department of medical physics, vu university, amsterdam, the netherlands (13). Tests were performed under three conditions: during spontaneous breathing over 3 min in the supine position (mean of all normal to normal r - r intervals [meannn], sd of all normal to normal r - r intervals [sdnn], low - frequency [lf] power, high - frequency [hf] power, and lf/(lf + hf)), during six deep breaths over 1 min in the supine position (expiration - inspiration [ei] difference and baroreflex sensitivity [brs]), and during an active change from lying to standing (sbp difference, r - r interval maximum [rrmax], and r - r interval maximum divided by minimum [rrmax / min]) (for further explanation of measurements, see supplementary table a1, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1544/dc1). We thus obtained results in four tests that reflect heart rate or blood pressure changes due to certain maneuvers, in this case deep breathing or standing up (i.e., ei difference, rrmax, rrmax / min, and sbp difference) (14), five tests of spectral analyses of heart rate variability (hrv) [i.e., meannn, sdnn, lf power, hf power, and lf/(lf + hf)] (15), and one brs measurement (16). In addition, these tests were grouped into those representing predominantly parasympathetic (i.e., ei difference, rrmax, and hf power), sympathetic (i.e., sbp difference), or both functions (i.e., mean - nn, sdnn, lf power, lf/(lf + hf), brs, and rrmax / min) (15,16). Bmi, waist - to - hip ratio (whr), sbp and diastolic blood pressure (dbp), levels of fasting plasma glucose; a1c; insulin; total, hdl, and ldl cholesterol; triglycerides; creatinine; and smoking status were measured as described elsewhere (5,9,12). Glomerular filtration rate was estimated by the short modification of diet in renal disease equation . Hypertension was defined as sbp 140 mmhg and/or dbp 90 mmhg and/or the use of antihypertensive drugs . Prior cardiovascular disease (cvd) was defined when individuals had (any of the following): a history of myocardial infarction, abnormalities on a resting electrocardiogram (minnesota codes 1.11.3, 4.14.3, 5.15.3, or 7.1), previous coronary bypass surgery or angioplasty, peripheral arterial bypass, or nontraumatic amputation, or an ankle - brachial index of <0.9 in either leg . Data on the participants' vital status up to 1 january 2005 were collected from the mortality register of the municipality of hoorn . Information on cause of death was extracted from the medical records of the general practitioners and the local hospital and coded according to the icd-9 . Cardiovascular mortality, including sudden death, was defined by icd-9 codes 390459 and 798 . Information on cause of death could not be obtained for 20 of the deceased individuals . All subjects were followed until death or end of follow - up, at which time they were censored . Baseline characteristics between survivors and nonsurvivors were compared with the use of student's t or tests . We computed z scores ([individual's observed value population mean]/sd) for each of the 10 measurements of c - ad . All z scores (except sbp difference) were inverted and averaged into a total score (c - ad total score), so that higher values reflect greater c - ad . The association between microalbuminuria and c - ad total score was investigated with multiple linear regression analyses . Kaplan - meier survival curves were plotted for cardiovascular mortality among individuals with normoalbuminuria versus microalbuminuria and similarly across tertiles of the c - ad total score, and differences between groups were tested with a log - rank test . Cox proportional hazards regression models were used to calculate the crude and adjusted relative risks (rrs) and respective 95% ci of microalbuminuria (versus normoalbuminuria) and of the c - ad total score (per sd [= 0.605] increase) for cardiovascular and all - cause mortality . Urinary albumin concentration was measured in a first voided sample by rate nephelometry (array protein system, beckman coulter, galway, ireland) with a detection threshold of 6.2 mg / l (intra- and interassay coefficients of variation of 5 and 8%, respectively). Microalbuminuria was present if the albumin - to - creatinine ratio (acr) was in the range of 2.030 mg / mmol (5). The average value of the acr was used if two representative samples (n = 154) were available . Excluding subjects with an acr> 30 mg / mmol (i.e., macroalbuminuria, n = 4) did not materially affect any of the results, which are therefore presented for the entire group . Cardiovascular autonomic function tests were performed as described in detail elsewhere (9). In brief, participants were asked to refrain from smoking and drinking coffee for at least 2 h before the assessments . A light meal> 1 h before the measurements was allowed . Tests took place between 8:30 a.m. and 4:00 p.m. in a quiet location with room temperature of 1922c after a resting period of at least 10 min . Ten parameters of cardiac autonomic function were derived from the r - r interval and finger systolic blood pressure (sbp) continuous recordings by a computerized data analyses system developed locally by the department of medical physics, vu university, amsterdam, the netherlands (13). Tests were performed under three conditions: during spontaneous breathing over 3 min in the supine position (mean of all normal to normal r - r intervals [meannn], sd of all normal to normal r - r intervals [sdnn], low - frequency [lf] power, high - frequency [hf] power, and lf/(lf + hf)), during six deep breaths over 1 min in the supine position (expiration - inspiration [ei] difference and baroreflex sensitivity [brs]), and during an active change from lying to standing (sbp difference, r - r interval maximum [rrmax], and r - r interval maximum divided by minimum [rrmax / min]) (for further explanation of measurements, see supplementary table a1, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1544/dc1). We thus obtained results in four tests that reflect heart rate or blood pressure changes due to certain maneuvers, in this case deep breathing or standing up (i.e., ei difference, rrmax, rrmax / min, and sbp difference) (14), five tests of spectral analyses of heart rate variability (hrv) [i.e., meannn, sdnn, lf power, hf power, and lf/(lf + hf)] (15), and one brs measurement (16). In addition, these tests were grouped into those representing predominantly parasympathetic (i.e., ei difference, rrmax, and hf power), sympathetic (i.e., sbp difference), or both functions (i.e., mean - nn, sdnn, lf power, lf/(lf + hf), brs, and rrmax / min) (15,16). Bmi, waist - to - hip ratio (whr), sbp and diastolic blood pressure (dbp), levels of fasting plasma glucose; a1c; insulin; total, hdl, and ldl cholesterol; triglycerides; creatinine; and smoking status were measured as described elsewhere (5,9,12). Glomerular filtration rate was estimated by the short modification of diet in renal disease equation . Hypertension was defined as sbp 140 mmhg and/or dbp 90 mmhg and/or the use of antihypertensive drugs . Prior cardiovascular disease (cvd) was defined when individuals had (any of the following): a history of myocardial infarction, abnormalities on a resting electrocardiogram (minnesota codes 1.11.3, 4.14.3, 5.15.3, or 7.1), previous coronary bypass surgery or angioplasty, peripheral arterial bypass, or nontraumatic amputation, or an ankle - brachial index of <0.9 in either leg . Data on the participants' vital status up to 1 january 2005 were collected from the mortality register of the municipality of hoorn . Information on cause of death was extracted from the medical records of the general practitioners and the local hospital and coded according to the icd-9 . Cardiovascular mortality, including sudden death, was defined by icd-9 codes 390459 and 798 . Information on cause of death could not be obtained for 20 of the deceased individuals . All subjects were followed until death or end of follow - up, at which time they were censored . Baseline characteristics between survivors and nonsurvivors were compared with the use of student's t or tests . We computed z scores ([individual's observed value population mean]/sd) for each of the 10 measurements of c - ad . All z scores (except sbp difference) were inverted and averaged into a total score (c - ad total score), so that higher values reflect greater c - ad . The association between microalbuminuria and c - ad total score was investigated with multiple linear regression analyses . Kaplan - meier survival curves were plotted for cardiovascular mortality among individuals with normoalbuminuria versus microalbuminuria and similarly across tertiles of the c - ad total score, and differences between groups were tested with a log - rank test . Cox proportional hazards regression models were used to calculate the crude and adjusted relative risks (rrs) and respective 95% ci of microalbuminuria (versus normoalbuminuria) and of the c - ad total score (per sd [= 0.605] increase) for cardiovascular and all - cause mortality . Subjects excluded from the analyses (n = 141) were older, more often had type 2 diabetes, and had a worse risk profile than the subjects included (n = 490) (data not shown). Median duration of the follow - up was 13.6 (range 0.5215.19) years . During the follow - up period, 141 (28.8%) participants died, of whom 53 (37.6%) died of cvd . At baseline, individuals who died had a worse c - ad total score, more often had microalbuminuria and were older, more often were men and had type 2 diabetes and hypertension, and had a higher a1c and whr as compared with the survivors (table 1). Baseline characteristics of survivors versus nonsurvivors data are presented as frequencies (%), means sd, or medians (interquartile range). For a detailed explanation of autonomic function tests, these include diuretics, -blockers, -blockers, calcium channel blockers, and other blood pressure lowering drugs but exclude ace inhibitors (because subjects using these drugs were excluded from the analyses). C - ad total score was associated with microalbuminuria (= 0.33 [95% ci 0.160.51]) (table 2, model 1). After adjustment for potential confounding factors, c - ad total score was still borderline associated with microalbuminuria (0.16 [0.01 to 0.33]) (table 2, model 3). Comparable results were obtained when tests were combined on the basis of their methodology or on the part of the autonomic nervous system they predominately represent . All individual c - ad tests, except lf/(lf + hf), were positively associated with microalbuminuria (although not all statistically significantly so) (data not shown). Association between microalbuminuria and c - ad * model 1: univariate analysis; model 2: adjusted for sex, age, and gts; model 3: model 2 plus adjustments for hypertension, whr, estimated glomerular filtration rate, ldl, hdl, triglycerides, smoking, and prior cvd . Indicates the difference in c - ad score between individuals with microalbuminuria vs. normoalbuminuria . Both microalbuminuria (rr 3.49 [95% ci 1.876.53]) and c - ad total score (2.54 [1.604.04]) these associations were attenuated after adjustment for age, sex, and glucose tolerance status (gts) (2.23 [1.164.29] and 1.81 [1.112.94], respectively; model 2). Further adjustment for other cardiovascular risk factors only slightly further attenuated these rrs (model 3), and when the c - ad total score or microalbuminuria was added to these models, the rr attributable to microalbuminuria remained practically unchanged and both microalbuminuria (2.13 [1.094.17]) and c - ad total score (1.76 [1.052.94]) were independently associated with cardiovascular mortality (model 4). Association of microalbuminuria or c - ad with cardiovascular and all - cause mortality * model 1: univariate analysis; model 2: adjusted for sex, age, and gts; model 3: model 2 plus adjustments for hypertension, whr, estimated glomerular filtration rate, ldl, hdl, triglycerides, smoking and prior cvd; model 4: model 3, with additional adjustment for, respectively, c - ad or microalbuminuria . Rr of microalbuminuria vs. normoalbuminuria or per sd (= 0.605) increase in c - ad total score . Kaplan - meier survival plots for cardiovascular mortality . Among individuals with normo- versus microalbuminuria (a) and across tertiles of c - ad total score (b). Both microalbuminuria and c - ad total score were also associated with all - cause mortality (table 3, model 1). However, after adjustment for age, sex, gts, and other cardiovascular risk factors, microalbuminuria (rr 1.33 [95% ci 0.832.13]), in contrast to c - ad total score (1.52 [1.112.09]), was not independently associated with all - cause mortality (model 3). The associations described above were also investigated by examining autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent (supplementary table a2, available in an online appendix). These additional analyses showed that overall all different groups of tests were associated with increased mortality risk, that these associations were attenuated when further adjusted for age, sex, gts, and other risk factors; and that none of the specific group of tests explained the association between microalbuminuria and mortality . The associations reported in table 3 did not materially change after additional adjustment for use of lipid- or blood pressure lowering drugs and the presence of leukocytes in urine (tested by microscopy and scored as positive whenever there were more than five leukocytes per high - power field) or when the dichotomous variable hypertension used in those models was replaced by either sbp or pulse pressure (a marker of arterial stiffness) or when analyses were adjusted for prior coronary heart disease instead of prior cvd (data not shown). Finally, we found no evidence of effect modification by gts in the association between cardiovascular mortality and c - ad total score or microalbuminuria (pinteraction> 0.4 in both cases). C - ad total score was associated with microalbuminuria (= 0.33 [95% ci 0.160.51]) (table 2, model 1). After adjustment for potential confounding factors, c - ad total score was still borderline associated with microalbuminuria (0.16 [0.01 to 0.33]) (table 2, model 3). Comparable results were obtained when tests were combined on the basis of their methodology or on the part of the autonomic nervous system they predominately represent . All individual c - ad tests, except lf/(lf + hf), were positively associated with microalbuminuria (although not all statistically significantly so) (data not shown). Association between microalbuminuria and c - ad * model 1: univariate analysis; model 2: adjusted for sex, age, and gts; model 3: model 2 plus adjustments for hypertension, whr, estimated glomerular filtration rate, ldl, hdl, triglycerides, smoking, and prior cvd . Indicates the difference in c - ad score between individuals with microalbuminuria vs. normoalbuminuria . Both microalbuminuria (rr 3.49 [95% ci 1.876.53]) and c - ad total score (2.54 [1.604.04]) were associated with cardiovascular mortality (table 3, model 1; fig . These associations were attenuated after adjustment for age, sex, and glucose tolerance status (gts) (2.23 [1.164.29] and 1.81 [1.112.94], respectively; model 2). Further adjustment for other cardiovascular risk factors only slightly further attenuated these rrs (model 3), and when the c - ad total score or microalbuminuria was added to these models, the rr attributable to microalbuminuria remained practically unchanged and both microalbuminuria (2.13 [1.094.17]) and c - ad total score (1.76 [1.052.94]) were independently associated with cardiovascular mortality (model 4). Association of microalbuminuria or c - ad with cardiovascular and all - cause mortality * model 1: univariate analysis; model 2: adjusted for sex, age, and gts; model 3: model 2 plus adjustments for hypertension, whr, estimated glomerular filtration rate, ldl, hdl, triglycerides, smoking and prior cvd; model 4: model 3, with additional adjustment for, respectively, c - ad or microalbuminuria . Rr of microalbuminuria vs. normoalbuminuria or per sd (= 0.605) increase in c - ad total score . Kaplan - meier survival plots for cardiovascular mortality . Among individuals with normo- versus microalbuminuria (a) and across tertiles of c - ad total score (b). Both microalbuminuria and c - ad total score were also associated with all - cause mortality (table 3, model 1). However, after adjustment for age, sex, gts, and other cardiovascular risk factors, microalbuminuria (rr 1.33 [95% ci 0.832.13]), in contrast to c - ad total score (1.52 [1.112.09]), was not independently associated with all - cause mortality (model 3). The associations described above were also investigated by examining autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent (supplementary table a2, available in an online appendix). These additional analyses showed that overall all different groups of tests were associated with increased mortality risk, that these associations were attenuated when further adjusted for age, sex, gts, and other risk factors; and that none of the specific group of tests explained the association between microalbuminuria and mortality . The associations reported in table 3 did not materially change after additional adjustment for use of lipid- or blood pressure lowering drugs and the presence of leukocytes in urine (tested by microscopy and scored as positive whenever there were more than five leukocytes per high - power field) or when the dichotomous variable hypertension used in those models was replaced by either sbp or pulse pressure (a marker of arterial stiffness) or when analyses were adjusted for prior coronary heart disease instead of prior cvd (data not shown). Finally, we found no evidence of effect modification by gts in the association between cardiovascular mortality and c - ad total score or microalbuminuria (pinteraction> 0.4 in both cases). In this prospective study we found that both microalbuminuria and c - ad (estimated from the mean of 10 standardized tests) were independently associated with cardiovascular mortality in a cohort of elderly, caucasian subjects with and without diabetes . Therefore, c - ad does not explain why microalbuminuria is related to cardiovascular mortality in either the general population or in diabetic subjects . Recently, hrv was found not to predict the decline of gfr but was independently associated with cardiovascular mortality in middle - aged type 1 diabetic subjects with overt nephropathy (17). In another study, investigating the independent predictive role of gfr, microalbuminuria, and cardiovascular autonomic neuropathy showed the latter to also be associated with all - cause and cardiovascular mortality in subjects with diabetes (18). Hitherto, no other prospective studies examining the relation of microalbuminuria and c - ad with cardiovascular mortality have been reported . The independent association of both microalbuminuria and c - ad with cardiovascular mortality reported herein indicates that they are linked to cardiovascular mortality by different biological pathways . Several mechanisms linking c - ad to cardiovascular mortality have been proposed (e.g., qt interval prolongation and silent myocardial ischemia) (19). Microalbuminuria may be linked to cvd by a common pathophysiological process (such as endothelial dysfunction or chronic low - grade inflammation) (3). However, the possibility that microalbuminuria reflects the presence of a set of undiscovered factors that are causally related to cvd remains to be further elucidated . Furthermore, aggressive treatment of microalbuminuria by inhibiting the renin - angiotensin - aldosterone system has been shown to decrease renal and cardiovascular risk in individuals with diabetes (20). There is some evidence that c - ad is also a useful risk indicator for cvd, especially in subjects at high risk (type 2 diabetes, hypertension, or prior cvd) (9,21), and this study reinforces that observation . However, whether the improvement in c - ad, e.g., with exercise training or blood pressure or lipid - lowering drugs (2224), leads to a better prognosis is unknown . In our study, additional adjustment for use of -blockers or lipid - lowering drugs had no effect on the associations reported . Therefore, the clinical relevance of treating c - ad in the general population remains unclear and needs to be further examined . The strengths of our study are the relatively large, truly population - based and prospective design with a long follow - up period . In addition, we characterized subjects' c - ad as comprehensively as possible by conducting 10 autonomic function tests, which are thought to reflect all aspects of cardiovascular autonomic function . Previous cross - sectional studies evaluating the relationship between microalbuminuria and c - ad used less extensive autonomic function tests (4,6). Because we included all three groups of gts, we were able to analyze possible effect modification of gts in the relationship between the main determinants, c - ad and microalbuminuria and cardiovascular mortality (but found none). First, each of the test parameters of autonomic function was measured with moderate levels of reproducibility (reliability coefficient 50%) (13). Therefore, the results were combined into a c - ad total score, which enabled us to circumvent, at least partially, this problem . Specifically, the results herein reported with the c - ad total score were more powerful than results obtained on the basis of each individual test (separately), because these results would be more strongly affected by misclassification (likely to be random), causing an underestimation of the strength of the associations . Indeed, the associations between each individual test result with cardiovascular mortality were in accordance, although with lower strength, with those as reported herein with the c - ad total score (data not shown). In addition, this approach had the advantage of circumventing the problem of multiple testing (type i errors) that would have occurred when the associations with each test were analyzed separately . However, because each test may represent different parts of autonomic function, some overlapping but others complementary, our c - ad total score does not allow the distinction of potential different contributions of sympathetic and parasympathetic functions to cardiovascular mortality . Therefore, we examined the autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent in the additional analyses . The results of these alternative combinations of autonomic function tests were comparable to those of the c - ad total score (supplementary table a2), but we acknowledge that these analyses may not have been specific enough, as most authors agree that a clear distinction between parasympathetic or sympathetic dysfunction cannot be made on the basis of these tests . Altogether, we feel that the advantages (i.e., reduced misclassification and avoidance of type i errors without impairment of etiological validity) of the approach used by us outweigh the disadvantages . Measurement of hrv during a longer period may be more reliable, although measurements obtained in time periods as short as 2 min correlate highly with 24-h measurements (25). Third, evaluation of subjects' c - ad was conducted at baseline only, and, therefore, we were not able to evaluate deterioration of autonomic function during follow - up . Fourth, for most participants microalbuminuria was defined on an acr measured in one urine sample only . Because microalbuminuria is quite variable from day to day we cannot fully exclude the possibility that some subjects were misclassified . However, we tried to avoid this problem by collecting urine overnight and expressing albumin excretion as acr . In addition, analyses in the 154 participants for whom two samples were available yielded essentially similar results (data not shown). Finally, we studied an elderly, caucasian population, and it is not known whether our results can be generalized to other ethnic groups or to younger individuals . In summary, we have shown that both microalbuminuria and c - ad are independently associated with cardiovascular mortality in an elderly, caucasian population of individuals with normal glucose metabolism, igm, and diabetes . These results suggest that microalbuminuria and c - ad are related to cardiovascular mortality by different biological pathways . Therefore, it may be useful to treat not only microalbuminuria but also c - ad in populations at high risk of cardiovascular mortality.
Cervical cancer is the third most common cancer after breast and colorectal cancer and the fourth leading cause of death among women in malaysia(1 - 3). It remains to be one of the major cancers that burden worldwide particularly in under - developed and developing countries (cervical cancer incidence rate increased with age after 30 years and has its peak at ages 65 - 69 years . According to ethnicity in malaysia, women of indian ethnic group have the highest incidence for cervical cancer followed by chinese and malay (3). Majority malays are muslims, thus lower incidence of this cancer was observed . In iran, there are almost no extramarital sexual relations and they depend strongly on family - based traditions (7) the five - year survival exceeded 70% in korea (810), 55% in turkey (11) and it can be as low as 17% which was in uganda (12). There were various prognostic factors established in many studies such as stage, age, lymph node involvement and tumor size (1316). In kentucky, between january 2001 and may 2010, a study of 381 cervical cancer patients who were referred to tertiary care centre found that stage of disease was a significant prognostic factor for overall survival (17). Meanwhile, a study of 44,182 patients diagnosed with cervical cancer between 1993 and 2002 in korea, found that both stage and histological type of cervical cancer were important factors for survival (18). To our knowledge, the analysis from published studies was often limited to the frequently used cox proportional hazard regression model when examining the relationship of the survival distribution to covariates (17, 1921). This is perhaps due to the fact that although baseline hazard is not specified in cox model, the parameter can still be estimated . The objective of this study was to determine the effects of explanatory variables on the survival of cervical cancer patients using parametric regression model . In some cases, parametric models are more informative than the cox model such as the baseline hazard and survival estimates are known (can be estimated). Several examples of parametric models can be found in the following studies (2227). Zhu et al . Have compared the cox and weibull model in modeling the gastric cancer data and found that the weibull model gave more a precise results in than the cox model (28). The weibull model has gained popularity in modeling survival data due to its flexibility in the shape parameter that can accommodate a decreasing, constant and increasing hazard . Furthermore, the suitability of the weibull model can be easily assessed using a log - cumulative hazard plot . In this study, a stratified weibull model was used since there was a time - dependent covariate that caused the proportional hazard assumption vio - lated . The study design was retrospective in nature in which patients records were reviewed retrospectively to obtain the data . The husm, located in kubang kerian, kelantan has been long regarded as the referral centre for the east coast region of malaysia . The inclusion criteria were histopathologically and clinically diagnosed with cancer of cervix between 1 july 1995 and 30 june 2007, and received at least one treatment related to cervical cancer in husm . Patients who were died due to other competing causes of death (not cervical cancer), or with incomplete data were excluded from this study . Ethical clearance was obtained from research and ethics committee of university sains malaysia (reference number: usmkk / ppp / jepem [205.4 (2.4)]). The variable of interest was time (in months) which was measured from the patient was diagnosed with cervical cancer up to the time of death . Factors that were considered in the analysis were ethnicity (non malay, malay), lymph node involvement (negative, positive), metastasis (with metastasis, without metastasis), histology (squamous cell carcinoma, adeno cell carcinoma), primary treatment (surgery, radiotherapy and/or chemotherapy), age at diagnosis (<40, 40 59, 60) and stage (stage i & ii, stage iii & iv). International federation of gynecology and obstetrics (figo) system was used in staging patients with cervical cancer . Data analysis was performed using the statistical package tibco spotfire s - plus version 8.1 . The suitability of the weibull model for the data was assessed using a plot of the log of the negative log of the estimated survivor function against log time or log - cumulative hazard plot . Univariate analysis was conducted using the simple weibull regression analysis to identify the possible prognostic factors individually . Significant factors from the univariate analysis were further analyzed by the weibull multivariate analysis to model the prognostic factors . Model selection procedure was based on the forward variable selection method with statistical significance set at = 0.10 . The proportionality of the hazards was assessed using the test based on schoenfeld (29). As the proportional hazard assumption was not satisfied, a stratified model was used instead (30) and in measuring the goodness of fit of the model, deviance residuals were used . 1 shows a study flow diagram which summarizes the steps of the statistical analysis performed . The study flow diagram the log - cumulative hazard plotshows a straight line which suggests that the distribution of survival time considered follows weibull distribution (fig . Patients characteristics and the incidence rate ratio were tabulated in table 1 . About 74% of total patients the ratio of the risk of death for patients who were diagnosed in stage iii & iv compared to the risk of death for patients in group stage i & ii was 1.44 . Meanwhile, the risk of dying of patients in with metastasis group was 1.55, greater than patients who had no metastasis . The results from the univariate analysis are presented in table 2 . From this analysis, based on the forward selection procedure in multivariate analysis, two variables namely stage and metastasis was found to be significant factors (table 3). However, it was noted that metastasis (p=0.032) did not satisfy the proportional hazard assumption and a stratified model was then considered . It is worthwhile to note that while the stratification allows separate baseline hazard function to each metastasis group, the coefficient of the predictor variables are assumed to be the same within each metastasis group (29). The first stratum consists of patients without metastasis and the second stratum were patients who do have metastasis . Parameter estimates of the stratified model were performed and the output is given in table 4 . The log - cumulative hazard plot characteristics of patients treated in husm (n=120) univariate analysis of weibull model with prognostic factors multivariate analysis of weibull model with prognostic factors output of weibull model under stratification the hazard ratio for variable stage stratified on metastasis the estimated scale parameters () for the first and second stratum were 1 = 1.11 and 2 = 0.733 respectively . The risks of death for cervical cancer patients were estimated by finding the regression coefficient (j) j=j, and the hazard ratio (j) j = exp(j) where j = 1,2 denotes the stratum . For the first stratum, patients who were diagnosed at stage iii & iv are at 2.30 times the risk of death as those in stage i & ii . Meanwhile, for the second stratum, patients in stage iii & iv group were more likely to die (3.53 times) than patients in stage i & ii . The index plot of deviance residuals (rdi) was performed to identify the presence of subjects who was poorly predicted by the model (31). 3 shows that the residuals are roughly symmetrically distributed around zero and most of the residuals are between -2 to 2 . Outliers and influential observations are not observed in the plot thus implying a good fit of the model . In this study, the weibull model was used instead of the cox model because the log - cumulative hazard plot has confirmed that the survival time followed the weibull distribution . Multivariate analysis through forward selection method found that the most feasible model to describe the survival of cervical cancer patients was dependent upon the covariates namely, stage and metastasis . As the metastasis variable did not meet the proportional hazard assumption, the stratified weibull model was applied . This study found that patients who were diagnosed at stage iii & iv have greater risk of death compared to those who were diagnosed at early stage, stage i & ii for both stratum, with and without metastasis . It is worthwhile to note that a study of 515 cervical cancer patients by dueas - gonzlez et al . Showed a significant result for advance stage iii & iv with adjusted hazard ratio of 1.54 (95% ci= 1.11- 2.14) (16), indicating that patients with advanced stage of disease had a 54% higher risk of progression or death at any time than earlier stage patients . After applying the stratified model, this study found that the prognosis of cervical cancer patients was dependent upon the stage at diagnosis . These findings provide useful knowledge on the understanding of the survival of cervical cancer patients . Besides, this study also demonstrates the solution to time - dependent covariates problem, or when the proportional hazards assumption is violated . Cervical cancer dataset that comprise of the national data may be obtained to give a more general prognosis and a better description of the survival pattern of all cervical cancer patients in malaysia . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
According to the european society of endodontology (1994), the assessment of endodontic treatment requires clinical as well as radiological follow - ups at regular intervals . The radiographic evidence of success is the presence of a normal periodontal ligament space around the root . If radiographs reveal that a lesion has remained the same or has only diminished in size, the treatment is not considered a success.1,2 it is generally accepted that the outcome of endodontic treatment is positively correlated with the technical quality of the root filling, expected to provide an hermetic seal against bacterial ingress.3,4 however, it has been suggested that the quality of the coronal restoration may also have an impact on the periapical health of root - filled teeth;5,6 when the restoration quality is good, this may allow for a favorable outcome even when the root filling quality is poor.7 attention has been focused on the prevalence and the technical quality of root fillings through the evaluation of intraoral810 or panoramic radiographs.2,11,12 epidemiologic studies have been performed on the root canal morphology in turkish populations.13,14 information about the prevalence and technical standard of root - canal treatment, and the occurence of periapical lesions in turkey are scarce.12,15 moreover, there is only one investigation about quality of root fillings and coronal restorations in turkish population.16 the aim of the present study was to relate the quality of root canal treatment and coronal restorations to the periapical status of root filled teeth in adult turkish population based on radiographic examination . The sample consisted of 400 subjects, aged 38.7013.80 years, 138 males (34.5%) and 262 females (65.5%), presenting consecutively as new patients seeking routine dental care in department of oral diagnosis and radiology, faculty of dentistry, marmara university, istanbul, turkey, between 2005 and 2006 . The criteria for inclusion in the study were that the patients should be attending for the first time . Patients younger than 20 years and patients having less than eight remaining teeth were excluded . All the patients gave written informed consent for the study . All participants underwent a panoramic radiograph and additional periapical radiographs of endodontically treated teeth were processed . All panoramic and periapical radiographs were taken with a veraviewpocs hi - sped (j. morita mfg . Kyoto, japan) and evolution x 3000 - 2c x - ray unit (new life radiology srl, italy), respectively . Two radiographers using the long - cone paralleling technique, setting of 70 kv, 10 ma, a film - focus distance of 30 cm took all periapical radiographs ., kg, bissingen, germany), kodak medical x - ray processor (eastman kodak company, rochester, ny, usa), general rapid medical developer and fixer (general a. ., the viewing conditions were standardized, using a view - box with fixed light intensity . The incidence of root fillings was recorded along with the periapical status of all teeth with the exception of third molars . Adequate if the root filling was 2 mm or less from the radiographic apex, when it was more than 2 mm from the apex, it was considered as an underfilling and when excess filling was beyond the radiographic apex, it was categorized as an adequate if the radiodensity of fill was uniform and appeared to be radiographically adapted to the root - canal walls periapical index (pai) score (orstavik et al18), recorded for each one of the roots . Pai scores 1 and 2 signified a nondiseased apical periodontium, and pai scores 3, 4, and 5 signified the presence of an apical radiolucency . The worst score of all roots was taken to represent pai score for multirooted teeth . In case of a root filling was present, the type of coronal restoration (crown or filling) was recorded . The quality of the restoration was assessed radiographically and classified as inadequate if the restoration was absent or if open margins, overhangs or secondary caries were detectable . Evaluation of the periapical status and the quality of root filling in the present study was made independently by two examiners (fnp, bg) using the same examination procedure after a calibration session . For calibration 20 radiographs were used and not included in main study . The cohen s kappa value for inter - examiner agreement of pai - values for all scored teeth was 0.81 . The inter - examiner agreement scores gave cohen s kappa of 0.68 for quality of filling and crown restoration, 0.83 for adaptation of fillings to canal walls, and 0.86 for length of root fillings . The data were analyzed with spss (statistical package for social sciences) for windows 10.0 statistical package software . Descriptive statistical methods (mean and standard deviation) were used for the evaluation of the data . P values of less than .05 were interpreted as significant, and the level in confidence intervals was 95% . The sample consisted of 400 subjects, aged 38.7013.80 years, 138 males (34.5%) and 262 females (65.5%), presenting consecutively as new patients seeking routine dental care in department of oral diagnosis and radiology, faculty of dentistry, marmara university, istanbul, turkey, between 2005 and 2006 . The criteria for inclusion in the study were that the patients should be attending for the first time . Patients younger than 20 years and patients having less than eight remaining teeth were excluded . All the patients gave written informed consent for the study . All participants underwent a panoramic radiograph and additional periapical radiographs of endodontically treated teeth were processed . All panoramic and periapical radiographs were taken with a veraviewpocs hi - sped (j. morita mfg . Kyoto, japan) and evolution x 3000 - 2c x - ray unit (new life radiology srl, italy), respectively . Two radiographers using the long - cone paralleling technique, setting of 70 kv, 10 ma, a film - focus distance of 30 cm took all periapical radiographs ., kg, bissingen, germany), kodak medical x - ray processor (eastman kodak company, rochester, ny, usa), general rapid medical developer and fixer (general a. ., istanbul, turkey)) were used in this study . The viewing conditions were standardized, using a view - box with fixed light intensity . The incidence of root fillings was recorded along with the periapical status of all teeth with the exception of third molars . Adequate if the root filling was 2 mm or less from the radiographic apex, when it was more than 2 mm from the apex, it was considered as an underfilling and when excess filling was beyond the radiographic apex, it was categorized as an adequate if the radiodensity of fill was uniform and appeared to be radiographically adapted to the root - canal walls . Periapical index (pai) score (orstavik et al18), recorded for each one of the roots . Pai scores 1 and 2 signified a nondiseased apical periodontium, and pai scores 3, 4, and 5 signified the presence of an apical radiolucency . The worst score of all roots was taken to represent pai score for multirooted teeth . In case of a root filling was present, the type of coronal restoration (crown or filling) was recorded . The quality of the restoration was assessed radiographically and classified as inadequate if the restoration was absent or if open margins, overhangs or secondary caries were detectable . Evaluation of the periapical status and the quality of root filling in the present study was made independently by two examiners (fnp, bg) using the same examination procedure after a calibration session . For calibration 20 the cohen s kappa value for inter - examiner agreement of pai - values for all scored teeth was 0.81 . The inter - examiner agreement scores gave cohen s kappa of 0.68 for quality of filling and crown restoration, 0.83 for adaptation of fillings to canal walls, and 0.86 for length of root fillings . The data were analyzed with spss (statistical package for social sciences) for windows 10.0 statistical package software . Descriptive statistical methods (mean and standard deviation) were used for the evaluation of the data . P values of less than .05 were interpreted as significant, and the level in confidence intervals was 95% . A total of 9460 teeth were examined, an average number of 23.634.35 remaining teeth per subject . The total number of root - filled teeth was 890 (9.39%); of which 658 (73.9%) teeth had ap . Apical periodontitis was found in 75.5% of teeth with adequate length of root filling, whereas if the filling was too short or long, periapical lesions were present in 72.8% and 96.2% of teeth, respectively . There was a significant correlation between the presence of periapical pathology and inadequate root canal fillings (p<.05). The relationship between the adaptation of root filling to canal walls and periapical status is shown table 2 . Root filled teeth without voids had ap in 72.9% of cases, whereas if voids were detected, ap was present in 74.7% of the teeth (p: 0.54, odds ratio: 1.09, ci: 0.811.48). The relationship between the quality of the prosthetic restoration and periapical status is presented in table 3 . 75.9% of the teeth with adequate coronal restorations had ap, whereas if the coronal restoration was inadequate, ap was present in 74.2% of the teeth (p: 0.70, odds ratio: 0.91, ci: 0.581.45). The relationship between the quality of the filling restoration and periapical status is presented in table 4 . 72.8% of the teeth with adequate filling restoration had ap, whereas if the filling restoration was inadequate, ap was present in 72.3% of the teeth (p: 0.92, odds ratio: 0.97, ci: 0.621.54). The subjects included in this study were adult patients attending for general dental treatment for the first time . The recruitment of subjects was the same as those used by others.8,11,17,19,20 there is no information available for this patient population, which makes it difficult to extrapolate the data obtained into the turkish population . However, the dental faculty attracts a patient population from numerous parts of the city and its surroundings, which eliminates the risk of only including patients previously treated by a limited number of practitioners . Some patients sought care because of the expense of prosthetic treatment, which in general is less at the dental faculty than in the private sector in turkey . In addition, extraction of teeth due to low socio - economic status gives rise to a limited number of root filled teeth epidemiologically . Patients with eight or fewer remaining teeth were excluded because they often had periodontal disease and it was impossible to determine the role played by the endodontic treatment in the occurrence of a radiographic periapical lesion.17 although studies comparing panoramic and periapical radiographs for detecting periapical lesions indicate similar overall diagnostic accuracy,21,22 panoramic radiographs are claimed to show lower sensitivity when detecting periapical lesions of the anterior teeth and mandibular molars compared with periapical radiographs.23 therefore, there is a possibility for loss of information when endodontic conditions are recorded based on panoramic radiographs only in some studies.2,11,17,2426 however, other epidemiological studies have used a combination of panoramic and periapical radiographs.4,8,9,27 as panoramic radiographs do not reveal details and inter - observer variability is greater with panoramic radiographs.28 in this investigation, therefore, all participants underwent a panoramic and additional periapical radiographs of affected teeth were processed . Criteria for ap vary among studies.2,10,29,30 in recent years, most of the studies on the prevalence of ap have used this index scoring to assess periapical status, so the results of this study can be more appropriately compared with them.4,18,25 therefore, in the present study, the pai was used to assess the periapical status . The reproducibility of the observer (cohen s kappa=0.81) was acceptable, probably because of prior calibration . A total of 890 teeth had root fillings, accounting for 9.39% of all teeth . The prevalence of root - filled teeth in the turkish population was found lower than previously done other studies about different countries (34%87%).4,5,9,10,19,27,29 this phenomenon can be explained by the fact that first, the survey population was not representative of the whole country, and secondly, the differences in health care services and socioeconomical factors in the various countries could account for these discrepancies . In contrast, other studies found the prevalence of root - filled teeth to range between 1.3% and 4.8%, similar to that found in this study.8,17,3032 the quality of endodontic treatment was disappointing . Root filling length was found to be adequate in 24.5% of the teeth and the adaptation of fillings to the canal walls in 27.1% of the teeth . In this respect, the data confirm the findings of several other investigators showing poor quality of root canal treatment.8,17,19 apical periodontitis was found in 75.5% of teeth with adequate length of root filling, whereas if the filling was too short or long, periapical lesions were present in 72.8% and 96.2% of teeth, respectively . Tsuneishi et al29 observed apical radiolucency most often in overfilled teeth (79.8%), followed by adequately filled teeth (41.6%) and then underfilled teeth (34.5%). Siquera et al33 indicated that poor outcome in case of overfillings is usually associated with concurrent endodontic infection, or as a result of previous overinstrumentation of an infected root canal that propelled infected dentin chips to the periradicular tissues or can be due to a lack of apical seal that allows the traffic of tissue fluids into the canal and of microorganisms and their products towards the periradicular tissues . In contrast, sunay et al12 reported 90.8% of root - filled teeth with apical periodontitis had inadequate root canal fillings, the majority of them being short of the radiographic apex . Dugas et al4 reported the prevalence of apical periodontitis close to 60% when the root filling was inadequate, regardless of whether the filling was too short or too long consistent with the observations in other studies.17,19,27 in the present study, the relationship between the type of coronal restoration within root filled teeth and the presence of apical periodontitis were investigated . It is indicated that the technical quality of the coronal restoration may be more important for periapical health than the technical quality of the root filling.34 although some researchers found a significant correlation between the radiographic quality of the coronal restoration and the periapical status of root filled teeth.4,5 on the other hand, hommez et al35 reported that the quality of the coronal restoration did not have a significant influence on the periradicular status when it was combined with the quality of endodontic treatment . The results of the present study revealed that the quality of the restoration did not significantly affect the treatment outcome . In the present study, the frequency of endodontically treated teeth was found lower than other studies . The technical quality of root fillings and restoration in a turkish subpopulation was poor and was consistent with a high prevalence of apical periodontitis.
The neighborhood has emerged as a prominent level of analysis in studies of contextual influences on health and wellbeing in human development . This owes to the social organization of life within neighborhoods, and to the availability of data at neighborhood - like levels of analysis (i.e., census tracts). Neighborhood context has also received greater attention due to ecological psychology's conceptualization of human development as the product of a dynamic interaction between the individual and multiple nested environments, of which the neighborhood is one of the most immediate environments . Contemporary scholarship on neighborhood factors has also highlighted the importance of understanding whether neighborhood variables add explanatory variance above and beyond individual differences . Neighborhood socioeconomic status (ses) is the most consistently reported neighborhood - level predictor of cognitive outcomes certainly in childhood, and potentially across the life span . Ses is also the strongest and most consistently reported neighborhood - level predictor of health outcomes among older adults a relationship which may have a cumulative effect across the life span . The association of neighborhood - level ses with health is in fact stronger among adults aged 6069 than among young and middle - aged adults, and during these years its association with health is comparable to or stronger than the relationship of individual ses to health . In addition to predicting poorer physical health, lower neighborhood ses is also associated with reduced rates of physical activity, increased incidence of depression and psychosocial stress, and less neighborhood - based social support and social engagement . Many of these outcomes have also been identified as correlates of late life cognition, yet few studies to date have specifically investigated the role of the neighborhood in cognitive aging . While neighborhood effects on late life cognition have received less attention, neighborhood effects on early - life cognitive development are relatively well documented . Neighborhood ses is positively correlated with cognitive development across the entire spectrum of child development, from the prenatal stages (in the form of reduced rates of congenital anomalies and neural tube defects among higher - ses neighborhoods, presumably due to reduced risk of exposure to environmental toxins; [6, 7]) and preschool years [8, 9] to academic achievement in the school age [10, 11] adolescent, and young adulthood years (e.g., high school graduation rates and college attendance). Four recent studies explored relationships between late life cognition and the neighborhood context on a broad scale . Wight and colleagues found that after controlling for individual - level education and area - level median household income, elders living in areas with low neighborhood - level educational attainment (defined by census tract area) achieved lower cognitive status scores compared to elders living in areas with high neighborhood - level educational attainment (as assessed using the mini - mental state examination or mmse). A united kingdom study reported a clear and significant downward trend in cognitive performance on the mmse and tests of verbal fluency and memory for older individuals living in neighborhoods with greater deprivation, after controlling for individual - level wealth, income, and education, across all age and gender groups . These effects were also robust after adjusting for the effects of individual systolic blood pressure, history of stroke, and duration of residence in the current neighborhood . Sheffield and peek moved beyond cross - sectional analysis to examine how neighborhood ses and ethnic composition (proportion mexican american) predicted 5-year change in mmse score in a us national sample . Independent of individual - level risk factors, odds and rate of incident cognitive decline increased as a function of lower neighborhood ses and decreased with the proportion of mexican american neighborhood residents . Finally, recent findings from the baltimore memory study suggest that the relationship between neighborhood and late life cognition may also depend on genotype . However, apolipoprotein e4 genotype was found to interact with high neighborhood psychosocial hazards, resulting in poorer cognitive performance on measures of processing speed and executive functioning after controlling for individual - level covariates . This suggests evidence of a gene - environment interaction in neighborhood's relation to late life cognition . The existing studies focused on broad differences in late life cognitive status (i.e., mmse score), but did not investigate the effect of neighborhood on different cognitive domains (see recommendations by cullum et al . ). Furthermore, the use of the mmse in these studies was not an ideal measure of cognition, as they targeted samples of initially healthy, independent elders . The mmse is not sensitive to differences among healthy older adults, having been designed to screen for dementia . Cognitive aging research has instead focused increasingly on the use of confirmatory factor analysis (cfa) and structural equation modeling (sem) to explore whether differences in late life cognitive performance are related to general or specific processes . It has also been noted that conceptualizing late life cognition using a single general factor (g) is a simplistic approach to a complex and dynamic cognitive system . This suggests neighborhood influences should be investigated at both general and domain - specific levels . Neighborhood effects may support some dimensions of cognition more strongly than others, in addition to influencing general cognitive status . The present study aimed to identify whether neighborhood effects on cognition were global, or specifically related to particular cognitive domains such as memory, reasoning, processing speed, everyday cognition, or vocabulary . Another innovation of the present study is the capacity to address whether neighborhood effects might also influence the magnitude of potential benefit from cognitive intervention . No study, in part because of a lack of data availability, has examined this question . Elucidating both the specificity of neighborhood effects on particular domains of cognition and the extent to which neighborhood affects cognitive training response may provide additional hints toward potential mechanisms by which the neighborhood exerts influence . For example, effects on the fluid cognitive abilities such as processing speed, memory, and reasoning (and their response to cognitive training) would suggest a more biologically mediated influence given their sensitivity to brain integrity . In contrast, effects on measures of crystallized cognitive abilities, especially vocabulary, might hint toward a more socioculturally mediated, and possibly lifespan accumulative influence, as crystallized cognitive abilities are developed with the accumulation of verbal knowledge, developed over a lifetime of experience in engaging with culture . As this lifelong accumulation is strongly influenced during childhood, adolescence, and young adulthood, effects of current neighborhood sep on crystallized abilities may also reflect, in part, effects of early - life neighborhood sep . The advanced cognitive training for independent and vital elderly (active) study was a randomized, controlled clinical trial examining the effects of three cognitive interventions for community - dwelling older adults . In active, older adults from six us catchment areas (baltimore / coastal maryland; the metropolitan areas of birmingham, boston, detroit, and indianapolis; central pennsylvania) completed a baseline assessment including multiple cognitive measures . Participants were then randomized to one of three ten - session cognitive intervention programs or a no - contact control condition . The present study sought to examine whether neighborhood socioeconomic position (sep) shows bivariate relationships with baseline cognitive level and immediate response to cognitive training, beyond individual - level predictors of cognition . If so, we aimed to discern whether neighborhood effects were significant for general cognitive ability (g), and whether effects were differential for specific cognitive abilities (memory, reasoning, speed, everyday cognition, and vocabulary). We hypothesized that the potential effect of neighborhood sep may most likely occur by way of sociocultural processes, and therefore would have significant positive associations with general cognitive ability and with crystallized cognitive domains, including vocabulary . Neighborhood sep was hypothesized to have relatively weaker, if detectable, associations with fluid cognitive domains, including memory, reasoning, speed, and everyday cognition . The primary objective of active was to test the effectiveness and durability of three cognitive interventions . The initial trial randomized individuals to one of three 10-session cognitive interventions designed to improve memory, reasoning, or processing speed performance or to a no - contact control condition . Following training there was an immediate posttest and follow - up assessments at 1-, 2-, 3-, and 5-years after intervention . The current study sought to examine only the immediate pretest - posttest data, focusing on the proximal outcome measures used . Information on potential covariates was collected, including demographic variables, mmse, recent depressive symptoms, and general health . Testers at all six sites were trained in standardized assessment protocols and quality control by both study investigators, and the coordinating center ensured fidelity to testing procedures . The initial sample of 2,802 participants was recruited through on - site presentations, letters to interested persons, newspaper advertisements, introductory letters, and follow - up telephone calls . Participants were cognitively healthy, community - dwelling older adults aged 65 to 94 years (mean age = 73.6 years, mean years of education = 13.5, mean mmse = 27, 75.8% female, 72% caucasian, 26% african american, and 2% other minority groups). The majority of participants (64%) were not married, and most reported good to excellent health (84.3%). Efforts were aimed at recruiting older adults independent of care at enrollment, as well as recruiting a diverse sample especially inclusive of african americans, who were previously under - represented in most cognitive training research . Exclusion criteria included (a) being under age 65 at the start of the study, (b) significant functional and/or cognitive decline at enrollment (e.g., impaired activities of daily living, mmse <23, and diagnosis of alzheimer's disease), (c) having a medical condition disposing the participant to imminent cognitive decline or to mortality within the next 2 years, (d) severe sensory or communicative difficulties precluding participation in assessments and training, (e) having had cognitive training, or (f) planning to be unavailable during the testing and training periods of the study . Active included multiple assessments of participants, but the present paper restricted itself to baseline cognitive performance and the immediate post - test . Neighborhood - level socioeconomic data from publicly available geographic datasets were then merged with active data at the individual level . Active did not aim a priori to include multiple measures of individual - level socioeconomic position . Education (highest level achieved) was assessed for all participants, but other person - level socioeconomic indices (e.g., income, occupation) were not collected at baseline; occupational status was collected at the second annual follow - up occasion . The cognitive domains measured in actve included memory, inductive reasoning, processing speed, everyday cognition, and vocabulary; descriptive statistics on the sample performance for measures within each domain are illustrated in table 1 . Memory was measured using the hopkins verbal learning test (hvlt,), a 12-word list memory task assessing immediate and delayed recall over 3 learning trials, the rey auditory verbal learning test (avlt,), a 15-word list memory task assessing immediate and delayed recall over 5 consecutive learning trials, and the rivermead behavioral memory test, a paragraph recall task assessing immediate verbal episodic memory . Reasoning was measured using letter series, a task requiring accurate identification of the next logical letter group in a series of letters (e.g., a m b a n b a o b), letter sets, an inductive reasoning task requiring participants to identify which of 4 sets of letters is unrelated to the others (e.g., eef hhi llm ysy), and word series, a task requiring accurate identification of the next logical word in a series . Processing speed was measured using the useful field of view test (ufov,), a computer - administered task measuring visual sustained, selective, and divided attention through four subtasks, and the complex reaction time test, a computer - administered task measuring the time taken to perform various motor behaviors and to complete each task . Everyday cognition was measured using the everyday problems test, a performance - based test of ability to solve everyday problems including medications, nutrition, phone use, shopping, and management of finances and household, the observed tasks of daily living (otdl), a timed task of problem solving in medication use, telephone use, and financial management, and the timed instrumental activities of daily living (tiadl), a timed test of ability to complete daily living tasks (e.g, find telephone number, make change, find and read ingredients on a can, find food items on a shelf, read instructions on medicine bottle). Vocabulary was measured using a multiple - choice measure of vocabulary attainment in which the participant is presented with a word and must choose one of four possible synonyms . Geocoding is a process by which a location (e.g., street address) is assigned cartesian mathematical coordinates, allowing for other levels of geographic information to be linked with that location . Geolytics, a commercial provider of geocoding services and census demographic data, (1) geocoded the active participant addresses, and (2) appended to these addresses data from the 2000 u.s . Census and 2002 economic census, creating a dataset that could be used to characterize the neighborhood environment . Neighborhood research, active participant addresses were geocoded and linked with their associated census tract numbers, which allowed for census tract - level data from the 2000 u.s . Census to be appended to the individual - level active data . Participants receiving mail by post office boxes were excluded as such addresses cannot be verified to represent the participant's actual place of residence . Addresses with invalid house numbers, street names, and zip codes were flagged for followup; invalid addresses or poorly matched addresses were dropped from the analysis (93% of the active addresses accurately matched to a us geological survey (usgs) geocode; final sample size = 2,521). Census tracts are subdivisions of a county, with an average size of approximately 4,000 residents designed to capture collectively agreed - upon areas approximating neighborhoods . That is, the boundaries of census tracts were agreed upon by local officials knowledgeable of the area and were intended to be homogenous with respect to population characteristics, economic status, and living conditions . Smaller measurement units of area - level ses (e.g., block group) have been shown to correlate highly with census - level measures of ses, and variability in ses among block groups is small relative to variability within their census tract . The association of area - level ses with the cognitive measures was tested at three different levels of area measurement (block group, census tract, and zip code), with no significant difference in the strength of associations across levels . Because the census tract has traditionally been the most frequently used and most strongly recommended unit of measurement for area - level effects in health research, especially in terms of sep [3, 35], and because it differed very little in model fit or regression coefficients from the other geographic levels, the census tract level of aggregation was selected for all final analyses involving sep . Neighborhood socioeconomic position was measured by creating a socioeconomic position (sep) index . Because ses variables such as income, education, and occupation are often strongly correlated and have been found to load onto a common factor at the census level, census - level data on these variables were combined into a weighted factor score to create a neighborhood sep index that parsimoniously represented multiple socioeconomic variables . The analytical framework for the study involved exploratory and confirmatory factor analysis, structural equation modeling, and repeated measures mixed effects modeling . In keeping with best practice in conducting neighborhood research, individual- and area - level effects would ideally have been examined using a multi - level modeling (mlm) approach, with samples of individuals clustered in neighborhoods allowing formal assessment of random variation within neighborhoods . However because active was not originally designed to sample data stratified by neighborhood but sampled widely across each region, the insufficient sample size within each neighborhood measured precluded the use of mlm . In keeping with recent scholarship, the current study can instead be classified as ecologic, in which neighborhood variables are measured for each person . Statistical package for the social sciences (spss) 18.0 and analysis of moment structures (amos) 18.0 were used to conduct all statistical analyses . Prior to analysis, all variables were inspected for consistency with the assumption of multivariate normality (to permit maximum likelihood estimation). Where that assumption was not met, blom transformations were applied to those variables to improve the normality of their distributions . While no data were missing for any neighborhood - level variables, in a few instances data were missing from the active dataset's cognitive variables (e.g., due to failure to complete a task or attrition between the baseline and posttraining testing occasions). Models were estimated using full information maximum likelihood (fiml), which uses all available data (thereby not eliminating participants with incomplete data). Preparatory to examining the relationship of neighborhood sep to active cognitive outcomes, factor analytic constructs representing neighborhood sep and baseline cognition were developed . Criteria for acceptable model fit indices included root mean square error of approximation (rmsea) <0.06, comparative fit index (cfi)> 0.95, normed fit index (nfi)> 0.95, and tucker - lewis index (tli)> 0.95 . Exploratory factor analyses were first conducted on the neighborhood sep variables, using promax rotation to allow optimal fit to the data . A weighted composite measure of sep was then estimated and optimized in amos based on a common factor identified in the exploratory factor analysis . The cognitive measurement model for the present study dimensionalized cognition into several domains, for which the active dataset was well designed, having captured at least three measures each of specific cognitive domains including memory, reasoning, processing speed, and everyday cognition, or measures of cognition related to everyday abilities . As stated in the introduction, cognitive aging research has increasingly emphasized conceptualization of cognition as both a general ability (g), and a multifaceted system of specific cognitive domains which may respond differently to neighborhood effects . Therefore the model also estimated effects on each of the above cognitive domains, as well as effects on a higher - order factor representing general cognition or g, which captures the shared variance among the five cognitive domains in the measurement model . The sep and cognition factor models were combined in a structural equation model estimating regression paths from sep to the each of the cognitive factors . Model covariates included age, quadratic age, years of education, gender, and race (white, african american). Measures of individual wealth and duration of residence in neighborhood were not collected in active, and could not be included as covariates . Neighborhood sep as a predictor of training gains was estimated as repeated - measures mixed effects models rather than as structural equation models because this provided a more flexible interface for the analysis of training gains, which was operationalized as a two - occasion difference score (and thus would not significantly benefit from a latent variable approach) while still permitting fiml estimation for maximum power in the presence of missing data (i.e., using all available cases). Separate models were estimated for each domain trained (memory, reasoning, or speed) to examine the gains of each training group relative to other participants who did not receive that training (i.e., the other two training groups and the no - contact controls). Each model tested sep as a continuous predictor and occasion (baseline, posttest) and training group (received training on the ability being examined or not) each as class variables . Each model also tested all possible two - way interactions and the three - way interaction of sep, occasion, and group . Interactions were estimated as residualized interaction terms to correct for collinearity with the model's main effects . Neighborhood sep was initially developed using exploratory factor analysis (efa) to combine 8 indicators of area - level ses: median household income,% households with income $150,000,% persons in poverty,% with 9th grade education,% with high school education,% with bachelor's degree,% unemployed, and% working in management or higher . The efa indicated all 8 variables loaded strongly onto one factor (eigenvalue = 5.97), explaining 71% of the variance (factor loadings ranged from 0.71 0.94). All indicators were then included in a confirmatory factor analysis, where modification indices (mi) suggested high intercorrelations of error variances among variables (e.g., ranging up to mi = 1325.42). To derive an optimally - fit, parsimonious set of variables capturing the shared variance of characteristics on which sep is typically based (e.g., income, education, and occupational attainment), redundant variables were progressively removed from the model . The final sep factor model (figure 1) consisted of four variables measuring socioeconomic advantage in income, education, and occupation (median household income,% with income $150,000,% with bachelor's degree, and% in management or higher). As two indices of area - level income would be expected to correlate, a correlation path was estimated between the error variances of median household income and% income $150,000 and was retained in all subsequent models . Model fit was good (cfi = 0.999, nfi = 0.999, tli = 0.996, /df ratio = 8.23 (p = 0.004), and rmsea = 0.05 (p = 0.36)). Notably, the variables loading on the latent sep factor are all indicators of neighborhood affluence or advantage rather than disadvantage; prior neighborhood research has suggested that measures of social advantage, compared to social disadvantage, may be especially important protective factors in neighborhood influences on development . Mean sep factor scores were found to differ across active catchment sites (f(5,2515) = 184.30, p <.001; table 2). Reestimating the models with catchment site as an additional covariate did not alter the pattern of effects, but reduced the fit of the model to an unacceptable extent . Thus, active catchment site was tested but not retained in the study models as a covariate . The present paper sought to characterize both general and specific domains of late life cognition . The measured variables (figure 2) related to memory were combined to represent a memory factor; measures of reasoning combined to form a reasoning factor; guided by preliminary exploratory analyses, measures of processing speed optimally first combined into separate factors based on test, which were then combined to form a speed factor, and individual measures of everyday cognition combined on a factor representing everyday cognition . Because these cognitive domains were represented by latent factors (making them purer representations of their domains), the vocabulary measure was also represented as a factor by using odd and even scores as indicators loading on a single vocabulary factor; this effectively transforms that factor into a construct representing the odd - even split half reliable variance of the measure . Model covariates included linear age, the quadratic effect of age, years of education, gender, and race . The error variances of the ufov 2 and 4 subtests, and the everyday problems test and vocabulary were also allowed to correlate in the process of optimizing model fit (table 3). As the first - order cognitive factors all correlated with one another (p <0.001), and as discussed earlier g, capturing the variance shared by the cognitive factors (table 4). Model fit was acceptable (cfi = 0.98, nfi = 0.97, rfi = 0.96, tli = 0.97, rmsea = 0.046, (p = 1.00), /df ratio = 6.40, and (p <0.001)). The sep and cognitive measurement models were combined in a structural equation model (figure 2) estimating regression paths from neighborhood - level sep to the cognitive constructs of g, reasoning, speed, everyday cognition, and vocabulary (a path could not be estimated for sep or any covariates to memory). Model covariates included linear age, the quadratic effect of age, years of education, gender, and race . Model fit was adequate (cfi = 0.96, nfi = 0.96, tli = 0.95, /df ratio = 6.52, (p <0.001), and rmsea = 0.05 (p = 0.99)). After controlling for individual - level predictors, sep remained a significant predictor of vocabulary alone (p <0.01; table 5). Repeated - measures mixed effects analyses of variance were used to estimate whether neighborhood sep predicted differences in training - related gains, beyond practice - related gains, on posttraining measures of memory, reasoning, and speed . Three separate models examined the gains of each training group relative to all other participants (control group plus members of other training groups) who did not receive that particular training (i.e., for the reasoning outcome, the reasoning training group was compared to the control plus memory plus speed training groups). Each model tested sep as a continuous predictor and occasion (baseline, posttest) and training group (received training on the ability being examined or not), all possible two - way interactions, and the three - way interaction of sep, occasion, and group . Significant effects for occasion, training group, and their interaction were observed as previously reported in the parent study . A main effect was found for sep, but for sep to predict differences in response to active training the three - way interaction would have to be significant; this was not found for any of the trained abilities (memory: f(1, 4594) = 0.66, p = 0.42; reasoning: f(1, 4847) = 0.001, p = 0.98; speed: f(1, 4793) = 0.001, p = 0.97). The present study sought to examine the relationship between current neighborhood - level socioeconomic position (sep) and cognitive level, including several specific cognitive domains in the active study, and response to cognitive training . The findings demonstrated that, after controlling for individual - level demographic predictors, census - defined neighborhood socioeconomic position independently predicted differences in late life vocabulary as measured in active, but not differences in general cognition (g), reasoning, everyday cognition, or speed . These findings differed from the similar prior study by brandt, which found neighborhood ses effects on a composite cognitive measure capturing cognitive status, verbal fluency, verbal learning, and prospective memory . The results also demonstrated that neighborhood sep does not predict individual differences in the immediate response to cognitive training in memory, reasoning, or processing speed . The lack of effect for neighborhood sep on any fluid cognitive ability (memory, reasoning, speed, and everyday cognition) or on cognitive plasticity (i.e., response to training) is somewhat surprising given the extensive research documenting neighborhood effects on factors affecting brain health, such as cardiovascular fitness and chronic diseases (e.g., [43, 44]). It would be reasonable to hypothesize that neighborhood could indirectly affect fluid cognitive abilities and training gains, which are more sensitive to compromised brain health than vocabulary, through differences in access to health care, nutrition, and opportunities for exercise; however, results suggest that if these indirect effects are present they may be relatively weak . Given the relatively good health of this cohort at baseline testing, the majority of respondents reported good or health, and all were independent of care, there also may not have been sufficient variability in baseline fluid cognition scores for neighborhood effects to be detectable . The specific association of neighborhood sep with vocabulary suggests neighborhood influences cognition more through sociocultural mechanisms, as vocabulary captures crystallized cognitive abilities, the dimension of cognition related to stored verbal knowledge, developed over a lifetime of engaging with culture . Crystallized knowledge is also the only domain that continues to improve in the presence of advancing age, compared to fluid cognitive abilities which become less efficient with age . A review of vocabulary in aging reported increasing vocabulary scores with advancing age, as well as with higher education (although in the present study neighborhood sep predicted vocabulary independent of education). Vocabulary measures like the one used in this study, requiring multiple - choice word recognition rather than production of word meanings, are especially robust to the effects of aging . Neighborhood may influence vocabulary by facilitating or constraining one's capacity for, and influencing the results of, sociocultural engagement in the community . It may do so by providing resources or facilities encouraging cultural interaction or by encouraging acculturation through modeling and social comparisons . That is, high - sep neighborhoods may support vocabulary because more neighbors with high educational and occupational attainment provide more social models of high achievement . This modeling may foster upward social comparisons [48, 49], pressuring or evoking desire in an individual to be more like his or her neighbors, resulting in greater engagement in activities enhancing cognitive skills and abilities . Certainly, other researchers have hinted toward social processes (i.e., social norms, interactions and ties, and collective efficacy) as the mechanism linking neighborhoods with developmental outcomes [2, 47]. While vocabulary is related to current neighborhood sep, it is important to consider that vocabulary is also highly correlated with childhood cognitive level . Vocabulary often reflects both cognitive reserve and premorbid ability level, as it is most robust to not only aging but physical insults to the brain, including head trauma, medical conditions, and exposure to neurotoxins . Expressed differently, when cognition is measured in late life, vocabulary is the strongest index of early life cognitive ability therefore, the current neighborhood - vocabulary association found in this study may reflect both historical and current relationships between neighborhood context and cognition; the well - established relationship between neighborhood and cognition in childhood supports this hypothesis . Thus, a part of the late life neighborhood effects on cognition observed here may represent an berkman and glymour found that the county of residence during primary schooling, by way of the laws guiding educational requirements in that county, predicted individual differences in both educational attainment and late life cognitive performance . Similarly, wilson and colleagues reported that parental education during childhood independently predicted cognitive activity across the lifespan and into old age . Therefore, it is likely that childhood sep, had it been collected in active, may be associated with both late life vocabulary and late life neighborhood sep . Finally, it was reported in the results that there were catchment site - level differences in mean sep . Clearly, an individual's neighborhood sep is also part of the general sep of the region in which that individual lived; the interaction between neighborhood and regional sep may influence how a neighborhood's sep relates to cognition . Normal to live in a lower - sep neighborhood; this experience might differ from the experience of living in a low - sep neighborhood within a high - sep region, and might differently affect cognition . Such issues have been explored on the level of individual ses - to - neighborhood ses interactions (e.g., low individual education predicts worse cognition for those living in low - education neighborhoods versus high - education neighborhoods,), but neighborhood - region sep interactions affecting cognition have not been described to our knowledge . This study was limited in several ways due to its nature as a secondary analysis . Several covariates would ideally have been included had they been collected in the parent study, including individual - level income and occupation (although this may later be examined in for the subset present at the 2nd - annual followup) and the length of time lived at current residence . As a consequence, this is an important limitation, as the observed neighborhood association may be attributable, partially or completely, to unmeasured differences in individual - level socioeconomic status . The possibility that the relationship between vocabulary and neighborhood sep can be explained by childhood neighborhood sep must also remain speculation, as this data was not originally collected in active . This study therefore was unable to examine measures of childhood socioeconomic status (e.g., mother's or father's education and income, neighborhood sep in childhood). It is likely that childhood sep is associated with both vocabulary and late life sep, in which case vocabulary's relationship with current neighborhood sep would be expected to diminish or disappear if childhood sep was accounted for in the study . The cross - sectional nature of effects also limits discussion to relationships among variables at a particular time rather than to causal or temporal relationships between variables . Longitudinal assessment of these relationships is an important next step that will be attempted in future studies . At the time this study was conducted, active collected its 10th annual follow - up testing occasion, allowing examination of the interaction between 2000 neighborhood and cognition with 2010 neighborhood and cognition . Future studies will also attempt to examine (a) neighborhood effects on change trajectories, and (b) whether there were neighborhood moves for participants, and whether such moves were associated with functional changes . Furthermore, at this follow - up data was collected documenting participant's county of primary schooling . National historical data will allow investigators to use county information to explore whether and how distal environmental influences (i.e., county - level sep and educational laws during primary schooling years) might predict contextual neighborhood characteristics and cognitive outcomes later in life . This paper adds to the body of research examining neighborhood - cognition associations in late life, and extends previous findings by looking beyond general cognition or cognitive status to examine effects of neighborhood across specific cognitive domains . The finding that neighborhood sep predicts crystallized cognitive abilities (specifically, vocabulary) suggests that neighborhood effects may be most related to sociocultural influences on cognitive development . There was a lack of association between neighborhood sep and fluid cognitive abilities, as well as between neighborhood sep and immediate cognitive change following training . As discussed above, future research should investigate how associations between early life neighborhood context and cognitive development may influence cognitive function and neighborhood selection in late life.
Cocaine and other amphetamine - like psychostimulants have been a significant part of the human pharmacopoeia for thousands of years . However, the appearance of these substances in western societies has been relatively recent, cocaine having debuted as both a local anesthetic and a psychostimulant in the 19th century . Over the course of the next century, it became increasingly clear that the amphetamine - like psychostimulants carried serious abuse liability, as well as producing a prominent paranoia - like syndrome among many individuals who chronically used this class of drugs . The abuse liability of these drugs has resulted in sociological use patterns that have been described as epidemics, such as the methamphetamine epidemic in japan in the 1950s, the cocaine epidemic in the united states in the 1980s, and the crack cocaine epidemic of the 1990s . The high abuse liability of this class of drugs relies on both pharmacological properties and the sociological characteristics of how the drugs are introduced into various societies around the world . This article will not significantly address the sociology of psychostimulant abuse, which involves diverse events ranging from the use of amphetamines by japanese soldiers in world war ii, to the formulation of crack as a less expensive version of cocaine in the united states, to the introduction of prescription formulations to regulate eating habits or to treat attention deficit - hyperactivity disorder . Rather, we will review the basic pharmacology of amphetamine - like drugs, integrate these molecular mechanisms into the brain circuitry of reward, and describe how these drugs are thought to create pathological changes in reward and learning circuitry finally, this knowledge will be amalgamated into a vision of future pharmacotherapies for treating psychostimulant addiction . The defining mechanism of action of amphetamine4ike psychostimulants as a class of drugs with high abuse liability is the ability to bind to dopamine transporters (dat). Dopamine transporters are a member of a class of proteins that eliminate monoamines, including dopamine, from the synaptic cleft after neuronal release . This protein has a high affinity for dopamine relative to other monoamines, such as norepinephrine or serotonin, and while all the readily abused psychostimulants bind to dat, they may also bind to the other monoamine transporters with greater or lesser affinity . To some extent, the relative profile of binding by individual drugs to the different transporter proteins explains different characteristics of the drugs . Most striking, for example, is 3,4-methylenedioxymethamphetamine (mdma) which has a relatively higher affinity for serotonin transporters, and is thereby a mild hallucinogen and neurotoxic to serotonin axon terminals, while methamphetamine binds more avidly to dat, which explains its greater toxicity at dopamine terminals, as well as its propensity to induce paranoid psychosis - like symptoms . While the binding to other monoamine transporters contributes to the antidepressant and hallucinogenic characteristics of some psychostimulants, it is the binding to dat that provides the major influence on abuse liability, which is the focus of this review . There are two major categories of interaction by ampetamine - like psychostimulants with dat, but in all cases the end result is to inhibit the elimination of dopamine from the synapse and thereby increase the quantity and half - life of synaptic and extrasynaptic dopamine levels . The first mechanism is typified by cocaine and methylphenidate that bind to dat, but are not transported into the presynaptic terminal as surrogate dopamine . Therefore, when these drugs bind to dat the increase in extracellular dopamine relies primarily on normal synaptic release, which is more amenable to physiological feedback regulation . The second mechanism is typified by amphetamines, and involves not only binding to dat, but also translocation into the cell in place of dopamine . In addition, these drugs enter dopamine synaptic vesicles, where the fact that these compounds are basically charged degrades the ph gradient necessary to sequester dopamine into the vesicle . This in turn results in a large buildup of dopamine in the cytosol, thereby reversing the direction of dat to release dopamine into the extracellular space rather than facilitating its removal regardless of the precise interaction with dat by individual amphetamine - like psychostimulants, this class of drugs dramatically elevates extracellular dopamine, and this action is thought to be the initiating molecular event that reinforces drugseeking behaviors, ultimately culminating in addiction . Thus, when an organism encounters a novel stimulus, whether a positive stimulus such as a food reward or a negative stimulus such as a stressor, the activity of dopamine cells in the ventral tegmental area, and dopamine release in axon terminal fields in the prefrontal cortex, nucleus accumbens, and/or amygdale, are altered . An important characteristic of this brain - environment interaction is that the ability of a given stimulus to increase dopamine cell firing and release decreases with repeated presentation of the stimulus . However, it can be shown that if a motivationally neutral stimulus (such as a light or tone) is associated with the motivational event in such a manner that the neutral stimulus predicts arrival of the motivational event, the ability of the motivational stimulus to release dopamine is transferred to the neutral stimulus . Thus, the neutral stimulus now causes dopamine release in a manner predicting arrival of a motivationally relevant event . Based upon these data, the role for dopamine release in the mesocorticolimbic brain regions is twofold: (i) to cue the organism that a novel motivationally relevant event is occurring and that adaptive behavioral responses need to be engaged (eg, approach a reward or avoid a stress); (ii) once the behavioral response is established, dopamine release is antecedent to the appearance of the motivationally relevant event and is triggered by environmental associations that the organism has made with the event as part of learning the adaptive behavioral response . In this way, dopamine serves to alert and thereby prepare the organism for an impending important event . The primary differences between psychostimulant - induced dopamine release and release associated with normal learning about important environmental events such as rewards and stressors is: (i) since psychostimulants block the elimination of dopamine through dat, the level of dopamine achieved far exceeds what is possible from a biological stimulus; (ii) in contrast to biological stimuli that cease to release dopamine once an approach or avoidance response to that stimulus has been learned, psychostimulants continue to release large amounts of dopamine upon every administration (with the possible exception of extended binging that can temporarily deplete dopamine stores). Thus, with psychostimulants, each administration releases dopamine into mesocorticolimbic regions, causing further associations to be made between the drug experience and the environment . In this way, it is thought that the more a psychostimulant is administered, the more learned associations are made with the environment and the more effective the environment becomes at triggering craving and drug - seeking . It is this overlearning of drugseeking behaviors by progressive associations formed between repeated drug - induced dopamine release and the environment that is thought to lead to increased vulnerability to relapse . As outlined above, psychostimulant - induced dopamine release is responsible for reinforcing behaviors designed to seek and administer the drugs . The dopamine projections involved in this process are outlined in figure 1a, and as indicated, the most critical projection in this regard is the projection from the ventral tegmental area dopamine cells to the nucleus accumbens . For example, if psychostimulant - induced release of dopamine in the nucleus accumbens is impaired, this affects the acquisition of drug - seeking behaviors, and can markedly influence the amount of drug taken in a well - trained subject . Thus, the learning of a task to obtain the drug and the amount of drug taken in a given session is strongly regulated by dopamine release in the accumbens . However, when an animal has been withdrawn from repeated psychostimulant use, and drug - seeking is initiated by an environmental stimulus such as a cue previously paired with drug delivery, or a novel stressor, it is dopamine release in the prefrontal cortex and amygdala, respectively, that mediates the reinstatement of drug - seeking . Thus, relapse can be induced by dopamine release in prefrontal and allocortical brain regions, and reflects the aforementioned physiological role of dopamine release as a predictive antecendent to stimulus (drug) delivery . What this implies is that chronic release of dopamine by repeated psychostimulant administration may be modifying cortical and allocortical regulation of behavior . Figure 1b shows that the cortical and allocortical regulation of behavior is primarily mediated by glutamatergic projections . These projections are to subcortical structures, such as the nucleus accumbens and dopamine cells in the ventral tegmental area, as well as between the cortical and allocortical regions . Thus, when dopamine is released into the prefrontal cortex or amygdala by a drug - associated cue or stressor, this is thought to stimulate glutamatergic projections between the prefrontal cortex and amygdala, as well as glutamatergic outputs to the accumbens and ventral tegmental area . A variety of studies have linked this activation of corticofugal glutamate transmission with craving in psychostimulant addicts or drug - seeking in animal models of addiction . The neuroimaging literature clearly shows metabolic activation of regions of the prefrontal cortex, including portions of the anterior cingulate and ventral orbital cortices, and the amygdala during cue - induced craving for amphetamine - like psychostimulants . Interestingly, while a cue or low dose of psychostimlant markedly increases metabolic activity in the prefrontal cortex and amygdala, in the absence of a learned drug association the prefrontal cortex is hypoactive . The reduction in basal metabolic activity is taken to indicate a potential deficit in cognitive ability to regulate relapse, and recent cognitive testing in psychostimulant addicts confirms the presence of certain cognitive dysfunctions related to impulse control and switching behaviors in an adaptive manner to changing environmental circumstances . A strong role for activation of both the prefrontal cortex and amygdala thus, pharmacological inhibition of either of these regions prevents the reinstatement of drug - seeking in animals withdrawn from drugs that have undergone extinction training . Moreover, a marked release of glutamate is measured in the nucleus accumbens of animals initiating drug - seeking in response to a stressor, and this glutamate is derived from increased activity in the projection from the prefrontal cortex to the nucleus accumbens . One final set of studies to be considered regarding cortical glutamate is the recent evidence that as drug - seeking becomes more compulsive there is a gradual shift to greater reliance on corticostriatal habit circuitry, and less involvement of prefrontal to accumbens circuitry . This possibility is supported by animal models in two ways: (i) if animals that have been trained to self - administer cocaine are left in abstinence for an extended period, drug - seeking is augmented, and in this case inhibition of the prefrontal cortex or amygdala no longer inhibits drug - seeking induced by drug - associated stimuli . However, inhibition of the dorsolateral striatum is still effective at blocking drug - seeking; (ii) as training of an animal in drug - seeking paradigms progresses it is possible to show a gradual increase in dopamine released into the caudate in favor of release into the nucleus accumbens . This is illustrated in figure 1a, showing that dopamine release into the caudate can regulate habitual behaviors . On one hand, these data point to the possibility that in treating compulsive relapse we should be focusing on regulation of corticostriatal habit circuitry, including glutamate input to the caudate from sensorymotor cortex and dopamine input from the substantia nigra . However, these studies have been conducted in rats in whom the frontal cortex is poorly evolved, and given the marked activation produced in the prefrontal cortex and amygdala by drug - associated stimuli in psychostimulant addicts, the conclusion that compulsive relapse is entirely derived from corticostriatal habit circuitry may be an oversimplification . Indeed, it has been argued that a primary role for therapy in treating addiction is to strengthen prefrontal regulation of drug - seeking behaviors, whether through psychosocial interventions or pharmacotherapy . Given the apparent critical role played by glutamatergic affrents to the nucleus accumbens in initiating drugseeking or craving, recent studies have identified a number of enduring cellular changes in glutamate transmission that may be critical pathological neuroadaptations to psychostimulant use, and may serve as targets for pharmcotherapeutic intervention . In general the neuroplasticity can be categorized as postsynaptic, presynaptic and nonsynaptic (ie, residing predominantly in glia). However, since these processes are intimately related to each other, it is perhaps best to consider all the adaptations as changes in glutamate homeostasis, the end result of which is a psychostimulant - induced enduring change in the fidelity of communication between the prefrontal cortex and the nucleus accumbens, and the regulation by this projection of corticostriatal habit circuitry . It has been proposed that this loss of fidelity results in a weakening or loss in the capacity of psychostimulant addicts to cognitively intervene in habitual behaviors, thereby making drug - seeking more difficult to control and increasing the vulnerability to relapse . As mentioned above, drug - seeking is associated with a large release of prefrontal glutamate into the nucleus accumbens . The large release of glutamate during drugseeking is all the more remarkable because it was discovered using microdialysis . Which is not a very sensitive measure of glutamate transmission . Indeed, when animals are trained to seek a biological reward, such as food, microdialysis cannot measure glutamate release . Thus, the large psychostimulant - induced release of glutamate has been hypothesized to be a pathological and perhaps critical mediator of relapse . This hypothesis is supported by the fact that treatments interrupting synaptic glutamate release also inhibit drug - seeking . This includes a variety of pharmacological treatments that have the potential to be developed into pharmacotherapeutic agents, as outlined below . Perhaps in part a consequence of the massive synaptic glutamate release occurring during psychostimulantseeking behavior, a number of marked changes in postsynaptic glutamate transmission have been measured in animals withdrawn from chronic cocaine or amphetamine administration . Perhaps among the most dramatic importantly, this appears to be accompanied by an increase in the insertion of a - amino3-hydroxy-5-methylisoxazole-4-propionic acid (ampa) glutamate receptors into the membrane of spiny neurons in the accumbens, and is associated with an increase in electrophysiological sensitivity to ampa receptor stimulation (as measured by the ampa: n - methyl d - aspartate [nmda] ratio). Moreover, a number of other proteins regulating the fidelity of postsynaptic glutamate transmission are altered after chronic cocaine use, including proteins that regulate the structure and function of the protein scaffolding in which the glutamate receptors are embedded, including postsynaptic density (psd)-95 and homer proteins, among others . Also, in addition to ampa ionotropic glutamate receptors, signaling through metabotropic glutamate receptors is downregulated . Finally, this psychostimulant - induced postsynaptic neuroplasticity is associated with changes in the biochemical machinery regulating spine formation, notably an increase in actin cycling and formation of factin (a primary structural protein regulating spine morphology and the insertion of proteins into and out of the membrane). Taken together, these findings indicate that significant changes have been produced by psychostimulants in the way that synaptically released glutamate will be interpreted by postsynaptic cells . Thus, there remain many apparent contradictions in the literature regarding changes in specific proteins, and in the overall direction of synaptic grading (ie, is postsynaptic glutamate transmission augmented or inhibited by chronic psychostimulant administration). Therefore, for now it is probably not prudent to speculate on the type of drug development that may arise from this particular direction of research into psychostimulant - induced changes in glutamate signaling . As outlined above, given our current state of knowledge it is more likely that pharmacotherapeutic restoration of normal glutamate release may be a more successful approach than manipulating postsynaptic proteins responsible for and/or associated with changes in the fidelity of postsynaptic glutamate transmission . In part, this is due to the relatively contradictory status of the emerging literature on postsynaptic plasticity . Moreover, it has been hypothesized that the adaptations in presynaptic glutamate release may be at least partly causal in the postsynaptic adaptations, posing the possibility that if the pathological release of glutamate can be successfully ameliorated, postsynaptic normalization may follow . Pharmacotherapeutic targets for regulating the pathological synaptic glutamate release seen in the accumbens of psychostimulant - seeking animals can be placed into two categories: (i) targets based upon psychostimulant induced changes in proteins regulating synaptic glutamate release; (ii) proteins that produce a general decrease in excitatory transmission . Compounds in the first category are likely to be the most specific for psychostimulant addiction, and perhaps carry the least number of unwanted side effects, while the latter category may be less selective not only regarding effects on other addictive drugs, but also in terms of unwanted side effects . Neuroplasticity produced by chronic cocaine administration that could potentially contribute to pathological glutamate release includes downregulation of cystine - glutamate exchange, downregulation of glial glutamate transporters, and downregulation of release - regulating presynaptic metabotropic glutamate receptors (mglur2/3). Importantly, these three changes are interrelated due to the cystine - glutamate exchanger and glutamate transporter regulating extrasynaptic glutamate tone on release regulating mglur2/3 . Drugs have been examined in animal models of psychostimulant addiction, and to a lesser extent in clinical trials with cocaine addicts that regulate one or more of these processes . For example, n - acetylcysteine upregulates cystine glutamate exchange, and has been shown in animal models to prevent synaptic glutamate release associated with drug - seeking, restore inhibitory tone on synaptic release through activation of mglur2/3, and to inhibit the desire for cocaine in a double - blind cue - reactivity trial in non - treatment - seeking cocaine addicts . Also, mglur2/3 agonists have proven effective at inhibiting cocaine seeking in animal models; however, unlike nacetylcysteine, food - seeking was inhibited at only a 3- to 10-fold increase in dose relative to inhibiting cocaineseeking . Although no studies have yet evaluated regulating glutamate transport in drug - seeking models of psychostimulant addiction, recent reports of the use of -lactam antibiotics to increase glutamate transporter membrane insertion poses an interesting possibility for pharmacologically overcoming the cocaine - induced downregulation of glutamate transporters . Finally, while the mechanism is not clear, modafinil has been reported to increase extracellular glutamate levels, which would restore tone on release inhibiting mglur2/3 . Notably, modafinil has been found to successfully decrease cocaine relapse in a number of clinical trials . The primary drugs in the category of nonspecific inhibitors of synaptic glutamate release include a variety of -aminobutyric acid (gaba)-mimetic compounds . These range from relatively specific agonists at gabab receptors, such as baclofen, which inhibit synaptic glutamate release to a host of less selective compounds known to increase gaba transmission via interactions with synthetic or elimination mechanisms, such as topiramate or vigabatrin . For all of these compounds there is preclinical and clinical data to support some potential efficacy . However, as predicted, especially for the nonselective gabamimetics untoward side effects, such as sedation, are reported . This review has endeavored to transport the reader from the initiating molecular actions of amphetamine - like psychostimulants on dopamine systems in the brain to enduring neuroplasticity produced in glutamate transmission responsible for communicating from prefrontal and allocortical brain regions through the nucleus accumbens to motor regulatory systems . Moreover, by examining molecular neuroplasticity produced in excitatory synapses by chronic psychostimulant administration, it is possible to make some deductions about potential pharmacotherapeutic interventions . Indeed, there already exists an emerging literature supporting this approach in developing potential pharmacotherapies for treating psychostimulant addiction . Importantly, this is a nascent and emerging science, and while much has been discovered, the cutting edge of discovery into the neuroplasticity produced by psychostimulants is understandably contradictory . As further discoveries are made that allow us to understand the nature of these contradictions, it should follow that additional targets will emerge to provide potential novel pharmacotherapies for treating psychostimulant addiction.
The roots of zapoteca portricensis is a common remedy in the treatment gastrointestinal disorders used by tradomedical practitioners in eastern nigeria . This study was aimed at evaluating the possible antiulcer activity of the root of this plant in experimental rats . Different groups of albino rats of male sex were given three doses (50, 100, 200 mg / kg) of the extract . The ethanol model produced an average ulceration in rats with reduction of ulcer (50%, 75% and 90%) seen in all the extract treatment . A dose dependent inhibition of ulcer was seen in all doses of the extract with doses 100 and 200 mg / kg produced a significant reduction compared with control . In the indomethacin model, inhibition of ulcer (57.1%, 65.7% and 80.0%) was seen in the treatments with the extracts in a dose dependent manner . This study has shown that roots of this plant (zapoteca portoricensis) possess potent antiulcer activity.
The online version of this article (doi:10.1007/s10815 - 012 - 9815-x) contains supplementary material, which is available to authorized users . The development of assisted reproductive technology (art) has recently enabled the direct observation of human oocytes, revealing various mysterious phenomena involving the beginning of life . However, it is undeniable that frequent microscopic examinations of human early embryos may have negative effects on them, making it difficult to obtain reliable detailed information of human embryonic development from still images . We therefore developed an in vitro culture system for time - lapse cinematography (tlc), based on payne et al . This system enables non - invasive and continuous imaging of human oocyte fertilization and embryonic development . Our previous dynamic analyses of the fertilization process in human oocytes and of human embryonic development using the in vitro tlc system confirmed for the first time the detailed time course of sequential events during embryonic development and revealed novel phenomena [fertilization cone, cytoplasmic strand, and splitting of the inner cell mass (icm)] under culture conditions in vitro [supplementary movie 1 (online resource 1)]. Our further observation of the movie revealed another novel phenomenon that is likely to be involved in the mechanism of polyspermy block, wherein once the leading sperm has penetrated the zona pellucida (zp) and attached to the oocyte membrane, any following sperm within the zp stop penetrating immediately . To date, two types of polyspermy block were thought to exist in marine animals and mammals, including human (oocyte membrane block and zona reaction). In addition, the oocyte membrane block occurs in seconds, while an oocyte membrane depolarization block has not been detected in studies of mammalian oocytes . We therefore consider that the phenomenon described herein differs from previously proposed mechanisms for polyspermy block, prompting us to re - analyze all of our tlc data covering the fertilization process . Herein, we provide the results of this second detailed tlc analysis, which we believe confirms the existence of a novel mechanism for the prevention of polyspermy . The in vitro culture system for tlc has been described in detail elsewhere . In brief, we used an inverted microscope (ix-71; olympus, tokyo, japan) with nomarski differential interference contrast optics (olympus) and a micromanipulator (narishige, japan), which was covered by a handcrafted acrylic chamber . Our system also contained a small acrylic chamber surrounded by a small waterbath on the stage of the microscope, into which a glass petri dish containing a microdrop of culture medium (5 l) was placed . The volume of flowing co2 and temperature within the chamber were adjusted to give the optimal values (temperature, 37.0 0.3 c; ph, 7.37 0.03). The inverted microscope was equipped with a ccd digital camera (roper scientific photometrics, tucson, az) connected to the computer and display by metamorph (universal imaging co, downingtown, pa) (fig . The upper two images are from the tlc system, and the lower two tables indicate imaging and culture conditions of the tlc time - lapse cinematography (tlc). The upper two images are from the tlc system, and the lower two tables indicate imaging and culture conditions of the tlc digital images of the cultured embryos were acquired for approximately 40 h by using an exposure time of 50 s . In total, approximately 2,0002,800 frames were taken during the observation period . We displayed movies at 30 frames per second to analyze the fertilization process and elucidate the mechanism of polyspermy block . Before the commencement of tlc observation, we mechanically and gently removed the cumulus cells from around the oocytes, so as not to damage the tails of the sperm that penetrated into the zp, at 1 h after the in vitro insemination (approximately 50 10 sperm per oocyte). Images were acquired at 10-s intervals for the first 2 h, and thereafter at 2-min intervals, based on our preliminary study that showed most of the sperm penetrating the zp within 3 h after the in vitro insemination . Oocytes were collected from 122 couples after receiving informed consent, from july 2004 to december 2011 . One oocyte was randomly selected from each couple and 122 oocytes were tested . In the tlc images obtained from these oocytes, penetration of the leading sperm into the zp and attachment to the oocyte membrane was confirmed in the 22 oocytes . Among these, only three oocytes showed both the leading and following sperms within the zp in the same frame, and these tlc data were therefore used to evaluate the dynamic changes in both sperms until the leading sperm attached to the oocyte membrane during the fertilization process . The ethics committee of the japanese institution for standardizing art (jisart) approved our study protocol . Of the 22 imaged oocytes, in which penetration into the zp and attachment to the oocyte membrane of the leading sperm were confirmed, the leading sperm attached to the oocyte membrane within an average of 96 min after insemination, and the sperm head disappeared an average of 37 min after attachment of the sperm to the oocyte membrane . There was no difference in the time course of the fertilization process between the three oocytes subsequently chosen to analyze the mechanism for prevention of polyspermy and the remaining 19 oocytes . Figure 2 shows the results of analyzing each tlc frame of the selected oocytes (oocyte 1, 2 and 3), in which the following sperm penetrated the zp together with the leading sperm . Penetration of the following sperm into the zp was arrested within 10 s after the leading sperm attached to the oocyte membrane, even though the tail of the following sperm was still actively moving in all three oocytes (fig . 2). Additional data are given in supplementary movies 2 to 4 (online resource 2 to 4).fig . The sperm fertilizing the oocyte (leading sperm) is indicated by a blue circle, while the sperm following the fertilizing sperm (following sperm) is indicated by a red circle among three oocytes (a). Sections of the images containing both leading and following sperms were magnified to facilitate observation of the process (b, c, d). The images were acquired in 10-s intervals . With regard to oocyte 1 shown in the upper panel, both the leading and following sperm penetrated into the zona pellucida from the beginning of imaging to shortly prior to the attachment of the leading sperm to the oocyte membrane (a, b). The leading sperm attached to the oocyte membrane 1,850 s (30.8 min) after the beginning of imaging (c), and the penetration of the following sperm was inhibited at 1,860 s (d), which is within 10 s after the attachment of the leading sperm to the oocyte membrane . With regard to oocyte 2 shown in the middle panel and oocyte 3 shown in the lower panel, the penetration of the following sperm was also inhibited within 10 s after the attachment of the leading sperm to the oocyte membrane (c, d) dynamics of the leading sperm and following sperm . The sperm fertilizing the oocyte (leading sperm) is indicated by a blue circle, while the sperm following the fertilizing sperm (following sperm) is indicated by a red circle among three oocytes (a). Sections of the images containing both leading and following sperms were magnified to facilitate observation of the process (b, c, d). The images were acquired in 10-s intervals . With regard to oocyte 1 shown in the upper panel, both the leading and following sperm penetrated into the zona pellucida from the beginning of imaging to shortly prior to the attachment of the leading sperm to the oocyte membrane (a, b). The leading sperm attached to the oocyte membrane 1,850 s (30.8 min) after the beginning of imaging (c), and the penetration of the following sperm was inhibited at 1,860 s (d), which is within 10 s after the attachment of the leading sperm to the oocyte membrane . With regard to oocyte 2 shown in the middle panel and oocyte 3 shown in the lower panel, the penetration of the following sperm was also inhibited within 10 s after the attachment of the leading sperm to the oocyte membrane (c, d) next, we analyzed the time course of the distance that the leading and following sperms traveled in the zp (fig . 3). The following sperm traveled at a similar velocity to the leading sperm, until the leading sperm attached to the oocyte membrane after penetrating the zp . However, once the leading sperm reached the oocyte membrane across the perivitelline space, the following sperm immediately ceased further penetration, within 10 s. the behaviors of the leading and following sperm were identical among the three oocytes.fig . 3change in the distance between the surface of the zona pellucida and leading or following sperm . The time course of the change in zona pellucida penetration distance of the leading sperm (blue line) and the following sperm (red line) shows that the zona pellucida penetration of the following sperm was inhibited when the leading sperm attached to the oocyte membrane (indicated by the arrow) after zona pellucida penetration in all oocytes (oocyte 1, 2 and 3) used in this analysis change in the distance between the surface of the zona pellucida and leading or following sperm . The time course of the change in zona pellucida penetration distance of the leading sperm (blue line) and the following sperm (red line) shows that the zona pellucida penetration of the following sperm was inhibited when the leading sperm attached to the oocyte membrane (indicated by the arrow) after zona pellucida penetration in all oocytes (oocyte 1, 2 and 3) used in this analysis we revealed a novel phenomenon that is likely to be associated with the polyspermy block in human oocytes . This new mechanism involved cessation of the following sperm penetrating the zp within 10 s of the leading sperm penetrating the zp and attaching to the oocyte membrane . This was despite the following sperm retaining active tail movement and both sperm traveling at a similar velocity within the zp before penetration . The behaviors of the leading and following sperm were identical among the three oocytes imaged and analyzed . The difficulty in obtaining tlc images in which both the leading and following sperm within the zp were clearly identified precluded analysis of their respective behaviors in more than three oocytes . Achieving normal fertilization of a single egg by a single sperm is core to life in many sexually reproducing animals, including humans . In most mammalian reproduction processes, huge numbers of sperm are ejaculated into the female reproductive tract (ratio of sperm to oocyte is approximately 10:1); however, the vast majority of sperm are rapidly eliminated from the female tract . It is thought that polyspermy is usually prevented by a decreased number of sperm reaching the fallopian tube and a block mechanism in the fertilized oocyte . In this context, it is also thought that in vitro fertilization (ivf) involves different mechanisms from the normal in vivo process in mammals . In particular, successful fertilization in vitro of a single oocyte requires a large number of motile sperms, and thus there is always the possibility that more than one sperm could penetrate the zp and reach the oocyte . However, between a healthy mature ovum and a healthy sperm, normal fertilization of a single egg by a single sperm occurs at a relatively high rate, which can also be confirmed easily in the environment of ivf . There are also many clinical cases of polyspermy that are attributable to the penetration of excess sperm after ivf . Hundreds of articles have been published on the process of fertilization and polyspermy block in marine animals and mammals including human [24]. Two types of mechanisms for polyspermy block have been reported: the oocyte membrane block to sperm penetration and the zona reaction . The former involves a depolarization of the oocyte membrane caused by the influx of na, which changes the potential of the oocyte membrane from negative to positive . This change in potential prevents any following sperm from attaching to the oocyte membrane, transiently contributing to polyspermy block . The oocyte membrane block or so - called fast block to polyspermy occurs in seconds [68]; however, it was unlikely to be involved in fast block to polyspermy in the fertilization process in mammals . The second proposed mechanism involves a ca oscillation event activated by attachment of the sperm to the oocyte membrane . The subsequent increase in intracellular ca concentration triggers the exocytosis of cortical granules (approximately 1 m in diameter) from just below the oocyte membrane into the perivitelline space . Enzymes such as hydrolase, proteinase, and peroxidase, which are contained in the cortical granules, prevent the penetration of following sperm by modifying the structure of the sperm receptors such as zp2 and zp3 (slow block to polyspermy) and by hardening the zp . This slow block occurs within approximately 5 to 8 min of oocyte activation and is considered the main mechanism of polyspermy block in humans; however, the mechanistic details of this process remain largely unclear in human . The entire fertilization process is extremely delicate and precise, and there is no doubt that multiple layers of safety mechanisms exist to ensure the achievement of normal fertilization . A failure of this safety mechanism is believed to instantly trigger abnormal fertilization, such as polyspermy . However, because it is so difficult to establish laboratory models of the human in vivo fertilization process and few such studies have been conducted, fertilization safety mechanisms operating in human oocytes remain unclear . In addition, dynamic morphological tactics such as tlc have not been applied to analyze the possible mechanisms of polyspermy block . It is true that the tlc analysis presented here still does not exactly represent the in vivo situation . Nevertheless, we believe that these morphological data reveal a novel system for polyspermy block that could occur in human oocytes, and that the images analyzed represent the true dynamic physiology of the human fertilization process . We believe that a novel mechanism of polyspermy block takes place in the zp, which differs from both the oocyte membrane block to sperm penetration (fast block to polyspermy) and the zona reaction (slow block to polyspermy) (fig . 4a proposed mechanism for a fast zona - block to polyspermy during the human fertilization process . The findings of our tlc analysis indicate the existence of a novel mechanism of polyspermy block, which takes place in the zona pellucida and differs from the oocyte membrane block to sperm penetration (fast block to polyspermy) and the zona reaction (slow block to polyspermy) a proposed mechanism for a fast zona - block to polyspermy during the human fertilization process . The findings of our tlc analysis indicate the existence of a novel mechanism of polyspermy block, which takes place in the zona pellucida and differs from the oocyte membrane block to sperm penetration (fast block to polyspermy) and the zona reaction (slow block to polyspermy) we are planning to elucidate the details of this novel mechanism by further observing the fertilization process of human oocytes using our tlc system . By use of our original tlc system, we have succeeded in demonstrating the possible existence of a novel mechanism of polyspermy block in human oocytes . However, the details of this mechanism of polyspermy block are not yet fully understood, and the mechanism we have revealed in this study may be a small part of a highly complicated system . We will continue to analyze the fertilization process of human oocytes by use of the tlc system to reveal the details of this novel mechanism of polyspermy block in human oocytes . Movie 1this is the first movie to successfully demonstrate the dynamic process of fertilization in human oocytes in vitro whereby once the leading sperm was attached to the oocyte membrane, the following sperm stopped further penetration within the zp . This phenomenon prompted our hypothesis that a novel mechanism could exist for the polyspermy block in the human fertilization process . (mpg 12897 kb) this is the first movie to successfully demonstrate the dynamic process of fertilization in human oocytes in vitro whereby once the leading sperm was attached to the oocyte membrane, the following sperm stopped further penetration within the zp . This phenomenon prompted our hypothesis that a novel mechanism could exist for the polyspermy block in the human fertilization process . (mpg 12897 kb) movie 2this movie represents the same tlc imaging used to make movie 1 . While the leading sperm (blue circle) traveled within the zp and moved across the perivitelline space, the following sperm (red circle) was also steadily penetrating within the zp . However, once the leading sperm was attached to the oocyte membrane, the following sperm stopped penetrating within the zp even though the following sperm was far away from the leading sperm attachment site . (mpg 2680 kb) this movie represents the same tlc imaging used to make movie 1 . While the leading sperm (blue circle) traveled within the zp and moved across the perivitelline space, the following sperm (red circle) was also steadily penetrating within the zp . However, once the leading sperm was attached to the oocyte membrane, the following sperm stopped penetrating within the zp even though the following sperm was far away from the leading sperm attachment site . (mpg 2680 kb) movie 3the penetration of the following sperm in this case was also inhibited within 10 s after the attachment of the leading sperm to the oocyte . (mpg 2243 kb) the penetration of the following sperm in this case was also inhibited within 10 s after the attachment of the leading sperm to the oocyte . (mpg 2243 kb) movie 4this movie represents the same tlc imaging data used to make movies 2 and 3 . It shows that even though the tail of the following sperm was still moving actively, the sperm did not penetrate further . (mpg 2680 kb) this movie represents the same tlc imaging data used to make movies 2 and 3 . It shows that even though the tail of the following sperm was still moving actively, the sperm did not penetrate further . This article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
Primary human schlemm's canal cell lines were grown to confluence in six - well plates (bd falcon, franklin lakes, nj, usa) containing dulbecco's modified eagle's medium (dmem; life technologies, grand island, ny, usa); 10% fetal bovine serum (mediatech, manassas, va, usa); and 1% penicillin / streptomycin (life technologies) in 5% co2 at 37c . Confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells . Cells were incubated with latanoprost (de - esterified free - acid form at 100 nm final concentration; cayman chemical, ann arbor, mi, usa) or vehicle (ethanol, final dilution 1:1000) in dmem containing 1% penicillin / streptomycin . Primary schlemm's canal cells were grown to confluence, treated with 100 nm latanoprost for 6 hours, and harvested . Total rna was extracted using an rna isolation kit (rneasy total rna isolation kit; qiagen, hilden, germany). Approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit (iscript cdna; bio - rad laboratories, inc ., hercules, ca, usa). Quantitative real - time polymerase chain (qpcr) reaction using stc-1 (forward, 5-aggcggagcagaatgactc-3; reverse, 5-gttgaggcaacgaaccactt-3) and glyceraldehyde 3-phosphate dehydrogenase (gapdh: forward, 5-cctctgacttcaacagc-3; reverse, 5-gctgtagccaaattcgt-3) primers were performed on a pcr system using a master mix (roche light cycler 480 with sybr green i; roche, indianapolis, in, usa). We performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c, annealing at 63c, and extension at 72c . Primary schlemm's canal cells were grown to confluence, treated with 100 nm latanoprost for 15 minutes, 1, 2, 4, or 6 hours, and harvested . Schlemm's canal cell pellets were suspended in ice - cold lysis buffer (50 mm tris ph 8.0, 0.5% sodium dodecyl sulfate, 0.5% triton x-100, 137 mm nacl, 3 mm kcl, 8 mm na2hpo4 - 7h2o, 1 mm kh2po4, protease inhibitors; roche) and passed repeatedly through a 21-gauge needle for homogenization . Lysate was centrifuged at 13,000 g for 10 minutes, and total protein was quantified by the bradford assay . Cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer (thermo fisher scientific, waltham, ma, usa) containing 15% 2-mercaptoethanol (sigma - aldrich corp ., st . Louis, mo, usa), heated and separated on a 4% to 15% sds - page gradient gel (bio - rad laboratories, inc . ). Proteins were transferred to polyvinylidene difluoride membrane (millipore corp ., billerica, ma, usa) in 1x transfer buffer (50 mm tris, 384 mm glycine, 0.01% sds, 20% methanol). Membranes were blocked in 20 mm tris (ph 7.5), 150 mm nacl, 0.05% tween-20, and 2% instant nonfat dry milk . Blots were probed with rabbit monoclonal anti - human stc-1 (novus biologicals, littleton, co, usa) and mouse monoclonal anti - human gapdh (novus biologicals). Secondary antibodies used were horseradish peroxidase linked anti - rabbit or anti - mouse, respectively (ge healthcare, piscataway, nj, usa). Antibody / antigen complexes were detected using ecl western blot signal detection reagent (ge healthcare). Chemiluminescence film (biomax xar; eastman kodak, rochester, ny, usa) was used to visualize protein signals . Each film was digitized with a photographic scanner (epson perfection 2400; epson america, inc ., long beach, ca, usa). The band intensities for western blot analysis were quantified using imagej software (http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health, bethesda, md, usa) and normalized to gapdh . All animal studies and treatment protocols were approved by the mayo clinic (rochester, mn, usa) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research . We obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory, baylor college of medicine, and bred at mayo clinic . A handheld rebound tonometer (icare tonolab; colonial medical supply, franconia, nh, usa) was used to measure iop in conscious mice . For iop measurements, the tonometer was held perpendicular to the cornea according to the manufacturer's instructions . The tonometer records six readings from the same eye, discards the highest and lowest values, and shows the average of the remaining four values as a single iop reading . Three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye . After 1 week of baseline iop measurements, stc-1 mice were treated with latanoprost (n = 10) or rho kinase inhibitor y27632 (enzo life sciences, farmingdale, ny; n = 10). Congenic wild - type controls were treated with latanoprost (n = 8) or y27632 (n = 10). Treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid (100 m dissolved in 1:1000 dmso in pbs) or 10 mm y27632 (dissolved in phosphate - buffered saline). In the contralateral eye, vehicle was added daily in the same proportion as the treated eye for 7 consecutive days . Additionally, wild - type mice (n = 7; charles rivers laboratories, wilmington, ma, usa) were treated with 5 l of topically administered recombinant human stc-1 (0.5 mg / ml; biovendor research & diagnostic products, asheville, nc, usa) or vehicle (phosphate - buffered saline) daily for 7 days to examine the effect of stc-1 on iop . In all animals, the right eye served as the vehicle control eye while the left eye received study drug (latanoprost, y27632, or stc-1). We recorded iop in both eyes three times daily at 1, 4, and 23 hours following treatment . Anterior segments from human donor eyes (age 75.5 17.5 years, range: 51 to 98 years; n = 8) were perfused in culture with dmem within 10.2 4.4 hours of death as previously described . After achieving a stable baseline pressure, one anterior segment from each pair received recombinant human stc-1 at concentrations of 5, 50, or 500 ng / ml (dissolved in h2o), while the fellow eye received vehicle and served as the control . We added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion . Hourly pressure readings were obtained from the average of 60, one - minute pressure measurements using a custom - designed software program . The experimental eye was typically the right eye and the control was the left eye . For human anterior segments, selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.2). Tissue wedges were dehydrated in a series of ascending ethanol concentrations, cleared with 100% acetone, infiltrated and embedded in epon araldite, and sectioned at 0.5 m . Eye tissue sections were stained with toluidine blue and examined using a light microscope (nikon corp ., additional tissue wedges were sectioned at 100 nm, placed on copper film grids, and stained with uranyl acetate and lead citrate . Tissue sections on copper film grids were examined using a transmission electron microscope (jeol-1400; jeol usa, inc ., peabody, ma, usa). For mouse eye histopathology eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.2) overnight, and processed for light microscopy as described above for human anterior segment wedges . Additional 100-nm sections were placed on copper film grids, and stained with uranyl acetate and lead citrate . Processed sections were obtained following examination under a light microscope (nikon corp .) And a transmission electron microscope (jeol usa, inc . ). Prior to initiation of statistical analysis, all data sets were evaluated for distribution assessment using the shapiro - wilk test . For animal studies, significance of iop change was assessed between experimental and vehicle - treated control eyes using student's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets . Variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes . Statistical calculations were performed using a statistical software package (jmp; sas institute, inc ., cary, nc, usa). For human anterior segment studies, the effect of latanoprost was expressed as the change in outflow facility (c) for each anterior segment . Results from each pair of anterior segments were combined into a group mean for each drug, and statistical significance was analyzed using a student's 2-tailed paired t test . Primary human schlemm's canal cell lines were grown to confluence in six - well plates (bd falcon, franklin lakes, nj, usa) containing dulbecco's modified eagle's medium (dmem; life technologies, grand island, ny, usa); 10% fetal bovine serum (mediatech, manassas, va, usa); and 1% penicillin / streptomycin (life technologies) in 5% co2 at 37c . Confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells . Cells were incubated with latanoprost (de - esterified free - acid form at 100 nm final concentration; cayman chemical, ann arbor, mi, usa) or vehicle (ethanol, final dilution 1:1000) in dmem containing 1% penicillin / streptomycin . Primary schlemm's canal cells were grown to confluence, treated with 100 nm latanoprost for 6 hours, and harvested . Total rna was extracted using an rna isolation kit (rneasy total rna isolation kit; qiagen, hilden, germany). Approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit (iscript cdna; bio - rad laboratories, inc ., hercules, ca, usa). Quantitative real - time polymerase chain (qpcr) reaction using stc-1 (forward, 5-aggcggagcagaatgactc-3; reverse, 5-gttgaggcaacgaaccactt-3) and glyceraldehyde 3-phosphate dehydrogenase (gapdh: forward, 5-cctctgacttcaacagc-3; reverse, 5-gctgtagccaaattcgt-3) primers were performed on a pcr system using a master mix (roche light cycler 480 with sybr green i; roche, indianapolis, in, usa). We performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c, annealing at 63c, and extension at 72c . Primary schlemm's canal cells were grown to confluence, treated with 100 nm latanoprost for 15 minutes, 1, 2, 4, or 6 hours, and harvested . Schlemm's canal cell pellets were suspended in ice - cold lysis buffer (50 mm tris ph 8.0, 0.5% sodium dodecyl sulfate, 0.5% triton x-100, 137 mm nacl, 3 mm kcl, 8 mm na2hpo4 - 7h2o, 1 mm kh2po4, protease inhibitors; roche) and passed repeatedly through a 21-gauge needle for homogenization . Lysate was centrifuged at 13,000 g for 10 minutes, and total protein was quantified by the bradford assay . Cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer (thermo fisher scientific, waltham, ma, usa) containing 15% 2-mercaptoethanol (sigma - aldrich corp ., st . Louis, mo, usa), heated and separated on a 4% to 15% sds - page gradient gel (bio - rad laboratories, inc . ). Proteins were transferred to polyvinylidene difluoride membrane (millipore corp ., billerica, ma, usa) in 1x transfer buffer (50 mm tris, 384 mm glycine, 0.01% sds, 20% methanol). Membranes were blocked in 20 mm tris (ph 7.5), 150 mm nacl, 0.05% tween-20, and 2% instant nonfat dry milk . Blots were probed with rabbit monoclonal anti - human stc-1 (novus biologicals, littleton, co, usa) and mouse monoclonal anti - human gapdh (novus biologicals). Secondary antibodies used were horseradish peroxidase linked anti - rabbit or anti - mouse, respectively (ge healthcare, piscataway, nj, usa). Antibody / antigen complexes were detected using ecl western blot signal detection reagent (ge healthcare). Chemiluminescence film (biomax xar; eastman kodak, rochester, ny, usa) was used to visualize protein signals . Each film was digitized with a photographic scanner (epson perfection 2400; epson america, inc ., long beach, ca, usa). The band intensities for western blot analysis were quantified using imagej software (http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health, bethesda, md, usa) and normalized to gapdh . Primary human schlemm's canal cell lines were grown to confluence in six - well plates (bd falcon, franklin lakes, nj, usa) containing dulbecco's modified eagle's medium (dmem; life technologies, grand island, ny, usa); 10% fetal bovine serum (mediatech, manassas, va, usa); and 1% penicillin / streptomycin (life technologies) in 5% co2 at 37c . Confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells . Cells were incubated with latanoprost (de - esterified free - acid form at 100 nm final concentration; cayman chemical, ann arbor, mi, usa) or vehicle (ethanol, final dilution 1:1000) in dmem containing 1% penicillin / streptomycin . Primary schlemm's canal cells were grown to confluence, treated with 100 nm latanoprost for 6 hours, and harvested . Total rna was extracted using an rna isolation kit (rneasy total rna isolation kit; qiagen, hilden, germany). Approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit (iscript cdna; bio - rad laboratories, inc ., hercules, ca, usa). Quantitative real - time polymerase chain (qpcr) reaction using stc-1 (forward, 5-aggcggagcagaatgactc-3; reverse, 5-gttgaggcaacgaaccactt-3) and glyceraldehyde 3-phosphate dehydrogenase (gapdh: forward, 5-cctctgacttcaacagc-3; reverse, 5-gctgtagccaaattcgt-3) primers were performed on a pcr system using a master mix (roche light cycler 480 with sybr green i; roche, indianapolis, in, usa). We performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c, annealing at 63c, and extension at 72c . Primary schlemm's canal cells were grown to confluence, treated with 100 nm latanoprost for 15 minutes, 1, 2, 4, or 6 hours, and harvested . Schlemm's canal cell pellets were suspended in ice - cold lysis buffer (50 mm tris ph 8.0, 0.5% sodium dodecyl sulfate, 0.5% triton x-100, 137 mm nacl, 3 mm kcl, 8 mm na2hpo4 - 7h2o, 1 mm kh2po4, protease inhibitors; roche) and passed repeatedly through a 21-gauge needle for homogenization . Lysate was centrifuged at 13,000 g for 10 minutes, and total protein was quantified by the bradford assay . Cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer (thermo fisher scientific, waltham, ma, usa) containing 15% 2-mercaptoethanol (sigma - aldrich corp . St . Louis, mo, usa), heated and separated on a 4% to 15% sds - page gradient gel (bio - rad laboratories, inc . ). Proteins were transferred to polyvinylidene difluoride membrane (millipore corp ., billerica, ma, usa) in 1x transfer buffer (50 mm tris, 384 mm glycine, 0.01% sds, 20% methanol). Membranes were blocked in 20 mm tris (ph 7.5), 150 mm nacl, 0.05% tween-20, and 2% instant nonfat dry milk . Blots were probed with rabbit monoclonal anti - human stc-1 (novus biologicals, littleton, co, usa) and mouse monoclonal anti - human gapdh (novus biologicals). Secondary antibodies used were horseradish peroxidase linked anti - rabbit or anti - mouse, respectively (ge healthcare, piscataway, nj, usa). Antibody / antigen complexes were detected using ecl western blot signal detection reagent (ge healthcare). Chemiluminescence film (biomax xar; eastman kodak, rochester, ny, usa) was used to visualize protein signals . Each film was digitized with a photographic scanner (epson perfection 2400; epson america, inc . The band intensities for western blot analysis were quantified using imagej software (http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health, bethesda, md, usa) and normalized to gapdh . All animal studies and treatment protocols were approved by the mayo clinic (rochester, mn, usa) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research . We obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory, baylor college of medicine, and bred at mayo clinic . Mice, aged 5 to 8 months, were utilized in the experiments . A handheld rebound tonometer (icare tonolab; colonial medical supply, franconia, nh, usa) was used to measure iop in conscious mice . For iop measurements, . The tonometer records six readings from the same eye, discards the highest and lowest values, and shows the average of the remaining four values as a single iop reading . Three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye . After 1 week of baseline iop measurements, stc-1 mice were treated with latanoprost (n = 10) or rho kinase inhibitor y27632 (enzo life sciences, farmingdale, ny; n = 10). Congenic wild - type controls were treated with latanoprost (n = 8) or y27632 (n = 10). Treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid (100 m dissolved in 1:1000 dmso in pbs) or 10 mm y27632 (dissolved in phosphate - buffered saline). In the contralateral eye, vehicle was added daily in the same proportion as the treated eye for 7 consecutive days . Additionally, wild - type mice (n = 7; charles rivers laboratories, wilmington, ma, usa) were treated with 5 l of topically administered recombinant human stc-1 (0.5 mg / ml; biovendor research & diagnostic products, asheville, nc, usa) or vehicle (phosphate - buffered saline) daily for 7 days to examine the effect of stc-1 on iop . In all animals, the right eye served as the vehicle control eye while the left eye received study drug (latanoprost, y27632, or stc-1). We recorded iop in both eyes three times daily at 1, 4, and 23 hours following treatment . Anterior segments from human donor eyes (age 75.5 17.5 years, range: 51 to 98 years; n = 8) were perfused in culture with dmem within 10.2 4.4 hours of death as previously described . After achieving a stable baseline pressure, one anterior segment from each pair received recombinant human stc-1 at concentrations of 5, 50, or 500 ng / ml (dissolved in h2o), while the fellow eye received vehicle and served as the control . We added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion . Hourly pressure readings were obtained from the average of 60, one - minute pressure measurements using a custom - designed software program . The experimental eye was typically the right eye and the control was the left eye . For human anterior segments, selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.2). Tissue wedges were dehydrated in a series of ascending ethanol concentrations, cleared with 100% acetone, infiltrated and embedded in epon araldite, and sectioned at 0.5 m . Eye tissue sections were stained with toluidine blue and examined using a light microscope (nikon corp ., additional tissue wedges were sectioned at 100 nm, placed on copper film grids, and stained with uranyl acetate and lead citrate . Tissue sections on copper film grids were examined using a transmission electron microscope (jeol-1400; jeol usa, inc ., peabody, ma, usa). For mouse eye histopathology, whole eyes were enucleated from euthanized mice following termination of the experiment . Eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.2) overnight, and processed for light microscopy as described above for human anterior segment wedges . Additional 100-nm sections were placed on copper film grids, and stained with uranyl acetate and lead citrate . Processed sections were obtained following examination under a light microscope (nikon corp .) And a transmission electron microscope (jeol usa, inc . ). Prior to initiation of statistical analysis, all data sets were evaluated for distribution assessment using the shapiro - wilk test . For animal studies, significance of iop change was assessed between experimental and vehicle - treated control eyes using student's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets . Variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes . Statistical calculations were performed using a statistical software package (jmp; sas institute, inc ., cary, nc, usa). For human anterior segment studies, the effect of latanoprost was expressed as the change in outflow facility (c) for each anterior segment . Results from each pair of anterior segments were combined into a group mean for each drug, and statistical significance was analyzed using a student's 2-tailed paired t test . Preliminary findings from confluent primary human schlemm's canal cell lines treated with latanoprost identified stc-1, a secreted phosphoglycoprotein hormone with multiple functions, as a gene consistently upregulated following latanoprost treatment . Quantitative pcr studies in human schlemm's canal cells showed an 18.1 4.3-fold (n = 3) induction . Cell lysates isolated from latanoprost - treated primary human schlemm's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours, correlating with increased mrna expression levels identified by qpcr (fig . 1). Induction of stc-1 in schlemm's canal cells following treatment with latanoprost . Because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs, we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls . Assessment of baseline iops for 6 days showed no significant difference between stc-1 mice (16.6 0.5, n = 10) and wild - type controls (16.2 0.3, n = 8; p = 0.1). Histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy, an open angle with appropriate trabecula within the trabecular meshwork, and intact inner and outer walls of schlemm's canal, all similar to wild - type controls (fig ., we proceeded to assess the effect of latanoprost treatment on iop in these mice . Topical eye treatment with latanoprost (100 nm) reduced iop in wild - type controls by 3.8 mm hg (p <0.001, n = 8), which correlated to a 22.0% 1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone (fig . Consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1, 4, and 23 hours after treatment (table). In contrast, stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment (0.5% 0.7%, p = 0.34, n = 10) at any time point throughout the 7-day treatment regime . To determine if this was unique to latanoprost, we treated stc-1 with y27632, a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway . Treatment of either wild - type (n = 10) or stc-1 (n = 10) mice with y27632 resulted in significant reductions of iop, 12.5% 1.2% and 13.1% 2.8%, respectively (p <0.0001, n = 10; fig . 4; table). These data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction . Top: light microscopy images of whole eye sections from wild - type and stc-1 mice . Wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . Middle: light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula, trabecular meshwork (tm) cells, and schlemm's canal (sc) volume . Bottom: transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm's canal inner walls (iw) and outer walls (ow). The daily iop was recorded as the average of iop measurements at 1, 4, and 23 hours following latanoprost treatment . While congenic control mice show iop reduction with latanoprost, stc-1 do not, suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction . Intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice . N = 10) and congenic controls (n = 10) were treated once daily with 10 mm y27632 . The daily iop was recorded as the average of iop measurements at 1, 4, and 23 hours following y27632 treatment . Both stc-1 mice and congenic controls showed iop reduction following treatment with y27632 . To determine if stc-1 would influence iop reduction by itself, we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours . Perfusion with either 5 ng / ml (0.15 0.04 to 0.15 0.04 l / min / mm hg, n = 2, p = 0.20) or 50 ng / ml (0.14 0.05 to 0.18 0.07 l / min / mm hg, n = 4, p = 0.20) had no significant effect on outflow facility . However, all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility (0.15 0.03 to 0.27 0.09 l / min / mm hg, n = 5, p = 0.02) compared to baseline (fig . 5). Paired controls treated with vehicle showed no change in outflow facility from their baseline values . To determine whether the changes in pressure could be secondary to morphologic changes morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm's canal of control and treated eyes (fig . No major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm's canal inner and outer walls were observed . These results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop . (a) outflow facility of human anterior segments (n = 8) following perfusion with 5, 50, or 500 ng / ml of recombinant human stc-1 . (b) representative graph of an eye pair perfused with 500 ng / ml stc-1 . (a, b) representative sections (3 m) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . (c, d) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes . Recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment . Ac, anterior chamber; jct, juxtacanalicular region; tm, trabecular meshwork; sc, schlemm's canal . To determine if stc-1 has ocular hypotensive activity in vivo, wild - type c57bl/6 mice were treated with recombinant stc-1 (0.5 mg / ml) or vehicle daily for 7 days (fig . Stanniocalcin-1 reduced iop by 15.2% 3.0% when compared to vehicle - treated contralateral eyes (range of iop reduction from 5.1% 5.2% these results suggest that stc-1 has iop - lowering properties when used as a standalone agent . Wild - type mice (n = 10) were treated topically with recombinant stc-1 (0.5 mg / ml) or vehicle (phosphate buffered saline). The daily iop was recorded as the average of iop measurements at 1, 4, and 23 hours following stc-1 treatment . Preliminary findings from confluent primary human schlemm's canal cell lines treated with latanoprost identified stc-1, a secreted phosphoglycoprotein hormone with multiple functions, as a gene consistently upregulated following latanoprost treatment . Quantitative pcr studies in human schlemm's canal cells showed an 18.1 4.3-fold (n = 3) induction . Cell lysates isolated from latanoprost - treated primary human schlemm's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours, correlating with increased mrna expression levels identified by qpcr (fig . 1). Induction of stc-1 in schlemm's canal cells following treatment with latanoprost . Because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs, we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls . Assessment of baseline iops for 6 days showed no significant difference between stc-1 mice (16.6 0.5, n = 10) and wild - type controls (16.2 0.3, n = 8; p = 0.1). Histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy, an open angle with appropriate trabecula within the trabecular meshwork, and intact inner and outer walls of schlemm's canal, all similar to wild - type controls (fig ., we proceeded to assess the effect of latanoprost treatment on iop in these mice . Topical eye treatment with latanoprost (100 nm) reduced iop in wild - type controls by 3.8 mm hg (p <0.001, n = 8), which correlated to a 22.0% 1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone (fig . Consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1, 4, and 23 hours after treatment (table). In contrast, stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment (0.5% 0.7%, p = 0.34, n = 10) at any time point throughout the 7-day treatment regime . To determine if this was unique to latanoprost, we treated stc-1 with y27632, a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway . Treatment of either wild - type (n = 10) or stc-1 (n = 10) mice with y27632 resulted in significant reductions of iop, 12.5% 1.2% and 13.1% 2.8%, respectively (p <0.0001, n = 10; fig . 4; table). These data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction . Top: light microscopy images of whole eye sections from wild - type and stc-1 mice . Wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . Middle: light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula, trabecular meshwork (tm) cells, and schlemm's canal (sc) volume . Bottom: transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm's canal inner walls (iw) and outer walls (ow). The daily iop was recorded as the average of iop measurements at 1, 4, and 23 hours following latanoprost treatment . While congenic control mice show iop reduction with latanoprost, stc-1 do not, suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction . Intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice . Stc-1 mice (n = 10) and congenic controls (n = 10) were treated once daily with 10 mm y27632 . The daily iop was recorded as the average of iop measurements at 1, 4, and 23 hours following y27632 treatment . To determine if stc-1 would influence iop reduction by itself, we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours . Perfusion with either 5 ng / ml (0.15 0.04 to 0.15 0.04 l / min / mm hg, n = 2, p = 0.20) or 50 ng / ml (0.14 0.05 to 0.18 0.07 l / min / mm hg, n = 4, p = 0.20) had no significant effect on outflow facility . However, all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility (0.15 0.03 to 0.27 0.09 l / min / mm hg, n = 5, p = 0.02) compared to baseline (fig . 5). Paired controls treated with vehicle showed no change in outflow facility from their baseline values . To determine whether the changes in pressure could be secondary to morphologic changes morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm's canal of control and treated eyes (fig . 6). No major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm's canal inner and outer walls were observed . These results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop . (a) outflow facility of human anterior segments (n = 8) following perfusion with 5, 50, or 500 ng / ml of recombinant human stc-1 . (b) representative graph of an eye pair perfused with 500 ng / ml stc-1 . P 0.05 . Histologic analysis of human anterior segment ocular tissue following treatment with recombinant stc-1 . (a, b) representative sections (3 m) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . (c, d) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes . Recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment . Ac, anterior chamber; jct, juxtacanalicular region; tm, trabecular meshwork; sc, schlemm's canal . To determine if stc-1 has ocular hypotensive activity in vivo, wild - type c57bl/6 mice were treated with recombinant stc-1 (0.5 mg / ml) or vehicle daily for 7 days (fig . Stanniocalcin-1 reduced iop by 15.2% 3.0% when compared to vehicle - treated contralateral eyes (range of iop reduction from 5.1% 5.2% [day 1] to 21.4% 5.3% these results suggest that stc-1 has iop - lowering properties when used as a standalone agent . Wild - type mice (n = 10) were treated topically with recombinant stc-1 (0.5 mg / ml) or vehicle (phosphate buffered saline). The daily iop was recorded as the average of iop measurements at 1, 4, and 23 hours following stc-1 treatment . Prostaglandin analogs like latanoprost are a first - line medical therapy for iop reduction due to their once daily dosing, greater response rates, and greater amount of iop reduction compared to other classes of pressure - lowering medications . Identifying the critical effector molecules in the signaling pathway of latanoprost will provide insights into additional molecules to target for iop reduction, potentially with fewer side effects . In the current study, we identified stc-1 as a transcript and protein that is highly induced following treatment of human schlemm's canal cells with latanoprost . Topical latanoprost significantly reduced iop in wild - type controls while stc-1 mice demonstrated no iop reduction to topical latanoprost . In contrast, rho kinase inhibitor y27632 demonstrated iop reduction in both wild - type and stc-1 mice, indicating that stc-1 is a unique and important downstream signaling molecule necessary for the ocular hypotensive properties of latanoprost . Additionally, recombinant stc-1 by itself also increased outflow facility in human anterior segments and reduced iop in vivo in wild - type mice . Together, these results suggest that stc-1 is a critical and unique effector molecule for the latanoprost signaling pathway that is necessary for latanoprost - induced iop reduction and that stc-1 by itself can act as an ocular hypotensive agent . Stanniocalcin was first described in fish as a 50-kda homodimeric glycoprotein that is secreted from the corpuscles of stannius into the bloodstream in the setting of hypercalcemia to regulate calcium excretion at the gills and gut . In mammals, two homologues of stc stc-1 is the most studied of the two mammalian forms, having a 50% amino acid homology with its fish counterpart . Stc-1 is a secreted, homodimeric phosphoglycoprotein that has preserved protein structure similarity between mammals and fish including the conservation of 11 cysteine residues . It is expressed in a wide variety of tissues most notably bone, skeletal muscle, heart, thymus, and spleen . Functionally, stc-1 has been associated with calcium uptake, hypoxic preconditioning, and antioxidative stress properties through suppression of reactive oxygen species . Additionally, stc-1 has also been shown to be neuroprotective for neurons, photoreceptors, and retinal ganglion cells, and has been linked to anti - inflammatory effects by inhibiting macrophage chemotaxis, modulating transendothelial migration of leukocytes and reducing t cell infiltration . In treating wild - type mice with topical latanoprost, we observed a reduction in iop by approximately 22.0% 1.9%, consistent with previous reports, while the stc-1 mice showed no response (0.55% 0.7%). Mice that were stc-1 were responsive to rho kinase inhibitor y27632, which confirmed that stc-1 mice have normal functioning outflow pathways, but that elimination of stc-1 expression in these mice renders topical latanoprost ineffective . The current study adds schlemm's canal cells to the list of cells that express stc-1 and shows that its expression is highly influenced by latanoprost . This is an important finding as little is known about the molecular events that connect latanoprost treatment with iop reduction . Latanoprost has been shown to phosphorylate myosin light chain kinase in the iris, indicating a role for latanoprost in cell relaxation . However, stc-1 is the first individual molecule that has been identified as a key downstream effector of latanoprost signaling . In addition to its variety of functional activities, our results also suggest that stc-1 has ocular hypotensive properties, since addition of recombinant stc-1 to human anterior segment cultures decreased pressure and increased outflow facility, and when applied topically to wild - type mice reduced iop in vivo . Despite the benefits of latanoprost in lowering iop, long - term treatment with latanoprost can often cause significant side effects, which can be contraindicative for its prolonged usage . Identification of stc-1 as a downstream signaling molecule through which latanoprost executes its iop - lowering effects, and the fact that recombinant stc-1 shows ocular hypotensive properties, makes this an attractive therapeutic target . Utilization of a specific downstream molecule such as stc-1 within the latanoprost signaling cascade may eliminate some of the major side effects while providing a specialized therapeutic strategy . In summary, additionally, stc-1 demonstrates ocular hypotensive properties when used by itself, mimicking the effect of latanoprost . Given its novel role in latanoprost - mediated iop reduction, stc-1 may be considered a promising candidate molecule for devising future therapeutic regiments to reduce iop.
Single - crystalline kinked nanowires were synthesized by the nanocluster - catalyzed vls method described previously18,19 in quartz tube connected to gas manifold and vacuum pump and heated by a temperature controlled tube furnace . Monodisperse gold nanoparticles (ted pella) were dispersed on sio2/si or sapphire growth substrates (au surface coverage: 0.010.1 particles/m), which were placed within the central region of the quartz tube reactor . The silicon (si) nanowires were synthesized at 450460 c using silane (sih4) as the silicon reactant source, hydrogen (h2) as the carrier gas, and phosphine (ph3, 1,000 p.p.m . In h2) and diborane (b2h6,100 p.p.m . In h2) as the n- and p - type dopants . In a typical synthesis of uniform n - type, 80 nm kinked silicon nanowires, the flow rates of sih4, ph3 and h2 were 12, 210 and 60 standard cubic centimetres per minute, respectively, and the total pressure was 40 torr and purge duration was 15 s; the minimum pressure during the purge cycle was ca . 3 10 torr . The dopant feed - in ratios (silicon: boron / phosphorus) in kinked p - n silicon nanowires were 500:1 for both p - and n - type segments . In n - kink - n - kink-(n - n) dopant modulated silicon nanowires, the silicon - phosphorus feed - in ratios were 200:1 and 10000:1 for n- and n - type segments, respectively, and the n- segment was grown for 30 s. germanium nanowires were synthesized at 270290 c, 40 torr, with germane (geh4, 10% in h2) and h2 as the reactant and carrier gas, respectively . Cadmium sulphide nanowires were grown in a three - zone furnace by evaporating cds power at 650720 c, with nanowire growth by gold nanocluster catalyzed vls method at 550 - 500 c . The purge cycle used to form kinks in the germanium and cadmium sulphide nanowires was typically 15 s. zeiss ultra55/supra55vp field - emission sems and jeol 2010 field emission tem were used to carry out sem and tem analyses, respectively . For sample preparation, kinked nanowires were gently sonicated in isopropyl alcohol and dispersed onto heavily doped silicon substrates (100 nm oxide/200 nm nitride, 110 cm resistivity, nova electronic materials, carrollton, tx) or lacey carbon grids (ted pella). Devices were fabricated on silicon substrates (nova electronic materials, n - type 0.005 cm) with 100 nm thermal oxide and 200 nm silicon nitride at the surface . Devices were defined by electron - beam lithography followed by ti / pd (1.5 nm/100 nm) contact deposition in a thermal evaporator . Current - voltage (i v) data were recorded using an agilent semiconductor parameter analyzer (model 4156c) with contacts to devices made using a probe station (desert cryogenics, model ttp4). Electrostatic force microscopy and scanned gate microscopy measurements were carried out with a digital instruments nanoscope iiia multimode afm and metal coated tips (nanosensors, ppp - nchpt). The electrostatic force microscopy surface potential maps and scanned gate microscopy conductance maps were acquired in lift mode with lift heights of 40 and 20 nm, respectively . In the surface potential measurements, the p - n diode was reverse - biased at 5 v and the tip voltage was modulated by 3v at the resonance frequency . In scanned gate measurements, the tip functions as a local gate vtip = 10 v, and the conductance versus position provides a measure of local accumulation or depletion of carriers in the device . Single - crystalline kinked nanowires were synthesized by the nanocluster - catalyzed vls method described previously18,19 in quartz tube connected to gas manifold and vacuum pump and heated by a temperature controlled tube furnace . Monodisperse gold nanoparticles (ted pella) were dispersed on sio2/si or sapphire growth substrates (au surface coverage: 0.010.1 particles/m), which were placed within the central region of the quartz tube reactor . The silicon (si) nanowires were synthesized at 450460 c using silane (sih4) as the silicon reactant source, hydrogen (h2) as the carrier gas, and phosphine (ph3, 1,000 p.p.m . In h2) and diborane (b2h6,100 p.p.m . In h2) as the n- and p - type dopants . In a typical synthesis of uniform n - type, 80 nm kinked silicon nanowires, the flow rates of sih4, ph3 and h2 were 12, 210 and 60 standard cubic centimetres per minute, respectively, and the total pressure was 40 torr and purge duration was 15 s; the minimum pressure during the purge cycle was ca . 3 10 torr . The dopant feed - in ratios (silicon: boron / phosphorus) in kinked p - n silicon nanowires were 500:1 for both p - and n - type segments . In n - kink - n - kink-(n - n) dopant modulated silicon nanowires, the silicon - phosphorus feed - in ratios were 200:1 and 10000:1 for n- and n - type segments, respectively, and the n- segment was grown for 30 s. germanium nanowires were synthesized at 270290 c, 40 torr, with germane (geh4, 10% in h2) and h2 as the reactant and carrier gas, respectively . Cadmium sulphide nanowires were grown in a three - zone furnace by evaporating cds power at 650720 c, with nanowire growth by gold nanocluster catalyzed vls method at 550 - 500 c . The purge cycle used to form kinks in the germanium and cadmium sulphide nanowires was typically 15 s. zeiss ultra55/supra55vp field - emission sems and jeol 2010 field emission tem were used to carry out sem and tem analyses, respectively . For sample preparation, kinked nanowires were gently sonicated in isopropyl alcohol and dispersed onto heavily doped silicon substrates (100 nm oxide/200 nm nitride, 110 cm resistivity, nova electronic materials, carrollton, tx) or lacey carbon grids (ted pella). Devices were fabricated on silicon substrates (nova electronic materials, n - type 0.005 cm) with 100 nm thermal oxide and 200 nm silicon nitride at the surface . Devices were defined by electron - beam lithography followed by ti / pd (1.5 nm/100 nm) contact deposition in a thermal evaporator . Current - voltage (i v) data were recorded using an agilent semiconductor parameter analyzer (model 4156c) with contacts to devices made using a probe station (desert cryogenics, model ttp4). Electrostatic force microscopy and scanned gate microscopy measurements were carried out with a digital instruments nanoscope iiia multimode afm and metal coated tips (nanosensors, ppp - nchpt). The electrostatic force microscopy surface potential maps and scanned gate microscopy conductance maps were acquired in lift mode with lift heights of 40 and 20 nm, respectively . In the surface potential measurements, the p - n diode was reverse - biased at 5 v and the tip voltage was modulated by 3v at the resonance frequency . In scanned gate measurements, the tip functions as a local gate vtip = 10 v, and the conductance versus position provides a measure of local accumulation or depletion of carriers in the device.
Arachnoid cysts are benign, non - neoplastic and extra - axial lesions; most of them are clinically silent and unchanged in size . In previous studies112131524), the incidence of arachnoid cysts has been estimated to range between 0.3% to 1.7% . However, the advanced neuroimaging techniques have facilitated the diagnosis of arachnoid cysts, which, in turn, has led to an increase in the number of patients2121415). In some cases, an arachnoid cyst arouses symptoms and its clinical presentations vary according to the size and location of the cyst . The most frequent symptom is headache, which is caused by the local mass effect, increased intracranial pressure (icp), and/or hydrocephalus15). Above this, seizures, cognitive dysfunction, developmental delay, and intracranial hemorrhage can be also presented . In this report, we describe a case where a patient with a posterior fossa arachnoid cyst complained of visual field defect, visual disturbance, and mild headache; no ataxia and unsteady gait were reported . The patient's visual symptoms rapidly deteriorated in a week and, consequently, the patient's vision got worse . A 39-year - old male patient presented at our emergency room after a traffic accident and the brain computed tomography (ct) scan indicated an incidental arachnoid cyst on posterior fossa . At that time, the patient complained only of facial abrasions and the left knee pain, but not of headache or visual disturbance . In eight months, however, he revisited our department of ophthalmology to check the decreased visual acuity, visual field defect, and headache . On the ophthalmologic examination, bilateral papilledema (fig . 2a) were found . After the transfer to the department of neurosurgery, we rechecked brain ct scans (fig . The diameter of the patient's arachnoid cyst did not change compared to the previous ct scans; however, the slightly enlarged third ventricle was compressing the optic chiasm and the suprasellar area . Since visual complications were expected, we recommended an emergency operation . One week later, he came back to the hospital with nearly complete visual loss . His right eye was completely blind, whereas his left eye recognized only light (fig . A conventional suboccipital craniectomy was performed on the patient lying on the operating table in the prone position . After the dural incision, the arachnoid cyst was exposed and cystic fluid was expelled . Arachnoid membrane was carefully dissected and the arachnoid cyst was fenestrated to cisterna magna for connecting with the cerebrospinal fluid (csf) flows . Arachnoid membrane which covering the foramen of magendie was also fenestrated and the csf flows was running properly (fig . 4). Excluding the brain tumor, the collected membrane was sent to the pathologic laboratory . In high power field microscopy under the hematoxylin and eosin stain, the cystic membrane was composed by membranous fibrocollagenous tissues without tumor cells . One week afterwards, we checked the brain ct scans and the removal of the cyst and the decreased ventricle size were confirmed . On the ophthalmologic examination, the papilledema and visual field were slightly improved; however, the patient's visual acuity did not improve . Upon the tenth day after the operation, the patient was discharged and, during the next one month, he was subject to the steroid therapy . In five months after the operation, papilledema (fig . Nevertheless, visual acuity and visual field improved slowly and slightly (fig . 2c). The blind right eye improved to recognize the light and the blurred left eye improved to acknowledge the silhouette of an object; thus, it did not fully recovered to the prior, normal condition . Their prevalence has been reported to amount to approximately 1% of all intracranial mass lesions112152430); however, advanced neuroimaging techniques and the widespread use of ct and mri imaging have led to an even higher incidence of report of these lesions1249121619203031). One recent study, al - holou et al.1), reviewed 48417 brain images and identified 661 (1.4%) arachnoid cysts . This report found that the prevalence of arachnoid cyst in children was 2.6%, slightly higher than in adults, and men (1.8%) have a high prevalence than women (1.1%). The most common locations were middle fossa (34%), retrocerebellar (33%), and cerebral convexity (14%)124). Despite the increased identification of arachnoid cysts, the natural history and mechanism of enlargement has not been well defined1131524). Nevertheless, when arachnoid cysts give rise to any symptom, headache is the most common complaint . In addition to headache, gait imbalance, seizure, and visual changes can also be presented according to the location and size of the arachnoid cyst . Various operation techniques, such as cyst excision1517), stereotactic aspiration1825), endoscopic cyst fenestration512), ventriculocystostomy26), and cystoperitoneal shunt810152728), have been introduced and the main purpose of such operations is similar, namely, a direct decompression of the arachnoid cyst or making the connection between the cyst and the csf flows for spontaneous resolution . Generally, visual symptoms occur in the patients with suprasellar arachnoid cysts, because the arachnoid cyst is adjacent to the optic nerves7). The patients with posterior fossa arachnoid cysts usually do not complain of visual symptoms, as the cyst is not adjacent to the optic nerves . However, a posterior fossa arachnoid cyst can indirectly disrupt the csf flows and, in turn, the disruption increases the whole ventricle size and the icp . In our case, the enlarged third ventricle was compressing the optic chiasm with cephalocaudal direction on the ct scan (fig . The compressed superior part of optic chiasm led to the defect of inferior visual field (fig ., visual acuity was affected by the papilledema, which was caused by the increased icp . Consequently, both visual acuity and visual field were influenced by the posterior fossa arachnoid cyst . When the patient first visited our department of ophthalmology, his visual symptoms and headache were not severe and the patient could walk and drive . Besides, the brain ct scan showed no changes as compared to previous ct scans, so we could not predict a risk of rapid deterioration . The patient's refusal to have an operation and the surgeon's wrong prediction delayed the appropriate on - time treatment; ultimately, the patient's vision was severely damaged during the critical seven days . After the operation, the patient's visual acuity, visual field, and papilledema were slightly improved; however, we regard that the patient's vision will unfortunately not fully recover . Unlike gait disturbance and ataxia, visual symptoms do not frequently occur in the patients with posterior fossa arachnoid cysts; this is so because the cyst is located away from the optic nerves . However, the disruption of csf flows can indirectly induce visual symptoms and these symptoms can rapidly deteriorate . Considering the results of our case, it should be emphasized that the patients with posterior fossa arachnoid cysts could develop a rapid neurologic deterioration . Therefore, in the event of the development of symptoms, an early intervention or close observation are crucial.
These techniques include closed reduction, percutaneous pin reduction, intramedullary pin reduction with elastic stable intramedullary nailing or kirschner - wires (k - wires), and open reduction with or without internal fixation . Our goals for treatment are to avoid incision, reduce the fracture adequately with no reduction loss, and achieve good postoperative function . We performed a closed reduction with a percutaneous leverage technique and internal fixation method with k - wires to treat angulated radial neck fractures in children . From january 2011 to april 2013, we treated 16 cases of angulated radial neck fractures in 12 boys and 4 girls . The types of fractures categorized by the obrien classification as follows: type i angulation <30; type ii angulation> 60. among our patients, 5 had type ii and 11 had type iii fractures . The left side was affected in 9 cases, right side was affected in 7 cases . Complications with ulnar olecranon fracture and medial epicondyle fracture were present in 3 cases and 2 cases, respectively . No blood vessel or nerve trauma was evident . The shortest time from trauma to surgery was 4 hours and the longest time was 6 days . A 1.6- or 2.0-mm k - wire was inserted from distal to proximal of the elbow percutaneously . In this procedure, the k - wire was inserted into the fracture site . The k - wire was used to lever the fracture and to orient the proximal part horizontally with the lateral condyle plane . Was attained, two 1.2 mm k - wires were percutaneously inserted from the articular surface through the fracture site to the opposite cortex for fixation (fig . The 3 cases with ulnar olecranon fractures were not treated because there was no obvious displacement . Our research was approved by the biomedical research ethics committee of hong hui hospital, xian jiaotong university college of medicine . (a) the kirschner wire (k - wire) was inserted percutaneously into the fracture site . (b) the inserted k - wire was used as a lever to reduce the fracture . (c) one 1.2 mm k - wires was percutaneously inserted from the articular surface passing through the fracture site to the opposite cortex . Typical case: femail, 12 years old, and right side . A long - arm cast or brace was used for 3 to 4 weeks after the procedures, with the elbow in 90 of flexion and the forearm in supination . A 1.6- or 2.0-mm k - wire was inserted from distal to proximal of the elbow percutaneously . In this procedure, the k - wire was inserted into the fracture site . The k - wire was used to lever the fracture and to orient the proximal part horizontally with the lateral condyle plane . Was attained, two 1.2 mm k - wires were percutaneously inserted from the articular surface through the fracture site to the opposite cortex for fixation (fig . The 3 cases with ulnar olecranon fractures were not treated because there was no obvious displacement . Our research was approved by the biomedical research ethics committee of hong hui hospital, xian jiaotong university college of medicine . (a) the kirschner wire (k - wire) was inserted percutaneously into the fracture site . (b) the inserted k - wire was used as a lever to reduce the fracture . (c) one 1.2 mm k - wires was percutaneously inserted from the articular surface passing through the fracture site to the opposite cortex . Typical case: femail, 12 years old, and right side . A long - arm cast or brace was used for 3 to 4 weeks after the procedures, with the elbow in 90 of flexion and the forearm in supination . Cases were followed up for an average of 3 years 7 months (range 2 years 6 months to 4 years 10 months). The postoperative reduction was assessed by roentgenograph within 1 week after surgery . No infection or nerve damage occurred in the short - term follow - up . No radial head necrosis or synostosis of the proximal ulna and radius was seen in the long - term follow - up . There was no disclosure of the epiphysis or valgus of the elbow during the follow - up . According to the metaizeau reduction classification, 12 cases were excellent, and 4 cases were good . According to the metaizeau clinical classification, 14 cases were excellent, and 2 cases were good . (a) the preoperative x - ray showed the severe tilt and displacement of radial neck . (c) the 2 years later follow - up x - ray picture showed the radial head normal . Most authors agree that fractures with slight angulation should be treated conservatively, but severely angulated fractures (> 60) require further treatment with different methods . The complications of these fractures reported in children include avascular necrosis, early physeal closure, elbow stiffness, periarticular ossification, and overgrowth of the radius head . The prognosis was not only related with the angulation of the radial neck fracture, but also with the age of the patient and extent of trauma . Metaizeau suggested that a residual tilt above 10 to 15 at 10 to 12 years of age or 20 to 30 at a younger age could not be remodeled by growth . Additionally, bernstein found that the determination of what constituted a significant angulation vari with the child's age, with greater angulation degrees acceptable for younger children . The compression and impaction extends to the lateral side of radial neck upon radial neck fracture . The redisplacement will be accomplished easily only if it is immobilized by cast as there is no support from the lateral portion of the radial neck for the radius head after reduction . Steinberg reported that 22 cases which occurred redisplacement in whole 28 cases which were immobilized by cast after reduction . One method uses an elastic stable intramedullary nailing to indirectly reduce and fix the fracture site . The other method uses an intramedullary k - wire to perform indirect reduction and internal fixation . The 3rd method uses a percutaneous k - wire to perform direct reduction and internal fixation . In comparing the 3 different methods, some authors recommended closed reduction and elastic intramedullary fixation to treat a fracture of the radius in children . However, we found that it was difficult to reduce the displaced radius head through rotating the distal part of the elastic intramedullary nail . This reduction approach usually requires an assistant to manipulate the percutaneous k - wire . It is challenging to fix the radial head with elastic intramedullary nail after a good reduction . The radial head was easily separated from the fracture site when penetrating with and rotating the nail . The functional lever arm was too long when the elastic intramedullary nail or intramedullary pin was used to fix the radial head . It was challenging to maintain a good position . There was no disclosure of the epiphysis or valgus of the elbow during the follow - up . Trauma was minor and there was no interference with the blood supply of the radial head . There was no postoperative scarring and the patients and their parents were satisfied with outcome . The insertion site should be carefully selected to avoid damage to the deep branch of the radial nerve . The k - wires should be appropriately sized, with a diameter no greater than 1.5 mm . We usually choose 1.2 mm k - wires to prevent damage to the epiphysis . We recommend the percutaneous leverage technique during closed reduction and internal fixation using k - wires for the treatment of angulated radial neck fractures in children . This is a simple method with little trauma to the patient and a satisfactory clinical outcome.
Cities around the world can look very different if you compare the living conditions for the residents . However, it is not only different cities that can have completely diverse standards of infrastructure and social security . Living in the slums compared to more wealthy neighbourhoods, will expose the inhabitants to different risks . Traditionally cities can offer many advantages compared to rural settings, but under certain circumstances they can rather be a health hazard . The rapid migrations of people to cities can lead to overcrowding, which can generate slums or shanty towns . These slums are characterized by poor housing, lack of fresh water, and bad sanitation facilities (2). All of these shortages can be a threat to the residents health and be a possible breeding ground for infectious diseases . The location of slums are often outside of the city centres, in more hazardous locations and the population feels a lack of social and economic opportunities compared with other residents . In sub - saharan africa, 62% of the urban population in 2012 lived in shanty towns (2). For example, in 2009, 96% of the urban population in central african republic lived in these slums (6). In kenya's capital nairobi, 60% of the population lives in slums, and child mortality there is 2.5 times greater than other parts of the city (9). The community and health care services have great challenges to provide the entire population with equal and adequate service . The collected parties need to be aware of the differences in threats with respect to infectious diseases, both at the local and governmental levels . Infections have been linked to slums in dar es salaam, tanzania, with high population density and low income (10). In several other countries, cholera incidence is the highest in urban regions with high population density (11, 12). Differences in prevalence of asymptomatic carriers of antimicrobial drug - resistant diarrhoeagenic escherichia coli have also been found in brazil between slum settlements and more wealthy parts of the community 13). The poor infrastructure in the slum can be a barrier for improvement, but at the same time targeted interventions for safer water and better sanitation facilities could potentially have a profound effect of the overall health . Overcrowded housing in high - density populations in the slums can be a breeding ground for infectious diseases such as tuberculosis . The rate of tuberculosis has traditionally been higher in urban centres compared to rural (14, 15). Studies in slum settlements in dhaka city, bangladesh, indicate a high prevalence of tuberculosis, which was almost twice as high compared to the overall national average and four times higher than the overall urban levels (16 . However, different patterns can be seen in different countries; for example, in poland the rates of tuberculosis have shown only slightly lower incidence in rural population compared to urban, 21.9 per 100,000 versus 22.4 per 100,000 respectively (17). Tuberculosis in the united states has declined in the twentieth century, and several factors such as improved nutrition status, socioeconomic status, overall public health, and new drug regimens have been thought to play a major role . However, in the mid-1980s a resurgence occurred which reached its peak in 1992, especially in urban areas among the homeless and incarcerated population (18). The knowledge regarding symptoms, transmission, and prevention has been shown to be greater among the urban population in pakistan's punjab province compared to the rural population . Health - seeking behaviour was also better among the urban population, in the aspect of when to seek medical advice for early diagnosis and potential treatment (19). Information about infectious diseases and how they spread in the community can help the individuals to protect themselves, but knowledge about the slums and the infectious diseases panorama is also crucial for local physicians . They need to know how to look for the correct diagnosis, even if their diagnostic tools might be limited . The rapid influx of migrants can lead to overcrowding and local governments might not be able to provide safe housing, drinking water, and adequate sewage facilities, all of which are potential health hazards and must be taken into account for safe city planning . Today more than half of the world's population, almost 4 billion people, have access to piped water connected to their homes . Since 1990, well over 2 billion people have gained improved drinking water facilities, and almost 2 billion people have access to improved sanitation . However, more than 700 million people still lack access to improved sources of safe drinking water, and in sub - saharan africa half of the population lack such facilities . Globally however, 1 billion people in the world still practice open defecation . In this group, 90% live in rural areas, but the actual amount of residents from urban settings is gradually increasing . Between 1990 and 2012, the group in urban settings which lacked sanitation actually significantly increased from 215 million to 756 million, which could be explained by population growth (20). Much of the hard work to improve sanitation facilities has benefited large population groups, but the rapid influx of new urban residents shows that there is still much hard work to be done . Residents who are subject to overcrowding and who lack access to safe drinking water or proper sanitation can be more susceptible to soil - transmitted helminths (21). These infections are among the most important causes of physical and intellectual growth retardation in the world and have a major impact on public health (22). Good hygiene practices and good sanitary conditions have lowered the prevalent levels of contamination . In the brazilian city of salvador, with a population of 2.5 million, an improvement of sewerage coverage from 26 to 80% of the households led to an estimated overall reduction of diarrhoeal diseases of 22% (23). Neglected tropical diseases can cause substantial health problems in developing countries, and some of these diseases have a faecal - oral transmission pathway . Examples of such diseases could be schistosomiasis, trachoma, and soil - transmitted helminthiases . In many countries, however, the focus is on treatment by medication and not improved sanitation . The reason could be that it would be much more expensive to carry out the necessary infrastructural improvements (24). Safe drinking water and proper sanitary facilities must be taken into account in city planning . Factors like this can potentially have a profound positive effect in lowering infectious diseases with a faecal oral route . One example of this is for chagas disease, which is a parasitic infection caused by the protozoan trypanosoma cruzi . An important mode of transmission is vectorial infected bites of triatomine bugs . Living in close contact to domestic animals and poor hygienic habits chagas disease affects an estimated 8 million people every year, and is an important health challenge in latin america . In recent decades, progress has been made to reduce the burden of disease, by vector control, screening blood donors, improved housing, and epidemiological surveillance . Chagas disease is a growing health problem in non - endemic areas because of population movements (26). It is estimated that 300,000 individuals in the united states are infected (27) and the most affected country in europe, spain, is thought to have 45,00067,000 cases (28). The example of chagas disease shows that physicians who practice in countries where the disease is not present must be aware of the travel history of the patient to connect the potential symptoms to the correct diagnosis . The environment in urban cities has proven to be favourable for the rat population (rattus spp .) And close encounters between rats and humans can lead to transmission of zoonotic infectious diseases ., rickettsia typhi, streptobacillus moniliformis, bartonella spp ., seoul hantavirus, and angiostrogylus cantonensis (29). New york city has one of the largest populations of rats in the united states . It has been shown that encounters between rats and humans have been linked to proximity to open public spaces and subway lines, the presence of vacant housing units, and low education of the population (30). Information like this can be useful for health officials when they launch specific control initiatives . The changes in human population with increased urbanization and urban poverty has also altered our perception of some zoonoses linked to the rat population . Leptospirosis has traditionally been perceived as a primarily rural disease, but the incidence in urban centres is increasing (31, 32). In chinese cities, the incidence of seoul hantavirus haemorrhagic fever with renal syndrome has been linked to urban growth, growing rat population, and increase rat human contact (33). Large megacities all over the world have large rat populations, but the surveillance and local knowledge seem to be inadequate . A better understanding of how to prevent uncontrolled growth in rat population can potentially lead to a decline of these zoonotic diseases . The growing trend of urbanization around the world has shifted some infectious diseases, which have traditionally been perceived as rural, to urban settings . The world health organization (who) has published a list of 17 neglected tropical diseases . Several of them have now become a reality in the urban environment, these diseases are something the practicing physicians in these areas have to be aware of (34). Many of the diseases on the list are present in the developing world, which sometimes lack the opportunity to solve these problems by themselves . One of the neglected infectious diseases is lymphatic filariasis (lf) with 2 billion people at risk, and 394.5 million in urban areas . One of the main reasons is the lack of proper sanitation facilities (35). Lf still has its major impact in rural settings, but the increasing urbanization in the developing world has made lf an infectious disease that also has to be considered elsewhere . One of the parasite species wuchereria bancrofti has been located in many urban areas and has the potential for transmission in this environment . Moreover, one of the vectors for the parasite is the mosquito culex quinquefasciatus, which thrives in these surroundings, especially in overcrowded areas with poor sanitary and draining facilities . However, within one city the transmission can vary substantially depending on the standard of the sanitary conditions . The mosquito vector culex spp can be found in large parts of central and south america, east africa, and asia (36). Another vector which has adapted to urban surroundings is the mosquito aedes aegypti, which is a key component for dengue transmission . Dengue is on who's list of neglected tropical diseases, and is on the rise worldwide . The number of infections has drastically increased in the tropical regions of the world in the last 40 years . Recent studies have estimated 390 million cases each year, and the burden is the highest in india with one - third of all the new infections (37). Several factors have played a big role in the escalation, such as urbanization, globalization, and lack of mosquito control . Aedes aegypti lay their eggs in artificial water containers made by humans, which is a key component in the urban transmission cycle . The adaption of dengue through its vector has made dengue an infectious disease on the clear rise (38). Thailand is a country with all four serotypes of dengue virus, and the epidemics of dengue haemorrhagic fever have shown a possible correlation to originate from the urban capital of bangkok and then spread geographically in an outward manner to more rural settlements and provinces . A model to understand this mechanism could lead to more effective use of the health systems in the affected areas (39). Dengue has become a global problem and is no longer restricted to the developing world . Despite better knowledge, it seems tough to control the vector, which has adapted to the urban environment and living close to people . An efficient vaccine is not yet commercially available, but could be a powerful factor in the fight against the global dengue epidemic . Often several different factors need to be favourable for a vector - borne disease to adapt to the conditions in an urban environment . For example, west nile virus (wnv) infection is an infectious disease which has become a reality in the urban environment . The primary vector is the mosquito culex pipens, which lay their eggs in water resources which are often man - made . However, for a successful transmission cycle wnv also need the american robin (turdus migratorius), which has several broods per season and hatchlings are more susceptible to wnv infection than adult birds (40). The county of dallas, texas, experienced an epidemic of wnv infections in 2012 . Surveillance reports revealed 25% of the cases in the united states were found in dallas county (41). It shows for a vector - borne disease to have a successful transmission cycle several different factors need to be in place to affect the human population . Leishmaniasis is a disease caused by the protozoa leishmania, which affects 12 million and threatens 350 million people in 88 different countries . When rural migrants bring their domesticated animals to urban settings, often slums, they create favourable conditions for an urban transmission (43). It has been shown that it is a growing health problem and the ongoing urbanization has contributed to the increase (44). If the different vectors can adapt to the urban environment and man - made resources, the potential health implications can be of major concern . Control programs and adequate surveillance is of importance, but in rapidly growing cities and slums it can be tough to implement such measures . Emerging infectious diseases can also make the jump to stable transmission in the urban surroundings and surveillance of these can potentially prevent major health concerns and high cost for the health care services . Who can play a major role in the fight for better control and knowledge . Many of the countries in the developing world do not have the proper resources and the problem is not concentrated to one region, but is a global concern . Numerous of the neglected tropical diseases play a major role in the developing world, which is currently experiencing a much faster pace of urbanization compared to the developed world . The who's call for help is important and, for example, dengue is now turning into a global crisis . Safe and targeted assistance can be a huge factor for overall health; such assistance could be an effective vaccine or safe and easy vector control programs . The basis of large population groups in a restricted area can provide the perfect conditions for different epidemics . International travel has connected the world in the last century, and this mobility creates a potential threat of many emerging diseases . International tourist arrivals have shown an exceptional growth from 25 million in 1950 to 1,087 million in 2013 . According to the latest forecast from the world tourism organization, international tourism arrivals will continue to increase, and in 2030 the figure is expected to be 1.8 billion (45). With the pace of modern travel, highly contagious infectious diseases can be a potential threat in a completely different setting compared to the original outbreak . Urban population and the density of residents can meet the criteria for a new epidemic and create a public health disaster, if not taken seriously . International trade and travel can potentially also contribute to the occurrence of a worldwide pandemic . Sars is thought to originate from the sars - like coronavirus (scov) of bats and reached the human host in china due to hunting and trading of bats for food (46). Investigations have found this scov from the himalayan palm civets in live - animal markets in the region . The first cases of sars reportedly occurred in individuals who handled these animals to prepare exotic food, and the virus is thought to have crossed over to their human host (47). It spread in urban dwellings in large cities and in well - equipped city hospitals . Public fear of travelling led to considerable economic losses that affected entire countries (48). The example of sars shows that food markets in southern china can be the origin of a worldwide health crisis . Travel routes around the world have connected the urban world and large megacities like never before . Accordingly it is important to take necessary preventive measures before the epidemic gets out of control, and here big organizations like who, but also governments, play an important role . Early action is of utmost importance, and functional surveillance programs needs to be in place . The zoonotic disease dengue is endemic in most tropical and subtropical regions, which often are also popular tourist destinations . The burden of disease is on the rise, and estimations are that in returning travellers from southeast asia, dengue is now a more frequent cause of febrile illness compared to malaria (49). Dengue is now an urban health problem, which is one of the major reasons why the rise is exceptional . The worldwide spread of certain antibiotic resistant staphylococcus aureus has been linked to tourism, which shows the potential impact on international health (50, 51). Faecal colonization with esbl - producing enterobacteriaceae has also been linked to international travellers in several studies (5254). The physician needs to take into account the recent travel activities of the patient to better evaluate the current condition and need for potential treatment and care . Global travel shows no signs of decline and the interconnected megacities around the world make global surveillance even more important when it comes to contagious infectious diseases . Measurements to stop the spread need to be taken at the original location, but knowledge about the specific disease needs to be passed on to the global community and local health workers in other parts of the world . This global surveillance and alert system needs to be fast and efficient to, if possible, reduce the impact . The expected rise of travel makes it critical for the future global health and the possibility to react in time for possible threats . Rapid and sometimes uncontrolled urbanization can, in certain circumstances, lead to closer encounters with wildlife . Human influence on the ecosystems creates meeting points for new and potential zoonotic diseases, which could have a profound impact for both local and global health . New housing in the outskirts of big cities can potentially be meeting points for new and already known zoonotic diseases . Of 335 emerging infectious diseases, which have been recognized between 1940 and 2004, more than 60% have been zoonotic diseases (55). Living in close contact to domesticated animals and hunt for bush - meat can also be risk factors for an infectious disease to make the jump from the animal host to humans . Major deforestation creates closer contact between humans and bats and even primates, who can potentially be host for new viruses . A better understanding, surveillance, and prevention of zoonotic diseases would be of great value, to both prevent and manage this upcoming threat for global health . Hot spots for this transmission have been found and they often correlate where the process of urbanization is on the clear rise (56). Even if it is not always the urban population who is at the front of new encounters with wildlife, it can still have an effect on urban health . The trend of people moving to cities are at the highest, where many of these new encounters with ecosystems take place, and infectious diseases can be introduced to these growing urban environments . The sometimes uncontrolled growth of cities pushes residents to untouched ecosystems when new housing expands . Ebola virus disease (evd) has had a profound impact on the world in 2014 . Since the spring of 2014, the hub of the epidemic has been the three countries in western africa: sierra leone, liberia, and guinea . It all began in december 2013 in guinea, in the providence of guckdou, in the eastern rainforest region . The disease transmission in the capital of conakry is thought to be the first major urban setting for evd (57). Who was first notified of the evd outbreak in march 2014, and on august 8, the who declared the current situation as evd outbreaks in central africa had been limited in size and geographical spread to a few hundred persons, mostly in remote areas and not large urban settings (59). The centre of the epidemic (guinea, liberia, and sierra leone) has, as many of their neighbouring countries, a large population living in rural settings; only 36, 49, and 40% of their population live in urban centres (6062). The population is, however, highly interconnected in these countries with travel and cross - border traffic, with good road access between rural and urban settings . These communications have made the magnitude of the evd epidemic possible . Despite cases of evd in nigeria and lagos, a megacity with 20 million inhabitants, the transmission has been limited, which proves that implementation of control measures can limit the transmission (63). The mortality rate has been high in previous outbreaks, up to 90% (64). The fatality rate in the west africa epidemic has been estimated to around 70% for guinea, liberia, and sierra leone when data for patients with recorded definitive clinical outcomes (63). This unprecedented epidemic points out the importance of better surveillance, understanding, and preventions measures for this potentially deadly virus . Ebola virus (ebov) is thought to be a zoonotic disease, and fruit bats are under investigation to be the natural reservoir . Ebov sequences have been found in these animals near the human outbreaks which implies where the virus might originate from (65, 66). Closer contact with humans and fruit bats are thus risks for a new global health crisis and the severity of an ebola epidemic has already been witnessed . The high costs, both from an economic and overall health perspective, have affected entire countries and have even cost lives on the other side of the earth . Different factors, such as education, direct primary care services, and the governments capacity for rapid response to upcoming health threats, can contribute to the opportunities in a city . However, in many cities the poor can find it difficult to access proper health care, due to the cost of such services . In more rural areas, the problem can instead be the distance to the nearest clinic, which in reality makes it impossible for prompt and efficient treatment (2). Malaria has historically been and is still a major health concern in large parts of the world . Who estimates 198 million cases (124283 million) of malaria and 584,000 deaths (367,000755,000) in 2013 . The highest mortality rates have been shown to be closely linked to poor countries with a low gross national income (gni) per capita (67). Estimations have been made that nearly 25% of the total african population, 200 million, currently live in urban settings where malaria transmission is a reality . The annual incidence is estimated at 24.8103.2 million cases of clinical malaria among the urban population in africa (68). The relationship between the malaria mosquito vector and the human host determines the burden of morbidity and mortality . This interface is dependent on many different factors and the degree of urbanization is an important one . Increased urbanization and decreased transmission have correlated in several different studies (69). However, whether it was the increased urbanization that led to a reduction in transmission or the malaria reduction that led to development that promoted urbanization of societies is a challenge to determine (70). A clear connection has been shown between reduced transmission of plasmodium falciparum and urbanization; however, for plasmodium vivax it is less obvious . For p. vivax, a connection has been found globally and in asia and africa; inconsistent results, however, were found in the americas . Several possibilities could explain these incoherent results, such as more widespread transmission of p. vivax, lower transmission intensity, the wide distribution in asia, and high prevalence of duffy negativity in africa, which protects against p. vivax (71). The overall decrease of the burden of malaria has been a positive effect of urbanization, but the exact mechanisms are not yet known . Coverage of measles vaccination in indonesia have shown to be 68.5% in rural areas, compared with 80.1% in urban regions (72). Studies in nigeria have shown that sometimes the coverage can actually be better in more rural areas, and it might be explained by better mobilization and participation in the delivery of immunization services (73). In a study in uganda, 58% of the urban group compared to 53% in the rural areas were fully immunized, but polio vaccine was given to 51% in the urban group and 52% in the rural group (74). Immunization coverage can also vary considerably among different settings, not only between rural and urban surroundings, but also between urban, rural, and slum settlements . In changdigarh, a union territory of india, full immunization of children at the age of 2 was 30% in slums, 74% in urban, and 62.5% in rural settings (75). It shows that there can be a wide variety of reasons for immunization status among the population in different regions and countries of the world . High coverage can prevent epidemics in large cities and save many lives; however, immunization needs to be available both for the rural and urban population to achieve the greatest benefit . A study in tanzania has compared the knowledge about certain zoonotic diseases among general practitioners in urban and rural areas . The rural practitioners had poor knowledge of how sleeping sickness is transmitted and clinical features of anthrax and rabies . Laboratories in rural areas are often poorly equipped and cannot always diagnose certain zoonotic diseases, which could limit the doctors capability for correct diagnosis and treatment (76). The knowledge about sexually transmitted diseases (stis) among bangladeshi adolescents was higher among people in urban areas compared to rural, both in general and hiv and aids (77). The same results about hiv and aids have been found among a canadian population (78). Studies in chengdu and shanghai, china, have shown risk perception about stis and hiv and aids is profoundly changed in rural - to - urban migrants (79, 80). The same result has been shown in a study among rural - to - urban migrants in ethiopia 81). The rapid influx of migrants moving to cities makes it hard to get adequate information to all the different groups in the society . To educate the public is one of the many challenges for local governments and health officials . Campaigns to improve the public knowledge are useful to fight the threat of infectious diseases . Residents need to be aware of symptoms of infectious diseases to gain knowledge about when to seek health care and when it is safe to treat yourself . Knowledge about food storage, waste management, vector control, and sanitary facilities are all aspects that can lower the burden of communicable diseases . These campaigns can sometimes be easier in the urban environment because of the density of the population . Urbanization is an ongoing process in the world at the moment, but the pace of the process is not universal . The developed countries, which have traditionally been thought of as high - income countries, are already urbanized, and it is in the developing world that the rapid rise is taking place . Infectious diseases still have a big impact on the global health, and urbanization is now altering the characteristics of these diseases . Living conditions in cities are overall better in urban environments compared to rural settings; better housing, sanitation, ventilation, and social services all play an important role in this improvement . Certain pathogens can, however, adapt to the different conditions and thus create a new challenge for both local governments and the global community . The capacity for surveillance, control programs, prevention, and public knowledge programs is far better in cities . But some countries do not have the resources and because these diseases can be of global concern, it is also the international community's responsibility to help and support with knowledge and resources . The rapid urbanization has also interfered in previously untouched ecosystems . These new settlements create new and closer encounters with wildlife, which can be a potential source of zoonotic diseases . These can be both previously known or new pathogens, which make the shift from their animal host to generate infections in humans . Surveillance is of primary importance to monitor the burden of disease and will give both local authorities and the global community a chance for a quick response to public health threats . The author have not received any funding or benefits from industry or elsewhere to conduct this study.
Inadequate blood flow to the brain, termed cerebral ischemia, will produce brain injury and a progression of pathophysiological responses associated with energy depletion that include cell swelling, cytotoxic edema, vasogenic edema and if sufficiently severe or prolonged, neuronal cell death . When brain regions are directly damaged by ischemia this can lead to secondary injury or degeneration in associated axonal connections such as the corticospinal tracts . Detection of such changes on early subacute diffusion mri have been described as early or such early signs of wallerian degenerative injury are predictive of specific functional outcomes and may improve prognostication, guide rehabilitation, and could represent both a selection criteria and potential novel target for clinical trials . In particular, if there is diagnostic evidence for early degenerative injury, such as increases in the intensity of the corticospinal tract in diffusion weighted magnetic resonance (mr) images, then prognosis for a good recovery in such infants has consistently been poor (domi et al ., 2009; understanding better the cellular responses that evolve with axonal degeneration following stroke are important considering both their potential contributions to diagnosis and the potential for providing novel targets for therapeutic intervention . Currently, the tissue alterations underlying these mr biomarkers are unknown and speculative because histopathology, if available, is usually performed at single and relatively chronic time points many weeks to months after an ischemic insult . Studies of the evolution of ischemic brain damage or infarction at subacute and chronic times has been assessed using standard mr imaging sequences such as t2 or diffusion weighted imaging (latchaw et al ., 2009). We recently demonstrated that following neonatal hypoxia with unilateral transient ischemia there is evidence for early wallerian degeneration visible as either decreases in the apparent diffusion coefficient (adc) and magnetization transfer ratio or increases in dw or t2 (lama et al . The early corticospinal axonal changes (e.g. In the cerebral peduncle) reflected several cellular alterations such as a reduced staining for phosphorylated neurofilament h and increased vacuolation in hematoxylin and eosin stained sections . Dti holds additional promise for detecting specific degenerative responses because of its potential sensitivity to certain ultrastructural cellular changes depending on the measured dti parameter i.e. Fractional anisotropy, mean diffusivity and parallel or radial diffusivity . Of the extensive cellular responses produced by ischemia, those considered most likely to affect the diffusion properties of water are those that involve morphological tissue changes such as necrosis, astrogliosis, loss of myelin, loss of axonal neurofilaments or cellular inflammation (i.e. Microglial / macrophage activation) (nucifora et al ., 2007; zhang et al ., 2012). Our group and others have shown that corticospinal tract dti alterations are strongly correlated with motor outcome in children with perinatal stroke (hodge, 2013; roze et al . However the progression of dti changes and whether there are differences between the evolution of cellular responses in direct ischemically injured brain versus associated connected tracts is not known . Differences are expected considering the different cellular compositions, for example neuronal rich cerebral cortex compared to axonal bundles within the descending corticospinal tract . A differential timing of dti alterations is also expected considering direct ischemic injury is thought to be followed by a delayed wallerian degeneration (lama et al ., 2011; tuor et al ., 2013). In order to better understand the unique cellular and dti imaging alterations associated with secondary wallerian degeneration relative to the onset and progression of direct ischemically damaged brain, it is necessary to compare their progression directly at multiple time points . In the current study we hypothesized that dti imaging would provide measures of progressive tissue changes within axonal tracts distal to the ischemic injury distinct from those in directly injured brain (e.g. Cerebral cortex) and these differences would correspond with specific tissue morphological alterations in response to ischemia this was investigated by using a neonatal rat model of unilateral transient cerebral hypoxia ischemia and measuring dti changes in the parietal cerebral cortex compared with its associated descending corticospinal tract axon fibers within the cerebral peduncle . Dti measurements were made at acute, subacute and chronic times after hypoxia ischemia along with processing of brains at each of these time points to assess immunohistochemically potential contributions of morphological modifications . We focused on using markers for detecting cell death in neurons, loss of myelin and loss of neurofilaments in axons, and, activation of astrocytes and microglia / macrophages . Fifty one pups delivered from 9 different pregnant wistar female rats (charles river laboratories, montreal, canada) were used in the study . Experiments followed the canadian council on animal care guidelines and were approved by a university of calgary animal care committee . A moderate unilateral ischemic lesion with hypoxia (vannucci and vannucci, 2005) was produced as described previously (lama et al . Briefly, pups (n = 38) on their 7th day of life had their right common carotid artery ligated under isoflurane anesthesia followed by a 60 minute exposure to hypoxia in a chamber containing 8% o2 and 92% n2 at 35.5 c . Sham control animals (n = 13) experienced a similar surgical procedure without carotid artery ligation and hypoxia exposure . The vannucci model of neonatal cerebral hypoxia ischemia (vannucci and vannucci, 2005) produces ischemic damage and infarction within the distribution of the middle cerebral artery territory . Thus, the parietal cortex was selected as a representative brain region of direct hypoxic ischemic damage or infarction . The posterior cerebral peduncle supplied by the posterior circulation was selected as a region of secondary injury remote to the hypoxia brain near the aqueduct in the posterior pons was selected as a control region generally unaffected by the hypoxia ischemia . Sham animals or animals subjected to cerebral hypoxia ischemia were anesthetized (1.52% isoflurane) and dti images in addition to anatomical scans were acquired at 3 h, 1 d, 2 d, 1 w or 4 w post - insult . Anatomical images were also acquired at 1 d post insult in the chronic animals (1 or 4 w) to confirm the extent of ischemic damage . Mr images were acquired using a 9.4 t bruker biospin mr imaging system and paravision 5.1 software . Throughout the scanning, respiration was monitored and maintained by adjustments in anesthesia and body temperature was maintained using a feedback heated air system (small instruments inc ., stony brook, ny). Images were acquired using a 3.5 cm diameter quadrature volume coil for radiofrequency transmission and reception . The head and body was restrained using custom designed swaddling and a head band or ear pins . Depending on the age of the animal, each mr imaging scan consisted of 2530 slices of 0.50.55 mm thick covering the cerebrum and medulla, a 2 2 cm or 2.5 2.5 cm field of view and a data matrix size of 128 128 . Anatomical t2 maps were first generated using a t2 imaging sequence consisting of a set of t2 weighted spin echo images with 32 echoes, repetition time of 10 s and echo time of 10 ms between echoes . For dti, a four - shot echo - planar imaging sequence was used to acquire four averages of sets of diffusion weighted images . These were acquired with b values of 0 (5 images) and 1000 s / mm (30 images in non - collinear directions) using a repetition time of 6500 ms and an echo time of 35 ms . Artifacts associated with imperfections in the radio frequency pulse, gradient stability, and gradient echo currents were removed using a navigator - echo phase correction . Nyquist ghost artifacts were suppressed in the image reconstruction using information acquired during the initial automatic receiver gain adjustment . Dti image acquisition time was approximately 1 h. t2 weighted images were visualized using local mr analysis software (marevisi, national research council of canada, winnipeg, canada). These images were used to assess the extent of ischemic damage and measure brain volumes (atrophy). Inspection of t2 images was also used to exclude from further analysis animals with no cortical t2 lesions or very large hyperintense lesions extending into the pons . They were also used to identify anatomical landmarks for selection of the regions of interest in subsequent dti analysis and histological analysis as identified using a rat brain atlas (paxinos and watson, 1998). These regions of interest were manually defined in the mr images and histological sections and included the cerebral peduncle (0.30.4 mm), pons (within the central gray and mesencephalic trigeminal nucleus regions, 0.60.9 mm) and parietal cortex (0.71.0 mm) for areas both ipsilateral and contralateral to the lesion containing hemisphere . Once the ischemic infarct had progressed towards cavitation and cyst formation (i.e. 1 and 4 w post - hypoxia ischemia), homologous measures of cortical tissue were not possible and therefore were taken adjacent to the cyst a peri - cyst cortical region (0.40.6 mm). Sizes and shapes were adjusted using the contralateral region (e.g. Cerebral peduncle) as a guide and areas of interest were adjusted if there was atrophy in order to minimize partial volume effects . Diffusion tensor images were imported into dti studio (jiang et al ., 2006) through the dti mapping function as philips rec files . Entire scans were excluded if less than eighteen artifact free images were available per slice . The minimum of eighteen directions was selected empirically as the cut - off following an analysis of 12 test data sets with repeatedly fewer b directions (e.g. 25, 22, 20, 18, 16 and 15 compared to 30) which demonstrated no significant effect of a reduction in the number of b directions except for fa and l3; and, those were altered only once the number of directions was 16 or less (p <0.05, repeated anova). For each image slice the dti program calculated the over - determined matrix, the adc vector and the pseudo - inverse of the matrix using singular value decomposition in order to compute the tensor for each pixel (jiang et al ., 2006). Imaging maps were then calculated for the mean diffusivity (adc; (1 + 2 + 3) / 3), the parallel diffusivity (1), the radial diffusivity ((2 + 3) / 2) and the fractional anisotropy (fa). Measures of the dti variables were obtained within regions of interest manually drawn ipsilateral and contralateral (non - ischemic damaged side) of the brain slice . Following their last mr scan, subgroups of animals were perfused transcardially with 10% formalin under deep pentobarbital anesthesia and their brains were embedded in paraffin . Brains from 44 rats were processed and stained for 5 different cellular or injury markers within sections containing the parietal cortex, posterior cerebral peduncle and central gray region of the pons . Thirteen of these animals were sham controls and 31 were animals subjected to hypoxia ischemia and euthanized at the 5 different times post hypoxia ischemia (n = 47/group). Sections (6 m thick) were cut at the level of the posterior cerebral peduncle and parietal cortex using the brain atlas and anatomical landmarks to match sections to the mr slices and measure staining changes in equivalent regions of interest to those for the mr images . Sections from each region were stained for a marker of cell death or degenerating neurons using fluorojade b (schmued and hopkins, 2000) according to the manufacturer's instructions (millipore, billerica, ma, usa). Briefly, deparaffinized sections were exposed to 0.06% potassium permanganate for 15 min washed in distilled water and then stained with 0.0004% fluorojade solution . Sections at each level were also processed immunohistochemically to stain for the following: neurofilaments using a pan - axonal neurofilament marker (monoclonal smi312), reactive astrocytes using anti - glial fibrillary acidic protein (gfap), activated microglia / macrophages using anti - cd68/ed1, and, myelin using an antibody against myelin basic protein (mbp). Sections were deparaffinized and processed for antigen retrieval by boiling in sodium citrate (ph = 6.0, 90 c) for 10 min then washed in pbs . Slides were subsequently blocked with 1% h2o2 in methanol for 5 min followed by 10% goat serum in 0.1 m pbs and then a pbs wash . Slides were then incubated for 1 h at room temperature with one of 4 different primary antibodies: smi312 antibody (1:1000 in bsa, covance, berkeley, ca, usa), polyclonal rabbit anti - gfap (1:500 in bsa, diagnostics canada inc ., on, canada), mouse anti - rat cd68/ed1 (1:500 in bsa, abd serotec, raleigh, nc, usa) or rabbit anti - mbp (1:500 in bsa, chemicon international, ca, usa). Following a pbs wash, slides were incubated overnight at 4 c with the appropriate secondary antibody, either biotinylated goat anti - rabbit igg or biotinylated goat anti - mouse igg (1:400 in bsa, jackson immunoresearch lab inc . This was followed by incubation for half an hour with horseradish peroxidase - streptavidin (1:400 in pbs, diagnostics canada inc ., on, canada). Finally, positive cells were visualized following an application of 3,3-diaminobenzidine (sigma aldrich, mo, usa) dissolved in 0.3% h2o2 in distilled water for approximately 3 min . The histological sections were assessed semi - quantitatively for changes in staining within the regions of interest either by measuring optical density or by quantifying numbers of positively stained cells per field of view . For optical density measures, sections stained with either smi312, gfap or mbp were first captured digitally using a scanner (nikon scan, version 4, nikon inc . ). These images were converted to gray images and mean gray levels (imagej, nih) were measured within the same regions of interest as those drawn for the mr brain slices . Regions with less intense staining had higher mean gray values . The ipsilateral versus contralateral differences in gray levels were converted to a percentage of the contralateral value . The cd68/ed1 and fluorojade stained sections were analyzed using digitized microscopy (microbrightfield, olympus, 40 magnification). The numbers of positively stained cells per field of view were estimated within regions corresponding to those selected from mr images . Three different fields of view were counted for positive staining cells by an investigator (m.s .) All group data is reported as mean sd and statistical comparisons were considered significant at p <0.05 . Measured values within different brain regions a two way repeated analysis was used to determine significant region and time interactions and these were followed by a holm statistical tests were performed using sigmaplot (systat software inc, san jose, ca). To convey clearly the differences for the cerebral peduncle, we noted in the graphs only the significant differences for this region . The correlation between histological changes and dti parameters was assessed using a pearson product correlation analysis within each time point or all time points with a bonferroni adjustment for multiple comparisons . Despite intensive investigation of dti's ability to detect brain injury, the interpretation of alterations in dti measures is usually speculative because of a lack of direct documentation of the corresponding histological changes . In the present study we report detailed differences in the progression and magnitude of dti and histological changes that provide a reference for identifying the potential contribution of various cellular responses to fa and axial, radial and mean diffusivity . The results are first to characterize the correspondence of the progression of cellular and dti changes within the cerebral peduncle of the descending corticospinal tract following hypoxic ischemic damage to the cortex . Novel is the demonstration of the differing progression of dti alterations in a directly damaged ischemic region such as parietal cortex versus that in associated subcortical tracts . Dti changes were less severe or delayed in the cerebral peduncle of the corticospinal tract and this in general corresponded to less severe and delayed cellular responses consistent with early wallerian degeneration . As discussed in more detail, the tissue alterations following ischemia and axonal degeneration had a complex relationship with changes in fa, parallel diffusivity and radial diffusivity . Regarding our assessment of progressive changes in the dti parameters, it is important to realize that superimposed on the effects of hi injury are well recognized developmental changes in the dti parameters that occur during development and aging (dubois et al ., 2013). Indeed, the contralateral hemisphere was similar to those in sham animals but developmental changes may have been differentially affected in the left and right hemispheres by the ischemic insult . Right differences needs to include an awareness that these can be accounted for by an injury modulation of cellular maturation processes in white matter, which we assessed in our histological sections . The region of cerebral peduncle measured was approx 0.30.4 mm consisting of approx 10 pixels of sizes 0.024 mm in the neonatal scans and 0.038 mm in 5 wk old animals . Partial volume effects could be greater in neonatal brain than older brain . However, because we compared left and right differences in values, we expect that there were similar ipsilateral and contralateral partial volume effects in the cerebral peduncle at each time point . Right differences but this did not appear to be an issue considering that many of our measures had the maximal changes in our neonates (e.g. 1 d post insult). There have been numerous studies regarding the evolution of adc or mean diffusivity following transient cerebral ischemia in regions of stroke . Adc alterations reported previously are comparable to those observed presently (e.g. Lama et al ., 2011; liu et al ., 2007). Notably, there were striking similarities between the adc and parallel diffusivity changes suggesting that both reflect similar cellular responses to injury . Very early adc and parallel diffusivity changes in ischemic cortex are likely due to a greater restriction of water diffusion as a result of cytotoxic edema, reduced extracellular space and increased tortuosity which in the present study was also accompanied by decreased neurofilament staining and increased neuronal cell death . As vasogenic edema and damage to cellular membranes proceeded, which histologically corresponded to a continuing cell death and microglial / macrophage activation and astrogliosis, the reductions in adc and parallel diffusivity normalized and this process occurred over several days . By 14 w, phagocytosis produced cysts in the infarct region and such cystic regions had an extremely large adc in all directions whereas peri - infarct regions generally had normalized adc and normalized parallel diffusivity despite the presence of inflammatory cells . In contrast, in the cerebral peduncle with indirect ischemic injury, by the first 3 h there were modest but significant adc and parallel diffusivity reductions . The hyperacute changes in adc and parallel diffusivity correspond to our previous observation of a reduction in smi31, demonstrating a decrease in phosphorylated heavy neurofilament subunits already hours after insult . Subsequently, the adc and parallel diffusivity reductions increased in magnitude and were maximal in the first few days . This corresponded well with the diminished staining observed for the pan - axonal (heavy, medium and light) neurofilament marker smi312 used presently . Loss of phosphorylation of neurofilaments promotes their disassembly and this corresponded to a decreased radial diffusivity as well as a decreased parallel and mean diffusivity of water . At chronic time points post - insult (14 w), a similar greater sensitivity for parallel diffusivity than adc in detecting wallerian degenerative changes in the corticospinal tract has been reported after an excision lesion of the feline cortex (qin et al ., 2012). Microglial / macrophage cell activation likely did not contribute greatly to the parallel diffusivity alterations observed, considering the rather modest positive ed1/cd68 staining observed in the cerebral peduncle . The occurrence of astrogliosis coincided with normalization of adc potentially contributing to some extent to these changes . However, as discussed below, there were also ipsilateral increases in radial diffusivity that likely contributed to the adc normalization . Reductions in radial diffusivity have been proposed to reflect well increases in myelin whereas elevations in radial diffusivity have been found to be associated with a loss of myelin (song et al ., 2002, 2004; the current study provides some support for this if one examines only chronic recovery times i.e. Times when the axons in the contralateral cerebral peduncle have become well myelinated . At 1 w and beyond, myelin within the cerebral peduncle ipsilaterally was reduced reflecting less ontogenic myelination of fewer axons following their degeneration and atrophy . This coincided with relative increases in radial diffusivity ipsilaterally and is consistent with increases in radial diffusivity reported in the corticospinal or pyramidal tracts at relatively chronic times (e.g. 1 w to 3 y) following an ischemic insult in children or adults (roze et al ., 2012; yu et al ., 2009; zanier et al ., 2013). At acute times post - insult we observed ipsilateral reductions in radial diffusivity within the cerebral peduncle indicating that in the absence of effects on myelination, early axonal injury such as that detected by decreases in phosphorylated neurofilaments were also associated with reduced radial diffusivity it is noteworthy that the neurophysiological correlates of alterations in parallel and radial diffusivity are potentially more complex in humans than rodents . A diffusion tensor analysis has an implicit assumption that the white matter fibers are cylindrical and when fibers have complicated microstructures or crossing fibers as occurs in human developing brain (dubois et al ., 2013; 2013) then in particular the directions of orientation for tractography require careful interpretation . Despite crossing fibers potentially complicating the interpretation of parallel diffusivity changes, transverse or radial diffusivity appears to consistently decrease with all maturational processes within white matter, including myelination (dubois et al ., 2013). Furthermore, emerging imaging methods exploring diffusion parameters beyond the tensor model may provide more in depth assessments to facilitate translation from animals to humans (dubois et al . The fa measures provided a sensitive measure of direct ischemic injury . In the cortex at hyperacute times, there was an increase in fa, as has been observed previously in rats and non - human primates, however, usually soon after permanent rather than transient occlusion (kim et al ., 2013; liu et al ., 2007; yang et al ., 1999). This suggests that the very early cortical ischemic changes were relatively severe and similar to those with permanent ischemia . Cellular changes likely involve previously established hyperacute cytotoxic cell swelling and reductions in extracellular space leading to cell death considering our histological evidence for hyperacute neuronal cell death in the cortex . By the first day post - insult, the fa increase became a decrease that persisted at subacute and chronic time points, confirming previous reports of a reduced fa commonly observed in damaged brain post - stroke (e.g. Kim et al ., 2013; liu et al ., 2007; yang et al ., 1999). This decrease in fa, reflecting diminished preferential diffusivity, is likely associated with a loss of membrane integrity and intracellular structure due to necrosis as supported histologically by continuing cell death and loss of neurofilament staining . The increase in astrogliosis between 3 and 1 d appears rather isotropic considering that there is a decline in fa between these times . The fa also provided a rather robust measure of degenerative changes in the cerebral peduncle . In contrast to ischemic cortex, fa in cerebral peduncle was decreased at all time points . Indeed, fa appeared to be the most sensitive dti parameter for detecting axonal degenerative changes in the cerebral peduncle independent of time post - insult and this corresponded well to the persistent decreases in axonal neurofilament staining . Similar reductions in fa have been reported at various select times in the corticospinal tract following neonatal stroke (ludeman et al ., 2008; puig et al ., 2013; tusor et al ., 2012). It is currently clear that at chronic times, fa is superior to adc or other standard imaging methods such as t2 imaging because lesions detected with these other sequences tended to pseudo - normalize at times post - insult (lama et al ., 2011; tuor et al ., the results demonstrate that in general, diffusivity changes and histological responses within directly damaged ischemic brain precede and are often larger in magnitude than corresponding alterations in a region of secondary axonal degeneration, such as the cerebral peduncle . With the progression of damage over one or more weeks, dti components such as fa and radial diffusivity are better than mean adc for detecting ischemia associated axonal injury indicative of wallerian degeneration . Also, differences in radial diffusivity in brain injured perinatally are likely related to a lack of ontogenic myelination on the injured side . Thus, the specific progression of dti alterations following an ischemic insult not only depends on the maturity of the subject but also appears dependent on other factors such as the type of insult (e.g. Spinal cord injury or brain resection) (brennan et al ., 2013; liu et al ., 2013; qin et al ., 2012). The inflammatory response investigated (microglial or astroglial activation) appears to have modest contributions to the evolution of axonal changes in diffusion tensor parameters . Thus, this systematic study is informative regarding the optimal dti mr imaging parameters to probe axonal injury following hypoxic / ischemic brain damage in developing brain in addition to aiding our interpretation of dti parameters for detecting various stages of wallerian degeneration.
The effect of family on workforce is an important though often overlooked issue for work organizations, with implications for the morale, stability and productivity of the workforce . Presumably men and women do not shed their family roles, relationships and experiences the moment they don their work clothes . Indeed, the logic underlying many corporations' decisions to offer employer - based family support, such as child care and flexible working schedules, may be that such benefits will enhance employees' abilities to handle family matters and in so doing will enhance their work performance, commitment and satisfaction . This line of reasoning views the relationship between work and family as a reciprocal process in which work and family influence each other in a circular or feedback fashion . Indian families are undergoing change, which in turn influences the environment in the basic unit . The family environment has been bombarded with new expectations, the media, high cost of living and a striving for better quality of life . Although not much literature is available on the aspect of how family stress has influenced the productivity of the indian workforce, the author is of the opinion that absenteeism, alcoholism, gambling, heavy debts among employees are a reflection of bruised familial systems . The dual - earning couple is a new prototype that reflects the increasing educational and career aspirations of women . A significant proportion of these women in the workforce comprises of wives and mothers whose employment status demands a radical change in their pattern, activities, commitments and responsibilities, requiring a reassessment of the family environment . In india the proportion of women in the workforce in 1981 was 19.67% and it rose to 22.73% in 1991, further rising to 25.68% in 2001 . On the flipside, psychosocial studies have shown that dual - earning couples in india have a poorer quality of marital life compared to single - earning couples,[46] in their study found that work - family conflict was expressed in 63% of thematic appreciation test (tat) stories written by women, whereas men's stories did not reflect this conflict . Sources of stress in the lives of working women emerged from a lack of time to attend to multiple roles, presence of young children (6 - 12 years) in the family and additional responsibility at work in the form of promotions . The most common outcome of stress for the working woman was found to be poor mental and physical health resulting in depression, anxiety, asthma and colitis. [911] rajadhyaksha[79] studied dual - career as opposed to dual - earning couples in two metropolitan cities in india and found that in a dual - career family where husbands and wives were presumably matched in terms of their career involvement, work - family role conflict and organizational role stress were not significantly different among husbands and wives . For instance, women experienced more conflict between their job and home roles, while men experienced more conflict between their job and spousal roles . In the present day, the young indian adult is exposed to an entirely new pattern of living and a new set of mores, values and standards that are being widely accepted but which stand in contrast to those which were promoted by their parents and grandparents . The ambiguous values children and adolescents observe today in india, coupled with the increasing gap between aspirations and possible achievement, have led to a greater sense of' alienation .' Parents too appear ill - prepared to cope with rapid social change, having grown up in hierarchically structured and interdependent social groups that included the extended family and kinship network, as well as caste groups that provided stability and solidarity . Parents who seem modern in their child - rearing practices get anxious when their offspring breach established social codes . Inter - generation conflicts related to marriage, career choice or separate living arrangements result in the tendency to fall back on tradition . Subtle changes in family patterns, especially with regard to the use of authority within the family, as well as an increased focus on individual autonomy,[1617] are also likely to influence members' expectations with regard to marriage and their choice of a spouse . Educated middle class families are now more hesitant to make decisions for their offspring with regard to marriage, education and employment . Changes have also been noticed with regard to a greater focus on the husband - wife relationship rather than the parent - child relationship . With an increased onus of responsibility falling on the individual rather than on the entire family, gore calls' choice anxiety' increased autonomy and increased choice that have led to increased anxiety . Ambiguity with regard to issues like respect for elders, role of the eldest son, love marriages, working wives, etc ., is an inevitable fallout of the changes within the indian family . Relationship - focused education for young adults in the workforce has not been on top of the agenda for india's policy makers . India's policies on family and child welfare have been focused more in terms of health care, nutrition, reproductive health, sexuality and venereal disease . However, one cannot forget that young adults of marriageable age from' first - generation urban - educated families' are facing relationship - based problems due to changing dynamics within the indian family . These include shifts in loyalty from filial obligations to lean more towards conjugal ties and issues of power and gender role allocations . Marriage is traditionally seen as means for procreation, but there are slow changes in perception among adolescents towards viewing it as a means of companionship . With couples migrating to cities for better job prospects, there is a decreased availability of the informal support provided by the extended family in the past, resulting in young couples having to develop greater problem - solving and coping skills on their own . This in turn weighs heavily on an employee's ability to focus completely on tasks at work . It is essential to bridge the gap between what their families of origin considered as an ideal or happy marriage and what they now desire from a spouse and family life . The author conducted individual interviews with family - intervention experts in bangalore city in order to better understand the changing family patterns that they were observing . Among the professionals, there were two clinical psychologists, two psychiatric social workers, two psychiatrists and one divorce lawyer . The key person interview (kpi) cue statements to initiate discussions with the experts were as follows [table 1]: description of key person interview participants changes that you are seeing in the nature of problems that families are coming to you with in comparison to the nature of problems brought up a decade agoname some culture - specific areas that need to be kept in mind while preparing the family life education programsome areas that you feel need to be essentially covered by a family life education program for single young adults . Changes that you are seeing in the nature of problems that families are coming to you with in comparison to the nature of problems brought up a decade ago name some culture - specific areas that need to be kept in mind while preparing the family life education program some areas that you feel need to be essentially covered by a family life education program for single young adults . It is interesting to note that among the changes noticed by family experts in relation to the aspect of family issues, is the ability to recognize issues early on in the marriage and to seek help for the same . Another important change noticed by the experts was that a larger number of young couples who were either engaged or in a relationship were now seeking professional help in order to help them make the right choice of a partner or to resolve issues that they were afraid would snowball into a larger issue post - marriage . An interesting perspective put forward by the legal expert in family law was that he was seeing an increasing number of divorces that were occurring as early as the third or sixth month of marriage, with both partners willing to go in for a divorce by mutual consent . This, he said, was in stark contrast to what he encountered some 10 years ago, when most couples would go through a long - drawn - out battle with much mud - slinging or would want to keep trying to make things work . The recognition of sexual dissatisfaction by, and the unwillingness to stay in a marriage on the part of, the female spouse was also another prominent change that experts were noticing . A long - distance relationship, with spouses staying in different cities and meeting on the weekends, was also another trend that was being noticed . Themes from key person interview with family experts leadership and decision - making issues in the joint family largely arose due to the inability to face up to, or communicate with, authority figures within the family, thus leading to frustration and passive aggression . Talking about one's sexual needs was another cultural peculiarity that often created rifts within couples or forced couples to try and work things out . It is also interesting to note that the family experts were able to clearly point out the need for any family life education program to be sensitive to indian culture in terms of how it viewed the role of the joint family . Experts also pointed out the program had to be careful about how it portrayed communication within the family and had to keep in mind the factor of' respect' that indian families often prescribe to . The third column clearly gives the areas that these family experts felt a family life education program should cover . The experts felt that adaptability was an important skill that the new indian spouse was quickly losing . Gender sensitivity in terms of acceptance of changing work and family roles of the female spouse by the male spouse was another important area that the experts talked about . Discussions with these family - intervention experts brought out the following areas as possible aspects to be included in family life education program for the younger - generation workforce: gender sensitivityparenting skillsconflict resolution skillsadaptability to changebalancing work and the familycommunication skillssocialization and social supportmoney managementchoosing a life partnersexuality and intimacydealing with violence and addiction conflict resolution skills adaptability to change balancing work and the family socialization and social support choosing a life partner sexuality and intimacy dealing with violence and addiction the experts definitely seemed to sense the need for some form of education or training for the young indian adult who is either contemplating family life or is a new entrant into marriage or parenthood . Family issues that eat into employees' ability to perform or affect his cognitive and emotional state of being need to be addressed . A promotive and preventive strategy rather than a curative one would have to be adopted for this purpose . Relationship - focused family life education programs for the target group of employees at the workplace may actually serve to fulfill this purpose, whereby employees, through group interaction and experiential learning, can be taught skills to deftly handle family - life conflicts either by themselves or through the support of mental health professionals . Thus a mental health professional need not be there merely to diffuse a difficult situation arising out of deviance of an employee . He or she may act as a preventive agent of avoiding such deviance from arising, by tackling the problem at the root by pre - equipping the family with the necessary psychoeducation and skills.
Macroscopically, renal cell carcinomas (rccs) generally present as either solid or cystic mass . Among cystic rccs, multilocular cystic rcc represents a rare entity that was recognized in 1982, with a reported incidence of 1 - 4% of all rccs . Renal leiomyomas are benign tumors of the kidney originating from smooth muscle cells of the renal capsule, pelvis, calices, or blood vessels . Although small renal leiomyomas can be found in about 5% of autopsy specimens, clinical incidence of these lesions is much lower . In this report, we discuss the first case of simultaneous occurrence of a multilocular cystic rcc and a leiomyoma in the same kidney . A 38-year - old man was referred for treatment of a right renal cystic mass found incidentally . Physical examination on admission revealed no palpable mass, and laboratory data were negative . Computed tomography of the abdomen showed a about 5.4 4.6 cm poorly enhancing cystic mass with enhancing septa in the polar area of the right kidney (fig . Since multilocular cystic rcc could not be excluded, the patient underwent right laparoscopic radical nephrectomy . Macroscopically, in the mid pole, a well - circumscribed mass showing a multicystic feature with thin fibrous septa and without expansile tumor nodules was noted . Rccs show cystic changes on imaging studies in 4 - 15% of cases [4, 5]. Since it is often difficult to differentiate cystic rccs from benign cystic lesions, a definite diagnosis can in most cases only be established by histopathologic examination . These include: (1) intrinsic multiloculated growth; (2) intrinsic unilocular growth; (3) cystic necrosis, and (4) origin from the epithelial lining in a pre - existing cyst . Because multilocular cystic rcc are more likely to be discovered incidentally, this feature may contribute to their excellent prognosis compared to that of conventional rccs . Since the majority of rccs originate from the proximal tubules, the characteristics and/or environment of the proximal tubules are considered to play an important role in the pathogenesis of rcc . Recently, imura et al . Carried out a detailed immunohistochemical analysis of multilocular cystic rcc cases . They found that in a high proportion of cases, multilocular cystic rcc reacted strongly with the distal nephron markers, but none reacted preferentially with proximal nephron markers . These results illustrate that multilocular cystic rcc originates from the distal nephron, although the precise pathogenesis of cystic formation in multilocular cystic rcc has not been elucidated clearly . Renal leiomyomas are benign tumors arising from the mesenchymal (or connective) tissue of the kidney . Tumors may be subcapsular (53%), capsular (37%), or located in the renal pelvis (10%). A variety of structural patterns have been described at imaging: solid, cystic, and both cystic and solid [9, 10]. In 1990, steiner et al . The first group comprises small cortical or subcortical neoplasms that are usually asymptomatic, less than 2 cm in size, and are often detected incidentally during autopsy or surgery . The second group consists of larger neoplasms that arise from the renal capsule or blood vessels and may be symptomatic . Unfortunately, it is almost impossible to clinically distinguish renal leiomyomas from their malignant counterparts . Because of their small size (usually less than 2 cm), these smooth muscle - containing tumors are commonly diagnosed during autopsy, and clinical manifestations become apparent only when they grow larger, hemorrhage, or undergo cystic or sarcomatous degeneration . Since the finding of primary synchronous renal neoplasms is very uncommon, especially when they have a different histogenesis, up to now, only a few primary, synchronous rccs with different histotypes have been reported . The coexistence of multilocular cystic rcc and leiomyoma has not been documented . This present case is the first report of synchronous renal neoplasms with different histogenesis (multilocular cystic rcc and leiomyoma) in a patient without any symptoms . The significance of the relationship between multilocular cystic rccs and leiomyomas is not well understood and needs further exploration.
The ganoderma strains listed in table 1 were obtained from the korean collection for type culture (ktct, jeongeup, korea), the american type culture collection (atcc, rockville, md, usa), the korean agricultural culture collection (kacc, suwon, korea), the mushroom division of the rural development administration (eumseong, korea), the centraalbureau voor schimmelcultures (cbs, utrecht, netherlands), incheon university (incheon, korea), and konkuk university (seoul, korea). Ganoderma species were cultured on potato dextrose broth (difco, detroit, mi, usa) and incubated at 30 for 2 wk . Cultured mycelia, filtered through 2 layers of miracloth (calbiochem, la jolla, ca, usa), were ground in liquid nitrogen, and genomic dna was extracted using the cetyltrimethylammonium bromide method . Pcrs were performed using a premixed polymerase kit (taq premix; tnt research, anyang, korea) in a 20 l reaction mixture containing 1 l of dna . Amplification of the its region was carried out using a thermal cycler (takara, tokyo, japan) at the following conditions: 5 min at 94 for initial denaturation, followed by 30 cycles of 30 sec at 94 for denaturation, 30 sec at 56 for primer annealing, and 1 min at 72 for extension, and 10 min at 72 for a final extension . Pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer . Pcr products were ligated into the pgem - t easy vector (promega, madison, wi, usa) according to the manufacturer's instructions . Ligation products were transformed into the escherichia coli dh5 competent cell (rbc, taiwan) by heat shock . Plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit (geneall, seoul, korea). Recombinant clones were identified, and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing (using the sp6 and t7 promoters; genotech, daejeon, korea). Nucleotide sequences were deposited in the national center for biotechnology information genbank database (table 1). The sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The phylogenetic trees were constructed by using the mega5 program and the neighbor - joining method . Confidence levels for individual branches of the resulting tree were assessed using the bootstrap test in which 1,000 replicate trees were generated from resampled data . The size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope (axio observer a1; carl zeiss, jena, germany) and a 5% koh solution as a mounting medium . The size of each spore was calculated, and the mean value was used in the description . The ganoderma strains listed in table 1 were obtained from the korean collection for type culture (ktct, jeongeup, korea), the american type culture collection (atcc, rockville, md, usa), the korean agricultural culture collection (kacc, suwon, korea), the mushroom division of the rural development administration (eumseong, korea), the centraalbureau voor schimmelcultures (cbs, utrecht, netherlands), incheon university (incheon, korea), and konkuk university (seoul, korea). Ganoderma species were cultured on potato dextrose broth (difco, detroit, mi, usa) and incubated at 30 for 2 wk . Cultured mycelia, filtered through 2 layers of miracloth (calbiochem, la jolla, ca, usa), were ground in liquid nitrogen, and genomic dna was extracted using the cetyltrimethylammonium bromide method . Pcrs were performed using a premixed polymerase kit (taq premix; tnt research, anyang, korea) in a 20 l reaction mixture containing 1 l of dna . Amplification of the its region was carried out using a thermal cycler (takara, tokyo, japan) at the following conditions: 5 min at 94 for initial denaturation, followed by 30 cycles of 30 sec at 94 for denaturation, 30 sec at 56 for primer annealing, and 1 min at 72 for extension, and 10 min at 72 for a final extension . Pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer . Pcr products were ligated into the pgem - t easy vector (promega, madison, wi, usa) according to the manufacturer's instructions . Ligation products were transformed into the escherichia coli dh5 competent cell (rbc, taiwan) by heat shock . Plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit (geneall, seoul, korea). Recombinant clones were identified, and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing (using the sp6 and t7 promoters; genotech, daejeon, korea). Nucleotide sequences were deposited in the national center for biotechnology information genbank database (table 1). The sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The phylogenetic trees were constructed by using the mega5 program and the neighbor - joining method . Confidence levels for individual branches of the resulting tree were assessed using the bootstrap test in which 1,000 replicate trees were generated from resampled data . The size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope (axio observer a1; carl zeiss, jena, germany) and a 5% koh solution as a mounting medium . The size of each spore was calculated, and the mean value was used in the description . The phylogenetic analysis of ganoderma species in this study was generally consistent with the findings reported by the group from china . The 62 ganoderma strains were divided into 7 groups, a to g (fig . In addition, ganoderma lucidum were largely divided into two groups (groups a and g). Group a included all the korean g. lucidum strains, as well as the g. lucidum strains from bangladesh and japan, and chinese g. sichuanense and g. lingzhi . G. meredithae (usa and unknown sources) in group b was closely related to g. lucidum in group a as evidenced by a high bootstrap value of 98% . G. multipileum (china and taiwan) was grouped within group c and g. tropicum (china and taiwan) within group d, with 96% and 99% bootstrap support, respectively . Group e includes three strains of g. resinaceum from the czech republic and uk (two strains), and was supported by very high bootstrap values of 99% . Strains of g. weberianum (australia, china, and philippines) and two other chinese g. sichuanense strains (cui 7691 and hmas 86597; holotype) were grouped within group f with 99% bootstrap support . Groups e and f were closely related with bootstrap values of 96% . It is worth noting that other g. lucidum strains from europe (france, finland, italy, sweden, and uk) and canada could be clustered into group g with g. tsugae strains from north america (canada and usa), supported by a very high bootstrap value of 99% . The findings of the current study are consistent with those of park et al . Who reported that g. lucidum strains from europe and north america could be clustered into one group together with g. tsugae based on both analyses of the its rdna gene and partial -tubulin gene sequences . Interestingly, korean g. lucidum strains could be clustered into a group together with g. sichuanense and g. lingzhi strains from china . Reported that g. lucidum is incorrectly recorded in china, as well as in other countries, and suggested that the name' g. lucidum' as used for the chinese species should be corrected as g. sichuanense . However, they did not obtain sequences from type specimens of g. sichuanense (the holotype; hmas 42798). Reported that korean g. lucidum (asi-7004) could be clustered into one group together with chinese g. lucidum and g. lingzhi based on the its2 sequences and rna secondary structures . Furthermore, cao et al . Reported that g. sichuanense (included the holotype; hmas 42798) was distantly phylogenetically related to g. lingzhi (included the holotype; wu 1006 - 38), but it was closely related to g. resinaceum . With regard to the morphological characteristics, it has been reported that chinese g. lucidum (g. lingzhi) has a yellow pore surface, european g. lucidum has a white pore surface, and korean g. lucidum has pale brownish thread - like tissues in the middle of the context . Cao et al . Found that the morphological characteristics of' g. lucidum' from east asia were consistent with those of g. lingzhi . Thus, they concluded that g. sichuanense was a distinct species since they display obvious morphological differences from g. lingzhi and suggested that the name' g. lucidum' should be corrected as g. lingzhi not g. sichuanense . It is commonly called " yeongji " in korea, " lingzhi " in china, and " reishi " or " mannentake " in japan . However, the ganoderma species from various countries including africa, oceania, america, asia (china, korea, and japan), and europe have been incorrectly reported as g. lucidum . In addition, moncalvo et al . Classified g. lucidum collections from different regions (asia and europe) into different species based on its and partial nuclear large subunit ribosomal dna sequences . This is similar to the results of our its nucleotide sequence analysis of g. lucidum . Likewise, hong and jung reported that the g. lucidum from korea and japan were monophyletic, and were distinguished from the g. lucidum from europe and north america based on sequence analysis of mitochondrial small - subunit ribosomal dna . As described above, the taxonomy of g. lucidum has been reported by many researchers . Nevertheless, the name g. lucidum has been misapplied to various species around the world . The results of this study also reveal that korean g. lucidum was clustered into one group together with chinese g. sichuanense and chinese g. lingzhi (both formerly g. lucidum), and it was clearly separated from g. lucidum from europe and north america . In addition, g. sichuanense were divided into two groups (groups a and f). G. sichuanense strains (cui 7691 and hmas 86597; holotype), including the holotype, could be clustered into group f together with g. weberianum, and it was closely related to g. resinaceum . Cao et al . Also reported that the size of basidiospores of g. lingzhi [(9.85 0.85) (6.4 0.6) m] differs from that of g. sichuanense [(8.3 0.9) (5.8 0.8) m]. Interestingly, the average size of the basidiospores from the korean cultivated yeongji strains [(10.65 0.65) (6.6 0.6) m for asi-7004, (10 1) (6.4 0.3) m for asi-7071] were similar to that of g. lingzhi (fig . The comparison of the its rdna sequences and the estimation of the basidiospores size presented in this study confirm previous findings and contribute additional evidence that suggests the naming korean cultivated yeongji strains of' g. lucidum' should be renamed as g. lingzhi.
Successfully conducted the first simultaneous liver - kidney transplant (slkt) in 1983 on a patient with chronic kidney allograft rejection and liver cirrhosis, this procedure has become the most effective method for the treatment of combined end - stage liver disease and renal failure . The number of patients with end - stage liver disease and renal insufficiency receiving slkt has increased remarkably, particularly since the introduction of the model for end - stage liver disease (meld) scoring system by the united network for organ sharing (unos) in 2002 . Statistics showed that the number of slkt recipients in the united states has quadrupled from 1998 to 2006 . In addition, the survival rate of slkt recipients has significantly increased following the adoption of meld . The primary causes of end - stage liver disease are cirrhosis caused by viral hepatitis, alcoholic cirrhosis, liver cancer, and congenital liver diseases . While hepatitis c is the leading cause in western countries, hepatitis b is the most prevalent type in china . Epidemiological studies have shown that more than 800 million people have been infected with hepatitis b in china . Up to 10.31% of the chinese population is hepatitis b surface antigen (hbsag)-positive . In china, up to 80% of patients with liver failure requiring liver transplant are due to hepatitis b - associated causes . Relapse of hepatitis b after liver transplant can reduce the patient s probability of survival by inducing graft dysfunction, recurrence of liver cancer, and lymphatic proliferation . A previous study reported that slkt for hepatitis c, non - alcoholic steatosis and hepatocellular cancer could be associated with worst outcomes compared with other slkt indications . However, few studies have reported the effectiveness of slkt and hepatitis b recurrence in hepatitis b carriers receiving slkt . Previous studies have compared the outcome of slkt mainly to outcomes of liver transplant (lt) patients with normal renal function as controls [1216], while comparison to lt patients with renal insufficiency has been largely ignored . A comparison between slkt recipients with hepatitis b infection and lt recipients with renal insufficiency and hepatitis b infection could provide a more objective assessment of the outcome of slkt associated with hepatitis b. in addition, which indicators can predict the long - term outcomes of liver transplant patients still need to be assessed . Therefore, this comparative study analyzed rates of postoperative infection, rejection, long - term survival, and relapse of hepatitis b in hepatitis b carriers receiving slkt at the organ transplant center of the shanghai first people s hospital . This was a retrospective analyses in 21 patients (male: 19, female: 2) who underwent slkt at the organ transplant center of shanghai first people s hospital, shanghai jiaotong university, between january 2001 and may 2005 . Recipients of slkt were selected based on the following criteria: 1) irreversibly compromised renal function confirmed by preoperative examination, including serum creatinine levels> 133 mol / l lasting for 1 month continuously; 2) high - risk factors for kidney diseases, such as diabetes and hypertension; and 3) massive proteinuria and/or requiring renal replacement therapy for more than 3 weeks . Liver and kidney allografts for the same patient came from the same donor, whom was not necessarily a relative . Panel reactive antibody (pra) class i of recipients was 028%, and class ii was 017% . The study period also included 25 patients who underwent lt alone with renal insufficiency (preoperative serum creatinine levels> 133 mol / l). Twenty - five recipients developed renal insufficiency due to hepatorenal syndrome without acute tubular necrosis and renal parenchymal disease . This study was approved by the ethical committee of the shanghai first people s hospital, and written informed consent was obtained from each patient . Written consent has been obtained from all donors at the time of their donation . Criteria for slkt: 1) end - stage liver disease and irreversible kidney failure; 2) congenital diseases involving the liver and kidney; 3) liver failure and chronic kidney disease (ckd) with gfr 30 ml / min; 4) acute kidney injury (aki) with creatinine levels 176.8 mol / l and dialysis 8 weeks; or 5) liver failure and ckd and biopsy demonstrating> 30% glomerulosclerosis or fibrosis . If there was no evidence to prove irreversible renal damage, liver transplantation alone was preferred . Whether patients with hepatorenal syndrome should undergo slkt was controversial, and each patient was discussed . When patients with hepatorenal syndrome were on dialysis for <4 weeks, liver transplant alone was performed . Biochemical indicators (including serum creatinine, total bilirubin, and aspartate aminotransferase (ast) levels) were followed up for 5 years in all transplant recipients . Lt patients were stratified according to the severity of aki as described by the risk, injury, failure, loss and endstage kidney disease (rifle) classification: risk, injury or failure . Early- and late - stage postoperative complications, postoperative infections, rejection, and long - term survival were also recorded . The diagnosis of acute renal allograft rejection was primarily based on recipients clinical presentation, biochemical indicators (a 25% increase of serum creatinine levels or more), and histopathological biopsy . The diagnosis of acute hepatic allograft rejection required increases in serum ast levels and/or in total bile acids, and was confirmed by liver biopsy . Treatment of acute allograft rejection mainly relied on methylprednisolone sodium succinate pulse therapy, an increase in the dose of immunosuppressants, and/or change of types of immunosuppressive agent . The induction regimen of anti - cd25 monoclonal antibody (daclizumab or basiliximab) was prescribed to all patients . Slkt recipients received a triple maintenance therapy of calcineurin inhibitor (cni; cyclosporine and tacrolimus), mycophenolate mofetil (mmf) and prednisolone (pred), while lt recipients received the steroid - free regimen of cni + mmf . Nucleoside analogues coupled with low - dose hepatitis b immunoglobulin (hbig) were given to prevent hepatitis b relapse . Preoperative nucleoside analogue therapy was followed by intraoperative hbig 2000 u. postoperative combined therapy was administered with maintenance hbig twice per week . Within 6 months of transplantation, survival rates of recipients and allografts were analyzed using the kaplan - meier and log - rank methods . Continuous data were analyzed using the student s t - test, and categorical variables using the chi - square test . This was a retrospective analyses in 21 patients (male: 19, female: 2) who underwent slkt at the organ transplant center of shanghai first people s hospital, shanghai jiaotong university, between january 2001 and may 2005 . Recipients of slkt were selected based on the following criteria: 1) irreversibly compromised renal function confirmed by preoperative examination, including serum creatinine levels> 133 mol / l lasting for 1 month continuously; 2) high - risk factors for kidney diseases, such as diabetes and hypertension; and 3) massive proteinuria and/or requiring renal replacement therapy for more than 3 weeks . Liver and kidney allografts for the same patient came from the same donor, whom was not necessarily a relative . Panel reactive antibody (pra) class i of recipients was 028%, and class ii was 017% . The study period also included 25 patients who underwent lt alone with renal insufficiency (preoperative serum creatinine levels> 133 mol / l). Twenty - five recipients developed renal insufficiency due to hepatorenal syndrome without acute tubular necrosis and renal parenchymal disease . This study was approved by the ethical committee of the shanghai first people s hospital, and written informed consent was obtained from each patient . Written consent has been obtained from all donors at the time of their donation . Criteria for slkt: 1) end - stage liver disease and irreversible kidney failure; 2) congenital diseases involving the liver and kidney; 3) liver failure and chronic kidney disease (ckd) with gfr 30 ml / min; 4) acute kidney injury (aki) with creatinine levels 176.8 mol / l and dialysis 8 weeks; or 5) liver failure and ckd and biopsy demonstrating> 30% glomerulosclerosis or fibrosis . If there was no evidence to prove irreversible renal damage, liver transplantation alone was preferred . Whether patients with hepatorenal syndrome should undergo slkt was controversial, and each patient was discussed . When patients with hepatorenal syndrome were on dialysis for <4 weeks, liver transplant alone was performed . Biochemical indicators (including serum creatinine, total bilirubin, and aspartate aminotransferase (ast) levels) were followed up for 5 years in all transplant recipients . Lt patients were stratified according to the severity of aki as described by the risk, injury, failure, loss and endstage kidney disease (rifle) classification: risk, injury or failure . Early- and late - stage postoperative complications, postoperative infections, rejection, and long - term survival were also recorded . The diagnosis of acute renal allograft rejection was primarily based on recipients clinical presentation, biochemical indicators (a 25% increase of serum creatinine levels or more), and histopathological biopsy . The diagnosis of acute hepatic allograft rejection required increases in serum ast levels and/or in total bile acids, and was confirmed by liver biopsy . Treatment of acute allograft rejection mainly relied on methylprednisolone sodium succinate pulse therapy, an increase in the dose of immunosuppressants, and/or change of types of immunosuppressive agent . The induction regimen of anti - cd25 monoclonal antibody (daclizumab or basiliximab) was prescribed to all patients . Slkt recipients received a triple maintenance therapy of calcineurin inhibitor (cni; cyclosporine and tacrolimus), mycophenolate mofetil (mmf) and prednisolone (pred), while lt recipients received the steroid - free regimen of cni + mmf . Nucleoside analogues coupled with low - dose hepatitis b immunoglobulin (hbig) were given to prevent hepatitis b relapse . Preoperative nucleoside analogue therapy was followed by intraoperative hbig 2000 u. postoperative combined therapy was administered with maintenance hbig twice per week . Within 6 months of transplantation, survival rates of recipients and allografts were analyzed using the kaplan - meier and log - rank methods . Continuous data were analyzed using the student s t - test, and categorical variables using the chi - square test . Meld scores, child - pugh scores, and incidence of preoperative dialysis were not significantly different between slkt and lt recipients . However, slkt recipients had lower egfr (p=0.003) and higher preoperative serum creatinine levels (p=0.004), lower serum ast levels, and lower total serum bilirubin levels than lt recipients (table 2). Four slkt recipients died: 2 died due to hepatitis b relapse and graft - versus - host disease within 2 months of surgery; 1 discontinued lamivudine against medical advice and died due to hepatitis b relapse and subsequent acute liver failure 14 months after surgery; and 1 died due to systemic infection 24 months after surgery . Recipients had a 1-year survival rate of 90.5%, a 3-year survival rate of 81.0%, and a 5-year survival rate of 81.0% (table 2). Eleven recipients died (table 3): 6 died due to fulminant hepatic failure and subsequent renal failure within 1 month of surgery, which is defined as a clinical syndrome developing as a result of massive necrosis of liver cells or following any other cause of sudden and severe impairment of hepatic function occurring in patients without pre - existing or at least well - compensated liver disease; 1 died due to primary graft dysfunction; 1 died due to portal vein thrombosis; and 1 died due to bleeding of an inferior vena cava aneurysm . Another 2 patients died due to tumor recurrence during long - term follow - up . Recipients had a 1-year survival rate of 60.0%, a 3-year survival rate of 56.0%, and a 5-year survival rate of 56.0% (table 2). Kaplan - meier survival analysis showed that slkt recipients had a higher survival rate than that of lt recipients (p=0.025), particularly at 1 year post - transplant (figure 1). Seven cases of hepatic allograft rejection occurred among the slkt recipients; 4 developed rejection within 1 month of surgery, and the others developed rejection at 12 months, 18 months, and 37 months after surgery, respectively . Four lt recipients developed allograft rejection (table 2): 2 cases of allograft rejection occurred within 1 month of surgery, while the other 2 cases occurred at 6 months and 12 months of surgery, respectively . Univariate analyses revealed that slkt recipients had a higher but insignificant incidence of infection at an early postoperative stage, compared with lt recipients . Slkt recipients had significantly longer stay in the intensive care unit (icu) and longer total length of hospital stay, compared with lt recipients . On the other hand, lt recipients demonstrated higher incidence of renal failure and higher mortality at an early postoperative stage compared to slkt recipients (p=0.005) (table 2). Slkt recipients and lt recipients demonstrated sero - clearance of hbsag within 3 months after surgery . However, slkt recipients experienced a significantly higher rate of hepatitis b relapse (38.1%, p=0.007) compared with lt recipients . Steroid therapy for other subjects was discontinued, and nucleoside analogues coupled with high - dose hbig (hbig 20 000 u / d14d) were given . Following the treatment, the patients had serologic hepatitis b virus (hbv) dna test results that showed undetectable levels of hbv (table 4). Risk factors for high mortality (32%) at an early stage after lt were analyzed using univariate and multivariate analyses . According to univariate analyses (table 5), patients who died at an early stage after surgery had significantly higher preoperative serum creatinine levels (239.5107.7 mol / l) than survivors (146.021.6 mol / l), and had significantly lower egfr levels (30.812.7 ml / min/1.73 m) than survivors (47.18.7 ml / min/1.73 m). The early death recipients after lt had higher rifle stage than survivors (75% vs. 11.8, p=0.004). Compared with survivors, patients who died during the study had significantly higher preoperative meld scores and child - pugh scores . In addition, a higher incidence rate of postoperative renal failure (24%) was associated with mortality compared with that of survivors (4%, p=0.001). Multivariate analysis revealed that postoperative renal failure (p=0.003; or 48; 95%ci: 3.65631.76) and higher rifle stage (p=0.012, or 8; 95%ci: 1.5638.22) were independent risk factors for mortality after liver transplant (table 6). Meld scores, child - pugh scores, and incidence of preoperative dialysis were not significantly different between slkt and lt recipients . However, slkt recipients had lower egfr (p=0.003) and higher preoperative serum creatinine levels (p=0.004), lower serum ast levels, and lower total serum bilirubin levels than lt recipients (table 2). Four slkt recipients died: 2 died due to hepatitis b relapse and graft - versus - host disease within 2 months of surgery; 1 discontinued lamivudine against medical advice and died due to hepatitis b relapse and subsequent acute liver failure 14 months after surgery; and 1 died due to systemic infection 24 months after surgery . Recipients had a 1-year survival rate of 90.5%, a 3-year survival rate of 81.0%, and a 5-year survival rate of 81.0% (table 2). Eleven recipients died (table 3): 6 died due to fulminant hepatic failure and subsequent renal failure within 1 month of surgery, which is defined as a clinical syndrome developing as a result of massive necrosis of liver cells or following any other cause of sudden and severe impairment of hepatic function occurring in patients without pre - existing or at least well - compensated liver disease; 1 died due to primary graft dysfunction; 1 died due to portal vein thrombosis; and 1 died due to bleeding of an inferior vena cava aneurysm . Another 2 patients died due to tumor recurrence during long - term follow - up . Recipients had a 1-year survival rate of 60.0%, a 3-year survival rate of 56.0%, and a 5-year survival rate of 56.0% (table 2). Kaplan - meier survival analysis showed that slkt recipients had a higher survival rate than that of lt recipients (p=0.025), particularly at 1 year post - transplant (figure 1). Seven cases of hepatic allograft rejection occurred among the slkt recipients; 4 developed rejection within 1 month of surgery, and the others developed rejection at 12 months, 18 months, and 37 months after surgery, respectively . Four lt recipients developed allograft rejection (table 2): 2 cases of allograft rejection occurred within 1 month of surgery, while the other 2 cases occurred at 6 months and 12 months of surgery, respectively . Univariate analyses revealed that slkt recipients had a higher but insignificant incidence of infection at an early postoperative stage, compared with lt recipients . Slkt recipients had significantly longer stay in the intensive care unit (icu) and longer total length of hospital stay, compared with lt recipients . On the other hand, lt recipients demonstrated higher incidence of renal failure and higher mortality at an early postoperative stage compared to slkt recipients (p=0.005) (table 2). Slkt recipients and lt recipients demonstrated sero - clearance of hbsag within 3 months after surgery . However, slkt recipients experienced a significantly higher rate of hepatitis b relapse (38.1%, p=0.007) compared with lt recipients . Steroid therapy for other subjects was discontinued, and nucleoside analogues coupled with high - dose hbig (hbig 20 000 u / d14d) were given . Following the treatment, the patients had serologic hepatitis b virus (hbv) dna test results that showed undetectable levels of hbv (table 4). Risk factors for high mortality (32%) at an early stage after lt were analyzed using univariate and multivariate analyses . According to univariate analyses (table 5), patients who died at an early stage after surgery had significantly higher preoperative serum creatinine levels (239.5107.7 mol / l) than survivors (146.021.6 mol / l), and had significantly lower egfr levels (30.812.7 ml / min/1.73 m) than survivors (47.18.7 ml / min/1.73 m). The early death recipients after lt had higher rifle stage than survivors (75% vs. 11.8, p=0.004). Compared with survivors, patients who died during the study had significantly higher preoperative meld scores and child - pugh scores . In addition, a higher incidence rate of postoperative renal failure (24%) was associated with mortality compared with that of survivors (4%, p=0.001). Multivariate analysis revealed that postoperative renal failure (p=0.003; or 48; 95%ci: 3.65631.76) and higher rifle stage (p=0.012, or 8; 95%ci: 1.5638.22) were independent risk factors for mortality after liver transplant (table 6). The effectiveness of slkt in patients with hepatitis viral infection had not been thoroughly examined previously . In western countries, hepatitis c virus is the primary cause of end - stage liver disease and subsequently renal impairment . Hepatitis c carriers who received slkt have demonstrated a 5-year survival rate of 68% . However, in asian countries, including china, most liver transplant recipients are hepatitis b virus carriers, in whom the outcome of slkt is less understood . This study reported the largest number of hepatitis b patients receiving slkt, in which the 1-, 3-, and 5-year survival rates were 90.5%, 81.0%, and 81.0%, respectively . Singal et al . Have reported that slkt for hepatitis c, non - alcoholic steatosis and hepatocellular cancer could be associated with worst outcomes compared with other slkt indications; however, they did not include patients with hepatitis b, which is the most common indication for liver transplantation due to liver failure in china . Slkt recipients had significantly higher 1-year survival rate (90.5%), compared with lt recipients with hepatitis b infection and renal failure . This finding suggested that slkt was effective in improving the survival rate of transplant recipients with renal insufficiency . Compared with lt alone, slkt was associated with significantly longer icu stay, longer total hospital stay, and higher incidence of postoperative early - stage infection (38.1% vs. 20%), which might be related to the greater surgical trauma and higher incidence of complications . In addition, the healthcare system in china requires that the patients are completely symptom - free at discharge, which could explain, at least in part, this longer hospital stay . The incidence of acute allograft rejection between slkt recipients (33.3%) and lt recipients (16%) did not reach statistical significance, suggesting that slkt failed to decrease the incidence of acute hepatic allograft rejection . These results are comparable with previous studies, but this comparison should be made with caution since these previous studies evaluated patients without hepatitis b virus infection [12,1416]. High mortality was observed in the lt group, which could be attributable to the fact that these patients were with kidney dysfunction, as shown by the multivariate analysis . In addition, even if the meld score was similar between the 2 groups, creatinine and bilirubin levels were higher in the lt group, which could be another part of the reason for the high mortality observed in this group . In addition, some other articles have reported a mortality rate from fulminant hepatic failure as high as 90% after transplantation . A number of factors may also be associated with death after liver transplantation, including infections and rejection . A previous study showed that primary liver graft dysfunction could predict kidney failure, but the present study was not designed to address this relationship . Our study revealed that slkt was associated with a higher rate of hepatitis b recurrence than lt (38.1% vs. 4%). This finding might be due to the stronger and longer immunosuppressive therapy, particularly long - term steroids, administered to slkt recipients compared with steroid - free treatment for lt recipients . The use of steroids has been reported as a risk factor for hepatitis b relapse and for poor survival and hepatitis c relapse . Glucocorticoids can directly stimulate an enhancer region and a glucocorticoid response element in hepatitis b virus dna, thereby inducing viral transcription . Glucocorticoid administration also suppresses the immune response by reducing the hbv - specific cytotoxic t cell response, and therefore hbv - dna titer increases during glucocorticoid treatment . Alternatively, slkt recipients higher hepatitis b relapse rate might be related to the effect of double transplantation on recipients immune function . Several previous studies have indicated that hbv can influence the survival of hepatic and renal allografts and lead to fulminant hepatic failure and renal allograft dysfunction . However, 2 subjects in our study died due to hepatic failure; they discontinued anti - hbv medicine against medical advice and died due to fulminant hepatic failure . For the 6 slkt recipients with hepatitis b relapse, hormone therapy was discontinued and replaced by nucleoside analogues and high - dose hbig (20 000 u / d14d) immediately after the relapse . Therefore, slkt recipients with hbv infection should be advised of the risks for hepatitis b relapse . Reasonable treatment can achieve hbv - dna sero - clearance in patients with hepatitis b relapse without affecting long - term survival . Criteria for slkt for potential lt recipients with renal insufficiency are still unclear . According to the study by hanish et al . On transplant recipients with dialysis history, slkt generates better outcome than single kidney transplantation, liver transplantation followed by dialysis, or staged liver and kidney transplantation . Reached similar conclusions . In the present study, lt recipients with renal insufficiency had higher mortality at an early stage after surgery than slkt recipients (lt vs. slkt: 32% vs. 0%). Risk factors for death in lt recipients included lower egfr and higher rifle stage, preoperative serum creatinine levels, meld score, child - pugh score, and postoperative renal failure . Although gfr is considered the best estimate of renal function than serum creatinine levels, gfr was not an independent risk factor for postoperative deaths . Therefore, a gfr less than 30 ml / min is not enough to qualify for slkt . Postoperative renal failure (or=48) and rifle stage (or=8) among lt recipients were an independent risk factor for postoperative deaths . It is reported that rifle criteria has been shown to predict clinical outcomes with a progressive increase in mortality with worsening rifle class . Therefore, patients who might inevitably develop postoperative renal failure or be in need of dialysis should opt for slkt when possible . It is essential that patients with liver failure with worsening rifle class should be considered for slkt . Indeed, this was a retrospective study that suffers from all the limitations inherent to this type of study . Furthermore, these patients were from a single center, and the sample size was relatively small . The small number of death events in the slkt group (4/21) prevented a multivariate analysis in this group . Despite longer hospital stay and higher infection rate at an early stage after surgery, slkt demonstrated satisfactory long - term survival rates in patients with hepatitis b infection . Despite a higher rate of hepatitis b relapse following slkt, timely and reasonable treatments were able to prevent hepatitis b relapse from affecting long - term prognosis . Therefore, slkt can achieve good long - term outcomes in patients with hepatitis b, end - stage liver disease, and renal insufficiency . For lt recipients with renal failure, postoperative renal failure and rifle stage were independent risk factors for postoperative deaths.
Although dinoflagellates are best known as the notorious cause of toxic red tides, they are also important contributors to the ocean's primary production . Photosynthesis in these organisms is typically carried out in plastids surrounded by three membranes (1), an evolutionary footprint reflecting their origin through secondary endosymbiosis (2). The evolutionary ancestor of the peridinin - containing plastids is suggested from molecular phylogenetic reconstructions using plastid - encoded genes to be a red alga (3,4), a conclusion supported by phylogeny of nuclear - encoded plastid - directed genes (5). In general, these findings are also consistent with phylogenetic reconstructions of the host cells as determined from non - plastid - directed genes (6,7). Despite widespread acceptance of a red - algal origin for the peridinin - containing plastid, these organelles display a number of peculiar characteristics that share no homology with any known extant plastids . For example, the carotenoid peridinin itself (8) and the unusual light harvesting peridinin - chlorophyll a - protein to which it binds (9) are found in no other organisms . Furthermore, the rubisco in peridinin - containing plastids, an unusual form ii enzyme, is dissimilar from the form i protein employed by all other plastids (10). The evolutionary provenance of these proteins is thus unknown, and as they are derived from nuclear - encoded genes, it seems possible that their history may be distinct from that the plastid itself . An additional issue having bearing on plastid evolution, and one more likely to reflect the plastids themselves, is the number and arrangement of the genes within the genome . Genome architecture may be different from the phylogeny of the plastid genes themselves, and in the case of the dinoflagellates, quite remarkably so . Indeed, the only known plastid genes so far identified have been located on generally unigenic plasmid - like minicircles . These minicircles have been found in at least five genera of peridinin - containing dinoflagellates including amphidinium (11), ceratium (12), heterocapsa (13), protoceratium (14) and symbiodinium (15). Each minicircle has regions conserved within a species, and extensive pcr amplification of the genes located between these conserved regions has been performed in several species . Taken together, these studies have led to the conclusion that the known minicircle gene complement has reached saturation (16) with a total of sixteen protein encoding genes (atpa - b; petb and petd; psaa - b; psba - e and psbi; ycf16 and ycf24; rpl28 and rpl33) in addition to the large (23s) and small (16s) ribosomal rna . The identification of this highly reduced set of plastid genes as comprising the plastid genome is also supported by recent results demonstrating that at least some of the missing genes (i.e. Ones normally found in plastids) are instead nuclear - encoded in several species (1719). These experiments suggest that dinoflagellates do not obey for the rules normally governing plastid gene transfer to the nucleus (20). However, it is still a formal possibility that additional genes may be encoded in the plastid in a form different from the minicircle format found so far . One method potentially applicable to the characterization of the plastid genome is to determine the spectrum of genes expressed, as gene expression should be independent from the form in which the genes are found . However, it is generally difficult to discriminate between organelle- and nuclear - encoded transcripts, as both can be modified by addition of a poly(a) tail at their 3 termini (21). We have found that, unlike other transcripts in the dinoflagellates, those encoded by known minicircle genes carry a homogenous polyuridine tract at their 3 termini . We have taken advantage of this unusual feature to characterize the dinoflagellate plastid transcriptome, and find that our analysis fully supports a highly reduced plastid genome for the peridinin - containing dinoflagellates . Furthermore, as it seems likely that polyuridylylation may be common in dinoflagellate plastids, it may be possible to rapidly characterize the transcriptome of many different species using the method described here . Amphidinium carterae (ccmp 1314) and lingulodinium polyedrum (ccmp 1936, formerly gonyaulax polyedra) were obtained from the provasoli guillard culture center for marine phytoplancton (boothbay harbor, maine) and cultured as described (22). Poly(a) rna was purified from lingulodinium and amphidinium using oligo(dt) chromatography (23) and hybridized to a psba and 23s probes as described (22). The 16s probe was prepared against an est sequence from our library (see below), while the atpb probe was prepared using previously described amino acid microsequence data (22) to design two degenerate oligonucleotides 5-ttyticargciggiwsigargt-3 and 5-acytcigciacraaraaiggytg-3; the 500 bp pcr product was confirmed as atpb by sequence analysis . The 3 end sequences of atpb and psba transcripts were obtained from cdna synthesized from poly(a) rna tailed in vitro with rgtp by poly(a) polymerase . Specific sequences were amplified using a d(c)10 oligonucleotide and 5-tatccaattggacaaggaag-3 (lingulodinium psba) 5-gtgtagcacaagatgtaagc-3 (lingulodinium atpb), 5-gtattcggtcaagaggatg-3 (amphidinium psba) and 5-ctatctcagccgttctttg-3 (amphidinium atpb). The genomic psba sequences from lingulodinium were obtained using tail - pcr (24) using three nested internal psba oligonucleotides (5-gctgcttggccagttattggtatctg-3; 5-tctggtttacagcacttggtgttag-3; 5-gtccataattgattcatcaggtcatc-3) to amplify a fragment from at - rich dna purified by bisbenzimide - cscl gradients . To obtain the 3 end of genomic sequences from amphidinium, minicircle dna was amplified by inverse pcr using outwardly directed oligonucleotides 5-gtattcggtcaagaggatg-3 and 5-caggagcaaggaagaaag-3 (psba) or 5-gtggtcgtaagattgagagg-3 and 5-gatgagagcgttggcatac-3 (atpb). For cdna preparation, 10 g poly(a)-enriched rna was used as a template for first strand synthesis using a commercial kit (stratagene) but replacing the usual oligo(dt) primer with 5-(ga)10actagtctcgag(a)18 - 3. sequences were identified using the blast algorithm () and have been deposited in genbank under the accession nos . Dq264844 through dq264867 . All sequence alignments and analyses were performed using macvector (accelrys) except for rna secondary predictions that were made using a web server (). Statistical analysis using rarefaction (25) to determine the likelihood that our sample size was sufficient to detect all different cdnas in the library was made on a web server (). We began our analysis of the dinoflagellate plastid transcriptome by fractionation of rna into poly(a)-enriched and depleted fractions by oligo(dt) chromatography . The plastid - encoded psba (22) in rna extracted from the dinoflagellates lingulodinium (figure 1a) and amphidinium (figure 1b), was found enriched by 10-fold in poly(a) fractions, while 23s rrna remained in the poly(a) fraction . The small size of the 23s rrna signal is suggestive of processing and has been previously observed in dinoflagellates (13). The 16s rna was similarly processed, but the full - length form was found to a greater extent in the polyadenylated fraction than was the 23s rna . The abundance of these transcripts in a poly(a) rich fraction was unexpected, as usually only a small fraction of plastid messages are polyadenylated (21). Furthermore, it seemed at odds with the lack of minicircle genes found in dinoflagellate est libraries (1719). We originally thought that the tails might be heterogeneous, similar to the 3 termini in chloroplasts and cyanobacteria rna formed by polynucleotide phosphorylase (pnp) (21), since the presence of nucleotides other than adenine in the 3 tails might inhibit cdna synthesis more than oligo(dt) chromatography . To test this, we guanylated our poly(a)-enriched rna using poly(a) polymerase, and performed rt pcr with a primer pair allowing specific amplification of the psba 3 end (figure 2a). Fourteen different psba clones were sequenced, and all contained a homogeneous 3 terminal stretch of thymidine residues . At least five of the sequences obtained were independent clones based on differences in tail length, which varied between 25 and 40 thymidine residues (figure 2b). A comparison to the genomic dna sequence, obtained by thermal asymmetric interlaced (tail) pcr (24), shows that these residues are added post - transcriptionally, and all appeared to have been added at the same site (between arrows, figure 2b). Poly(t) tracts of similar length were found on the 3 termini of atpb transcripts from lingulodinium (data not shown) and on atpb transcripts from the dinoflagellate amphidinium (figure 2c). We propose that binding of these polyuridylylated mrnas to the longer and more prevalent poly(a) tails of nuclear transcripts is responsible for their presence in poly(a)-enriched rna fractions obtained by oligo(dt) cellulose chromatography . The unusual 3 terminal polyuridylylation, if a common feature of dinoflagellate plastid transcripts, suggested that analysis of cdna synthesized using an oligo(da) instead of the usual oligo(dt) primer would provide a straightforward method to catalog the plastid gene complement . Roughly 60 ng of cdna was synthesized from 10 g poly(a)-enriched rna, a yield 30-fold less than that obtained with an oligo(dt) primer in similar experiments . Polyuridylylated transcripts are thus a small but significant proportion of cellular rna . To characterize the library, several hundred clones were selected at random and sequenced . Some gc - rich and unidentified sequences were found, but none were polyuridylylated and were presumably derived from hairpin priming of the predominant gc - rich nuclear - encoded transcripts . In contrast, all polyuridylylated sequences were at - rich and were identified as transcripts from the minicircle genes found in other dinoflagellates (16) (table 1). The majority of the transcripts correspond to photosystem ii components (psba - d) and 16s rna . This latter may appear more frequently in our oligo(da)-primed library than the 23s rna because the full - length16s rna appears more abundant in a poly(a) enriched sample (figure 1a). To assess the likelihood that other low abundance polyuridylylated transcripts might be present in the library, we performed rarefaction analysis (25). Originally developed to compare species richness in biodiversity collections of different sizes, this technique estimates the number of different cdnas that would be obtained if smaller sample sizes were taken . The analysis of the data in table 1 indicates that the identification of twelve different sequences could have been possible with only 120 random clones sequenced; the three hundred clones reported here represent a significant excess of this minimum value . The calculations can also be used to illustrate the progression in the number of random sequences required to reveal each additional sequence: while 10 different cdnas are expected in 65 random sequences, almost twice as many sequences are required to uncover the eleventh sequence, and roughly six times more sequences to yield the twelfth cdna (figure 3 inset). This analysis suggests that to recover a potential new plastid transcript, well over a thousand random clones would have to be sequenced . To further test the contention the twelve genes recovered are likely to represent saturation coverage of the oligo(da), roughly a thousand different cdna clones were randomly selected and streaked on a new petri plate in a grid configuration . Colony lifts were then hybridized with a probe prepared against a mixture of psba - d or 16s sequences . From this, 50 cdnas that hybridized weakly or not at were selected and sequenced . Only 14 sequences among the 50 sequenced were at - rich and polyuridylylated, and these included two atpa, four 23s rna, a petb, three psbb, three psbc and a psbd . These later three photosystem ii components may have been recovered following the virtual subtraction because of poor bacterial growth or poor transfer to the membrane . More importantly, no new polyuridylylated genes were identified, again suggesting that our coverage of the library had reached saturation . As it seems unlikely that alternative 3 modifications might be found within the same organelle, we conclude that the genome of this photosynthetically active chloroplast is likely to encode only the transcripts identified here . To address the mechanism underlying site selection for poly(u) addition secondary structure is the principal determinant of polyadenylation site selection in prokaryotes (27). However, computer generated secondary structure predictions from rna complementary to the genomic sequence surrounding the polyuridylylation site of either lingulodinium psba or amphidinium atpb did not show any reasonably stable stem loop structures (data not shown). However, two loosely conserved primary structure motifs are located within 50 bp of the modification site in all twelve transcripts (figure 4). These motifs (agaaa and aauua) might thus constitute primary structure elements signalling the polyuridylylation site in a manner similar to the use of aauaaa in determining polyadenylation sites in nuclear encoded transcripts (28). We were also curious about the identity of the enzyme that might be used to catalyze the polyuridylylation reaction . As polyuridylylation of protein coding transcripts has been observed in organelles undergoing extensive uridine insertion editing (29,30), we thus checked for uridine insertion in comparisons of genomic and cdna sequences using lingulodinium psba, petb and 16s rna (2.5 kb total sequence). Our data reveals no evidence for uridine insertion although numerous examples of substitutional editing in dinoflagellate plastid transcripts (principally a to g) were observed (table 2, supplementary figures s1 and s2). These results agree with a previous report of substitutional editing in the dinoflagellate ceratium (31). We have found that the plastid transcripts in two species of peridinin - containing dinoflagellates are characterized by an unusual 3 polyuridylylation . This modification differs dramatically from the poly(a) tails of nuclear - encoded transcripts, and so provides a facile method for cataloging the plastid transcriptome . We report here that an oligo(da)-primed cdna library has a remarkably low complexity, with only 12 different clones identified in 300 randomly selected polyuridylylated sequences (table 1). Interestingly, all the sequences identified from the library were previously identified as minicircle genes in other dinoflagellate species . This concordance between two independent methods (characterization of the minicircular genome and the transcriptome analysis reported here) suggests that the minicircular gene format is likely to be the only genome architecture in the plastid . Our results thus strongly support the contention that the dinoflagellate genome is the smallest of any functional chloroplast . Furthermore, since the dinoflagellate amphidinium also contains polyuridylylated transcripts, it is possible that the technique described here could be used to catalog the plastid transcriptome from a range of other species . Is it likely that the transcriptome contains other low abundance transcripts that were undetected in our relatively small sample size? The chance of finding a specific transcript in a single random sample is a function of its relative proportion, or frequency of occurrence within the bank . However, if many other sequences were present, then the chance of finding any other sequence would also depend on the number of additional sequences present and on their relative proportions within the library . To estimate our coverage of the library, we used rarefaction analysis to determine if smaller sample sizes would also have recovered the same twelve genes . Our analysis suggests that it is likely the same twelve genes would have been recovered using a smaller sample size, indicating that the number of clones sequence was in excess of that required . Furthermore, the progression in the number of clones sequenced as a function of the number of different clones identified (figure 3) suggests that well over a thousand random clones would have to be sequenced to find an additional clone if it were indeed present in the library . Taken together with the virtual subtraction of psba - d and 16s rna sequences, these results strongly suggest that coverage of the library has reached saturation . The selective forces that serve to maintain genes in the chloroplast are hotly debated, and thought to reflect either difficulties in targeting or transport of proteins that are extremely hydrophobic or a relationship between control of gene expression and the redox state of the organelle (32). With respect to the former, the twelve protein encoding genes found in our library are not the most hydrophobic of known thylakoid proteins . Indeed, a better explanation for the retention of this particular gene set in the plastid may lie in the length of the protein rather than hydrophobicity . As recently shown by analysis of arabidopsis thylakoid proteins (33), the proteins encoded by the dinoflagellate plastid genes are generally the longest of the thylakoid proteins . However, even a combination of length and hydrophobicity is insufficient to explain transfer of some genes to the nucleus, such as the shorter yet relatively hydrophobic protein encoded by the atpi gene recently reported in an oligo(dt) primed est bank from the dinoflagellate alexandrium . If instead of hydrophobicity, the redox control of gene expression were the determining factor, this would suggest the set of proteins encoded by the dinoflagellate plastids are most sensitive to changes in redox potential . The proposal that it is genes requiring editing may that are conserved in plastids (34) is supported by analysis of the petb gene of lingulodinium, as editing removes a stop codon in the middle of the derived protein sequence (supplementary figure s2). With respect to the unusual nature of the modification itself, there is precedent for polyuridylylation in organelles experiencing extensive editing, such as the mitochondria of trypanosomes or myxomycetes . In some cases, 3 polyuridine tails have been observed not only for the guide rnas used to determine the site of uridine insertion but for rrna and mrna as well (29,30). However, a comparison of 2.5 kb genomic and cdna sequences showed only substitutional editing (table 2). Our experiments thus provide the first example of extensive polyuridylylation occurring in the absence of rna editing . It is tempting to speculate that the poly(u) tracts may result from a terminal uridylyltransferase (tut) (35) acting in the absence of guide rna to define a site for uridine insertion . However, an alternative possibility is that the plastids may contain a type poly(a) polymerase with specificity for uridine residues instead of adenine . Recently, it has also been shown that microrna - directed cleavage can result in addition of non - encoded oligonucleotides (mostly uridine) to 3 termini (36). However, in this case the polyuridylylated transcripts are intermediates in an rna degradation pathway . It seems unlikely that the extensive polyuridylylation of the plastid transcripts is used as a signal for rna turnover, since the majority of psba transcripts appear modified as judged by their co - purification on oligo(dt) chromatography . In contrast to the full - length protein coding transcripts, the small fragments hybridizing to 16s and 23s rna on northern analyses appear unmodified by these criteria (figure 1). Interestingly, the apparent stability of the polyuridylylated plastid transcripts thus suggests that this particular 3 end modification has a different function from that in either cyanobacteria or the plastids of higher plants, where transcripts are marked for degradation by 3 end polyadenylation (21). Despite extensive molecular phylogenetic studies pointing to a common evolutionary origin for both host cells (7) and plastids (37) of the chromalveolates, no other plastids or cyanobacteria thus the mechanism and function of the dinoflagellate plastid transcript 3 end modification are as unique as their form ii rubisco (10) and peridinin chlorophyll a - protein (9). The major challenge for plastid phylogeny underscored by our results is to reconcile the many unique features of the dinoflagellate plastids with their phylogenetic relationships to red algae . In addition, given the concurrence of several lines of evidence supporting the highly reduced nature of the dinoflagellate plastid genome, it will be of interest to reinvestigate the nature of the selective forces maintaining the current plastid genome size in higher plants . Dinoflagellate plastid messages are located in poly(a)-enriched rna . Total rna samples (t) from the dinoflagellates lingulodinium (a) and amphidinium (b) were resolved into fractions enriched (a+) and depleted (a) for poly(a) rna by chromatography on oligo(dt) cellulose . Rna blots were challenged with gene probes for either psba, 23s rna or 16s . (a) lingulodinium rna samples enriched for poly(a) rna were tailed with guanine residues, and transcript 3 end sequences amplified using a specific internal oligonucleotide and an oligo(dc) oligonucleotide . (b) sequences of the 3 end of fourteen psba cdnas yielded five clones with different numbers of thymidine residues . The polyuridylylation site is defined to the stretch of thymidine residues encoded by the genomic sequence (arrows). (c) amphidinium rna samples were treated similarly except that atpb specific primers were used in the amplification . Estimates of the number of different sequences expected for different numbers of random clones sequenced were made by rarefaction analysis of the data in table 1 . The estimated number of different cdna sequences expected with smaller sample sizes shows a statistical possibility that the twelve different clones could have been identified with only 120 different sequences . The progression of the number of random sequences as a function of the number of different clones (inset) suggests over a thousand sequences would be required to identify any potentially new clone in the library . All different polyuridylylated 3 terminal sequences corresponding to sequences identified in the library were aligned at the site of the poly(u) modification and at each of two potential conserved sequences (underlined). Only transcripts from known minicircle genes are polyuridylylated in lingulodinium various patterns of substitutional editing are found in dinoflagellate plastid transcripts
Several prospective, randomized, controlled clinical trials regarding cervical artificial disc replacement (c - adr) have been published . However, the number of c - adr patients with long - term follow - up for more than 4 years is still sparse . The purpose of this systematic review is to provide a summary of the available literature reporting long - term follow - up of c - adr and to elucidate whether the favorable outcomes seen in the short - term continue after 4 to 5 years . Does single - level unconstrained, semiconstrained, or fully constrained c - adr improve health outcomes compared with single - level acdf in the long - term? Search: pubmed, cochrane collaboration database, and national guideline clearinghouse databases; bibliographies of key articles . Inclusion criteria: (1) fda trials comparing c - adr with anterior cervical discectomy and fusion (acdf); (2) follow - up 4 years . Exclusion criteria: (1) non - fda trials comparing c - adr with acdf; (2) follow - up <4 years . Outcomes: neck disability index (ndi), pain in the neck and arm (visual analog scale [vas]), quality of life (sf-36 physical component score [pcs]), adjacent segment disease (asd), neurological success, subsequent surgeries, and complications . Study design: systematic review . Search: pubmed, cochrane collaboration database, and national guideline clearinghouse databases; bibliographies of key articles . Inclusion criteria: (1) fda trials comparing c - adr with anterior cervical discectomy and fusion (acdf); (2) follow - up 4 years . Exclusion criteria: (1) non - fda trials comparing c - adr with acdf; (2) follow - up <4 years . Outcomes: neck disability index (ndi), pain in the neck and arm (visual analog scale [vas]), quality of life (sf-36 physical component score [pcs]), adjacent segment disease (asd), neurological success, subsequent surgeries, and complications . Two randomized, multicenter fda trials comparing outcomes following c - adr and acdf with follow - up> 48 months were found (fig . 1). Inclusion and exclusion criteria and demographic information for each study are listed in table 1 . Overall, a total of 1004 adult patients (47% male) with a mean age of 44 years were included . All patients were diagnosed with single - level degenerative disc disease between c3 and c7 and had failed a minimum of 6 weeks conservative treatment . Function, pain, and health - related quality of life (table 2) at 48 months in the bryan trial, patients in the c - adr group showed significantly greater mean improvement in ndi, vas neck and arm pain, and sf-36 pcs measured at 48 months compared with patients in the acdf group . At 60 months in the prestige trial, only mean improvement in ndi scores was significantly greater in the c - adr group (38.4 vs 34.1, p = .022); however, for the remaining three outcomes, mean improvements were slighter greater following c - adr versus fusion . Success, asd, and return to work (table 3) at 48 months in the bryan trial, overall success and ndi success, were achieved in a significantly greater proportion of c - adr patients compared with acdf patients (p = .004 and .003, respectively). At both 48 months in the bryan trial and 60 months in the prestige trial, more patients achieved overall neurological success (maintenance or improvement) and were working following c - adr compared with acdf, although these differences were not significant . The rate of asd at 48 months in the bryan trial was identical between groups (4.1%); at 60 months in the prestige trial the rate was 2.9% in the c - adr group versus 4.9% in the acdf group, however these differences were not statistically significant . Both studies reported preserved segmental range of motion in the cervical spine following c - adr compared with acdf: 8.5 versus 1.1 (48 months, bryan) and 6.5 versus 0.4 (60 months, prestige). Subsequent operations (table 4) no significant differences between groups were reported for rates of revisions, hardware removal, supplemental fixation, use of bone growth stimulators, or reoperation at 48 months postoperatively in the bryan trial . At 60 months, a significant difference was seen between the prestige c - adr and acdf groups, respectively, in revisions (0% vs 1.9%; p = .028), supplemental fixation (0% vs 1.9%; p = .028), and the use of external bone growth stimulator (0% vs 2.6%; p = .007). The bryan study only reported more severe who grade 3 or 4 events that occurred after 24 months and up to 48 months follow - up in the c - adr and acdf groups, respectively: any, 24.3% vs 26.1%; severe arm and neck pain, 1.7% vs 3.6%; and new neurological deficits, 0% vs 1.4% . In patients with complete radiographic follow - up at 60 months in the prestige trial, subsidence (loss of> than 2 mm in functional spinal unit height) was seen in 2.8% and 1.4% of patients in the c - adr and acdf groups, respectively; bridging bone was reported in 3.2% of the c - adr patients . One guideline was found, published by the north american spine society (nass) in 2010, entitled diagnosis and treatment of cervical radiculopathy from degenerative disorders . Among the major recommendations listed were the following statements relevant to the topic of this review: acdf and total disc arthroplasty (tda) are suggested as comparable treatments, resulting in similarly successful short term outcomes, for single level degenerative cervical radiculopathy . (grade: b; fair evidence level ii or iii studies) surgery is an option for the treatment of single level degenerative cervical radiculopathy to produce and maintain favourable long term (> 4 years) outcomes . (grade c; poor quality evidence level iv or v studies) results of literature search . Adults> 18 years of age single - level symptomatic ddd between c3 - 7 intractable radiculopathy, myelopathy or both vas neck pain scores 20 preserved motion at the symptomatic level found in all included patients unresponsive to 6 weeks conservative treatment or progressive neurological worsening despite conservative treatment no previous procedures at the operative level negative for several radiographic findings, medications, and diagnoses multilevel symptomatic ddd or evidence of cervical instability sagittal plane translation of> 3.5 mm or sagittal plane angulation of> 20 at a single level symptomatic c2-c3 or c7-t1 disc disease previous surgery at the involved level severe facet joint disease at the involved level medical condition that required long - term use of medication, such as steroid or nonsteroidal antiinflammatory drugs that could affect bone quality and fusion rates ddd at single level between c3 and c7 disc herniation with radiculopathy, spondylotic radiculopathy, disc herniation with myelopathy, or spondylotic myelopathy 6 weeks minimum unsuccessful conservative unless myelopathy requiring immediate treatment ct, myelography and ct, and/or mri demonstration of need for surgical treatment preoperative ndi 30 and minimum one clinical sign associated with level to be treated willing to sign informed consent and comply with protocol significant cervical anatomical deformity moderate to advanced spondylosis any combination of bridging osteophytes, marked reduction, or absence of motion collapse of intervertebral disc space of> 50% normal height, radiographic signs of subluxation> 3.5 mm, angulation of disc space> 11 greater than adjacent segments, significant kyphotic deformity or reversal or lordosis axial neck pain as solitary symptom previous cervical spine surgery metabolic bone disease active systemic infection or infection at operative site known allergy to components of titanium, polyurethane, ethylene oxide residuals concomitant conditions requiring steroid treatment daily insulin management medical condition which may interfere with postoperative management program or may result in death before study completion current or recent alcohol and/or drug abuser signs of being geographically unstable fda indicates us food and drug administration; ddd, degenerative disc disease; ndi, neck disability index; vas, visual analog scale; ct, computed tomography; and mri, magnetic resonance scan . C - adr indicates cervical artificial disc replacement; fda, us food and drug administration; ndi, neck disability index; sf-36, short - form 36 questionnaire; and pcs, physical component score . P values compare the mean improvement in scores from baseline to each follow - up time - point between c - adr and fusion . Asd indicates adjacent segment disease; c - adr, cervical artificial disc replacement; fda, us food and drug administration; ndi, neck disability index; and ns, not statistically significant . Composite measure in which patients had to achieve all the following: an improvement of 15 points on ndi, neurological improvement, no serious (who grade 3 or 4) adverse events related to the implant or surgical implantation procedure, and no subsequent surgery or intervention that would be classified as treatment failure . Improvement of 15 points in ndi from baseline . Defined as maintenance or improvement of all three neurological parameters (motor and sensory function, and reflexes). C - adr indicates cervical artificial disc replacement; fda, us food and drug administration; and ns, not statistically significant . A 43-year - old woman presented with cervical myelo - radiculopathy that did not respond to medical treatment for 6 weeks . The magnetic resonance images demonstrated a large herniated disc at the level of c5/6, eccentric to the right side (fig . Her symptoms improved significantly after surgery and x - rays taken 2 years postoperatively demonstrated very good range of motion at the index level (fig . Her vas arm pain score improved from a preoperative score of 8 to a postoperative score of 1 at 2-year follow - up . Preoperative axial (a) and sagittal (b) magnetic resonance images of a 43-year - old woman with myeloradiculopathy due to a c5/6 disc herniation . Postoperative flexion (a) and extension (b) x - rays of the patient 24 months after surgery . Limitations: a small number of studies with long - term data comparing c - adr with cervical acdf were available . In both prospective studies, the c - adr cohort maintained statistical improvement in validated clinical outcome measurements at 48 and 60 months and preserved segmental motion at the operated level . Rates of revision and supplemental fixation surgeries were lower in the c - adr group . These studies demonstrate the durability of the c - adr procedure; however, it may still be too early to detect implant - related failures . Future studies should examine issues such as wear - related failures, device fatigue, or delayed spinal instability . The follow - up rates were 68.7% (318/463) in the bryan disc study at 4 years and 50.1% (271/541) in the prestige st study at 5 years . These high rates of lost to follow - up may alter the study results if those patients lost to follow - up had late - onset clinical or radiographic issues . Significantly greater mean improvement from baseline in all outcomes in the bryan c - adr group compared with the acdf group at 48 months . Only the ndi showed greater mean improvement from baseline in the prestige c - adr group compared with the acdf group at 60 months . Overall success and ndi success, which were only reported by the bryan trial, were achieved in a significantly greater proportion of c - adr patients compared with acdf patients at 48 months . At both 48 (bryan) and 60 months (prestige) the rates of asd at 48 (bryan) and 60 months (prestige) were not statistically different between treatment groups . At both 48 (bryan) and 60 months (prestige), more patients were working following c - adr compared with acdf . Significantly fewer revisions, supplemental fixations, and use of external bone growth stimulators were reported in the prestige c - adr compared with the acdf group at 60 months . Rates of who grade 3 or 4 adverse events were similar between groups at 48 months in the bryan trial . Cervical arthroplasty is a viable treatment option for cervical herniated disc / spondylosis with radiculopathy . The inclusion / exclusion criteria of us fda trials should be followed . C - adr achieves neural decompression and preserves normal segmental motion at the operated level at 4 to 5 years follow - up . Adjacent level degeneration may be decreased with arthroplasty versus acdf, but further study is warranted on this topic.
Zeolite is a natural porous mineral described as hydrated aluminosilicates containing exchangeable alkali and earth alkaline cations (na, k, ca, or mg) and could be synthesised from the aluminium silicate materials as clay minerals and pumice by alkali attack [13]. Zeolites have a large surface area, microporous structure, and high affinity for metal ions, providing an efficient scavenging pathway for heavy metals in toxic systems [4, 5]. Some of the attractive applications of natural zeolites are listed by taffarel and rubio and babel and kurniawan . Most of these research efforts are undertaken on laboratory scale and are at early stages of investigations to develop the synthesis methods . Synthetic zeolites are carried out in many industrialized countries in europe, east asia, and north america . Na - p1 zeolite is one of the synthetic zeolites which are a class of highly porous materials . The unique structural features of these crystalline microporous solids that contain pores and cavities in the order of molecular dimensions (310) are the main reason for their application in the realms of catalysis, separation, purification, ion exchange, and radioactive waste clean - up . More novel applications for na - p1 zeolites are expected in electrochemistry, photochemistry, pharmaceutical, engineering, membrane science, and nanotechnology as their structures are expanded and more suitably engineered in a foreseeable future . It is well established that these materials that are equivalently called molecular sieves are mainly hydrated aluminosilicates . Zeolite na - p1 contains two - dimensional pore system with two intersecting 8-ring channels . A synthetic zeolite na - p1 has similar pore structure to the natural zeolite phillipsite . Na - p1 zeolite is classified as high silica zeolites where it has si / al ratio equal to or greater than about 3 . It is considered as a hydrated sodium aluminosilicate zeolite that could be synthesised from the naturally occurring alkaline volcano with naoh attack of varying concentrations at different volcanic temperatures . Pumice is a natural rock of volcanic origin, formed by gases released during the solidification of acidic lava . The vesicular structure of pumice is formed during the eruption of gases contained in the molten viscous lava on cooling . In egypt, pumice occasionally occurs in few locations, at the northern coast of the mediterranean sea at el - arish, north sinai . The heavy metals contamination of water by the discharge of industrial wastewater of electroplating, metallurgy, chemical manufacturing, mining, and battery manufacturing is a worldwide environmental problem . Adsorption process provides an attractive alternative treatment to other removal techniques because it is more economical and readily available [1012]. A good number of adsorbents have been used for cd(ii), fe(ii), cu(ii), and co(ii) removal from water . The adsorption of cd(ii) on red mud fitted langmuir isotherm model having a maximum adsorption capacity of 10.57 mg g . Wang used maize cobs for adsorption of fe(iii) from aqueous solution with the langmuir adsorption capacity and the freundlich adsorption capacity being 2.53 mg g and 0.104 l g, respectively . The maximum adsorption capacity for cu(ii) on red mud gupta et al . Used spirogyra species for removal of cu(ii) with the maximum adsorption capacity of 133.3 mg g. use of adsorbents like mg pellets has also been reported for co(ii) removal from water and the adsorption capacity has been found to be 15.8 mg g. the aim of this work is to study the best condition of temperature, sodium hydroxide concentration, and time required for synthesis of na - p1 zeolite from the local pumice raw material and utilize the synthetic zeolite as an adsorbent for removing (cd, fe, cu, and co) ions from synthetic solutions in a batch laboratory system . Also, the effect of experimental conditions such as contact time and initial zeolite dose was described . The main material used in this work is pumice grains received from el - nile mining company from the mediterranean sea coast at el - arish province, north sinai, egypt . In addition, commercial naoh is used for alkaline hydrothermal attack for synthesis of zeolite from pumice . Hydrated chloride salts of cu, co, fe, and cd of analytical grade (ar) supplied by merck as analytical - grade reagents and deionized water are used . All adsorption experiments were carried out using an aqueous solution of cucl22h2o, cocl26h2o, fecl2, and cdcl2h2o . The stock solution was prepared by dissolving accurately weighted salt in distilled water to the concentration of 500 mg / l . The initial concentrations of solution were done by diluting the stock solutions in appropriate proportions to different initial concentrations . Na - p1 has been prepared from pumice, which is naturally occurring alkaline volcanic rock with naoh attack . A series of experiments were conducted to synthesis na - p1 zeolite from locally produced pumice raw material at temperatures (80 to 120c), naoh solution concentrations (13 molar), and crystallization periods (12 h, one week, and up to one month . Pumice powder was mixed with naoh solution in a well - sealed teflon cylindrical vessel, heated, and stirred for 12 hours at 80c, and then the vessel was kept in the dryer at 8090c for one week . The produced gel was filtered and washed well till becoming free from sodium hydroxide; the wet powder was dried for 24 h in the dryer at 110c . The optimum conditions required for synthesis of na - p1 zeolite from locally produced pumice raw material are one molar naoh concentration, temperature at 80c, and one week as a crystallization time . Several investigations for the adsorption of the heavy metal were carried out, including effect of contact zeolite dose, effect of time, and effect of metals concentration . The physical and chemical characteristics of prepared adsorbents such as slurry ph, point of zero charge (phpzc), bulk density, and moisture were performed to determine the capability of prepared adsorbents in removing the metals co, cu, fe, and cd from an aqueous media . For ph determination, 0.2 g of the dry, grinded adsorbent was mixed with 25 ml of distilled water and allowed to boil for 30 min in stoppered glass bottle . After cooling, the ph of the adsorbent was measured using a digital ph meter (hanna, model ph20), allowing 5 min for the ph probe to equilibrate . Also, the point of zero charge (pzc) for the samples was determined by the following procedure: 200 ml of deionized water was added to an erlenmeyer flask, which was then capped with stoppered glass . The deionized water was heated until boiling for 20 min to eliminate the co2 dissolved in the water . The co2-free water was cooled down as soon as possible and the flask was immediately capped . On the other hand, 0.5 g of each sample was weighed and placed in a 25 ml erlenmeyer flask to which 20 ml of co2-free water was added . The flask was sealed with a rubber stopper and left in continuous agitation for 72 h at 25c . Then the solution ph was measured and this value is the point of zero charge . The bulk density was estimated by a standard procedure by weighing a known volume of gently tapped adsorbent granules . The apparent density was calculated from the volume of the graduated cylinder closely packed with the powdered sample and from the sample weight in triplicate . For moisture measurement, about 0.5 g of powdered air - dried adsorbent sample is weighed and taken in a crucible . The crucible is placed inside an electric hot - air oven and heated at 150c for 3 hours . It is then taken out, cooled in a desiccator, and weighed . From this, the percentage of moisture can be calculated as follows: percentage of moisture = (loss in weight of adsorbent / weight of air - dried adsorbent taken) 100 . Surface characteristics of prepared zeolite sample were characterized by scanning electron microscope (sem) using sem model philips xl 30 attached with edx unit, with accelerating voltage 30 k.v ., magnification 10x up to 400.000x, and resolution for w. (3.5 nm). X - ray powder diffractograms, xrd, of the samples were recorded using a bruker diffractometer (bruker d8 advance target) and the scan rate was fixed at 8 in 2 min for phase identification . The patterns were run with cu k and secondly with monochromator (= 0.1545 nm) at 40 kv and 40 ma . Mineralogical and chemical study of the pumice were done by using x - ray fluorescence spectrometer (xrf) to identify the chemical composition . Xrf, a spectrometer with wavelength dispersion pw 2400 philips, and centrifuge hermle were used . Xrf shows that quartz, cristobalite, and albite are the main constituents in the pumice (figure 2). Sio2, al2o3, and na2o are the main oxides in the pumice under investigation (93.69%) confirming the mineralogical constituents, whereas tio2, mno, mgo, fe2o3, and cao analyzed in the pumice under are calculated as 3.88% (table 1). The concentrations of heavy metals in all samples were determined according to apha using atomic absorption spectrometer unicam model 939 with graphite furnace accessory, equipped with deuterium arc background corrector . Precision of the metal measurement was determined by analyzing (in triplicate) the metal concentration of all samples and for each series of measurements an absorption calibration curve was constructed, composed of a blank and three or more standards . Accuracy and precision of the metals measurement were confirmed using external reference standards from merck . The adsorption isotherm shows the equilibrium relationship of concentration in the adsorbate - adsorbent system at constant temperature . Such adsorption isotherms may be used for scaling up batch type processes with moderate success . The shape of the equilibrium adsorption isotherm provides us with information about the adsorbent surface whether it is homogeneous or heterogeneous . Also, its study is helpful in evaluating the maximum adsorption capacity of adsorbate for the given adsorbent . For adsorption isotherms models they differ in their assumption, shape of the isotherm, and nature of the adsorbent surface . Langmuir (1918) model assumes that uptake of metal ion occurs on a homogeneous surface by monolayer adsorption without any interaction between adsorbed ions . The model is also based on the assumption that all the sorption sites are energetically identical and sorption occurs on a structurally homogeneous sorbent . Thus, a linear plot of ce / qe versus ce confirms the validity of the langmuir giving correlation coefficients (r) close to unity, and kl is a constant related to the free energy of adsorption as expressed (kle). Deviations from the basic assumption of the langmuir model do limit interpretation of the values for qm and kl in terms of absolute surface areas and sorption free energies . The free energy of adsorption, g, can also be evaluated from the parameter kl according to the expression g = rtlnkl . Values of qm and kl are obtained from slope and intercept of ce / qe versus ce plot, respectively . A further analysis of the langmuir equation can be made on the basis of a dimensionless equilibrium parameter, rl (known as the separation factor which is considered as a more reliable indicator of adsorptions). This parameter (rl) can be expressed by weber and chakkravorti (1974): (2)rl=11+bci, where ci is the initial concentration metal ion (mg / l) and b (l mg) is the langmuir constant described above . Also, the influence of the isotherm shape on whether the adsorption is unfavorable at highest initial concentration of the dye, co, can be described by a term rl, a dimensionless constant separation factor in equation; calculated value for rl indicates the nature of adsorption process as given: irreversible (rl = 0), favorable (0 <rl <1), linear (rl = 1), and favorable (rl> 1). Freundlich isotherm is an empirical equation that can be described as the reversible adsorption onto heterogeneous surface at sites with different energy of adsorption and is not restricted to the formation of the monolayer of adsorbate . The nonlinear form of this model is expressed as (3)qe = kfce1/n, where kf is the freundlich constant (mg / g)/(l / mg)1/n and also referred to as adsorption capacity, while n is the heterogeneity factor and related to adsorption intensity . The parameter n gives an indication of the adsorption type whether is linear (n = 1) or a physical process (n> 1) is favorable, or a chemical process (n <1). On the other hand, the value of 1/n <1 or 1/n> 1 indicates a normal langmuir isotherm and cooperative adsorption, respectively . Freundlich model can be represented by linear form as follows: (4)lnqe = lnkf+1nlnce . A plot of lnqe versus lnce gives a straight line, where the values of kf and 1/n are determined from the intercept and the slope, respectively . The kinetic tests were carried out following the same procedure used for the equilibrium tests . Aqueous samples were taken at different intervals of time and the concentrations of adsorbent were measured at the same intervals . The amount of absorbed onto the developed adsorbents at time t (min), qt (mg / g) was calculated by means of the expression below: (5)qe=(c0ct)vm, where co and ct are the liquid - phase concentrations at an initial and predetermined time t (mg / l), respectively, v is the volume of solution (l), and m is the dry weight of the added adsorbent (g). The kinetic data were then carried out for the pseudo - first - order and pseudo - second - order . The rate constant of adsorption was determined from the pseudo - first - order equation: (6)logqeqt = logqek1t2.303, where qe and qt (mg / g) are the amounts of adsorbed (mg / g) at equilibrium and at time t (min), respectively, and kt is the adsorption rate constant (min). The pseudo - second - order equation based on the equilibrium adsorption is expressed as (7)tqt=1k2qe2+tqe, where k2 (g / mgmin) is the rate constant of second - order adsorption . Some physical and chemical features of pumice and na - zeolite p1 are listed in table 2 . It is evident that the treatment with naoh has a significant efficiency on the physicochemical properties of synthesized adsorbent na - zeolite p1 . The ph of the aqueous slurry and phpzc of adsorbents may give a good indication about the surface oxygen complexes and the electronic surface charges of adsorbents . The complexes on adsorbent surface are generally classified as acidic, basic, or neutral . Based on the slurry ph, the nature of surface oxygen groups on the support and the dominant complexes can be deduced . From table 2, it is clear that the ph is higher than the phpzc; the surface of the adsorbent is negatively charged, favoring the adsorption of cationic species (co, cu, fe, and cd). Bulk density of the pumice and na - zeolite p1 decreased from 0.85 to 0.50 mg / g and the moisture is 0% . This finding is due to the generation of porous structure and the increase in binding sites after chemical modification . Chemical and phase composition as well as microstructure of the pumice raw material have been studied . The data obtained from the xrf and xrd are discussed before . Table 1 illustrates that sio2 is the major component (~71.0%) with about 14.20% al2o3 as well as a total of about 12.4% fluxing oxides . Xrd pattern in figure 1 confirms the glassy phase in addition to minor crystalline quartz phase, which is the only phase . The amorphous nature of the silicate pumice as well as the high content of the pore size with variable distributions is ensured by the sem in figure 3(b). Phase composition and microstructure of the synthetic zeolite studied by xrd (figure 2) and sem reveal that the zeolite p1 is the only phase formed during these conditions of alkali treatment during the alkaline hydrothermal attack of locally produced pumice raw material at the optimum condition of synthesis . The micrograph of synthetic zeolite shows very fine agglomerated crystalline grains (gray) figure 3(a). Xrd analysis was used to prove the formation of na - zeolite p1 before and after treatment with naoh . The x - ray diffractogram of pumice shows that it was considered a glass because it has no crystal structure . Pumice is an unusually light rock due to the many bubbles inside it, figure 3(b). Pumice varies in density according to the thickness of the solid material between the bubbles; many samples float in water . It is formed by volcanic eruptions when molten lava is shot in the air with many bubbles of gas in it . The treatment with naoh leads to the formation of na - zeolite p1 which has higher degree of crystallinity, figure 3(a). Xrd analysis was used to prove the formation of na - zeolite p1 before and after treatment with naoh . Adsorption isotherm and kinetics of co, cu, fe, and cd on the synthetic zeolite were investigated using batch experiments . Equilibrium and kinetic studies were carried out on the removal of co, cu, fe, and cd from aqueous solution using synthetic zeolite p1 . Adsorption test on prepared adsorbent was studied using a batch process . For this purpose, the effects of adsorbents dose (0.11 g), contact time (18 min), and metal concentration (300700 mg / l) on the adsorption of co, cu, fe, and cd (25 ml of 100 for all the above - mentioned equilibrium and kinetic studies, the mixture of adsorbent and solution was magnetically stirred at 150 rpm for 30 min as determined from kinetic tests . Batch adsorption experiments were conducted by mixing 50 cm solutions of 500 ppm containing one metal of co, cu, fe, and cd with a constant dose of na - zeolite p1 (0.1 g, 0.25 g, 0.5 g, 0.75 g, or 1 g) in 100 cm pyrex erlenmeyer flasks with cap . Flasks were shaken (150 rpm) at 25 1c using a brunswick scientific g25kc incubator orbital shaker . It is observed that the percentage adsorption for co, cu, fe, and cd ions increases with increasing zeolite weight in aqueous solutions as illustrated in figure 4 . The maximal exchange levels attained were as follows: cd> fe> cu> co. the heavy metal uptake may be attributed to different mechanisms of ion - exchange processes as well as to the adsorption process [1820]. During the ion - exchange process, metal ions had to move not only through the pores of the na - zeolite p1 mass, but also through channels of the lattice, and they had to replace exchangeable cations (mainly sodium). Diffusion was faster through the pores and was retarded when the ions moved through the smaller diameter channels . In this case the metal ion uptake 6 could mainly be attributed to ion - exchange reactions in the microporous minerals of the na - zeolite p1 samples . By varying the contact time (1, 2, 4, 6, and 8 min) while keeping all other parameters constant with respect to ph, temperature, and dose of na - zeolite p1, 0.25 gm, the influence of contact time on the zeolite capacity for different metal ions is shown in figure 5 . Removal efficiency increases for cadmium from 95 to 98%, for copper from 71 to 76%, for iron from 64 to 76%, and for cobalt from 58 to 61% as the time increases from 1 to 8 min . Figure 6 represents the variation in metal ion adsorption with time; however the adsorption rate becomes slower with passage of time up to 4 min . The initial faster rate of removal of each metal ion may be due to the availability of the uncovered surface area of the adsorbents . The slight increase in the percentage of adsorption of each metal adsorbent is cd> fe> cu> co. this may be attributed to the difference in nature between metal ions with respect to na - zeolite p1 surface . That may be attributed to different mechanisms of ion - exchange processes as well as the adsorption process [1820]. The uptake of cd(ii), cu(ii), ni(ii), pb(ii), and zn(ii) onto na - zeolite p1 as a function of their concentrations was studied at 25 0.1c by varying the metal concentrations 300, 400, 500, and 700 mg / l while keeping all other parameters constant with respect to dose and time . For adsorption isotherms models were used in this work, langmuir and freundlich isotherms . They differ in their assumption, shape of the isotherm, and nature of the adsorbent surface . Langmuir (1918) isotherm which models the monolayer coverage of the sorbent surface assumes that sorption occurs at specific homogeneous sorption site . The model is also based on the assumption that all the sorption sites are energetically identical and sorption occurs on a structurally homogeneous sorbent [1820]. Figure 7 shows that the langmuir model effectively and significantly described the sorption data with all r values 0.96 . Table 3 shows the relation between the ionic radius, electronegativity, and langmuir constant . The maximum sorption capacity (kl) represents monolayer coverage of sorbent; according to the kl (mg / g) parameter, monolayer capacities of zeolite are arranged in the following sequence: cd> fe> cu> co. these metals seem to reach saturation, which means that the metal had clogged possible available sites in zeolite and further adsorption could take place only at new surfaces . The sequence of adsorption of several heavy metals are not correlated either with the sequence of ionic radii or with the sequence of electronegativity . There is, however, a parallel relation between the adsorption sequence and the hydrolysis properties of the heavy metal cations, as pointed out by several investigators [2224]. This isotherm developed by freundlich (1926) describes the equilibrium on heterogeneous surfaces and does not assume monolayer capacity . This isotherm gives an expression encompassing the surface heterogeneity and the exponential distribution of active sites and their energies . A linear plot is obtained when log ce is plotted against log qe over the entire concentration range of metal ions investigated and the values of kf (mg / g) and n can be calculated from the intercept and the slope of this straight line, respectively (figure 8). The arrangement of heavy metals according to dose required for the reduction of metal concentrations in freundlich equation is cd> fe> cu> co. the adsorption isotherm data were fitted well to the langmuir model from langmuir model equations . In order to investigate the adsorption kinetics of heavy metals on the prepared zeolite adsorbent the calculated parameters from pseudo - first - order and pseudo - second - order models (figures 9 and 10) are summarized in table 4 . According to the pseudo - first - order model, the values of k1 and qe were calculated from the slope and intercept from the plots of log (qe qt) versus t. it was found that the experimental values were not adequate with the calculated ones (table 4), indicating that the first - order model does not reproduce the adsorption kinetic of heavy metals on the prepared adsorbents . In case of the pseudo - second - order model, the calculated values of kinetic studies showed that the zeolite is a good potential as adsorbent for (co, cu, fe, and cd) ions follows the pseudo - second - order, and, hence, this model is more likely to predict the behavior over the whole experimental range of h m adsorption more than pseudo - first - order model . Also, the values of correlation coefficients (2) are close to unity for pseudo - second - order model rather than the pseudo - first - order one; that is, the kinetic removal of h m is quite described by pseudo - second - order model . The alkaline treatment of very cheap local pumice leads to the formation of na - zeolite p1 which has the high degree of crystallinity, the ph is higher than the phpzc, and the surface of the adsorbent is negatively charged, favoring the adsorption of cationic species (co, cu, fe, and cd). The adsorption kinetics study of the heavy metals followed a pseudo - second - order model . Both the langmuir and freundlich models fit equilibrium data well implying the existence of a monolayer adsorption and a heterogeneous surface existence in na - zeolite p1, showing a strong adsorption capacity for (co, cu, fe, and cd) ions reaching a maximum capacity for cd . Heavy metal ions uptake is the result of a combination of several interfacial reactions, namely, ion exchange, chemisorption, and adsorption as potential determining ions . Results found showed that the modified zeolite shows a good potential as adsorbent for (co, cu, fe, and cd) ions.
Surgical resection of tumors is a common therapeutic procedure, especially for early - stage localized, and potentially curative, disease . While surgery can ultimately cure many patients, such as those with early - stage disease, distant macroscopic metastasis can emerge in others months to years later (demicheli et al . It has been reported that 25%30% of colorectal cancer patients who have no visible metastasis at the time of diagnosis will develop distant metastases within 5 years after primary tumor resection, which in some cases may be related to the effects of the surgery (van der bij et al ., 2009). Similarly, high risk of recurrence for early - stage breast cancer patients following mastectomy has been reported in an analysis of 1,173 patients who underwent mastectomy with no subsequent adjuvant systemic therapy (demicheli et al ., 1996). Mechanisms to explain distant metastasis following primary tumor resection include (1) the presence of residual tumor cells or tissue at the resected site (ando et al ., 2003, minsky et al ., 1988), (2) the recruitment of inflammatory cells and platelets to the resected site that promote wound healing and cell proliferation (ceelen et al ., 2014, hofer et al ., 1999, retsky et al ., 2012), and (3) increased local and systemic effects that can induce an angiogenic switch in remote dormant tumors (bono et al ., 2010, retsky et al ., 2012, takemoto et al ., 2012). Previous studies have revealed that hypoxic tumor cells stimulate angiogenic - related factors via hif1-, leading to increased tumor invasion (paraskeva et al ., 2006, hif1- expression in tumors can also upregulate lysyl oxidase (lox) (erler et al ., 2006), a member of the secreted copper - dependent amine oxidases known to covalently crosslink collagens and elastin in the extracellular matrix (ecm) (barker et al ., 2012). Lox is expressed by different cell types, including tumor cells and stromal cells within the tumor microenvironment (decitre et al ., 1998). It has been shown that increased lox expression in tumors accounts for the recruitment of cd11b bone - marrow - derived cells (bmdcs) at distant organs, contributing to the formation of a niche and facilitating a pre - metastatic microenvironment for tumor cell seeding (erler et al ., 2009). However, the contribution of lox to tumor cell seeding and subsequently to metastasis soon after surgery is unknown . The host response to anti - cancer therapy and its contribution to tumor (re)growth and metastasis has been evaluated following chemotherapy (daenen et al ., 2011, gingis - velitski et al ., 2011), radiotherapy (barcellos - hoff et al ., 2005, 2015), and various molecularly targeted drugs (beyar - katz et al ., 2016) (for a review, see shaked, 2016). Here, we evaluated remote (pulmonary) changes in lox expression and activity in response to surgery and their contribution to tumor cell seeding and metastasis . Increased metastases after localized tumor resection in some cases could be due to systemic changes that affect various host tissues in response to surgery . Previous clinical studies indicated that both systemic and local angiogenesis are induced in response to surgery when compared to laparoscopy (bono et al ., 2010). To test whether our surgical mouse model induces angiogenesis, we performed a surgical procedure in non - tumor - bearing mice involving a 1 cm incision in the peritoneum followed by suturing . Thereafter, we evaluated the levels of circulating bone - marrow - derived proangiogenic cells over time and the extent of local angiogenesis following surgery . A significant increase in the number of viable circulating endothelial cells (cecs) and endothelial progenitor cells (ceps) likewise, increases in microvessel density in matrigel plugs, in vitro human umbilical vein endothelial cell (huvec) microvessel tube formation, and microvessel sprouting from murine aortic rings were observed in the presence of plasma obtained from post - surgery mice compared to control (figure s1b the surgical procedure in our mouse model mimics host angiogenic effects reported in certain clinical circumstances . We next evaluated the host - derived effects of surgery on tumor cell seeding and growth in metastatic sites . We employed an experimental lung metastasis assay using the murine emt/6-gfp+ breast cancer cell line . Control mice and mice that previously underwent surgery were intravenously injected with tumor cells, which subsequently seeded in the lungs . Mortality rates were increased in post - surgery mice compared to controls (figure 1a). Similarly, recipient mice injected with 100 l plasma from donor mice that had undergone surgery exhibited an increased mortality rate in comparison to mice injected with control plasma (figure 1b), indicating that a host effect, expressed within the plasma, in response to surgery rather than the actual surgical procedure is responsible for the observed effects . To directly assess tumor cell seeding in the lungs, regardless of any host responses that may affect tumor cells per se while seeding and proliferating at the metastatic sites, a pulmonary metastatic assay (puma) was performed using the emt/6-gfp+ cell line . In this assay, the potential of tumor cell seeding is solely dependent on tumor cells binding to the lungs and not systemic effects that could affect tumor cell proliferation . The number of gfp+ tumor cells present in the lungs of mice that underwent surgery was substantially elevated in comparison to control mice (figures 1c and 1d). Taken together, our results suggest that enhanced tumor cell seeding is a result of remote changes occurring, in part, in the pre - metastatic (lung) tissue in response to surgery . Ecm remodeling is one of the main factors that can facilitate tumor cell seeding; lox mediates collagen crosslinking and therefore is a key regulator of ecm remodeling (barker et al ., 2012). Recent studies have shown that lox is expressed by tumor cells, and serves as a key enzyme promoting metastasis by contributing to a pre - metastatic niche (cox et al ., 2015, erler et al ., 2006, erler et al ., 2009). In our model, we found that lox expression and activity were significantly upregulated at the hypoxic surgical site in the wounded peritoneum in comparison to control peritoneum (figures 2a2c). Specifically, high - magnification images of the surgical site revealed intracellular lox expression of peritoneal myofibers (figure s2a). Furthermore, lox expression and activity, quantity of newly synthesized collagen, and fibrillar collagen expression were all significantly higher in the lungs of mice that underwent surgery than in the lungs of control mice (figures 2d2i). Notably, high lox extracellular staining was located mainly in the lung stroma surrounding the bronchioles in post - surgery lungs when compared to control lungs (figure 2j). However, high - magnification images detected intracellular lox expression in different cell types, with no noticeable differences in expression pattern between control and post - surgery lungs, suggesting that the major source of lox in the lungs of post - surgery mice is from the remote surgical site (figure s2b). In addition, a significant decrease in lox expression and activity was found in the liver of mice that underwent surgery compared to control mice, whereas no significant changes were observed in the spleen (figures s2c and s2d). Importantly, 24 hr after surgery, the percentage of cd11b cells in the pre - metastatic lungs of post - surgery mice was comparable to that in control mice (figures s2e and s2f), ruling out the possibility of the formation of a reported pre - metastatic niche at this early time point (erler et al ., 2009, kaplan et al ., 2005, these results indicate that the immediate effects of surgery are primarily associated with ecm remodeling . In order to strengthen the hypothesis that lox is a major contributor to metastatic seeding in the lungs within hours of surgery, a puma, which measures cell seeding in the lungs, was carried out on mice 24 hr after they were injected with recombinant lox, the function of which was evaluated by a lox activity assay (figure s2 g). An increased number of metastatic cells was found in the lungs of mice injected with recombinant lox compared to control mice injected with pbs (figures 2k and 2l). The seeding of cells in collagen and ecm is regulated in part by integrin signaling pathways (larsen et al ., 2006). One of the major readouts of integrin signaling is paxillin, a scaffold protein connecting focal adhesion kinase to the actin cytoskeleton (turner et al ., 1990, zaidel - bar et al ., 2007). We therefore sought to determine the level of phospho - paxillin (p - pax) in mcf-7 breast carcinoma cells, a cell line with minimal lox expression (kraft - sheleg et al ., 2016), in response to plasma from control or post - surgery mice . Mcf-7 cells were seeded on collagen, laminin, or fibronectin, all of which are ligands of integrins (heino and kpyl, 2009), in the presence or absence of plasma from control or post - surgery mice . Increased expression of p - pax was observed with all three ligands in the presence of plasma from post - surgery mice compared to control, with laminin producing the weakest effect (figures 3a and 3b). In addition, when the same experiment was performed using plasma from post - surgery mice depleted of lox, the expression of p - pax was found to be lower than the post - surgery non - depleted plasma group (figures 3c and 3d), suggesting that lox expression mediates focal adhesion signaling . Furthermore, expression of p - pax was higher in the lungs of mice that underwent surgery than in control mice, an effect that could be inhibited by treating the mice with -aminoproprionitrile (bapn), an irreversible competitive inhibitor of all lox family members (bondareva et al ., 2009) it should be noted that all three ligands are substrates of lox as assessed by the oxidation assay, whereas laminin was the least effective substrate, in line with the results shown in figures 3a3d (figure s3a). Taken together, increased lox activity in the lungs soon after the mice undergo surgery contributes to tumor cell seeding via focal adhesion signaling, suggesting interactions between integrins and lox ligands in the remodeled ecm . As lox contributes to the seeding of tumor cells in the lungs, we next studied the effects of lox inhibition in mice using bapn . Plasma from control mice or mice that underwent surgery was injected into recipient naive mice . After 24 hr, the recipient mice were injected with emt/6-gfp+ cells to obtain the experimental lung colonization metastasis assay described above . Bapn monotherapy did not affect the percentage of gfp+ cells in the lungs of control mice . In contrast, the increase in percentage of gfp+ cells in the lungs of mice injected with plasma obtained from the post - surgery mice was completely abolished by the bapn therapy (figures 4a and 4b), and their mortality rate was reduced, as assessed in a parallel experiment (figure 4c). No significant changes in mortality rate and percentage of gfp+ cells in the lungs of control mice treated with bapn or pbs control were found (figures 4b and 4c). Importantly, escalating doses of bapn in a range of 010 mg / ml did not contribute to tumor cell apoptosis (figure s3b), suggesting that the therapeutic effect of bapn is related to the inhibition of lox activity . Furthermore, inhibiting lox using specific anti - lox activity - inhibiting antibodies similarly reduced tumor cell seeding in the lungs of mice injected with plasma from post - surgery mice, indicating that the effect is largely regulated by lox . Of note, it is not clear why blocking lox in control mice increased metastatic cell seeding (figures 4d4f). It is possible that other bypass pathways may play a role similar to mmp9 inhibition in control mice as previously described (gingis - velitski et al ., 2011, moreover, fibrillar collagen and newly synthesized collagen quantities in the lungs of mice 3 days after localized peritoneal surgery were significantly increased when compared to lungs obtained from post - surgery mice treated with bapn or anti - lox antibodies (figures 4g4i). Of note, both anti - lox and bapn reduced lox activity in the lungs, while control rabbit immunoglobulin g (igg) had no effect (figures s3c and s4, respectively). To further strengthen our results, we wished to rule out the possibility that inflammation - induced pulmonary vessel permeability mediates the increased tumor cell seeding in the lungs at this early time point . Evans blue assay performed on mice 24 hr after they underwent surgery revealed that pulmonary vessel permeability was not significantly different in post - surgery mice compared to control mice (figure s5a), suggesting that tumor cell seeding was not affected by inflammation . Additionally, the number and percentage of tumor cells (emt/6-gfp+) seeded in the lungs 20 min after tail vein injection of 2 10 cells (10-fold higher than the number of cells injected in the puma) were significantly higher in post - surgery mice than in control mice . This effect was dramatically reduced when the mice were also treated with bapn as assessed by microscopic imaging of lung specimens and flow cytometry of single - cell suspensions from dissociated lungs (figures s5b and s5c). Taken together, lox - induced ecm remodeling accounts for tumor cell seeding in the lungs of post - surgery mice . The fact that plasma derived from post - surgery mice had the same effect on tumor cell seeding as surgery itself suggested it entails the factor mediating this activity . Therefore, lox levels were evaluated in the plasma of control and post - surgery mice . The plasma of post - surgery mice exhibited significantly elevated lox levels compared to plasma from control mice, suggesting that lox upregulation in the plasma may affect tumor cell seeding in the lungs (figure 5a). To test this possibility, we depleted lox from plasma of control mice and mice that underwent surgery and assessed tumor cell seeding using puma in recipient mice injected with the different plasma samples . Whereas lox depletion did not affect tumor cell seeding in the control group, it significantly decreased tumor cell seeding in the surgery group (figures 5b and 5c). In a parallel experiment, plasma was obtained from untreated wild - type mice or bapn - treated lox heterozygous mice (mki et al ., the plasma was then injected into mice, and survival was assessed in an experimental lung metastasis assay . However, in the surgery group, survival times were increased when lox was inhibited (figures 5d and 5e; statistical significance [p = 0.043] was reached when comparing surgery with surgery + lox depletion groups). Collectively, our results suggest that lox in the plasma from post - surgery mice promotes tumor cell seeding, and its depletion inhibits collagen synthesis and ecm remodeling . To bolster the possibility that our results may be clinically relevant, we employed two different experimental approaches . In the first, we used a more clinically relevant tumor mouse model of metastasis, in which spontaneous metastases appear in the lungs after resection of a primary orthotopic 4t1 murine breast cancer . To inhibit lox, bapn - treated mice exhibited significantly extended survival and reduced lung metastasis in comparison to mice that did not receive treatment after surgery (figures 6a6d). In the second approach, plasma was drawn from colorectal cancer patients at baseline and 24 hr after they underwent abdominal surgery (n = 6). The plasma was injected into mice, and metastases were evaluated using emt/6-gfp+ cells in an experimental lung metastasis assay . In five out of six cases, the post - surgery plasma induced higher numbers of lung metastases in comparison to baseline plasma (figures 6e and 6f). Such post - surgical plasma also induced ecm remodeling and increased fibrillar collagen expression as well as lox activity in the lungs in a manner similar to that found in mice that underwent abdominal surgery (figures 6 g, s6a, and s6b). Importantly, pooled plasma obtained from patients that underwent surgery that was depleted of lox did not increase metastatic seeding in the lungs as assessed by puma (figures 6h and s6c). The notion that tumor cells remaining at the site of a surgical resection can re - grow and sometimes even spread may contribute to inferior outcomes in both clinical and preclinical scenarios (coffey et al ., 2003, delisser et al . We find that the host, in response to surgery, conditions potential metastatic sites to facilitate tumor cell seeding . We demonstrate that mice that undergo abdominal surgery and are subsequently injected with tumor cells via the tail vein succumb to extensive metastatic lesions earlier than controls . These mice exhibit structural changes in the ecm at the site of metastasis due to an increase in newly synthesized collagen . Lox activity and expression are substantially enhanced in the lungs of mice following surgery, as well as in mice pre - conditioned with plasma from donor mice that underwent surgery . Accordingly, we find that the surgery is associated with release of lox to the circulation from the hypoxic wounded site, which in turn induces the formation of the pre - metastatic sites . Such pro - metastatic effects are abolished following lox inhibition (e.g., using bapn, neutralizing antibodies for lox, or lox depletion from plasma). Notably, antibodies only neutralize extracellular lox and therefore act primarily on the ecm rather than on lox intracellular targets . Lox has been implicated as a key enzyme contributing to the pre - metastatic niche promoting the recruitment of cd11b cells to future metastatic sites measured within weeks after primary tumor cell implantation (erler et al ., 2009). Furthermore, increased activity of lox and collagen crosslinking have been shown to promote fibrosis and subsequent metastasis (cox et al ., 2013). In our study, we focused on the immediate contribution of lox to metastasis by means of ecm remodeling, which promotes the seeding of tumor cells in the lungs within hours following surgery . At this time point, no changes in cd11b cells colonizing the pre - metastatic sites were observed, and there was no significant difference in pulmonary permeability, suggesting that inflammation at this early time point did not contribute to tumor cell seeding . However, it should be noted that clinically, the use of ketorolac (an analgesic drug with non - steroidal anti - inflammatory properties) in surgery resulted in superior disease - free survival of breast cancer patients who underwent mastectomy compared to patients who underwent mastectomy but were not treated with ketorolac (retsky et al ., 2012). Have demonstrated that lox is a key regulator of metastasis that is induced by hypoxia . Lox expression in hypoxic tumors correlates with increased incidence of metastasis in patients with breast and head and neck cancers . Accordingly, the blockade of lox in mice bearing orthotopic breast cancers results in decreased tumor cell invasiveness and metastasis (erler et al ., 2006). In addition, bone metastasis of triple negative breast cancer cells is associated with hypoxia and lox activity (cox et al ., 2015). Family members promote endothelial cell activity and angiogenesis in tumors and support ecm remodeling (baker et al . Therefore, antiangiogenic therapy, which induces hypoxia in tumors (blagosklonny, 2001), may lead to increased lox expression . As a result, alterations in ecm may take place in distant sites (e.g., lungs or liver) that can decrease the therapeutic response to therapy . These effects may also explain why, at least pre - clinically, antiangiogenic drugs sometimes promote metastasis (ebos et al ., 2009, pez - ribes et al ., 2009, the question remains why lox has a significant effect on ecm remodeling in the lungs while its expression and activity in other organs are reduced or do not change in post - surgery mice when compared to control mice . It is possible that the high oxygen concentration in lung tissue contributes to lox enzymatic activity . Abundant microvessel networks in the lungs contribute to increased oxygen availability, which in turn promotes lox enzymatic activity (barker et al . This may explain the increased lox enzymatic activity in the lungs compared to other organs . Thus, the level of ecm remodeling could be higher in lung stroma than in other tissues . Mice bearing tumors that spontaneously metastasize exhibit a reduced mortality rate when lox is inhibited following surgery . Additionally, mice preconditioned with plasma from post - surgery colorectal cancer patients exhibit an increased number of metastatic cells in the lungs compared to mice injected with the same plasma that was depleted of lox or plasma at baseline (before surgery). Taken together, in addition to the known metastatic effects of lox (barker et al ., 2012), our study reveals that lox induction contributes to the rapid formation of a permissive niche for metastatic cell seeding in response to surgery . Murine lewis lung carcinoma (llc), emt/6 and 4t1 mammary adenocarcinoma, and human mcf7 breast carcinoma cell lines were purchased from the american type culture collection (atcc). All cells were grown in dmem, supplemented with 10% fetal calf serum, 1% l - glutamine, 1% sodium pyruvate, and 1% streptomycin, penicillin, and neomycin in solution (10 mg / ml, biological industries). Some of the cell lines (as indicated below) were stably transfected with gfp or mcherry vectors (clontech laboratories, 632379 and 631985, respectively). The cells were passaged in culture for no more than 4 months after being thawed from authentic stocks and were regularly tested and found to be mycoplasma - free . The use of animals and experimental protocols were approved by the animal care and use committee of the technion . Balb / c female mice (harlan), 810 weeks of age, were orthotopically injected with 0.5 10 mcherry - expressing 4t1 (4t1-mcherry+) to the mammary fat pad . Tumor size was assessed regularly with vernier calipers using the formula, width length 0.5 . An experimental pulmonary metastatic model was obtained using female balb / c or c57bl/6 mice, 810 weeks of age, intravenously injected through the tail vein with 2.5 10 gfp - expressing emt/6 (emt/6-gfp+) or gfp - expressing llc (llc - gfp+) cells, respectively . Mice were not randomized after surgery except in the case of the clinically relevant tumor model (4t1 mouse model). The experiments were not blinded to the investigator; however, they were blinded to the mouse health care attendant who carried out daily health checks and reported on mice at endpoint . 10-week - old female lox c57bl/6 heterozygous mice described previously (mki et al ., 2002) were used in some experiments . Mice were anesthetized using an induction of 4% isoflurane, followed by maintenance anesthesia with 1.5% isoflurane . A 11.5 cm incision in the peritoneum was then performed, followed by suturing with silk . For resection of primary 4t1 tumors, when tumors reached 150200 mm, . Of note, all mice (control and surgery groups) received anesthesia and analgesic buprenorphine at a concentration of 0.04 mg / kg for three sequential days according to the institutional ethical protocols . 8- to 10-week - old balb / c or c57bl lox mice underwent the surgical procedure (as above) or left as control . After 24 hr, blood was drawn by cardiac puncture using citrate tubes, and plasma was separated . Plasma was injected intraperitoneally into recipient mice at a volume of 100 l / mouse . In some experiments, bapn (sigma - aldrich) was injected intraperitoneally at a dose of 100 mg / kg daily for seven consecutive days, or as indicated in the text, as previously described (bondareva et al ., 2009). The antibodies were intraperitoneally injected at a dose of 20 mg / kg as previously described (erler et al ., 2006). Control rabbit igg antibodies (jackson immunoresearch laboratories) were injected intraperitoneally at a dose of 20 mg / kg . Recombinant lox (origene) was injected intraperitoneally at a dose of 25 g / kg, as previously described (cox et al ., 2015). The human study was approved by the ethic committee at the european institute of oncology (eio), milan, italy, and all patients signed an informed consent . Plasma from colorectal cancer patients, who underwent open abdominal surgery, was provided by the department of pathology at the eio . Blood from the patients (n = 6) was collected at baseline (before surgery) and 24 hr after surgery . Blood was drawn from anaesthetized mice by cardiac puncture and collected in edta tubes, and red blood cells were lysed . Lung samples or matrigel plugs were prepared as single cell suspensions as previously described (adini et al ., 2009, gingis - velitski et al ., 2011). Cecs, ceps, emt/6-gfp, and 4t1-mcherry+ were analyzed by flow cytometry as previously described (shaked et al ., 2005). Frozen lung sections or matrigel plugs were immunostained as previously described (gingis - velitski et al ., 2011). Plates coated with collagen (20 g / ml), fibronectin (20 g / ml), or laminin (20 g / ml) were incubated with plasma from control, post - surgery mice or plasma from post - surgery mice depleted of lox . After 4 hr, the plasma was washed and mcf7 cells (7 10/well) were seeded and cultured for 24 hr . Mcf7 cells were then removed, and lysates were prepared to evaluate paxillin and p - pax expression by western blotting . The assay was performed as previously described (mendoza et al ., 2010). Collagen production in lung lysates was quantified using the sircol collagen assay kit (biocolor) in accordance with the manufacturer s instructions . Details are provided in supplemental experimental procedures . To visualize ecm and collagen, a two - photon microscopy and a second harmonics generation system lungs from the different groups as indicated in the text were frozen in optimal cutting temperature (oct) and sliced at a thickness of 150 m in pbs . The slices were imaged using a two - photon microscope 2pm: zeiss lsm 510 meta nlo, equipped with a broadband mai tai - hp - femtosecond single box tunable ti - sapphire oscillator, with automated broadband wavelength tuning 7001,020 nm from spectraphysics, for two - photon excitation . For second harmonic imaging (shg) to detect collagen, a wavelength of 800 nm was used (detection at 400 nm). Data were collected (n = 3 mice / group). For quantification measurements, images were analyzed using imagej 1.41k . To avoid edge effects (attenuation of the shg signal at the top and bottom of the section), only the central image of each z mean gray value limited to threshold of each image was calculated for each image and averaged over a set of at least five fields of view . Lox activity was evaluated as previously described (siegel, 1974, siegel et al ., 1970). Briefly, a reaction solution containing 50 mm sodium borate (ph = 8.2) and 4 u / ml horseradish peroxidase was mixed with lung extracts to obtain a protein concentration of 250 g / ml . The lysate extracts were obtained from mice treated with bapn, anti - lox, recombinant lox, or rabbit polyclonal antibody using the same concentrations as above . Furthermore, lysate extracts of lungs or peritoneum obtained from control mice or mice that underwent surgery were treated with bapn, anti - lox antibodies, recombinant lox, or rabbit polyclonal antibody and used as controls for the lox activity assay . The enzymatic reaction was started by adding substrate mixture containing 50 mm sodium borate (ph = 8.2), 100 mm n - acetyl-3,7-dihydroxyphenoxazine (amplex red; molecular probes, invitrogen), and 20 mm 1,5-diaminopentane . In some experiments, 500 m bapn was added to the mixture as a negative inhibitory control . The production of h2o2 by lox results in fluorescent resorfurin production, which can be measured (excitation at 540 nm and emission at 580 nm wavelengths). The fluorescent reaction was measured every 5 min for 1.5 hr at 37c using a fluorimeter (fluo star galaxy). Lox expression in plasma and organ lysates (50 g protein) was evaluated by elisa (cloud - clone - corp . Sec580mu) in accordance with the manufacturer s instructions . To analyze the activity of lox on different substrates, the oxidation assay was performed as previously described (kraft - sheleg et al ., 2016, zaffryar - eilot et al ., 2013). Briefly, the lox activity assay was performed as above using lung lysates from control mice at a concentration of 200 g / ml . The only modification is that the substrate (1,5-diaminopentane) used in the lox activity assay was replaced with collagen (1 mg / ml), fibronectin (1 mg / ml), or laminin (1 mg / ml). The fluorescent reaction was measured every 5 min for 1.5 hr at 37c using a fluorimeter (fluo star galaxy). A representative plot out statistical significance of the in vitro experiments was determined by either two - tailed student s t test for a comparison between two groups or one - way anova for multiple groups followed by tukey ad hoc statistical test using graphpad prism 5.0 . For the lox activity assay and human experiment results, a comparison between control / baseline and the related group was calculated based on paired student s t test . The number of replicates for each experiment is provided in supplemental information and/or figure legends . In the in vitro experiments, estimate of variance was performed and parameters for the statistical test were adjusted accordingly . In the in vivo / ex vivo studies, n = 37 mice / group (as specified in the figures) was used to reach statistical significance . The sample size for each experiment was designed to have 80% power at a two - sided of 0.05 . For the calculation of mouse survival, a kaplan - meier survival curve statistical analysis was performed in which the uncertainty of the fractional survival of 95% confidence intervals was calculated . C.r .- t ., p.h ., and y.s . Conceived and designed all experiments.
These conditions may be life - threatening (e.g. Oral cancers) or not, progressing (caries, periodontitis, etc .) Or not, dealing with aesthetics (staining in anterior teeth such as molar incisor hypomineralisation (mih)) or pain (pulpitis, mih in posterior teeth, etc . ). Ohrqol is highly subjective and has to be assessed within the framework of patients conditions, sociocultural environments and own experiences and states of mind: because ohrqol is related to daily life and is unique to each individual, even patients with severe conditions can report having good quality of life . Furthermore, quality of life is by itself multi - faceted, showing variation over time for each individual . Ohrqol should therefore be assessed longitudinally to take into account changes over time, using versatile tools . This means having relevant questions with well - defined items and being able to analyse answers in a good way . Many limitations can be found to the current validation testing, including relevance of the questions, validity and sensitivity to change, risk of misinterpretations (role of the ethnocultural environment), problems of translation of english questionnaires and difficulty to interpret the significance of a psychometric measurement when reported simply as a numerical score or a mean [1, 2]. This latter point is of importance since the same score can be obtained from people answering in a different way to a majority of questions . Finally, patient - based outcome measures (as named by fitzpatrick et al .) Should provide the opportunity to measure the extent or intensity of the changes in ohrqol . Various psychometric instruments have also been used to measure ohrqol (tables 1 and 2) [1, 4]. These are based on different criteria that enable them to be more or less patient- or expert - centred . Some are generic (ohip1 - 49, ohip-14, oidp, oh - qol, sf-362) and can be considered as core indicators; others are adapted to specific conditions / domains (orthognathic qol questionnaire, sooq for orthodontic surgery, ohip - aesthetic,3 ohrqol for dental hygiene) or populations (cohqol and child - oidp for children, gohai for elderly people, etc. ).table 1conceptual and structural basis of psychometric instruments used in dentistry (adapted from brondani and mcentee) instrumentsacronymstructural originsempirically basedconnotation of questionsnumber of questionssocial impacts of dental diseasesiddsipyesn14oral health impact profileohipicidhyesn49geriatric (generic) oral health assessment indexgohaiicidh and sipyesn and p12oral health - related qol instrumentohrqlmultiplenon36oral impact on daily performancesoidpicidhnon8dental impact on daily livingdidlsipyesn and nt and p36dental impact profiledipsipyesn and nt and p25oral health - related quality of life measureohqolmultiplenon3oral health quality of life inventoryoh - qolsipunclearp15rand dental questionnaireunspecifiedsipnon3oral health questionnaireunspecifiedicidhunclearn and nt and p70oral health quality of life ukohqol - ukicidh2yesn and p16subjective oral health status indicatorssohsimultiplenon and nt34liverpool oral rehabilitation questionnairelorqunclearnon40self - rated oral healthsrohicidhnon and p3dentaldentalunclearnon15dental health status quality of life questionnaireds - qolgeneric qol instrumentnon and punclearn 1/4 negative, nt neutral, p positive, sip sickness impact profile, icidh international classification of impairments, disabilities and handicapsinformation derived from open - ended interviewssome indicators present shorter or extended forms other than the original versionhealth - related models: natural history of disease model and sipdeveloped from existing measures (rand, oral facial pain index, etc. ).table 2oral health outcome measures developed before 2007 (adapted from locker and allen) pre-1997 (presented at the 1997 conference) social impacts of dental diseasegeneral (geriatric) oral health assessment index (gohai)dental impact profile (dip)oral health impact profile (ohip)oral impacts on daily performances (oidp)subjective oral health status indicators (sohsi)oral health - related quality of life measuredental impact on daily living (didls)oral health quality of life inventoryrand dental questionspost-1997ohqol - ukchild oral health quality of life questionnaire (cohqol)child oidpohrqol for dental hygieneorthognathic qol questionnairesurgical orthodontic outcome questionnaire (sooq) conceptual and structural basis of psychometric instruments used in dentistry (adapted from brondani and mcentee) n 1/4 negative, nt neutral, p positive, sip sickness impact profile, icidh international classification of impairments, disabilities and handicaps information derived from open - ended interviews some indicators present shorter or extended forms other than the original version health - related models: natural history of disease model and sip developed from existing measures (rand, oral facial pain index, etc . ). Oral health outcome measures developed before 2007 (adapted from locker and allen) the ohip, also called ohip-49, is the most widely used, and this has enabled investigators to modify forms that can be subsequently adapted to populations or conditions . The initial 49-question form was constructed to assess the social impact of oral disorders . Each of the set of 49 statements represented one of seven domains: it is mainly expert - centred and constructed to select items according to their fit with a conceptual framework rather than on the basis of their importance to the patients from whom they were derived . A shorter version of ohip restricted to 14 items (ohip-14) was later proposed . One major question is to know if we need to use either a generic questionnaire, an adapted form of a generic questionnaire or to construct a new questionnaire specific to the population or condition to be studied . Constructing or using one of these specific questionnaires may lead to many questions, for example, (1) is it made specifically for the purpose of research or for clinical practice? Or (2) how to adapt each questionnaire to local languages and cultures? This may subsequently lead us to consider the impact of dentin hypersensitivity (dhs) or exposed cervical dentin (ecd) on ohrqol of those individuals being assessed . Very few studies have been devoted to this aspect of dhs / ecd as recently shown, with only two papers written in english specifically dedicated to the evaluation of ohqol in dhs / ecd patients . One paper provided results using a generic questionnaire and the second paper constructed a specific questionnaire to evaluate ohqol in dhs / ecd patients but provided no epidemiological results . These studies are more extensively described in an accompanying paper . In the future, studies using validated questionnaires specifically constructed to evaluate the impact of the condition on ohqol should be employed . These questionnaires should be patient - centred and derived from interviews with patients who are expected to complete the questionnaire [4, 10]. Furthermore, if these studies also attempt to evaluate the efficacy of desensitising agents in reducing dhs / ecd and its subsequent impact on ohqol, then it is imperative that the condition should be clearly diagnosed by trained and calibrated dentists experienced in conducting clinical studies using recognised and accepted clinical criteria for the evaluation of dhs / ecd . Due to the cultural and language differences between countries, there is also a need of norm or reference value(s) for each population to be studied . For example, when constructing a questionnaire for a non - english - speaking population, the questionnaire should be initially written in english, then translated by two people of the designated native (foreign) language and subsequently translated back into english by two native english - speaking people to identify any potential issues that may have arisen from the translation . Finally, as indicated above, any future study attempting to evaluate the efficacy of a desensitising agent in reducing dhs / ecd and its subsequent impact on ohqol should be conducted by experienced and calibrated examiners using established guidelines for conducting dhs / ecd clinical studies . Such studies should also be based on a randomised clinical study design and include both placebo or control groups . Patients suffering from dhs / ecd have been reported to have a significantly impaired ohrqol; this may however be improved following treatment with a desensitising agent as reported by several authors . It is therefore of the upmost importance that the use of the ohrqol as a quality control tool in the dental office be established in robust clinical studies . Furthermore, because of its ability to reflect a patient s satisfaction with any proposed treatment, it may prove to be a valuable asset for practitioners when assessing their patients quality of life before, during and after treatment of various clinical conditions such as dhs / ecd.
Male sprague - dawley rats (n = 24) weighing approximately 250 g were used for the study . Permission for the experimental work was obtained from the institutional animal ethics committee (767/iaec/13 dated 1 - 1 - 2014). Rats were divided into 4 equal groups and injected physiological saline (twice daily subcutaneously in morning and evening; group i), morphine sulfate (10 mg / kg twice daily subcutaneously; group ii), fosaprepitant (30 mg / kg once by intraperitoneal route; group iii), and finally, the last group was co - administered both morphine and fosaprepitant (group iv) in the same doses as in group ii and iii, respectively, for a week . In the last group, fosaprepitant was injected 30 min before the morphine injection . The dose of fosaprepitant (30 mg / kg) was higher than that used in an earlier study (25 mg / kg). The rationale for selecting a higher dose was the rapid conversion of fosaprepitant to aprepitant (half - life ~30 min) in the rat compared to dogs and humans . Hot plate latency or the thermal escape behaviour was determined in the morning, 40 min after saline (group i), fosaprepitant (group iii), or morphine injection (group ii and iv). The hot plate test is commonly employed for screening the putative antinociceptive property of drugs . The advantages of this test are the brief nature of the nociceptive stimulus, which does not produce any tissue damage and that multiple testing can be done in the same animal . The predictability of this test to clinical situations is high for morphine and related opioid substances . Testing was done in a quiet room with the ambient temperature between 22c and 25c . On the day of the experiment, the testing platform of the hot plate apparatus (stoelting, usa) was set at a constant temperature of 52.5c . The rats were placed on the hot plate, and the behavioral end points were either licking of the hindpaw or jumping . The test was repeated thrice at 57 min intervals and the average of these values was the latency period (sec). A 40 s, cut - off was fixed to prevent damage to the completely analgesic paw after morphine injection . Transformation of these values was done by calculating the percent maximum possible effect (% mpe) as follows: ([drug induced latency basal response time]/[40 s basal response time]) 100 on day 8 (morning), rats were anesthetized by pentobarbital injection (100 mg / kg intraperitoneal). This was followed by intracardiac perfusion with 4% paraformaldehyde solution in 0.1 m phosphate buffer saline . Transverse sections of the spinal cord (20 m thick) were obtained in a cryostat and processed for immunohistochemical localization of sp and cgrp using specific antibodies (anti - sp antibody, abcam, uk; anti - cgrp antibody, calbiochem, usa; 1:500 titer) using the avidin - biotin complex method (vector labs, usa). Representative sections (3/rat; systematic random sampling) were later viewed under a microscope and the images captured . The expression of sp and cgrp in the superficial laminae (rexed's laminae i - ii) of the gray matter was quantitated by image j software (nih, usa). Specific expression was obtained after deducting background staining in the white matter (lateral funiculus) from the total value obtained from the superficial laminae . Some of the cryostat sections were stained with 0.5% cresyl violet for localization of neurons in the dorsal horn . Statistical evaluation of data was done by graphpad prism version 5 (graphpad software, la jolla, san diego, usa). Values of latency period of the different groups of animals were independently analyzed at each time point by one - way analysis of variance followed by bonferroni multiple comparison test . The various groups of rats had different baseline values, which affected the subsequent comparison of the antinociceptive effect of the drugs [figure 1a]. Hence, normalization of the data was done by calculating the% mpe [figure 1b]. The% mpe values of control and the fosaprepitant - treated groups did not show any statistically significant change during the experiment . Morphine injection produced significantly higher antinociceptive effect on day 1 (p <0.001) and to a lesser extent day 4 (p <0.05) compared to the control group . The sharp decrease in the antinociceptive effect from day 2 in the morphine treated group indicated the development of tolerance . Morphine + fosaprepitant combination delayed the onset of tolerance in comparison to morphine treated group on days 1 (p <0.01) and 3 (p <0.001). Compared to control, antinociception was higher between days 1 and 4 (p <0.001) for this group . (a) please note the variability of basal latency period and hence these values were not considered in the experimental results (b)% maximum possible effect shows rapid development of tolerance after morphine treatment . However, co - administration of fosaprepitant with morphine increased the antinociceptive effect of morphine (days 14) and also attenuated tolerance (days 1 and 3). Values are represented as mean standard error of mean p <0.05, * * p <0.01, * * *,,p <0.001 cresyl violet staining showed the dorsal horn neurons arranged in various laminae [figure 2]. Immunohistochemical study revealed the expression of sp and cgrp over the superficial laminae (laminae i - ii) of the dorsal horn [figure 2]. Quantitative image analysis revealed increased sp expression in the morphine + fosaprepitant treated group compared to others [figure 3]. Statistically significant difference was present between morphine and morphine + fosaprepitant co - treatment groups (p <0.001). Cresyl violet staining showed the different laminae of the gray matter of the cervical region of the spinal cord (top panel). Location of the box showed the area of interest (laminae i - ii). Expression of both substance p and calcitonin gene - related peptide was noted over the superficial laminae (i - ii). Each group had 6 animals quantitative image analysis of the expression of substance p and calcitonin gene - related peptide (cgrp) shows that morphine + fosaprepitant treatment led to higher substance p expression compared to all other groups including the morphine treated group . P the a and c groups of nerve fibers carry pain from the periphery to the spinal cord . The peptidergic subgroup of these nerve fibers contains neuropeptides such as sp and cgrp, which are released into the synaptic cleft following noxious stimulation . These neuropeptides diffuse to the postsynaptic terminal, where they bind to the corresponding receptors . Morphine - induced antinociception is linked to decreased release of glutamate, sp, cgrp, etc ., from presynaptic terminals and greater postsynaptic hyperpolarization due to the activation of inwardly rectifying potassium channels . Co - administration of fosaprepitant possibly interfered with the binding of sp to the nk1rs expressed by postsynaptic neurons in the spinal cord . The results of this study show that combining morphine with fosaprepitant (group iv) delayed the development of morphine tolerance and concurrently increased the antinociceptive effect compared to the control group . As mentioned earlier, this could be due to decreased binding of sp to the nk1r . Evidence from electrophysiological experiments indicates that noxious stimuli produce slow and prolonged excitatory potentials in postsynaptic dorsal horn neurons, which are inhibited by nk1r antagonist . Since fosaprepitant treatment (group iii) alone did not result in an antinociceptive effect, the interaction between morphine and fosaprepitant is likely synergistic in nature . Previously, injection of a bifunctional peptide having both -opioid receptor agonist and nk1r antagonist properties relieved pain in neuropathic rats without producing tolerance . In a different study, tumati et al . Reported that intrathecal administration of both morphine and a nk1r antagonist reduced subsequent opioid withdrawal - induced hyperalgesia . Pharmacokinetic interaction between fosaprepitant / aprepitant and morphine could also contribute to the enhanced antinociceptive effect . Hence, morphine concentration in the nervous system could be increased by concurrent administration of these drugs . Significant alterations in the expression of sp and cgrp were not observed except in the group treated with morphine + fosaprepitant combination (group iv). This was unexpected because prevailing evidence suggests that nk1r does not regulate the release of sp in the spinal cord . However, contrary to this, nk1r has been recently reported to be crucial for release of sp from dorsal root ganglion neurons . Moreover, it has been speculated that there could be sp autoreceptors on presynaptic terminals, which can modulate sp release . But, fosaprepitant treatment alone did not increase sp immunoreactivity in the present work . As noted earlier, morphine inhibits the release of sp under acute conditions but rats chronically treated with morphine in our study did not show a statistically significant increase in sp expression . Presumably, both nk1 autoreceptors as well as morphine - induced inhibition of sp release might have contributed to the increased sp expression in group iv [figure 4]. A limitation of this study was that the expression of sp and cgrp was evaluated at the end of the observation period and not between days 1 and 4, when the antinociceptive effect was maximum . Another limitation was the use of a fixed - dose combination of morphine and fosaprepitant . Diagrammatic representation of the dorsal horn of the spinal cord (a) primary sensory afferents carrying nociceptive sensation ends in the dorsal horn of the spinal cord . (b) the presynaptic terminals of the primary sensory afferents contain excess substance p following morphine + fosaprepitant treatment (red). - receptor is present on both the presynaptic and postsynaptic terminals the antinociceptive effect of morphine decreased rapidly following daily administration, indicating development of tolerance . The antinociceptive effect of morphine is likely due to decreased release of pronociceptive neurotransmitters such as glutamate and neuropeptides such as sp from presynaptic nerve terminals which could be lost during tolerance . One of the factors responsible for tolerance could be counter - adapting processes, which maintains the status quo in the spinal cord . Similarly, the potentiation of the antinociceptive effect of morphine by fosaprepitant is also lost on continued administration (day 5 onward). Identical result was reported with a chimera possessing both opioid agonist and nk1r antagonist properties . Moreover, the factors can differ depending upon the specific -opioid receptor agonist used for producing the tolerance . The results indicate that addition of fosaprepitant to morphine can delay morphine tolerance for a limited period of time . To the best of our knowledge, this is the first report on the novel antinociceptive effect of morphine + fosaprepitant combination . Further studies are required to further elucidate this novel antinociceptive effect of morphine + fosaprepitant combination.
The direct conversion of solar energy to a chemical fuel is an essential part of future sustainable energy systems that are independent of fossil reserves . Hydrogen is an environmentally benign energy carrier of high - energy density and can be produced by photocatalytic water reduction . Platinum and other noble metals can serve as heterogeneous hydrogen evolution catalysts; however, their limited earth abundance and cost precludes further development and/or large - scale applications . On the other hand, organometallic compounds are attractive catalysts for this transformation considering the variety of complexes that can be prepared, the synthetic ease with which electronic properties (and hence catalyst activity) can be modulated, and the ability to study their mechanisms in detail . In nature, hydrogenase enzymes, especially those containing fe2 active sites, efficiently catalyze proton reduction to molecular dihydrogen . Models of the [fefe]-hydrogenases can act as biomimetic hydrogen evolution catalysts, although they often suffer from limited stability, especially when catalysis is driven photochemically in conjunction with photosensitizers . The necessity of an external matrix to stabilize the catalyst is thus evident . Metal organic frameworks (mofs), also referred to as porous coordination polymers, have emerged as an intriguing class of microporous crystalline materials due to their intrinsic topology and porosity and have been studied for a range of applications in gas storage / separation, chemical sensing, drug delivery, and catalysis . Unlike other porous materials such as zeolites, the organic ligand component of mofs allows for functionalization of internal channels and/or cavities either through direct solvothermal synthesis or by postsynthetic modification reactions that include the metathesis of metal ions and organic linkers under relatively mild conditions . Hupp, cohen, and others have reported on this postsynthetic exchange (pse) phenomenon (also termed solvent - assisted linker exchange, sale), including in highly robust mofs, such as the materials of the institute lavoisier, zeolitic imidaozolate framework, and university of oslo (uio) materials . All of these materials are considered to be inert with exceptionally high thermal and chemical stability, and can provide a robust platform for the incorporation of potentially labile molecular catalysts . Incorporation of catalytic sites into mofs has resulted in heterogeneous catalysts that promote a wide range of organic reactions . The heterogeneous nature of mof catalysts allows for their easy separation, reusability, and enhanced stability . In the context of light - to - fuel conversion schemes, homogeneous photosensitizers such as ir polypyridine complexes and porphyrins have been incorporated in mofs and were shown to drive photochemical hydrogen production catalyzed by pt nanoparticles . Organometallic ir and re catalysts have been incorporated as the ligand linker part of the mof and were shown to catalyze ce - promoted water oxidation and photochemical co2 reduction, respectively . Although excellent proof - of - concept studies, in both cases, resource - limited precious metal catalytic sites were used . Moreover, the scope of these reports is somewhat limited, as the solvothermal procedures that were used for the synthesis of the mofs require organometallic units that are thermally robust . Herein, we describe the incorporation of an organometallic fe2 complex that bears structural resemblance to the active site of [fefe] h2ases into a mof . [fefe](bdt)(co)6 (2) (bdt = benzenedithiolate) has previously been shown to be an effective proton reduction catalyst in electro- and photochemical schemes . Decoration of complex 2 at the bdt ligand with two carboxylates results in complex 1 which can be introduced into mofs by pse of 1,4-benzenedicarboxylate (bdc) ligands, which is a common ligand linker in many mofs (figure 1). Pse thus allowed for the introduction of a thermally unstable [fefe](bdt)(co)6 moiety into the thermally stable zr(iv)-based uio-66 mof (figure 2). X - ray absorption spectroscopy (xas) was used to confirm the coordination environment of the fe2 site in the mof . Importantly, uio-66-[fefe](dcbdt)(co)6 was found to be a highly active hydrogen production catalyst in photochemical arrays with [ru(bpy)3] as a photosensitizer and ascorbate as an electron donor . The catalytic performance of the mof exceeds that of an analogous homogeneous reference system . [fefe] hydrogenase active site model complexes [fefe](dcbdt)(co)61 and [fefe](bdt)(co)62 and the bdc ligand 2,3-dimercaptoterephthalic acid was prepared from benzene-1,2-dithiole via lithiation and carboxylation and directly converted to complex 1 ([fefe](dcbdt)(co)6, dcbdt = 1,4-dicarboxylbenzene-2,3-ditiolate) by combining the ligand with fe3(co)12 in thf . Single crystal x - ray analysis of complex 1 shows the usual distorted octahedral coordination sphere around the fe ions, with the dcbdt ligand perpendicular to the fe fe bond vector (see also esi). The uio-66 framework, consisting of zr(iv)-based secondary building units (sbus) and bdc ligand (zr6o4(oh)4(bdc)6), was chosen for the incorporation of complex 1 because of its exceptionally high structural stability with respect to water and weak acids . Highly crystalline uio-66 was synthesized under solvothermal conditions using zrcl4, bdc, and benzoic acid (as a crystal growth modulator) in dmf for 24 h, followed by washing with meoh and activation under vacuum . Field - emission scanning electron microscopy (fe - sem) shows an octahedral morphology of the resultant uio-66 crystals with a particle size ranging from 200 to 500 nm (figure 3). Fe - sem image of (a) uio-66 and (b) uio-66-[fefe](dcbdt)(co)6 . Scale bar is 1 m . Attempts to directly include 1 during solvothermal synthesis (> 50 c) resulted in decomposition of the cluster, presumably due to the labile bonds between the fe centers and the highly electron - deficient dcbdt ligand . Taking advantage of the structural homology of the bdc and dcbdt ligands in complex 1 (figure 1), we employed pse as a mild functionalization approach to introduce complex 1 into uio-66 (figure 2). Optimization of the pse conditions revealed that the use of deoxygenated, ultrapure water (room temperature for 24 organic solvents, including meoh, dmf, and chcl3, gave lower incorporation, consistent with previous observations on the solvent dependence of pse . As expected from the attempted solvothermal syntheses, pse at elevated temperatures (> 50 c) gave results that also suggested partial decomposition of 1 . Interestingly, it was found that pse was facilitated by using a highly microcrystalline form of uio-66 that was synthesized in the presence of a benzoic acid modulator . In contrast, conventionally synthesized uio-66 (without modulator) resulted in a less crystalline material and a less efficient exchange process . The linker - exchanged material, uio-66-[fefe](dcbdt)(co)6, was isolated as an orange microcrystalline powder after washing thoroughly with fresh meoh and activation under vacuum . G / cm, measured with nitrogen at 77 k. this value is close to the bet surface area of pristine uio-66 (1475 89 g / cm), suggesting a true pse process between 1 and the framework, and not simple trapping of the iron complex in the mof pores (which would be expected to produce a much lower surface area). N2 absorption / desorption isotherms of uio-66 and uio-66-[fefe]dcbdt(co)6 do indicate a modest decrease in pore size distribution (figure s1). Uio-66 is known to possess two pore types, tetrahedral and octahedral cages, with pore widths of 8 and 11, respectively (figure s2). Pristine uio-66 gave a median pore width of 11.8, while after incorporation of 1, a reduction in the median pore width to 10.9 was observed, consistent with incorporation of the [fe2s2] functionality in uio-66-[fefe]dcbdt(co)6 . Powder x - ray diffraction (pxrd) patterns before and after pse confirmed the retention of the crystalline uio-66 framework (figure 4a). Fe - sem showed that uio-66-[fefe](dcbdt)(co)6 possesses a nearly identical particle size and octahedral particle morphology compared to uio-66, again indicative of a pse mechanism (figure 3). Asterisks indicate remaining benzoic acid (modulator) and the black square indicates dcbdt . The degree of pse was characterized by energy - dispersed x - ray spectroscopy (edx), elemental analysis (ea), thermogravimetric analysis (tga), and proton nuclear magnetic resonance spectroscopy (h nmr). The ratio of heavy elements in uio-66-[fefe](dcbdt)(co)6 was determined to be 3.52:1:1.01 (zr: fe: s, normalized to fe) via edx, which suggests that 14% of bdc in uio-66 was exchanged for [fefe](dcbdt)(co)6 (figure s3). The expected 1:1 fe / s ratio determined by edx also further supports that the cluster is intact within the mof . Treatment of uio-66-[fefe](dcbdt)(co)6 with dilute hf / d - dmso was used to digest the mof but also decomposed [fefe](dcbdt)(co)6 to dcbdt . Integration of the proton resonances for bdc and dcbdt in the h nmr confirmed the degree of pse at 14% (figure 4), giving an overall formula for uio-66-[fefe](dcbdt)(co)6 as zr6o4(oh)4(bdc)5.1([fefe](dcbdt)(co)6)0.92ch3oh . Based on this given formula, c / h / n / s elemental analysis also confirmed the degree of functionalization [c(%): 32.75 (obs), 32.80 (calcd); h(%): 1.75 (obs), 1.68 (calcd); n(%): 0.00 (obs), 0.00 (calcd); s(%): 2.97 (obs), 2.84 (calcd)]. Unlike pristine uio-66, which displays only one major decomposition step at 400 c, the tga trace of uio-66-[fefe](dcbdt)(co)6 exhibits two decomposition steps at 80200 and 350400 c, respectively (figure 4). The first mass loss is likely due to partial thermal liberation of the carbonyl ligands attached to fe centers (obs: 7%; calcd: 7.2%). Both bdc and [fefe](dcbdt)(co)6 start to decompose at 350 c, leading to a combination of zro2 and fe2o phases (obs: 46.3%; calcd: 43.6%, percent weight residual mass). To confirm that compound 1 was being incorporated into the framework lattice to give uio-66-[fefe]dcbdt(co)6, additional experiments were performed to exclude the possibility that compound 1 was merely trapped in the pores of the mof . In one experiment, compound 2 (figure 1) contains the same cluster core but lacks the coordinating carboxylate ligands required for mof formation and hence pse . Incubation of uio-66 with compound 2 showed no evidence of substantial incorporation into the mof as shown by a lack of color change (figure s4) and a low iron content in the edx analysis (figure s5). In a second experiment, pse between uio-66 and 1 was performed in d2o, and the presence of bdc was observed in the reaction solution, as determined by h nmr (figure s6), indicative of displacement of bdc by 1 . Importantly, uio-66 in d2o in the absence of 1 does not show release of free bdc into solution . These nmr observations are also indicative of a ligand metathesis process and argue against simple inclusion of 1 into the pores of the mof . Finally, as stated above, performing pse between uio-66 and 1 in other solvents (meoh, dmf, and chcl3) was not efficient, achieving negligible incorporation (<2%), consistent with the known solvent dependence of pse processes . If complex 1 was only being included into uio-66 via sorption into the pores, then inclusion would not be expected to be strongly solvent dependent . Taken together, these experiments provide strong evidence, consistent with reported pse studies, that the iron cluster is becoming part of the uio-66 framework via a ligand pse process and that the data do not support simple inclusion of the cluster into the pores of the mof . In order to further demonstrate the incorporation of the intact fe2s2 dinuclear cluster into the mof, we employed fourier - transformed infrared spectroscopy (ftir) and diffuse reflectance uv vis spectroscopy . Ftir of uio-66-[fefe](dcbdt)(co)6 exhibited three prominent co stretching vibration bands at 2078, 2038, and 2001 cm, while no such absorption bands were observed for pristine uio-66 material between 2100 and 2000 cm (figure 4). Moreover, the relative intensity of these three characteristic bands was identical to that of free 1, suggesting the dinuclear cluster is intact in the mof . Solid - state uv vis spectroscopy of uio-66-[fefe](dcbdt)(co)6 also showed a characteristic absorption at 350 nm, which is consistent with the spectral features of 1 (figure s7). Due to the potentially labile nature of [fefe](dcbdt)(co)6, we sought to provide data to confirm the coordination environment of fe2s2 core in the mof . Fe k - edge extended x - ray absorption fine structure spectroscopy (exafs) was performed on both 1 and uio-66-[fefe](dcbdt)(co)6 . As shown in figure 5, fourier - transformed exafs in r space revealed nearly identical coordination environments of the fe centers in [fefe](dcbdt)(co)6 before and after pse into the uio-66 framework . Both sets of data were best fit using the first and second neighboring atoms of fe from the single - crystal x - ray structure obtained for [fefe](dcbdt)(co)6, where fe centers occupy a distorted octahedral geometry (see esi). Exafs of uio-66-[fefe](dcbdt)(co)6 suggests three carbon atoms from carbonyl groups and two sulfur atoms bridging the dinuclear fe2 center at distances of 1.7961.814 and 2.2832.285, respectively (table 1). Importantly, these bond lengths are in good agreement with the crystallographic data of 1 (see esi), showing 1.7971.811 (fe c) and 2.2552.257 (fe s). In addition, x - ray absorption near - edge structure indicates a common fe(i) oxidation state of the cluster within the framework and in 1 (figure s8). Fe k - edge exafs fourier transforms and exafs spectra (inset) for (a) 1 and (b) uio-66-[fefe](dcbdt)(co)6 . Solid black lines show the experimental data, dashed red lines show the fits based on crystallographic data of 1, and dotted gray lines show the fitting window . Fitting data having observed that pse could be used to incorporate complex 1 into a robust mof, we explored the suitability of this material as a catalyst in photochemical hydrogen production schemes . Thus, uio-66-[fefe](dcbdt)(co)6 was suspended in a 1.0 m acetate buffer solution of [ru(bpy)3] photosensitizer (0.5 mm), and ascorbate electron donor (100 mm) at ph 5 . As depicted in figure 6, the projected sequence for photocatalytic proton reduction by uio-66-[fefe](dcbdt)(co)6 commences with the reductive quenching of photoexcited [ru(bpy)3] by the electron donor ascorbate with a rate constant of 1 10 m s. following the analysis of schmehl et al ., 14% of * [ru(bpy)3] excited states can be expected to form the [ru(bpy)2(bpy)] reductant ([ascorbate] = 0.1 m; (*[ru(bpy)3]) = 500 ns). Charge recombination between photogenerated [ru(bpy)3] and oxidized ascorbate can be expected to occur close to the diffusion limit and will compete with the productive heterogeneous electron transfer (et) between [ru(bpy)3] and uio-66-[fefe](dcbdt)(co)6 . The driving force for the et can be estimated at 300 mv from the electrochemically obtained reduction potentials, assuming that the reduction potential of the fe2 site in uio-66-[fefe](dcbdt)(co)6 is similar to that obtained for complex 1 in solution . From solution studies, it is well established that the electrochemical reduction of 1, and its bdt derivative 2, is a two - electron process due to inverted electrochemical potentials . Assuming that the fe2 complex in uio-66-[fefe](dcbdt)(co)6 shows similar reductive chemistry as complex 2 in solution, the driving force for electron transfer from a second photogenerated [ru(bpy)3] to the previously produced monoreduced fe2 site in the mof will be even> 300 mv . The dianionic fe2 site 1 a second plausible pathway to 1 is through disproportionation of two singly reduced 1 in the mof . This disproportionation is thermodynamically feasible as evidenced by the inverted electrochemical potentials of the 1/1 and 1/1 couples . Reaction scheme for the photocatalytic reduction of protons . As demonstrated in figure 7, uio-66-[fefe](dcbdt)(co)6 is indeed a proton reduction catalyst . Under the reaction conditions described above, hydrogen production is observed and can be quantified with a hydrogen specific solid - state sensor (see esi for details). It is thus clear that heterogeneous electron transfer between photogenerated [ru(bpy)3] and uio-66-[fefe](dcbdt)(co)6 can compete with homogeneous charge recombination with oxidized ascorbate . The fe2 sites within the mof can be reduced in a light - driven reaction and are themselves catalysts for the reduction of protons to molecular hydrogen . As the rate of electron transfer from photogenerated [ru(bpy)3] decreases exponentially with distance, it can be assumed that only fe2 sites that reside within a few nm from the surface of the mof particles will be viable acceptor sites . The de facto concentration of operating catalysts in the mof may thus be substantially smaller than the total concentration of 1 in the mof . Comparing the activity of uio-66-[fefe](dcbdt)(co)6 with that of a homogeneous reference system that contains complex 1 at concentrations similar to the total amount of fe2 complex in uio-66-[fefe](dcbdt)(co)6 shows that the activity of the former is not only preserved but actually exceeds that of the latter, both in terms of initial rate as well as overall amount of produced hydrogen . Control experiments without uio-66-[fefe](dcbdt)(co)6 or with unmodified uio-66 (which does not contain [fefe](dcbdt)(co)6) do not show meaningful amounts of hydrogen generation (figure 7). Photocatalytic hydrogen production in the presence of uio-66-[fefe](dcbdt)(co)6 (blue trace, 5 mg mof, 0.59 mol catalyst), complex 1 (red, 0.59 mol), uio-66 (black, 5 mg mof), and background (gray). Conditions: 1 m acetate buffer ph 5, 100 mm ascorbic acid, 0.5 mm [ru(bpy)3]. Quantitative comparison between the homogeneous and heterogeneous hydrogen production systems must be done with great care, as hydrogen production in both systems is not limited by an intrinsic step of the catalytic cycle but by insufficient photoproduction of the [ru(bpy)2(bpy)] reductant . Nevertheless, figure 7 clearly shows that the heterogeneous system outperforms the homogeneous one both in overall hydrogen production as well as initial rate . As shown in a recent study, the photoproduction of [ru(bpy)2(bpy)] is not strongly influenced by homogeneous complex 2(39) and probably also not by uio-66-[fefe](dcbdt)(co)6 . The amount of available reductant can thus considered to be comparable in both systems . Also, the heterogeneous et rate constant ket, het in figure 6 is presumably not higher than the corresponding ket, hom in the homogeneous reference system . Therefore, the reasons for the superior catalytic performance of uio-66-[fefe](dcbdt)(co)6 compared to that of the homogeneous reference system must be due to differences in the catalyst . A trivial but nevertheless important rationale for the increased hydrogen production yield in the mof is the stabilization of the catalyst when inside the framework . Supporting this notion, it was found that uio-66-[fefe](dcbdt)(co)6 recovered after 1 h of photocatalysis still shows the characteristic co bands in the ir spectrum (figure s10). In contrast, and in accordance with published work, complex 1 decomposes under identical photocatalysis conditions, as evidenced by the lack of any ir signals in the typical co region . As for all photochemical reduction schemes based on [ru(bpy)3], one - electron photochemistry needs to be coupled to a two - electron catalytic process . With the limited availability of reductant, most productive et events will produce singly reduced fe2 sites (1), while dianionic fe2 species (1) are unlikely to be formed by an encounter with a second equivalent of [ru(bpy)2(bpy)] due to the low concentrations of both species . As discussed above, it is thermodynamically feasible that two 1 sites disproportionate to form the catalytically active 1 site that reacts with two protons to form hydrogen . Disproportionation as well as protonation needs to occur before the reduced species recombine with oxidized ascorbate . Here, the fe2 site in uio-66-[fefe](dcbdt)(co)6 has an undisputable advantage over the homogeneous system, as its incorporation into the mof spatially protects from unproductive charge recombination . Moreover, the presence of many fe2 sites within the mof may promote disproportionation as soon as two monoreduced sites are present . It is these kinetic advantages that explain the higher initial hydrogen production rates in uio-66-[fefe](dcbdt)(co)6 . We employed pse as an efficient and mild approach to obtain the first mof that contains a multinuclear, organometallic, nonprecious - metal - based proton reduction catalyst . The resulting uio-66-[fefe](dcbdt)(co)6 is a hybrid material that combines the advantages of molecular catalysts with a highly ordered and stable inorganic support . [fefe](dcbdt)(co)6 (1) is among the most complex structure that has ever been introduced into a mof, and its presence and molecular integrity within the uio-66 framework could be confirmed by exafs, ftir, and other methods . Uio-66-[fefe](dcbdt)(co)6 exhibits high efficiency for photochemical hydrogen evolution, exceeding that of the homogeneous reference system in terms of rate and total hydrogen production yield . Incorporation of the fe2 complex in uio-66-[fefe](dcbdt)(co)6 results in a higher stability under the photocatalysis conditions, protects reduced species from nonproductive charge recombination, and may promote disproportionation reactions to produce catalytically active dianion 1.
Aging is part of the natural process of human development, and it brings multiple biopsychosocial changes that cause a slow and progressive deterioration of body functions that are essential to life . Among these functions that undergo important mostly irreversible changes is hearing.1 hearing loss is considered one of the three most common conditions in the elderly,2 with occurrence of 5 to 20% in 60-year - olds, increasing to 60% in people over 65 years old.1 3 in brazil, 60% of the elderly have some degree of hearing loss.4 the term used to describe hearing loss associated with aging is presbycusis, which causes a decrease in hearing sensitivity and a reduction in the ability to understand speech . This occurs due to several degenerative and physiologic changes that affect the inner ear, impairing hearing at high frequencies and affecting communication, especially in noisy environments.1 2 4 5 6 7 other factors associated with the natural degeneration process, such as exposure to loud noise, ototoxic agents, and sequelae of otitis media caused by medical problems and treatments, can contribute to lifelong aggravation of the loss.4 8 9 difficult communication associated with hearing loss can lead to social and emotional consequences, especially for the elderly, who have several limitations related to aging that can impact their quality of life, increasing social isolation in addition to causing emotional disorders such as depression.1 4 5 8 10 11 hearing loss associated depression can be even more harmful in the elderly . Thus, recommended intervention by hearing aid adaptation contributes to the prevention of emotional disorders such as depression.12 depression is defined as a mental disorder that causes feelings of sadness, pessimism, hopelessness, difficulty concentrating, and difficulty making decisions and initiating tasks, among other symptoms.13 despite being very common in the elderly, it cannot be attributed solely to aging . Studies in so paulo (brazil)14 and florianpolis (santa catarina, brazil)15 showed that the factors associated with depression in the elderly are: female, unmarried or widowed, altered cognitive function, dependence, use of many medicines, very old (over 80 years), low education, poor economic status, cognitive impairment, fair or poor self - rated health, functional dependency, and chronic pain . Other causes include smoking, comorbidities (endocrine, vascular, neurologic, oncological), and negative changes in relationships with family and friends.15 hearing loss may be associated with depression to be a debilitating, chronic disorder.16 17 18 the main goal of an auditory rehabilitation program in elderly patients is to minimize the effects caused by sensory deprivation of hearing and reinstate the patients in their family and society, helping them cope with the disadvantages and limitations caused by hearing loss.3 monaural adaptations are commonly performed even in cases of bilateral hearing loss, whereas the most appropriate should be binaural adaptation . The reasons for that may be related to factors such as rejection to the use of two hearing aids, financial issues, aesthetic reasons, problems with manual dexterity, very asymmetric losses, and central processing issues.19 given such reality, this work intends to analyze the effects of unilateral adaptation of hearing aids on the symptoms of depression and the social activity constraints of elderly subjects with hearing impairment . This is a prospective interventional study of elderly individuals of both sexes with recommended use of hearing aids at a hearing center in porto alegre . Inclusion criteria were signature of the free and informed consent; 60 years or older; good health (seniors able to commute to the research location, conduct tests, and answer questionnaires); new hearing aid users with monaural fitting; and participation in all phases of the research . Exclusion criteria were initiation of antidepressant therapy during the study and a history of cognitive and/or neurologic disorders . The study was divided into two phases . In phase 1, subjects responded to an interview with questions on demographics and health (general and hearing). Pure tone audiometry was performed to determine the airway tone thresholds (frequencies 250 to 8,000 hz) and bone conduction (frequencies 500 to 4,000 hz) in a vocal booth, using an ad-229e interacoustics audiometer (assens, denmark). The presence and degree of hearing loss were classified according to the world health organization by analyzing mean airway tone threshold in the frequencies of 500, 1,000, 2,000, and 4,000 hz.20 after that, seniors were asked to answer two questionnaires individually, without the intervention of third parties: the geriatric depression scale (gds) and the hearing handicap inventory for elderly short version (hhie - s). The gds, an instrument validated for portuguese and composed of 15 yes - or - no questions, is used to detect depression symptoms in the elderly . Thus, scores less than 5 points indicate absence of symptoms, scores of 5 to 10 indicate mild to moderate symptoms, and scores of 11 or more indicate severe symptoms of depression.21 after completing the first phase, participants answered a summarized version of the hhie - s . The questionnaire was prepared in 198222 and translated and adapted to brazilian portuguese.23 it contains 10 questions that aim to assess the impact of hearing loss on the emotional and social status of elderly people . All 10 questions provide three choices of answers: for each yes answer, 4 points is assigned; each sometimes, 2 points; and each no, 0 points . The total score ranges from 0 to 40 points, and the higher the score, the greater subjects' self - perception of social activity constraints: 0 to 8 points indicate no social activity constraints; 10 to 23 points, mild to moderate social activity constraint; and 24 to 40 points, significant social activity constraints.8 22 after testing, the participants were subjected to the selection and unilaterally hearing aid adaptation processes thirty days after purchasing the device, they returned to the hearing center to participate in the second phase of the research . In phase 2 the research project was approved by the research committee and by the ethics and research committee of the institution (protocol 266.060). Patients who agreed to participate signed an informed consent and had their rights guaranteed for confidentiality, nonidentification, and withdrawal of participation . The results were analyzed using the statistical package for social science version 20.0 (ibm, ny, usa). To examine associations of categorical data, the chi - square and student t test for paired samples were used . All statistical tests were nonparametric tests, and always observed exact p value (not asymptotic), which are the best measures to statistically evaluate a study when the sample is restricted . The chi - square test considered the p value of fisher exact test, and for the kappa coefficient of agreement, the exact p value (not asymptotic) was observed . The sample consisted of 13 elderly patients with hearing loss, ages from 60 to 90 years (mean 72.85 11.05 years), 10 women (76.9%) and 3 men (23.1%). Table 1 shows data on the characterization of sample in terms of age, gender, mean thresholds, and degree of hearing loss . Table 2 depicts an association analysis between the gds classification of phases 1 and 2, indicating that there was no significant association between the variables in the periods before and after the adaptation (p = 0.615). Thus, of the 6 patients (46.2%) who initially had mild symptoms of depression, only 2 (15.4%) of them continued to have mild symptoms . All seniors who initially had severe symptoms of depression (15.4%) also started to lose some symptoms of depression in phase 2 . Hhie classification analysis in phases 1 and 2 of the study are presented in table 3 . No significant association was found for these variables in the periods before and after the adaptation (p> 0.999). Thus, the only senior who had mild to moderate social activity constraints in the first phase showed reduction in the second phase . After prosthetization, participants who had severe social activity constraints in phase 1 started to show no social activity constraints or mild to moderate social activity constraints . Only 1 (7.7%) the lack of association between the classification of the gds and hhie on the two phases is an important point, because it shows that there were different results in both assessments (before and after hearing aid adaptation)that is, on the sample studied, unilateral prosthetization resulted in benefits for patients . Table 4 shows the scores obtained by the subjects in the instruments used for evaluation in the two phases of the research . There was significant difference between the gds (p = 0.031) and hhie (p <abbreviations: gds, geriatric depression scale; hhie - s, hearing handicap inventory for elderly short version . The analysis of the survey data showed that the age of the individuals analyzed varied from 60 to 90 years . Another study that examined the degeneration of the auditory system in the course of aging had a sample with similar age and results.4 we had a greater number of female participants . Although it has been found that men often have more problems related to hearing loss than women,24 they also have a lower perception of their disability compared with women,1 25 26 which explains why women seek health services more often26 27 and why there is a greater number of female participants in this study . Another fact to be noted is that, in terms of population, there is a greater number of older women in brazil, a fact known as feminization of aging.28 there was a prevalence of moderate hearing loss in both the right and the left ears, agreeing with other published studies that refer to this as being the most commonly found degree of hearing impairment in the elderly.2 9 25 the data show that many seniors had some degree of depression symptoms before prosthetization (table 2). After unilateral hearing aid adoption, there was improvement in symptoms for most of them . Thus, a hearing aid, even when unilateral, promoted elimination or reduction of symptoms of depression in the sample studied . This result had already been described in the literature,2 but we emphasize that the result was obtained with bilateral prosthesis . Thus, the adaptation of a single amplifying device can also generate benefit for elderly patients, which is important for the reduction of one of the most prevalent problems in the aging population, depression . The data show that all participants had some degree of social activity constraints before using a unilateral hearing aid (table 3). After 30 days of hearing aid use, 12 (93.3%) and 13 (100%) participants showed absence or reduction of social activity constraints, respectively . Results show evidence of the positive effects of hearing aid use, even if unilateral, helping to reduce the impact that hearing loss has on social relationships and on the quality of life of the elderly.9 results are consistent with another study that examined the short - term benefits of amplification in new users.29 the data also show a significant difference in scores obtained in phases 1 and 2 of the gds (p = 0.031) and hhie - s (p <0.001), confirming a reduction or elimination of depression symptoms and social activity constraints in elderly patients, agreeing with other published studies (table 4).2 8 11 29 30 again, it is important to highlight that in the studied group, unilateral prosthesis resulted in substantial benefits not only regarding hearing but also in other matters essential for the well - being and quality of life of individuals . The findings reinforce the importance of hearing aid use in elderly patients, because many times family and social relationships are affected by the hearing impairment . The adaptation to amplification devices, albeit unilaterally, has proved to be effective in reducing symptoms of depression and social activity constraints . Moreover, the length of time between assessments was 30 days, shorter than that considered ideal for auditory acclimatization, and even then, benefits occurred, confirming the importance of auditory rehabilitation in elderly . Results show that unilateral hearing aid contributed to the elimination or reduction of depression symptoms and of social activity constraints in elderly participants in this research sample group.
Ameloblastomas are rare benign odontogenic tumours, most commonly found in a patient s lower jaw . Although rarely metastatic, the growth of these lesions can result in defects in the jaw easily destroying surrounding bony tissues . For this reason, the treatment is often excision of the lesion, with wide excision margins, resulting in the absence of a part, or all, of the mandible . The surgical approach for correction of this mandibular defect is often reconstruction of the mandible in view of inserting dental implants at a later date . Despite surgical advancements,, bone grafting with non - vascularized bone remains a good option where facilities for soft tissue flaps are not readily accessible . Creating an effective wound closure and reducing the incidence of infection is an important stage in the success of the operation, and has been combated here by tissue expanding the submucosa, using osmed pellets, prior to surgery . This is a relatively new technique to the mandibular region and has the potential to revolutionize the success of microsurgeries in this area . In this report, i describe a woman who underwent a repair of her mandibular defect by a combination of innovative techniques, in order to reduce the risk of complications and get her back to an aesthetically pleasing level . A 35-year - old lady presented to the craniofacial surgeons looking for a permanent fixture to the bony defect in her lower - right mandible . In 1991 she had her first operation to remove the ameloblastoma and then had it further excised 6 years later due to a reoccurrence . This resulted in an absence of teeth from the canines back and removal of bone down to the inferior alveolar nerve . The patient has since been clear of any further reoccurrences and wears lower dentures for aesthetic reasons . She has no significant past medical history nor family history, nor does she have any known drug allergies . She does not smoke or drink alcohol . On clinical examination, the patient experiences mild parasthesia in the distribution of her right inferior dental nerve, particularly the lateral aspect of the lower lip . On examination, there is clear absence of teeth on the lower - right side from the canines back (to include the premolar, molar and wisdom teeth) and presence of healthy oral mucosa with laxity to move the submucosa medially . There is a loss of the vertical height of the posterior body of the mandible of about 50% and the vestigial remnants of the lingual plate are just palpable and visible on plain radiograph and ct . Scans show that the superior margin of the resection is well corticated under the inferior dental nerve (fig ., the patient wishes to get dental implants . The surgical approach taken, prior to the implants, (b) ct scan of head imaging of the patient's mandibular defect pre - surgically . (a) radiolocal image of patient's jaw . (b) ct scan of head the patient was initially given the option between a bone graft or vascularized tissue (free fibular flap) and informed of the complications a bone graft carries a risk of rejection and vascularized tissue needs an increased amount of post - operative care to ensure that the tissue remains vascularized . The patient was not keen on a free flap and given the fact that the bony deformity was <5 cm, non - vascularized iliac bone graft was deemed to be sufficient to carry out this operation . The first step was achieving significant tissue expansion (> 2 cm) using osmed pellets, guided into the submucosa and kept over the alveolar ridge for several months (fig . (c) osmed pellets before insertion into the submucosa image of patient's oral cavity during insertion of osmed pellets . (a) incisions made in the submucosa . (c) osmed pellets before insertion into the submucosa two months later, the patient underwent the main bulk of her operation . The osmed pellets were removed from the subperiosteum (fig . 3a and b) and the iliac bone graft was taken, made up of cortical and cancellous bone and placed to one side (fig . An epidural catheter was placed inside the soft tissue of the graft site and left overnight . It had a continuous fusion of a local anaesthetic to improve post - operative pain and encourage early mobilization . (b) open site where iliac bone graft is harvested from (c) cortical and cancellous iliac bone submucosa after adequate tissue expansion . (b) open site where iliac bone graft is harvested from (c) cortical and cancellous iliac bone previously, using ct scans, a titanium construct was made to guide the outline of the bone graft and complete the missing height and width of the mandible . Once fitted to the patients jaw, it was packed with the bone graft and screwed down into position (fig . A slight groove was made on the mandible to allow space for the inferior dental nerve to lay, so that the construct did not compact the nerve and cause functional problems . Figure 5:titanium construct packed with iliac bone graft and screwed into position on the lower mandible titanium construct packed with iliac bone graft and screwed into position on the lower mandible to complete the surgery, the periosteum was sutured over the construct and then the buccal muscosa was sutured over again to form a double - layered closure (fig . 6a c).the purpose of this was to prevent infection and rejection, by ensuring that there was an increased distance between the foreign object (titanium construct) and the outside environment . The dental implants are scheduled to be inserted several months after this operation . Figure 6:image of patient's surgical site . (c) image of post - surgical site image of patient's surgical site . Bony defects in the maxillofacial area are a huge problem in clinical orthodontics, caused by a wide range of injuries and diseases presenting as either cosmetic or functional defects and in some instances both . It is the importance of accounting for the anatomical, functional and aesthetic aspects that make the reconstruction so challenging . Often patients suffering from ameloblastomas undergo resection as treatment and are left with bony defects that require reconstruction . In this case, the intention was to realize an aesthetically pleasing appearance for the patient and it has been shown that bone grafting with subsequent dental implants is a successful method . As in this case, it is recommended that non - vascularized autogenous bone grafting can be used when a two - layer watertight closure is attainable . Sources of bone could have been harvested from either local or distant sites but in this case, an extra - oral site (iliac bone) was used as a moderate amount of bone was needed . The surgery lasted 5 h and incorporated several novel techniques to improve the outcome of the operation . The use of osmed pellets was innovative, as pellets of this size have only recently been experimented on goats maxilla . As well as allowing for space to put the construct, the pellets allowed for a tension - free closure . Otherwise, the result would have been a high - tension closure, leading to rapid breakdown of the graft providing an entrance for infection, a common complication occurring when titanium plates are exposed . Advantages of the osmed tissue expanders are that the material is safe with a low complication rate and low risk of infection due to small incision site and minimal trauma . The titanium construct is another relatively novel approach and was invaluable in the success of this surgery, as it guided reconstruction of the mandible, allowing for dental implants to be put in at a later date . At present, an alloplastic material such as titanium is often used for mandibular reconstruction as it is resistant to corrosion, adaptable and biocompatible . Although the insertion of dental implants is the end goal for this surgery, the importance of this case report is to highlight the novel techniques involved in making this surgery a success . The use of small osmed pellets in the submucosa of the mouth to cause sufficient tissue expansion and create a tension - free closure, combined with the titanium construct packed with grafted iliac bone to rebuild the jaw was an approach recommended by the doctors for the repair of bony defects of <5 cm . I suggest prudent management of future cases post - operatively to ensure a continued success of the graft.
Glucagonlike peptide1 (glp1), the most potent incretin hormone, stimulates glucoseinduced insulin secretion and inhibits glucagon secretion, which consequently leads to a decrease in hepatic glucose production and blood glucose levels . In addition to its ability to modulate insulin and glucagon secretion, glp1 inhibits gastric emptying and gastric acid secretion, which suppress appetite and energy intake in obese individuals, and in patients with type 2 diabetes . Glp1 is secreted from intestinal l cells in response to ingestion of nutrients, including carbohydrate and lipids . A previous study showed that alphalinolelic acid (la) promoted glp1 secretion through the stimulation of gproteincoupled receptor (gpr) 120, which is abundantly expressed in the intestine, in mice . Furthermore, a recent study has shown that eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), metabolites of la, stimulate endogenous glp1 secretion in mice . These reports show that 3 polyunsaturated fatty acids (pufa), including la, epa and dha, increase glp1 secretion in mice . However, no evidence that 3 pufa ingestion increases glp1 secretion is available in humans . As compared with americans and europeans, japanese eat more fish, which is a major source of epa and dha . Hence, serum epa or dha concentrations are higher in japanese than those in americans or europeans . Evidence that fish intake prevents atherosclerosisrelated cardiovascular disease have been accumulating . A recent study has shown a significant inverse correlation between serum epa or dha concentrations and cardiovascular risk . Furthermore, some studies showed that fish intake reduced the rates of incidence for diabetes in patients with metabolic syndrome, and reduced the rates of death in patients with type 2 diabetes . However, the direct effects of epa or dha consumption on glycemic control in patients with type 2 diabetes have remained uncertain . Dpp4 inhibitor increases endogenous active glp1 levels through inhibition of dpp4 enzyme activity, leading to increased circulating insulin levels and decreased blood glucose levels . The circulating glp1 levels were reported to be lower in patients with type 2 diabetes than in nondiabetic patients . Therefore, dpp4 inhibitor might be more effective in increasing circulating glp1 levels in patients with type 2 diabetes if they have low glp1 levels . Based on these lines of evidence, we hypothesized that dpp4 inhibitor would be effective in patients with type 2 diabetes with high serum 3 pufa concentrations . In the present study, therefore, we investigated the relationship between serum epa or dha concentrations and a decrease in hemoglobin a1c in patients with type 2 diabetes prescribed dpp4 inhibitors . Serum epa and dha concentrations were measured in 62 consecutive patients with type 2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine, kyoto, japan, who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents (oha). Patients with advanced renal dysfunction (serum creatinine concentration was equal to or more than 2.0 mg / dl) were excluded from the present study . Sitagliptin at a daily dose of 50 mg, which was the standard dose in japanese patients, was continuously given once daily during the observation period . The prior diet, exercise program and dosage of oha in the study patients were maintained during the observation period . After 24 weeks of treatment with dpp4 inhibitor, we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations, as well as age, sex, body mass index (bmi), hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs . Type 2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus . Approval for the study was obtained from the local research ethics committee, and informed consent was obtained from all patients . Serum total cholesterol, highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods . Hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society skewed variables, such as epa and dha, are presented as the median (interquartile range), and continuous variables are presented as the mean standard deviation (sd). Paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment, and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software (version 5.0; sas institute, cary, nc, usa). Because epa and dha showed skewed distributions, logarithm (log) transformation was carried out before performing correlation and regression analysis . The relationship between log epa, log dha or log (epa + dha) and a decrease in hemoglobin a1c was examined by linear regression analysis . In multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c, we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses . Serum epa and dha concentrations were measured in 62 consecutive patients with type 2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine, kyoto, japan, who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents (oha). Patients with advanced renal dysfunction (serum creatinine concentration was equal to or more than 2.0 mg / dl) were excluded from the present study . Sitagliptin at a daily dose of 50 mg, which was the standard dose in japanese patients, was continuously given once daily during the observation period . The prior diet, exercise program and dosage of oha in the study patients were maintained during the observation period . After 24 weeks of treatment with dpp4 inhibitor, we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations, as well as age, sex, body mass index (bmi), hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs . Type 2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus . Approval for the study was obtained from the local research ethics committee, and informed consent was obtained from all patients . Serum total cholesterol, highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods . Hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society . Means, medians or frequencies of potential confounding variables were calculated . Skewed variables, such as epa and dha, are presented as the median (interquartile range), and continuous variables are presented as the mean standard deviation (sd). Paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment, and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software (version 5.0; sas institute, cary, nc, usa). Because epa and dha showed skewed distributions, logarithm (log) transformation was carried out before performing correlation and regression analysis . The relationship between log epa, log dha or log (epa + dha) and a decrease in hemoglobin a1c was examined by linear regression analysis . In multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c, we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses . Characteristics of the 62 patients with type 2 diabetes enrolled in the present study are shown in table 1 . The median (interquartile range) serum epa concentration was 62.6 g / ml (39.588.0) and the median (interquartile range) serum dha concentration was 139.1 g / ml (109.4165.7). Among 62 patients, 16 patients were given dpp4 inhibitor as a monotherapy and 46 patients were given it as an addon therapy (34 sulfonylurea, 25 metformin, 8 pioglitazones, 5 alphaglucosidase inhibitor). Data are n, mean sd or median (interquartile range). Mean hemoglobin a1c was significantly decreased from 8.1 1.1% at baseline to 7.2 1.0% at 24 weeks after administration of dpp4 inhibitor . Correlation between log epa, log dha or log (epa + dha) and other variables are shown in table 2 . Log epa and log (epa + dha) correlated with a decrease in hemoglobin a1c . Log dha tended to correlate with a decrease in hemoglobin a1c, but it did not reach statistical significance . Dha, docosahexaenoic acid; epa, eicosapentaenoic acid . A decrease in hemoglobin a1c correlated with bmi (r = 0.396, p = 0.0013), age (r = 0.275 p = 0.0032), hemoglobin a1c at baseline (r = 0.490, p <0.0001), log epa (r = 0.285, p = 0.0246) and log (epa + dha) (r = 0.260, p = 0.0411). There were no significant differences in decrease in hemoglobin a1c between male and female patients (1.0 0.8% vs 1.0 0.9%, p = 0.9758). Decrease in hemoglobin a1c was greater in patients treated with antidiabetic concomitant drugs compared with that in patients without those (1.1 0.9% vs 0.6 0.4%, p = 0.0342). In the multiple regression analysis to examine the effects of variables on the decrease in hemoglobin a1c, we included the following independent variables, which were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses: age, bmi, hemoglobin a1c at baseline, usage of antidiabetic concomitant drugs and log epa . Multiple regression analysis showed that bmi, hemoglobin a1c at baseline and log epa were independent determinants of a decrease in hemoglobin a1c (table 3). Presence of antidiabetic concomitant drugs (no = 0, yes = 1). In the present study, serum epa concentrations correlated with a decrease in hemoglobin a1c . This correlation remained significant after adjustment for age, bmi, hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs . The median (interquartile range) serum epa concentration was 62.6 g / ml (39.588.0; the mean serum epa concentration was 68.6 34.1 g / ml), which was almost consistent with previous reports of the epa concentration in general japanese . Serum epa concentrations increase with age in apparently healthy japanese . In the present study, however, there was no significant correlation between log epa and age in patients with type 2 diabetes . Dpp4 inhibitor increases endogenous active glp1 levels through prevention of glp1 degradation, leading to an increase in circulating insulin levels and a decrease in blood glucose levels . Because of the pharmacological action of dpp4 inhibitor on insulin secretion, dpp4 inhibitor is effective in type 2 diabetic patients with impaired insulin secretion, which is a characteristic of japanese patients with type 2 diabetes . Indeed, as compared with american and european patients with type 2 diabetes, japanese patients with type 2 diabetes might be higher responders to dpp4 inhibitor . Interestingly, the present study has shown a strong correlation between bmi and decrease in hemoglobin a1c, suggesting that dpp4 inhibitor is more effective in lean patients who generally have impaired insulin secretion, rather than in obese patients with insulin resistance . This is consistent with a recent study reporting the association of bmi with efficacy of the dpp4 inhibitor, sitagliptin . Previous studies showed that dpp4 inhibitor was effective as an addon therapy to some antidiabetic drugs, including metformin, pioglitazone and sulfonylurea . The profile of diabetic therapy before dpp4 inhibitor administration was as follows: 16 used diet, 15 received sulfonylurea monotherapy, 12 received metformin monotherapy, eight received sulfonylurea and metformin, five received sulfonylurea and pioglitazone, two received sulfonylurea and alphaglucosidase inhibitor, and four took three drugs . We analyzed the correlations between serum epa concentrations and a decrease in hemoglobin a1c in patients with dpp4 inhibitor monotherapy, dpp4 inhibitor addon therapy to sulfonylurea, to metformin, and to sulfonylurea and metformin . Log epa tended to correlate with a decrease in hemoglobin a1c only in patients with dpp4 inhibitor addon therapy to metformin, although it did not reach statistical significance (r = 0.526, p = 0.0796). There were no significant correlations between log epa and decrease in hemoglobin a1c in other treatment groups . It seems to be difficult to show these results because of the small sample size in each treatment group . In basic studies have shown that gpr120 functioned as a receptor for unstructured longchain fatty acids, and that the stimulation of gpr120 with la promoted glp1 secretion in vitro and in vivo . Furthermore, gpr120 has been shown to be abundantly expressed in the intestine both in humans and mice . . However, the mechanism of glp1 secretion by 3 pufa has been less clear . In the present study, furthermore, serum dha concentrations did not show a significant association with the efficacy of dpp4 inhibitor, although they tended to positively correlate with a decrease in hemoglobin a1c . Further studies are required to clarify the mechanism of 3 pufastimulated glp1 secretion . Taken together with these previous studies, and the ability of dpp4 inhibitor to increase endogenous active glp1 levels, the present result that serum epa concentrations correlated with the efficacy of dpp4 inhibitor might provide indirect evidence that epa has a potential to promote glp1 secretion in humans . Limitations of the present study included a small sample size and a short observation period . Therefore, the direct link between serum epa concentration and glp1 or insulin concentrations could not be assessed . Furthermore, because ingested fatty acids are generally absorbed within the small intestine, epa might be expected to act on gpr120 inadequately after oral administration . There are no reports of a linear relationship between epa intake and serum epa concentrations . However, serum epa concentrations positively correlated with epa intake assessed by a selfadministered diet history questionnaire in a subgroup of patients with type 2 diabetes (n = 40, r = 0.419, p = 0.0066). Therefore, we hope that serum epa concentrations could be a marker of epa intake . To our knowledge, this is the first report of the relationship between serum epa concentrations and the efficacy of dpp4 inhibitor in patients with type 2 diabetes, and suggests new avenues for research into incretin therapies . Large interventional studies are required to better assess the contributions of epa to the efficacy of dpp4 inhibitor in patients with type 2 diabetes . In conclusion, dpp4 inhibitor is effective in patients with type 2 diabetes with high serum epa concentrations.
Pregnancy course was regular, except for maternal consumption of mebendazol at 5 weeks' gestation to treat a pinworm infection . Maternal history was negative for consumption of any known teratogenic class drug during the whole pregnancy . Clinical examination at birth was normal, except for complete congenital anonychia of left middle finger (fig.1). X - ray was performed, showing an absence of the left middle finger distal phalanx (fig.2).the rest of the nails on the fingers and toes were normal . Parents were not consanguineous and have normal fingernails and toenails . The baby was discharged on the third day of life, and was found healthy and thriving at three - month follow - up visit . Final diagnosis was that of a sporadic co. x - ray showing absence of left middle finger distal phalanx . Congenital onychodystrophy (co), also called as iso kikuchi syndrome, was first described by iso in 1969 1 and later by kikuchi in 1974 2 as a clinical syndrome involving dysplasia / absence of fingernails with underlying bone abnormalities . Since these first observations, clinical criteria have been expanded to include a number of additional associated conditions derived from small series, case reports, and retrospective reviews over the next 30 years . Co clinical criteria are the following: unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 (up to total anonychia of hands and feet); radiographic abnormalities of the distal bony phalanx of the affected fingers; congenital occurrence, which can be both sporadic or hereditary 4,5 . Unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 (up to total anonychia of hands and feet); radiographic abnormalities of the distal bony phalanx of the affected fingers; congenital occurrence, which can be both sporadic or hereditary 4,5 . Genetic loci responsible for the condition are still under investigation: linkage to the known keratin gene clusters on 12q12 and 17q21 has been excluded by krebsova et al . In 2000; 7 a putative isolated congenital nail dysplasia locus, designated ndic, has been identified on 17p13, although the identified region harbors no genes known to be involved in skin or nail abnormalities 7 . Several acquired isolated nail disorders which may represent differential diagnoses of co are presented in table1 8 . Differently from all these conditions, co presents as a congenital hypoplasia, dysplasia, or absence of one or more fingernails, and is typically accompanied by underlying phalanx bone disease . Acquired isolated nail disorders 8 several heterogeneous multisystem pathologies may also come with ungueal abnormalities; these are summarized in table2 813 . 813 triangular nail lunula fingernails and toenails hypoplasia 8 absent or hypoplastic patellae elbow dysplasia, often involving posterior subluxation of the radial head iliac horns dysplasia arrested or reduced nail growth thickened and over - curved nails with absence of cuticle differently from the above - mentioned conditions, classic co does not come with involvement of systems other than nails and relative phalanges . Kikuchi originally suggested fetal grip as a causative mechanism of nail and phalanx ischemia, leading to dysplasia or even complete resorption of such structures 14 . This theory, however, is not consistent with the recent acquisitions on developmental biology stating that limbs and bones develop early in fetal life, prior to fetus' ability to exert a significant grip . More recently, several pathogenetic mechanisms have been proposed for this condition: selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury: the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15, which should be more protected than the ulnar one;16 in utero dysplastic change in the crescent - shaped cap of the distal phalanx, a theory that would account for the frequent y shape of the dysplastic distal phalanx, when present;17 genetic mutations causing impairment of the wnt signaling pathway, an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development, growth regulation of multiple tissues, and cancer development;18 fetal exposure to teratogens, particularly antiepilectic drugs: phenytoin 19, valproate, and carbamazepine20 selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury: the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15, which should be more protected than the ulnar one;16 in utero dysplastic change in the crescent - shaped cap of the distal phalanx, a theory that would account for the frequent y shape of the dysplastic distal phalanx, when present;17 genetic mutations causing impairment of the wnt signaling pathway, an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development, growth regulation of multiple tissues, and cancer development;18 fetal exposure to teratogens, particularly antiepilectic drugs: phenytoin 19, valproate, and carbamazepine20 a literature research showed no proven association between the use of mebendazole during pregnancy and co. conversely, helminth infection during pregnancy is associated with poor cognitive and gross motor outcomes in infants, so that measures to prevent helminth infection during pregnancy should be reinforced;21 anthelminthic therapy is actually recommended in infected pregnant women, and it has been associated to a decreased rate of maternal anemia and low birth weight 22 . Nevertheless, clinical follow - up is needed in order to exclude other conditions associated with multisystem pathology; a complete family history may also be warranted to determine sporadic or hereditary transmission of such a condition.
Glycogen synthase kinase-3 (gsk-3), an evolutionarily conserved ubiquitous serine threonine kinase consisting of and isoforms, is a multifaceted protein with diverse cellular and neurophysiological functions . The main structural difference between gsk-3 and gsk-3 isoforms lies in the n- and c - terminal regions, while their sequences within the kinase domain are highly homologous . A growing body of evidence indicates that gsk-3 is pro - apoptotic and that its dysfunction may be linked to the pathophysiology of mood disorders, schizophrenia, diabetes, and various neurological / neurodegenerative diseases, among others (for review, meijer et al ., 2004;, 2007; chiu and chuang, 2010; li and jope, 2010). Gsk-3 inhibition has attracted widespread attention as one of the critical therapeutic targets whereby lithium exerts its effects on mood stabilization, neurogenesis, neurotrophicity, neuroprotection, anti - inflammation, and others (for review, rowe and chuang, 2004; rowe et al ., 2007; beurel et al ., 2010). Pharmacological inhibition or gene knockout / knockdown of this kinase mimics the anti - depressant and anti - manic effects of lithium observed in rodent models (gould et al ., 2004; kaidanovich - beilin et al, 2004, 2009; obrien et al ., 2004; rosa et al ., 2008; omata et al ., 2011). The activities of gsk-3 are negatively regulated by phosphorylation of gsk-3 at ser21 and gsk-3 at ser9 . Gsk-3 can be inhibited by lithium through direct binding to the atp - dependent magnesium - sensitive catalytic site of the enzyme (klein and melton, 1996; stambolic et al ., 1996), and/or indirectly through enhanced serine phosphorylation of gsk-3 isoforms by multiple mechanisms (figure 1). Lithium has been shown to enhance gsk-3 serine phosphorylation by activation of protein kinase a (pka; jope, 1999; liang et al ., 2008), or phosphatidylinositol 3-kinase (pi3-kinase)-dependent akt (chalecka - franaszek and chuang, 1999) and protein kinase c- (kirshenboim et al ., 2004 it has also been reported that lithium can disrupt the -arrestin-2pp2a akt complex that dephosphorylates / inactivates akt, thereby enhancing gsk-3 serine phosphorylation (beaulieu et al ., 2005). Moreover, it has been proposed that lithium can interrupt auto - regulation of gsk-3 via disinhibition of the inhibitory action of inhibitor-2 complex on protein phosphatase-1 (pp-1; zhang et al ., 2003). This article reviews the findings supporting the role of gsk-3 inhibition in mediating lithium s neuroprotective effects against excitotoxicity in both cultured neurons and animal models of ischemic stroke . Lithium, a competitive inhibitor of magnesium, directly inhibits atp magnesium - dependent catalytic activity of gsk-3 . Lithium can indirectly increase this serine phosphorylation of gsk-3 through pi3-kinase - mediated phosphorylation / activation of akt, pi3-kinase - mediated activation of pkc, and camp - dependent activation of pka . Lithium can also increase the serine phosphorylation of gsk-3 by disrupting the -arrestin-2 (arr2)pp2a akt complex that dephosphorylates and inactivates akt . In addition, by disinhibiting the inhibitory action of inhibitor-2 (i-2) on protein phosphatase - l (pp-1) that dephosphorylates gsk-3 at serine residues, lithium s direct inhibition of gsk-3 interrupts this auto - regulation of gsk-3 and further decreases gsk-3 activity . Lines with solid arrows represent stimulatory connections; lines with flattened ends represent inhibitory connections . We have designed isoform - specific small interfering rnas (sirnas) to distinguish the functional and regulatory differences between the two gsk-3 isoforms in rat cerebral cortical neuronal cultures (liang and chuang, 2007). Transfection with sirna for gsk-3 or gsk-3 or with dominant - negative mutants specific for either isoform produced almost complete protection against glutamate - induced, n - methyl - d - aspartate (nmda) receptor - mediated excitotoxicity . The sirna - induced neuroprotection was associated with enhanced n - terminal phosphorylation in both gsk-3 isoforms . Moreover, transfection with or isoform - specific dominant - negative mutants of gsk-3 mimicked lithium - induced neuroprotection against glutamate excitotoxicity . These results strongly suggest that both gsk-3 and are involved in glutamate - induced neuronal death and that both isoforms are the initial targets of lithium - elicited neuroprotection . Gsk-3 has also been implicated in neuronal development, maturation / differentiation, and aging in the mammalian cns (spittaels et al ., 2002; kim et al . Substrates phosphorylated by gsk-3 include metabolic, signaling, and structural proteins as well as transcription factors . It is known that inhibition of gsk-3 results in activation, and sometimes suppression, of an array of transcription factors (for review, grimes and jope, 2001; jope and roh, 2006; chiu and chuang, 2010). Among the long list of transcription factors regulated by gsk-3 are cyclic amp response element binding protein (creb), nuclear factor-b (nf-b), activating protein-1 (ap-1), heat - shock factor-1 (hsf-1), -catenin, t - cell factor (tcf)/lymphoid enhancer factor (lef), and p53 . Dysfunction of gsk-3-mediated phosphorylation of transcription factors is believed to relate with the pathophysiology of various pathological conditions (for review, chiu and chuang, 2010). We found that gsk-3 silencing activated camp response element (cre)- and nf-b - responsive transcription more robustly than gsk-3 silencing (liang and chuang, 2006). Dna array further identified two novel gsk-3-regulated transcription factors, early growth response-1 (egr-1) and smad3/4, both of which play important roles in growth, differentiation, survival, and plasticity of brain cells (harada et al ., 2001; derynck and zhang, 2003; lee and kim, 2004; droguett et al ., 2010). Specifically, the binding activity of egr-1 was down - regulated by sirna for gsk-3, but was up - regulated by sirna for gsk-3 (liang and chuang, 2006). By using sirnas or dominant - negative mutants specific to gsk-3 isoforms, inhibition of gsk-3 the differential roles of gsk-3 isoforms are further supported by the opposite effects of gsk-3 and sirnas on the protein levels of plasminogen activator inhibitor type-1 (pai-1), a smad3/4-regulated gene product . These results demonstrate that selective silencing or inhibition of the two gsk-3 isoforms could produce different and sometimes opposite effects on the regulation of certain transcription factors including novel gsk-3 targets (liang and chuang, 2006). For example, the disruption of gsk-3 in mice is embryonic lethal, despite the normal expression of gsk-3, indicating that the presence of isoform cannot compensate for the loss of isoform (hoeflich et al ., 2000). Transfection and sirna studies suggested that gsk-3 inhibition decreased the processing of -amyloid (a) precursor protein to form a140 and a142, while gsk-3 appeared to have a lesser role (phiel et al ., 2003). In addition, gsk-3, but not gsk-3, is required for interferon--induced activation of signal transducer and activator of transcription-3 (beurel and jope, 2009). Together, these findings underscore important similarities and differences between the roles of gsk-3 isoforms and in cell survival as well as transcription, and suggest that the development of isoform - specific inhibitors may be essential for therapeutic intervention of gsk-3-related neuropathological conditions . We have also explored lithium s effects on smad3/4-dependent transcriptional activity and the underlying mechanisms . Smad3/4 is a down - stream mediator of the signaling pathway triggered by transforming growth factor- (tgf-), and plays a prominent role in regulating the expression of proteins involved in neuronal survival, differentiation, and synaptic plasticity (for review, gomes et al ., 2005). Treating cultured cortical neurons with therapeutically relevant concentrations of lithium significantly decreased smad3/4-dependent transactivation and protein levels of pai-1, a tgf--responsive smad3/4-dependent gene product (liang et al ., 2008). Of particular relevance to the therapeutic efficacy of lithium, pai-1 has been implicated in the etiology and progression of neurodegenerative diseases and mood disorders (for review, pawlak et al ., 2003; lithium s effects on smad3/4 likely result from cross - talk of signaling pathways between camp / pka and pi3-kinase / akt / gsk-3. We have shown that lithium - induced smad3/4 suppression involved gsk-3 inhibition through the activation of pka and cell survival factor akt followed by the phosphorylation of gsk-3 at ser9 and creb at ser133 (liang et al ., 2008). Creb binding protein (cbp) and p300 are known to be co - activators of creb . Our data further demonstrated that over - expression of p300, but not cbp, completely antagonized lithium - induced reduction of pai-1 promoter activity . A series of experimental data support the notion that, in smad3/4 signaling, the inhibitory effects of lithium are due to complex formation of activated creb and p300, which results in limited interactions of p300 with the transcription factors / smad complexes . This in turn prevents efficient smad3/4-dependent transcription of smad3/4-dependent genes such as pai-1 and p21 (figure 2). Transcriptional activations triggered by stimulation of cell surface tgf- and bdnf receptors are mediated by smad3/4- and pi3-kinase / akt - dependent pathways, respectively . Lithium treatment - induced inhibition of gsk-3, directly and indirectly via camp - dependent activation of pka as well as bdnf - stimulated activation of pi3-kinase / akt pathways, potentiates bdnf - induced phosphorylation / activation of creb . This in turn increases cre - mediated transactivation and expression of survival factors such as bdnf and bcl-2 . Enhanced gene transcription triggered by bdnf, via sequestration of transcriptional co - activator p300, suppresses smad3/4-dependent transactivation and subsequently decreases the expression of tgf--responsive genes, pai-1, and p21 . Lines with solid arrows represent stimulatory connections; lines with flattened ends represent inhibitory connections . Dashed lines represent pathways with reduced activity as a result of lithium treatment . Lithium - induced neuroprotection against glutamate excitotoxicity was first noted in rodent primary neuronal cultures of cerebellar granule cells (cgcs), cerebral cortical neurons, and hippocampal neurons (nonaka et al ., 1998). This experimental paradigm was selected because glutamate - related excitotoxicity has been implicated in many neurodegenerative diseases including stroke (for review, chuang, 2004; chiu and chuang, 2010). Our pioneering studies have shown that glutamate - induced, nmda receptor - mediated excitotoxicity was robustly reduced by extended lithium chloride pretreatment (57 days) in cultured rat cgcs and cortical neurons, partly via inhibition of nmda receptor - mediated calcium influx (nonaka et al ., 1998; hashimoto et al ., 2002a). Moreover, these effects of lithium were likely due to the attenuation of constitutive phosphorylation at tyr1472 of the nr2b subunit of nmda receptors, possibly as a result of inhibiting src tyrosine kinase (hashimoto et al ., 2002a, 2003). Although glutamate - induced excitotoxicity in cultured cortical neurons was blocked by treatment with either lithium or mk-801 (an nmda receptor antagonist), the src kinase inhibitor su6656 only partially diminished this toxicity (hashimoto et al ., 2003), suggesting that other components are involved . In cgcs, lithium - induced neuroprotection against glutamate excitotoxicity was associated with up - regulation of the anti - apoptotic protein bcl-2, down - regulation of the pro - apoptotic proteins p53 and bax, and suppressed release of cytochrome c from mitochondria (chen and chuang, 1999), whereas the involvement of gsk-3 in the regulation of nmda signaling by lithium treatment is currently unclear and requires further investigations . Cyclin - dependent kinase 5 (cdk5) also regulates signaling mediated by nmda receptors, either directly through phosphorylation of the nr2b subunit or indirectly through phosphorylation of psd-95 (morabito et al ., 2004; zhang et al ., 2008 when it binds to p25 (the product of calpain - mediated cleavage of p35), cdk5 becomes pro - apoptotic and its activity is dysregulated (lee et al ., p25 accumulation was observed in neurons in response to glutamate or oxidative stress, and also in the brains of several animal models of neurodegenerative diseases . Sustained activation of cdk5 in neurons has been implicated in many neurodegenerative diseases (cruz and tsai, 2004; dhariwala and rajadhyaksha, 2008). In cultured rat cgcs, lithium pretreatment prevented colchicine - induced apoptosis and associated increase in cdk5 expression and fragmentation of p35 into p25 (jorda et al . . Additionally, pretreatment with lithium also attenuated intracellular calcium increase, calpain activity, cdk5 activation, and cellular death in primary cultured hippocampal neurons and rat striatum following the treatment of 3-nitropropionic acid (crespo - biel et al ., 2009), a succinate dehydrogenase inhibitor (for review, brouillet et al ., 1999). Therefore, lithium - induced inhibition of calpain and cdk5 activation may also contribute to protection against glutamate excitotoxicity . Prior to changes in gene expression, lithium rapidly and transiently activated the cell survival pi3-kinase and its down - stream target, akt-1, through phosphorylation at ser473, thereby reversing glutamate - induced inactivation of this signaling pathway in cgcs (chalecka - franaszek and chuang, 1999). Activated akt is known to affect several anti - apoptotic targets including bcl-2 associated death promoter (bad), creb, members of the forkhead family, and procaspase-9 (for review, neri et al ., 2002; nicholson and anderson, 2002; huang and reichardt, 2003). In addition, lithium also triggered ser21 phosphorylation of the isoform of gsk-3 (and hence resulted in inhibition), and this effect was prevented by a pi3-kinase inhibitor (chalecka - franaszek and chuang, 1999). Another signaling pathway affected by lithium is the mitogen - activated protein (map) kinase pathway . One of the down - stream targets of map kinase is creb, a transcription factor that is involved in learning and memory, and promotes the expression of bcl-2 as well as brain - derived neurotrophic factor (bdnf; for review, finkbeiner, 2000). In cgcs, toxic concentrations of glutamate - induced an nmda receptor - dependent decrease in creb phosphorylation at ser133 and creb - driven transcriptional activity (kopnisky et al ., 2003). Concurrent with its neuroprotective effects, long - term (but not acute) lithium treatment suppressed glutamate - induced dephosphorylation of creb . We also found that glutamate rapidly activated c - jun - n - terminal kinase (jnk) and p38 kinase in cgcs, resulting in a robust increase in ap-1 binding (chen et al ., 2003a). These two kinases are also activated by a variety of apoptotic insults (for review, mielke and herdegen, 2000), and ap-1 has been known to be activated by different stress factors as well . Experiments using lithium and curcumin, a selective ap-1 inhibitor, suggest that nmda receptor - mediated apoptotic death requires concerted action of jnk and p38 to enhance ap-1 binding, and that lithium s neuroprotection is mediated, at least in part, by suppressing the jnk and p38 kinase pathways . As one of the major neurotrophins, bdnf is essential for cortical development, synaptic plasticity, and neural survival, and is likely a key mediator of the clinical efficacy of anti - depressants and anxiolytic drugs (for review, woo and lu, 2006). The notion that bdnf plays a key role in neuronal survival is supported by our observation that bdnf and neurotrophin-4 (nt-4), but not nt-3, completely protected immature cgcs from apoptosis induced by cytosine arabinoside (leeds et al ., 2005). It was first reported that chronic treatment with lithium increased the expression of bdnf in the rat brain (fukumoto et al ., 2001), and we have documented that bdnf protein levels were increased in cortical neurons following lithium treatment (hashimoto et al ., 2002b). We hypothesized that this bdnf up - regulation and subsequent activation of its receptor trkb might play a critical role in mediating the neuroprotective effects of lithium . In confirmation of this hypothesis, we found that lithium s neuroprotection against glutamate excitotoxicity was blocked by a trkb inhibitor, k252a, or by a neutralizing antibody against bdnf, and was mimicked by exogenous bdnf in rat cortical neurons . In addition, lithium increased intracellular levels of bdnf and this was followed by activation of trkb . Furthermore, lithium - induced neuroprotection was prevented in cortical neurons from heterozygous (+ /) or homozygous (/) bdnf knockout mice (hashimoto et al ., 2002b). Rodent bdnf has a complex genomic structure that makes it an ideal target for multiple and complex regulation . We found that treatment of rat cortical neurons with therapeutic concentrations of lithium (e.g., 1 mm) caused a significant increase in the levels of bdnf exon iv - containing mrna, while levels of exon i, ii, or vi - containing mrna remained unchanged (yasuda et al ., 2009). It is known that exon iv - containing bdnf transcripts are expressed in response to kcl - induced depolarization in rat cortical neurons (tao et al ., 2002). This transcriptional activation requires utilization of the promoter region 80 bp up - stream from the transcription initiation site of exon iv - containing three calcium responsive elements (cares; chen et al ., 2003b). We generated various bdnf promoter iv deletion constructs to investigate whether lithium treatment causes an increase in bdnf promoter iv activity, and, if so, which region of promoter iv confers the sensitivity to this drug . We identified that the drug - induced up - regulation of exon iv - containing bdnf transcript was associated with a significant increase in the activity of bdnf promoter iv and total bdnf protein . To our surprise, the lithium - responsive element(s) in promoter iv resides in a region up - stream from the cares responsible for depolarization - induced bdnf induction (170 to 704 bp). Moreover, activation of bdnf promoter iv occurred in cortical neurons depolarized with kcl and depletion of these three cares failed to abolish lithium - induced activation . Importantly, we found that lithium - induced activation of promoter iv was mimicked by pharmacological inhibitors of gsk-3 (sb216763, sb415286, inhibitor i, and inhibitor vii) or by transfection with specific sirna for gsk-3 or gsk-3. Additionally, their dominant - negative mutants also mimicked lithium - induced activation of promoter iv . These results demonstrate that gsk-3 is the initial target of lithium to selectively activate bdnf promoter iv and that bdnf induction by lithium involves a novel responsive region in promoter iv of the bdnf gene . Lithium - induced, gsk-3-dependent bdnf promoter iv activation could be a part of the molecular mechanisms underlying its neuroprotective effects and as such, possibly accounts for the therapeutic actions in bipolar patients . It should be noted that in addition to lithium, other gsk-3 inhibitors have been shown to almost completely block glutamate - induced excitotoxicity in rat cortical neuronal cultures (liang and chuang, 2007). These include atp - competitive inhibitors, sb216763 and sb415286, and atp - non - competitive inhibitors, inhibitor i and vii . As mentioned in the preceding section, glutamate - induced death of cortical neurons was mitigated by silencing of gsk-3 and/or, or both isoforms, or inhibition of gsk-3 activity via transfection with dominant - negative mutants of gsk-3/ isoforms (liang and chuang, 2007). Studies from other laboratories also supported the roles of gsk-3 inhibition in protecting neurons from glutamate neurotoxicity . For example, stimulation of nmda receptors in cultured rat hippocampal or cortical neurons activated gsk-3 by pp-1-mediated serine dephosphorylation of gsk-3 (szatmari et al ., 2005). Treatment of primary rat cortical neurons with -amino-3-hydroxy-5-methyl-4-isoxazole propionate (ampa), lithium or sb216763 blocked glutamate - induced caspase-3 activation and excitotoxicity, and the protective effects of ampa required pi3-kinase akt - dependent serine phosphorylation of gsk-3 (nishimoto et al ., 2008). Further, in organotypic cultures of chick embryo spinal cord, lithium prevented kainate - induced excitotoxic death of motoneurons by targeting gsk-3, and this neuroprotection was associated with cytopathological changes (caldero et al ., 2010). Stroke is the third leading cause of death in the united states and a major global cause of serious long - term disability in adults . Ischemic strokes represent approximately 87% of all cases, while the rest are hemorrhagic strokes (roger et al ., 2011). In addition to physical deficits, stroke victims also suffer from vascular depression and dementia, both of which are difficult to treat with conventional medicine . It is becoming clear that there is a substantial increase in extracellular glutamate in the brain following cerebral ischemia, and that a significant portion of ischemia - induced brain damage is mediated by over - stimulation of nmda receptors . Shortly after ischemia, the interruption of cerebral blood flow depletes oxygen and glucose and subsequently prevents atp production . Inadequate atp supply will cause the malfunction of atp - dependent ion pumps and alter the ion concentration gradient across the neuronal membranes . The resulting failure to transport glutamate leads to an accumulation of glutamate in the extracellular space and over - stimulates nmda receptors, which leads to a toxic influx of calcium and in turn drives the activation of damaging calcium - mediated intracellular enzymes . This cascade of events ultimately results in mitochondrial failure, production of reactive oxygen species, neuroinflammation, and cell necrosis and apoptosis (allen and bayraktutan, 2009; deb et al ., 2010). Gsk-3 has been strongly implicated in the neuronal cell death caused by cerebral ischemic insult . One study in rats subjected to transient middle cerebral artery occlusion (mcao) demonstrated a rapid increase in the expression of cytoplasmic and nuclear gsk-3 protein in ipsilateral lamina i, ii, v, and vi in young rat brains, whereas in lamina v and vi in old rat brains (sasaki et al . Although the phosphorylation status of gsk-3 was not mentioned, these findings implicate a role of gsk-3 in cerebral ischemic injury . It is well known that gsk-3 can be phosphorylated at serine and tyrosine residues in which ser9 phosphorylation renders it inactive, while tyr216 phosphorylation is necessary for its functional activity (hughes et al ., 1993). Discrepancies exist in the literature regarding changes of ser9 and tyr216 phosphorylation levels following cerebral ischemia . The phosphorylation levels of gsk-3 at ser9 and akt at ser473 were reported to be markedly enhanced in the vulnerable hippocampal ca1 region, but not in the ischemia - resistant ca3 region in rats subjected to transient global cerebral ischemia, while there was no change in levels of tyr216 phosphorylation or total gsk-3 (endo et al ., 2006). Levels of gsk-3 ser9 phosphorylation were also increased shortly after permanent focal cerebral ischemia and decreased to basal levels or even lower 24 h after ischemic onset (sasaki et al ., 2006; gao et al ., 2008 however, it has also been reported that transient focal cerebral ischemia in rats caused an increase in gsk-3 tyr216 phosphorylation in degenerating cortical neurons with no alteration in ser9 phosphorylation (bhat et al ., 2000). This discrepancy may stem, in part, from the difference of ischemic severity and ischemic models across various studies . It appears that transient focal cerebral ischemia tends to activate gsk-3 and subsequently to induce apoptotic cell death . In contrast, gsk-3 is inactivated shortly after permanent focal cerebral ischemia or global cerebral ischemia, which in turn may promote survival of vulnerable neurons . Ischemia both gsk-3 and gsk-3 mediated the expression of a lethal protein, neuronal pentraxin 1 (russell et al ., 2011). In light of these findings the beneficial effects of lithium in rodent cerebral ischemic models demonstrated by us and others support this notion . In an initial study, long - term lithium pretreatment at therapeutically relevant doses decreased brain infarct volume, reduced apoptotic cell death and improved behavioral performance after permanent cerebral ischemia - induced by mcao (nonaka and chuang, 1998; xu et al ., 2003). In a subsequent study, we demonstrated that subcutaneous injection of rats with lithium at therapeutic doses (e.g., 0.5 and 1.0 meq / kg) after the onset of transient mcao markedly decreased infarct volume, reduced tunel - positive dna damage, and suppressed neurological deficits measured by sensory, motor, and reflex tests (ren et al ., 2003). The time window for these beneficial effects was at least 3 h after the onset of ischemia . Heat - shock protein 70 (hsp70), a well - established cytoprotective factor against apoptosis, was induced in the ischemic penumbra where neuronal recovery takes place . Post - insult treatment with lithium increased the dna binding activity of hsf-1 to the heat - shock element, superinducing hsp70 which inhibits brain ischemia - induced apoptosis (ren et al ., 2003). Lithium - elicited gsk-3 inhibition is likely associated with hsf-1 activation and hsp70 induction (bijur and jope, 2000). Notably, post - insult lithium treatment mitigated apoptosis and brain damage by preventing gsk-3 and erk dephosphorylation, suppressing calpain and caspase-3 activation, and inhibiting mitochondrial release of cytochrome c and apoptosis - inducing factor in a neonatal hypoxic these findings suggest that lithium - induced gsk-3 inhibition contributes to its anti - apoptotic effects under ischemic conditions . In addition, it was found that lithium pretreatment largely suppressed ischemia - induced exploratory behavioral changes and memory impairments in gerbils after global cerebral ischemia (bian et al ., 2007). These behavioral benefits were associated with an increase in the number of viable cells and a decrease in apoptotic cells in the ca1 hippocampal area of ischemic gerbils . Moreover, lithium - induced neuroprotection in the ischemic brain was accompanied by down - regulation of pro - apoptotic p53 in the ca1, and up - regulation of anti - apoptotic bcl-2 and hsp70, both of which are targets of gsk-3 . It is likely that lithium protection against ischemia - induced injury involves multiple mechanisms . In the rat hippocampus, lithium was reported to inhibit ischemia - induced nmda receptor hyperactivation by inhibiting nmda subunit 2a tyrosine phosphorylation and its interactions with src and fyn through psd-95 (ma and zhang, 2003). Lithium also attenuated hypoxia - induced serine dephosphorylation of gsk-3 and in the mouse brain (roh et al ., 2005). Additionally, in organotypic cultures of rat hippocampus subjected to oxygen and glucose deprivation, lithium showed neuroprotection in conjunction with hsp27 activation (cimarosti et al ., 2001). Post - ischemic inflammation is a dynamic process involving a complicated set of interactions between inflammatory cells and molecules (iadecola and alexander, 2001). A recent study documented the anti - inflammatory effects of lithium in a neonatal rat hypoxic ischemic model . Post - insult lithium treatment significantly reduced total tissue loss following hypoxia ischemia, and this beneficial effect of lithium was associated with inhibiting microglia activation and attenuating levels of pro - inflammatory cytokines or chemokines, such as interleukin-1 and chemokine ligand 2 (li et al ., 2011). Hsp70 over - expression can inactivate the key inflammatory transcription factor nf-b by stabilizing the nf-b - ib complex, and thereby preventing nuclear translocation of activated nf-b subunits in a mouse mcao model (zheng et al ., 2008). Besides anti - inflammation, lithium also increased proliferation and differentiation of hippocampal neural progenitor cells in both non - ischemic and ischemic brains without altering the relative levels of neuronal and astrocytic differentiation, and this effect lasted at least 7 weeks after hypoxia ischemia in neonatal rats (li et al ., in line with this finding, chronic lithium pretreatment was found to increase the generation and survival of newborn cells in the hippocampal dentate gyrus, and did not affect the neuronal or astrocytic differentiation of these newborn cells in a transient four - vessel occlusion model (yan et al . Erk1/2 phosphorylation following ischemia was enhanced by lithium treatment, while erk1/2 inhibitor u0126 prevented the effects of lithium in increasing brdu - positive cells and improving spatial learning and memory (yan et al ., 2007). In fact, chronic lithium treatment has been demonstrated to increase activity in the mek / erk pathway in vivo, and lithium s neuroprotection has been suggested to depend on the induction of this signaling pathway (einat et al . In addition, it was reported that activation of erk associates with and phosphorylates gsk-3 at the thr43 residue, which primes this kinase for its subsequent phosphorylation at ser9 by p90rsk, resulting in inactivation of gsk-3 and up - regulation of -catenin (ding et al ., 2005). Therefore, lithium might affect gsk-3 phosphorylation through the mek / erk pathway, which in turn inhibits this kinase via rsk . Gsk-3 is also negatively regulated by the wnt/-catenin signaling pathway and, accordingly, activating the canonical wnt pathway has been shown to contribute to adult hippocampal neural progenitor cell proliferation triggered by lithium treatment (wexler et al ., 2008). In a collaborative study, the neurohemodynamic aspects of recovery induced by delayed chronic lithium treatment were assessed using functional magnetic resonance imaging (mri; kim et al ., 2008). Rats were subjected to transient mcao and then injected with lithium (licl, 1 meq / kg, s.c .) This delayed lithium injection was followed by daily injections, and on day 15, an mri scan was performed to monitor changes in blood oxygen level dependence (bold) and functional cerebral blood volume (fcbv) responses using electric stimulation of forelimbs . The mean activated volume ratio and total activation magnitude ratio between ipsilateral and contralateral cortices for both bold and fcbv were significantly higher in the lithium - treated than in the saline - treated rats . The lithium - induced increase in fcbv in the peri - infarct regions suggests a possible vascular transformation . Indeed, the size and distribution of immunohistochemical staining of cd31, a microvasculature marker, were enhanced by lithium treatment in the peri - infarct regions . Co - localized with cd31, the tissue staining of matrix metalloproteinase-9 (mmp-9) was also much more pronounced following lithium treatment, suggesting mmp-9-dependent neurovascular remodeling in the recovering brain area . Moreover, treatment of cultured rat brain endothelial cells with lithium in a follow - up study was also found to increase the protein levels of vascular endothelial growth factor (vegf) via a mechanism involving the pi3-kinase and gsk-3 signaling pathways (guo et al ., since vegf has been linked to angiogenesis, neurogenesis, and neuroprotection (for review, fan and yang, 2007), vegf over - expression may contribute to lithium s ability to promote neurovascular remodeling and to induce functional recovery after ischemic stroke . Ample evidence supports the therapeutic potential of mesenchymal stem cells (mscs) in several human diseases including stroke . However, it is increasingly recognized that the effectiveness of msc transplantation is limited by their poor migration toward disease target sites such as ischemic brain regions . In a recent study, we investigated whether treatment of mscs with lithium and another mood stabilizing drug, valproic acid (vpa), would enhance cell migration (tsai et al ., 2010). We found that treatment of mscs with lithium (2.5 mm for 1 day) selectively elevated the transcript and protein levels of mmp-9 and its enzymatic activity . These effects were mimicked by pharmacological inhibition or gene silencing of gsk-3. Lithium treatment also potentiated stromal cell - derived factor-1 (sdf-1)-dependent msc migration across the extracellular matrix, which was suppressed by two mmp-9 inhibitors, doxycycline and gm6001 . Short - term (3 h) exposure of mscs to a relatively high concentration (2.5 mm) of vpa markedly increased the transcript and protein levels of cxc chemokine receptor 4 (cxcr4). Vpa - induced cxcr4 expression required its ability to inhibit histone deacetylases (hdacs), including the hdac1 isoform, and involved histone hyperacetylation at the cxcr4 gene promoter . Vpa treatment enhanced sdf-1-mediated msc migration, which was completely blocked by amd3100, a cxcr4 antagonist . Notably, combining lithium and vpa treatment further increased msc migration, and the additive enhancement of migration was completely blocked by the co - presence of amd3100 and gm6001 . Our results suggest that lithium and vpa stimulate msc migration through distinct targets and mediators: gsk-3mmp-9 and hdac cxcr4, respectively (tsai et al ., 2010). In a follow - up in vivo study, mscs were primed with lithium and/or vpa and then injected into the tail vein of transient mcao rats 24 h after ischemic onset . Priming with lithium or vpa increased the number of mscs homing to the cerebral infarcted regions such as the cortex and striatum 2 weeks after transplantation, and co - priming with lithium and vpa further enhanced this migratory effect (tsai et al ., 2011). Mcao rats receiving lithium- and/or vpa - primed mscs showed improved functional recovery, reduced infarct volume, and enhanced angiogenesis in the infarcted penumbra regions . These beneficial effects of lithium and vpa priming were reversed by pharmacological inhibition of mmp-9 and cxcr4, respectively, suggesting that these effects were likely mediated by lithium - induced mmp-9 up - regulation and vpa - induced cxcr4 over - expression . Together, these findings raise the potential utility of using mscs primed with inhibitors of gsk-3 and hdac to enhance the migration and homing capacity for transplantation into stroke victims . In addition to lithium, other pharmacological gsk-3 inhibitors have been shown to exert neuroprotective effects against cerebral ischemia by various groups . A specific gsk-3 inhibitor, chir025, was demonstrated to protect cultured hippocampal neurons from glutamate excitotoxicity and to attenuate death of cortical neurons following oxygen glucose deprivation, an in vitro model of cerebral ischemia (kelly et al ., 2004). Moreover, chir025 reduced infarct size in focal cerebral ischemic rats, but did not affect tunel - positive neurons or caspase-3/9 activities, although bcl-2 expression was increased . Gsk-3 enzymatic activity was markedly elevated after transient mcao in rats, and this gsk-3 activation was blocked by jugular vein injection of gsk-3 inhibitor viii (koh et al ., 2008). Pre- or post- (up to 2 h) mcao injection with inhibitor viii also reduced blood glucose levels, infarct size, caspase-3 activity, and water content in the ipsilateral brain hemisphere . Furthermore, ischemia - induced inflammation - related signals such as cox-2 over - expression and neutrophil infiltration were alleviated by this gsk-3 inhibitor . Prophylactic or therapeutic administration of a gsk-3 inhibitor tdzd-8 reduced infarct volume and cerebral injury in the rat hippocampus after transient ischemia (collino et al ., 2008). This was accompanied by suppression of ischemia - induced oxidative stress, apoptosis, and neuroinflammation . Delayed treatment with compound i, a gsk-3 and cdk inhibitor, decreased tunel - positive cells in the ipsilateral hippocampus and striatum of adult (but not juvenile) mice subjected to hypoxic these neuroprotective effects of compound i were associated with long - lasting functional recovery . Finally, gsk-3 inhibition by sb216763 counteracted oxygen glucose deprivation - induced mitochondrial biogenesis impairment and reduced mitochondrial reactive oxygen species generation in primary cortical neurons (valerio et al ., 2011). When systematically administrated to permanent mcao mice, sb216763 decreased infarct volume and restored the loss of mitochondrial dna, thus supporting a novel role of gsk-3 inhibitors in stimulating the renewal of functional mitochondria following ischemic stroke . A growing body of evidence supports that lithium, a mood stabilizer used to treat bipolar disorder, has neuroprotective properties in both cellular and in vivo experimental settings . One of the major targets of lithium is gsk-3, a serine / threonine kinase implicated in the pathogenesis of diverse cns disorders . Lithium inhibits gsk-3 activity by direct binding to the enzyme or indirectly by enhancing serine phosphorylation of both and isoforms through multiple mechanisms . Lithium has been used as a prototype drug to seek evidence for the involvement of gsk-3 inhibition in lithium - induced protection against excitotoxicity in cultured neurons and animal models of cerebral ischemic stroke . Lithium at therapeutically relevant concentrations robustly protected primary brain neurons from glutamate - induced, nmda receptor - mediated excitotoxicity . The neuroprotective effects of lithium were associated with gsk-3 inhibition, and were mimicked by other pharmacological gsk-3 inhibitors, by silencing gsk-3 and/or isoforms, or by expression of isoform - specific dominant - negative mutants . These results support the roles of gsk-3 inhibition in lithium - elicited protection against excitotoxicity . Lithium rapidly activated the cell survival pi3-kinase akt signaling pathway to enhance gsk-3 serine phosphorylation and to block glutamate - induced akt inactivation as well as apoptosis . Lithium also caused an increase in the expression of cytoprotective bcl-2 and suppressed glutamate - induced up - regulation of pro - apoptotic p53 and bax, resulting in blocking cytochrome c release from mitochondria . Bdnf promoter iv was selectively activated by gsk-3 inhibition using lithium or other drugs or through gene silencing / inactivation of either isoform . This effect on however, there is a gap in the understanding of how gsk-3 inhibition causes an increase in bdnf promoter activity . In addition, lithium s neuroprotective effects were associated with inhibition of nmda receptor - mediated calcium influx and suppression of p38/jnk and ap-1 activation, thus reducing apoptosis . This effect appears to stem from inhibition of src / fyn kinase to suppress nr2b tyr1472 phosphorylation of the receptor . It remains to be explored as to whether this lithium - induced action on nmda receptors is related to gsk-3 inhibition . The potential roles of these other targets in mediating the neuroprotective effects of this drug also deserve future investigation . It is well known that glutamate overflow and nmda receptor hyper - stimulation are early events following cerebral ischemia . In rodent ischemic models, pre- or post - insult treatment with therapeutic doses of lithium decreased infarct volume, caspase-3 activity and apoptotic cells in the injured brain . The beneficial time window of lithium is at least 3 h after the ischemic onset . Up - regulation of hsp70 and bcl-2 as well as down - regulation of p53 likely contributed to the protective effects of lithium in the ischemic conditions, thus supporting similar underlying neuroprotective mechanisms in the excitotoxic cellular models and animal models of ischemic stroke . Limited data suggested that lithium might also display anti - inflammatory effects by inhibiting ischemia - induced microglia activation and pro - inflammatory factors release . Delayed and chronic injections of lithium improved functional mri responses such as increases in bold and fcbv . Indeed, lithium was found to induce two pro - angiogenic factors, mmp-9 and vegf in a gsk-3-dependent manner . Lithium has also been reported to stimulate erk1/2 activity and to enhance proliferation of hippocampal neural progenitor cells and memory performance after ischemia . Finally, lithium promoted migration of mscs in vitro by up - regulation of mmp-9 through gsk-3 inhibition and this migratory effect was potentiated by co - treatment with vpa, another mood stabilizer . Notably, transplantation of lithium vpa co - primed mscs into ischemic rats markedly increased msc migration to the injured brain regions, decreased infarct size and improved the neurological performance . Lithium - induced stem cell migration, neurogenesis, and angiogenesis all likely contribute to functional recovery . Figure 3 illustrates proposed molecular events leading to lithium - induced beneficial effects following cerebral ischemia . It should be noted that several other gsk-3 inhibitors have also been reported to exert beneficial effects in rodent ischemic models and their actions were accompanied by suppression of ischemia - increased gsk-3 activity . Accordingly, gsk-3 inhibitors have therapeutic potential to treat stroke and other excitotoxicity - related neurodegenerative diseases . Lithium has been used in bipolar patients over 60 years and its clinical profiles are well understood . Therefore, lithium is a prime candidate for use in clinical trials of new therapies for stroke victims . The neuroprotective effects of lithium against cerebral ischemia are proposed to result from its interactions with cell survival and apoptotic machinery . A significant portion of brain damage following cerebral ischemia is caused by an increase in extracellular glutamate and subsequent over - stimulation of nmda receptor - mediated toxic increase in intracellular calcium . This signaling pathway plays a critical role in mediating glutamate - induced caspase activation and apoptosis . Lithium at therapeutically relevant concentrations inhibits nmda receptor - mediated calcium influx, which in turn decreases subsequent activation of jnk, p38 kinase, and transcription factor ap-1 . Inhibition of intracellular calcium increase also attenuates the activity of calpain and calpain - mediated activation of pro - apoptotic cdk5/p25 kinase . On the other hand, lithium can directly and indirectly reduce the activity of constitutively activated gsk-3 by multiple mechanisms, leading to disinhibition of several transcription factors, such as creb and hsf-1, and resulting in induction of major cytoprotective proteins such as bdnf, vegf, mmp-9, hsp70, and bcl-2 . A decrease in gsk-3 activity further reduces the activity of pro - apoptotic protein p53 and its downregulating effect on bcl-2 . Bdnf, via activating its cell surface receptor and the down - stream erk and pi3-kinase / akt pathways, induces neuroprotective effects in part by inhibiting gsk-3 and stimulating creb . Induction of bdnf is an early and essential step for neuroprotection and is involved in lithium - induced neurogenesis . In addition, superinduction of hsp70 by lithium treatment not only inhibits brain ischemia - induced apoptosis, but also contributes to the anti - inflammatory effects of lithium through inactivation of nf-b . Counteraction of gsk-3 inhibition of vegf and mmp-9 by lithium enhances angiogenesis and neurovascular remodeling . Taken together, these effects of lithium in reducing apoptosis, suppressing inflammation, enhancing angiogenesis and neurogenesis, contribute to behavioral improvement and functional recovery after ischemia . Lines with solid arrows represent stimulatory connections; lines with flattened ends represent inhibitory connections . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Studies have shown the incidence of injury from squash to range from 35.5 to 80.9 per 100,000 players . Trauma to the head (including the eyes) is the most common cause of squas - related hospital presentation, comprising 48.7% of all emergency department presentations . Craniofacial and spinal fractures typically result from high - energy blunt force to the skeleton, as seen in motor vehicle accidents . This is the first case reported of a patient sustaining such extensive head, neck and spinal injuries from a squash - related injury . A 55-year - old man was brought in by ambulance after colliding with the corner of the wall and floor of a squash court, without the use of protective eye - glasses or mouth - guard . He was chasing a ball toward the corner of the court when he fell and hit the junction of the wall and floor with his head . On examination in the emergency department, the patient had a glasgow coma scale (gcs) of 14, with confusion to time and place . Initially, the patient reported diplopia at primary position but examination by the surgical team elicited normal eye movements with nil complaint of diplopia . There was significant boggyness to palpation of the forehead with a palpable defect of the frontal bone . The cervical spine was slightly tender over / c1 - 2 and c5 - 6 . Upper limb examination was limited by pain maximal over the distal radii but bilateral arm weakness was noted with nil sensory dysfunction . Primary and secondary surveys did not elicit any further findings . A non - contrast computed tomography (ct) imaging of the facial bones [figure 1] showed a le fort type ii fracture, with comminuted fractures of the roofs of the orbit bilaterally and a depressed fracture of the left orbital floor . There was also fracture of the right maxilla at the junction of the maxilla and zygomatic arch, extending towards but not involve the right orbital floor . On the left, there was fracture of the anterior part of the left zygomatic arch which extended into the anterolateral wall of the left maxillary sinus . A large, depressed fracture of the frontal bone was also evident, extending superiorly from the superior lateral wall of the right orbit and extending across the frontal bone and inferiorly toward the mid - portion of the roof of the left orbit . The fracture involved the inner and outer tables of the frontal bone and was depressed by at least 3.5 mm . There was also a comminuted fracture of the nasal bone, which was not significantly displaced . Anterior and anterolateral views of three - dimensional ct reconstructions demonstrating lefort type ii, frontal, orbital and zygomatic fractures ct brain showed tiny subarachnoid or intraparenchymal haemorrhages but there was no evidence of extensive intra- or extra - axial haemorrhage . Imaging of the cervical spine [figure 2] showed an undisplaced fracture of the posterior arch of c1 on the right side and a type 3 dens fracture with posterior displacement of 4 mm but no contact with the spinal cord . Anterolateral view 3 dimensional ct reconstruction demonstrating type 3 dens fracture with posterior displacement and fracture of c7 spinous process subsequent ct of the thoracic spine showed a comminuted t6 vertebral body fracture with loss of vertebral height and extension into the left costovertebral joint . There was a small bony fragment projecting posteriorly into the spinal canal and making contact with the cord, but there was nil evidence of compression . Associated with this finding were two to three hyperdense foci within the spinal cord likely to represent small contusions . The above findings were confirmed on magnetic resonance imaging (mri) of the spine, which also demonstrated a small cortical buckle at the t6 level without significant cord impingement . The patient underwent craniotomy, obliteration of the frontal sinus and open reduction internal fixation (orif) of the frontal bone . Orif of the maxilla bilaterally, orif of the infra - orbital rim and supra - orbital rim bilaterally and inter - maxillary screw fixation was also performed . The patient was admitted to the neurosurgical intensive care unit post - operatively and underwent cervical fusion with odontoid screw fixation of the c2 fracture one week after surgical correction of the head injuries . Post - operative ct scans demonstrated complete bony union in the c2 fracture site [figure 3] and uncomplicated fixation of the facial bone fractures [figure 4]. Post - operative lateral x - ray of the cervical spine in extension demonstrating union of dens fracture with overall alignment in - tact post - operative coronal ct demonstrating surgical correction of multiple craniofacial fracture sites our patient suffered injuries to multiple sites in the cranium, facial bones and the vertebrae that have previously not reported in the literature . The thinnest and weakest segment of the orbit is the floor, which is the most common site of fracture . Our patient suffered a le fort type ii fracture, which is pyramid shaped and passes through the posterior alveolar ridge, lateral walls of maxillary sinuses, inferior orbital rim and nasal bones . He also suffered a depressed frontal bone fracture involving the inner and outer tables, an extremely rare complication of sport seen in only 1.3% of sports related craniofacial fractures . Spinal injury is associated with sports in approximately 8.7% of cases, usually in relation to contact sports such as wrestling . Our patient had a fracture of the posterior arch of c1 on the right side and a type 3 dens fracture, which required cervical fusion . There was also fracture of the c7 cervical process, from forceful flexion of the cervical spine . We report the first case of extensive head and spinal injury resulting from collision with the wall of a squash court . The injuries ranged from the frontal bone to the thoracic vertebra and are usually seen in high velocity trauma.
Lung cancer is a leading cause of cancer mortality in the usa and other developed countries.1 the two major forms of the disease are non - small cell lung cancer and small cell lung cancer, which account for 85% and 15% of all lung cancers, respectively.2 despite advances in preliminary detection and standard treatment with combination chemotherapy and radiotherapy, the prognosis for patients with lung cancer has not improved significantly in the last 20 years,3,4 and survival rates have changed little over the past 2 decades . Currently, an enormous amount of research is aimed at understanding the molecular and cellular biology of lung cancer; however, much more work is needed to better understand the correlation between gene regulation and lung cancer . Growing evidence shows that aberrant hypermethylation in 5-cpg islands in the promoter regions is a major mechanism for silencing tumor suppressor or other cancer - associated genes in many kinds of human cancer.58 loss of function in cancer suppressor genes may hinder inhibition of growth of cancer cells, which leads to malignant transcription and translation during replication of dna . A number of genes, including the cyclin - dependent kinase inhibitor (p16), the tumor suppressor gene ras association domain family protein 1a, kelch - like ech - associating protein 1, the dna repair gene mgmt, and the cystic fibrosis transmembrane conductance regulator are demonstratively methylated in lung cancer.912 cdh1, a member of the transmembrane glycoprotein family, also known as cadherin-1, cam 120/80, epithelial cadherin (e - cadherin), or uvomorulin, is encoded by the cdh1 gene (16q22.1).13 cdh1 is a calcium - dependent cell - cell adhesion glycoprotein containing three domains, ie, five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail.14 cdh1 as a tumor suppressor gene plays an essential role in maintaining cell adhesion and adherent junctions in normal tissues . Cdh1 expression is frequently absent in a variety of epithelial tumors, and loss of normal intercellular junctions results in promotion of cancer invasion and metastasis and is correlated with several types of cancers.1517 however, the association between and clinicopathological significance of cdh1 promoter hypermethylation and lung cancer remains unclear . In this study, we systematically investigate studies of cdh1 promoter hypermethylation and lung cancer, and validate the association between cdh1 promoter hypermethylation and lung cancer using meta - analysis methods . In addition, we summarize these findings and discuss tumor suppressor function, as well as the clinicopathological significance of cdh1 in lung cancer . A systematic literature searching was performed using pubmed, embase, and the web of science up to august 13, 2014 without any language restrictions . The following keywords and terms were used: [methylation or dna methylation or hypermethylation or de - methylation] and [cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin (e - cadherin) or uvomorulin] and [lung cancer or lung carcinoma or lung tumor]. The published scientific articles were restricted to english language, and conference abstracts were excluded due to lack of sufficient data . Titles, abstracts, and key words in the articles were initially evaluated for inclusion criteria . Details and additional information were identified and collected from the full text of these articles . A study included for meta - analysis needed to have: evaluated the correlation between cdh1 methylation and lung cancer; included a clinical cohort and controls; included at least three patients and controls; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression; and used tissue data rather than blood data . Studies that did not meet our inclusion criteria were excluded . When the same groups of patients were reported in multiple papers, two researchers independently collected the information and extracted the data regarding authorship, year, source of publication, inclusion criteria, cdh1 methylation frequency, sex status, smoking history, pathological type, clinical staging, degree of differentiation, lymph node metastasis, epidermal growth factor receptor status, and prognosis in patients and controls . Methodological evaluation was assessed by the researchers according to the remark (reporting recommendations for tumor marker prognostic studies) guidelines and elcwp (european lung cancer working party) score.18,19 the meta - analysis was performed using reviewer manager 5 (cochrane collaboration, oxford, uk). Pooled odds ratios (ors) and confidence intervals (cis) were calculated to assess the correlation between cdh1 methylation and lung cancer . Cochran s q test and i 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i 2 test> 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters . Otherwise, a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 (statacorp, college station, tx, usa). A systematic literature searching was performed using pubmed, embase, and the web of science up to august 13, 2014 without any language restrictions . The following keywords and terms were used: [methylation or dna methylation or hypermethylation or de - methylation] and [cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin (e - cadherin) or uvomorulin] and [lung cancer or lung carcinoma or lung tumor]. The published scientific articles were restricted to english language, and conference abstracts were excluded due to lack of sufficient data . Titles, abstracts, and key words in the articles were initially evaluated for inclusion criteria . Details and additional information were identified and collected from the full text of these articles . A study included for meta - analysis needed to have: evaluated the correlation between cdh1 methylation and lung cancer; included a clinical cohort and controls; included at least three patients and controls; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression; and used tissue data rather than blood data . Studies that did not meet our inclusion criteria were excluded . When the same groups of patients were reported in multiple papers, only the most recent and complete paper was selected to avoid overlap . Two researchers independently collected the information and extracted the data regarding authorship, year, source of publication, inclusion criteria, cdh1 methylation frequency, sex status, smoking history, pathological type, clinical staging, degree of differentiation, lymph node metastasis, epidermal growth factor receptor status, and prognosis in patients and controls . Methodological evaluation was assessed by the researchers according to the remark (reporting recommendations for tumor marker prognostic studies) guidelines and elcwp (european lung cancer working party) score.18,19 the meta - analysis was performed using reviewer manager 5 (cochrane collaboration, oxford, uk). Pooled odds ratios (ors) and confidence intervals (cis) were calculated to assess the correlation between cdh1 methylation and lung cancer . Cochran s q test and i 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i 2 test> 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters . Otherwise, a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 (statacorp, college station, tx, usa). The process followed to select the papers used in this report is shown in figure 1 . Ninety papers were identified by electronic database searching and 20 further papers by manual searching . Eighty - five papers were excluded for being duplicate publications, being an irrelevant title or abstract, or being a relevant title and abstract but not published in the english language . After retrieval of the full - text articles, eleven papers were excluded for not having a large enough study population or for being reviews . Thirteen further studies were screened out (involving 866 cases and 757 controls) based on the inclusion and exclusion criteria in the pooled analysis . The characteristics of the studies are shown in table 1.2436 six papers were used to study the correlation between cdh1 methylation and sex status . Five papers provided smoking history data, allowing an investigation of the influence of smoking on cdh1 methylation . Three studies investigated the effect of clinical stage, ie, stage i, ii, iii, or iv . One paper discussed lymph node metastasis and another discussed degree of differentiation . Analyzing tissue samples from 657 patients and 593 controls, the mean frequency of cdh1 methylation was 32% (range 8.33% to 66.32%) in tumor tissue and 9% (range 0.00%27.78%) in tissue from controls . This result indicates that the occurrence of cdh1 methylation is higher in tumor tissue than in normal tissue . Using the fixed model, meta - analysis showed that 657 cases and 593 controls from 12 studies were pooled or as shown in figure 2 (or 3.89, 95% ci 2.875.27, p<0.00001). These findings indicate that cdh1 methylation is a key molecular event in tumor tissue but not in normal tissue . The results also show heterogeneity across the included studies (i 2 is 61%, ie, more than 50%). Given this significant heterogeneity, a subgroup analysis was performed to investigate sex status, smoking history, pathological type, clinical stage, differentiation degree, and lymph node metastasis to observe the relationship between cdh1 methylation and clinical characteristics (see figure 3). However, there was no correlation between cdh1 promoter methylation and any of these factors (sex status, or 0.78, 95% ci 0.411.50, p=0.46; smoking history, or 0.97, 95% ci 0.531.79, p=0.93; pathological type, or 0.97, 95% ci 0.591.60, p=0.91; clinical stage, or 1.48, 95% ci 0.812.68, p=0.2; lymph node metastasis, or 0.68, 95% ci 0.133.63, p=0.65; and differentiation degree, or 1.01, 95% ci 0.343.02, p=0.99). The or ranged from 0.78 to 3.89, which was not a significant change, suggesting that the results of our meta - analysis were not significantly unstable . The funnel plot shown in figure 4 is partially symmetric, indicating low publication bias regarding cdh1 methylation in lung cancer . Hypermethylation of tumor suppressor genes and hypomethylation of oncogenes are two essential components of the molecular mechanism involved in the epigenomic regulation of initiation and progression of cancer . As a tumor suppressor gene, cdh1 maintains cell - cell adhesion and keeps epithelial cells arranged in normal arrangement and layer . In vitro studies demonstrate that loss of expression or function of cdh1 can activate transcription factors associated with epithelial - mesenchymal transition, leading to metastasis of cancer cells.37 cdh1 methylation has been detected in several types of carcinoma, including breast cancer, gastric cancer, and lung cancer.3840 a comprehensive evaluation of markers of methylation in lung cancer is needed to better understand the relationship between cdh1 methylation and lung cancer . Although a large number of studies have demonstrated a possible relationship between cdh1 methylation and lung cancer, a meta - analysis can summarize the relevant studies and compare different subgroup characteristics . In this meta - analysis we analyzed data from 13 studies that included 657 tumor tissue samples and 593 control samples . The results show that the cdh1 methylation level in the cancer group was significantly higher than in the control group . The pooled or using the fixed - effect model was 3.89 (95% ci 2.875.27 versus the control group). Cdh1 methylation plays a key role in the induction of lung cancer due to silencing of the tumor suppressor gene cdh1 . This conclusion is consistent with that of a previous study.36 since changes in cdh1 promoter hypermethylation are reversible, drug treatment promoting demethylation may be useful for delaying carcinogenesis and progression . Treatment with 5-aza-2-deoxycytidine showed that migration of a549 cells decreased markedly upon restoration of cdh1.41 these preclinical studies show the therapeutic potential of restoration of tumor suppressor expression via epigenetic modulation . We further determined the clinicopathological significance of cdh1 promoter hypermethylation in patients with lung cancer . For smoking history, the summary or was 0.97 (95% ci 0.531.79) in the 65 cases and 27 controls . Smoking may target other specific genes for methylation or mutation, for example, methylenetetrahydrofolate reductase.33 other subgroup meta - analysis was performed, including for sex status (or 0.78, 95% ci 0.411.50), pathological type (or 0.97, 95% ci 0.591.60), clinical stage (or 1.48, 95% ci 0.812.68), lymph node metastasis (or 0.68, 95% ci 0.133.63), and degree of differentiation (or 1.01, 95% ci 0.343.02). Cdh1 methylation determined by clinical staging shows a slightly more significant association than the other subgroups . Interestingly, cdh1 methylation was detected much more frequently in stages iii and iv than in stages i and ii . However, cdh1 methylation itself does not correlate with pathological type, sex status, lymph node metastasis, or degree of differentiation . Possible reasons for this finding might be the widely heterogeneous results for the subgroups or the lack of cases and controls in the subgroups . Other potentially significant factors may need to be investigated, such as patient age, tumor size, and biopsy sample control.42 cdh1 methylation might be a biomarker of lung cancer, with potential value in predicting the prognosis of the disease, and warrants further studies involving more clinical cases for meta - analysis in the future . In addition, the potential variables on cdh1 methylation from different group database are still not clear due to the limitation of the statistical power of meta - analysis.
Nerve conduction studies (ncs) are an objective, quantitative, and reproducible measure of peripheral nerve function and are widely used in the diagnosis of neuropathies [1, 2]. They can be used to monitor neuropathic disease progression and the efficacy of interventions in clinical trials . However, non - uniform electrophysiologic test procedures degrade reproducibility . Potential sources of variation include the use of different emg instruments at different test sessions or sites, inconsistent placement of recording and stimulating electrodes, use of non - standardized distance measurements, use of sub - maximal electrical stimuli, poor skin preparation resulting in high skin impedance, and failure to maintain limb temperature within an acceptable range or to compensate for temperature . All these factors may compromise the repeatability of ncs measurement and lead to erroneous diagnostic conclusions . Aside from true physiological changes, factors that influence repeatability of ncs measurements are broadly grouped into two categories: inter - tester variability and intra - tester variability . Inter - tester variability refers to variability of a test parameter measured on a single individual when repeat test measurements are made by two or more examiners . Intra - tester variability refers to variability of a test parameter when repeat test measurements are made by a single examiner . In the absence of physiological changes, both inter - tester and intra - tester variability are influenced by electrodiagnostic examination technique . Automated ncs instruments may improve ncs repeatability by utilizing prefabricated electrode arrays and automating evoked waveform analysis . The objective of this study is to quantify ncs repeatability utilizing the nc - stat (neurometrix, inc ., waltham, massachusetts), an automated ncs system [810]. We hypothesized that use of the nc - stat system would yield highly reproducible ncs results . All were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy . The study was approved and monitored by an independent review board (copernicus group, cary, nc, usa). Each upper and lower extremity nerve was tested twice (7 days apart) by the same technician, utilizing the nc - stat (neurometrix, inc ., waltham, ma, usa), an automated ncs instrument . Shown in figure 1 is a photograph of the nc - stat system components: pre - fabricated electrode arrays specific to peroneal nerve motor testing; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis; a communication port to transmit data for report generation . The nc - stat system is fda 510(k) cleared for the performance of motor studies of the median, ulnar, peroneal and tibial nerves, and sensory studies of the median, ulnar and sural nerves . Tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used . Each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . A technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks . The electrode arrays incorporated stimulating, recording, and ground electrodes, as well as a temperature sensor . The device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . The evoked compound muscle (cmap) or compound sensory nerve (snap) action potentials were recorded following a series of supramaximal stimuli . Supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities (step size varies between 2.5 and 20 ma depending on nerve and stimulus duration). Snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count . Time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . It consists of three components: pre - fabricated electrode arrays (biosensor, left panel) for a peroneal motor nerve conduction study, an electronic monitor (connected to a biosensor during testing), a report generation system (not shown) based on electrophysiological data collected and processed by the monitor and transmitted via docking station (a telecommunication port for secure data transfer via telephone line). It consists of three components: pre - fabricated electrode arrays (biosensor, left panel) for a peroneal motor nerve conduction study, an electronic monitor (connected to a biosensor during testing), a report generation system (not shown) based on electrophysiological data collected and processed by the monitor and transmitted via docking station (a telecommunication port for secure data transfer via telephone line). The median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . A volume - conducted median motor response generated by abductor pollicis brevis was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease . Concurrently, an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal (pip) joint (active electrode), with the inactive electrode 3 cm distal . The ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease . The ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist . The ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal . The stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line . Responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe, over the vicinity of the extensor digitorum brevis muscle (see figure 1). For tibial nerve testing, stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus . A ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases . At the conclusion of ncs for a given subject, motor responses (cmap and f - wave) were recorded with filter settings of 15 hz high pass and 3 khz low pass; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters . Supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz . For recording snaps, 615 individual waveforms were averaged depending upon the signal - to - noise ratio . Up to 10 f - wave responses (with 12 traces as a maximum) up to 20 peroneal and tibial f - wave responses (with 40 traces as a maximum for personal, and 24 traces as a maximum for tibial) were acquired for lower extremity tests . 2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . Top left panel shows the ulnar sensory nerve action potential (snap) with distal sensory latency (dsl) marked by an open circle . Bottom left panel shows the ulnar compound muscle action potential (cmap) with distal motor latency (dml) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . Pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . Dotted line is the calculated mean f - wave latency for the f - wave set . Sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . Top left panel shows the ulnar sensory nerve action potential (snap) with distal sensory latency (dsl) marked by an open circle . Bottom left panel shows the ulnar compound muscle action potential (cmap) with distal motor latency (dml) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . Pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . Dotted line is the calculated mean f - wave latency for the f - wave set . All ncs parameters were determined by automated computer algorithms [7, 9, 10]. For motor studies, distal motor latency (dml) was the time difference between stimulus onset and initial negative deflection (marked as + in the lower left panel of figure 2). Dml values from four cmap waveforms were averaged and reported (after temperature correction). Cmap amplitude was measured baseline to negative peak (upward deflection, identified by the upper triangle) based on the averaged cmap . F - wave onset latency was identified for each trace with an identified f - wave response (traces with +, right panel of figure 2), and their average was reported as the mean f - wave latency (vertical dotted line). The distal sensory latency (dsl) was measured from stimulus onset to the initial negative peak (upward deflection) of the snap (open circle in the upper left panel of figure 2). The snap amplitude was measured peak to peak (negative - positive, or vertical distance between open and closed circles). All motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values (32 c upper extremity, 30 c lower extremity) with a linear correction formula: latency(corrected) = latency (raw))corrcoef(temperature - reference). The temperature correction factor corrcoef was previously determined in an independent study population (150 subjects, data on file), which also found dependence of cmap and snap amplitude on temperature to be not statistically significant . Statistical measures used to quantify ncs repeatability mirrored those used in prior studies [2, 4, 5]. The pearson product - moment correlation (cc) was used to assess the association between ncs parameters obtained 7 days apart . Intraclass correlation coefficient (icc) was used to determine the agreement between the two tests . Coefficient of variation (cov) of test - retest ncs parameters was calculated and the average over all nerves was reported . Relative intertrial variation (riv) the riv is the difference between two tests as a percentage of the average of the two tests . Because of the small sample size, 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . To facilitate comparison, the 5th and 95th percentile values, as reported by others, were also obtained for selected parameters . The mean and standard deviation of the difference between the test and retest results paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean . All were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy . The study was approved and monitored by an independent review board (copernicus group, cary, nc, usa). Each upper and lower extremity nerve was tested twice (7 days apart) by the same technician, utilizing the nc - stat (neurometrix, inc ., waltham, ma, usa), an automated ncs instrument . Shown in figure 1 is a photograph of the nc - stat system components: pre - fabricated electrode arrays specific to peroneal nerve motor testing; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis; a communication port to transmit data for report generation . The nc - stat system is fda 510(k) cleared for the performance of motor studies of the median, ulnar, peroneal and tibial nerves, and sensory studies of the median, ulnar and sural nerves . Tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used . Each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . A technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks . The electrode arrays incorporated stimulating, recording, and ground electrodes, as well as a temperature sensor . The device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . The evoked compound muscle (cmap) or compound sensory nerve (snap) action potentials were recorded following a series of supramaximal stimuli . Supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities (step size varies between 2.5 and 20 ma depending on nerve and stimulus duration). Snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count . Time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . It consists of three components: pre - fabricated electrode arrays (biosensor, left panel) for a peroneal motor nerve conduction study, an electronic monitor (connected to a biosensor during testing), a report generation system (not shown) based on electrophysiological data collected and processed by the monitor and transmitted via docking station (a telecommunication port for secure data transfer via telephone line). It consists of three components: pre - fabricated electrode arrays (biosensor, left panel) for a peroneal motor nerve conduction study, an electronic monitor (connected to a biosensor during testing), a report generation system (not shown) based on electrophysiological data collected and processed by the monitor and transmitted via docking station (a telecommunication port for secure data transfer via telephone line). The median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . A volume - conducted median motor response generated by abductor pollicis brevis was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease . Concurrently, an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal (pip) joint (active electrode), with the inactive electrode 3 cm distal . The ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease . The ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist . The ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal . The stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line . Responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe, over the vicinity of the extensor digitorum brevis muscle (see figure 1). For tibial nerve testing, stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus . Both the active and inactive electrodes were located just distal to the medial malleolus . A ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases . At the conclusion of ncs for a given subject, test data were uploaded to a central database . Motor responses (cmap and f - wave) were recorded with filter settings of 15 hz high pass and 3 khz low pass; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters . Supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz . 615 individual waveforms were averaged depending upon the signal - to - noise ratio . Up to 10 f - wave responses (with 12 traces as a maximum) were recorded for the median and ulnar nerves . Up to 20 peroneal and tibial f - wave responses (with 40 traces as a maximum for personal, and 24 traces as a maximum for tibial) were acquired for lower extremity tests . 2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . Top left panel shows the ulnar sensory nerve action potential (snap) with distal sensory latency (dsl) marked by an open circle . Bottom left panel shows the ulnar compound muscle action potential (cmap) with distal motor latency (dml) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . Pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . Dotted line is the calculated mean f - wave latency for the f - wave set . Sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject . Top left panel shows the ulnar sensory nerve action potential (snap) with distal sensory latency (dsl) marked by an open circle . Bottom left panel shows the ulnar compound muscle action potential (cmap) with distal motor latency (dml) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation . Pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response . Dotted line is the calculated mean f - wave latency for the f - wave set . All ncs parameters were determined by automated computer algorithms [7, 9, 10]. For motor studies, distal motor latency (dml) was the time difference between stimulus onset and initial negative deflection (marked as + in the lower left panel of figure 2). Dml values from four cmap waveforms were averaged and reported (after temperature correction). Cmap amplitude was measured baseline to negative peak (upward deflection, identified by the upper triangle) based on the averaged cmap . F - wave onset latency was identified for each trace with an identified f - wave response (traces with +, right panel of figure 2), and their average was reported as the mean f - wave latency (vertical dotted line). The distal sensory latency (dsl) was measured from stimulus onset to the initial negative peak (upward deflection) of the snap (open circle in the upper left panel of figure 2). The snap amplitude was measured peak to peak (negative - positive, or vertical distance between open and closed circles). All motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values (32 c upper extremity, 30 c lower extremity) with a linear correction formula: latency(corrected) = latency (raw))corrcoef(temperature - reference). The temperature correction factor corrcoef was previously determined in an independent study population (150 subjects, data on file), which also found dependence of cmap and snap amplitude on temperature to be not statistically significant . Statistical measures used to quantify ncs repeatability mirrored those used in prior studies [2, 4, 5]. The pearson product - moment correlation (cc) intraclass correlation coefficient (icc) was used to determine the agreement between the two tests . Coefficient of variation (cov) of test - retest ncs parameters was calculated and the average over all nerves was reported . Relative intertrial variation (riv) the riv is the difference between two tests as a percentage of the average of the two tests . Because of the small sample size, 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . To facilitate comparison, the 5th and 95th percentile values, as reported by others, the mean and standard deviation of the difference between the test and retest results were also reported . Paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean . Their ages ranged from 24 to 52 years (mean 37.1, sd 8.6 years). Height varied from 157 to 180 cm (mean 173, sd 7.9 cm). Total of 15 subjects (5 females) volunteered for the lower extremity repeatability study and nine of them had also enrolled in the upper extremity study . Their age range was 2247 years (mean 32.6, sd 8.2 years) and height range was 157180 cm (mean 172, sd 8.2 cm). The rivs for mean fwl at 5th and 95th percentiles were [11.4%, 9.4%], [5.1%, 3.3%], [4.9%, 4.5%], and [2.8%, 4.0%], respectively, for median, ulnar, peroneal, and tibial nerves . At p = 0.05 level, paired t - tests indicated that the means of all test and retest ncs parameters were the same . Pearson ccs for all latency parameters were greater than 0.80 with the exception of median nerve mean fwl (cc = 0.69). All upper extremity amplitude parameters had ccs greater than 0.85 . For lower extremity nerves, all latency results were based on temperature corrected latencies . Without temperature compensation, latency parameters exhibited lower repeatability . For example, the cc values for ulnar nerve dml, mean fwl, and dsl without temperature correction were 0.72, 0.93, and 0.72, respectively . Standard deviation of the difference between test and retest results would have been 0.31, 1.04, and 0.34 . Table 1statistical measures of nerve conduction study parameter repeatabilityriv percentiles(day 8day 1)nerveccicccov10th (%) 90th (%) meanstdevpaired t - testdistal motor latency (dml)median0.8720.8700.0329.55.10.0550.1940.289ulnar0.8890.8320.0458.311.00.0260.1900.605peroneal0.8640.8450.06115.48.10.1240.4530.307tibial0.8500.8240.0386.88.80.0160.2390.799mean f - wave latency (fwl)median0.6920.6790.0316.64.40.0371.9700.943ulnar0.9490.9210.0173.91.10.2860.9150.246peroneal0.8960.9010.0174.73.40.1631.5630.693tibial0.9430.9400.0131.83.50.2781.1450.363distal sensory latency (dsl)median0.8870.8790.0287.24.00.0560.1890.270ulnar0.8150.7970.0375.79.00.0120.2070.825compound muscle action potential (cmap) amplitudemedian0.8790.8620.10919.219.90.0310.1980.550ulnar0.9810.9740.0455.710.70.2880.6090.088peroneal0.3920.3310.29830.899.50.8491.9370.112tibial0.7140.7280.14830.925.40.1651.6010.695sensory nerve action potential (snap) amplitudemedian0.9540.9540.05811.87.80.6738.4020.761ulnar0.9900.9820.0424.410.91.4855.0630.275cc: pearson product moment correlation to assess the association between test - retest results . Riv: relative interval variation defines the difference between test - retest as a percentage of average . Paired t - test results indicate the means of test and retest are not statistically different . Dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference . Dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference . Fwl is the arithmetic mean of individual f - wave latencies in an f - wave set . Statistical measures of nerve conduction study parameter repeatability cc: pearson product moment correlation to assess the association between test - retest results . Riv: relative interval variation defines the difference between test - retest as a percentage of average . Paired t - test results indicate the means of test and retest are not statistically different . Dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference . Dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference . Fwl is the arithmetic mean of individual f - wave latencies in an f - wave set . Several studies (summarized in table 2) have examined ncs parameter repeatability [2, 4, 5]. In, the repeatability of ncs was evaluated for 60 sites participating in a clinical trial, with ncs oversight performed by an experienced, insightful and knowledgeable core lab . Our study yielded similar results, though our study demonstrated a higher cov for peroneal cmap amplitude (9% vs. 29.8%). In, 132 healthy subjects were retested at a time interval of 14 weeks and icc and riv were used to measure the test - retest repeatability . In comparison with that study, the riv (5th and 95th percentile) for tibial nerve f - wave latency was tighter in our study ([2.8%, 4.0%] vs. [4.6%, 5.7%]) while the relationship was reversed for median nerve fwave latency results ([11.4%, 9.4%] vs. [6.7%, 6.7%]). Decreased repeatability of median nerve f - wave latency may be attributed to the lower amplitude signal - to - noise ratio as median nerve f - waves were acquired via a volume - conduction recording technique . Indeed, latency repeatability was much higher for ulnar f - waves recorded directly over the muscle . The icc for ulnar f - wave latency was 0.92 in our study, higher than the icc of 0.59 reported in a similar study of 49 healthy adults . Salerno and colleagues studied upper extremity sensory test - retest repeatability based on 158 active workers who were tested by a neurologist and a physiatrist . Our results fell into the upper range of their repeatability results as measured by cc and icc . Table 2repeatability results reported in the literaturesnerveccicccovdistal motor latency (dml)median0.8260.04/0.08> peroneal0.05/0.08tibial0.6170.08minimum f - wave latency (fwl)ulnar0.59tibial0.921distal sensory latency (dsl)median0.820.920.820.920.04ulnar0.370.640.320.63compound muscle action potential (cmap) amplitudemedian0.7770.07/0.11peroneal0.09tibial0.846sensory nerve action potential (snap) amplitudemedian0.830.880.834/0.810.880.08ulnar0.680.850.680.85 132 healthy subjects retested in 14 weeks . Results for control subjects from 60 sites with core lab support in a clinical trial . 33 subjects retested in 12 week, results for small electrode size (0.78 cm) and all three nerves combined . Repeatability results reported in the literatures 132 healthy subjects retested in 14 weeks . Results for control subjects from 60 sites with core lab support in a clinical trial . 33 subjects retested in 12 week, results for small electrode size (0.78 cm) and all three nerves combined . It is possible that the small recording electrodes (surface areas 2 cm) used in our study degraded repeatability, since tjon - a - tsien and colleagues noted that when electrode size was changed from 0.78 to 7.65 cm, the cov of cmap amplitude improved from 11 to 7% (lower cov is associated with higher repeatability). Dml repeatability was less affected by electrode size in both our study and the study of . However, control of skin temperature is often difficult to achieve, especially in different testing environments . In the present study, skin temperature adjacent to the recording site was acquired automatically using an embedded probe and latencies were adjusted to the reference temperature . As expected, temperature correction improved ncs repeatability . For example, the cc increased from 0.72 (without temperature correction) to 0.89 (with correction) for both motor and sensory latencies of the ulnar nerves . Temperature correction also reduced the standard deviation of the test - retest latency difference by as much as 39% (ulnar dml). The principal limitation of the current study was the small number of subjects evaluated relative to several prior investigations . The short test - retest interval of 1 week reduced the likelihood of intervening physiological changes . It would be valuable to quantify repeatability of the automated ncs instrument in neuropathic nerves . At the time of this study, temperature correction may improve the repeatability of the amplitude parameters, especially for sensory responses . The reproducibility of individual ncs parameters followed the same pattern as those observed in traditional settings . F - wave latency had the greatest repeatability, followed by dml, dsl, and snap amplitude . The cmap amplitude was the least reproducible parameter, with peroneal cmap amplitude repeatability the lowest among all nerves . A larger recording electrode size should improve its repeatability . F - wave latency repeatability for median nerve was lower due to the lower signal - to - noise ratio inherent in volume - conduction recordings . As a result, over - muscle recording would be expected to enhance f - wave repeatability, which was the case for the ulnar nerve . Further studies with longer test - retest intervals and a higher subject count would be valuable in confirming the high reproducibility of ncs parameters measured in this study . Extending the study to pathologic nerves and validating the equivalent or superior performance of automated ncs instrument should increase the adoption rate of the automated ncs technology in patient management
This study was conducted within the north central cancer treatment group, a national cancer cooperative group that is funded in part by the national cancer institute . Questionnaire and clinical trial data from two separate cancer therapeutic trials, n9921 and these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006, respectively . The present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials . Eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents, gefitinib, paclitaxel, and carboplatin, which were used in one or both of the trials mentioned above . These criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following: 1) patient aged 65 years or older; 2) eastern cooperative group performance score (an often - utilized score that describes the level of function of cancer patients) of 0, 1, or 2; 3) histologic or cytologic confirmation of non - small cell lung cancer; 4) unresectable cancer; 5) no prior chemotherapy; 6) physician - anticipated life expectancy of 12 weeks or longer; and 7) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial . In addition, all patients had to have had adequate organ function, as demonstrated by an acceptable hemogram, liver function tests, and serum creatinine at the time of study enrollment . Other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive, such as, for example, an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel . All eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy . This 12-item instrument has been previously validated, has been tested in a variety of settings, and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens: 1) weekly carboplatin and paclitaxel (intravenous); 2) weekly carboplatin and paclitaxel (intravenous) potentially for four months followed by daily gefitinib (oral); or 3) daily gefitinib (oral). Patients were to continue cancer therapy until evidence of cancer progression, until unacceptable side effects occurred, or, for a maximum of four months, if they were receiving conventional chemotherapy . They were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria, version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed . The sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials, not for the social support aims . Nonetheless, for the current study, we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender, performance score, and severe adverse events . The sample size of 113 (56 in each group) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points (60 versus 72) using a two - sided alpha of 0.007 . Scores from the lubben social support network (items 110 on table 2) were summed for analyses and then transformed to a 100% scale . A score of 0 represents the lowest possible level of social support; whereas, a score of 100 represents the highest possible level of social support . To transform the scores to a 100% scale, a patient s individual score was divided by the patient s possible range accounting for any missed items (maximum minus minimum) and multiplied by 100 . A rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender, age (> = 70), and performance status . Similarly, a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events (grade 3 or worse) versus those who did not . Time - to - cancer progression was defined as the time from registration to documentation of cancer progression . Survival time was defined as the time from study registration to death due to any cause . A cox proportional hazards model, which adjusted for cancer treatment, performance score, age, gender, and sum of target lesions at baseline, was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . In order to illustrate the influence of social support on overall survival, we divided the cohort by social support score quartiles . No widely accepted cut - points exist to separate social support into categories, so quartiles were used to distribute evenly the 113 patients into four groups . A logistic regression model, which adjusted for the same baseline factors mentioned above, was used to assess relationships between social support and adverse events . Because of the exploratory nature of these analyses and because of the number of statistical tests performed, a p - value of <0.01 was considered statistically significant . This decision to utilize this p - value was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment . All statistical analyses were conducted with sas software (sas institute, cary, nc, usa). This study was conducted within the north central cancer treatment group, a national cancer cooperative group that is funded in part by the national cancer institute . Questionnaire and clinical trial data from two separate cancer therapeutic trials, n9921 and these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006, respectively . The present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials . Eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents, gefitinib, paclitaxel, and carboplatin, which were used in one or both of the trials mentioned above . These criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following: 1) patient aged 65 years or older; 2) eastern cooperative group performance score (an often - utilized score that describes the level of function of cancer patients) of 0, 1, or 2; 3) histologic or cytologic confirmation of non - small cell lung cancer; 4) unresectable cancer; 5) no prior chemotherapy; 6) physician - anticipated life expectancy of 12 weeks or longer; and 7) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial . In addition, all patients had to have had adequate organ function, as demonstrated by an acceptable hemogram, liver function tests, and serum creatinine at the time of study enrollment . Other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive, such as, for example, an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel . All eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy . This 12-item instrument has been previously validated, has been tested in a variety of settings, and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens: 1) weekly carboplatin and paclitaxel (intravenous); 2) weekly carboplatin and paclitaxel (intravenous) potentially for four months followed by daily gefitinib (oral); or 3) daily gefitinib (oral). Patients were to continue cancer therapy until evidence of cancer progression, until unacceptable side effects occurred, or, for a maximum of four months, if they were receiving conventional chemotherapy . They were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria, version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed . The sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials, not for the social support aims . Nonetheless, for the current study, we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender, performance score, and severe adverse events . The sample size of 113 (56 in each group) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points (60 versus 72) using a two - sided alpha of 0.007 . Scores from the lubben social support network (items 110 on table 2) were summed for analyses and then transformed to a 100% scale . A score of 0 represents the lowest possible level of social support; whereas, a score of 100 represents the highest possible level of social support . To transform the scores to a 100% scale, a patient s individual score was divided by the patient s possible range accounting for any missed items (maximum minus minimum) and multiplied by 100 . A rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender, age (> = 70), and performance status . Similarly, a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events (grade 3 or worse) versus those who did not . Time - to - cancer progression was defined as the time from registration to documentation of cancer progression . Survival time was defined as the time from study registration to death due to any cause . A cox proportional hazards model, which adjusted for cancer treatment, performance score, age, gender, and sum of target lesions at baseline, was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . In order to illustrate the influence of social support on overall survival, we divided the cohort by social support score quartiles . No widely accepted cut - points exist to separate social support into categories, so quartiles were used to distribute evenly the 113 patients into four groups . A logistic regression model, which adjusted for the same baseline factors mentioned above, was used to assess relationships between social support and adverse events . Because of the exploratory nature of these analyses and because of the number of statistical tests performed, a p - value of <0.01 was considered statistically significant . This decision to utilize this p - value was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment . All statistical analyses were conducted with sas software (sas institute, cary, nc, usa). A total of 113 patients met the eligibility criteria, received chemotherapy on a clinical trial, and therefore participated in the current study . One patient did not receive chemotherapy, and two did not meet the eligibility criteria described earlier; thus, these three patients are not included in the analyses . Forty - two percent of the cohort consisted of women, and the median age (range) was 74 years (65, 91). At the time of study entry, a performance score of 0, 1, or 2 was observed in 29%, 55%, and 16% of the cohort, respectively . Lubben social network scale data are summarized in table 2 . Of note, 44% of patients reported that they see nine or more relatives at least once a month, and 27% reported that they see five to eight . In addition, 73% reported that when they have an important decision to make, they always have someone who can take them to their medical appointments, and 71% reported that they always have someone from whom they can seek help if they have medication side effects . The median transformed social support score for the entire cohort (range) was 72 (30100). It should be noted that, although normative reference data are not readily available, a community - based study among a group of 7,524 older individuals observed a similarly - derived, average social support score of 63.19 among women and men in the present study, the average score was not statistically different: 72 in both (rank sum, p = 0.87) (figure 1). Additionally, there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2: median score 74 and 70 (rank sum, p = 0.06). Although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression (p = 0.02; hazard ratio [hr] = 1.02; 95% confidence interval [ci]: 1.001.03), these scores did not predict overall survival (p = 0.34; hr = 1.01; 95% ci: 0.991.02) (figure 2). There was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not: median score 74 and 70 (rank sum, p = 0.28) (adjusted logistic regression p = 0.70; odds ratio [or] = 1.00; 95% ci: 0.971.02) (table 3). A total of 113 patients met the eligibility criteria, received chemotherapy on a clinical trial, and therefore participated in the current study . One patient did not receive chemotherapy, and two did not meet the eligibility criteria described earlier; thus, these three patients are not included in the analyses . Forty - two percent of the cohort consisted of women, and the median age (range) was 74 years (65, 91). At the time of study entry, a performance score of 0, 1, or 2 was observed in 29%, 55%, and 16% of the cohort, respectively . Lubben social network scale data are summarized in table 2 . Of note, 44% of patients reported that they see nine or more relatives at least once a month, and 27% reported that they see five to eight . In addition, 73% reported that when they have an important decision to make, they always have someone to talk with . Always have someone who can take them to their medical appointments, and 71% reported that they always have someone from whom they can seek help if they have medication side effects . The median transformed social support score for the entire cohort (range) was 72 (30100). It should be noted that, although normative reference data are not readily available, a community - based study among a group of 7,524 older individuals observed a similarly - derived, average social support score of 63.19 among women and men in the present study, the average score was not statistically different: 72 in both (rank sum, p = 0.87) (figure 1). Additionally, there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2: median score 74 and 70 (rank sum, p = 0.06). Although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression (p = 0.02; hazard ratio [hr] = 1.02; 95% confidence interval [ci]: 1.001.03), these scores did not predict overall survival (p = 0.34; hr = 1.01; 95% ci: 0.991.02) (figure 2). There was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not: median score 74 and 70 (rank sum, p = 0.28) (adjusted logistic regression p = 0.70; odds ratio [or] = 1.00; 95% ci: 0.971.02) (table 3). The present study was designed to assess and evaluate the role of social support in a group of geriatric non - small cell lung cancer patients who were enrolled in two different cancer therapeutic clinical trials . This study found that this cohort of geriatric patients had high levels of social support, as suggested by the fact that 81% reported that they always had someone who could take them to medical appointments, and 71% reported that they always had someone from whom they could seek help if they had medication side effects . However, social support was not associated with improved survival or diminished side effects from chemotherapy . Moreover, no differences were observed between men and women in terms of baseline social support . Thus, although previous studies have reported that social support carries a positive impact on clinical outcomes and although gender differences in extent of social support have been hypothesized, the present study did not observe such findings . Why might the results of this study be divergent from the others that preceded it? First, relying on a clinical trial to answer such questions provides a solid infrastructure that ensures reliable outcome data . The fact that all clinical trial participants were followed prospectively in such a thorough and systematic fashion makes it unlikely that patients particularly those who have poor social support and who are thereby especially vulnerable to being lost to follow up would, in fact, be lost to follow up . Such artificially compromised outcomes were side - stepped by building the study around two clinical trials, as was done here . Thus, the present study s meticulous follow up may have allowed for a more definitive and accurate assessment of the predictive capabilities or lack thereof of social support . Second, and more importantly, one might argue that patients willingness to participate in a clinical trial is associated with a high level of social support and that this selection bias may have contributed to the lack of differences between groups . The fact that 82% of patients reported that they always had someone to bring them to medical appointments speaks to the possibility that this group as a whole has a high level of social support . Similarly, the median social support score of 72 in this study appears greater than the 63 score derived from a community - based study, an observation that also suggests cancer clinical trial participants have high levels of social support.19 in short, although the findings from this study suggest that social support does not influence clinical outcomes, these findings might pertain to only a culled group of geriatric clinical trial participants . Future studies may choose to focus further on the extent of social support among geriatric patients who participate in clinical trials, and further studies may also choose to study further whether social support is a predictive factor for the patients who can and do enroll in cancer clinical trials . Two final points merit further discussion . First, although the current study did not find that social support was predictive of outcomes, there are a variety of other potentially clinically relevant factors that had not been captured and adjusted for . These include socioeconomic status, ongoing tobacco use, use of nutritional supplements, as well as several other factors . Other investigators may choose to further study the issue of social support in the context of these other factors . Second, although this study found that social support is not associated with clinical outcomes among elderly lung cancer patients, there remains no question that these patients have major needs that are likely exacerbated by a lack of social support . Patients with metastatic non - small cell lung cancer suffer from a poor prognosis, as seen from both studies presented here where the prognosis was poor, and previous studies have demonstrated that elderly patients suffer from a wide constellation of cancer - related symptoms, including depression, guilt, debility, pain, and dyspnea.1013 although the present study did not observe an association between social support and clinical outcomes, there nonetheless remains a strong need for maximizing social support to help with some of the cancer - related challenges these patients face.
Port - wine stains (pwss) are congenital vascular malformations comprised of ectatic dermal capillaries and affecting 0.3 - 1% of newborn infants . Acquired pwss are an uncommon entity developing later in life, but morphologically and histologically similar to the congenital lesions . We hereby report a case of 41-year - old male patient who acquired pws about a year ago . A 41-year - old male presented to the outpatient department of dermatology of our tertiary care hospital with complaint of asymptomatic reddish patches involving the lower part of right leg for 10 months . The patch started from near the ankle and gradually spread to involve whole of the medial aspect of right leg over a period of 10 months . We could not elicit any history of antecedent mechanical or thermal trauma, drug intake, topical application or excessive ultraviolet exposure . Dermatological examination revealed a diffuse partially blanchable erythema extending from the right ankle and involving chiefly the medial aspect of right leg up to the knee [figure 1]. There were also a few discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg [figure 2]. A diffuse partially blanchable erythema extending from the right ankle and involving the medial aspect of right leg up to the knee discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg on the basis of the clinical examination, a provisional diagnosis of acquired pws was made and the patient investigated . The histopathological examination revealed multiple dilated, variably sized capillaries lined by a single layer of endothelial cells throughout the dermis showing lobular arrangement at places [figure 3]. Color doppler of the limb did not reveal any abnormality of the underlying arteries and veins . (a) multiple dilated variably sized capillaries in the dermis (h and e, 40, arrows). (b) high power showing groups of capillaries lined by a single layer of endothelial cells (h and e, 250, arrow) laser therapy was discussed with the patient, but he declined treatment for the lesion . Pwss are congenital vascular malformations usually presenting at birth in the form of pink - red to purple macules, which become darker, raised, and nodular as the person ages . The most accepted hypothesis is a defective embryological maturation of the sympathetic fibers, resulting in a loss of normal sympathetic control of the cutaneous vessels which leads to ectasia . Various factors have been proposed for the occurrence of acquired pwss . Of these, trauma has been found to be the most important causative factor in majority of the cases . Trauma - induced pws was first described by fegeler in 1949 and hence called fegeler syndrome . In a review of 59 patients with acquired pws, trauma was found to be a causative factor in 17 (29%) cases . It has been proposed that injury may result in loss of a previously effective sympathetic regulation of the cutaneous blood flow, leading to development of the lesions . Reports of acquired port - wine stain few cases of acquired pws have also been proposed secondary to oral medications like isotretinoin, oral contraceptive pills, simvastatin, and metformin . Isotretinoin causes skin fragility and frictional trauma, while simvastain and metformin have been shown to promote angiogenesis by upregulation of vascular endothelial growth factor . Isolated cases of acquired pws following frostbite injury, obstruction of the peritoneovenous shunt, herpes zoster infection, cerebral arteriovenous malformation, spinal root compression, and solid brain tumor have also been reported . No definite cause was found for the development of lesion in our patient . In such cases, a likely possibility of having a latent vascular anomaly which manifests clinically after unnoticed trauma an unusual finding in our patient was the presence of a lobular arrangement of dilated vessels in the dermis which has not been observed in previous cases of congenital or acquired pws . Interestingly, there are multiple reports of development of pyogenic granulomas (pg) within pws which also shows a lobular pattern of dilated vessels . It has been proposed that the pg probably results from an underlying arteriovenous anastamosis frequently associated with the pws . Though, there was no clinically appreciable pyogenic granuloma like lesion, the distinct histological features seen in our patient might be attributed to a similar pathogenetic mechanism . As regards therapy, it includes treatment with krypton, argon, argon - pumped, tunable dye, neodymium: yttrium - aluminum garnet, copper vapor, and pulsed dye lasers . Acquired pws tends to have a quicker and better response to pulsed dye therapy than congenital pws . The case report demonstrates a rare occurrence of a vascular malformation in an adult patient . Although no definite cause could be appreciated for the development of lesion in our patient, further reports and future insights into the pathogenesis of such cases would broaden our knowledge regarding this infrequently reported phenomenon . This is the first report of an acquired port - wine stain from india and previous reports of such cases have also been reviewed.
Ionizing radiation is a well - known carcinogen in humans, and breast is one of the most sensitive organs to radiogenic cancer . The rate of breast cancer in postwar japan was among the lowest in the world, but breast cancer contributed a disproportionately large fraction of the radiation - related cancer burden in atomic bomb survivors [2, 3]. The data from the hiroshima and nagasaki survivors provides strong evidence for increased breast cancer following single acute doses of 20 cgy and linearity with increasing dose [36]. Also, an increase in the incidence of breast cancer has been observed in areas affected by the chernobyl accident, which resulted in radioactive contamination of large areas of belarus and ukraine . A twofold increase in risk was observed when comparing the most (> 40 msv cumulative dose) and least contaminated regions . Interestingly, the increase appeared 10 years after exposure and was most prominent in women exposed at younger age . More than 50,000 women in the united states have been treated with chest radiation (20 gy) for a pediatric or young adult cancer . Children treated from cancer with radiotherapy have a 2.9 relative risk of subsequent malignancy compared to those who were not [8, 9]. A systematic review of 14 studies concluded that risk of breast cancer increased as early as 8 years following chest radiation and did not plateau with increasing length of follow - up . Studies estimating low - dose radiation - induced cancer risk from diagnostic x - rays and ct scans have found a small but significant increased lifetime risk [11, 12]. While the benefits of diagnostic x - ray and ct scans outweigh potential individual lifetime risk, their use should be justified and alternatives considered . We know remarkably little of molecular mechanisms that may be protective or risky for breast cancer after exposure to low - dose ionizing radiation (ldir). Identification of transcriptomic changes induced by ldir in mammary tissue will be valuable to elucidate the molecular mechanisms associated with radiation - induced breast cancer . Long noncoding rnas (lncrnas), which initially were thought of as transcriptional noise, are emerging as key regulators of a multitude of cellular processes by taking part in epigenetic, transcriptional, and posttranscriptional regulation of gene expression [13, 14]. The lncrnas have a weaker evolutionary constraint and lower levels of expression compared to the protein - coding transcripts [15, 16] but exhibit more tissue specific expression than the protein - coding genes . Recently, a number of studies have shown that lncrna expression can be deregulated in human cancers [17, 18]. As the functions of individual lncrnas in cancer are beginning to be elucidated, they are being categorized and referred to as either tumor suppressor or oncogenic lncrnas, in the same way as traditional protein - coding cancer genes . However, the relevance of lncrnas in the cell and tissue response to ionizing radiation has not yet been characterized . In this study, we used agilent sureprint g3 microarrays to profile lncrna and mrna from mammary glands of balb / c mice 2, 4, and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10 cgy of x - radiation . We identified lncrna and mrna expression signatures for each time point after irradiation in comparison to sham . Of the total 1338 lncrnas identified to be differentially expressed after ldir in either balb / c or spret / eij, 1337 had a significantly correlated expression pattern with at least one mrna that was also differentially expressed after ldir . Our results indicate lncrnas may exert a partial or key role in the regulation of coding rna expression induced by radiation . Balb / c and spret / eij mice were purchased from jackson laboratory, housed four per cage under a 12 hr light and 12 hr dark cycle, and fed with lab diet 5008 chow and water ad libitum . The mice were irradiated whole body at 8 - 9 weeks of age to a single dose of 10 cgy using a precision x - ray inc rad320 320 kvp x - ray machine, operated at 300 kv, 2 ma . Mammary tissues were collected for gene expression profile at 2, 4, and 8 weeks after irradiation . All animal experiments were performed at lawrence berkeley national laboratory and the study was carried out in strict accordance with the guide for the care and use of laboratory animals of the national institutes of health . The animal use protocol was approved by the animal welfare and research committee of the lawrence berkeley national laboratory . Agilent sureprint g3 mouse ge 8x60k microarrays were used according to the manufacturer's protocol (arrays contained 39,430 entrez gene rnas and 16,251 lncrnas). Genes that were differentially expressed between sham and irradiated were identified by the unpaired student's t - test with a p value cut - off of 0.05 (p value <0.001 for baseline strain comparison) and a fold - change criteria of more than 1.5 . For each of the 8 experimental treatment groups, the average expression values of the 3 biological replicates significantly correlated pairs of mrna and lncrna were calculated using a standard permutation test . In brief, for each potential mrna and lncrna pair, the 8 mrna values were randomly rearranged and a correlation coefficient was calculated between the 8 mrnas and 8 lncrnas values . The p value reported in this work represents the percentage of permutations leading to a higher correlation than the original correlation between the 8 mrnas and 8 lncrnas values . In other words, the lower the p value is, the more likely the mrna and lncrna pair is not randomly associated . Gene lists were annotated with biological functions using ingenuity pathway analysis (ipa), kegg pathway analysis (http://bioinfo.vanderbilt.edu/webgestalt/) and david go gene ontology (http://david.abcc.ncifcrf.gov/; p 0.05). Annotations for the various shapes used in the ipa networks in figures 13 are shown in figure s3 in supplementary material available online at http://dx.doi.org/10.1155/2015/461038 . To identify potential lncrnas and mrnas that may determine susceptibility to radiation - induced breast cancer, we profiled two inbred strains of mice with differing genetic susceptibilities: balb / c mice as more sensitive and spret / eij as more resistant . Balb / c mice carry two dna - pkcs polymorphisms with reduced catalytic subunit activity and defective nonhomologous - end - joining of double strand breaks . We identified 195 lncrnas as upregulated and 95 lncrnas as downregulated in balb / c in comparison to spret / eij (fold - change 1.5; p value <0.001) (figure 1(a); table s1). Additionally, 582 mrnas were upregulated and 402 mrnas were downregulated in balb / c in comparison to spret / eij (fold - change 1.5; p value <0.001) (figure 1(b); table s1). Gene ontology analyses of differentially expressed genes between balb / c and spret / eij showed significant enrichment for metabolic processes (p = 5.9e 06), ion binding (p = 3.00e 08), and chemokine signaling (p = 0.02) (figure 1(c); table s2). Our analyses identified significant strain differences in gene expression between balb / c and spret / eij mammary tissues . We found significant differences in the expression of a number of chemokines including cxcl10, ccl6, and ccl25, which were expressed at higher levels in mammary tissues of the more sensitive and susceptible balb / c mice and which have previously been associated with breast cancer progression when over expressed . To identify lncrna and mrna expression changes induced by low - dose ionizing radiation (ldir), we profiled lncrna and mrna expression from mammary glands of balb / c mice 2, 4, and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10 cgy of x - radiation . For balb / c mice, a total of 357, 480, and 335 lncrnas and 550, 911, and 389 coding rnas were identified to be differentially expressed at weeks 2, 4, and 8 after ir in comparison to sham (fold - change 1.5; p value <0.05), respectively (figure 2(a); table s1). For spret / eij, a total of 327 lncrnas and 424 mrnas were identified as differentially expressed at week 4 after irradiation in comparison to sham (fold - change 1.5; p value <0.05) (figure 3(a); table s1). Few coding - rnas and lncrnas were found to be differentially expressed at different time points (figure s1(a) and s1(b)) and between balb / c and spret / eij (figure s1(c)). To determine the biological functions associated with the ldir response, we excluded genes whose levels fluctuate in the mouse mammary gland across the estrous cycle . We recently mapped transcript - level changes across the estrous cycle in the murine mammary gland using rna sequencing and defined a comprehensive estrous variable gene signature of 3893 genes whose levels fluctuate in mammary glands of balb / c mice . Comparison of our mapped ldir genes in mammary glands of balb / c and spret / eij mice with the estrous signature revealed an approximate 20% overlap in balb / c (figure 2(b)) and 9% overlap in spret / eij mice (figure 3(b)). Nonoverlapping and differentially expressed ldir genes for each of the time points were then computationally mapped to biological functions, pathways, upstream regulators, and networks . These analyses suggested that the ldir response signatures in mammary glands of balb / c mice transitions between time points and is distinct from the ldir response in spret / eij mice . Two weeks after ldir exposure pathways and biological functions significantly enriched in mammary glands of balb / c mice compared to sham irradiated mice included chemokine signaling (p = 0.01), ccr3 signaling in eosinophils (p = 0.05), cellular movement (3.92e 04 <p <3.41e 02), and cell death and survival (1.29e 03 gene interaction networks were enriched for tissue and endocrine system injury (figure 2(c) top panel) and significant predicted upstream regulators (table s3) include gli2 (p = 5.29e 04) and satb1 (p = 1.98e 03). Similar to the two - week ldir response, gli2 was predicted to be an upstream regulator (p = 4.52e 03; table s3). Gata3 and stat6 were among other significant upstream regulators associated with the four - week low - dose response (table s3). We furthermore observed that the mammary gland of balb / c mice four weeks after ldir was enriched for inflammatory response genes (1.84e 05 <p <1.45e 02), cell - cell signaling (2.79e 05 <p <1.60e 02), while gene interaction networks were enriched for lipid metabolism (figure 2(c) middle panel). Interestingly, similar responses were observed in mammary glands of spret mice at 4 weeks after ldir including inflammatory response functions (1.69e 03 <p <4.49e 02), cell - cell signaling (5.54e 04 <p <4.49e 02), suggesting that the functional response is similar across strains and is independent of the gene transcript response . At 8 weeks after ldir we observed downregulation of genes involved in mammary gland development including progesterone receptor, prolactin, amphiregulin, and wnt4 (figure 2(c) bottom panel; table s3). To identify lncrnas potentially regulating the expression of coding rnas in response to radiation, correlation coefficients on expression data were calculated for each coding rna and lncrna that were identified as differentially expressed after ldir . A permutation - based algorithm was then used to determine which correlations were statistically significant (p <0.05; table s4). We observed that nearly all ldir modulated lncrnas were correlated with at least one of the ldir modulated coding mrnas (figure 4(a)). To determine whether these correlations were driven by estrous variations, we only considered genes whose expression levels were not overlapping with our previously determined estrous signature (figure 2(b)). Again, we observed that nearly all differentially expressed lncrnas were correlated with at least one differentially expressed mrna suggesting that estrous cycling does not affect the strong correlation between lncrna and mrna expression after ldir . To test the robustness of these correlations, we compared the number of lncrnas associated with at least one mrna at three different p values (p <5e 02, p <5e 03, and p <5e 04). At p <5e 02 or p <5e 03, nearly all (97100%) differentially expressed lncrnas were found to be correlated with at least one mrna (table s5). At p <5e 04, corresponding to a correlation coefficient> 0.9, we still observed a significant fraction (6381%; table s5) of lncrnas correlated with mrnas . Representative correlation networks of lncrnas are shown in figure 4(b) (p <5e 03) for each of the timepoints . We furthermore observed that the same lncrna correlates with different gene sets across different time points (figure s2). Taken together these data show that ldir induces coordinated changes in lncrna and mrnas and suggests a critical role for lncrnas in mediating the low - dose radiation response . In this study, we demonstrate that genetic background strongly influences the expression of lncrnas and their response to low - dose radiation by transcriptomic analysis of mouse mammary glands using microarrays that contain both lncrnas and coding rnas . We have identified a number of lncrnas that are significantly changed after exposure to ldir at three different timepoints after radiation exposure . Moreover, the changes in the expression of lncrnas are significantly correlated with the expression of coding rnas, suggesting that lncrnas may coordinate the tissue response to radiation via regulation of coding mrnas . However, the specific regulatory mechanism of this control requires further investigation, and knock - out and overexpression of the lncrna genes in mice and other model systems should be performed to increase our understanding of the regulatory mechanisms in response to ldir.
Women with psychiatric diseases are treated with antipsychotic medication(s) with limited evidence to support the safety of their use during pregnancy.olanzapine, a second - generation antipsychotic, is a us food and drug administration pregnancy category c drug with no unequivocal evidence of harm to the fetus.we report the first case of tracheo - esophageal fistula as a possible teratogenic effect of olanzapine exposure . Women with psychiatric diseases may become pregnant and are treated with antipsychotics without any proven evidence of safety . Due to ethical issues, pregnant women are rarely included in clinical trials, leading to a dearth of data available on the safety of antipsychotic drugs in this population . There have been reports of congenital anomalies in newborns of mothers exposed to antipsychotic medications during pregnancy [14]. Olanzapine, a second - generation antipsychotic, is a us food and drug administration (fda) pregnancy category c drug [3, 5] with no unequivocal evidence of harm to the fetus . A literature search revealed that maternal exposure to olanzapine appears to be associated with lumbar meningomyelocele, dysplastic kidney, hip dysplasia, atrioventricular canal defect, club foot, microcephaly, ventricular septal defect, absent fingers, craniosynostosis, cleft lip, encephalocele, aqueductal stenosis, etc . The possibility of further teratogenic effects of olanzapine cannot be ruled out . In this case report, we report the first case of tracheo - esophageal fistula (tef) as a possible teratogenic effect of olanzapine exposure . We report the case of a 29-year - old married woman with bipolar disorder with mixed episodes who had been treated with olanzapine in the psychiatric outpatient department of our institute (lady hardinge medical college, new delhi, india) for the last 7 years who delivered a full - term female baby with tef in september 2013 . One week after her marriage in february 2008, the patient developed psychotic symptoms such as excessive talking, episodes of crying and laughing, and forgetting things frequently . Following initial unsuccessful local treatment, she was taken to the psychiatry department of a tertiary care hospital . After initial management of acute symptoms there, she continued her treatment from our institute, where she was prescribed olanzapine (oleanz 10 mg tablet orally once daily). In december 2008, she had a miscarriage at 4 months gestation, following which she stopped taking the medication of her own choice . A careful history / enquiry from the parents failed to reveal any data available regarding congenital malformation of the aborted fetus . In 2009, she conceived again (it is noteworthy that the patient was not taking olanzapine during this pregnancy) and delivered a full - term healthy female child in january 2010 . Eight months after delivery of this baby the patient had a recurrence of psychotic symptoms for which she was prescribed the same treatment . In december 2012, she conceived a third time but due to persistent psychiatric symptoms, olanzapine was continued throughout the pregnancy by the treating psychiatrist, who considered there to be a favorable benefit: risk ratio for the patient at that time . She received regular antenatal care and was compliant with all prescriptions and instructions, including prophylactic folic acid and two doses of tetanus toxoid . There was no history of maternal alcohol intake, smoking, or exposure to other potential teratogenic drugs or exogenous hormones throughout the pregnancy . All baseline hematological (hemoglobin 13.8 g / dl, total leukocyte count 6000/l, platelets 2.3 lac/l, etc .) And biochemical investigations (random blood sugar 98 mg / dl, blood urea 27 mg / dl, etc .) Tests for trisomy 21 and 18 (nuchal translucency scan and dual marker tests) and rubella were negative ., she delivered a full - term female baby by vaginal delivery . Immediately after delivery, the baby experienced respiratory distress and excessive salivation, for which she was admitted to the neonatal intensive care unit . On examination, a red rubber catheter could not be negotiated beyond 10 cm in the esophagus . Other concomitant anomalies including vertebral, anal, renal, limb (components of vacterl [vertebral defects, anal atresia, cardiac defects, tracheo - esophageal fistula, renal anomalies, and limb abnormalities]), eye, ear, and nasal defects were ruled out . The tef was repaired using esophageal esophageal anastomosis with a drainage tube in the fifth intercostal space . On the second post - operative day for this reason, cervical esophagotomy was performed and a gastrostomy was inserted for feeding purposes . The baby was discharged in a stable condition with advice for full breast milk feeding through gastrostomy, some required medications (multivitamin syrup, calcium, anti - emetic and antibiotics) for an appropriate duration, and regular vaccination . However, although the baby was well until this point, she was re - hospitalized at 11 months of age because of diarrhea and unfortunately died on 19 august 2014, probably because of widespread infection (no definitive record of the last hospitalization is available and a post - mortem was not performed).fig . 1chest x - ray posteroanterior view of the newborn baby, who was born to a mother with antenatal exposure to olanzapine, suggestive of tracheo - esophageal fistula chest x - ray posteroanterior view of the newborn baby, who was born to a mother with antenatal exposure to olanzapine, suggestive of tracheo - esophageal fistula a number of drugs are clearly known to be associated with teratogenicity, such as thalidomide and isotretinoin, but the majority of drugs do not have conclusive data concerning their effect on fetal development and growth . Due to strict ethical constraints, at - risk populations (including pregnant women) are not included in most clinical trials, limiting the availability of data on the use of drugs (including antipsychotics) in these populations, and thus spontaneous reporting of suspected adverse drug effects constitutes an essential tool for generating data on the safety of drugs, especially in the population of pregnant women . According to a retrospective analysis of data from the lilly safety database, of the 610 prospectively identified pregnancies exposed to olanzapine with an available outcome, there were 401 (66%) normal births, 60 (9.8%) premature births, 57 (9.3%) spontaneous abortions, 49 (8%) perinatal complications, 27 (4.4%) congenital anomalies (cleft lip, encephalocele, and aqueductal stenosis), and 16 (2.6%) other outcomes (post - perinatal condition, ectopic pregnancy, post - term birth, and still birth). Other reported congenital anomalies with olanzapine included meningocele, ankyloblepharon, hip dysplasia, acheiria, atrioventricular canal defect, and unilateral club foot [1, 68]. A link between teratogenicity and antipsychotics is also supported by the fact that these drugs readily cross the placental barrier [1, 4, 9], with 23.8% placental passage for quetiapine and 72.1% for olanzapine [2, 4, 9]. Newport et al . Measured placental passage of medication from mother to fetus by measuring levels in the umbilical cord serum and found that olanzapine has the highest rate of placental passage, compared with haloperidol, risperidone, and quetiapine [3, 9]. The overall incidence of esophageal atresia / tef ranges from one in every 2500 to 4500 live births . However, a careful literature search revealed a paucity of data on the incidence of tef in children born to women with psychiatric disease and the problem of confounding by indication cannot be ruled out . It has been proposed that antipsychotics interfere with the action of calmodulin, which has an important role in organogenesis . Esophageal atresia and abnormalities of pharyngeal glands have also been proposed as being related to neural crest cells . The drugs (olanzapine in this case) associated with such anomalies may be hypothesized to cause neural crest cell damage during early fetal development as a causative factor . Although the neural crest cell damage - related esophageal abnormalities in many cases are also associated with cardiovascular anomalies, these were absent in the present case . We tried to establish the causality between the adverse outcome (tef) and use of olanzapine during pregnancy according to the who - umc (world health organization uppsala monitoring centre) adverse drug reaction causality assessment scale and the naranjo causality assessment scale . Causality is probable (score + 5) as per the naranjo scale, whereas it is one of the important reasons for lower scores of causality is the non - applicability of many questions in these scales . The current case report of tef associated with antenatal olanzapine exposure demonstrates the need for large clinical studies to generate more conclusive data concerning use of this drug during pregnancy . On the basis of the present case and the literature, it is suggested that antenatal olanzapine exposure may be related to the development of tef in newborns . However, the present case report can only be considered as preliminary evidence and further observational prospective studies of women with antenatal exposure to olanzapine should be conducted to explore the strength of association between tef and olanzapine . Knowledge of the teratogenic potential of a drug is essential to prevent / reduce the morbidity and mortality associated with a congenital anomaly (tef in this case). Such post - marketing reports are effective tools to disclose the adverse effects of drugs, which are difficult to detect in clinical trials and may factually change the safety status of a drug . Written informed consent was obtained from the infant s father for publication of this case report.
Olfactory neuroblastoma (onb) is a rare malignant tumor arising from the olfactory epithelium of the nasal cavity and the paranasal sinuses . The anatomic origin in the superior nasal cavity leads to nonspecific symptoms that make early diagnosis difficult . Direct extension of the tumor into the anterior cranial fossa, either at presentation or at disease recurrence, is common . Onb is mostly a locally aggressive tumor that can spread to lymph nodes but metastatic involvement of bone, bone marrow, and other organs was also reported . The most commonly used staging system proposed by kadish is currently based on computer tomography and magnetic resonance imaging . In stage a, the tumor is limited to the nasal cavity, in stage b the tumor extends to paranasal sinus, and in stage c the tumor is extending over the nasal cavity and sinus and/or metastasized distantly . A histological grading system based on microscopic findings proposed by hyams at 1988 showed a clear correlation of survival with histological differentiation of disease . However most authors believe that craniofacial resection followed by rt offers the gold standard treatment in this disease . Despite aggressive therapies, local, regional relapse, and distant metastasis occur frequently and often after extended periods of follow - up . Data for this case series were acquired retrospectively from the files of division of medical oncology, cerrahpasa medical faculty, istanbul university . Additional information regarding the clinical course and outcome was collected from the patients' charts and phone calls to the patients, their relatives, and their general practitioners . In this study, we have reviewed 19 cases of olfactory neuroblastomas treated over 24-year period and analyzed the clinical features, treatment outcomes, and prognostic factors . The data of nineteen patients of olfactory neuroblastomas treated and followed up between 1986 and 2010 at division of medical oncology, cerrahpasa medical faculty, istanbul university, were analyzed retrospectively . Patient characteristics, initial symptoms, tumor extent, histologic features, primary therapy, tumor recurrence, and treatments of recurrent disease were determined . Patients were staged according to the kadish staging system . A single pathologist with expertise in head and neck neoplasms reviewed the histological specimens . Local ethics committee's approval and patients' or their next of kin's informed consent were taken before the study . Treatment response was assessed by clinical examinations and computed tomography (ct) or magnetic resonance imaging (mri). Survival rates were calculated from diagnosis to the death of the patient using the kaplan - meier life table method . The madian age at diagnosis was 46 (range: 19 to 68 years). Tumor staging was kadish stage a in 3, stage b in 5, and stage c in 11 patients . Four patients had metastasis of cervical lymph nodes, two brains, one bone marrow, one bone and lung metastases, and one bone, mediastinal lymph node, and liver metastases at the time of diagnosis . Tumor localization, clinical symptoms, and presentations at the time of diagnosis are listed in table 1 . Median time from onset of first symptoms until diagnosis was 8.8 months (range: 1.829 months). The common presenting symptoms were nasal obstruction (53%), epistaxis (31%), rhinorrhea, and facial pain (26%). Patient treatments consisted of surgical resection, radiation therapy, and chemotherapy (ctx) (table 3). Initial treatments included surgery alone in three patients, radiotherapy (rt) with or without ctx in five, surgery plus postoperative rt in seven and multimodality therapy (surgery plus postoperative ctx plus postoperative rt) in three, and finally ctx alone in one . In 14 patients, four of these patients had also radical neck dissection due to cervical lymph nodes involvement . Nasal tumor resection in 2 patients, maxillectomy plus orbital exenteration in 1, and external ethmoidectomy (ee) in 1 were also used as surgical treatments . Of these 14 patients who underwent surgical tumor resection, 4 were treated with surgery alone, 5 with adjuvant radiation therapy after surgery, 2 with adjuvant ctx after surgery, and the remaining 3 with radiation therapy and ctx after surgery . Of the remaining 5 patients, four in advanced stage and one in early stage refused surgical treatment . Radiation therapy was administered to two of the patients, radiation therapy followed by ctx to the other two, and ctx alone to one patient . The average radiation dose was 57.9 gy and the ctx was chosen from cisplatin - based regimens . As a part of the primary treatment modality, 12 patients underwent radiation therapy, so did the five relapsed patients . As a part of primary treatment modality 8 patients received ctx, so did the three relapsed patients . Fifteen patients died of tumor progression during the follow - up, of whom 9 were at an advanced stage (stage c) by the time of diagnosis and 6 at an early stage (stage a / b). Kadish stage c patients had a significantly poorer outcome compared to the stage a / b patients (2-year survival rate: 71 versus 25%, resp . Histopathologically high - grade patients (grade 3/4) had a significantly poorer outcome when compared to the lower grade (grade 1/2) patients (2-year survival rate: 16 versus 50%, resp . Primary tumor extensions to orbital area had a significantly poorer outcome compared to the tumors with no extension to orbital area (2-year survival rate: 22 versus 60%, resp . There was no statistically significant difference in survival rate between male and female patients (2-year survival rate: 40 versus 44%, resp . Patients with age <45 years had no poorer outcome compared to patients with age> 45 years (2-year survival rate: 41 versus 43%; p = .059). Brain and bone marrow involvements showed poorer outcome compared to those without such involvement (median survival rate: 9.1 versus 28.8 months, resp . ; survival rate was not significantly different among pateints of kadish c when stratified according to initial treatment (p = .8). Locoregional recurrence developed in 6 and distant metastasis in 6 patients . During the follow - up period, these groups exhibited distant metastasis to the brain, spinal cord, bone, bone marrow, meninges, and lungs . As a treatment for recurrence and metastasis, reoperation of primary site plus radiation therapy in 4 patients and radiation therapy alone in 4 patients were offered . Onb is a rare malignant tumor that comprises 4,65% of malignant nasal and paranasal tumors . Some 1000 cases have been reported in the english literature since it was first introduced in 1924 . The incidence rate is bimodal, with peaks in the second and third decades of life and in the sixth and seventh decades of life and equal among females and males . As for our study, the age at diagnosis was most common in the fourth decade and female - to - male ratio was equal . The average time between the appearance of the first symptom and the diagnosis was reported as 6 months and this period was similar also in our group . The most common symptoms at the initial diagnosis were defined as unilateral nasal obstruction and epistaxis in the literature . This tumor may be misdiagnosed as an undifferentiated small cell carcinoma, melanoma, rhabdomyosarcoma, or other small blue cell neoplasms . Hyam proposed a grading system with grades 14 based on the presence or absence of seven different histopathological parameters . These are growth, architecture, mitotic activity, necrosis, nuclear pleomorphism, rosette formation, and fibrillary stroma . . Staining paterns for neuron - spesific enolase, chromogranin, synaptophysin, s-100, and epithelial markers can be helpful . There is evidence that histological grade of onb influences biologic behavior, particularly because it relates to disease progression, local recurrence, and metastasis . For the well - differentiated tumors a slower disease progression and less tendency for local recurrence have been described . In our study, the 2-year survival rate of this grade i / ii - iii / iv disease was 16 versus 50%, respectively . More aggressive treatment, such as surgery, radiation, and intensive ctx, may be useful for patients with grade iii / iv disease . A meta - analysis showed that the median overall survival at 5 years is 45% in 390 patients . The distribution of onb from 21 studies according to kadish staging system was 12% at stage a, 27% at stage b, and 61% at stage c. the mean 5-year survival is 75% for stage a, 68% for stage b, and 41% for stage c . The conclusions of the university of virginia stated that kadish stage is predictive of disease - related mortality . However, in the series of the mayo clinic it was found that kadish stage did not affect the outcome . Danish clinicopathological study found that the staging system of kadish was able to stratify into prognostically significant groups . In our study, the 5-year survival rate of these tumors was 26% and median survival time was 23 months . The distribution of onb according to kadish staging system was 16% at stage a, 26% stage b, and 58% stage c. however, in our study, the 5-year survival rates of these stage groups were inferior compared to those reported in other studies . The cause of this poor result was presentation of 5 patients with distant metastases (26%; 2 brain, 1 bone marrow, 2 other sites), 4 patients (21%) with cervical lymph node metastasis, 12 patients (63%) with high - grade histology at the time of diagnosis, and inability to evaluate the surgical margins of the resected tumors . The survival rate of node positive patients is 29% compared with 64% of node negative patients . Distant metastases have been described in the pancreas, the liver, mediastinum, bone marrow, lungs, leptomeninges, skin, and breasts [11, 12]. Cervical nodal metastases can develop in 17 to 33% of patients and distant metastases in 10 to 40% of patients over the course of disease metastatic disease at presentation occurs in 10 to 50% of patients, depending on the study [6, 13, 14]. We found the local recurrence rate to be 31.5% and distant metastasis 31.5% after initial surgery . During the follow - up periods these groups exhibited distant metastases to the brain, spinal cord, bone, bone marrow, meninges, and lungs . Patients who had brain and bone marrow metastases had poorer prognosis than others having metastatic deposits of other sites . Involvement of the orbit is a serious prognostic factor, but when the orbital periosteum is affected without penetration into the orbital structures themselves, the eye may be preserved by resection and grafting of the periosteum without an adverse effect on survival as compared with orbital clearance . Irrespective to the most sophisticated imaging, involvement of the dura and orbital periosteum can accurately be determined only at surgery . In our study, this group's (8 patients) survival rate is poorer than those with no involvement of the orbita (p = .042). The institute of laryngology and otology, university college london, published a study of 42 patients treated over 23 years . Overall and disease - free survival rates were 77 and 61% at 5 years, and 53 and 42% at 10 years, respectively . Late recurrences were seen . Based on this experience, combined therapy with craniofacial resection and radiation therapy is recommended by this group . In a retrospective study of 47 patients treated at multiple centers in germany from 1979 to 2001, the 5-year overall survival and event - free survival rate were 64% and 50%, respectively . Patients who received multimodality therapy had a significantly better event - free survival rate compared with those who did not receive multimodality therapy (74 versus 41%). The authors recommend a combination therapy including ctx, surgery, and postoperative radiation therapy for kadish stage c patients . In our study, initial treatment included surgery alone in three patients, rt with or without ctx in five, surgery plus postoperative rt in seven, and multimodality therapy (surgery plus postoperative ctx plus postoperative rt) in three, only ctx in one patient . However, in our study choice of treatment modality did not show a statistically significant difference in survival rate . The reasons for that might be including low number of patients in the study, being 5 distant site metastasis, 4 cervical lymph node metastasis at the time of diagnosis, only one orbital exentration (although there were 8 orbital involvement), and 12 patients exhibiting high grade histology and inability to evaluate surgical margins in resected tumors . In conclusion, advanced tumor stage, the initial histopathologic grade of iii / iv, and involvement of orbita, brain, or bone marrow metastasis were the statistically significant poor prognostic factors, whereas age, sex, and treatment modality were not prognostic factors.
Unrepaired dsbs are usually lethal for dividing cells due to the subsequent loss of the acentric fragment and the unstable break - end of the centric fragment . Dsbs can be caused enzymatically, by ionizing irradiation, or by other s phase - independent clastogens . Dsbs with only one break - end may appear when a replication fork meets a single - strand break or a repair - mediated parental single - strand discontinuity . Mostly however, the break is extended by a 5-specific exonuclease activity generating 3-overhanging ends . As soon as overhanging ends meet at short stretches of complementary bases (single - strand annealing, ssa), the distal free ends are resected, and the break becomes ligated, resulting in a (micro-)deletion (figure 1a). Alternatively, the break - ends may invade undamaged homologous template strands and a limited dna synthesis across the break region is followed by re - ligation of both strands (synthesis - dependent strand annealing, sdsa). The result is genetically not detectable when the template was the undamaged sister chromatid; it appears as a gene conversion if an allele of the homologous chromosome served as a template (figure 1c). If for break - end elongation an ectopic or an extra - chromosomal (partially) homologous sequence is used, the result may be linked with an insertion into the break (figure 1b and puchta, 2005). When the holiday junction (caused by break - end invasion into a template double strand) is resolved in connection with an exchange of the flanking region (figure 1c), then, depending on the template double helix involved, a sister chromatid exchange, a crossing over, or a reciprocal translocation results . Pathways of dsb misrepair via single - strand annealing(ssa) or via synthesis - dependent strand annealing (sdsa). (a) deletion via exonucleolytic 5-end resection, ssa at complementary overhang sequences, resection of the non - aligned ends, and ligation of break - ends . (b) insertion into a dsb by break - end invasion and elongationalong an ectopic and partially homologous (vertical bars) template. (c) re - synthesis of break - ends after invasion into a homologous template double - strand without (gene conversion) or with exchange of flanking regions due to appropriate resolution of holiday junctions (greenarrow heads). Based on genetic experiments on budding yeast, a further mechanism, break - induced replication (bir;, 1996, 2005; morrow et al ., 1997; haber, 1999; for review haber, 2006; mceachern and haber, 2006; llorente et al ., 2008) has been postulated . With the exception of a direct ligation of clean break - ends, all dsb repair events are linked with a limited replication step, bir is claimed to extend replication from the proximal break - end up to the end of the template chromatid, using a (homologous) undamaged double helix as template . Mechanistically, bir appears as a non - reciprocal translocation event (bosco and haber, 1998) which can be considered as a type of gene conversion extending from the breakpoint up to the telomere . Simultaneously, the original distal fragment (if occurring) of the broken double helix is lost (figure 2). Schematic models of replication and chromosome labeling patterns after bir at proximal dsb ends in s and g2 . (a) bir through conservative replication of a one ended dsb during s phase . The dsb appears when the replication fork arrives at a single - strand break (arrow head). Conservative replication occurs via recurrent strand invasion (or via unidirectional fork migration) without resolution of the holiday junction(s) using the parental double strand as a template . (b) bir during g2 phase, through conservative replication at the proximal end of a dsb (arrow head) via recurrent strand invasion and/or via unidirectional fork migration without resolution of the holiday junction(s) using the undamaged sister double helix as a template . (c) bir during g2 phase through semiconservative replication achieved by resolution of the holiday junction (green arrow head) after invasion of the elongating break - end into the template double strand . Full lines unlabeled; broken lines labeled by edu . The distal fragment of the broken double helix in (b, c) gets lost . When a homologous or a heterologous chromatid instead of the sister chromatid is involved in bir, a non - reciprocal translocation between homologous or heterologous chromosomes is mimicked . Microhomology - mediated bir involving heterologous chromosomes leads to duplication of the template region and deletion of the region distal the original dsb . The problem of bir before or after regular replication in species with large chromosomes is that breakage occurring distantly to the arm end requires replication of the involved region (larger than a chromatin unit of jointly regulated replication comprising 1 mb, zink et al ., 1998) more than once between two nuclear divisions, thus overriding the licensing mechanism which allows only one round of replication between two divisions . Bir occurring during s phase (figure 2a and hastings et al ., 2009) requires a recurrent 3-end invasion into a still unreplicated template chromatid (most likely the sister chromatid), or a continuous migration of the replication fork toward the arm end . Resolution of the holiday junction results in a reciprocal exchange of the distal regions between the chromatids involved . Bir during s phase without resolution of the holiday junction circumvents the dsb, yielding a conservative replication pattern with regard to the broken chromatid distal to the breakpoint . To test whether bir may occur within chromosomes of higher plants after dsb induction, we treated field bean root tip meristems with the s phase - independent clastogen bleomycin during s or g2 phase and checked for the expected chromatid labeling by incorporation of the base analog ethynyldeoxyuridine (edu, see kotogany et al ., 2010) also semiconservative bir at terminal chromatid regions (figure 2c) was not observed after breakage in the presence of the base analog during g2 . Our results suggest that bir either does not occur or is so infrequent that its role within the concert of dsb repair mechanisms can at best be a minor one in organisms with a large dna content (> 1 mb) per chromosome arm . The field bean, vicia faba l., karyotype acb with six large and individually distinguishable chromosome pairs (dbel et al ., 1978) is much more comfortable for aberration scoring than for instance a. thaliana with smaller chromosomes and less mitoses per root meristem . Seeds were germinated for 3 days on wet filter paper at room temperature in the dark . About 12 cm long primary roots were incubated for 18 h in aerated hoagland solution, ph 5.5, containing 1.25 mm hydroxyurea which synchronizes cells by blocking the cell cycle in early s phase . For mutagen treatment during s phase, the roots were incubated for 1.5 h in hoagland solution containing 10 g / ml bleomycin with or without 20 m edu (invitrogen) followed by incubation in hoagland solution for 4.5 h and then 0.05% colchicine for 2.5 h (to arrest metaphases) before fixation in ethanol: acetic acid (3:1) overnight . For scoring of chromatid aberrations, roots were washed for 10 min in distilled water, hydrolyzed for 11 min in 1 n hcl at 60c, stained for 3040 min in feulgen solution and squashed in a drop of 45% acetic acid . Only complete metaphase cells were scored for chromatid aberrations: chromatid and isochromatid breaks (one or both sister chromatids with terminal deletion), interstitial deletions, duplication deletion (the deleted part of one chromatid is inserted into a break of the sister chromatid), and reciprocal chromatid translocations . Alternatively, the root tips were fixed in 4% formaldehyde for 20 min for isolation of metaphase chromosomes (schubert et al ., 1993) and subsequent detection of edu incorporation using the click.it edu imaging kit from invitrogen according to manufacturer's instruction . Microscopic analysis of chromosomes was performed with an epifluorescence microscope (zeiss axiophot) using a 100/1.45 zeiss alpha - plan - fluar objective and a sony (dxc-950p) camera . Images of fluorescent chromosomes were captured using filter sets f36 - 513 for dapi and f36 - 750 for alexa fluor 594 (ahf analysentechnik, germany). Dapi and alexa fluor 594 azide images were merged using adobe photoshop 6.0 software (adobe systems, san jose, ca, usa). For mutagen treatment during g2 phase, primary roots were incubated in hoagland solution with hydroxyurea as above, then in hoagland solution for 4.5 h and subsequently in hoagland solution with bleomycin, with edu, or both for another 1.5 h followed by 2.5 h in colchicine before fixation, staining, and evaluation . The s phase - independent clastogen bleomycin induces dna breaks directly . A dose of 10 g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems (heindorff et al ., 1987). The same bleomycin concentration applied for 1.5 h to (mainly) g2 cells of root tip meristems, in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations . Treatment with edu (20 m, 1.5 h) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase . Bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells (table 1). Although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells, no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin . Chromatid - type aberrations induced by edu, bleomycin, or both during g2 or s phase in field bean root tip meristems (summary of two independent experiments). T = reciprocal translocation, i = isochromatid break, d = interstitial deletion, b = chromatid break, see figure 3a . When root tips were incubated in 20 m edu 8.57 h before fixation, most metaphase chromosomes (87.4%) were completely labeled and some (12.5%) were labeled along their euchromatin regions (table 2), indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems (schubert and meister, 1977). No chromosome showed asymmetric labeling up to the chromosome end, i.e., an unlabeled terminal region of one of the sister chromatids . Only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid . Type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment (two independent experiments summarized). * (see figures 2a and 3b), * * (see figures 2b and 3c), * * * in () maximum percentage of cells harboring an asymmetrically labeled chromosome . When s phase cells were treated simultaneously with edu and bleomycin, all 2130 observed chromosomes were labeled (81.4% completely and 18% along their euchromatin). None of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix, or if it replicated till the end of the template by unidirectional fork migration (figure 2a and hastings et al ., 2009). Thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b). Since 2130 chromosomes correspond to 178 cells, in about 7.3% of cells such an asymmetrically unlabeled spot occurred, representing an eightfold increase compared to the variant with edu treatment alone . (a) chromatid - type aberrations after bleomycin treatment . Left cell: isochromatid break (arrow head), the centric, and the acentric chromatid fragments are surrounded by black dots, the homologous undamaged chromosome is surrounded by white dots . Middle cell: symmetric reciprocal chromatid translocation (arrow) and two terminal chromatid breaks (arrow heads). The latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid (left) or being at least 90 apart from the other break - end as in case of the broken secondary constriction (right). Right cell: interstitial deletion (arrow), the deleted fragment remains attached to the undamaged sister chromatid, the chromosome involved is surrounded by black dots . (b) interstitial asymmetric chromatid labeling (arrows) after bleomycin treatment in the presence of edu during s phase . (c) interstitial asymmetric chromatid labeling (arrows) after bleomycin treatment in the presence of edu during g2 . The asymmetric signals appear on chromosomes ii, iv, v, and vi, respectively, at interstitial heterochromatic regions composed of homologous tandem repeats (fuchs et al ., 1994). When metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5 h before fixation, the corresponding cells should have been in g2 during edu treatment . Indeed, most of the chromosomes (81.3%) were unlabeled while 18.3% revealed late replication pattern (symmetric labeling of heterochromatic regions) thus indicating they were in late s phase during edu treatment . No chromosome showed asymmetric labeling of one chromatid up to the arm end, as expected in the case of a conservative bir (figure 2b). Also symmetric terminal labeling of sister chromatids, as expected in the case of semiconservative bir (figure 2c) was not observed . Single transversal interstitial fluorescence signals on one of the sister chromatids (figure 3c) were observed in 10 (0.4%) chromosomes . After simultaneous exposure to edu and to bleomycin 42.5 h before fixation, again most of the chromosomes (85.5%) were completely unlabeled and thus were in g2 during treatment; 13.2% of chromosomes showed late replication patterns, indicating that the corresponding cells were in late s during treatment . However, there was a> 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal (20 signals per 130 cells), usually in heterochromatic regions . The s phase - independent clastogen bleomycin induces dna breaks directly . A dose of 10 g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems (heindorff et al ., 1987). The same bleomycin concentration applied for 1.5 h to (mainly) g2 cells of root tip meristems, in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations . Treatment with edu (20 m, 1.5 h) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase . Bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells (table 1). Although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells, no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin . Chromatid - type aberrations induced by edu, bleomycin, or both during g2 or s phase in field bean root tip meristems (summary of two independent experiments). T = reciprocal translocation, i = isochromatid break, d = interstitial deletion, b = chromatid break, see figure 3a . When root tips were incubated in 20 m edu 8.57 h before fixation, most metaphase chromosomes (87.4%) were completely labeled and some (12.5%) were labeled along their euchromatin regions (table 2), indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems (schubert and meister, 1977). No chromosome showed asymmetric labeling up to the chromosome end, i.e., an unlabeled terminal region of one of the sister chromatids . Only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid . Type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment (two independent experiments summarized). * (see figures 2a and 3b), * * (see figures 2b and 3c), * * * in () maximum percentage of cells harboring an asymmetrically labeled chromosome . When s phase cells were treated simultaneously with edu and bleomycin, all 2130 observed chromosomes were labeled (81.4% completely and 18% along their euchromatin). None of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix, or if it replicated till the end of the template by unidirectional fork migration (figure 2a and hastings et al ., 2009). Thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b). Since 2130 chromosomes correspond to 178 cells, in about 7.3% of cells such an asymmetrically unlabeled spot occurred, representing an eightfold increase compared to the variant with edu treatment alone . Isochromatid break (arrow head), the centric, and the acentric chromatid fragments are surrounded by black dots, the homologous undamaged chromosome is surrounded by white dots . Middle cell: symmetric reciprocal chromatid translocation (arrow) and two terminal chromatid breaks (arrow heads). The latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid (left) or being at least 90 apart from the other break - end as in case of the broken secondary constriction (right). Right cell: interstitial deletion (arrow), the deleted fragment remains attached to the undamaged sister chromatid, the chromosome involved is surrounded by black dots . (b) interstitial asymmetric chromatid labeling (arrows) after bleomycin treatment in the presence of edu during s phase . (c) interstitial asymmetric chromatid labeling (arrows) after bleomycin treatment in the presence of edu during g2 . The asymmetric signals appear on chromosomes ii, iv, v, and vi, respectively, at interstitial heterochromatic regions composed of homologous tandem repeats (fuchs et al ., 1994). When metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5 h before fixation, the corresponding cells should have been in g2 during edu treatment . Indeed, most of the chromosomes (81.3%) were unlabeled while 18.3% revealed late replication pattern (symmetric labeling of heterochromatic regions) thus indicating they were in late s phase during edu treatment . No chromosome showed asymmetric labeling of one chromatid up to the arm end, as expected in the case of a conservative bir (figure 2b). Also symmetric terminal labeling of sister chromatids, as expected in the case of semiconservative bir (figure 2c) was not observed . Single transversal interstitial fluorescence signals on one of the sister chromatids (figure 3c) were observed in 10 (0.4%) chromosomes . After simultaneous exposure to edu and to bleomycin 42.5 h before fixation, again most of the chromosomes (85.5%) were completely unlabeled and thus were in g2 during treatment; 13.2% of chromosomes showed late replication patterns, indicating that the corresponding cells were in late s during treatment . However, there was a> 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal (20 signals per 130 cells), usually in heterochromatic regions . If bir occurs in higher plants in the same way as postulated for yeast (i.e., as replication extending from the break position up to the corresponding chromosome arm end) conservative replication in s or g2 (figures 2a, b) or semiconservative replication in g2 (figure 2c) should be detectable . Of the chromatid aberrations observed after bleomycin and bleomycin plus edu treatment during s and g2, reciprocal translocations (25), isochromatid breaks (36), and interstitial deletions (7) require two dsbs each, while simple chromatid breaks (87) representing terminal deletions, go back to a single dsb . Therefore, within 600 g2 and 750 s phase cells, bleomycin caused 223 dsbs which underwent misrepair . Thus, on average at least one dsb per six cells was induced . Considering that a fraction of dsbs might have been repaired via pathways not resulting in microscopically detectable chromatid - type aberrations (for instance by sister chromatid exchange, intrachromosomal ssa, or gene conversion), the actual number of dsbs may be higher . However, in a number of chromosomes corresponding to 308 s and g2 cells, treated simultaneously with the chromosome - breaking agent bleomycin and the base analog edu, not a single chromosome showed a labeling pattern indicative of bir . In budding yeast, more than half of diploid cells repair ho - endonuclease - induced dsbs by bir using the homologous chromosome as template (bosco and haber, 1998); meaning more than half of the dsbs are repaired via bir if the endonuclease ho causes one dsb per diploid yeast cell . Had bir occurred with a similar efficiency in the field bean, we should have observed> 223 instead of no chromosomes with asymmetric terminal edu signal . Our result does not exclude the occurrence of bir in plants, however, the frequency of such events is below 0.45% (less than 1 out of 223) of the dsbs causing chromosome structural aberrations, i.e., more than 100-times lower than in yeast . Unidirectional replication forks that override distal replication origins have to migrate up to the arm ends, or, alternatively, the requirement of recurrent invasion of 3-ends into other double helices, are presumptions for which experimental evidence is lacking so far . Long range replication in addition to the regular round of replication between two nuclear divisions, represent a further unproven assumptions . To our knowledge no experimental evidence as to the occurrence of bir in animals bas been shown (zhang et al ., 2009). Taken together these arguments imply that the existence of a bir pathway in higher eukaryotes is unlikely . Even when such events occur, their frequency is much lower than in yeast arguing against their importance as a pathway for dsb repair at least in higher plants such as the field bean . Because the 12 megabase pair (mb) genome size of saccharomyces cerevisiae is distributed over 32 chromosome arms, the risk that bir interferes with replication licensing at the replicon level is much smaller than for species with orders of magnitude larger genomes with several mb per arm . Also complex chromosome rearrangements (e.g., difilippantonio et al ., 2002; zhang et al ., 2009) can more easily be explained by other well - documented pathways (zhang et al ., 2009; schubert and lysak, 2011) rather than by bir via recurrent invasion of a dsb - induced 3-end into different template double helices . The more so as experimental approaches even in yeast cannot differentiate between bir and gene conversion events involving g2 cross over, followed by appropriate chromatid segregation nevertheless, the increased frequency of interstitial asymmetric labeling patterns of chromosomes exposed to bleomycin and edu during s or g2 might indicate that conserved replication, although not reaching arm ends, could be linked with dsb repair to result in microscopically detectable stretches of gene conversion extending hundreds of kilobases (possibly, up to the junction with the next replication unit, but not covering dna stretches of> 1 mb up to the arm ends). Such extended gene conversion - like repair might be considered as bir, if the postulation up to the chromosome terminus is excluded . Similar events could potentially have been responsible for the zebra structure of the long arm of chromosome z5a observed in a backcross progeny between elymus trachycaulus gould ex shinners and triticum aestivum l. (zhang et al ., 2008). The preferential appearance of local asymmetric labeling within heterochromatic regions of the field bean suggests that for late replicating repetitive dna the units of replication regulation are larger or more variable than for euchromatin . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Is an effort to manipulate the metabolism of an organism worthy and reasonable, knowing that this cellular process has been continuously modified and refined through evolution and natural selection for adapting, in the most convenient manner, to the ongoing environmental conditions? The answer to this question seems obvious when three broad areas of research and development are identified in which manipulation of metabolic pathways is relevant: (a) drug design to treat diseases, (b) genetic engineering of organisms of biotechnological interest, and (c) genetic syndromes therapy . Historically, drug design was the first area in which modification of metabolism was tried: the primary goal of drug administration is the inhibition of essential metabolic pathways, for example, in a parasite or a tumor cell . Thus, any metabolic pathway can be a potential therapeutic target . In the absence of a solid theoretical background that may build a strategy for the rational design of drugs, the pharmaceutical industry has applied the knowledge of inorganic and organic chemistry for the arbitrary and rather randomized modification of metabolic intermediaries by replacing hydrogen atoms in a model molecule with any other element or compound . The era of rational drug design probably started in the 50s when hans krebs proposed that, after having an exact description of a metabolic pathway, the pacemaker enzyme or rate - limiting step had to be identified . This approach certainly decreased the amount of intermediaries to be chemically modified, focusing only on the substrates, products, and allosteric effectors of the rate - limiting step, instead of dispersing efforts on all the metabolic pathway intermediates . The experimental approaches used in the identification of the pacemaker, key enzymes, bottlenecks . Limiting steps, or regulatory enzymes [1, 2] were inspection of the metabolic pathway architecture: due to cell economy and for reaching the highest efficiency, pathway control must reside in the enzymes localized at the beginning of a pathway or after a branch (teleological approach);determination of nonequilibrium reactions: those reactions in which the quotient between the mass action ratio () and its equilibrium constant (keq) is low, /keq 1 (thermodynamic approach);identification of the steps with the lowest maximal rates (vmax) in cellular extracts: the key enzyme of the pathway is the one that has the lowest rate (kinetic approach);enzymes with sigmoidal kinetics: steps that are susceptible to alteration in their kinetic properties by compounds different from substrates and products and which may coordinate the entire metabolism (nadh / nad; nadph / nadp, atp / adp; acetyl coa / coa; ca / mg; high ph / low ph) or at least two metabolic pathways (citrate, pi, amp, malonyl - coa);crossover theorem . Comparing the intermediary concentrations between a basal and an active steady - state pathway flux, the rate - limiting step in the basal condition will be that for which its substrate concentration diminishes and its product concentration increases when the system changes from the basal to the active state or vice versa (crossover point on a histogram of each intermediary versus its normalized variation in concentration);the shape of the metabolic flux inhibition curve: a sigmoidal curve on a plot of inhibitor concentration versus flux shows that the sensitive step to the inhibitor exerts no control, that is, there is not proportionality between enzyme activity inhibition and pathway flux inhibition because there is an excess of enzyme . On the other hand, a hyperbolic curve indicates that the enzyme susceptible to the inhibitor controls the flux . Inspection of the metabolic pathway architecture: due to cell economy and for reaching the highest efficiency, pathway control must reside in the enzymes localized at the beginning of a pathway or after a branch (teleological approach); determination of nonequilibrium reactions: those reactions in which the quotient between the mass action ratio () and its equilibrium constant (keq) is low, /keq 1 (thermodynamic approach); identification of the steps with the lowest maximal rates (vmax) in cellular extracts: the key enzyme of the pathway is the one that has the lowest rate (kinetic approach); enzymes with sigmoidal kinetics: steps that are susceptible to alteration in their kinetic properties by compounds different from substrates and products and which may coordinate the entire metabolism (nadh / nad; nadph / nadp, atp / adp; acetyl coa / coa; ca / mg; high ph / low ph) or at least two metabolic pathways (citrate, pi, amp, malonyl - coa); crossover theorem . Comparing the intermediary concentrations between a basal and an active steady - state pathway flux, the rate - limiting step in the basal condition will be that for which its substrate concentration diminishes and its product concentration increases when the system changes from the basal to the active state or vice versa (crossover point on a histogram of each intermediary versus its normalized variation in concentration); the shape of the metabolic flux inhibition curve: a sigmoidal curve on a plot of inhibitor concentration versus flux shows that the sensitive step to the inhibitor exerts no control, that is, there is not proportionality between enzyme activity inhibition and pathway flux inhibition because there is an excess of enzyme . On the other hand, a hyperbolic curve indicates that the enzyme susceptible to the inhibitor controls the flux . Once a site in a metabolic pathway has been identified with at least one of the criteria described above as the rate - limiting step, researchers have frequently concluded that such enzyme or transporter is the only limiting step of the metabolic flux and extend this conclusion to all cell types and to all conditions . For example, inspection of the glycolytic pathway (teleological approach) suggests that hexokinase (hk) and phosphofructokinase-1 (pfk-1) (which are at the beginning and after a branch of the pathway) are the key steps of glycolysis . However, all studies on glycolysis in the 60s, 70s, and 80s were performed by taking into account only the intracellular reactions from hk to ldh (i.e., without including the glucose transport reaction through the plasma membrane) and by considering glycolysis as a linear pathway without branches . To this regard, it is recalled that the glucose transporter (glut) includes a family of proteins and genes that are susceptible of regulation . Thus, if the extracellular glucose is considered as the initial glycolytic substrate, then another potential key step would be glut . Hence, if all the branches of the pathway are considered (figure 1), then according to the teleological approach there will be additional potential rate - limiting sites . Application of the thermodynamic and kinetic approaches to glycolysis reveals that hk, pfk-1, and pyruvate kinase (pyk) are the rate - limiting steps because in the living cell they catalyze reactions that are far away from equilibrium (/keq = 1010), and they are also the slowest enzymes in the pathway by at least one order of magnitude (they have the lowest vmax values). The use of the enzyme cooperativity approach has established that the regulatory steps of glycolysis are (i) pfk-1 and pyk because they are allosteric enzymes and (ii) hk because it is inhibited by its products (g6p and adp, or amp as an adp - analogue). The application of the crossover theorem (approach no . V) to glycolysis has shown a consistent variation in the pfk-1 substrate (f6p) and product (f1,6bp). Up to now, there are few studies on control of glycolysis using the shape of the inhibitor titrating curve (approach no . Vi), due to the lack of specific inhibitors for any of the three presumed key steps . An exception is iodoacetate which is indeed a potent inhibitor of gapdh, but also of other highly reactive cysteine - containing enzymes [35]. By using iodoacetate as specific inhibitor, both gapdh activity and flux showed identical titration curves, leading to the conclusion that gapdh was the rate - limiting step of glycolysis in streptococcus lactis and s. cremoris (see, however, section 3.2; glycolysis in lactobacteria below). All together, these results constitute the main reason why many intermediary metabolism researchers, including the authors of biochemistry text books, have proposed hk, pfk-1, and pyk as the rate - limiting steps of glycolysis . In consequence, to vary the glycolytic flux, one of these enzymes has to be modified . Although the above - described experimental approaches are qualitative, full control has been automatically assigned to the key steps because the concept of the rate - limiting step assumes that there is only one single enzyme controlling the metabolic pathway flux (and the concentration of the final product of the pathway) and, in consequence, assigns values of zero to the control exerted by the other enzymes and transporters . However, as analyzed for glycolysis, researchers have commonly identified more than one limiting step . In the case of oxidative phosphorylation (oxphos), in the 70s and 80s some researchers considered cytochrome c oxidase as the rate - limiting step, whereas others preferred the atp / adp translocator or the krebs cycle ca - sensitive dehydrogenases (for a review, see). Rephrasing the initial question, which could be the aim of manipulating a metabolic pathway such as glycolysis, knowing its universal distribution in the living organisms? From a clinical standpoint, the inhibition of glycolysis is relevant for the treatment of human parasitic or pathological diseases such as cancer . The glycolytic reactions are almost identical in all organisms; in addition, the enzymes catalyzing these reactions are highly conserved throughout the evolutionary scale (their amino acid sequences are highly similar). In mammals, the genes of the 12 glycolytic enzymes are scattered throughout the genome, generally in different chromosomes, whereas in bacteria many of the glycolytic enzymes are clustered in operons . However, there are organisms (like some human parasites) that contain enzymes with remarkable differences in their biochemical properties (substrate selectivity, catalytic capacity, stability, and oligomeric structure), or in genetic expression regulation in comparison to the human enzymes, which could be considered as drug targets . Furthermore, some glycolytic products are of commercial interest such as ethanol for wine, beer, and other alcoholic beverages; co2 for bread manufacturing; and lactic acid and other organic acids for cheese production . Thus, from a biotechnological standpoint, it is convenient to accelerate the pathway flux to diminish the processing time and it is also desirable to increase the concentration of the metabolite to obtain robust commercial products . Here, it is important to emphasize that the metabolic pathways are designed to attain changes in flux with minimal disturbances in the intermediary concentrations . For example, the glycolytic flux in skeletal muscle can increase from rest to an active state by 100 fold, without large changes in metabolites . Then, it is physiologically more common to change a metabolic flux and the production of the final metabolite in the pathway than varying the intermediary concentrations . However, we will see that, by using a suitable approach of metabolic control analysis, it is possible to design strategies to manipulate not only fluxes but also metabolic intermediary concentrations . When the yeast saccharomyces cerevisiae is exposed to high glucose (> 2%; 0.11 m), the genes of all glycolytic enzymes are induced (pdc and eno increase their expression by 20 fold; pgk, pyk, and adh, 310 times; and the others, 2 fold in average) [811]. However, when the methodological development of genetic engineering allowed modulating the expression of enzymes within cells, researchers turned to the rate - limiting step concept to manipulate a metabolic pathway to increase flux and/or its intermediates, hypothesizing that the overexpression of only one, or of a few key glycolytic genes, should increase the flux . Historically, heinisch in germany was the first author to obtain a 3.5 fold overexpression of pfk-1 in s. cerevisiae, but surprisingly he observed that the rate of ethanol production was not modified . Subsequent experiments for increasing the ethanol production rate by overexpressing either each of the presumed limiting steps, or in combination with other glycolytic enzymes (table 1), have been unsuccessful and, even in some cases, a slight decrease in flux has been attained . For instance, the simultaneous overexpression of seven enzymes of the final section of glycolysis induced only a 21% increase in ethanol production after 2 hours of culture (table 1). This was accompanied by a 1020% decrease in pfk-1 expression, which might have attenuated the flux increase . In yeasts, hk is not product inhibited by g6p or adp; instead, it is strongly feedback inhibited by trehalose-6-phosphate (tre6p). Deletion of the tre6p synthase gene does not bring about an increased ethanol production, but it rather induces a defective cellular growth on glucose and fructose and a lowered ethanol production, as a result of a highly active hk that leads to hyperaccumulation of hexose phosphate metabolites (particularly f1,6bp) and fast depletion of atp, pi, and downstream metabolites . The explanation for this event is that, in the tre6p synthase mutants, the rate of glucose phosphorylation exceeds the rate of glycolytic atp synthesis (named turbo effect). Heterologous expression of a tre6p - insensitive hk does not recover completely the wild - type phenotype . Furthermore, deletion of the tre6p synthase gene in the tre6p - insensitive hk strain did affect growth, suggesting other interactions and functions of tre6p synthase in the control of sugar metabolism, at least in schizosaccharomyces pombe . Davies and brindle obtained a 5-fold overexpression of pfk-1 in s. cerevisiae, but the increase in ethanol production was not attained under anaerobic conditions . There was a slight increase in ethanol production in resting cells in aerobic conditions, under which the mitochondrial metabolism contributes to the atp supply . In all these works, it may be noted that enzyme overexpression indeed affects the concentration of several intermediaries, but this effect has not been further examined . It is worth noting that the experiments described in table 1 do not rigorously reproduce the physiological situation, in which overexpression of all the enzymes should be carried out in the proportions found in the organisms . The rationale behind this observation is that overexpression of only one limiting step leads to a flux control redistribution, a condition at which other steps now become rate limiting . Thus, the concept of rate - limiting step offers no simple answer to the question of increasing the yeast glycolytic flux, and it rather makes this problem to appear as a difficult task to solve . In contrast, it seems that all relevant controlling steps have to be overexpressed, thus reproducing what natural selection has already successfully accomplished . In addition to s. cerevisiae, overexpression of glycolytic enzymes in other organisms such as e. coli [15, 16], lactobacteria, tomato, potato, and hamster ovary cells has been accomplished, although without increasing flux (table 1). It is somewhat surprising to note that in the glycolytic enzyme overexpression experiments, the strong inhibitory effect of g6p (or tre6p in s. cerevisiae), and citrate on hk and pfk-1, respectively, have been neglected . This regulatory mechanism does not disappear in the cells overexpressing the enzymes but, on the contrary, it is exacerbated . Then, what would be the aim of overexpressing hk, pfk-1 or any other allosteric, or strongly product - inhibited enzyme if they will be more inhibited? A successful experiment of increasing the glycolytic flux was performed in primary cultures of rat hepatocytes . The transformed hepatocytes showed higher activity of 18.7- and 7.1-times for hk and gk, respectively, at 3 mm glucose, and of 6.3- and 7.1-times at 20 mm glucose . However, at 20 mm glucose, the flux to lactate was not modified in hk - transformed cells, just like the experiments described above (table 1). In contrast, with gk overexpression, a 3-fold increase in flux was achieved . The mechanistic difference is the hk inhibition by g6p (10 mm g6p inhibits hk activity by 90%), whereas gk is not product inhibited . The end products, lactate and h, are expelled and acidify the external medium which contributes to cheese flavor and texture and inhibits the growth of other bacteria . Similarly to yeast, the lack of carbon source in lactobacteria promotes a metabolic change that leads to the production of formic and acetic acids, ethanol, and, in a lower proportion, l - lactic acid, altering the product quality . Thus, from a commercial point of view, it does not seem important to know what controls the flux to lactate (because its rate of production is adequate), but what controls the branching flux . To understand the process, and to eventually inhibit the production of secondary acids, andersen et al . Constructed ldh mutants, using a synthetic promoter library for tuning the gene expression . In mutants lacking this enzyme, most of the pyruvate was transformed into acetic and formic acids (figure 1). In turn, flux to lactate was affected in mutants expressing only 10% or less of wild - type ldh levels, which indicated that ldh exerts no control of the glycolytic flux in wild - type bacteria . Only with a normal content of this enzyme (100%), therefore, the flux to formic and acetic acids is negatively controlled by ldh, and positively by pyk [17, 25]. As in s. cerevisiae, overexpression of pfk-1, pyk, or gapdh in lactobacteria did not increase the flux to l - lactic acid [17, 25]. Similarly to e. coli glycolysis, glycolysis in l. lactis was controlled by the atp demand when working below its maximum capacity [27, 28], whereas, under high - rate conditions, the glucose and lactate transporters exerted the main flux control . Furthermore, this kind of observations indicates that the flux control may reside outside the pathway [2729], and it also supports the proposal by hofmeyr and cornish - bowden that the end - product demand (which is usually overlooked in studies of metabolism because these metabolites are frequently not considered as part of the pathway) might be essential in flux control . Glutathione (-glu - cys - gly; gsh) is the most abundant nonproteinaceous thiol compound (110 mm) in almost all living cells . Gsh is involved in the oxidative stress processing, xenobiotic detoxification, and, in some plants and yeasts, in the inactivation of toxic heavy metals (for a recent revision see). Gsh is synthesized by two enzymes: -glutamylcysteine synthetase (-ecs) and glutathione synthetase (gs) (figure 2), which catalyze reactions with high - equilibrium constants (keq> 1000). Under a low gsh demand (unstressed conditions), the producing block of enzymes has to receive information from the last part of the pathway to (i) avoid the excessive and toxic accumulation of the intermediary -ec and (ii) reach a stable steady state . This information transfer is mediated by gsh, which exerts strong competitive inhibition of -ecs (figure 2). Gsh and cys also exert inhibition on the atp - sulfurylase (atps) and on sulfate transporters (figure 2) (for a review, see). The feedback inhibition of -ecs has led several researchers to propose that this enzyme is the rate - limiting step of gsh synthesis [3335]. Although there are no studies about the pathway's behavior under stressed conditions, which means under a high gsh demand, the proposal that -ecs is the key enzyme has been automatically extended to any environmental condition such as heavy metal exposure . By assuming that -ecs is the rate - limiting step, many research groups have tried to increase, in plants and yeasts, the rate of synthesis and the concentration of gsh and phytochelatins (pcs) with the aim of fortifying their heavy metal resistance and storage capacity, mainly toward cd . The development of organisms able to grow in soils and water systems contaminated with heavy metals, which may have the ability of accumulating toxic metal ions, is of biotechnological interest for bioremediation strategies . With this goal in mind, researchers have then overexpressed -ecs and other pathway enzymes, including phytochelatin synthase (pcs) (table 2). Some of these experiments have been partially successful in increasing gsh levels, although this has been rather marginal with no correlation between enzyme levels and gsh concentration . Unfortunately, these overexpression experiments have not been accompanied by determinations of fluxes or other relevant metabolite concentrations such as pcs or cys . On the other hand, the overexpression of pcs has surprisingly induced oxidative stress and necrosis instead of increasing cd accumulation and resistance . This result suggests that, under high gsh demand (i.e., for pcs synthesis and for direct heavy metal sequestration by gsh), the gsh concentration does not suffice for maintaining the other essential gsh functions such as oxidative stress management and xenobiotic detoxification . Another problem in the study of gsh biosynthesis for its eventual manipulation is that the pathway has been analyzed considering only the gsh - synthetic reactions without taking into account the gsh - consuming reactions (figure 2), . The analysis of an incomplete pathway leads to misleading conclusions about the control of flux . Metabolic modeling has shown that only with the incorporation of the consuming reactions of the pathway end products, a true steady state can be established . In conclusion, without a solid theoretical framework, the overexpression of only one enzyme (the rate - limiting step), or of many arbitrarily selected enzymes (tables 1 and 2), the problem of increasing the flux or metabolite concentrations cannot be solved . There are some successful examples of the genetic engineering approach to manipulate metabolism: overexpression (approx . 23 fold) of the five genes of the tryptophan synthesis pathway in s. cerevisiae, to increase (9-fold) flux; increase in amino acids (trp, ile, lys, val, thr) and trehalose production in corynebacterium glutamicum, in which some proteins of each metabolic pathway are simultaneously overexpressed, but some of them with mutations that confer insensitivity to feedback inhibition [4853]. In these transformed bacteria, the end products are indeed overproduced and their excretion is accelerated;overexpression of pfk and pyk to increase ethanol production by 35% in e. coli, although lactic acid formation was not modified; mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase mannitol production by 2750% in ldh - deficient lactococcus lactis; increase in sorbitol production (5 fold) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol 6-phosphate dehydrogenase (activity up to 250 fold in mutants versus wild type); overexpression of pfk (14 fold) or ldh (3.5 times) to increase 2 - 3 times the homolactic fermentation flux in lactococcus lactis growing on maltose, and in parallel decrease fluxes toward secondary acids and ethanol . Overexpression (approx . 23 fold) of the five genes of the tryptophan synthesis pathway in s. cerevisiae, to increase (9-fold) flux; increase in amino acids (trp, ile, lys, val, thr) and trehalose production in corynebacterium glutamicum, in which some proteins of each metabolic pathway are simultaneously overexpressed, but some of them with mutations that confer insensitivity to feedback inhibition [4853]. In these transformed bacteria, the end products are indeed overproduced and their excretion is accelerated; overexpression of pfk and pyk to increase ethanol production by 35% in e. coli, although lactic acid formation was not modified; mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase mannitol production by 2750% in ldh - deficient lactococcus lactis; increase in sorbitol production (5 fold) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol 6-phosphate dehydrogenase (activity up to 250 fold in mutants versus wild type); overexpression of pfk (14 fold) or ldh (3.5 times) to increase 2 - 3 times the homolactic fermentation flux in lactococcus lactis growing on maltose, and in parallel decrease fluxes toward secondary acids and ethanol . Several glycolytic enzymes are overexpressed in at least 70% of human cancers . Except for glucose transporter 1 (glut-1), the other 11 glycolytic enzymes (hk to ldh) are overexpressed in brain and nervous system cancers . Prostate and lymphatic nodule cancers (hodgkin and non - hodgkin lymphomas; myelomas) overexpress 10 glycolytic enzymes (except for hk; in prostate cancer glut1 is also overexpressed). There is a second group of cancers that overexpresses 68 glycolytic genes (skin, kidney, stomach, testicles, lung, liver, placenta, pancreas, uterus, ovary, eye, head and neck, and mammary gland). A third group includes those cancers overexpressing 1 or 2 glycolytic genes (bone, bone marrow, cervix, and cartilage). In animals, gene expression of glycolytic enzymes is regulated (both coordinately and individually) under hypoxic conditions by hypoxia - responsive transcription factors such as hif-1 (hypoxia - inducible factor 1), sp family factors, ap-1, and possibly mre (metal response elements) [8, 5961]. . There are binding sites (consensus sequence acgt) for hif-1 in the promoters of genes for hk, pfk-1, aldo, gapdh, pgk, eno, pyk, and ldh (reviewed in). Tpi and perhaps hpi and pgam are also induced by hypoxia, but it is not clear whether hif-1 mediates this induction, and whether this factor regulates other metabolic pathways associated with glucose catabolism . For example, although glycogen phosphorylase is overexpressed under hypoxia in human tissues, the role of hif-1 has not been demonstrated . If direct manipulation of pathway genes becomes difficult, then the overexpression or repression of transcription factors such as hif-1, ap1, and mres might solve the problem of changing flux, although overexpression of transcription factors may also be difficult due to the numerous upstream and downstream factors involved . The kinetoplastid parasites trypanosoma cruzi, trypanosoma brucei, and leishmania are the causative agents of chagas disease, african trypanosomiasis, and leishmaniasis, respectively . The available drugs to treat these diseases are highly toxic for humans . Moreover, the parasites may become resistant, and hence the search for new drugs and drug targets is relevant for solving these public health problems . In these parasites, the metabolism is organized in a peculiar way; they have a subcellular structure called glycosome in which several metabolic pathways take place: gluconeogenesis, reactions of the pentose phosphate pathway, purine salvage and pyrimidine biosynthesis, -oxidation of fatty acids, fatty acid elongation, biosynthesis of ether lipids, and the first seven steps of glycolysis . Glycosomal glycolytic enzymes have unique structural, kinetic, and regulatory features not found in their human counterparts, and therefore have been the subject of extensive biochemical studies to use them as drug targets . The rationale behind this is to synthesize inhibitors that affect mainly the parasitic enzymes with relatively low effect on the human enzymes since the infective parasite stages rely mostly on glycolysis for atp supply . There are reports on the design of presumed specific inhibitors for some of the t. brucei glycolytic enzymes: glut (bromoacetyl-2-glucose), hk, hpi, pfk, aldo, tpi, gapdh, pgk, pyk, and glycerol-3-phosphate dehydrogenase . Although the purified enzymes display very low ki values for these inhibitors and some of them inhibit parasite growth or infective capabilities, their effect on inhibiting the glycolytic flux has not been explored . Therefore, it is not yet possible to directly ascribe the effects seen in parasite culture with the in vitro effects on the isolated enzymes . To identify the best drug targets, determination of the flux control steps of glycolysis in t. brucei has been recently initiated . Trypanothione (tsh2) is a reducing agent present in trypanosomatids that is synthesized from one spermidine and two gsh molecules by tsh2 synthetase (trys) (figure 3). This metabolite and its reducing enzyme, tsh2 reductase (tryr), replace the antioxidant and metabolic functions of the more common gsh / gsh reductase system present in mammals . In fact, most of the antioxidant metabolism of these parasites depend on tsh2 (figure 3) [69, 70]. Thus, the enzymes of this metabolic pathway have been proposed as drug targets for killing the parasites . Diminution in its gene transcription yields a loss of activity between 5690%, depending on the genetic technique [7275]. In knockdown t. brucei cells (i.e., when tryr activity has diminished to less than 10% of the wild - type level), the parasites show growth diminution and higher sensitivity to h2o2 in culture and loss of infectiveness in mice . However, tsh2 and thiol compound contents were not affected . Tryr downregulation by> 85% in leishmania species causes inability to survive under oxidative stress inside macrophages [7274]. In contrast, when tryr is 14- and 10 fold overexpressed in leishmania and t. cruzi, respectively, there are no significant differences in h2o2 susceptibility between control and transfected cells; both types of cells are also equally resistant to the oxidative stress - inducers gentian violet, and nitrofurans . Intriguingly, the cellular levels of tsh2, gsh, and glutathionyl - spermidine, determined in both types of experiments (tryr suppression and overexpression) were similar in control and transformed cells . Knockdown of trys by sirna in procyclic t. brucei causes (i) viability impairment and arrest of proliferation when tsh2 levels decrease to 15% of the wild - type level, (ii) increased sensitivity to h2o2 and alkyl hydroperoxides, (iii) damage to the plasma membrane, and (iv) diminution of the tsh2 content and accumulation of gsh and glutathionyl - spermidine . A similar metabolite variation (lower tsh2; higher gsh) was attained with a trys knockdown induced by sirna in the bloodstream form of t. brucei . This trys knockdown also induced an increased sensitivity to different compounds that affect tsh2 metabolism such as arsenicals, melarsen oxide, trivalent antimonials, and nifurtimox . Indeed, western blot analysis showed, in addition to the expected (10-fold) decrease in trys protein, a 2 - 3-folds increase in -ecs and tryr . The changes in expression of other enzymes suggest unveiled compensatory or pleiotropic effects on tsh2 metabolism . Other researchers have selected -glutamylcysteine synthetase (-ecs), the presumed rate - limiting step of gsh synthesis, as an alternative drug target of tsh2 synthesis in t. brucei (figure 3). Knockdown of -ecs gene in the parasite induces cell death and depletion of gsh and tsh2 only after 80% decrease in the enzyme content . The -ecs knockdown cells are rescued from death by adding external gsh, which elevates the cellular gsh and tsh2 levels . Glutathione synthetase (gs) has not been manipulated in trypanosomatids, or in any other organism, perhaps because it has been considered as a nonrate - limiting step of gsh and tsh2 biosynthesis . However, dna microarray analysis of antimonite - resistant leishmania tarentolae shows increased transcription of -ecs, gs, and p - glycoprotein a rnas . Although it was not evaluated whether increase in gene transcription correlated with an increase in enzyme activity, it may be possible that under high gsh demand (i.e., under oxidative stress conditions) gs might exert control of tsh2 synthesis . On the other hand, ornithine decarboxylase (odc) overexpression in t. brucei (the presumed limiting step of spermidine synthesis) causes no change in tsh2 levels . Therefore, odc does not seem to be a controlling step of tsh2 synthesis . Although almost full inhibition (> 80%) of gene transcription or activity of any of these enzymes results in parasite death, the question remains of how tsh2 metabolism is affected when the enzymes are less inhibited . For example, in the therapeutic treatment of patients it is certain that drugs have to be administered for long periods of time . If the parasites are not completely cleared from the patient, disease recurrence and generation of drug - resistant parasites are possible . The results described above indicate that each enzyme by itself has low control on tsh2 synthesis and concentration; therefore, highly specific and very potent inhibitors have to be designed in order to attain the required full activity blockade to affect tsh2 metabolism in these parasites . The metabolic control analysis (mca) was initially developed by kacser and burns in scotland [82, 83] and by heinrich and rapoport in east germany [84, 85]. This analysis establishes a theoretical framework that explains the results observed with the enzyme overexpression and downregulation experiments . In addition, it helps to identify and design experimental strategies for the manipulation of a given process in an organism (heavy metal hyperaccumulation; increased production of ethanol, co2, lactate or acetate; or inhibition of a metabolic pathway flux with therapeutic purposes). Mca rationalizes the quantitative determination of the degree of control that a given enzyme exerts on flux and on the concentration of metabolites . Different experimental approaches have been developed to detect and direct what has to be done and measured, in order to identify and understand why an enzyme exerts a significant or a negligible control on flux and metabolite concentration in a metabolic pathway . Experiments for identifying and manipulating the conceptually wrong rate - limiting step . To understand how a metabolic pathway is controlled and could be manipulated, its control structure has to be evaluated . The control structure of a pathway is constituted by the flux control coefficient (cvi), which is the degree of control that the rate (v) of a given enzyme i exerts on flux j; the concentration control coefficient (cvi), which is the degree of control that a given enzyme i exerts on the concentration of a metabolite (x); and the elasticity coefficients . The control coefficients are systemic properties of the pathway that are mechanistically determined by the elasticity coefficients (x), which are defined as the degree of sensitivity of a given enzyme vi (i.e., the enzyme's ability to change its rate) when any of its ligands (x: substrate, products or allosteric modulators) is varied . Viojo, in which the expression dj / dvi describes the variation in flux (j) when an infinitesimal change is done in the enzyme i concentration or activity . In practice, the infinitesimal changes in vi are undetectable, and hence measurable noninfinitesimal changes are undertaken . If a small change in vi promotes a significant variation in j, then this enzyme exerts an elevated flux control (figure 4, position 1). In contrast, if a rather small or negligible change in flux is observed when vi is greatly varied, then the enzyme does not exert significant flux control (figure 4, position 2). To obtain dimensionless and normalized values of cvi the scaling factor vio / jo is applied, which represents the ratio between the initial values from which the slope dj / dvi is calculated . If all cvi of the pathway enzymes and transporters are added up, the sum comes to one (summation theorem). The mca clearly distinguishes between the control exerted by a given enzyme on flux (flux control coefficient) and on the metabolite concentration (concentration control coefficient). Thus, an enzyme can have significant control on a metabolite concentration but not on the pathway flux . On one hand, the use of the rate - limiting step concept for manipulating metabolic pathways does not make such differentiation, which probably has contributed to the many unsuccessful experiments reported in the literature; on the other hand, it should be clearly defined whether the aim of the project is to increase flux and/or a metabolite concentration since mca establishes for each aim a different experimental design . To determine the flux control coefficient of a given enzyme, small variations in the enzyme content, or preferentially, in activity are required, without altering the rest of the pathway, and then the changes in flux are determined . The experimental points are plotted as shown in figure 4 to calculate the slope at the reference point vio / jo . This experiment, apparently easy to perform, has demanded great intellectual and experimental effort . Several experimental strategies have been developed to determine cvi: formation of heterokarionts and heterocygots (classical genetics),titration of flux with specific inhibitors, elasticity analysis, mathematical modeling (in silico biology),in vitro reconstitution of metabolic pathways, genetic engineering to manipulate in vivo protein levels . Formation of heterokarionts and heterocygots (classical genetics), titration of flux with specific inhibitors, mathematical modeling (in silico biology), in vitro reconstitution of metabolic pathways, genetic engineering to manipulate in vivo protein levels . The arginine biosynthesis in neurospora crassa was the first metabolic pathway in which flux control coefficients were experimentally determined by kacser's laboratory . This fungus forms multinucleated mycelia that facilitate the generation of polyploid cells . By mixing different ratios of spores containing genes encoding wild (active) and mutant (inactive) enzymes of this pathway, it was possible to generate heterokaryont mycelia with different content, and activity, of four pathway enzymes . The authors built plots of enzyme activity versus flux (see figure 4) for acetyl - ornithine aminotransferase, ornithine transcarbamoylase, arginine - succinate synthetase, and arginine - succinate lyase . All the experimental points of these heterokaryonts localized near to position 2 of figure 4 with cvi = 0.020.2 (flux control by these enzymes was only 220%), which indicated that none of these enzymes exerted significant control on arginine synthesis . The authors did not determine the remaining flux control (75%), which might reside in carbamoyl - phosphate synthetase i (this mitochondrial ammonium - dependent isoform can be bound to the mitochondrial inner membrane or form complexes with ornithine transcarbamoylase [87, 88]) and in mitochondrial citruline / ornithine transporter, both of which have been proposed as limiting steps, or might be in the arginine demand for protein synthesis . Organisms with many alleles of one enzyme may form homo - and heterozygotes expressing different activity levels . Drosophila melanogaster has three adh alleles encoding for isoforms with different vmax . When three natural homozygotes, a null mutant, and some heterozygotes were generated, different adh activities were attained but the ethanol consuming rate did not change (figure 4, position 2). Oxidative phosphorylation (oxphos) is the only pathway for which specific and potent inhibitors for many enzymes and transporters are available . Oxphos is divided in two segments (figure 5): the oxidative system (os) formed by substrate transporters (pyruvate, 2-oxoglutarate, glutamate, glutamate / aspartate, dicarboxylates), krebs cycle enzymes, and the respiratory chain complexes; and the phosphorylating system (ps) constituted by the atp / adp (ant) and pi (pit) transporters, and atp synthase . When the flux (atp synthesis) is titrated by adding increasing concentrations of each specific inhibitor, plots are generated in which the enzyme activity is progressively diminished by increasing inhibitor concentration . Hence, the cvi value depends on the type of inhibitor usedfor irreversible inhibition,(2)cvij = (imaxjo)(djdi)[i]0, for simple noncompetitive inhibition,(3)cvij = (kijo)(djdi)[i]0, for simple competitive inhibition,(4)cvij = (-ki[(1+s)/km]jo)(djdi)[i]0, where jo is the pathway flux in the absence of inhibitor; imax, minimal inhibitor concentration to reach maximal flux inhibition; k i, inhibition constant; s, substrate concentration; km, michaelis - menten constant; and d j / d i, initial slope ([i] = 0) of inhibition titration curve . For irreversible inhibition,(2)cvij = (imaxjo)(djdi)[i]0, for simple noncompetitive inhibition,(3)cvij = (kijo)(djdi)[i]0, for simple competitive inhibition,(4)cvij = (-ki[(1+s)/km]jo)(djdi)[i]0, to estimate flux control coefficients from inhibitor titration of adp - stimulated (state 3) respiratory rates (i.e., mitochondrial o2 consumption coupled to atp synthesis), (2) for irreversible inhibitors was used because researchers assumed that mitochondrial inhibitors such as rotenone, antimycin, carboxyatractyloside, and oligomycin were however, under this assumption flux control coefficients were usually overestimated [90, 91]. To solve this problem, gellerich et al . Developed (5) for noncompetitive tightly - bound inhibitors and, by using nonlinear regression analysis, it was possible to include all experimental points from the titration curve thus increasing accuracy in calculating cvi:(5)j = [n(joji)2encojoeon[(n co)jo (nji)en]] + ji e2+(kd+ieo)ekdeo=0, in which jo and ji are the respiration fluxes in the noninhibited (e = eo) and inhibited (e = 0) states; k d is the dissociation constant of the enzyme - inhibitor complex, and n is an empirical component that expresses the relationship between substrate concentration and the reaction catalyzed by the enzyme e. the analysis of data in table 3 shows that oxphos is not controlled by only one limiting step, but the flux control is rather distributed among several enzymes and transporters . It is worth noting that the value of the flux control coefficient depends on the content of enzyme or transporter, which varies from tissue to tissue . Perhaps the atp / adp translocase in as-30d hepatoma mitochondria might reach the status of being the oxphos limiting step with a ca nt = 0.70, or the pi transporter in kidney mitochondria, or the atp / adp translocase and the respiratory chain complex 3 in liver mitochondria, but it should be noted that other steps also exert significant control (table 3). Although the distribution of control varies between tissues, the flux control mainly resides in the ps of organs with high atp demand such as the heart (cpt+ant+atpsynthase = cps = 0.73), kidney (cps = 0.75; cos = 0.31), and fast - growing tumors (cps = 0.98). In contrast, in the liver (cos = 0.80; cps = 0.65) and brain (cos = 0.35; cps = 0.41), the control is shared by both systems . Determined that the oxphos control was shared by the ps (cps = 0.62) and the atp demand (purified atpase). In turn, rossignol et al . Concluded that the os exerted the main control (cos = 0.68), but these authors apparently used low - quality mitochondria (low respiratory control values that lead to low rates of atp synthesis associated with high rates of respiration) that were not incubated under near physiological conditions (10 mm pyruvate, 10 mm malate, 10 mm pi, ph 7.4 in tris buffer), and the authors incorrectly assumed that rotenone and antimycin were irreversible inhibitors . It is notorious that in all works shown in table 3 at least one of these mistakes is evident . There are some inhibitors for enzymes and transporters from other pathways, but they are not quite specific and may affect other sites . Due to the fact that there are no inhibitors for every step in these pathways, examples of these inhibitors are 6-chloro-6-deoxyglucose for glucose transporters in bacteria, 2-deoxyglucose for hpi, iodoacetate for gapdh, 1,4-dideoxy-1,4-imino - d - arabinitol for glycogen phosphorylase, oxalate and oxamate for ldh, 6-amino nicotinamide for the phosphate pentose pathway, amino - oxyacetate for aminotransferases and kirureninase (tryptophan synthesis), norvaline for ornithine transcarbamylase, mercaptopycolinate for pep carboxykinase, acetazolamide for carbonic anhydrase, and isobutyramide for adh (compiled by fell). Potential uses of the experimental approachmitochondrial pathologies are a heterogeneous group of metabolic perturbations characterized by morphological abnormalities and/or oxphos dysfunction . Mitochondrial dna analysis has revealed specific mutations for some mitochondriopathies . Although the specific oxphos mutations causing the disease may appear in all tissues, the functioning of only some of them is altered . The organ's sensitivity might be related to the different flux control coefficients of the mutated enzyme in the different tissues (table 3) and to their atp supply dependence from oxphos versus glycolysis.mca allows for the analysis of a metabolic flux or intermediate concentration by focusing either on one step or by grouping enzymes in blocks or in pathways . Thus, a comparative analysis of oxphos control distribution reveals that heart, kidney, some fast growing tumors (rat as-30d hepatoma, mouse fibrosarcoma, human breast, lung, thyroid carcinoma, melanoma), and perhaps skeletal muscle are more susceptible to mitochondrial mutations in atp synthase, which is the only ps site with subunits encoded in the mitochondrial genome . On the other side, liver and brain might be more susceptible to mitochondrial mutations of the respiratory chain enzymes (see table 3). Considering that the brain is a fully aerobic organ, whereas the liver depends on both oxphos (7080%) and glycolysis (2030%) for atp supply, then it can be postulated that the brain is more sensitive to mutations in the mitochondrial genome than the liver because subunits of complexes i, iii, and iv are encoded by the mitochondrial genome.titration of flux with specific inhibitors to determine the flux control coefficients of oxphos has been applied to intact tumor cells . The results showed that the flux control resided mainly in site 1 of the respiratory chain (csitel = 0.30), whereas the other evaluated sites exerted a marginal control . This observation could have therapeutic application if site 1 does not exert control in healthy cells, leading to less severe side effects.the use of inhibitors in intact cells to determine control coefficients might pose two problems: hydrophilic inhibitors such as carboxyatractyloside (for ant) and -cyano-4-hydroxy - cinammate (for pyruvate transporter) cannot readily enter the cell due to the presence of the plasma membrane barrier; the other problem is that hydrophobic but slow inhibitors, such as oligomycin, require long incubation times to ensure the interaction with the specific sites . These problems can be solved by incubating the cells for long periods of time and taking care of cell viability, for instance, as-30d hepatoma cells are fairly resistant to this mechanical manipulation as they maintain high viability after a lengthy incubation under smooth orbital agitation of 1 h at 37c . Mitochondrial pathologies are a heterogeneous group of metabolic perturbations characterized by morphological abnormalities and/or oxphos dysfunction . Mitochondrial dna analysis has revealed specific mutations for some mitochondriopathies . Although the specific oxphos mutations causing the disease may appear in all tissues, the functioning of only some of them is altered . The organ's sensitivity might be related to the different flux control coefficients of the mutated enzyme in the different tissues (table 3) and to their atp supply dependence from oxphos versus glycolysis . Mca allows for the analysis of a metabolic flux or intermediate concentration by focusing either on one step or by grouping enzymes in blocks or in pathways . Thus, a comparative analysis of oxphos control distribution reveals that heart, kidney, some fast growing tumors (rat as-30d hepatoma, mouse fibrosarcoma, human breast, lung, thyroid carcinoma, melanoma), and perhaps skeletal muscle are more susceptible to mitochondrial mutations in atp synthase, which is the only ps site with subunits encoded in the mitochondrial genome . On the other side, liver and brain might be more susceptible to mitochondrial mutations of the respiratory chain enzymes (see table 3). Considering that the brain is a fully aerobic organ, whereas the liver depends on both oxphos (7080%) and glycolysis (2030%) for atp supply, then it can be postulated that the brain is more sensitive to mutations in the mitochondrial genome than the liver because subunits of complexes i, iii, and iv are encoded by the mitochondrial genome . Titration of flux with specific inhibitors to determine the flux control coefficients of oxphos has been applied to intact tumor cells . The results showed that the flux control resided mainly in site 1 of the respiratory chain (csitel = 0.30), whereas the other evaluated sites exerted a marginal control . This observation could have therapeutic application if site 1 does not exert control in healthy cells, leading to less severe side effects . The use of inhibitors in intact cells to determine control coefficients might pose two problems: hydrophilic inhibitors such as carboxyatractyloside (for ant) and -cyano-4-hydroxy - cinammate (for pyruvate transporter) cannot readily enter the cell due to the presence of the plasma membrane barrier; the other problem is that hydrophobic but slow inhibitors, such as oligomycin, require long incubation times to ensure the interaction with the specific sites . These problems can be solved by incubating the cells for long periods of time and taking care of cell viability, for instance, as-30d hepatoma cells are fairly resistant to this mechanical manipulation as they maintain high viability after a lengthy incubation under smooth orbital agitation of 1 h at 37c . Xovio, which is a dimensionless number that show the rate variation v of a given enzyme or transporter i when the concentration of a ligand x (substrate s, product p or allosteric modulator) is varied in infinitesimal proportions . The elasticity coefficients are positive for those metabolites that increase the enzyme or transporter rate (substrate or activator), and they are negative for the metabolites that decrease the enzyme or transporter rates (product or inhibitor). An enzyme working, under a steady - state metabolic flux, at saturating conditions of s or p, is no longer sensitive to changes in these metabolites . Thus, its elasticity is close to zero (figure 6, x = 0). In turn, an enzyme working at s or p concentrations well below the michaelis constant (kms or kmp) is expected to be highly sensitive to small variations in these metabolites (figure 6, x = 1). If the kinetic parameters of an enzyme are known (vmf, vmr, kms, and kmp), then the enzyme elasticity for any given metabolite concentration may be calculated as shown in the following equations . For substrate,(7)svi = s / kms1 + s / kms + p / kmp + 11 /keq, and for product,(8)pvi = p / kmp1 + s / kms + p / kmp /keq1 /keq, in which is the mass action ratio, and keq is the equilibrium constant preferentially determined under physiological conditions . An enzyme with low elasticity cannot increase (or decrease) its rate despite large variations in s (or p) concentration; in consequence, such enzyme exerts a high flux control . In turn, an enzyme with a high elasticity can adjust its rate to the variation in s or p concentrations, and thus it does not interfere with the metabolic flux, exerting a low flux control . This inverse relationship between the elasticity and the flux control coefficients is expressed in a formal equation denominated connectivity theorem . A metabolic pathway can be divided in two blocks around an intermediary x: the producing (synthetic, supply) and the consuming (demand) enzyme blocks of x are i1 and i2, respectively . Thus, the connectivity theorem for this two - block system is(9)cv1jcv2j = xv2xv1 . The negative sign of the right part of the equation cancels with x, which is negative because x is a product of enzyme block i1 (figure 6). To obtain the flux control coefficients many strategies have been designed [90, 103108], but the most used and probably more trustworthy is that in which the initial pathway metabolite (so) concentration is varied to increase the x concentration (any intermediary in the pathway), and measuring in parallel the variation in flux . Under steady - state conditions, the flux rate is equal to the rate of end - product formation (i.e., lactate or alcohol for glycolysis; oxygen consumption for oxphos) and to the rate of any partial reaction . The slope, calculated at the reference coordinate (xo, jo) that is equivalent to (so, vio), yields the elasticity coefficient of the consuming block of x. in another set of experiments, an inhibitor is added to block one or more enzymes after x. the x concentration and flux are determined and plotted as shown in figure 7, from which the elasticity coefficient of the producing block is calculated . The flux control coefficients are determined by using the connectivity theorem and considering that the sum of the control coefficients comes to 1, c1 + c2 = 1 (summation theorem):(10)cv1j = xv2xv2 xv1,cv2j = xv1xv2 xv1 . This method for determining cvi using the elasticities of the two blocks was called double modulation by kacser and burns . Years later, brand and his group [103, 104] renamed this method as top - down approach . By applying the procedure shown in figure 7 and using (10) for different metabolites along the metabolic pathway, it is possible to identify those sites that exert a higher control (which may be the sites for therapeutic use or biotechnological manipulation) and those that exert a negligible control under a given physiological or pathological situation . Elasticity analysis has been used to evaluate the oxphos control distribution in tumor cells . Almost all studies on this subject have been carried out with isolated mitochondria incubated in sucrose - based medium at 25 or 30c or with the more physiological kcl - based medium but still at 30c (table 3). Furthermore, these studies did not consider that the product, atp, never accumulates in the living cells, which does occur in experiments with isolated mitochondria . Under such a condition, a steady state in atp production can never be reached as in living cells . In other words, the distribution of control in mitochondria (table 3) has been determined in the absence of an atp - consuming system . A remarkable exception to this incomplete experimental design was the work done by wanders et al ., in which isolated liver mitochondria were incubated with two different atp - consuming systems (or adp - regenerating systems): hk + glucose and creatine kinase (ck) + creatine . Under this more physiological setting, the oxphos flux control distributed between ant and the atp - consuming system; however, flux control by the other pathway components was not examined . Therefore, to accurately evaluate oxphos control distribution, mitochondria should be incubated in the presence of an atp - consuming system or in their natural environment (i.e., inside the cell). The rate of oxphos in intact cells is determined from the rate of oligomycin - sensitive respiration: in the steady state, the enzyme rates are the same and constant; in branched pathways the sum of the branched fluxes equals the flux that supplies the branches . The global elasticity of the atp - consuming processes (e.g., synthesis of protein, nucleic acid, and other biomolecules, as well as ion atpases to maintain the ionic gradients, mechanical activity such as muscular contraction or flagellum and cilium movement, and secretion of hormones, digestive enzymes and neurotransmitters) is estimated by inhibiting flux with low concentrations of oligomycin or a respiratory chain inhibitor . To determine the elasticity of the atp - producing block, flux, and [atp] are varied with streptomycin, an inhibitor of protein synthesis (figure 7). The elasticity coefficients are calculated from the initial coordinate slopes (without inhibitors) of each titration . With this procedure, it has been determined that the atp - consuming block exerts a significant flux control of 34% . Remarkably, this flux control value obtained in cells is quite similar to the flux control coefficients of the atp - consuming system (hk or ck) reported by wanders et al . With isolated mitochondria . Elasticity analysis by enzyme blocks allows the inclusion of the end - product demand as another pathway block . The conclusions obtained from this analysis have formulated the supply - demand theory, which proposes that when flux is controlled by one block (demand), the concentration of the end - product is determined by the other block (supply). The ratio of elasticities determines the distribution of flux control between supply and demand blocks . For instance, if x> x (i.e., demand becomes saturated by the end - product x, and hence its elasticity is near zero), then the demand block exerts the main flux control . For concentration control, at larger x x, smaller absolute values of both csupply and cdemand are attained; hence, under demand saturation, the supply elasticity fully governs the magnitude of the variation in the end - product concentration . On the other hand, when demand increases, it loses flux control and induces a diminution in the end - product concentration . In turn, supply gains flux control and loses concentration control . In the presence of feed - back inhibition, the system can maintain the end - product concentration orders of magnitude away from equilibrium (at a concentration around the k0.5 of the allosteric enzyme). As mentioned before, the demand is not usually included in the pathway because it is erroneously thought that it is not part of it . But then, is it valid to analyze the control of a metabolite synthesis if its demand is not considered? When the demand block is not included, it is assumed that the metabolic pathway produces a metabolite at the same rate regardless whether the metabolite demand is high or low . This reasoning is incorrect because a metabolic pathway indeed responds to changes in the metabolite demand and, more importantly, a pathway without end - products consumption reactions is unable to reach a steady state . The intermediary x linking the two blocks is one of the end - products of the producing block (e.g., pyruvate or lactate or ethanol, and atp for glycolysis). The variation in rate of the two blocks in response to a variation in x can be theoretical or experimentally determined (figure 8(a)). It is worth noting that, for this supply - demand approach, it is not necessary to know the kinetics of each pathway enzyme because the rate response of each block reflects the global kinetics of all participating enzymes . When the x concentration is increased, the rate of the supply block decreases (i) because x is its product and (ii) because usually an enzyme within this block receives information from the final part of the pathway, decreasing its rate through feedback inhibition . In turn to better visualize the effect of large rate changes, the kinetics of both blocks are plotted in a logarithmic scale . Figure 8(b) shows the kinetics described in figure 8(a) converted to natural logarithm . The intersection point between kinetic curves, at which the supply and demand rates are identical, represents the pathway steady - state flux (in the y axis) and end - product concentration (in the x axis). Since the elasticity is also defined as x = dlnvi / dlnx, the slope at the intersection point represents the elasticity of each block towards the intermediary x. here, the use of the scalar factor is not necessary because it is included in the logarithmic equation . With the elasticity coefficients calculated from plots like those shown in figure 8, and the connectivity theorem, the example in figure 8(b) shows that the demand exerts a high flux control (and has low elasticity) and the supply block exerts low control (and has high elasticity). The fact that the demand may exert a high flux control in metabolite pathways has at least three important implications: (a) the supply block responds to variations in the demand (high elasticity); (b) the demand block has information transfer mechanisms towards the supply block that avoid the unrestricted intermediary accumulation under a low demand, particularly when the supply block has reactions with large keq (> 100; g> 3 kcal mol at 37c); and (c) if the main flux control resides in the demand block, then the supply block may only exert control on the intermediary concentration but not on the flux [30, 32]. This last conclusion explains why it is incorrect to consider that an enzyme that controls flux must also control the intermediary concentration . Regulatory mechanisms of enzyme activity are modulation of protein concentration by synthesis and degradation, as well as covalent modification and variation in the substrate or product concentrations (which are components of the pathway). In addition, another regulatory mechanism is the modulation by molecules that are not part of the pathway, that is, through allosteric interaction with cooperative (sigmoidal kinetics) or noncooperative enzymes (hyperbolic kinetics) (e.g., ca activates some krebs cycle dehydrogenases; citrate inhibits pfk-1; malonyl - coa inhibits the mitochondrial transporter of acyl - carnitine / carnitine; or the initial substrate of a pathway that has not entered the system). For these last cases, kacser and burns proposed the use of the response coefficient r which is defined by the following expression:(11)rm j = cvijmvi, where m is the external modulator of the i enzyme . The response coefficient is dj / dmmo / jo . If the elasticity of the sensitive enzyme toward the external effector is also determined, then it is possible to calculate cvi by using (11). Unfortunately, due to the experimental complexity for determining the elasticity coefficient, this coefficient is often calculated in a theoretical way by using the respective rate equation (michaelis - menten or hill equations) and the kinetic parameters km and vmax determined by someone else under optimal assay conditions, which are commonly far away from the physiological ones . Therefore, for this theoretical determination of elasticity only the value of the external modulator concentration is required . It is convenient to emphasize that the determination of the flux control coefficients becomes more reliable when they are calculated from several experimental points (figure 7), instead of only one, as occurs with the theoretical elasticity analysis . Determined the flux control distribution of gluconeogenesis from lactate in hepatocytes by using both theoretical and experimental elasticity analysis and the response coefficient . These authors concluded that gluconeogenesis stimulated by glucagon was controlled by the pyruvate carboxylase (cpc = 0.83); in the absence of this hormone, the control was shared by pc, pyk, eno - pgk segment, and tpi - fructose-1,6-biphosphatase segment . Elasticity analysis has been applied to elucidate the flux control of atp - producing pathways in fast - growing tumor cells . For oxphos, this approach showed that respiratory chain complex i and the atp - consuming pathways were the enzymes with higher control (cvi = 0.7). For glycolysis, the main flux control (cvi = 0.71) resided in glut + hk reactions because hk is strongly inhibited by its product g6p despite extensive enzyme overexpression . Examples of elasticity analysis on other pathways are photosynthesis, ketogenesis, serine and threonine synthesis in e. coli, glycolysis in yeast, glucose transport in yeast, dna supercoiling, glycogen synthesis in muscle, and galactose synthesis in yeast . In conclusion, the elasticity analysis is the most frequently used method for determining flux control coefficients because it does not need a group of specific inhibitors for all the enzymes and transporters of the pathway, neither does it require knowledge of the inhibitory mechanisms or kinetic constants . It is only necessary to produce a variation in the intermediary concentration x by using an inhibitor of either block or by directly varying the x concentration . In agreement with fell, it seems impossible for a researcher to analyze one by one the rate equation of each enzyme in a metabolic pathway to predict and explain the system behavior as a whole . To deal with this problem, in the last three decades some scientists have constructed mathematical models for some metabolic pathways using several software programs . Thus, the specific variation of a single enzyme activity without altering the rest of the pathway (figure 4), which has been an experimentally difficult task for applying mca, becomes easier to achieve with reliable computing models . There are two basic types of modeling: (a) structural modeling and (b) kinetic modeling . The former is related to the pathway chemical reaction structure and does not involve kinetic information . The information obtained with structural modeling is the description of the following: the exact determination of which reactions and metabolites interact among them;the conservation reactions . There are metabolites for which their sum is always constant or conserved (e.g., nadh + nad; nadph + nadp; ubiquinol + ubiquinone; atp + adp + amp; coa + acetyl - coa). The identification of conserved metabolites might not be obvious;enzyme groups catalyzing reactions in a given relationship with another group of enzymes;elemental modules, which are defined as the minimal number of enzymes required to reach a steady state, which can be isolated from the system (for a review about structural modeling; see). The exact determination of which reactions and metabolites interact among them; the conservation reactions . There are metabolites for which their sum is always constant or conserved (e.g., nadh + nad; nadph + nadp; ubiquinol + ubiquinone; atp + adp + amp; coa + acetyl - coa). The identification of conserved metabolites might not be obvious; enzyme groups catalyzing reactions in a given relationship with another group of enzymes; elemental modules, which are defined as the minimal number of enzymes required to reach a steady state, which can be isolated from the system (for a review about structural modeling; see). In addition to an appropriate computing program, this approach requires the knowledge of the stoichiometries, rate equations, and keq values of each reaction in the pathway (or the vmax in the forward and reverse reactions), as well as the intermediary concentrations reached under a given steady state . Some currently used softwares are copasi (http://www.copasi.org/tiki-index.php) based on gepasi (http://www.gepasi.org/;); metamodel; winscamp and jarnac (both available at http://www.sys-bio.org/); and pysces (http://pysces.sourcesforge.net/;). For other programs and links, go to http://sbml.org/index.psp . To reach a steady - state flux, it is necessary to fix the initial metabolite concentration to a constant value and the irreversible and constant removal of the end products . Except for the final reactions in which their products have to be removed from the system, all pathway reactions have to be considered as reversible, notwithstanding whether they have large keq (if there is an irreversible reaction under physiological conditions, then a reversible rate equation that includes the keq suffices to maintain the reaction as practically irreversible). Care should be taken to include the enzyme's sensitivity toward its products because this property is related with the enzyme elasticity and hence with its flux control; omission of this parameter may very likely lead to erroneous conclusions . It should be pointed out that the purpose of kinetic modeling is not merely to replicate experimental data but also to explain them . Thus, pathway modeling is a powerful tool that allows for (i) the detection of those properties of the pathway that are not so obvious to visualize when the individual kinetic characteristics of the participating enzymes are examined; and (ii) the understanding of the biochemical mechanisms involved in flux and intermediary concentration control . Modeling requires the consideration of all reported experimental data and interactions that have been described for the components of a specific pathway, thus allowing for the integration of disperse data, discarding irrelevant facts . Although all models are oversimplifications of complex cellular processes, they are useful for the deduction of essential relationships, for the design of experimental strategies that evaluate the control of a metabolic pathway, and for the detection of incompatibilities in the kinetic parameters of the participating enzymes and transporters, which may prompt the experimental revision of the most critical uncertainties . With the model initially constructed, the simulation results do not usually concur with the experimental results; in consequence, the model normally requires refinement, a point at which the researcher's thinking and knowledge of biology plays a fundamental role in modifying the structure and parameters of the model . The discrepancies observed between modeling and experimentation unequivocally pinpoint what elements or factors have to be re - evaluated or incorporated so that the model approximates more closely reality (i.e., experimental data). The comparison of the experimentally obtained intermediary concentrations and fluxes with those obtained by simulation is an appropriate validating index of the model; this index indicates whether the model approximation to the physiological situation is acceptable or whether re - evaluation of the kinetic properties of some enzymes and transporters and/or incorporation of other reactions or factors is required . A reason to why the results obtained by modeling may substantially differ from the experimental results is that the kinetic parameters of the pathway enzyme and transporters and the keq values used were determined by different research groups, under different experimental conditions and in different cell types . Moreover, enzyme kinetic assays are carried out at low, diluted enzyme concentrations (thus discarding or ignoring relevant protein - protein interactions), and at optimal (but not physiological) ph and room temperature (which may be far away from the physiological values). In addition, no experimental information is usually available regarding the reactions reversibility and the product inhibition of the enzymes and transporters (particularly for physiological irreversible reactions, i.e., reactions with large keq). With worrisome frequency, the researcher has to adjust the experimentally determined vm and km values to achieve a model behavior that acceptably resembles that observed in the biological system . Apparently, this type of limitations as well as the sometimes overwhelming amount of kinetic data necessary for the construction of a kinetic model has restricted the number of reliable models that can be used for the prediction of the pathway control structure . Once the kinetic model stability, robustness, structural and dynamic properties have been evaluated, and experimentally validated, the model may become a virtual laboratory in which any parameter or component can be modified or replaced and any aspect of the pathway behavior can be explored within a wide diversity of circumstances or limits . At this stage, glycolysis in s. bayanus, s. cerevisiae [113, 123, 124], and trypanosoma brucei [125, 126] is the metabolic pathway that has been more extensively modeled . Both cell types have a very active glycolysis and are fully dependent on this metabolic pathway for atp supply, under anaerobiosis and aerobiosis, respectively . One advantage of modeling glycolysis in these cell types is that most of the kinetic parameters used have been experimentally determined by the same groups under the same experimental conditions . However, the kinetics of the reverse reactions has not been determined and thus these authors used kmp and keq values reported by others and obtained in other cell types under rather different experimental conditions, or they were adjusted to improve model fitting . Nevertheless, the simulation results yielded relevant information on the control of the glycolytic flux . In both cases, the enzymes traditionally considered the rate - limiting steps, hk, atp - pfk-1, and pyk did not contribute to the flux control, whereas the main control resided in glut (54% in the parasite and 85100% in yeast). Under some conditions, hk may exert some control (15%) in s. cerevisiae and some nonallosteric enzymes such as aldo, gapdh, and pgk may also exert some flux control in t. brucei . Mca through kinetic modeling has been applied to several pathways: glycolysis in erythrocytes in which flux control distributes between hk (71%) and pfk-1 (29%);carbohydrate metabolism during differentiation in dictyostelium discoideum with cellulose synthase (86%) as the main controlling step;sucrose accumulation in sugar cane with hk, invertase, fructose uptake, glucose uptake, and vacuolar sucrose transporter having the most significant flux control; glycerol synthesis in s. cerevisiae with gapdh (85%) as the main control step; penicillin synthesis in penicillium chrysogenum controlled (7598%) either by d-(a - aminoadipyl) cysteinylvaline synthetase (short incubation times <30 hour) or isopenicillin n. synthetase (long incubation times> 100 h); calvin cycle controlled by gapdh (50%) and sedoheptulose-1,7-bisphosphatase (50%);threonine synthesis in e. coli controlled by homoserine dehydrogenase (46%), aspartate kinase (28%), and aspartate semialdehyde dehydrogenase (25%); lysine production in corynebacterium glutamicum mainly controlled by aspartate kinase and permease; nonoxidative pentose pathway in erythrocytes mainly controlled by transketolase (74%); egf - induced mapk signaling in tumor cells controlled by ras - activation by egf (21%), ras dephosphorylation (43%), erk phosphorylation by mek (44%), and mek phosphorylation by ras (143%); aspergillus niger arabinose utilization with flux control shared by arabinose reductase (68%), arabitol dehydrogenase (17%), and xylulose reductase (14%); glycolysis in l. lactis in which several end products are generated (lactate, organic acids, ethanol, acetoin). Model predictions indicated that flux toward diacetyl and acetoin (important flavor compounds) was mainly controlled by ldh but not by acetolactate synthetase, the first enzyme of this branch . Glycolysis in erythrocytes in which flux control distributes between hk (71%) and pfk-1 (29%); carbohydrate metabolism during differentiation in dictyostelium discoideum with cellulose synthase (86%) as the main controlling step; sucrose accumulation in sugar cane with hk, invertase, fructose uptake, glucose uptake, and vacuolar sucrose transporter having the most significant flux control; glycerol synthesis in s. cerevisiae with gapdh (85%) as the main control step; penicillin synthesis in penicillium chrysogenum controlled (7598%) either by d-(a - aminoadipyl) cysteinylvaline synthetase (short incubation times <30 hour) or isopenicillin n. synthetase (long incubation times> 100 h); calvin cycle controlled by gapdh (50%) and sedoheptulose-1,7-bisphosphatase (50%); threonine synthesis in e. coli controlled by homoserine dehydrogenase (46%), aspartate kinase (28%), and aspartate semialdehyde dehydrogenase (25%); lysine production in corynebacterium glutamicum mainly controlled by aspartate kinase and permease; nonoxidative pentose pathway in erythrocytes mainly controlled by transketolase (74%); egf - induced mapk signaling in tumor cells controlled by ras - activation by egf (21%), ras dephosphorylation (43%), erk phosphorylation by mek (44%), and mek phosphorylation by ras (143%); aspergillus niger arabinose utilization with flux control shared by arabinose reductase (68%), arabitol dehydrogenase (17%), and xylulose reductase (14%); glycolysis in l. lactis in which several end products are generated (lactate, organic acids, ethanol, acetoin). Model predictions indicated that flux toward diacetyl and acetoin (important flavor compounds) was mainly controlled by ldh but not by acetolactate synthetase, the first enzyme of this branch . We modeled the gsh and pcs biosynthesis (figure 2) to determine and understand the control structure of the pathway and thus be able to identify potential sites for genetic engineering manipulation that might lead to the generation of improved species in heavy metal resistance and accumulation . Two models were constructed, one for higher plants and the other for yeast, both exposed to high concentrations of cd . Due to the similarity in the results, only the plant results are analyzed below . An interesting conclusion from the gsh - pcs synthesis modeling is that control of flux (and gsh concentration) is shared between the gsh supply and demand under both unstressed and cd exposure conditions (table 4). This observation strongly differs from the idea that -ecs is the rate - limiting step [3335]. For many researchers, the concept of -ecs being the key controlling step has seemed to be correct because (a) -ecs receives information from the final part of the pathway, as it is potently inhibited by gsh, the pathway end - product; and (b) -ecs is localized in the first part of the pathway (figure 2). In addition however, in most of the studies on the control of gsh synthesis, the gsh demand has not been considered . The gsh synthesis modeling shows that under a physiological feedback inhibition of -ecs by gsh a small increase in demand increases flux because the gsh concentration decreases and the -ecs inhibition attenuates . In contrast, if the demand remains constant, then an increase in -ecs activity or content (by overexpression) does not increase flux because the gsh inhibition is still there and operates on both new and old enzymes . The same pattern is also observed when hk is overexpressed to increase glycolytic flux since it is still inhibited by g6p (see section 3). On the other hand, -ecs indeed exerts significant concentration control on gsh, which means that a -ecs increase results in higher gsh concentration (table 4). This last observation demonstrates that an enzyme controlling a metabolite concentration does not necessarily control the flux . Cd exposure promotes a high gsh demand because significant oxidative stress surges, thus causing oxidation of gsh through gsh peroxidases, and because gsh and pcs are used for sequestering the toxic metal ion; hence, a higher gsh consuming rate sets up . Under this condition, modeling predicted that control was almost equally shared between the supply and demand blocks, but particularly between -ecs and pcs (see figure 2). Modeling was also able to explain why pcs overexpression can have toxic effects on the cell . An increase in the gsh demand (pcs overexpression) under high - demand conditions (cd stress) leads to gsh depletion that severely compromises other processes such as the oxidative stress control and xenobiotic detoxification . The conclusions drawn by this model led us to propose that, to significantly increase the cd resistance and accumulation, -ecs and pcs should be simultaneously overexpressed (table 4; figure 9). This particular manipulation promotes an increase in the rate of gsh and pcs synthesis (determined by the high - to - low transition of their flux control coefficients) and in the gsh and pcs concentrations (determined by their high concentration control coefficients). The model predicts that a 2-fold increase in the simultaneous overexpression of -ecs and pcs brings about a 1.92.4-fold increase in flux to gsh (jgs) and pcs (jpcs) and in pcs concentration (figure 9); a 5-fold overexpression further increases by 4.58.1 times the fluxes and pcs concentration . This proposed enzyme overexpression should not exceed the gs and the complex pc - cd (or gs - cd - gs) vacuolar transporters' maximal activities, in order to keep the cell away from a severe oxidative stress caused by gsh depletion or -ec accumulation . Indeed, the concentration of gsh was maintained high and constant although -ec accumulated with the simultaneous overexpression (figure 9). Furthermore, this enzyme manipulation should avoid the increase of the pc - cd and gs - cd - gs complexes in cytosol to toxic levels . In other words, excessive enzyme overexpression should be avoided, unless this is accompanied by compensating overexpression of consuming enzymes (gs for -ecs overexpression and pcs vacuolar transporters for that of pcs). In yeasts and plants, cd is ultimately inactivated by the additional interaction with s and the subsequent formation of stable high molecular weight complexes with pcs, cd, s, and gsh [138, 139]. In parallel to the -ecs and pcs overexpression, moderate repression of gsh - s - transferases, which compete for the available gsh (figure 2), may also promote an increase in gsh concentration and pcs formation flux . In contrast, gene overexpression induces large changes in activity; hence, further theoretical background has been developed for predicting the effect on flux and metabolite concentrations induced by large enzyme changes . Such a theoretical background was initially developed by small and kacser, who depicted (12) based on the flux control coefficients to predict the effect promoted by large changes in enzyme activity:(12)fejmrj = 11 i = jm(cvi0jo (ri 1)/ri), in which f is the amplification factor (the flux increase), and r represents how many times the enzyme is overexpressed . To predict the flux changes, promoted by identical overexpression of two enzymes (same r value) with different cvi, the equation is (13)fejmrj = 11 (cij + cjj) ((r 1)/r). Figure 10 shows the effect on flux when one or more enzymes with different cvi are changed by the same r factor . If the sum of cvi of one or more enzymes is less than 0.25, the impact on flux is discrete when the expression increases 5 folds (which is the most common variation in the overexpression experiments analyzed in section 2). But for a 3-fold overexpression of a group of enzymes, for which their sum of cvi is more than 0.5, then a significant flux change is achieved . If the sum of cvi is 1, the flux varies in a linear proportion with the degree of overexpression . It has to be remarked, however, that the predicted change in flux (figure 10) will be valid until certain degree, the limits of which being determined by the other pathway enzymes that should stay as noncontrolling steps . Figure 10 also shows the effect on flux of decreasing an enzyme activity (third quadrant). This segment plot is useful when inhibition of pathway flux is being pursued for therapeutic purposes or for understanding the molecular basis of the genetic dominance and recessivity . Like in the enzyme overexpression experiment, only a significant effect on flux is achieved when the enzymes with high cvi values are inhibited . For an enzyme or group of enzymes with cvi of 0.25, greater than 80% inhibition has to be attained to decrease 50% the pathway flux . In this context, it seems feasible to explain why knockdown of enzymes involved in tsh2 synthesis has to be almost total to detect an effect on tsh2 content or to alter functional or pathogenic properties of the parasites (section 4.3). The knockdown or knockout experiments in trypanosomatids suggest that -ecs, trys, and tryr most probably have low flux control and concentration - control coefficients since their contents or activities have to be reduced> 80% of the normal levels to reach changes in intermediary levels or in oxidative stress handling . Contrary to the several unsuccessful overexpression experiments carried out to increase the flux or metabolites of a metabolic pathway, modeling may allow for a more focused and appropriate design of experimental strategies of genetic engineering to increase flux or a given metabolite, and for selecting drug targets to decrease flux or metabolite concentration . For these predictions, modeling considers that overexpression of a controlling enzyme or transporter may promote flux or metabolite control redistributions . Thus, a low - control step may become a controlling point when overexpressing another step and, in consequence, the prediction shown in figure 10 based on (11) and (12) may be inaccurate . By considering the whole pathway components, modeling is also a powerful tool for predicting the effects on flux and metabolite concentration of varying an enzyme activity (by overexpression or drug inhibition). Model predictions to inhibit a pathway fluxkinetic modeling has been used to identify the flux controlling steps in trypanosoma brucei glycolysis for drug targeting purposes . Interestingly, modeling has predicted controlling steps for the parasite pathway different from those described for glycolysis in human host cells [125, 126]. The parasite lacks functional mitochondria and has neither krebs cycle nor oxphos enzyme activities . Therefore, substrate level phosphorylation by glycolysis is the only way to generate atp for cellular work . An important difference in amebal glycolysis in comparison to glycolysis in human cells is that it contains the pyrophosphate (ppi)-dependent enzymes phosphofructokinase (ppi - pfk) and pyruvate phosphate dikinase (ppdk), which replace the highly modulated atp - pfk and pyk present in human cells . Moreover, both have been proposed as drug targets by using ppi analogues (bisphosphonates).we recently described the construction of a kinetic model of e. histolytica glycolysis to determine the control distribution of this energetically important pathway in the parasite . The model was constructed using the gepasi software and was based on the kinetic parameters determined in the purified recombinant enzymes, as well as the enzyme activities, fluxes, and metabolite concentrations found in the parasite . The results of the metabolic control analysis indicated that hk and pgam are the main flux control steps of the pathway (73 and 65%, resp .) And perhaps glut . In contrast, the ppi - pfk and ppdk displayed low flux control (13 and 0.1%, resp .) Because they have overcapacity over the glycolytic flux . The amebal model allowed evaluating the effect on flux of inhibiting the pathway enzymes . The model predicted that in order to diminish by 50% the glycolytic flux (and the atp concentration; data not shown), hk and pgam should be inhibited by 24 and 55%, respectively, or both enzymes by 18% (figure 11). In contrast, to attain the same reduction in flux by inhibiting ppi - pfk and ppdk, they should be decreased> 70% (figure 11). Therefore, the kinetic model results indicate that hk can be an appropriate drug target because its specific inhibition can compromise the energy levels in the parasite . They also indicate that although ppi - pfk and ppdk remain as promising drug targets because of their divergence from the human glycolytic enzymes, highly potent and very specific inhibitors should be designed for these enzymes in order to affect the parasite's energy metabolism . Kinetic modeling has been used to identify the flux controlling steps in trypanosoma brucei glycolysis for drug targeting purposes . Interestingly, modeling has predicted controlling steps for the parasite pathway different from those described for glycolysis in human host cells [125, 126]. The parasite lacks functional mitochondria and has neither krebs cycle nor oxphos enzyme activities . Therefore, substrate level phosphorylation by glycolysis is the only way to generate atp for cellular work . An important difference in amebal glycolysis in comparison to glycolysis in human cells is that it contains the pyrophosphate (ppi)-dependent enzymes phosphofructokinase (ppi - pfk) and pyruvate phosphate dikinase (ppdk), which replace the highly modulated atp - pfk and pyk present in human cells . Moreover, both have been proposed as drug targets by using ppi analogues (bisphosphonates). We recently described the construction of a kinetic model of e. histolytica glycolysis to determine the control distribution of this energetically important pathway in the parasite . The model was constructed using the gepasi software and was based on the kinetic parameters determined in the purified recombinant enzymes, as well as the enzyme activities, fluxes, and metabolite concentrations found in the parasite . The results of the metabolic control analysis indicated that hk and pgam are the main flux control steps of the pathway (73 and 65%, resp .) And perhaps glut . In contrast, the ppi - pfk and ppdk displayed low flux control (13 and 0.1%, resp .) Because they have overcapacity over the glycolytic flux . The amebal model allowed evaluating the effect on flux of inhibiting the pathway enzymes . The model predicted that in order to diminish by 50% the glycolytic flux (and the atp concentration; data not shown), hk and pgam should be inhibited by 24 and 55%, respectively, or both enzymes by 18% (figure 11). In contrast, to attain the same reduction in flux by inhibiting ppi - pfk and ppdk, they should be decreased> 70% (figure 11). Therefore, the kinetic model results indicate that hk can be an appropriate drug target because its specific inhibition can compromise the energy levels in the parasite . They also indicate that although ppi - pfk and ppdk remain as promising drug targets because of their divergence from the human glycolytic enzymes, highly potent and very specific inhibitors should be designed for these enzymes in order to affect the parasite's energy metabolism . Another experimental approach for determining the enzyme control coefficients is the in vitro reconstitution of segments of metabolic pathways . It is recalled that for determining the flux control coefficient exerted by a given step on a metabolic pathway the enzyme activity has to be varied, without altering the other components in the system, and the flux variations are to be measured (figure 4). Such an experiment can be readily made if a pathway is reconstituted with purified enzymes . Some advantages of this approach are that the pathway structure is known, in which the components concentration may be manipulated and analyzed separately, and the enzyme effectors can be assayed . As the system composition is strictly controlled, the results may be highly reproducible . The main disadvantage is that the enzyme concentrations in the assays are diluted and thus the enzyme interactions are not favored . If this interaction is important for activity, the in vitro reconstitution may limit the extrapolation to the metabolic pathway inside the cell . There are not many studies describing this type of experiments, most probably due to the fact that for applying mca the pathway must be working under steady - state conditions . In a reconstituted system, only a quasi steady state may be reached because there is net substrate, and cofactors consumption, as well as product accumulation, since it is difficult to attain a constant substrate supply and release of products . One of the first experimental reports on control coefficient determination in a reconstituted system was carried out for the upper glycolytic segment with the commercially available rabbit muscle hk, hpi, pfk-1, aldo, and tpi . Each enzyme was separately titrated and the flux variation to glycerol-3-phosphate (by coupling the reconstituted system to an excess of -gpdh) was measured in the presence of ck to maintain the atp concentration constant . The results showed that pfk-1 and hk exerted the main flux control (65% and 20%, resp . ), whereas the remaining 15% resided in the other enzymes . These authors observed that the addition of f1,6bp, a pfk-1 activator slightly diminished the flux control exerted by pfk-1 and increases that of hk . The lower glycolytic segment has also been reconstituted with commercial enzymes for determining the flux control coefficients . The results showed that flux was mainly controlled by pyk (60100%), although under some conditions control was shared with pgam; eno did not contribute to the flux control . Another important limitation of the reconstitution experiments is that the commercial availability of the purified enzymes from the same organism is restricted or inexistent . However, by using the information from the genome sequence projects and the recombinant dna technology, it is now possible to access all the enzyme genes from a metabolic pathway in the same organism, thus facilitating their cloning, overexpression, and purification . With this strategy, we cloned, overexpressed, and purified the 10 glycolytic enzymes of entamoeba histolytica for studying the flux control distribution in this organism by using kinetic modeling and pathway reconstitution . The reconstitution experiments of the lower amebal glycolytic segment, under near physiological conditions of ph, temperature, and enzyme activity (figure 12) showed that pgam and, to a lesser extent, ppdk exert the main flux control (these amebal enzymes are genetically and kinetically different from their human counterparts) with eno exhibiting negligible control . In turn, reconstitution of the upper amebal glycolytic segment has revealed that hk and, to a much lesser extent hpi, ppi - pfk, and ald, exerted the main flux control, with tpi having negligible control . These results strongly correlate with the enzyme catalytic efficiencies previously reported, in which hk is highly sensitive to amp inhibition, ald, and pgam have the lowest catalytic efficiencies among the glycolytic enzymes, leading to high flux control coefficients and thus becoming suitable candidates for therapeutic intervention . The reconstitution results also agree with the pathway modeling predictions previously analyzed (section 5.4), in which hk and pgam are two of the main controlling steps . The in vitro reconstitution experiments are also useful for studying the effect on control redistribution of an enzyme modulation that is particularly difficult to manage in vivo; the main controlling steps identified with the reconstitution experiments should be further analyzed with other experimental strategies such as elasticity analysis in the in vivo systems . This experimental approach for determining the control coefficients could be part of the genetic approach analyzed in section 5.1, but it was separated due to its recent methodological development and because it actually belongs to the molecular genetics rather than to the mendelian genetics . This approach is based on the in vivo modulation of the enzyme levels using the rna antisense technology . There are at least three strategies to inhibit gene expression: (a) the use of single stranded antisense oligonucleotides, which form a double stranded rna that might be degraded by rnase h; (b) target rna degradation with catalytically active oligonucleotides, known as ribozymes that bind to their specific rna; and (c) rna degradation using sirnas (2123 nucleotides). The rna antisense technology was applied for control coefficient determination of the ribulose - bisphosphate - carboxylase (rubisco) that fixes co2 in the plant calvin cycle . This enzyme considered the rate - limiting step of the calvin cycle and of the whole photosynthetic process, despite its high concentration (4 mm) in the chloroplasts stroma that compensates its low catalytic efficiency . Attempts to make rubisco a nonlimiting step, either by modifying its catalytic efficiency or by overexpressing it, have been unsuccessful . The plants were transformed with dna antisense against the mrna of the enzyme's small subunit, thus promoting its degradation . For calvin cycle enzymes, the pleiotropic effects were minimal . The results showed that rubisco may indeed be the photosynthesis limiting step with a crubisco = 0.690.83 when plants are exposed to high illumination (1050 mol quanta ms), high humidity (85%), and low co2 concentrations (25 pa). However, this flux control decreases to 0.050.12 under moderate illumination or high co2 levels . Unfortunately, the authors did not determine the control coefficients of the other pathway enzymes or the branches fluxes which may be significant . As described in section 5.4, the results of the t. brucei glycolysis modeling indicated that glut was the main flux control step (cglut 50%), [125, 126]. This model predicted a large overcapacity for hk, pfk-1, aldo, gapdh, pgam, eno, and pyk over the glycolytic flux leading to low flux control coefficients [125, 126]. To validate the modeling results, the concentrations of hk, pfk-1, pgam, eno, and pyk these knockdown expression experiments showed overcapacity of hk and pyk over the flux, although at lower levels than predicted by the model . A good correlation for pgam and eno was obtained between model predictions and experimental results . However, a large difference (9 folds) was obtained for pfk-1 . This discrepancy is perhaps related to pleiotropic effects of pfk-1 downregulation, as these mutants also displayed diminution in the activities of other enzymes (hk, eno, and pyk). The combination of these two approaches, in silico modeling and in vivo experimentation, is complementary: on one hand, modeling identifies the enzymes (out of 19 that contain the model) that display the highest flux control coefficients, whereas in vivo experimentation validates the accuracy of the model to establish predictions about the pathway's behavior . The knockdown experiments described above usually yield only two experimental points of the plot shown in figure 4: the wild - type and the knockdown strain protein levels or enzyme activities . Thus, with such an approach high levels of inhibition (> 80%) are mostly analyzed, whereas intermediate levels of downregulation (if obtained) are generally overlooked . Therefore, knockdown experiments are not very useful to obtain the complete set of experimental data (above and below the wild - type levels of enzyme activity with the corresponding flux) for determining reliable control coefficients . A strategy to determine flux control coefficients from several protein levels has been developed by using adenovirus - mediated glucose-6-phosphatase (g6pase) overexpression under the control of the cytomegalovirus promoter in rat hepatocytes . A 2-fold g6pase overexpression did not alter cg6pase or cgk (gk, glucokinase). However, if g6pase is overexpressed by 4 folds, then cgk diminished from 2.8 to 1.8 and there was a 35% lowering in glycogen synthesis . However, this approach allows titration of flux only above the basal enzyme activities found in the cell, but not below . These experimental inconveniences have been circumvented by using inducible gene expression systems based in the lac, lambda, nisin, gal, tetracycline, and other inducible promoters, in bacteria and yeast [151, 152]. However, a problem frequently encountered with inducible promoters is that a steady - state of protein expression is difficult to attain [151, 152]. Recently, jensen and hammer described the design of synthetic promoter libraries (spl), in particular for l. lactis metabolic optimization . These promoters maintain constant the array of the known consensus sequences for l. lactis gene transcription (10 and 35 boxes), while the nucleotide sequence between these boxes (a spacer sequence of 17 1 bp) is randomized, thus producing a set of promoters with different transcriptional strength . These promoter libraries allow the transcription and protein expression several folds above and below the wild - type levels of enzyme activity, thus enhancing the usefulness of this approach for mca studies . The control distribution of glycolysis in e. coli and l. lactis, as discussed in section 3.2 [17, 24, 27, 151], has been determined by using the spl technology . Spl for yeast, mammalian and plant cells are also under development [151, 152]. Certainly, the advances in genetic engineering in combination with mca allow better experimental designs for metabolic optimization of micro - organisms of biotechnological interest . Concluding remarks the frequently recurred idea of manipulating the key enzyme or rate - limiting step (a concept based on a qualitative and rather intuitive background) to change metabolism is incorrect . As mca has demonstrated, flux control is shared by multiple steps and it is not usually localized in only one step . Mca determines quantitatively the control that a given enzyme exerts on the flux and on intermediary concentration and helps to explain why an enzyme does or does not exert control . A metabolic pathway is manipulated to change the rate of the end - product formation (i.e., the flux) or the concentration of a relevant intermediary . As it is demonstrated in many unsuccessful experiments, it is not enough to overexpress one enzyme (the rate - limiting step) or many arbitrarily selected sites of the pathway . Mca proposes an initial experimental analysis that determines the structural control of the pathway and identifies the sites (enzymes and transporters) with higher control coefficients values (i.e., targets to be manipulated). For example, if there is a system composed of six enzymes and three of them have flux control coefficients with values of 0.2 or higher and the other three with values of 0.1 or lower, the three enzymes with high control coefficients must be overexpressed (if a flux increase is desired) or repressed (if flux inhibition is the objective) and not only one of them . If one of the selected enzymes is strongly inhibited by its product or has allosteric inhibition, the overexpression of this enzyme might not be enough to increase the flux, as it may also be necessary to moderately vary the product and allosteric modulator consuming enzymes.if the aim of the researcher is a metabolite concentration increase, which is not the end product of the pathway, mca suggests the overexpression of those enzymes or transporters in the supply block with the highest control coefficients and/or the repression of those enzymes in the demand block with the highest control coefficients . These manipulations may become complicated if the metabolite of interest has allosteric interactions with enzymes and transporters (inhibition and activation) of both the supply and demand blocks . It is recalled that ethanol production in yeast and lactate and acetate production in lactobacteria do not increase by overexpressing pfk-1, an allosteric enzyme and the presumed rate - limiting step of glycolysis . However, the analysis of these results reveals that the f1,6bp concentration is indeed increased many times over the control level . Another strategy for eliminating the feedback inhibition might be the introduction of mutations on the enzymes that are closer to the metabolite of interest . The frequently recurred idea of manipulating the key enzyme or rate - limiting step (a concept based on a qualitative and rather intuitive background) to change metabolism is incorrect . As mca has demonstrated, flux control is shared by multiple steps and it is not usually localized in only one step . Mca determines quantitatively the control that a given enzyme exerts on the flux and on intermediary concentration and helps to explain why an enzyme does or does not exert control . A metabolic pathway is manipulated to change the rate of the end - product formation (i.e., the flux) or the concentration of a relevant intermediary . As it is demonstrated in many unsuccessful experiments, it is not enough to overexpress one enzyme (the rate - limiting step) or many arbitrarily selected sites of the pathway . Mca proposes an initial experimental analysis that determines the structural control of the pathway and identifies the sites (enzymes and transporters) with higher control coefficients values (i.e., targets to be manipulated). For example, if there is a system composed of six enzymes and three of them have flux control coefficients with values of 0.2 or higher and the other three with values of 0.1 or lower, the three enzymes with high control coefficients must be overexpressed (if a flux increase is desired) or repressed (if flux inhibition is the objective) and not only one of them . If one of the selected enzymes is strongly inhibited by its product or has allosteric inhibition, the overexpression of this enzyme might not be enough to increase the flux, as it may also be necessary to moderately vary the product and allosteric modulator consuming enzymes.if the aim of the researcher is a metabolite concentration increase, which is not the end product of the pathway, mca suggests the overexpression of those enzymes or transporters in the supply block with the highest control coefficients and/or the repression of those enzymes in the demand block with the highest control coefficients . These manipulations may become complicated if the metabolite of interest has allosteric interactions with enzymes and transporters (inhibition and activation) of both the supply and demand blocks . It is recalled that ethanol production in yeast and lactate and acetate production in lactobacteria do not increase by overexpressing pfk-1, an allosteric enzyme and the presumed rate - limiting step of glycolysis . However, the analysis of these results reveals that the f1,6bp concentration is indeed increased many times over the control level . Another strategy for eliminating the feedback inhibition might be the introduction of mutations on the enzymes that are closer to the metabolite of interest . The frequently recurred idea of manipulating the key enzyme or rate - limiting step (a concept based on a qualitative and rather intuitive background) to change metabolism is incorrect . As mca has demonstrated, flux control is shared by multiple steps and it is not usually localized in only one step . Mca determines quantitatively the control that a given enzyme exerts on the flux and on intermediary concentration and helps to explain why an enzyme does or does not exert control . A metabolic pathway is manipulated to change the rate of the end - product formation (i.e., the flux) or the concentration of a relevant intermediary . As it is demonstrated in many unsuccessful experiments, it is not enough to overexpress one enzyme (the rate - limiting step) or many arbitrarily selected sites of the pathway . Mca proposes an initial experimental analysis that determines the structural control of the pathway and identifies the sites (enzymes and transporters) with higher control coefficients values (i.e., targets to be manipulated). For example, if there is a system composed of six enzymes and three of them have flux control coefficients with values of 0.2 or higher and the other three with values of 0.1 or lower, the three enzymes with high control coefficients must be overexpressed (if a flux increase is desired) or repressed (if flux inhibition is the objective) and not only one of them . If one of the selected enzymes is strongly inhibited by its product or has allosteric inhibition, the overexpression of this enzyme might not be enough to increase the flux, as it may also be necessary to moderately vary the product and allosteric modulator consuming enzymes . If the aim of the researcher is a metabolite concentration increase, which is not the end product of the pathway, mca suggests the overexpression of those enzymes or transporters in the supply block with the highest control coefficients and/or the repression of those enzymes in the demand block with the highest control coefficients . These manipulations may become complicated if the metabolite of interest has allosteric interactions with enzymes and transporters (inhibition and activation) of both the supply and demand blocks . It is recalled that ethanol production in yeast and lactate and acetate production in lactobacteria do not increase by overexpressing pfk-1, an allosteric enzyme and the presumed rate - limiting step of glycolysis . However, the analysis of these results reveals that the f1,6bp concentration is indeed increased many times over the control level . Another strategy for eliminating the feedback inhibition might be the introduction of mutations on the enzymes that are closer to the metabolite of interest.
Network - driven spindle - like oscillations are a functional hallmark of the developing cerebral cortex . During late prenatal and early postnatal stages of development, spontaneous spindle - like oscillations have been identified as physiological activity patterns in various neocortical areas of different mammalian species [14]. In humans, so - called delta brushes can be observed in eeg recordings from preterm babies already at gestational week 28, that is ~12 weeks before normal birth of a full - term neonate (for review see). In ac - filtered eeg recordings, delta brushes are brief rhythmic delta waves (0.31.5 hz) of 50300 v amplitude with a superimposed burst of fast rhythm (> 8 hz, the brush). It has been suggested that delta brushes in human preterm infants correlate with so - called spindle bursts recorded in rodents during the early postnatal period . From a developmental point of view, these early spontaneous activity patterns in developing rodent and human cerebral cortex are probably of similar or even identical origin . Rats and mice are altricial - born in a far less mature condition than humans . In rodents the degree of neocortical development at the day of birth (postnatal day [p] 0) can be compared to the stage of human cortex between gestational weeks 28 and 32 and the cerebral cortex of a p12p14 rat is comparable to that of the full - term newborn human baby [6, 8]. In preterm infants born between gestational weeks 28 and 32, milh et al . And colonnese et al . Recorded eeg signals containing spontaneous and stimulus - evoked delta brushes with oscillatory activity in the frequency range of 8 to 25 hz, suggesting that spontaneous delta brushes may represent a physiological neocortical activity pattern of the human fetus in utero . In the cerebral cortex of rodents, spindle bursts (beside short gamma oscillations) constitute the majority of spontaneous activity during the first postnatal week (figure 1). These spindle bursts resemble in their appearance spindles recorded in the adult brain during sleep . These sleep spindles are one of the hallmarks of human stage 2 sleep for comprehensive overviews the reader is referred to recent reviews by lthi and by mccormick et al . . However, in humans sleep spindles appear 4 to 9 weeks after birth, which is much later than the disappearance of delta brushes around the end of the first postnatal week, thus excluding the hypothesis that delta brushes or spindle bursts gradually develop into sleep spindles . In addition, the frequency profile of spindle bursts and delta brushes displays a rather broad frequency distribution up to 25 hz, whereas sleep spindles present oscillations in a narrower band of ~1015 hz . The present review focuses on spindle burst activity in the cerebral cortex of the developing rat during the first postnatal week and summarizes our current understanding (i) on the functional properties of spindle bursts, (ii) the mechanisms underlying their generation, (iii) the synchronous patterns and cerebral networks associated with spindle bursts, and (iv) the physiological and pathophysiological role of spindle bursts during early cortical development . Two distinct activity patterns can be observed in the neonatal rat cerebral cortex in vivo: gamma oscillations and spindles bursts (figures 1 and 2) [1419]. Gamma oscillations have a duration of 100 to 300 ms, a frequency of 30 to 40 hz and appear spontaneously every 10 to 30 s (figures 1 and 2(b)). The properties and functional role of gamma oscillations as well as the mechanisms underlying their generation spindle bursts are characterized by their spindle - like oscillatory appearance, have a duration of 0.5 to 3 s and a frequency in the range of 8 to 30 hz, and occur spontaneously every ~10 s (figures 1 and 2(a)). As recognized in full - band direct - current (dc) coupled recordings, spindle bursts are nested in slow (delta) waves, which in infant rats, and preterm human babies [22, 23] have been termed spontaneous activity transients (sats). Spindle bursts have been described in primary somatosensory cortex (s1) including barrel cortex [14, 1619], in primary visual cortex (v1) [10, 15], in primary motor cortex (m1), and in prefrontal cortex of anesthetized and awake rats during the first postnatal week . In s1, spontaneous and stimulus - evoked spindle bursts can be observed as early as p0 [17, 18]. In rats, the incidence of spindle bursts declines during the second postnatal week and sporadic spindle bursts are obscured by the ongoing neocortical activity . Likewise in humans, delta brushes are replaced by more continuous eeg activity at birth of a full - term infant . This gradual developmental shift from a highly synchronized state of spontaneous activity to a desynchronized state seems to be a fundamental network property of the cerebral cortex during early neonatal stages . Spindle bursts synchronize the activity of a local neuronal network of 200 to 400 m in diameter, which resembles the dimension of a whisker - related neocortical column in the immature barrel cortex (figure 3) [18, 25]. Using a combination of voltage - sensitive dye imaging and high - density multielectrode recordings in p0-p1 rat barrel cortex in vivo, yang et al . Could demonstrate that this early spontaneous activity constitutes the later emergence of the whisker - related barrel field map . These data indicate that spontaneous activity patterns at this early age form functional precolumns, supporting the concept of the existence of ontogenetic columns in the radial unit hypothesis . Further support for this hypothesis comes from in vivo two - photon calcium imaging in the barrel cortex of both anesthetized and nonanesthetized newborn mice, demonstrating highly synchronous spontaneous burst activity, reminiscent of spindle bursts, in local networks of 100 to 200 m in diameter . These data strongly indicate that early spindle bursts, and probably also gamma oscillations (for review), synchronize early neocortical networks into functional columns at a developmental stage when the upper layers 2/3 have not even been formed (i.e., in rats at p0). At this developmental stage, thalamocortical afferents have not reached layer 4 and instead transiently innervate the subplate (for review [28, 29]). Thalamic afferents form transient glutamatergic synapses with surprisingly mature properties including ampa and nmda receptors [3032]. In vitro studies in acute brain slice preparations and in intact whole cortical hemisphere preparations have demonstrated that oscillatory network activity in the frequency range of spindle bursts depend on an intact subplate [34, 35]. Selective removal of the subplate in s1 in vivo causes a significant decline in the occurrence of spontaneous spindle bursts and disturbances in the development of the cortical architecture in the barrel cortex . These data further support the hypothesis that spindle bursts in developing cerebral cortex fulfill an important role in the maturation of the neocortical architecture . In the next section we will discuss the network and molecular mechanisms underlying the generation of spindle bursts in neonatal cerebral cortex . The rodent cerebral cortex develops rapidly during late prenatal and early postnatal stages and at least four different activity patterns may occur sequentially between birth and the end of the first postnatal week (for review). Furthermore, the cortex shows a mediolateral and anterior - posterior gradient in development and within the same neocortical area neurons in upper layers 2/3 are ~2 days younger compared to lower cortical layers . Many inconsistencies in the literature on the properties of spontaneous activity patterns in newborn rodents and their underlying mechanisms can be explained by the fact that these important developmental differences are often ignored . In addition, 2 days in early rodent cortical development make a large difference, so that the neocortex of a p0 rat (without layer 2/3) cannot be compared to that of a p2 rat (with almost complete lamination). Already in newborn (p0-p1) rat barrel cortex in vivo, mechanical stimulation of a single whisker elicits in field potential recordings and to some extent detectable also in voltage - sensitive dye imaging (vsdi) a sequence of an early gamma oscillation followed by a spindle burst (figure 3(b)). At this age, the thalamocortical activity reaches the developing cortical network largely via the subplate [17, 31, 32] and is amplified by an intrinsic gap - junction coupled network within the subplate and cortical plate [28, 34]. Spontaneous and evoked delta brushes can be observed in premature human neonates of 2832 weeks postconceptional age, a developmental stage when the human cerebral cortex resembles that of a newborn rat . Impressive examples of large delta brushes are provided in the supplementary eeg videos of milh et al ., demonstrating that a single touch elicits a large oscillatory response in the somatosensory evoked potential (sep) recorded above the contralateral parietal cortex . In mature human cortex, seps with smaller amplitudes and shorter durations can be only obtained after averaging of at least 100 epochs . Thus, in both species, rats and humans, at a comparable stage of cortical development mechanical stimulation of the sensory periphery elicits in s1 spindle bursts and delta brushes, respectively . Simultaneous multielectrode recordings in the barrel cortex and in the ventral posteromedial nucleus (vpm) of the somatosensory thalamus of p0-p1 rats in vivo have demonstrated that the majority of spontaneous cortical spindle bursts and also gamma oscillations are not generated within s1, but rather in subcortical structures or outside of s1 (figures 4(a) and 4(b)). At this age a local, functionally defined lesion in the vpm blocks the whisker stimulation - induced cortical responses and also profoundly reduces the spontaneously occurring cortical burst activity, further demonstrating that the majority of the spontaneous spindle bursts in p0-p1 rat barrel cortex are generated in subcortical structures of the whisker - to - barrel cortex pathway (for further information see). Silencing the sensory periphery by injection of lidocaine into the whisker pad causes a significant reduction in the occurrence of spontaneous spindle bursts and gamma oscillations by ~50% (figure 4(c)), indicating that during this developmental period at least half of the spontaneous burst activity in s1 is related to activity in the sensory periphery . A similar peripheral generation of spontaneous burst activity may occur in human preterms and fetuses between gestational weeks 28 and 32 [9, 22]. Similar to the spindle bursts in s1, spontaneous spindle bursts in v1 are also largely generated in the sensory periphery . (for review), provide the primary drive for spindle bursts in newborn rat v1, as demonstrated by simultaneous recordings from the retina and v1 . Intraocular injection of forskolin, which augmented retinal waves, increased the occurrence of v1 spindle bursts, and removal of the retina reduced the spindle bursts frequency . As in the somatosensory system, spindle bursts in v1 can be also evoked by stimulation of the sensory periphery . However, since rod- and cone - mediated visual signaling is not functional in rats during the first postnatal week, spindle bursts cannot be evoked by light flashes before p8 . At that age, the neocortical response in v1 consists of an early visual evoked response followed by an evoked spindle burst . Similar responses could be observed in v1 of preterm infants once photoreceptor mediated light responses occur in the retina (for review). Whereas a large amount of experimental data has shown that retinal waves provide the main trigger for the cortical v1 spindles bursts, it is not completely understood which pacemaker drives the spontaneous activity in s1 and how spontaneous activity is generated in the somatosensory periphery . In p3p6 rats spontaneous whisker movements occur during active sleep and are correlated with activation of whisker - related cortical columns in the barrel cortex . In newborn rats the proprioceptive feedback from self - generated myoclonic movements trigger spindle bursts in s1 (for review). Spontaneous limb movements of the human fetus during the third trimester of gestation, or those of the preterm infant associated with delta brushes in s1, are similar to these twitching movements of the neonatal rat and can be also triggered by sensory feedback . Kreider and blumberg have demonstrated in 1-week - old rats that the mesopontine region plays a central role in the generation of myoclonic twitching . Have shown in newborn rats that spatially confined spindle bursts in s1 are triggered in a somatotopic manner by sensory feedback signals from spontaneous muscle twitches . These spontaneous movements are generated by neuronal networks in the spinal cord, but spindle bursts persisted at a reduced frequency after sensory deafferentation (spinal cord transection) in s1, indicating that spindle burst activity can be also generated in neocortical or thalamocortical circuits during this early period of development . This assumption is also supported by the observation that silencing of the sensory periphery causes only a ~50% reduction in the occurrence of spontaneous spindle bursts (figure 4(c)). However, since it cannot be excluded that the remaining portion of spindle bursts actually conveys activity from adjacent or distant sensory areas (transmitted via inter- and intrahemispheric connections, see next chapter), the outcome of this experiment may underestimate the contribution of the sensory periphery . Simultaneous monitoring of forepaw movements, vsdi, and extracellular multielectrode recordings in s1 and m1 of p3p5 rats under light urethane anesthesia have demonstrated that tactile forepaw stimulation triggers spindle bursts in s1, followed by gamma and spindle bursts in m1 (figure 5). Focal electrical stimulation of corticospinal tract neurons in layer 5 of m1 mimicking physiologically relevant 40 hz gamma or 10 hz spindle burst activity reliably elicited forepaw movements, indicating that m1 cortical spindle bursts are capable of triggering muscle twitches at this age . However, only 23.7% of the spontaneous bursts in m1 triggered forepaw movements and were followed by spindle bursts in s1 (figures 5(b)(a) and 5(c)), indicating that only a fraction of m1 activity transients triggers motor responses directly . In 40.7% of the cases, spontaneous movements preceded the burst activity in m1 and s1 (figures 5(b)(b) and 5(c)), suggesting that this activity may arise from subcortical regions in the brainstem or spinal cord . The remaining 35.6% of the m1 bursts were unrelated to any movements (figure 5(c)). The finding that 23.7% of the movements were triggered by m1 bursts as observed by an et al . Is in contrast to previous observations, which demonstrated that dissection of neocortical inputs fails to suppress muscle twitches in rat pups . In summary, these data indicate that neocortical spindle bursts in newborn rodents (and delta brushes in human fetus during the third trimester or in preterms) are generated by central pattern generator (cpg) circuits in spinal cord, brainstem, and motor cortex (for cpg circuits in mature brain see [4244]). Neuropharmacological studies provided insights into the molecular mechanisms underlying the generation and modulation of neocortical spindle bursts . In vivo and in vitro data suggest that gabaergic synapses are not crucial for the generation of spindle bursts or spindle burst - like activity, respectively, but are essential for their spatial confinement to a cortical (pre-) column . In contrast, spindle bursts depend on intact glutamatergic synapses including alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (ampa) and n - methyl - d - aspartate (nmda) receptors [34, 45]. Carbachol - induced spindle - like oscillations in p0p3 mouse neocortex in vitro and spindle bursts in p0p2 rat in vivo are blocked or significantly reduced by different gap junction blockers, indicating that electrical synapses are critically involved in the generation of spindle bursts at this neonatal period . However, minlebaev et al . Reported for p1p3 rats a significant increase in the occurrence of spontaneous spindle bursts in s1 following application of the gap - junction blocker mefloquine . Single - cell recordings revealed additional insights into the mechanisms underlying the generation of spindle bursts . Spindle bursts in v1 and s1 are accompanied by a barrage of glutamatergic and gabaergic postsynaptic currents (pscs) that are phase - locked to the spindle oscillations (figure 6). In prefrontal cortex, glutamatergic and gabaergic synaptic inputs to excitatory pyramidal neurons are phase locked to the theta - band component of the spindle, while pscs of inhibitory interneurons are phase locked to the higher beta and gamma frequencies, suggesting that excitatory and inhibitory neurons differentially modulate the distinct components of the spindle bursts . Further, the application of cnqx eliminates the glutamatergic pscs and completely blocks the occurrence of spindle bursts, indicating a major causal role of ampa - receptor mediated glutamatergic inputs in the generation of spindle bursts . Additional insights have been obtained by experiments in which subplate cells were selectively ablated in s1 shortly after birth . In these animals these in vivo results support previous in vitro studies, in which spindle bursts elicited by cholinergic stimulation were suppressed after removal of the subplate (see below) [34, 35]. Taken together, these data suggest that thalamocortical inputs relayed and amplified by the subplate [28, 29]play an important role in the generation of spindle bursts (e.g., [17, 45]). Spindle bursts in the cerebral cortex of newborn rats can be elicited [34, 35] and modulated by cholinergic mechanisms . In vitro, spindle burst - like oscillations can be reliably induced by activation of muscarinic acetylcholine receptors, predominantly of the m1 and m5 type . In vivo, spindle bursts in v1 are decreased by ~50% following the application of the muscarinic receptor antagonist atropine . Furthermore, blockade of acetylcholine esterase with physostigmine or direct stimulation of the cholinergic basal forebrain nuclei augmented the occurrence v1 spindle bursts, indicating that the cholinergic system facilitates spindle burst activity in developing cerebral cortex . As discussed above, spatially confined spindle bursts in newborn rat cortex synchronize a local neuronal network resembling a neocortical (pre-) column (figure 3). Beside these intra - areal synchronization, spindle bursts are also synchronized between different cortical regions within one hemisphere (intrahemispheric). As described above, a tight functional correlation in spontaneous and stimulus - evoked spindle burst activity exists between s1 and m1 cortex (figure 5). Spindle bursts with similar properties as those in v1 and s1 have been also recorded in vivo in the prefrontal cortex of urethane - anesthetized rats older than p2 . The same authors demonstrate that the hippocampus drives this early activity in the prefrontal cortex . Beside this intrahemispheric synchronization between different cortical regions, spindle bursts also interact between both hemispheres (interhemispheric). In vivo simultaneous recordings of spontaneous activity in homotopic cortical areas in both hemispheres at the same stereotaxic coordinates and depth have demonstrated that the amount of interhemispheric synchronization in newborn rats is initially rather low and increases during the first postnatal week . This interhemispheric communication of spindle burst activity depends on an intact corpus callosum . In this regard, in unanesthetized newborn rats callosotomy doubled the occurrence of spontaneous spindle bursts, suggesting that the corpus callosum modulates functionally inhibitory interactions between homotopic regions in both hemispheres during the occurrence of spindle burst activity . Experiments in p2p15 rats demonstrated that this callosotomy - induced disinhibition is a transient feature of early development that disappears abruptly after p6 . It is not surprising that intra- and interhemispheric interactions of spontaneous activity in the spindle burst frequency range can be also observed in developing human cerebral cortex at early stages . Using eeg and functional magnetic resonance imaging (fmri) omidvarnia et al . Electric resting - state network that shows functional intra- and interhemispheric interactions in the 815 hz frequency range . In summary, several in vitro and in vivo studies have demonstrated that spindle bursts represent elementary states of intra- and interhemispheric synchronization in the very immature cerebral cortex . An increasing amount of experimental and clinical data strongly indicate that spindle bursts play an important role in the physiological development of the cerebral cortex . Experimental evidence indicates that spindle bursts may be particularly suited to interfere with early neurodevelopmental processes, and thus disturbances in spindle burst activity may cause long - term structural and functional disorders . At early stages of development spontaneous and sensory - evoked activity patterns influence a variety of developmental processes, such as neurogenesis, apoptosis, neuronal migration, cellular differentiation, network formation, and myelination (for review, see). It is not completely understood how electrical activity controls these different developmental processes and whether distinct activity patterns, such as spindle bursts, play a specific role . However, for the control of apoptotic cell death of immature neurons in vitro and in vivo the essential role of spontaneous network bursts to provide antiapoptotic signals has been demonstrated [52, 56, 57]. For this activity - dependent control of neuronal survival the phosphatidylinositol 3-kinase pathway plays an important role, while the mapk / extracellular signal - regulated kinase or the calcium / calmodulin - dependent protein kinase pathway is not directly involved . Since one spontaneous spindle burst is associated at the single neuron level with 510 action potentials and the frequency of spontaneous spindle bursts is ~5 per minute [16, 17], a single neuron discharges with 2550 action potentials per minute . Under in vitro conditions this discharge frequency supports neuronal survival of developing neocortical neurons, suggesting that spontaneous spindle bursts in vivo provide an important physiological signal for the control of neuronal survival versus apoptosis in the neonatal cerebral cortex . Notably, spindle burst and gamma activity provides an ideal physiological stimulus for the activity - dependent release of bdnf, an important antiapoptotic signal . Balkowiec and katz demonstrated for neuronal cultures that 3060 min of electrical burst stimulation (50 pulses at 2050 hz at intervals of 20 s) increased extracellular bdnf levels by 20-fold, whereas stimulation patterns at lower frequency (albeit producing the same number of extracellular electric shocks) were ineffective (for review, see). These data indicate that spontaneous spindle bursts represent a physiological trigger for the release of bdnf, which plays an important role in several aspects of development (for review, see). Using in vitro and in vivo models it has been recently shown that an experimentally induced inflammation by application of the endotoxin lipopolysaccharide induces rapid (<2 h) alterations in the pattern of spontaneous spindle bursts and gamma oscillations in neonatal rodent cerebral cortex, which subsequently leads to increased apoptotic cell death . These inflammatory effects are specifically initiated by the microglia - derived proinflammatory cytokine tumor necrosis factor alpha and to a lesser extent by the chemokine macrophage inflammatory protein 2 . Thus, inflammation causes a fast dysfunction in the pattern of spontaneous burst activity, which subsequently leads to increased apoptotic cell death, most likely by disturbances in the release of survival factors such as brain - derived neurotrophic factor (bdnf) acting on neurotrophin tropomyosin - related kinase b / c receptor . Furthermore, removal of the subplate massively reduced spindle burst activity and led to a persistent loss of the typical barrel - like whisker representation within layer 4, indicating that spindle bursts play a role in the development of the neocortical architecture . In summary, these experimental data suggest that any disturbances in the spontaneous activity of the developing cerebral cortex (including spontaneous spindle bursts) induce acute dysfunctions, which may cause long - term disorders . However, it remains to be elucidated whether spindle burst in particular can be causally related to neurodevelopmental disturbances . It has been recently shown in extremely preterm infants that the properties of neocortical bursts recorded with eeg and their scaling relationships correlate significantly with later cognitive development . These clinical data suggest that analyses of burst shapes obtained in eeg recordings from preterm and full - term newborn babies may have diagnostic use in neonatal intensive care units and predict the clinical outcome . While it is generally accepted that early electrical activity shapes the maturation of neocortical circuits, it remains an open question whether the specific properties of spindle burst are required or fulfill a distinct role in development . In this regard, it is possible that the spatiotemporal patterns of spindle bursts translate into a local molecular signal which fulfills an important developmental role . In particular as gaba does not seem to be necessary for the generation or maintenance of spindle bursts, and since gaba is essential for neuronal differentiation [64, 65], it is tempting to speculate that spindle bursts control the spatially confined release of gaba in developing local networks . Another unresolved issue is that both spindle bursts and gamma oscillations can be observed during the same period of early development . They can be observed in early postnatal rodent brain from the day of birth (; also see) and occur spontaneously as well as after sensory stimulation . Moreover, both can be observed mainly during the critical periods of the primary sensory areas, although spindle bursts probably persist for slightly longer periods . In addition, spindle bursts and early gamma oscillations in newborn cortex are proposed to rely on thalamocortical inputs, in contrast to gamma oscillations in adult cortex which depend on perisomatic gabaergic inhibition . Beside their differences in duration, occurrence, and frequency (see above), spindle bursts and gamma oscillations in newborn cortex reveal a number of additional distinctions . In the immature barrel cortex the vast majority (> 90%) of spontaneously occurring spindle burst spans several barrel - related columns, whereas the majority (~70%) of spontaneous gamma oscillations are restricted to a single or two barrel related columns . In line with these observations, gamma oscillations evoked by tactile stimulation are also closely related to a single functional column, while evoked spindle bursts span over more than one column . Thus, one reason for the coexistence of spindle bursts and gamma oscillations during early postnatal development might be a distinct role in neocortical maturation . Gamma bursts may reflect local information processing mostly within a single functional column, thus supporting the maturation of a column related network . In contrast, spindle bursts probably reflect larger local network events and may thus serve to promote the connectivity between neighboring columns . Although spindle bursts as well as gamma oscillations are spatially confined to a small network in one neocortical area, it is unclear to what extent and how this activity connects to other cortical and subcortical regions (e.g., see [18, 19]). Further, it remains to be studied whether the immature brain shows a spindle burst related resting state and how this network state is altered by sensory activation or by pathophysiological events . Finally, it would be most interesting and important to correlate specific patterns of spontaneous activity (e.g., delta brush) recorded by means of full - band direct - current eeg in preterm und full - term human neonates with the acute functional state and with the further development of the child, as impressively done by vanhatalo and colleagues [62, 67]. Spatially confined spindle bursts and delta brushes represent the most prominent physiological activity patterns in the developing cerebral cortex of newborn rodents and preterm human infants, respectively . Spontaneous and stimulus - evoked spindle bursts can be observed in various neocortical areas of different mammalian species and play important roles in the early development of cortical networks . However, it remains to be studied in more detail how exactly spindle bursts influence the maturation of the cortex and how a potential long - term dysfunction due to disturbances in spindle burst activity can be prevented by early intervention . Since this important type of brain activity is already present in the human fetus in utero (either spontaneously occurring or related to sensory inputs from the uterine environment), a better understanding of the physiological relevance of spindle burst oscillations is of major clinical relevance.
Atrazine, 6-chloro - n2-ethyl - n4-isopropyl-1, 3, 5 atrazine, 4-diamine, is a selective herbicide that has been extensively used in corn production to control many broad - leaf and some grassy weeds . Atrazine has long - term reproductive and endocrine - disrupting effects and a probable human carcinogen . International agency for research on cancer (iarc) has concluded atrazine as a group 2b carcinogen . The maximum contaminant level (mcl) for atrazine in drinking water established by the usepa is 3.0 gl . Atrazine is moderately persistent in the environment and despite its low solubility, water resources contamination, it has become an international issue . The major dissipation route for atrazine is biodegradation, runoff, and leaching [4, 5]. The rate of biodegradation of atrazine is reduced due to the adsorption, and desorption, and its bioavailability is the rate - limiting step in biodegradation . Bioaugmentation is the addition of acclimated indigenous bacteria that can degrade the contaminant at accelerated rates . The bacteria are isolated from the heavily polluted soil in the laboratory through the enrichment process . The enrichment culture technique developed a mixed consortium of bacteria that are able to degrade the herbicide . A research was done by dehghani and colleague confirmed that atrazine biodegradation was enhanced in kavar corn field soil compared to the other soils that had not been exposed to the herbicide . Therefore, several applications of atrazine on soil resulted in an enhancement of atrazine degradation . The addition of nutrient causes an increase of microbial populations, thereby, increasing the number of indigenous microorganisms capable of degrading the pollutant . N - containing compounds such as atrazine have been shown to serve as sources of nitrogen . In theory, addition of high c / n should induce nitrogen limitation and increase selective pressure for utilization of recalcitrant n sources like s - triazines, which contain n that can be used by certain bacteria and microbial consortia . Cometabolic biotransformation can be enhanced by an increase in microbial activity which is stimulated by addition of organic matter . In the past few decades, intensive use of agricultural fertilization and herbicides has contributed to increasing concentration of n and herbicides . Mixed microbial consortia [8, 11] and strains have been isolated from soils and with a capability to complete the mineralization of the ring . The agrobacterium strain j14, rhodococcus erythropolis, pseudaminobacter, and nocardioides can use atrazine as sole carbon and nitrogen source [1214]. Laboratory experiments indicated that addition of carbon to soil inhibited atrazine biodegradation, but inorganic phosphate stimulates atrazine biodegradation . Atrazine degradation rate was increased by addition of carbon sources to pure and mixed bacterial culture . Ostrofsky and his coworkers (2001) found that cyanuric acid amendment increased atrazine mineralization by stimulation of general microbial population and activity . In liquid culture and in the presence of simple carbon sources, (1996) showed that the effect of n addition varies with the form and amount of added n . However, another study showed that the utilization of atrazine by pseudomonas adp in the presence of exogenous nitrogen (nh4, no3, urea and glycine) was not affected in a great amount by the presence of exogenous nitrogen . Degradation of atrazine (about 87%) by p. adp and a. radiobacter was unaffected by the presence of n source, whereas no degradation occurred with bacterium m91 - 3 in media containing urea or nh4-n . Another study reported that mineralization of atrazine by indigenous soil bacteria was inhibited by the addition of inorganic nitrogen . A study showed that addition of other organic amendments containing nitrogen suppressed atrazine . However, there are some limitations to the use of acclimated microbial cultures to degrade organic compound in a real field . Physiochemical conditions of soils, and competition with native microorganisms, may destroy or reduce the inoculums and limit its degradative capacity . Brandon and his coworkers (1997) showed that atrazine biodegradation was higher in liquid cultures than soil . He found that bacterial consortium in soil culture degraded 78% and 21% of atrazine at initial concentrations of 0.046 and 0.23 moles in 100 days . However, in liquid cultures, 90% and 56% of atrazine degraded in 80 days, respectively . In liquid culture, since fars is an agricultural province and enjoys the top rank in wheat and corn production in the country in recent years, atrazine has been widely used as a selective herbicide to control broad - leaf and grassy weeds in agricultural corn fields . The high incidence of atrazine to contaminate water resources and the increasing concern about the toxicological properties of atrazine has made researches directed toward bioremediation of atrazine in polluted sites . Therefore, the main objectives of this research are evaluating the effects of carbon and nitrogen sources on atrazine biodegradation using mixed bacterial consortium isolated from corn field soil located in south of shiraz (kavar) and determining the efficiency of atrazine biodegradation process in soil culture . Kavar corn field soil with a long history of atrazine application in fars province of iran has been explored for their potential of atrazine biodegradation . Soil samples for liquid cultures were taken from south of shiraz (kavar) corn field with a long history of atrazine application (more than 10 years) in fars province . Soil samples for soil cultures were also taken from a field in bajgah which has been under alfalfa cultivation for 3 years and has not received atrazine in the past 10 years . Disturbed soil samples were collected from 0 to 20 cm of soil depth with a hand - driven soil auger and stored at 4c until they were used . The soil samples were air - dried and passed through 2 mm sieve to be prepared for further microbiological examinations . Other soil characteristics such as soil solution ph and organic matter content were determined . The soil texture in kavar corn field was loam and the amount of sand, silt, and clay distribution were in order of 47.44%, 31.5% and 17.06% . The native soil characteristics at kavar site was fine loamy, mixed, thermic typic haploxerepts . At bajgah field, the soil texture was clay - loam and the amount of sand, silt and clay distribution were in order of 28.7%, 33.3% and 32.0% . The native soil characteristics at bajgah site was fine loamy, mixed, and thermic, typic calcixerepts . The soil samples were transported to the laboratory in zipped plastic bags and were kept frozen at 20c until they were ready for chemical analysis . The soil samples were thawed and air - dried at dark in room temperature and screened through a 2.0 mm sieve for maintaining homogeneity of soil in order to reduce the variability of adsorption data . 30 ml of dichloromethane was added to 10 g of the soil sample and shaken in a reciprocal shaker for 20 minutes . After filtration, the organic phase was transferred to a separating funnel and then atrazine was back extracted with 20 ml hcl (0.01 n). Afterwards, the liquid phase was collected and transferred to a 15 ml glass vial and stored in a refrigerator prior to electrochemistry analysis . Square wave voltammetry with the hanging mercury drop electrode (auto lab type analyzer equipped with metrohm va stand 663 and gpes 4.9 software) was used in this study to determine atrazine residual concentration in soil samples . Atrazine recovery percent from soil with this method of extraction was 98% . In order to isolate mixed bacterial consortiums capable of growth on atrazine as a carbon source, the selective enrichment culture and basal salt medium were prepared as described in rousseaus, . Ten grams of wet soil was inoculated into 90 ml of atrazine medium and supplemented with sodium citrate and delvocid (25 mg l) after autoclaving . Delvocid was used to prevent the growth of fungi and ph was also adjusted to 7.5 . Cultures were incubated aerobically on a reciprocal shaker (150 rpm) at room temperature in dark to preclude photolysis reactions . All enrichment cultures were subcultured on the same medium at one - week interval . From a one - week - old culture, 10 ml after culture was subculture for 30 and 300 days under conditions of nitrogen limitation, the remained atrazine after inoculation of the media for 10 days was quantified by electrochemistry . The bacterial consortium was harvested by centrifugation (6000 g at 40c for 20 minutes) washed twice with 0.1 ml phosphate buffer (ph = 7.3). Different carbon compounds such as glucose (g), sodium citrate (sc), sucrose (su), starch (st) with three replications each at a concentration of 2 g l, and also the combination of these mentioned carbon sources such as g + sc, st + su, su + sc, st + sc, su + g, and st + g each at a total concentration of 2 after inoculation of the media supported with carbon sources, they were incubated at room temperature and placed in dark for 10 days . Control and blank without bacteria inoculation and no carbon sources (ncs) were also used for this study . After 10 days, the remained atrazine was measured . To measure the influence of nitrogen sources on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture, nitrogen sources as routine fertilizers were added to atrazine mineral salt broth containing sucrose and sodium citrate . Ammonium nitrate and urea as fertilizers were applied to corn field at a concentration of 600825 and 200400 kg ha, the nitrogen percent for these fertilizers were 34% and 46%, respectively . Urea was added to atrazine minimal salt media at a concentration of 138690 mg after inoculation of the media supported with nitrogen sources, they were incubated at room temperature in dark for 10 days . A laboratory experiment was arranged to measure the influence of ph on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture . A different ph from 5.5 to 8.5 with three replications was used in atrazine mineral salt broth containing sucrose and sodium citrate and no nitrogen sources . Atrazine degradation rate by the bacterial consortium was measured in 100 ml capped erlenmeyer flask containing soil samples . Ten grams of soil sample was brought to the desired soil moisture (7% and 25%) by the addition of sterile deionized water . The experimental design consisted of 36 flasks with 12 treatment and three replications for each treatment . The initial atrazine concentrations of 1.3 and 6.7 mg g soil are corresponded to 0.5 and 2.5 kg ha, respectively . However, atrazine concentration of 20 mg g soil is related to 7.5 kg ha(4.43 kg a. i. ha) which is considered a relatively high atrazine concentration and might have occurred due to an accidental spillage . After one day of incubation to allow herbicide sorption to the soil, inoculation with 300 l phosphate buffer containing the selected bacterial consortium was added to soil to yield 7.5 10 bacterial cell g soils as determined by plating on soil extract agar . The soil samples were mixed until they were homogeneously wet and then incubated at room temperature in dark until the end of the experiment . Soil moisture was maintained constant through the incubation by weighing and correcting for any weight loss by adding sterile deionized water . To maintaining high population of atrazine degraders, the inoculation with the consortium was made every 5 days over a 30-day period for a total of 6 inoculations . Soil samples at the initial atrazine concentration of 6.7 mg g soil were extracted at the incubation time of 2, 10, 20, and 30-day and analyzed to determine the remained atrazine concentration at each time interval . By using a mixed bacterial consortium with a high capability of atrazine degradation isolated from kavar corn field soil, the effect of different carbon sources on atrazine biodegradation was studied (figure 1). According to this figure, the percentage of atrazine biodegradation rate for different carbon sources was in the range of 9.47% to 87.72% . A blank sample did not adequately support the growth of consortium bacteria (due to low turbidity), and the rate of atrazine degradation is lass than 3% . Linear regression showed that there was a significant difference between different carbon sources and atrazine biodegradation rate (p <0.001). The effect of different nitrogen sources on atrazine biodegradation was shown in tables 1 and 2 . According to table 1, the percent of atrazine biodegradation rate decreased from 87.72% to 29.58% as the ammonium nitrate concentration increased from 0.0 to 900 kg ha soil . The rate of atrazine biodegradation for ammonium nitrate decreased quickly when the concentration of ammonium nitrate increased from zero to 600 kg ha soil, after that the reduction rate of atrazine degradation was getting slower . The same trend has been observed for atrazine biodegradation in the presence of urea . According to table 2, the percentage of atrazine biodegradation rate decreased from 87.72% to 26.76% as the urea concentration increased from 0.0 to 500 kg ha soil . For urea, atrazine biodegradation rate under nitrogen amendment showed an initial sharp decreasing slope and then reaching constant with relative slower rate . Variations of ph on atrazine degradation rate were examined when sucrose and sodium citrate were used as a carbon source, while there was no nitrogen source available (figure 2). According to regression analysis, it can be concluded that there was a significant difference between ph and atrazine biodegradation (p <0.05). Table 3 showed the effect of initial atrazine concentration and soil moisture on the biodegradation of the inoculated soil . According to data on this table, after 30 days of incubations atrazine degradation rate for noninoculated soil samples was low and atrazine reductions were only 7% to 19% . However, for inoculated soil samples, degradation rate was higher and its reductions were 19.572% . The inoculated soil samples at 25% soil moisture at initial concentration of 1.3, 6.7, and 20 mg g soil, atrazine reductions were 69.5%, 60.5% and 30.5% within 30 days of incubation, respectively . Figure 3 showed that atrazine biodegradation rate during the different time intervals in soil culture . Figure 4 depicted the plot of the semilogarithm of initialized atrazine concentration (c / c0) versus time . During the 30 days of incubation period, atrazine concentration decreased from an initial concentration of 6.7 mg gsoil to a final concentration of 2.67 mg gsoil . Only a 5% decrease in atrazine concentration however, figure 3 showed that at 20 days 40% of the atrazine had degraded . After that a very slow increase in atrazine degradation was observed and remained constant until the end of the incubation period . Assuming a pseudo - first - order reaction for the disappearance of atrazine, a plot of the natural logarithm of initialized atrazine concentration (c / c0) versus time resulted in a rate constant equal to 0.0328 d (figure 4). The t1/2 for atrazine calculated from the plot was 21.13 d. the initial slow degradation rate of atrazine was followed by a much faster degradation rate that lasted about 5 day and then the degradation rate became slower and finally remained constant until the end of incubation period . Carbon sources of sodium citrate and sucrose had the highest atrazine biodegradation rate . According to data, mandelbaum and his coworkers (1993a) used sodium citrate and sucrose as the carbon source . According to data in this research, the percentage of atrazine biodegradation rate with no carbon sources was only 5.5% . Past studies showed that carbon in atrazine ring cannot be used by many bacteria as energy sources; however, carbon in alkylated group provided carbon for bacterial growth . The low amount of atrazine cannot support bacterial growth, and therefore, a supplemental carbon was needed . According to data on tables 1 and 2, atrazine utilization is repressed under nitrogen - sufficient growth condition and activated under nitrogen limitation . Many investigations also showed a negative effect of nitrogen amendment on atrazine biodegradation by indigenous populations in soils [34, 35]. However, other study reported that organic nitrogen supplied in dairy manure increased atrazine mineralization . Data regarding the effect of ph shows that as ph increased from 5.0 to 7.0, the rate of atrazine biodegradation increased . However, as ph increased from 7.0 to 8.5 this caused a reduction rate in atrazine biodegradation . Atrazine biodegradation rates were significantly enhanced in the inoculated soils as compared to uninoculated control soils . After 30 days, the percent of atrazine reduction was only 12% for uninoculated soils at initial atrazine concentration of 6.7 mg g soil and the soil moisture of 25% . However, soil that was inoculated every 5 days with the bacterial consortium had reduced atrazine up to 60.5% at the same initial atrazine concentration and the soil moisture by day 30 (table 3). As initial atrazine concentration increased from 1.30 to 20 mg g soil, the percentage of atrazine reduction decreased from 69.5% to 30.5% (inoculated soils and soil moisture 25%). The decrease in atrazine reduction at 20 g g soil was possibly due to the result of complex interaction between microbial activity and nutrient availability . Therefore, unbalanced nutrient supply and not atrazine toxicity was probably responsible for the decrease . Figure 3 showed that atrazine degradation rate increased as time passed . According to (table 3), enhanced atrazine biodegradation rates occurred with an increase in soil moisture from 7% to 25% . The percent of atrazine reduction increased from 32.5% to 69.5% as the soil moisture increased from 7% to 25% (at initial atrazine concentration of 1.30 mg g soil). Many researchers found that higher atrazine biodegradation rate occurred with an increase in soil moisture . Soil moisture influences microbial processes through direct effect (e.g., water availability) or indirect effects, for example, solute diffusion, chemical availability, and aeration . Therefore, the positive effect of increasing soil moisture was probably due to increased microbial mobility, solute diffusion, and chemical availability which all had an indirect effect on atrazine degradation . In this study, more than 60% of atrazine was degraded in 30 days of incubation periods (figure 3). Atrazine degradation exhibited a half - life of approximately 21.13 d. in conclusion, our results confirmed that atrazine biodegradation was higher in liquid cultures than soil . The mixed bacterial consortium in soil culture degraded 69.5% of atrazine at initial concentrations of 1.3 mg g soil in 30 days . However, in liquid cultures, 87.72% of all atrazine degraded in 10 days . Results of this study suggested that atrazine bioremediation in soil utilizing atrazine - degrading bacterial consortium could be accomplished across a wide range of atrazine concentration if the soil moistures swere enough and also nutrient availability was balanced, too . The bacterial consortium with the ability to degrade atrazine provided a good opportunity for increasing the degradation rate of this herbicide through bioaugmentation . It is very important to note that these bacteria in the controlled laboratory conditions (with the addition of carbon and nitrogen sources and without the interaction of environmental factors) had been able to use atrazine . However, there are some limitations to the use of acclimated microbial cultures to degrade the herbicide in a real field . Therefore, it is highly recommended that the feasibility of atrazine degradation to be studied by the bioaugmentation of mixed bacterial isolates to the real contaminated soil . The mixed bacterial consortium successful in laboratory studies may fail in the real field because of their sensitivity to high concentrations of other compounds.
Neurons, oligodendrocytes (ols), and astrocytes are unique cell types in the central nervous system (cns) of vertebrates.1 neurons process and transmit information electrochemically.2 ols generate myelin sheaths around most axons of the vertebrate cns, enabling a faster conduction of the nerve impulses . Astrocytes provide support and nutrients for the nerve tissue.3 the different cell types in the nervous system are regulated by the precise spatiotemporal expression of specific genes.4,5 transcriptional regulation plays an important role in the determination of neural cell differentiation.6,7 olig genes belong to the basic helix loop helix transcription factor family, which encode ol lineage transcription factors 1, 2, and 3 (olig1, olig2, and olig3). With the exception of olig3, olig genes are specifically expressed in the cns, and play a critical role in cns development by controlling differentiation and maturation of ols, motor neurons (mns), and astrocytes.8,9 olig2 null mice die at birth from a lack of mns.10 both gain- and loss - of - function studies were performed in an olig1 null mouse with normal myelin during development, but which were unable to remyelinate on experimental challenge.11 a second olig1 null mouse with less compensatory effect by olig2 had a more severe phenotype and died around postnatal day 14 from a complete lack of myelin . This mutant had mature ols, but failed to wrap myelin or even deposit lipid around axons.12 knocking out olig1 and olig2 individually or together affected differentiation and maturation of ols, suggesting functional overlap in the cns.10,13,14 until now, the role of olig2 during development of spinal cord attracted more attention . However, studies investigating the expression and function of olig1 in development and disease are limited . Although it is widely known that olig1 promotes the differentiation and maturation of ols, it is unclear how these occur during development . Exploring temporal and spatial expression and distribution of olig1 will contribute to our understanding of the role of olig1 in specialization of neural cells during development . Therefore, in this study, we determined the expression pattern of olig1 in neural cells during rat spinal cord development . Sprague dawley rats were obtained from the laboratory animal center, bengbu medical college (bengbu, people s republic of china). All experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . To produce embryonic and newborn rats, one female was cohabited with two males, and gestational age (embryo, e) was designated as day 0.5 (when vaginal plugs in female rat were observed). Eighty rats were randomized to eight groups and subgroups: embryonic day 14.5 (e14.5) (n=10), e18.5 (n=10), postnatal day 0 (p0) (n=10), p3 (n=10), p7 (n=10), postnatal 2 weeks (p2w) (n=10), p4w (n=10), and adults (n=10). Each group was randomized into two subgroups equally . In the first subgroup (n=5), the spinal cords were immunohistochemically stained, and in the second subgroup (n=5), the spinal cords were subjected to western blot . Spinal cords from embryos (e14.5 and e18.5) were dissected following cervical dislocation of the pregnant rats . Spinal cords were dissected from postnatal rats (p0, p3, p7, p2w, p4w, and adults) and perfused intracardially with phosphate - buffered saline (pbs), followed by 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). For immunohistochemistry, spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution (sigma, st louis, mo, usa) at 4c for another 57 days . Using a cryostat, transverse 15 m thick sections were obtained, mounted onto gelatin - coated slides (fisher scientific, fairlawn, nj, usa), and stored at 80c until use . For western blot, spinal cord tissues were stored immediately at 80c until use . For immunohistochemistry staining, the frozen slides were air - dried at room temperature (rt) for 10 minutes and rinsed with pbs for 10 minutes . They were blocked with 10% donkey serum in tris - buffered saline (tbs) containing 0.3% triton x-100 (tbst) for 1 hour at rt . The following primary antibodies were used: monoclonal mouse anti - olig1 (igg, 1:100, millipore, billerica, ma, usa), monoclonal rabbit anti - myelin basic protein (anti - mbp; 1:300, millipore), rabbit anti - glial fibrillary acidic protein (anti - gfap; 1:100, millipore), monoclonal mouse anti - o4 (igm, 1:100, millipore), and rabbit anti--tubulin (1:100, millipore). The next day, the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm, igg or goat anti - rabbit igm, igg (all 1:200; jackson immunoresearch lab, west grove, pa, usa) for 1 hour at 37c . The slides were washed three times with pbs and coverslipped with gel aqueous mounting media (biomeda, foster city, ca, usa) containing hoechst 33342 (sigma - aldrich) to counterstain the nuclei . At least five randomly selected fields with a total of more than 500 cells were counted . In all experiments, western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages . Briefly, tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride (pmsf) (10 mg / ml) and thoroughly homogenized for 5 minutes on ice, and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant . The protein concentrations of the supernatant were determined using a bca protein assay kit (pierce, rockford, il, usa). The protein concentration was determined using the bio - rad dc protein assay (every 10 l sample contain 120 g protein). Protein samples containing an equal amount of protein (20 g) were electrophoresed on sodium dodecyl sulfate (sds)-polyacrylamide gels, and transferred to polyvinylidene difluoride filters (millipore). The filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies (in tbst-5% bovine serum albumin [bsa]) including olig1 (1:200, millipore) and -actin (1:400, sigma) as markers of differentiated neural cells . After rinsing with tbst, the membranes were incubated with the appropriate horseradish peroxidase (hrp)-conjugated secondary antibody (all from kpl, gaithersburg, md, usa) for 1 hour at rt . To visualize the immunoreactive proteins, the enhanced chemiluminescence (ecl) kit (pierce, rockford, il, usa) was used, following the manufacturer s instructions . Films were digitized, and densitometry was performed using gel - pro analyzer (media cybernetics, silver spring, md, usa). The intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin . The data were analyzed using spss16.0 (ibm, armonk, ny, usa) software . The data with three or more groups were analyzed by one - way analysis of variance (anova) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups . Sprague dawley rats were obtained from the laboratory animal center, bengbu medical college (bengbu, people s republic of china). All experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . To produce embryonic and newborn rats, one female was cohabited with two males, and gestational age (embryo, e) was designated as day 0.5 (when vaginal plugs in female rat were observed). Eighty rats were randomized to eight groups and subgroups: embryonic day 14.5 (e14.5) (n=10), e18.5 (n=10), postnatal day 0 (p0) (n=10), p3 (n=10), p7 (n=10), postnatal 2 weeks (p2w) (n=10), p4w (n=10), and adults (n=10). Each group was randomized into two subgroups equally . In the first subgroup (n=5), the spinal cords were immunohistochemically stained, and in the second subgroup (n=5), the spinal cords were subjected to western blot . Spinal cords from embryos (e14.5 and e18.5) were dissected following cervical dislocation of the pregnant rats . Spinal cords were dissected from postnatal rats (p0, p3, p7, p2w, p4w, and adults) and perfused intracardially with phosphate - buffered saline (pbs), followed by 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). For immunohistochemistry, spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution (sigma, st louis, mo, usa) at 4c for another 57 days . Using a cryostat, transverse 15 m thick sections were obtained, mounted onto gelatin - coated slides (fisher scientific, fairlawn, nj, usa), and stored at 80c until use . For western blot, spinal cord tissues were stored immediately at 80c until use . For immunohistochemistry staining, the frozen slides were air - dried at room temperature (rt) for 10 minutes and rinsed with pbs for 10 minutes . They were blocked with 10% donkey serum in tris - buffered saline (tbs) containing 0.3% triton x-100 (tbst) for 1 hour at rt . The following primary antibodies were used: monoclonal mouse anti - olig1 (igg, 1:100, millipore, billerica, ma, usa), monoclonal rabbit anti - myelin basic protein (anti - mbp; 1:300, millipore), rabbit anti - glial fibrillary acidic protein (anti - gfap; 1:100, millipore), monoclonal mouse anti - o4 (igm, 1:100, millipore), and rabbit anti--tubulin (1:100, millipore). The next day, the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm, igg or goat anti - rabbit igm, igg (all 1:200; jackson immunoresearch lab, west grove, pa, usa) for 1 hour at 37c . The slides were washed three times with pbs and coverslipped with gel aqueous mounting media (biomeda, foster city, ca, usa) containing hoechst 33342 (sigma - aldrich) to counterstain the nuclei . At least five randomly selected fields with a total of more than 500 cells were counted . In all experiments, western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages . Briefly, tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride (pmsf) (10 mg / ml) and thoroughly homogenized for 5 minutes on ice, and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant . The protein concentrations of the supernatant were determined using a bca protein assay kit (pierce, rockford, il, usa). The protein concentration was determined using the bio - rad dc protein assay (every 10 l sample contain 120 g protein). Protein samples containing an equal amount of protein (20 g) were electrophoresed on sodium dodecyl sulfate (sds)-polyacrylamide gels, and transferred to polyvinylidene difluoride filters (millipore). The filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies (in tbst-5% bovine serum albumin [bsa]) including olig1 (1:200, millipore) and -actin (1:400, sigma) as markers of differentiated neural cells . After rinsing with tbst, the membranes were incubated with the appropriate horseradish peroxidase (hrp)-conjugated secondary antibody (all from kpl, gaithersburg, md, usa) for 1 hour at rt . To visualize the immunoreactive proteins, the enhanced chemiluminescence (ecl) kit (pierce, rockford, il, usa) was used, following the manufacturer s instructions . Films were digitized, and densitometry was performed using gel - pro analyzer (media cybernetics, silver spring, md, usa). The intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin . The data were analyzed using spss16.0 (ibm, armonk, ny, usa) software . The data with three or more groups were analyzed by one - way analysis of variance (anova) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups . Western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood . Our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points . However, interestingly, the expression of olig1 increased again from p2w to a high level until adulthood (figure 1). To determine the expression and localization of olig1 in different types of neural cells, we performed double immunostaining using the antibody against olig1 and another antibody against o4, mbp, -tubulin, and gfap, respectively . Our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells (opcs; figure 2) and -tubulin - positive neurons (figure 3) at all time points . Furthermore, we failed to observe expression of mbp in the spinal cord during the embryonic period (data not shown). Mbp expression was not observed prior to p3, and no cellular coexpression of mbp and olig1 was observed (figure 4). The coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood . We also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development . Western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood . Our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points . However, interestingly, the expression of olig1 increased again from p2w to a high level until adulthood (figure 1). To determine the expression and localization of olig1 in different types of neural cells, we performed double immunostaining using the antibody against olig1 and another antibody against o4, mbp, -tubulin, and gfap, respectively . Our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells (opcs; figure 2) and -tubulin - positive neurons (figure 3) at all time points . Furthermore, we failed to observe expression of mbp in the spinal cord during the embryonic period (data not shown). Mbp expression was not observed prior to p3, and no cellular coexpression of mbp and olig1 was observed (figure 4). The coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood . We also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development . The cns originates in the neuroepithelial stem cells (nscs), which ultimately generate the three major cell types: neurons, ols, and astrocytes.1 in this process, transcriptional regulation plays an important role.6,7 in mice, genetic studies revealed that olig transcription factors play key roles in the genesis of mns and ols . Gain- and loss - of - function analysis confirmed the fundamental role of olig1 in the survival and maturation of opcs.12,1517 regulation of olig1 gene expression in spinal cord of demyelinating disease promotes myelin repair after spinal cord injury . However, the distribution and expression of olig1 in neural cells during development is unclear . In this study, we systematically investigated the expression pattern of olig1 in neural cells during rat spinal cord development . First, using the western blot, we demonstrated the differential expression of olig1 in rat spinal cord development . Olig1 expression increased and peaked at e18.5, which may contribute to the differentiation of a large number of neural cells into opcs and mn precursors at this stage . Recently, it has been reported that olig1 protein expression was significantly decreased during mouse ol progenitor differentiation.18 however, the expression of olig1 increased again from p2w, which was consistent with a recent report that olig1 was expressed in human ols during process outgrowth and maintained at a high level prior to myelination.19 next, we identified the expression of olig1 in neural cells at several time points during spinal cord development . We confirmed that olig1 was expressed not only in opcs but also in neurons from e14.5 to adulthood . Cns development starts from nscs, which generate the three major cell types (neurons, ols, and astrocytes) of the cns.20,21 however, the intermediate steps in the differentiation of nscs in vivo have not been clarified . In the embryonic ventral spinal cord, mns and ols are derived from a common progenitor pool, suggesting that these lineages share intrinsic and extrinsic regulatory mechanisms.10,13,21,22 ol progenitors were more closely aligned with neuronal subtype progenitors than with astrocytes, at least in the developing cns.23 a study demonstrated that olig2 was more likely an early - stage factor for ol precursor cells, while olig1 played a critical role in late - stage ol maturation and myelin formation.19 however, jakovcevski and zecevic24 found olig1 protein expressed in primitive neuroepithelia that can give rise to interneurons in the human fetal brain . A recent study suggests that olig1 regulates neuron - glial fate choice in the embryonic telencephalon.25 in conclusion, olig family members do not act independently but interact with other transcription factors to tightly control cns development.17,26 our findings raise the possibility that olig1 has some function in the formation of mns during cns development . Mbp is a major myelin protein, which is expressed in mature ols.27,28 mbp is the major protein component of myelin at 2 weeks after birth in the rat brain, expressed a week later than in spinal cord.28 in this study, we observed that mbp expression commenced from postnatal period, and olig1 was not coexpressed with mbp during rat spinal cord development . Opcs undergo two types of differentiation, resulting in ols in the absence of serum or type ii astrocytes in the presence of serum in vitro.2931 fate - mapping studies have shown that opcs generate a subset of astrocytes during perinatal development.32 most neurogenic bhlh transcription factors, for example, mammalian achaete - scute homologue 1, mammalian atonal homologue, and neurogenins 1 and 2, are only expressed transiently in progenitor cells as needed.23 in this study, we observed the transient expression of olig1 in gfap - positive cells at e14.5, suggesting that ols and astrocytes may derive from the same progenitor cell . Both olig1 and olig2 occur in the nucleus of ols and their progenitors occur on p1 in mouse brain.33 olig2 continues to be nuclear at all developmental stages, whereas olig1 is almost completely cytoplasmic in the adult mouse brain . Changes in nuclear or cytoplasmic olig1 location correspond with altered morphology of opcs.11 recently, othman et al19 reported that o1-positive multipolar opcs with nuclear expression of olig1 exhibited fewer processes, while opcs with cytoplasmic olig1 manifested more extensive membrane expansion . Nuclear olig1 facilitated mbp expression, but with greatly diminished membrane expansion.19 however, these experiments were performed in mouse and human . Our results demonstrated that olig1 was expressed in o4-positive opcs and -tubulin - positive neurons, but was localized in the cytoplasm during the period from e14.5 to adulthood . We speculate that the cause for this inconsistency may be attributed to differences between animal species or other unknown factors . In conclusion, we systematically explored the expression pattern of olig1 in neural cells during rat spinal cord development . Our results showed that olig1 was coexpressed with o4-positive opcs and -tubulin - positive neurons at all time points during development . Olig1 was localized in the cytoplasm of o4- or -tubulin - positive cells during the transition from e14.5 to adult . Our results contribute to understanding the mechanism of developmental regulation of neural cells by olig1.
Neck pain is a common musculoskeletal disorder in modern society that can produce severe pain . 67% of the population suffers from it at least once in a lifetime and the prevalence is about 23%1 . The pain exacerbates and fades periodically, and many patients do not fully recover from the symptoms2 . Neck pain occurs in the upper thoracic spine area including the shoulder, and it is mechanical pain caused by bad postures and habits in most cases3 . According to sharon, the upper thoracic spine is involved in the physiologic motion of the neck4 . The decreased movement of the upper cervical spine can cause excessive movement of the lower cervical spine, increase fatigue in the sternocleidomastoid, anterior scalenus, and upper trapezius, cause changes of neck postures and breathing patterns, and a decrease in the range of motion5 . Patients with chronic neck pain experience functional impairments including weakening of deep bending neck muscles due to the activation of neck surface muscles6, increased deformity of the forward head posture7, proprioception impairment8, and poor balance9 . In addition, decreased movement of the cervical spine restricts the range of motion of the spine and decreases breathing function10, 11 . Slightly bent positions like the forward head posture may cause mechanical neck pain, cause a greater load by affecting the mobilization order of the muscles operating when the arm is raised, and restrict the range of motion12 . These changes reduce the ability to maintain balance and increase the risk of falls and injury of the musculoskeletal system9 . Physiotherapists are using methods such as electrotherapy, therapeutic exercise, and manual therapy to intervene in the neck pain13 . In a cochrane review study of neck pain, stretching exercises for the neck and upper limbs, strengthening exercises, static and dynamic stabilization exercises maitland mobilization grade 3 and 4 treatment for the cervical spine and upper spine significantly decreases neck disability index (ndi), the pain index15, and increases the range of motion16 . In addition, an the intervention combining therapeutic exercise and manual therapy was significantly more effective compared to manual therapy alone17 . Recent evidence - based studies report that treatment combining therapeutic exercise and manual therapy is more effective18, but research of combined interventions is sparse . The purpose of this study was to apply joint mobilization and therapeutic exercise to the cervical spine and upper spine, investigate the effects on functional impairments caused by the neck pain, and examine differences between groups by comparing the intervention group with the group to which only therapeutic exercises were applied . This study was implemented with non - specific neck pain patients with no medical findings who had visited the cheongju st . All participants received verbal and written information about the study and signed a consent form . The patients who had undergone or would have surgery in the spine, and those who had neurological damage, a cervical spine fracture, osteoporosis, arthritis, a malignant neoplasm, a vascular disease, or a psychiatric problem were excluded . The subjects were randomly assigned to one of two groups of nine people each . Group i was the therapeutic exercise group, and group ii was the group to which joint mobilization was applied in combination with therapeutic exercise (table 1table 1.general characteristics of the subjects (n=18)group igroup iigender (female)99age (years)58.0 1.659.0 2.4height (cm)158.0 3.9157.0 4.7weight (kg)58.0 4.659.0 6.6values are expressed as mean sd . * significant difference between pre - post test (p<0.05). Group i (therapeautic exercise), group ii (mobilization & therapeautic exercise)). The pretest included the visual analog scale (vas), neck disability index (ndi), active cervical range of motion (acrom), static balance ability, muscle tone of the upper trapezius, and respiratory function . After the pretest, group i performed therapeutic exercise and group ii performed both therapeutic exercise and joint mobilization for 60 minutes a day, three times a week for two weeks under the guidance of a physical therapist . The participants did not receive any other interventions associated with the neck pain while this research was being conducted . The post - test was carried out with the same protocol as the pretest after two weeks . Values are expressed as mean sd . * significant difference between pre - post test (p<0.05). Group i (therapeautic exercise), group ii (mobilization & therapeautic exercise) therapeutic exercises involved enhancing mobility, stability and muscular strength of the neck, improving proprioception, and performing reeducation of movement, and the intensity of exercises was adjusted according to the physical abilities of the individuals . For the joint mobilization, after the painful sites were located by examination, the active movement test was conducted to find where joint mobilization would be applied, and the amount and quality of motion were examined . Afterwards, passive physiological intervertebral movement (ppivm) testing and passive intervertebral accessory movement (paivm) test were conducted to find which joints had restricted or excessive movement, and determine where joint mobilization would be applied . The vas was employed to measure the intensity of pain19, the ndi was used to measure neck pain disability20, and crom instrument to assess the cervical range of motion21 . In order to check static balance, myoton pro (myoton as, estonia) measured the muscle tone of the upper trapezius22, and a spirometer (pony fx spirometer; cosmed, rome, italy) measured the respiratory function23 ., an ibm company, chicago, il, usa). For the normality test, the shapiro wilk test was conducted, confirming that the data are normally distributed . Assuming homogeneity between groups, the independent samples t - test was performed, and the paired sample t - test was conducted for the within - group comparison of the measurements before and after the interventions . In addition, the independent samples t - test was conducted to compare the differences between groups . Null hypotheses of no difference were rejected if p - values were less than 0.05 . Vas and ndi led to significant changes in both groups, and group ii improved significantly more than group i (tables 2table 2.visual analog scale of the subjectsgroup igroup iipre4.8 0.44.9 0.3post2.7 0.5 * 1.4 05*significant difference between pre - post test (p<0.05). Significant difference between groups (p<0.05), 3table 3.neck disability index of the subjectsgroup igroup iipre15.1 2.317.2 3.1post8.9 1.5 8.6 1.9*significant difference between pre - post test (p<0.05). The acrom increased significantly in both groups, and group ii improved significantly more on the right lateral flexion and rightward rotation than group i (table 4table 4.active cervical range of motion of the subjects ()group igroup iiflexionpre49.2 8.748.9 4.7post54.2 4.5 56.1 6.8extensionpre54.9 4.356.4 4.9post 64.3 3.2 * 67.6 3.9rt . Rotationpre60.8 3.259.7 5.1post 67.6 3.6 67.7 4.2*significant difference between pre - post test (p<0.05). Both groups improved significantly in static balance, with no difference between groups (tables 5table 5.center of gravity sway velocities of each group under different conditions (/s)group igroup iifirm - eyes openpre0.04 0.00.05 0.0 post0.03 0.00.04 0.0firm - eyes closedpre0.15 0.00.15 0.0post0.14 0.00.14 0.10.37 0.1post 0.30 0.1 0.32 0.1*significant difference between pre - post test (p<0.05). Significant difference between groups (p<0.05), 6table 6.center of gravity total sway distances of each group under different conditions (/s)group igroup iifirm - eyes openpre294.6 64.1304.6 97.9 post270.4 66.4281.9 109.2foam - eyes closedpre618.2 82.7687.3 182.8post 529.1 88.2 548.4 172.5*significant difference between pre - post test (p<0.05)significant difference between groups (p<0.05)). Muscle tone in the left and right upper trapezius muscle improved significantly in both groups, and there was no difference between groups (table 7table 7.muscle tone of the subjectsgroup igroup iirt . Upper trapeziuspre16.0 1.5 16.0 1.2post13.7 1.3 14.2 1.4*significant difference between pre - post test (p<0.05). Significant difference between groups (p<0.05) significant difference between pre - post test (p<0.05). Significant difference between groups (p<0.05) * significant difference between pre - post test (p<0.05). Significant difference between groups (p<0.05) * significant difference between pre - post test (p<0.05). Significant difference between groups (p<0.05) * significant difference between pre - post test (p<0.05) significant difference between groups (p<0.05) * significant difference between pre - post test (p<0.05). Chronic neck pain patients suffer diverse functional impairments, including a deterioration of balance ability, respiratory function, range of joint motion, and proprioception11 . Research related to neck pain suggests that there are more efficient interventions based on evidence through systematic reviews, and the interventions combining manual therapy and therapeutic exercise are described as more effective in recent literature18 . In this study, therapeutic exercises were conducted that are reported to be effective for patients with mechanical neck pain, namely, stretching exercise for the neck and upper limbs, static and dynamic stability exercise, strengthening exercise, and exercise reeducation14 . In addition, as manual therapy, mobilization to reduce pain and improve the movements of the soft tissue exhibiting contracture and restricted motions of joints (maitland grade iii and iv)13, 24 were applied to group ii . The effect of the two kinds of interventions, and group differences based on those interventions were examined . In this research, a significant reduction in the vas and ndi was observed in both groups, and the comparison between the groups showed that the intervention was more effective in group ii . This result is different from studies that found no difference between groups based on a short - term intervention17 . These differences may result from our smaller number of subjects and differences of individual characteristics . For the acrom, both groups improved significantly overall, and group ii improved more on right lateral flexion and rightward rotation . These results are consistent with studies that reported that mobilization applied to both the neck and spine at the same time improved cranial vertical angle and cranial rotation angle25 and research that showed that endurance exercise of the neck, stabilization exercise, and strength training improved the joint range of motion26 . In this study, static balance was measured under a variety of conditions, including hard or soft bearing surfaces and open or closed eyes . A significant decrease in the sway velocity of the center of gravity and in the sway distance occurred only under eyes closed, and there was no difference between groups . Although it was reported that patients with neck pain and forward head posture may exhibit reduced balance ability9, it was possible to measure the static balance ability most accurately when the soft bearing surface was used and eyes were closed to block the visual feedback . Muscle tone of the upper trapezius decreased significantly in both groups, and there was no difference between the groups . This may indicate that the tension and fatigue of the upper trapezius were reduced due to pain reduction, enhancement of mobility of the neck joints, and change in the mobilization sequence of movements caused by afferent information activation of proprioception5 . Deterioration of mobility of the spine and thoracic cage was reported in patients of chronic neck pain, and respiratory function was also affected27 . However, in this research, no significant change in the respiratory function was observed . This may be because the patients were not given direct training for functional improvement of breathing muscles, although the range of motion of the cervical and upper thoracic spine was improved through the study interventions . The number of subjects was small, and since the period of intervention was not long, it was difficult to produce a mechanical change of muscles . Therefore, future research is required to investigate the effect of joint mobilization and therapeutic exercise on the functional impairments caused by chronic neck pain using diverse subjects and intervention periods, and research on the persistence of the effect also needs to be conducted . In conclusion, joint mobilization and therapeutic exercise for functional impairments caused by chronic neck pain significantly improved several types of functional impairment . In addition, in the group to which both joint mobilization and therapeutic exercise were applied, significantly more improvement in the pain index, neck disability levels, and acrom was seen than in the group that received only therapeutic exercise.
Data on the adult (over 20 years) population has been reported elsewhere (5). The present analysis focused on albertan youth <20 years of age . Flagged in the databases and were defined as any alberta inhabitant registered under the federal indian act and entitled to treaty status with the canadian government . The status aboriginal identifier captures first nations and inuit peoples both on- and off - reserve with treaty status, but not mtis individuals or aboriginal individuals without treaty status who are included in the general population comparison group . Alberta law requires that all residents and dependants register with the alberta health care insurance plan, thus the alberta central stakeholder registry functioned as a denominator . Diabetes cases were identified by applying the national diabetes surveillance system (ndss) algorithm (13), which has recently been validated for the determination of pediatric diabetes rates (14). The ndss methodology of identifying has been validated in non - aboriginal populations that include persons of varying geographical locations (15,16), and also within an aboriginal community in alberta (17). The algorithm requires an individual to have either 2 physician visits or 1 hospitalization for diabetes (icd-9 codes starting with 250 and icd-10 codes e10e14) within 2 years to be labelled as case of diabetes (13). In contrast to the ndss case definition, pregnant women that may have had gestational diabetes were not excluded in the current analysis due to the elevated risk of subsequent diabetes (18). Also, since the numbers of diabetes cases are small among this population, only crude, unadjusted rates are provided . For both populations, the denominators were based on the health insurance registry (which provincial law requires all residents and dependants to register) for the entire population as of june 30 in each year . The first year in which an individual met the criteria for diabetes (with no diabetes claims in the preceding 2 years) was considered an incident year, and considered a prevalent case in subsequent years . Prevalence was determined using the following group - specific formula: total number of youth with diabetes in the calendar year / the total population for the calendar year . Incidence was determined using the following formula: total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases). Odds ratios (or) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . Or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . To examine trends over time, average annual percent changes (aapc) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 (rockville, md) for crude values . The aapc provides a relative summary measure of the trend over a pre - specified fixed interval . Tests of parallelism were then performed to determine if trends over time differed by group or sex . The denominators were based on the health insurance registry (which provincial law requires all residents and dependants to register) for the entire population as of june 30 in each year . The first year in which an individual met the criteria for diabetes (with no diabetes claims in the preceding 2 years) prevalence was determined using the following group - specific formula: total number of youth with diabetes in the calendar year / the total population for the calendar year . Incidence was determined using the following formula: total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases). Odds ratios (or) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . Or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . To examine trends over time, average annual percent changes (aapc) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 (rockville, the aapc provides a relative summary measure of the trend over a pre - specified fixed interval . Tests of parallelism were then performed to determine if trends over time differed by group or sex . Based on alberta health and wellness administrative databases, 853,733 youth (51.2% male) were living in the province as of 2007, of which 50,930 were status aboriginal (51.2% male). Among these youth a total of 2,589 prevalent diabetes cases were apparent in 2007, with 140 cases occurring amongst status aboriginal youth . The 2007 crude prevalence rate of diabetes was 0.27 and 0.31% in the status aboriginal and general population youth populations respectively (fig . 1, table i). Incidence was 0.59 per 1,000 for status aboriginal and 0.49 per 1,000 for general population . No discernible differences were seen in diabetes prevalence or incidence with respect to group or sex in 2007 (fig . 1, crude rates of diabetes among status aboriginal and general population youth in alberta, 19952007 (adss). A = prevalence; b = incidence . Crude diabetes prevalence and incidence among status aboriginal and general population youth, 2007 crude rates of both diabetes prevalence and incidence increased in status aboriginal and general population youth over the observation period (fig . 1, table ii). However, prevalence grew to a greater extent among status aboriginal youth (aapc 6.98) compared to those in the general population (aapc 3.93; p <0.01), with status aboriginal males experiencing the largest increase in diabetes prevalence (aapc 9.18). Ethnic differences in diabetes incidence trends were only observed among the male population, which saw a significantly larger rise in incidence for status aboriginal (aapc 11.65) compared to general population males (aapc 4.62; p <0.01). Male youth, regardless of group, experienced greater increases in both prevalence and incidence over time than that of respective female youth (table ii). Age - specific crude prevalence and incidence of diabetes by group are shown in fig . 2 . Age specific crude rates of diabetes among status aboriginal and general population youth, 1995 and 2007 . Ethnicity comparisons of aapc (average annual percent change) in diabetes prevalence and incidence among status aboriginal and general population youth, 19952007 p <0.05 for aapc ethnicity comparison . Paralleling adult populations, diabetes among youth is increasing worldwide (20). While type 1 diabetes remains the majority of cases in youth, type 2 diabetes (a disease typically regarded as an adult phenomenon) is becoming progressively more common in children and adolescents (20). We report that youth - onset diabetes is an increasing problem in alberta, especially among status aboriginals . Though our results are in line with international data showing increasing trends (20,21), they contradict the recent ndss reports (12,22), which found diabetes prevalence and incidence rates remained stable at 0.3 and 0.4 (per 1,000) respectfully among general population youth between 20012006 . It is possible that differences in ethnicities and/or socioeconomic parameters contribute to these differences . Among indigenous populations especially, including american indian / alaskan natives, australian aborigines, the maori of new zealand, and canadian aboriginals, youth - onset diabetes is increasingly being identified (14,2327). In canada, longitudinal trends of diabetes in aboriginal youth had previously only been explored in manitoba . Dean and colleagues (10) showed the crude type 2 diabetes incidence rate rose to 0.55/1,000 (for ages 019) in 2001 . Similarly, crude diabetes incidence increased from 0.22 per 1,000 to 0.59 per 1,000 over a 12 year time span in the current analysis . The observed accelerated increase in diabetes among status aboriginal youth is consistent with reports of a decrease in the age of diagnosis of diabetes among aboriginal adults . In saskatchewan type 2 diabetes incidence was highest among status aboriginal adults aged 4049, while most new diabetes cases among the general population were in those> 70 years of age (4). Also, as indicated by the first nations and inuit regional health survey, the majority (53%) of those with diabetes in aboriginal communities were <40 years of age (28). Compared to general population, diabetes grew approximately twice as much among status aboriginal youth . Ethnic comparisons have remained largely unexplored in youth, despite numerous studies documenting the divergence in diabetes epidemiology in adult populations . In the beginning of adss observation 1995, incidence and prevalence of diabetes were distinctly higher among general population . By 2007 however, status aboriginal youth have caught up to the point where no detectable differences were apparent, likely (as mentioned above) due to type 2 diabetes . Reinforcing this notion, a recent national surveillance study amongst practicing physicians found the incidence of type 2 diabetes among aboriginal children (0.23/1,000) was strikingly 46-fold higher than caucasian children (0.005/1,000) over a 24 month period (29). We observed an increased rise of diabetes among young status aboriginal males, confirming earlier studies . Sex ratios of prevalence and incidence (female: male) of approximately 4:1 and 2:1 in 1995 decreased to 1:1 and 1:1 respectively in 2007 . Additionally, the aapc in prevalence was highest amongst status aboriginal males (aapc 9.18) compared to females (aapc 5.70). Over a 15 year time period in manitoba, the early predominance of youth diabetes cases among aboriginal females (8-fold compared to males) disappeared by 2001 (10). From 1990 to 1998, american indian and alaskan native young males also experienced relative increases in diabetes prevalence almost double that of their female counterparts (30). Similarly, the gap in diabetes prevalence between adult status aboriginal females and males is also diminishing (4,5). Reasons for the accelerated rise of diabetes among status aboriginal males have not been explored, but may be due in - part to escalating childhood overweight and obesity (31) with a possible gender - based lag in the obesity epidemic where it may have occurred earlier in time amongst females . In countries with universal healthcare, administrative data have become common and invaluable sources for the population - based surveillance of many chronic diseases . Ndss methodology has been validated in adults and youth, and is a standard for diabetes monitoring at the population level (16,18,32,33). For instance, guttmann et al . (33) recently found the ndss algorithm in youth (019 years of age) in ontario was associated with 100% sensitivity and 94% specificity . Using a similar algorithm (4 physician claims over a 2-year period), diabetes prevalence rose from 0.18 to 0.24 and incidence (per 1,000) increased from 0.25 to 0.32 among ontarian youth between 1994 and 2003 (32). In support, general population youth in the current study experienced nearly identical increases in prevalence and incidence between 1995 and 2003: 0.19 to 0.25 and 0.22 to 0.32 (per 1,000) respectively, with rates progressively increasing until the end of the study period in 2007 . We are unable to distinguish the type of diabetes in our administrative data . However, our clinical experience suggest that the majority of the diabetes in aboriginal youth is type 2 diabetes, whereas in the non - aboriginal population it is heavily dominated by type 1 diabetes . Moreover, rates of type 1 diabetes are much lower among american indian and canadian aboriginal youth compared to the general population historically (34). Future research aimed at describing the contribution of both type 1 and type 2 diabetes to the observed trends is warranted . Possible gestational diabetes cases were included and may have inflated rates in status aboriginal as gestational diabetes has been shown to be more common in adult aboriginal populations (35). It is possible that increased awareness and screening in youth may have contributed to the observed increases, however for various projects (http//:www.braiddm.ca) we have screened 799 youth at risk (mostly aboriginal) in rural alberta since 2001 and have found only 3 (0.4%) of those had undiagnosed diabetes (unpublished). By contrast, the rates of undiagnosed diabetes are approximately 4% in at risk adults . Thus, it appears that diabetes in youth is not often asymptomatic, and therefore is less likely to be detected by increased awareness and screening . Our results cannot be generalized to non - registered aboriginal or mtis youth, whom could not be identified and were included in the general population group . Also, due to the small number of diabetes cases in youth, age - standardized prevalence and incidence rates were not calculated and only crude rates were reported . Lastly, amendments to the indian act in 1985 are possibly increasing the status aboriginal denominator (bill c-31) and likewise some descendants of status aboriginal people are losing their status through the three generations rule (36). Taken together, prudence is needed when interpreting the observed epidemiological findings . In conclusion, diabetes prevalence and incidence has increased among all alberta youth from 1995 to 2007 . Status aboriginal youth, males in particular, experienced a disproportionate growth in diabetes . If unabated, increasing diabetes in youth will likely only further perpetuate the diabetes epidemic in the status aboriginal population . Type 2 diabetes is typically the tip of the iceberg as it is often preceded by several co - morbidities (obesity, hypertension, metabolic syndrome, pre - diabetes, etc), rendering its increased diagnosis in youth a potential public health crisis . This work was supported in part by a team grant to the alliance for canadian health outcomes research in diabetes (achord; reference #: otg-88588), sponsored by the canadian institutes for health research institute of nutrition, metabolism and diabetes . This study is based in part on de - identified data provided by the alberta health and wellness.
One disability that can sufficient to interfere with activities of daily living is non - syndromic hearing loss (nshl) (1, 2). People with even mild nshl have problems hearing speech when there is background noise and identifying the sounds sources (3, 4). Nshl is the most common sensory deficit in humans, with an incidence of about 1 in 1,000 newborns . The prevalence increases during childhood, reaching a rate of 2.7 per 1,000 children before the age of 5 years and 3.5 per 1,000 adolescents . Two thirds of people with nshl worldwide live in developing countries (5, 6). The transmembrane channel - like 1 (tmc1) gene is considered a member of a gene family predicted to encode transmembrane proteins (7, 8). Mutations in the tmc1 gene have been associated with profound prelingual deafness (dfnb7/b11) and progressive postlingual hearing loss (dfna36); thy have been reported in different populations (9). The dfna36 and dfnb7/b11 loci are located on chromosome 9q13 - q21 (7). Tmc1 and tmc2 are members of a gene family predicted to encode transmembrane proteins and are located on p13 of chromosome 20 . The tmc1 gene encodes a sodium sensor and may function as ion transport channel or pump . Tmc1 mrna is specifically expressed in neurosensory hair cells of the inner ear, and it is required for normal function of cochlear hair cells, although the molecular and cellular functions of the tmc1 protein are unknown (8). Since no report has yet determined the frequency of tmc1 gene mutations in the iranian population, the present study was performed to screen and identify the mutations of this gene associated with nshl using polymerase chain reaction single - stranded conformation polymorphism (pcr - sscp) and heteroduplex analysis (ha). This experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . In the present study, we investigated the mutations of the tmc1 gene, locus dfnb7/11, in a cohort of 100 patients with nshl in iran . The 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid (edta)-containing tubes (sarstedt) from 10 provinces of iran, namely semnan, sistan & baloochestan, fars, khozestan, kohgilooye va boyer ahmad, kordestan, chaharmahal & bakhtiari, booshehr, golestan, and gilan . Finally, 100 patients (one proband from each family) were selected (table 1). Nshl informational questionnaires were filled out for all families . In previous work, these patients had no mutations in the cx26 gene (10). Known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study, so families with the possibility of exposure to these factors were excluded from the research . Total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure (11). The quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer (thermo scientific inc ., wilmington, de, usa) at a wavelength of 260/280 nm according to the method described by sambrook and russell (12) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr, two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 (hastings software, inc ., hastings, ny), and primer sequences were blasted in the national center for biotechnology information s (ncbi s) genbank . The details of the designed primers are shown in table 2 . Mutant primers created by site - directed mutagenesis (sdm) as positive control, tmc1-m (tmc1 mutant primer). Site - directed mutagenesis (sdm) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples . Standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler (corbett research, australia). The pcr reaction consisted of 0.2 pm of each primer, 2.5 mm mgcl2, 200 m dntps mix, 5 m of 10 x pcr buffer (200 mm tris - hcl [ph 8.4], 500 mm kcl), 1 unit of taq dna polymerase (all fermentas, germany), and 100 ng of template dna . Pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c, 50 seconds of annealing at 57.5c (exon 7) or 60c (exon 13), 40 seconds of extension at 72c, and a final extension at 72c for 5 minutes . The pcr amplification products (2 l / lane) were loaded on 8% polyacrylamide gel (29:1 acrylamide: bis - acrylamide) electrophoresis (page) in 1 x tbe buffer (10.8 g of tris - base 89 mm, 5.5 g of boric acid 2 mm, edta (ph = 8.0) 4 ml of 0.5 m edta (ph = 8.0), combined all components in sufficient h2o and were stirred to dissolve) at 85 v for 30 minutes, and the gels were stained using the silver nitrate staining method . For sscp, microtubes containing pcr products were mixed with an equal volume of formamide loading dye, heated to 96c for 15 minutes, and chilled on ice for 5 minutes before loading on the polyacrylamide gel . The electrophoresis tank was filled by tbe buffer 0.6 x, and tbe 1 x was used in the gel; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours . First, 3 l of edta (0.5 m) was added to 2 l of pcr products . Then, the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . After mixing with 6 x triple dye loading buffer at a volume ratio of 1:5, the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours (table 3). Finally, the heteroduplex fragments were visualized using standard silver nitrate staining . The samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer (applied biosystems) by macrogen inc . For this study, the regional research ethical committee of shahrekord university of medical sciences (grant number 91 - 3 - 2, january 2011) approved the protocol and informed consent forms . Informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki (doh). All data were collected in the statistical program for the social sciences software (spss, inc ., the mean difference between groups was calculated using a t test . In this study, a p value of 0.05 was considered statistically significant . This experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . In the present study, we investigated the mutations of the tmc1 gene, locus dfnb7/11, in a cohort of 100 patients with nshl in iran . The 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid (edta)-containing tubes (sarstedt) from 10 provinces of iran, namely semnan, sistan & baloochestan, fars, khozestan, kohgilooye va boyer ahmad, kordestan, chaharmahal & bakhtiari, booshehr, golestan, and gilan . Finally, 100 patients (one proband from each family) were selected (table 1). Nshl informational questionnaires were filled out for all families . In previous work, these patients had no mutations in the cx26 gene (10). Known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study, so families with the possibility of exposure to these factors were excluded from the research . Total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure (11). The quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer (thermo scientific inc ., wilmington, de, usa) at a wavelength of 260/280 nm according to the method described by sambrook and russell (12) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr, two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 (hastings software, inc ., hastings, ny), and primer sequences were blasted in the national center for biotechnology information s (ncbi s) genbank . The details of the designed primers are shown in table 2 . Mutant primers created by site - directed mutagenesis (sdm) as positive control, tmc1-m (tmc1 mutant primer). Site - directed mutagenesis (sdm) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples . Standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler (corbett research, australia). The pcr reaction consisted of 0.2 pm of each primer, 2.5 mm mgcl2, 200 m dntps mix, 5 m of 10 x pcr buffer (200 mm tris - hcl [ph 8.4], 500 mm kcl), 1 unit of taq dna polymerase (all fermentas, germany), and 100 ng of template dna . Pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c, 50 seconds of annealing at 57.5c (exon 7) or 60c (exon 13), 40 seconds of extension at 72c, and a final extension at 72c for 5 minutes . The pcr amplification products (2 l / lane) were loaded on 8% polyacrylamide gel (29:1 acrylamide: bis - acrylamide) electrophoresis (page) in 1 x tbe buffer (10.8 g of tris - base 89 mm, 5.5 g of boric acid 2 mm, edta (ph = 8.0) 4 ml of 0.5 m edta (ph = 8.0), combined all components in sufficient h2o and were stirred to dissolve) at 85 v for 30 minutes, and the gels were stained using the silver nitrate staining method . For sscp, microtubes containing pcr products were mixed with an equal volume of formamide loading dye, heated to 96c for 15 minutes, and chilled on ice for 5 minutes before loading on the polyacrylamide gel . The electrophoresis tank was filled by tbe buffer 0.6 x, and tbe 1 x was used in the gel; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours . First, 3 l of edta (0.5 m) was added to 2 l of pcr products . Then, the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . After mixing with 6 x triple dye loading buffer at a volume ratio of 1:5, the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours (table 3). The samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer (applied biosystems) by macrogen inc . For this study, the regional research ethical committee of shahrekord university of medical sciences (grant number 91 - 3 - 2, january 2011) approved the protocol and informed consent forms . Informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki (doh). All data were collected in the statistical program for the social sciences software (spss, inc ., the mean difference between groups was calculated using a t test . In this study, in this study, blood samples of 100 patients with hearing loss (mean age 16.5 2.01 years, 49.2% men and 50.8% women, 74.15% married) were collected from 10 provinces in iran . The connexin 26 gene is responsible for a large proportion of deafness (about 14.6%), and these patients had no mutations in this gene (10, 13). Extracted dna with a 260/280 nm absorbance ratio of 1.8 - 2 was subjected to gene amplification . Exons 7 and 13 of the tmc1 gene were amplified using the pcr technique with specific and mutated oligonucleotide primers . After page, fragments with a length of 187 and 250 bp were revealed for exons 7 and 13, respectively, of the tmc1 gene . Pcr products were used for sscp and ha for investigation of mutations in the sequences . Neither pcr - sscp nor ha showed mutations in exons 7 and 13 of the tmc1 gene (figures 1 and 2). Line m is a 100 bp dna ladder (fermentas, germany), line 5 is a sample amplified by mutant primers with typical shift (positive control), and lines 2 - 4 and 6 - 13 are deafness patients samples . All specimens have the same template bands without shifts in the sscp bands and after ha . Line m is a 100 bp dna ladder (fermentas, germany), line 1 is positive control, line 3 is a suspected sample containing different bands compared to other samples, and lines 2 and 4 - 15 are samples from deaf patients . In the present study, to increase the accuracy of the sscp reaction, ha and mutant control (positive control created by sdm) were performed . Only a number of suspected fragments in exon 7 showed a different banding pattern, but after sequencing, the mutations were not confirmed (figure 3). In various human populations, different dominant (e.g., coch, dfna5, and pou4f3) and recessive genes (e.g., gjb2 and slc26a4) have been reported as the cause of hearing loss worldwide (7, 9, 14 - 16). The tmc1 gene is an autosomal recessive gene and a common cause of hearing loss in many countries, such as india, pakistan, algeria, iraq, lebanon, sudan, tunisia, and turkey (15, 17 - 19). The prevalence of non - syndromic hearing impairment due to tmc1 in the pakistani population is 4.4%, and one study has indicated that the tmc1 protein might have an important function in the k channels of the inner hair cells (16). 2002) detected a 1.6 kb genomic deletion encompassing exon 14 of tmc1 in a recessive deafness mouse mutant, which lacks auditory responses and has hair - cell degeneration (7). In the present study, the association of exons 7 and 13 mutations of tmc1 gene locus dfnb7/11 in deaf patients after gene amplification, neither sscp nor ha showed mutations in exons 7 and 13 of this gene related to hearing loss in these patients . Only a suspected sample in exon 7 had a different banding pattern, but after sequencing, mutations in this exon were not identified . The strength of this study was the number of samples, as 890 blood samples were provided; however, due to a lack of financial resources, we chose two exons of this gene . Via the molecular analysis of the tmc1 gene in korean patients, one study showed that this gene was not the cause of nshl in the korean population (20). In our study, as in the research in korea, a relation between the tmc1 gene and nshl in the iranian population was not observed . (2005) investigated four novel tmc1 (dfnb7/dfnb11) mutations in turkish patients with congenital autosomal recessive nshl (arnshl), and they indicated that tmc1 mutations account for at least 6% (4/65) of arnshl cases in gjb2-negative turkish families from the northeast and east of turkey; however, in our study, mutation in exons 7 and 13 of tmc1 gene was not observed (14). In another study in sudan, it was shown that tmc1 mutations contribute to deafness; this confirmed and extended previous reports on the role of tmc1 in recessive non - syndromic deafness (20, 21). Meanwhile, in the present study on the iranian population, no significant relation between exon 7 and 13 mutations of the tmc1 gene were observed . In pakistan, 2007) identified 10 new families segregating dfnb7/b11 deafness and tmc1 mutations, including three novel alleles; moreover, they identified a c.100c> t mutation in exon 7 (16). 2008) identified four new mutations in the tmc1 gene and suggested an additional deafness gene at loci dfna36 and dfnb7/11 in 51 familial of turkish patients with autosomal recessive hearing loss; these results implied the presence of mutations outside the coding region of this gene, or alternatively, at least one additional deafness - causing gene in this region (9). Another study in turkey reported five novel mutations in the tmc1 gene related to arnshl (19). In comparison with the other populations discussed, in a review study in iran by mahdieh et al . The frequencies and distributions of nshl included gjb2, gjb6 (large deletion), tecta, slc26a4, and pejvk mutations . The researchers indicated that mutations in gjb2, slc26, tecta, and pjvk genes have an important role in deafness in iran, and a screening test should be generated for better intervention and diagnostic programs (22). The study of hildebrand et al . In iranian families showed that two families are related to locus dfnb7/11, and one of them had a c.776 + 1 g> a mutation in exon 7 (23). In 2014, lin et al . Identified novel compound heterozygous mutant alleles of tmc1 responsible for arnshl in a tibetan chinese family (24); meanwhile, in the present work, we did not observe a relation with mutations of exons 7 and 13 of the tmc1 gene in iranian nshl . The findings of the current study indicate that mutations in exons 7 and 13 of tmc1 gene are not related to hearing loss in the iranian population . Therefore, the tmc1 gene may not related to nshl, but further studies investigating related mutations in other parts of this gene in iranian population are necessary and could help in the genetic counseling of patients and design of practical strategies for the management of auditory disorder.
Microleakage between the root canal filling and root - canal walls may adversely affects the results of root - canal treatment.1 apical leakage is considered to be common cause of endodontic failure.2 hence, different endodontic filling materials, sealers and techniques have been introduced to the dental community in an attempt to improve apical seal.1 various materials have been used in root canal treatment in an attempt to achieve success . But, a combination of gutta - percha and a sealer are used most commonly . Gutta - percha is considered an impermeable core material; therefore, leakage through an obturated root canal is expected to take place at the interfaces between the sealer and dentin or the sealer and gutta - percha, or through voids within the sealer.3 apical sealing is desirable to prevent passage of bacteria and their endodotoxin apically . In vitro evaluation of apical dye penetration is used to estimate the sealing ability which is corresponding to in vivo amount of micro leakage with particular sealer.2 many techniques were used to evaluate the leakage of sealers such as; colored dye penetration radio labeled tracer penetration dissolution of hard tissue clearing of teeth, spectrometry of radioisotopes electrochemical and gas chromatography . However, many studies showed no significant difference between these techniques.1 the aim of this in vitro study was to quantitatively evaluate the sealing properties of three different root - canal sealers; tubliseal, sealapex and ah26 using a spectrophotometric method . Thirty - six extracted sound mandibular molar natural teeth specimens with complete root and free from caries or cracks were collected, stored, disinfected and handled as per the recommendations and guidelines laid down by occupational safety and health administration and centers for disease control and prevention.4 the teeth were then placed in 0.9% physiologic saline solution for ten days prior to access cavity preparation . The samples were divided into three experimental groups; group i: sealapex, group ii: tubliseal and group iii: ah26 with 12 samples in each group . The samples were then coated with nail varnish all over the root surface except 2 mm around the apical foramen . 2 ml of freshly prepared 2% methylene blue dye was taken in each vial and the apical third of the root was suspended in the dye for 72 h. samples were washed with distilled water, nail varnish removed and then placed in 20 ml of 35% nitric acid for 72 h. standard solutions of 1%, 0.5%, 0.2%, 0.05%, 0.02% and 0.01% of methylene blue in 35% nitric acid were prepared and stored for 72 h. the standard solutions and the nitric acid solutions were filtered and centrifuged for 1 min after 72 h. the supernatant was subjected to spectrophotometric analysis using a filter of 670 nm . The amount of leakage was extrapolated from a standard linear regression curve constructed from stock standard methylene blue dye solutions . Obtained data were statistical analyzed with kruskal wallis and mann whitney u - tests using spss software version 20 (ibm). The results of the quantitative evaluation of the sealing properties of the three root - canal sealers are shown in table 1 ., tubliseal showed a significant difference (p> 0.005). In comparison to sealapex comparisons made between tubliseal and ah26 showed no significant difference (table 2, chart 1). Mean and standard deviation values of volumetric dye penetration in groups with reference to transmission . Three - dimensional obturation of the root canal system with a fluid impervious seal is an important factor for successful endodontic therapy . The root canal filling should seal the canal both apically and coronally to prevent the passage of microorganism to apex or vice versa.5 most reliable method is the use of gutta - percha cones with sealer cement . Sealers based on zinc oxide - eugenol (tubliseal), calcium hydroxide (sealapex), epoxy resins (ah26) were included in the present study . In present quantitative dye leakage study, tubliseal demonstrated least dye leakage in comparison with other experimental groups (table 1). Our results were in contrary to study by masoud and saleh where they found more microleakage in tubliseal group than other groups.6 sealapex is a calcium hydroxide type sealer . Calcium hydroxide used as root canal sealer since it stimulates periapical tissues in order to maintain health or promote healing and secondly for its antimicrobial effects.7 it has been observed in some studies that, calcium hydroxide sealers showed a significant volumetric expansion during setting because of water absorption, which increases its solubility . The present in vitro investigation indicated maximum leakage value with sealapex among the experimental groups . In a contradictory to our results, cobankara et al . (2006) observed sealapex with better apical sealing than the other sealers (ah plus and rc sealer) at 7, 14, and 21 days.3 ah26 is an epoxy resin based sealer that provides easy handling characteristics, good flow, good sealing to dentin and prominent antimicrobial activity.6 kumar et al . Observed more micro leakage with zinc oxide - based sealer and least with resin based sealers, this is contradictory to our study.8 in our study, there was no statistical significant difference between tubliseal and ah26 in micro leakage . Observed least amount of dye penetration for ah plus and endorez group.9 even though, the current study did not indicate any statistically significant difference between ah26 and sealapex, it is time to question the overall efficacy of calcium hydroxide sealers on the grounds that the reparative and the calcification capabilities attributed to calcium hydroxide are generally desirable before completing the obturation . Ah26, resin based sealer provided a better apical seal when compared with sealapex even though the results were statistically not significant . It mixes easily, flows well, and has ample working time, good radio - opacity, comparable solubility, good adhesion and good biocompatibility.3 in the present study, there was no significant difference between group ii and iii and group i and iii . Did nt find any significant difference between the tested three groups; sealer 26, enfoflas and resin group . But they observed higher microleakage in sealer 26 group compared with control.11 joseph and sing evaluated the apical sealing with four root canal sealers; ah26, sealapex, endoflas fs and ah plus and observed no significant differences between all groups except between ah plus and endoflas.5 nagas et al . Observed significantly lower overall leakage with ah plus group, whereas no difference was found between master cone points.12 kopper et al . Observed significant dye penetration for ah plus, endofill and sealer 26.13 cobankara et al . Observed better sealing values for roekoseal after 21 days when compared to ketac - endo and ah plus, and there was no statistically significant difference.1 dultra et al . Found no statistical difference between groups for apical leakage (endofill, ah plus, endorez and epiphany).14 in addition, before accepting a new material for routine clinical use further experiments should also be performed to evaluate the other aspects of the materials physical and biological properties such as biocompatibility, solubility, disintegration, radio - opacity and dimensional stability . However, these in vitro studies do provide comparative information of the relative performance of sealers tested under the same conditions in each particular study and clinicians can use this information to possibly choose a better sealer . In the present study, tubliseal sealer showed least microleage compared with sealapex and ah26 sealer . It is important to remember before declaring any root canal sealer as most acceptable that the results of the dye penetration studies indicate only the relative sealing ability of root canal fillings in vitro and they do not indicate their ability to prevent the penetration of bacteria into filled root canals in vivo.
Targeting type 4 phosphodiesterase (pde4) has been recognized as a promising approach to managing copd by relieving the symptoms, slowing the progress of the disease, increasing exercise tolerance, reducing exacerbation rate, and improving quality of life (giembycz 2001, 2005; mehats et al 2003; spina 2003, 2004; lagente et al 2005; lipworth 2005; soto and hanania 2005) the pressing need to develop drugs that control symptoms and reduce mortality (pauwels et al 2001; gold 2005) and the billion - dollar marketing potential for management of copd have pushed the r&d of pde4 inhibitors into the product development pipelines of major pharmaceutical companies in the recent years . The early clinical trial data for the second - generation pde4 inhibitors cilomilast (ariflo, glaxosmithkline, usa) and roflumilast (daxas, altana, germany) all pointed to a successful introduction of a novel non - steroid anti - inflammatory therapy to clinicians in combating severe copd (gamble et al 2003; rabe et al 2005) nevertheless, while the progression of developing cilomilast has idled at the approvable stage for more than two years, the announcement of the termination of the agreement to develop roflumilast between altana and pfizer has raised concerns about the therapeutic efficacy of selectively inhibiting one or two isoenzymes in the pde4 family for copd management (pharmiweb 2005). In the early six - month record phase iii trial, roflumilast (500 mg daily) clearly improved lung function (ie, increased fev1 by + 97 ml) and significantly reduced exacerbations (acute worsening of symptoms) compared with placebo (rabe et al 2005). However, in the follow - up one - year phase iii trials using exacerbations as one of the key endpoints, the results from the european copd ratio study that included 1513 patients with severe and very severe copd have failed to repeat the previously claimed efficacy . In addition, the new trial data confirmed that the pde4 inhibitor roflumilast s efficacy was considerably lower than the approved therapies such as fluticasone / salmeterol (a combination therapy of glucocorticosteroid and long - acting 2-agonist) and tiotropium bromide (long - acting anticholinergic). The unexpectedly low long - term efficacy on exacerbation rate from roflumilast therapy made the r&d community re - examine the role of targeting pde4 in copd because one of the highest unmet needs in treating the disease is to reduce or eliminate exacerbations (pharmiweb 2005). In november of 2005, altana announced the withdrawal of the european marketing authorization application (maa) for roflumilast and decided to wait for more clinical trial data for submission of a future maa (altana 2005a). This holdup no doubt sets back the r&d of the most promising pde4 inhibitor in development for copd . Copd is a complex disease with pathophysiological features including inflammation (neutrophils, macrophages, cd8 + lymphocytes infiltration, and inflammatory mediator tnf- and il-8 release), airway obstruction (smooth muscle contraction, elevated cholinergic tone), respiratory bronchiolar alveolar vasculature remodeling (loss of elastic recoil, alveolar destruction, and fibrosis), pulmonary hyperinflation, gas - exchange abnormalities, and pulmonary hypertension . The progressive loss of lung function leads to reductions in patients quality of life and results in exacerbations, cor pulmonale, and death . It is believed that the chronic non - infectious inflammation underlies the pathogenesis and the steady progression of the disease (pauwels 2001; gold 2005). The pathological changes in the patients with copd are not fully reversible and it often takes many years for a patient at risk (cough, sputum production) to progress into suffering from mild airflow limitation, to moderate, severe, and very severe copd (with chronic respiratory failure). In the absence of a magical therapy that can stop the disease progression and reverse the abnormalities of pulmonary function, the management, including drug therapy, for copd is long - term care . Inhibition of pde4 has been established as an effective and reliable approach to increasing intracellular camp (conti et al 2003) that underlines the signaling mechanisms for the treatment of copd . In recent years, numerous in vitro, in vivo, and clinical trial studies demonstrated that pde4 inhibitors (eg, rolipram, cilomilast, and roflumilast) relax airway smooth muscles to increase air flow (holbrook et al 1996; bundschuh et al 2001) and improve pulmonary circulation (schermuly 2000; de witt 2000), inhibit bronchiolar alveolar vasculature remodeling, and fibrosis (kumar et al 2003), reduce neutrophils macrophages / cd8 + t cells infiltration and pro - inflammatory mediator release (kumar et al 2003; profita et al 2003; wollin et al 2005), improve patients exercise capacity and quality of life, and prevent the progressive loss of pulmonary function (rabe et al 2005; gamble et al 2005). With all these preferred outcomes, it seems that the pde4 inhibitors in development (cilomilast and roflumilast) would be an ideal armory for the healthcare community to combat copd . Why, then, has the long - term trial with roflumilast failed to produce the expected results? It could be due to a dose regimen (500 mg daily) that was effective for patients with moderate to severe copd (rabe et al 2005) but not adequate for those patients suffering from severe and very severe copd (altana 2005b) or the intrinsic low efficacy of the narrow - spectrum pde4 inhibitors . Developing pde4 inhibitor as a therapy for copd is based on the fact that theophylline dilates airway smooth muscles and improves pulmonary function by inhibition of pde activity (barnes 2003; spina 2004) the dose - limiting adverse reactions (nausea, emesis, cardiac arrhythmias) with the non - selective pde inhibitor theophylline and the first - generation pde4 inhibitor rolipram (huang et al 2001; lagente et al 2005) directed the r&d of pde inhibitors to discover the second - generation of pde4 inhibitors cilomilast and roflumilast that have been successfully brought to the final stage for administration approval (spina 2003, 2004; lipworth 2005) based on the fact that the emetogenic reaction to pde4 inhibition is due to reticence of the pde4d isoenzyme (lamontagne et al 2001; robichaud et al 2002), several researchers in the field proposed to develop isoform - specific pde4 inhibitors that reduce or completely avoid disturbing pde4d activity and therefore do not trigger the emetic responses in the nervous system (giembycz 2002; robichaud et al 2002; card et al 2004). Structural studies have provided evidence that the folding of catalytic domains of pde4 has a conformation involved in binding of selective inhibitors with a common scheme: (i) a hydrophobic sub - pocket sandwiching an inhibitor in the active site; (ii) hydrogen bond(s) to an invariant glutamine controlling the orientation inhibitor binding (therefore the affinity or potency) (lee et al 2002; huai et al 2003; card et al 2004) (figure 1). The scaffold of individual pde4 isoenzyme and the structure of a given selective inhibitor govern isoenzyme - selective inhibition, depict the binding affinity, and determine the therapeutic window and rank order of potency in clinical use for the treatment of copd . Enhancement of isoenzyme selectivity is critical for reducing side - effects of the pde4 inhibitors . The strength of the interaction (hydrogen bond) between the oxygen group(s) of an inhibitor and the amide nitrogen group of glutamine (gln) 369 for pde4d and gln 443 for pde4b plays a pivotal role in determination of the potency and isoenzyme selectivity of an inhibitor (lee et al 2002; huai et al 2003; card et al 2004). In addition, selective pde4 inhibitors such as cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space of the pocket to yield extra binding energy (increasing potency) and result in greater isoenzyme selectivity (lee et al 2002; huai et al 2003; card et al 2004) for example, while cilomilast s additional functional groups interact with 10 almost identical residues that form the hydrophobic sub - pocket in pde4d and pde4b, the oxygen atoms of the cyclopentyloxy and methoxy groups of cilomilast form two hydrogen bonds with the gln369 of pde4d, whereas there is only one hydrogen bond being formed between the methoxy group of cilomilast and the gln 443 of pde4b (card et al 2004; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1). This difference may partially explain the fact that cilomilast is approximately 10-fold more selective for pde4d than for pde4b, despite over 90% identity between the pde4b and pde4d catalytic domains . (giembycz 2001; odingo 2005) roflumilast shows a better fitting to the hydrophobic sub - pocket in the pde4d catalytic site than cilomilast (card et al 2004; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xoq&template=ligands.html&l=2.1), which depicts the experimental finding that roflumilast inhibits pde4d 338-fold more potently than cilomilast (table 1). As for pde4b inhibition, roflumilast s cyclopropylmethoxy and difluoromethoxy oxygen groups form two hydrogen bonds with gln 443 of pde4b (card et al 2004; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xmu&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1), which may partially explain its 585-fold greater inhibition potency than cilomilast toward pde4b (table 1). The dichloropyridyl substitution of roflumilast also increases its potency compared with cilomilast in terms of pde4b inhibition (card et al 2004). The rank order of potency for inhibition of pde4 activity and lipopolysaccharide - stimulated tnfa release, for relaxing bronchoconstriction in guinea pigs, and the daily doses for treatment of copd by roflumilast, cilomilast, rolipram, and theophylline are summarized in table 2 . By improving the inhibition ratio on pde4b / pde4d, roflumilast indeed lowers the emetic profile without compromising its therapeutic efficacy in comparison to cilomilast (huang et al 2001; spina 2003) the pde4b - specific single - molecule targeting can undoubtedly reduce or eliminate an inhibitor s unwanted effects . However, this approach may compromise the efficacy of a pde4 inhibitor because airway and vascular smooth muscles express multiple pde4d isoforms (eg, d1, d2, and d5) and pde4d plays a crucial role in bronchoconstriction and vascular smooth muscle contraction (liu et al 2000; baillie et al 2001; mehats et al 2003). An agent without 4d inhibition may end up lacking sufficient therapeutic efficacy for controlling copd (spina 2004). The beneficial effects on copd via relaxing airway smooth muscles and anti - inflammation mediated by pde4 inhibition with cilomilast and roflumilast have been emphasized and reviewed extensively (spina 2004; lagente et al 2005; lipworth 2005; soto 2005). Improving pulmonary circulation has not been considered as an important therapeutic approach for managing patients with copd . However, the facts are 1) during copd exacerbations, pulmonary hypertension (ph) is increased, 2) the presence of ph is recognized as the single strongest indicator of prognosis in copd patients among the numerous clinically used lung function parameters (doi et al 2003; alp et al 2006), and 3) clinically, the higher the pulmonary arterial pressure, the shorter the life expectancy of copd patients (barbera et al 2003). Although inhaling nitric oxide vasodilator can worsen gas exchange because of altered hypoxic regulation of ventilation - balance (barbera 1996) in patients with stable copd, and vasodilators are considered as contraindications for copd patients, in their preliminary clinical trial, alp et al (2006) demonstrated that reduction of pulmonary vasculature resistance using the pde5 inhibitor sildenafil was able to significantly improve the exercise capacity (6-minute walk test) of patients suffering from severe copd . A double - blinded, placebo - controlled, crossover clinical trial of sildenafil in patients (n=10) with copd is going on to evaluate the effect of pde5 inhibition on patients exercise function, pulmonary function, and quality of life (national institutes of health). Although there is a lack of reports about cilomilast- or roflumilast - caused pde4 inhibition on the improvement of pulmonary circulation, in isolated perfused lung preparations, intravascular or transbronchial administration of subthreshold doses of the wide - spectrum pde4 inhibitor rolipram synergistically amplified the pulmonary vasodilatory response to inhaled pgi2 and concomitantly improved ventilation more interestingly, in anesthetized cats, de witt et al (2000) found that rolipram was more potent than either siguazodan (pde3 inhibitor) or zaprinast (pde5 inhibitor) in reducing pulmonary lobar arterial pressure . When the tone in the pulmonary vascular bed was raised to a high steady level with a constant infusion of the thromboxane mimic u46619 (9,11-dideoxy-11, alpha9alpha - epoxymethano prostaglandin f2), intralobar injections of rolipram caused dose - related decreases in systemic arterial pressure and pulmonary arterial pressure . The preference reduction in pulmonary arterial pressure by inhibition of pde4 suggests that administration of pde4 inhibitors may benefit copd patients by increasing ventilation (airway relaxation) and improving alveolar perfusion (vasodilation), therefore improving blood gas exchange capacity and patients pulmonary function . Thus, it is highly likely that the maximal therapeutic efficacy of targeting pde4 in the treatment of severe copd depends upon three effectors of downstream intracellular camp elevation: 1) anti - inflammation (lipworth 2005), 2) airway relaxation (spina 2004), and 3) vasodilation . Sacrificing any one of these effectors with an isoenzyme - specific narrow - spectrum pde4 inhibitor will compromise the effectiveness of the therapy . How, then, is the dose - limiting dilemma (nausea, vomiting, diarrhea, and arteritis) (giembycz 2005; spina 2004; lipworth 2005) associated with the wide - spectrum pde4 inhibition overcome? The fact that there are over 60 pde isoenzymes encoded by 21 human pde genes and at least 16 pde4 isoenzymes from 4 pde4 genes (soderling and beavo 2000; conti et al 2003; houslay and adams 2003; huai et al 2004) may render it highly possible that searching for isoenzyme - specific pde4 inhibitors will yield low - efficacy agents . In addition, mechanisms for upregulation of pde4 activity by camp - induced pde4 gene expression and pka - catalyzed phosphorylation activation of pde4 isoforms (conti et al 2003; wallace et al 2005) may very likely reverse an isoenzyme - specific, pde4 inhibitor - produced elevation of intracellular camp level, and therefore the associated biological beneficial effects . Noticing the low therapeutic ratio and insufficient clinical efficacy of the current generation of pde4 inhibitors (cilomilast and roflumilast), giembycz (2005) assumed that one potential means of improving the therapeutic ratio and safety of pde4 inhibitors may lie in the development of compounds that have broader phosphodiesterase specificity and suggested dually targeting pde4 and pde1, pde3, or pde7 to enhance clinical efficacy . This approach seems to revisit the previously well - described pde inhibitors such as theophylline or zardvarine (schmidt et al 2000; barnes 2003). It may lead to the dose - limiting drawback cycle again, because it is known that targeting camp - specific pde3 is associated with an increase in morbidity and mortality in heart failure patients (packer et al 1991). In a news feature on pharmiweb.com, there is a remark about pfizer s development of an inhaled dual - action pde4/spiriva (long - lasting muscarinic antagonist tiotropium) combination product for copd (pharmiweb 2005). This combined dual action modality is a favorable approach for managing patients with severe copd considering the presence of bronchoconstricting and inflammatory pathologies in the disease . In general, moderately targeting two mechanisms to reach the therapeutic goal should be more effective and safer than exploiting a single mechanism to its extended degree . We have proposed to co - administer ca channel antagonist (cca) to overcome pde4 inhibitor - caused adverse effects, especially emetic responses (wang and wang 2005) because 1) stimuli that upregulate the camp pathway can increase the excitability of the neurons in the locus coeruleus (lc) that plays an important role in mediating neuronal emesis (nestler et al 1999; takeda et al 2001); 2) the pde4d isoform is localized to neurons in the structures of the medulla including lc (lamontagne et al 2001), which are consistent with a role for pde4d in the emetic response; and 3) the lc neurons fire action potentials spontaneously, resulting from endogenous properties of the membrane conductance to a persistent inward ca current, which can be blocked by diltiazem (williams et al 1984; tokuyama and ho 1996; filosa and putnam 2003). Thus, in the presence of cca, even when a complete inhibition of pde4d leads to an elevation of camp in the lc neurons, the lc cells will not be able to fire action potentials due to a blockade of the depolarizing l - type ca currents, therefore eliminating the intrinsic dose - limiting emetic pharmacological profile of the wide - spectrum pde4 inhibition . In addition, ccas also relax airway smooth muscles and exhibit anti - inflammatory effects, which may synergistically augment a pde4 inhibitor s therapeutic effects on copd (worley and kotlikoff 1990; szabo et al 1997; brown et al 2004). The clinical use of ccas in treatment of pulmonary hypertension in patients with copd further support the combination therapy of a pde4 inhibitor and a cca (sajkov et al 1997). One concern regarding the combined therapy is the possible difference between the pharmacokinetics of the two drugs that may compromise the expected outcomes . This pitfall can be eliminated by developing an agent with two pharmacophores in one chemical structure, therefore being able to simultaneously target two therapeutic mechanisms such as l - type ca channels and pde4s (wang and wang 2005). We believe that it is worthwhile to carry out a randomized clinical trial to evaluate the safety and efficacy of dually targeting pde4 and ca channels in managing the patients with severe copd . The unsatisfied efficacy of using pde4 inhibitor roflumilast in treatment of severe and very severe copd has raised concerns in the r&d community about the administrative approvability for the highly expected novel therapeutic modality in combating copd . The extensive in vitro, in vivo, and clinical trial study data and the established clinical beneficial effects (anti - inflammation, bronchorelaxation, pulmonary vasodilation) associated with pde4 inhibition strongly validate targeting pde4 for controlling copd . Development of an inhaled dual - action therapy such as pde4 inhibitor and muscarinic antagonist may be a correct approach to bringing a pde4 inhibitor to the demanding market . The other approach is using cca to overcome pde4 inhibitor - caused adverse effects, especially emetic responses and simultaneously enhance pde4 inhibitor s anti - inflammatory and bronchorelaxation, pulmonary vasodilation effects . Of course, development of a dual agent that has with two pharmacophores in one chemical structure, therefore being able to target pde4 and l - type ca channels should also be able to improve the therapeutic window of pde4 inhibition and may make available a new therapeutic approach to managing copd.
Long - acting beta-2 agonists such as salmeterol and formoterol are important controller medications in the maintenance therapy of chronic stable asthma, used as an add - on drug to inhaled corticosteroid . However, their usefulness in the management of acute attacks of asthma has recently been recognized, and they are approved for use as reliever medication in europe . Short - acting 2 agonists are the preferred drugs as the initial bronchodilator for acute asthma because of their rapid onset of action . However, due to short duration of action they require frequent administration . Formoterol is a unique bronchodilator having rapid onset and long duration of action with a favorable safety profile . So it can be an ideal alternative to short - acting 2 agonists in the management of acute asthma exacerbation by providing rapid bronchodilatation and reducing the need for frequent administration . Onset of action of formoterol is similar to salbutamol (13 min), and 8090% of brochodilatation occurs by 510 min of inhalation . Duration of action is up to 12 h. however, duration of systemic effects with formoterol is found to be as short as salbutamol . Arformoterol ([r, r] formoterol) is a single isomer form of formoterol, which is available in india recently . This study compares the efficacy and safety of arformoterol and salbutamol delivered by nebulization in the management of acute non - severe asthma . A total of 50 patients attending the emergency room or chest opd of a tertiary care hospital with acute non - severe asthma over a 6-month period from january 2010 to june 2010 were selected for this study . The inclusion criteria were as follows: (a) age> 18 years; (b) british thoracic society definition of acute non - severe asthma; and (c) ability to perform forced expiratory maneuver . The following patients were excluded from the study: (a) patients presenting with acute severe / acute life - threatening / near - fatal asthma; (b) patients with a previous diagnosis of chronic obstructive pulmonary disease (copd); (c) history of hypersensitivity to 2 agonists; and (d) pregnant or lactating women . The study was approved by the institutional ethics committee, and informed consent was obtained from each patient . The age, sex, duration of asthma symptoms, and present controller medications were recorded . The pulse rate, respiratory rate, and spo2 (measured with fingertip pulse oximeter, model 6500, nidek medical, kolkata, india) were also recorded at the time of initial assessment . Baseline peak expiratory flow rate (pefr) was measured with a peak flow meter (peak flow master, cipla ltd ., patients were then assigned by random number allocation to either of the two groups, that is, salbutamol or arformoterol group according to the medication given to them . In the salbutamol group, 5-mg salbutamol respules were administered through oxygen driven (6 l / min) nebulizer (pulmo mist ii nebuliser, nidek medical, kolkata, india) at 20-min intervals for 1 h amounting to a total of 15 mg . In the arformoterol group, total 45 g of the drug was administered as 15 g respules every 20 min through the same nebulizer . The drugs were administered in a double - blind manner . To eliminate the effects of other drugs on the treatment outcome pefr was also measured 5 min after each dose . At each time, best of the three pefr measurements was recorded . Differences in mean pefr between the two groups were analyzed by unpaired t - test . One - sample test was used to compare pefr before and after each dose of the drug to determine whether there was significant improvement in either group . Among 50 patients satisfying the inclusion criteria, 25 patients received salbutamol and 25 patients received arformoterol therapy . Baseline characteristics of the patients (n = 50) receiving salbutamol and arformoterol therapy from the table, it is evident that the demographic profile and baseline pefr in the two groups were comparable . In both the groups, pefr showed significant increase over the baseline values and the increase was evident after each dose of the drug . The increases in pefr after the first and the second dose were significantly more with arformoterol than with salbutamol, but the increase in pefr after the third dose was similar in these two groups . The comparison of improvement following the therapy with salbutamol and arformoterol is shown in table 2 . Improvement in pefr after each dose of bronchodilator when compared with the baseline both drugs were well tolerated; no major adverse effect was noted in either group . As arformoterol is the only long - acting 2 agonist available in india as a nebulizing solution, we have used this drug considering the fact that the clinical pharmacology will be similar with formoterol . Our study has shown that both salbutamol and arformoterol are equally effective as a reliever medication as nebulizing solution in acute non - severe asthma . Improvement in pefr was demonstrated in both the groups and following each dose of the drugs . The absolute increase in the pefr after the first and the second dose were more with arformoterol than with salbutamol, but the increase in the pefr after the third dose was similar with these two drugs . Relatively, hydrophilic drugs such as salbutamol have a rapid onset of action due to their ability to reach the 2 receptor from the aqueous phase . The aqueous portion rapidly activates the 2 receptor, whereas the lipophilic portion is taken up into the cell membrane from which it diffuses slowly to stimulate 2 receptor over a prolonged period . Several studies have shown the efficacy of formoterol in acute non - severe asthma, acute severe asthma, exercise induced bronchospasm, childhood asthma, and copd. [1317] most of the studies have used formoterol with terbuhaler, aerolizer, or metered dose inhaler . However, studies using formoterol nebulization are lacking . Although few studies have shown clinically significant improvement in lung function (fev1) with formoterol when compared with salbutamol, most studies have shown a comparable efficacy . Safety of formoterol is also well documented even at high doses in patients with asthma and copd . Although it is a long - acting drug, systemic effects with formoterol are as short as salbutamol . In few studies, the impact of high - dose formoterol on heart rate, blood pressure, serum potassium, electrocardiogram changes, and arterial blood gas were assessed which showed identical changes comparable with salbutamol we did not study these parameters but evaluated only the side effects reported by the patients . The side effects were very few, non - severe, and comparable between these two drugs . Dryness of mouth was the most reported side effect of formoterol inhalation in one study, but we found oral irritation and headache as common side effects . The sample size is relatively small which limits our ability to detect small but potentially significant differences . This study is restricted to patients of acute non - severe asthma only; efficacy and tolerability of arformoterol in other acute settings such as acute severe asthma and acute exacerbation of copd are yet to be explored . The side effect profile studied by us was only on the basis of the self - reported symptoms of the patients, and objective assessment and laboratory abnormalities were not assessed . We also did not follow the patients after discharge; so long - term efficacy and safety were not studied . Further studies are thus needed to clarify the efficacy, actual dose, dose frequency, cost - effectiveness, and long - term safety of arformoterol in acute exacerbations of asthma and copd with different levels of severity.
Hepatic angiomyolipoma (haml) is a rare benign mesenchymal liver tumor first described by ishak in 1976; it belongs to a group of perivascular epithelioid cell tumors called pecoma . Until date, approximately 300 cases have been reported [310]; however, its natural history has not been clarified . The tumor composed of blood vessels, smooth muscle, and adipose cells and due to the variety of predominance of these tissues, its patterns in imaging studies have resulted in a difficulty in diagnosis and misdiagnosis of the tumor as hepatocellular carcinoma (hcc) in some cases [6, 11]. Therefore, the preoperative correct diagnosis has been difficult; however, recent advances in imaging diagnosis through a combination of ultrasonography (us), computed tomography (ct), magnetic resonance imaging (mri), and angiography and specific immunohistochemical analysis of this tumor using human melanoma black-45 antigen (hmb-45) staining have resulted in accurate diagnosis and it is reported that the current accurate preoperative diagnosis was made in 25%52% of cases [8, 9]. The majority of these tumors are believed to be clinically benign during a mean follow - up period of 6.8 years; however, an increasing number of cases and aggressive changes including growth in size, recurrence after surgical resection, metastasis, and invasive growth pattern into the parenchyma and along the vessels have been reported . In this paper, we have focused on the characteristic features of this tumor shown in imaging studies and by histological analysis, summarized these cases showing aggressive patterns, and discussed management of the patients and indications for surgical treatment . Most cases were found as incidental liver tumors upon health screening or imaging examinations for other diseases . It usually follows a benign clinical course while some patients visited hospitals with unspecific symptoms of abdominal discomfort, fullness, and other such complaints . More than half of the renal aml are considered to be associated with tuberous sclerosis which features the loss of heterozygosity at tsc1 (9q34) and tsc2 (16p13), while it is estimated to be 515% in the liver . Thus the etiology of most of these tumors in the liver is unknown; most cases have no history of liver diseases or specific symptoms, and no changes in laboratory data are seen . Moreover, serum levels of the tumor markers alpha - fetoprotein, protein induced by vitamin k absence or antagonist ii, carcinoembryonic antigen, and carbohydrate antigen 19 - 9 were normal . Since this tumor is composed of various tissues, such as lipomatous, myomatous, and angiomatous tissue in various proportions, the imaging studies show a wide array of features depending on the predominance of each tissue . The tumor showing the predominance of lipomatous tissue is likely to be correctly diagnosed; however, myomatous and angiomatous variant poses diagnostic problems since it is difficult to be distinguished from malignant tumors . Us images may vary depending on the tissue components affected by the tumor . High echogenic lesions can be observed because of lipomatous and myomatous tissue, and angiomatous tissue may result in low echoic lesions in tumor images (figure 1(a)). If the tumor has predominance of lipomatous tissue, the differential diagnosis with hepatic hemangioma is difficult by sonography alone . Color doppler sonography shows punctiform or filiform vascular distribution pattern if the tumor has predominance of angiomatous tissue . Recent reports showed the diagnostic effectiveness of contrast - enhanced us (ceus) [10, 13]. Reported that ceus revealed the typical imaging characteristics of haml, that is, an inhomogeneous hyperenhancing pattern in the arterial phase and prolonged enhancement during the portal and kupffer phases . Plain ct showed homogeneous or heterogeneous low - density lesions, and contrast - enhanced dynamic ct showed highly enhanced lesions in the arterial phase, prolonged enhancement in the portal phase, and, occasionally, defective lesions in the late venous phase depending on the component of the tumor tissue . A density of less than 20 hounsfield units in plain ct is useful to determine the involvement of lipomatous tissue . The difficulty is, however, to diagnose the tumor that is myomatous tissue and angiomatous variant . For this point, modification of size of region of interest in ct was reported to be effective for accurately diagnose renal angiomyolipoma from renal cell carcinoma . Abdominal angiography showed marked tumor staining in the arterial phase, which remained in the portal phase (figure 1(b)). In some tumors, drainage of the hepatic veins can be observed in the late vascular phase . The first phase of ct during hepatic arteriography showed significant hypervascular lesions in the tumor (figure 1(c)), and the second phase showed the remains of staining and defective lesions in other areas (figure 1(d)). Ct during arterial portography showed areas of defective tumors (figure 1(d)). Mri is considered to be the best modality to determine the components of the tumor . Hyperintensity on the t2-weighted image and hyper- or hypointensity on the t1-weighted image are observed depending on the component of tumor tissue [810]. Lipomatous lesions may be determined as hyperintensity on the t1-weighted image; they may also be determined by the chemical shift imaging technique . Contrast - enhanced dynamic mri using gadolinium or the hepatocyte - specific contrast agent gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid showed early enhancement in the arterial phase followed by the prolonged enhancement in the portal phase and defective lesions in the hepatobiliary phase . Macroscopic and magnifying glass view of the tumor showed a soft, white to yellow, and well - circumscribed tumor and range in size from 0.1 cm to greater than 36 cm . No signs of chronic inflammation or fibrosis were seen in the surrounding liver tissue . Because haml comprises lipomatous, myomatous, and angiomatous tissues, microscopic examination showed a mixture of blood vessels, specialized smooth muscle cells, and adipose cells with atypical changes in classic haml (figures 2(a) and 2(b)). This variation of mixture levels in one tumor reflected the differences in imaging studies and made accurate diagnosis difficult . It is believed that majority of the hamls behave in a benign fashion and even if some cases showed invasive growth pattern into the adjacent hepatic parenchyma, portal triad (figure 2(c)), and hepatic vein . Tumor cells, especially myomatous cells, were stained positive for hmb-45 in most tumors (figure 2(d)), for cd34 in the endothelial cells of the blood vessels and for smooth muscle actin in spindle smooth muscle cells . Hmb-45 is an antibody that reacts with an oncofetal premelanosome - associated glycoprotein 2, found in neoplastic melanocytes . Also cd63, cd67, desmin, s100, and ema may also be positive but not specific . These cells were negative for cytokeratin 18, cytokeratin 19, cam 5.2, hepatocyte paraffin-1 . Therefore, the identification of lipomatous, myomatous, and angiomatous tissue by a positive reaction to hmb-45 currently provides the only evidence of haml [46, 18] and can be useful for defining from the other liver tumors such as hcc after the tumor biopsy and surgical resection followed by these immunohistochemical stainings . Although favorable prognosis can be expected for this tumor since the majority of the tumors are benign, however recently, the number of reports of haml showing malignant potential has increased [3, 5, 8, 9, 12, 1624] which revealed significant growth, recurrence, metastasis, and poor prognosis summarized in table 1 . Among those cases, deng et al . Reported in their case that haml showed atypical angiomatous, epithelioid components with pleomorphic and frequent mitosis in the center of the large tumor displayed p53 immunoreactivity, and mutation at exon 7 for p53 and resulted in vascular invasion, distant metastasis, and fatal outcomes . These results indicate that the hmb-45 staining, mitotic analysis by mib-1 (ki67), and p53 reactivity following the fine - needle biopsy of the tumor might be useful for the diagnosis of the tumor and its malignant potential . Since haml usually follows benign clinical courses, the majority of the cases can be conservatively treated . However, as not a few cases showed aggressive pattern in their courses [3, 5, 8, 9, 12, 1624] and because of the low level of accurate diagnoses by imaging studies, and because of the possibility of dissemination of tumor cells into the peritoneal cavity if a tumor is malignant, tumor biopsy has been avoided, and many hamls have been surgically resected [3, 8, 9]. With the increasing number of resected samples, careful comparisons of imaging studies and pathological findings therefore, once haml has been diagnosed by imaging studies, a fine - needle biopsy should be performed to make an accurate diagnosis and to confirm the predominance of the tissue components, the existence of pleomorphic nuclei with high proliferation activity, p53 immunoreactivity, and mutation in p53 if possible . If the aggressive patterns such as vascular invasion, high proliferation of the tumor cells, p53 immunoreactivity were marked, or when the imaging findings and biopsy cannot provide a definitive diagnosis, or if the patients have abdominal symptoms, surgical resection should be considered . Haml is considered to be a benign mesenchymal tumor, and nonsurgical treatment with conservative management involving close followup is therefore suggested for patients with asymptomatic tumors smaller than 5 cm, which have been proved to be a typical haml by fine - needle aspiration biopsy . As a fact the renal angiomyolipoma often showed perirenal invasion, involvement of the renal vein, and the inferior vena cava, that are not considered as signs of malignant behavior and the angiomatous, epithelioid monophasic or pleomorphic variant might be associated with the aggressive behavior and cellular atypia, mitotic activity, and metastatic lesions are the criteria for the malignant renal angiomyolipoma . However, due to the difficulties in diagnosis by imaging studies, many hamls have been surgically resected and have been followed closely for a long period . In addition, by careful analysis of these recent cases [3, 5, 8, 9, 12, 1624] summarized in table 1, it was revealed that, although rare, hamls may have malignant potential, which may be distinguished by aggressive patterns characterized by (1) significant changes in size in short period; (2) a change of tumor composition (cases 1, 2, 5, and 6) [9, 16, 17, 19]; (3) metastases to the other organs (cases 1, 9, and 10) [17, 22, 23]; (4) recurrence after curative surgical resection (cases 3, 7, 9, 10, 11, 12, and 13) [3, 8, 18, 20, 2224]; and (5) invasive growth into the vessels (cases 2, 4, 8, 9, and 10) [12, 17, 2123]. Ohmori et al . Reported the first possibly malignant case, which showed that a significant increase in the tumor size resulted in liver dysfunction . Fatal progression was observed in nine cases listed by multiple recurrences [3, 8, 18, 20, 2224] and metastases to the liver, peritoneum, retroperitoneal region, gastrohepatic omentum, pancreas, and lung [17, 22, 23]. The cases reported by chang, rouquie, and us underwent surgical resection, with a suspicious of aggressive patterns and no recurrence has occurred . We also reported that portal thrombosis, that is, high - grade portal vein invasion, found in five cases in table 1 (cases 2, 8, 9, 10, and 11) [3, 17, 2123] may be a clinical marker of the malignant potential and transformation of haml as it resulted in a significantly aggressive disease and fatal course in 4 cases with multiple recurrences and metastases . This finding might be able to be detected by imaging studies as similar to hcc . In addition, pathological findings of atypical epithelioid component with high proliferation activity, p53 immunoreactivity, and mutation in p53 might be predictive markers for malignant transformation . Based on these reports, as recommended by previous papers [3, 8, 9], long - term follow - up of haml is necessary after its diagnosis by imaging studies and biopsy specimens and curative surgical treatment . The majority of hamls behave as a benign tumors and conservative follow - up may be recommended; however, with increasing number of the reports showing potentially malignant behavior, prompt surgical resection is essential for better prognosis of this tumor . We have reviewed noninvasive imaging studies and the role of histological diagnosis showing distinctive characteristics of haml to increase the rate of accurate diagnoses . In addition, we summarized the cases that showed progressive patterns of the tumor and concluded that a careful followup of the tumor even after the final diagnosis is necessary . We propose that tumor resection is indicated in the following scenarios: (1) the patients show symptoms; (2) the tumor shows an aggressive growth; (3) the tumor shows invasive growth into the vessels evidenced by fine - needle biopsy or imaging studies; (4) the component of the tumor shows atypical epithelioid pattern, high proliferation activity, and/or p53 immunoreactivity; and (5) a definitive diagnosis cannot be made by imaging and pathological studies from malignant tumors.
Considering the therapeutic consequences, involvement of the sentinel node by metastatic disease in breast cancer patients is of great interest to the breast surgeon in order to plan the surgical approach . Unfortunately, the physical examination of the axillae can be inaccurate in identifying pathologic lymph nodes . The same is also true for the pre - treatment imaging (with or without interventional procedures on the axillary node), characterized by a poor negative predictive value [48]. Using gamma probe combined with ultrasound- (us) guided percutaneous core needle biopsy (cnb) could increase the accuracy of identifying the sentinel node and allow a proper histopathologic diagnosis in order to avoid further slnb . The purpose of this pilot study was to assess this new pre - operative diagnostic approach . Main inclusion criteria were: women 18 years old, invasive breast carcinoma, with no clinically positive axillary node and no history of any other malignancy . Multicentric cancer and previous excisional biopsy were exclusion criteria . The sentinel node occult lesion localization (snoll) was performed in all patients the day before surgery . A dose of 510 (generally 7) mbq of technetium 99-labelled human albumin nanocolloid particles in 0.3 ml saline were administered by us - guided percutaneous injection in the area immediately surrounding the breast lesion, followed by injection of 0.2 ml saline using a 22 g needle . We used nanocolloid particles with a size range of 5100 nm (nanocoll, ge healthcare). After injection, antero - posterior and lateral lymphoscintigraphic projections were obtained to identify the presence of a sentinel node and to define whether the radiocolloid has shifted to other possible sites of drainage, such as the internal mammary, intramammary, contralateral axillary or supraclavicular nodes . The hot spot was identified over the skin by the handheld gamma probe (navigatog gps rmd) and was examined using ultrasound logos hi vision gold (hitachi) using a breast - dedicated linear array transducer . The radiologist performing the us - guided biopsy established which lymph node corresponds to the hot spot under gamma probe guidance . We evaluated suspect us findings such as cortical thickening, especially if focal, markedly hypoechoic cortex, absence of the fatty hilum, large expansion, irregular shape, round shape, extracapsular tumoral extension or increased peripheral blood flow at power doppler . Lymph nodes with at least 2 of these criteria were considered pathologic, and we excluded suspicious lymph nodes . Patients were placed in a supine or contralateral - side - down oblique position on the table, with the ipsilateral hand placed behind the head . Biopsies were performed in all patients by a single radiologist with more than 10 years experience in ultrasonography . Cnb was performed with an automatic 18 g needle (bard core biopsy instrument, tempe, usa) after local anesthesia with 2% mepivacaine . All biopsies with a free - hand technique were performed under us guidance with direct visualization of the needle entering into the cortex of the node to confirm position of the needle tip in the appropriate location . Only 1 pass the percutaneous biopsy was performed only if the lymph node corresponded to the hot spot . We considered the biopsy procedure to be successful when the obtained sample contained a large portion of solid non - fatty tissue and/or sank in formalin to be stained with haematoxylin and eosin . The skin above the first radioactive node was marked by the radiologist with an indelible ink tattoo to assist the surgeon . The puncture zone was compressed and the presence of complications such as local pain or hematoma was evaluated . The next day, in the operating room, radioactive lymph nodes were detected in all cases using the same gamma probe that was used to detect the hot spot for the us - guided pre - operative imaging . Surgical slnb was performed in all cases, followed by alnd in cases of positive percutaneous biopsy . The surgeon was able to recognize the sentinel lymph node biopsy marked by the radiologist with the skin mark and correlate with the results of the standard slnb (validate the ability of the preoperative diagnostic method to correctly identify the sentinel node). Because the most frequent tumor site is the upper - outer quadrant of the breast, the surgeon generally used the same type of incision of the breast to identify the sentinel node using the gamma probe guide when the conservative surgery was performed . In different cases, the surgeon used a separate incision guided by the skin marker performed by the radiologist . The final histopathological diagnosis of slnb was compared with the results of the cnb of the sentinel node . The accuracy of percutaneous biopsy guided by us and gamma probe over the skin in the pre - operative staging was correlated with final pathologic reports of slnb . Information on histological type and grade and biological characteristics (eg, receptor status or peritumoral vascular invasion of the primary carcinoma) were obtained from the pathology report of the breast specimen . For the pathological staging of the axillae, this study used the guidelines and terminology proposed by the seventh edition of the american joint committee on cancer staging manual . Main patient characteristics are shown in table 1 and include age, type of surgery, location of primary breast cancer, nodal stage at percutaneous biopsy, pathological nodal stage, and biological features of the breast tumor . Sentinel node identification was successful in all patients, although there were no suspicious nodes at us examination . The hot spot was detected over the skin by gamma probe in all 10 patients, and was examined using us - guided cnb in all patients . Multiple sentinel nodes were not detected and no patient had the sentinel node draining to different possible sites . There were no major complications (eg, clinically important bleeding, nerve injury, or infection) related to the cnb procedure . One patient experienced a small amount of bleeding, which was stopped by simple local compression; this was considered a minor complication . In all patients, nodal metastases were found at final diagnosis in 2 of 10 patients (table 1). The definitive histopathological report of slnb expressed the presence of 1 micrometastasis that was not observed in the frozen section at the extemporary analysis . In the second patient, micrometastases were observed in the para - sentinel node at slnb, but no macrometastasis in the sentinel node were found . In all the surgically excised sentinel lymph nodes, the pathologist documented the sign of the previous core needle biopsy, verifying that the biopsied lymph node was really the sentinel node . In women with early breast cancer, slnb has proved to be a safe and accurate method for evaluating axillary disease, and is associated with less morbidity than complete alnd . Nevertheless, slnb is not a problem - free procedure . Patients with documented lymph node involvement at the slnb usually require further alnd as a second step during the same surgical procedure, favoring scar tissue formation and edema, and increasing the rate of complications and the surgery time . Hospital stays are also increased in case of slnb + alnd compared with alnd alone as suggested by goyal et al . . Pre - operative knowledge of an axillary metastatic sentinel lymph node could avoid the intraoperative slnb . In fact, in cases with positive preoperative lymph node cnb, a complete alnd can be performed directly as a primary procedure . The obvious advantages of this approach are suggested by several authors that used different methods to localize the abnormal lymph node before surgery . Had avoided slnb in 30% of patients with a final pathology of metastatic lymph node by performing fine needle aspiration cytology (fnac) of the abnormal sonographic axillary node . Sever et al10 proposed the use of intradermal peritumoral microbubble us contrast agents injection and lymphatic imaging to identify and localize the sentinel node in the preoperative stage . Many studies report a moderate diagnostic sensitivity of the percutaneous biopsy of a morphologically abnormal axillary lymph node and an even lower sensitivity in cases where the biopsied lymph node has an unaltered appearance and the procedure is performed randomly . But how can you differentiate the sentinel node from a regular lymph node especially when the pathological sentinel node is not always the largest one or the morphologically abnormal one? . Found metastatic involvement in 12% of the lymph nodes with normal sonographic appearance, and declared that us - guided biopsy would miss the sentinel node in 36% of the cases, resulting in false - negative axillary cnb, partly due to failure to identify the lymph node or the abnormal region of the lymph node to biopsy . Furthermore, there is little information in the literature regarding the accuracy criteria for performing an axillary us . Adding gamma probe over the skin could increase the sensitivity of percutaneous us - guided biopsy, providing a good approximation of detection of true sentinel lymph node . Theoretically, it is well established that a negative sentinel lymph node is currently equivalent to disease - free axillae . The majority (about 70%) of women with clinically negative axillae will prove to be microscopically negative as well . On the other hand, it is also documented that a certain percentage of the false - negative sentinel node cnbs are for micrometastases or isolated tumor s cells . There is no current evidence showing that submicroscopic metastases predict an adverse outcome or that they require treatment . Several authors [1820] demonstrated the absence of axillary recurrence during long - term follow - up in patients with sentinel node micrometastasis in which alnd was not performed . Several studies were done, and one of the most recent addressing this question participated in an ibcsg trial that compared complete alnd (used in cases with positive slnb) to follow - up (in patients with node - free macrometastasis disease). Similarly, we speculate that patients with micrometastases and false - negative sentinel node proved by cnb, gamma probe and us - guided biopsy, may not need intraoperative slnb, particularly since follow - up with ultrasonography, and eventually pet, could detected early axillary disease that can be adequately treated later by therapeutic axillary dissection . Nevertheless, avoiding the morbidity of slnb must be weighed against the risk of harboring axillary micrometastases that may potentially seed occult metastatic disease after a percutaneous biopsy . In the clinical context, considering a patient s expected life span and associated health problems, this situation might be defined as a minimal acceptable risk . Slnb has also a false - negative rate (5% according to veronesi et al .) And this is the reason why these patients are generally subject to regular follow - up with clinical and us examination of the axilla . We also suggest that cases with negative sentinel node percutaneous gamma probe and us - guided biopsy should be similarly monitored . Several studies reported that the sensitivity of us in identifying axillary adenopathy has been increased by cytology sampling of the suspicious lymph node; the approach is limited by the high rate of false - negatives results of the aspiration tecnique . In other words, the unacceptably high rate of false - negatives results makes nodal fnac an unreliable method that cannot be used to avoid intraoperative slnb . As known, there are a number of possible reasons for a false - negative result at fnac: inadequate specimen sampling, a low number of metastatic lymph nodes, small - sized metastasis, and failure to visualize true sentinel lymph node during the us examination of the axilla . The aim of this study was to verify the feasibility of this innovative method to detect and to biopsy the sentinel node in order to improve confidence in identification of the metastatic lymph node in the preoperative phase . In our opinion the decision to use large cnb rather than fnac was made because the amount of tissue taken is greater than with the latter, providing a high negative predictive value . It is reasonable that increasing the volume of tissue obtained from 1 step by cnb can be more effective than multiple samples of fnac to obtain a final diagnosis . The sensitivity of the method could be increased by performing several cnbs (23 or 35 samples according to garcia - ortega) from the cortex of the presumed sentinel node . Do not recommend the use of cnb because of the potential complications of this procedure (eg, bleeding and nerve damage); previous reports have shown that such interventional procedures in all breast cancer patients are not cost - effective . On the other hand, complications can be reduced if the procedures are performed by an experienced, dedicated breast radiologist (within the breast unit). This feature is of major importance because, in early the stage, the lymph nodes usually have normal findings at us, and they are frequently small in size . Consequently, the cutting needle must be sampling only the cortical layer if performed in a technically adequate way . The second element is identification of the axillary sentinel lymph node preoperatively using the gamma probe over the skin . Combining the use of the gamma probe, with which the true sentinel node can be identified with reasonable confidence, and the us - guided cnb that can avoid the sampling error dictated by the reasons mentioned above (in case of fnac), the diagnostic sensitivity should, theoretically, increase . As a minimally invasive staging procedure, us - guided lymph node biopsy under percutaneous gamma probe surveillance seems attractive because it provides information during the preoperative period that could allow avoidance of intraoperative slnb if the sentinel node is negative, and hence, avoid alnd in case of micrometastasis or isolated tumor s cells . The most relevant predictive factor for sentinel node metastases is the primary carcinoma peritumoral vascular invasion . It could be suggested that whenever this pathology (or other findings) finding is found in association with a negative percutaneous sentinel node biopsy performed under us gamma probe guidance, intraoperative slnb should be mandatory . Have reported the pre - operative use of gamma probe over the skin to detect axillary sentinel node under us guidance . The latter had evaluated the sentinel node s location using the gamma probe and then marked it with a hook wire, while motomura used the gamma probe combined with fnac to obtain the sample . To our knowledge, no other published study evaluated gamma probe - assisted sonographic localization combined with cnb of the presumed sentinel node as a one -step procedure in the pre - treatment phase of breast cancer patients . First, this is a pilot study that analyzed the feasibility of a new pre - treatment method proposed for detecting the sentinel node . Second, the patient population is small because this is a pilot study, hence the results do not have a statistical impact . However, the concepts presented could improve the quality of lymph node sampling, particularly in early breast cancer patients . This could be particularly useful in cases that need slnb before neoadjuvant chemotherapy . The pre - treatment sentinel node evaluation technique described in this pilot study had shown a perfect concordance with the histological findings, even if the number of subjects enrolled in this study was relatively low . The accuracy and the clinical implications of the method in conclusion, gamma probe - assisted sonographic localization associated with cnb of the sentinel node in early breast cancer patients could be a feasible and accurate new method . Further studies should investigate the definitive role of this method in pre - treatment breast cancer staging . Particularly, it is necessary to investigate the clinical impact of this method in avoiding slnb, especially in cases when percutaneous sentinel node cnb is negative, without primary carcinoma peritumoral vascular invasion, in clinically node - negative breast disease patients.
Since the first fontan procedure was introduced by fontan in 1971, the surgical technique has evolved and the survival rate has shown dramatic improvement . Recently, fontan surgery has been performed as an operation of the final stage for congenital heart disease with single ventricle physiology1). In fontan circulation therefore, the pulmonary vascular resistance (pvr) must be lower than the antegrade transpulmonary flow resistance2). Thus, even a mild increase in the pvr may interfere with pulmonary circulation which in turn may result in decreased preload of ventricle, diminished cardiac output, and declined exercise tolerance3,4). Loss of pulsatility of pulmonary flow, reduced growth of the pulmonary vasculature, micro and macro thromboembolism in pulmonary circulation and reduced ventricular function are underlying mechanisms that induce the high pvr in fontan circulation5,6,7). Several studies8,9,10,11,12) reported the pulmonary vasodilating therapy had an effective influence on fontan patients so we want to determine whether inhaled iloprost as a pulmonary vasodilator might have a role in the clinical symptoms, hemodynamics and exercise capacity of fontan patients . Ethical approval for this study and permission for acquiring, analyzing and reporting of patient's data were obtained in accordance with the guidelines of the institutional review board of gachon university gil medical center (no . An 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion (doe). A physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area, liver and spleen were not palpable . Recently, she felt more difficult to climb the stairs due to shortness of breath . The inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks . Pre and post medication, laboratory test - including pro brain natriuretic peptide, cardiac catheterization, bicycle ergometer test, nyha class, and 6-minute walking test - were performed . After 12 weeks, the patient's systemic blood pressure and o2 saturation were increased . Six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii ., the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr . The results are shown in table 1 . A 22-year - old male with double outlet right ventricle after 12 weeks, the patient's systemic blood pressure, o2 saturation, and nyha class were not changed significantly ., the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr . An 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion (doe). A physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area, liver and spleen were not palpable . Recently, she felt more difficult to climb the stairs due to shortness of breath . The inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks . Pre and post medication, laboratory test - including pro brain natriuretic peptide, cardiac catheterization, bicycle ergometer test, nyha class, and 6-minute walking test - were performed . After 12 weeks, the patient's systemic blood pressure and o2 saturation were increased . Six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii ., the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr . A 22-year - old male with double outlet right ventricle status post fontan surgery at age of five years complained of mild doe only . After 12 weeks, the patient's systemic blood pressure, o2 saturation, and nyha class were not changed significantly ., the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr . Despite the increased survival rate of patients who has congenital heart disease with single ventricular physiology due to advanced surgical technique, the fontan surgery is still a palliative procedure . Development of the fontan failure state might occur gradually over time; ventricular dysfunction, aggravated cyanosis, hepatic dysfunction, coagulopathy, protein loosing enteropathy, plastic bronchitis, arrhythmia etc.1). Progressive decreasing cardiac output, and increasing central venous pressure can be also observed in fontan physiology with age1). Most patients who underwent cardiac transplant long after fontan surgery had elevated pvr before and immediately after the operation13). Indeed, pvr is an important factor in the progressive decline in efficiency of the fontan physiology . The loss of pulsatile blood flow of pulmonary circulation of fontan patients is positively correlated with high pvr . The lowering of pvr may increase pulmonary blood flow and preload reserve in fontan physiology, and for this reason the selective pulmonary vasodilating therapy is interesting as a means to improve the fontan circulation8). In addition, nonpulsatile pulmonary blood flow may reduce capillary recruitment and endothelial function . As a result of endothelial dysfunction, impairment in production of nitric oxide, prostacyclin and prolonged overexpression of endothelin-1 level can be observed5,14). Most studies were based on high level of endothelin-1 in fontan patients9,10), the endothelin receptor antagonist induced improvement of clinical, hemodynamic parameters and exercise capacity . Experiences with sildenafil therapy on fontan patients have also been reported11,12). In a recent study, rhodes et al.15) reported on the effect of inhaled iloprost on exercise function of fontan patients . In this article the authors administered a single treatment with iloprost nebulizer just before exercise and concluded that iloprost improves the peak oxygen pulse and peak vo2 of patients with fontan physiology . In our two case studies, we found that 12 weeks of iloprost therapy showed an association with improvement of clinical, hemodynamic parameters and exercise capacity in patients with fontan physiology . First case was considered fontan failure because of aggravated doe, functional class and pitting edema . Unlike previous study using iloprost once, we used iloprost for three month period and positive results were achieved without major side effects . In addition, we found that iloprost was effective for fontan patients with or without symptoms . Although more advanced curable treatments of fontan physiology have not been developed, pulmonary vasodilator therapy on fontan circulation is one of good suggestion for symptomatic treatment of fontan failure and improvement of the cardiac output.
Angiomyolipoma is a benign tumor that is histologically composed of groups of mature adipose tissue intermixed with convoluted thick - walled blood vessels, interlacing bundles, and irregularly arranged sheets of smooth muscle, with the kidney as the most frequent site of involvement . Extrarenal angiomyolipoma can occur in organs such as the liver, lung, uterus, and skin . Only two instances of this tumor arising in the tongue have previously been reported [1, 2]. A 61-year - old woman was referred to our hospital with a mass in the left proglossis that was painless, but had enlarged slowly for 5 years . The mass measured 20 mm 20 mm and was soft and dome shaped with a dark violet - colored surface (figure 1). The mass was well - demarcated and easily dissected with an ultrasonic surgical aspirator (figure 2). Histopathological examination showed an encapsulated lesion composed of a proliferation of an intricate mixture of mature adipose tissue, blood vessels, and smooth muscle (figure 3(a)). The patient's postoperative course was uneventful, and there were no signs of recurrence at followup at 18 months (figure 4). Koizumi et al . Reported a case of angiomyolipoma in the centre of the tongue . Described a case of a angiomyolipomatous hamartoma arising in the left lateral border of the tongue . In the present case, angiomyolipoma arose in the proglossis and it had a diagnosis of hemangioma because of its hardness and color . Because the three principal components in angiomyolipoma, regardless of its location, vary greatly in proportion and distribution, its heterogeneity may cause diagnostic confusion . The differential diagnosis includes lipomatous or myolipomatous tumours, angiomyoma, angiolipoma, hemangioma, fibroma, and fibrolipomatous hyperplasia . Angiomyolipoma could be considered a hamartoma, but there is no consensus that these lesions are a single entity . Ide et al . Reported that the present lesion may not be a classic oral angiomyolipoma because it was poorly circumscribed and not encapsulated . They suggested the term angiomyolipomatous hamartoma to designate this lesion . In agreement with ide et al ., we diagnosed the present case as an angiomyolipoma because the mass had enlarged gradually with active proliferation and was well - encapsulated . In particular, tongue cancer and hemangioma are tumors with many blood vessels in the proposed area of surgical excision . For preservation of the nervous system and avoidance of bleeding, an ultrasonic surgical aspirator is effective in the resection of these tongue tumors [35]. In our case,
In particular, osteoimmunology investigates how the immune system impacts on bone turnover in physiological and pathological conditions through the immunoskeletal interface . Researchers recently hav - egained better understanding of the dialogue between immune and bone cells and a new reading register of bone remodeling is emerging, in which the various phases of bone formation and resorption, that coexist in a dynamic equilibrium, are under strict immunological control . Bone and immune system are functionally integrated through complex homeostatic networks and, in all respects, osteoporosis could be considered a chronic immune mediated inflammatory disease which shares clinical and biological features with many other inflammatory conditions, as well as of other immune mediated diseases . In this context immune and bone systems appear to be integrated and sharing signaling pathways and regulatory mechanisms whose understanding provides a framework for obtaining new insights for the discovery of novel treatments for diseases related to both systems . Thus osteoimmunology appears definitely as an interdisciplinary research and clinical field which also allowed new pathogenetic and clinical interpretations of well - known and common diseases, such as osteoporosis . Its fields of interest are constantly expanding, thus enriching with an increasing number of translational implications, even in clinical practice and in various branches of medicine . Here, the most important concepts in osteoimmunology are addressed in the context of physiopatological states bridging these two organ systems, including osteoporosis, ageing, menopause, inflammatory arthritis, cancer, dysmetabolism and neurological disorders . Purpose of this review is to outline a new panorama of osteoimmunology that is not limited to immune mediated bone turnover but also consider, in the light of the latest findings in this field, interesting connections with other systems and regulatory functions over bone remodeling . Bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells, osteocytes (ocy), osteoblasts (ob) and osteoclasts (oc). In addition, many other types of cells take part in bone composition, including cartilage, stromal, hematopoietic and mesenchimal stem cells, all linked by a dense network of signals . Antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment . Bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis . Bone turnover requires two coordinated processes: bone formation, driven by ob and bone resorption, mediated by oc . Ocy, through a complex network of tiny channels, transmit mechanical and microtraumatic signals leading to the activation of repair processes . Moreover, ocy synthesize the bone matrix proteins which, along with the mineral component, determine the quality of the bone . Ob are the precursors of ocy, i.e, the structural cells in the bone, and interact with oc to drive their differentiation and function . The mesenchymal stem cell (msc) from which the ob originate, can also give rise to chondrocytes, marrow stromal cells, and adipocytes . There are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells (mspcs), that have specific relationships with the different kinds of niche, i.e. The surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [7 - 9]. The stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular (ocr) stem cell that generate ob, chondrocytes, and reticular marrow stromal cells, but not adipocytes . They are characterized by the expression of the bone morphogenetic protein (bmp) antagonist gremlin 1 (grem 1). The perisinusoidal msc population also contains nes - gfp, leptin receptor (lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential . The osteoblast precursor cells (obp) after increasing the osteopontin receptor (cd44) and the receptor for stromal cell - derived factor 1 - sdf1 (cxcr4) expression, migrate and become mature ob, attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation . Oc are multinucleated myeloid cells, specialized to remove mineralized bone matrix through the production of lysosomal enzymes, such as tartrate - resistant acid phosphatase (trap) and catepsin k, against which a selective inhibitor (odanacatib) has been recently synthesized to be employed in osteoporotic patients . They derive from a bone marrow precursor which gives rise also to professional antigen presenting cells (apc), i.e. Dendritic cells and macrophages . Ob, ocy and oc continuously communicate with each other to optimize the quality of the bone . For example, ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor (m - csf), receptor activator of nuclear factor - kb (nf - kb) ligand (rankl) and other co - stimulatory factors . The binding of rank receptor on oc and their precursors by its ligand rankl, expressed by ob and stromal cells, is the main activation signal for bone resorption . The ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors (ocp), enabling the rank / rankl signal . Osteoprotegerin (opg) inhibits osteoclastogenesis by acting as a decoy receptor of rankl, thus preventing bone resorption . Rank receptor on oc, through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 (tr - af6), bound to its cytoplasmic tail, activates nf - kb and other transcription factors, such as mapks, c - fos, activator protein 1 (ap1), up to nuclear factor of activated t cells (nfatc1), the hub of various signaling pathways . Simultaneously, the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins, such as the immunoreceptor tyrosine - based activation motif (itam) and fc - receptor common gamma (fcr) subunit . In the nucleus nfatc1, together with other transcription factors, such as ap1, pu.1, microphthalmia - associated transcription factor (mitf) and cyclic amp responsive - element - binding protein (creb), induces oc specific genes, including those codifying for calcitonin receptor, cathepsin k and trap, leading to oc differentiation and proliferation . Many other receptor pathways interact with rank, some costimulators and amplificators, others inhibitors and modulators, and many of these are shared by immune cells . An inhibitor receptor system for rank signal is ephrin (eph) b2/b4 . Ephb2 receptor on oc, stimulated by ephb4 ligand on ob, inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade . A peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals: the cell expressing the receptor and the one that expresses the ligand influence each other at the same time . Therefore, ephb4 activation on ob, through the induction of osteogenetic regulatory genes, contemporaneously favours the coupling of bone formation and resorption . The canonical wingless (wnt)/ catenin pathway, involved mainly in the response to mechanical load, promotes differentiation, proliferation and mineralization activity of ob and also inhibits their apoptosis . It encompasses a family of proteins that bind to complex transmembrane receptors, formed by the association of frizzled (fz) proteins and low density lipoprotein related receptors (lrp-5, lrp-6) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation . Wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages, stimulating the differentiation towards osteoblastogenesis . Wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy . The bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . At the same time, various natural inhibitors, produced both by ob and ocy, exert a negative feed - back control on wnt system, such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 (dkk-1), the secreted frizzled related protein (sfrp) and the sclerostin, synthesized by the sost gene, which binds to lrp5/6 receptors . The inflammatory cytokine tumor necrosis factor- (tnf-) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression . Cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis . An anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis . Other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules, now in the experimental stage . Bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells, osteocytes (ocy), osteoblasts (ob) and osteoclasts (oc). In addition, many other types of cells take part in bone composition, including cartilage, stromal, hematopoietic and mesenchimal stem cells, all linked by a dense network of signals . Antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment . Bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis . Bone turnover requires two coordinated processes: bone formation, driven by ob and bone resorption, mediated by oc . Ocy, through a complex network of tiny channels, transmit mechanical and microtraumatic signals leading to the activation of repair processes . Moreover, ocy synthesize the bone matrix proteins which, along with the mineral component, determine the quality of the bone . Ob are the precursors of ocy, i.e, the structural cells in the bone, and interact with oc to drive their differentiation and function . The mesenchymal stem cell (msc) from which the ob originate, can also give rise to chondrocytes, marrow stromal cells, and adipocytes . There are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells (mspcs), that have specific relationships with the different kinds of niche, i.e. The surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [7 - 9]. The stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular (ocr) stem cell that generate ob, chondrocytes, and reticular marrow stromal cells, but not adipocytes . They are characterized by the expression of the bone morphogenetic protein (bmp) antagonist gremlin 1 (grem 1). The perisinusoidal msc population also contains nes - gfp, leptin receptor (lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential . The osteoblast precursor cells (obp) after increasing the osteopontin receptor (cd44) and the receptor for stromal cell - derived factor 1 - sdf1 (cxcr4) expression, migrate and become mature ob, attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation . Oc are multinucleated myeloid cells, specialized to remove mineralized bone matrix through the production of lysosomal enzymes, such as tartrate - resistant acid phosphatase (trap) and catepsin k, against which a selective inhibitor (odanacatib) has been recently synthesized to be employed in osteoporotic patients . They derive from a bone marrow precursor which gives rise also to professional antigen presenting cells (apc), i.e. Dendritic cells and macrophages . Ob, ocy and oc continuously communicate with each other to optimize the quality of the bone . For example, ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor (m - csf), receptor activator of nuclear factor - kb (nf - kb) ligand (rankl) and other co - stimulatory factors . The binding of rank receptor on oc and their precursors by its ligand rankl, expressed by ob and stromal cells, is the main activation signal for bone resorption . The ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors (ocp), enabling the rank / rankl signal . Osteoprotegerin (opg) inhibits osteoclastogenesis by acting as a decoy receptor of rankl, thus preventing bone resorption . Rank receptor on oc, through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 (tr - af6), bound to its cytoplasmic tail, activates nf - kb and other transcription factors, such as mapks, c - fos, activator protein 1 (ap1), up to nuclear factor of activated t cells (nfatc1), the hub of various signaling pathways . Simultaneously, the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins, such as the immunoreceptor tyrosine - based activation motif (itam) and fc - receptor common gamma (fcr) subunit . In the nucleus nfatc1, together with other transcription factors, such as ap1, pu.1, microphthalmia - associated transcription factor (mitf) and cyclic amp responsive - element - binding protein (creb), induces oc specific genes, including those codifying for calcitonin receptor, cathepsin k and trap, leading to oc differentiation and proliferation . Many other receptor pathways interact with rank, some costimulators and amplificators, others inhibitors and modulators, and many of these are shared by immune cells . An inhibitor receptor system for rank signal is ephrin (eph) b2/b4 . Ephb2 receptor on oc, stimulated by ephb4 ligand on ob, inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade . A peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals: the cell expressing the receptor and the one that expresses the ligand influence each other at the same time . Therefore, ephb4 activation on ob, through the induction of osteogenetic regulatory genes, contemporaneously favours the coupling of bone formation and resorption . The canonical wingless (wnt)/ catenin pathway, involved mainly in the response to mechanical load, promotes differentiation, proliferation and mineralization activity of ob and also inhibits their apoptosis . It encompasses a family of proteins that bind to complex transmembrane receptors, formed by the association of frizzled (fz) proteins and low density lipoprotein related receptors (lrp-5, lrp-6) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation . Wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages, stimulating the differentiation towards osteoblastogenesis . Wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy . The bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . At the same time, various natural inhibitors, produced both by ob and ocy, exert a negative feed - back control on wnt system, such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 (dkk-1), the secreted frizzled related protein (sfrp) and the sclerostin, synthesized by the sost gene, which binds to lrp5/6 receptors . The inflammatory cytokine tumor necrosis factor- (tnf-) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression . Cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis . An anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis . Other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules, now in the experimental stage . Both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling . Inflammation and bone turnover share the same mediators, such as cytokines and transcription factors . Various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk . T lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone (pth), in physiological and pathological conditions . During inflammatory diseases or in conditions characterized by low - grade systemic inflammation, such as menopause and aging, oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes . However, bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells . Two mechanisms are involved: the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors, and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage . The final effect of t lymphocytes on bone depends on their activation state and their specific phenotype . The prevailing bone marrow t cells are activated central memory cd8 + lymphocytes, secreting relatively high levels of effector cytokines, mainy tnf-. These cells are abundant in postmenopausal women with osteoporotic fractures . T helper (th17) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis, rheumatoid arthritis, periodontal disease, and inflammatory bowel disease . Th17 cells induce osteoclastogenesis by secreting interleukin (il)-17, rankl, tnf-, il-1, and il-6, along with low levels of ifn-. Moreover, il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc . Treg exert anti - osteo - clastogenic activity by producing suppressor cytokines, including il-4, il-10, and tgf- . For example it is interesting to note how even the cells of the immune sytem, mainly activated t and b lymphocytes and dendritic cells, express rankl . Moreover, ran - kl, the principal osteoclastogenic mediator, stimulates bone resorption through the nfatc1, which is also a crucial factor in the immune system regulation . Rankl, initially regarded as activator of apc by t lymphocytes, also plays an important role in the generation of treg, which suppress the development of cd8 + lymphocytes into cytotoxic cells . The expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus . Thus, depending on the context in which it acts, rankl can stimulate or suppress immune reactions . Other examples of shared receptor signals are the immunoglobulin (ig)-like receptors which amplify the nfatc1 signal . The toll like receptors (tlr), stimulated by pathogen associated molecular patterns (pamp), utilize traf6 in their cascade signaling . Tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells, leading to bone resorption stimulation . Their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases . The osteoclast associated receptor (oscar), which belongs to the ig - like receptor family, mediates interactions between ob and oc and is also involved in the regulation of both the adaptive and innate immunity . It associates with the adaptor molecule fcr subunit, which harbors an immunoreceptor tyrosine - based activation motif (itam) critical for calcium signaling activation in the immune system . Also, the itam - harboring adaptor dap12 plays a role in oc differentiation and function . Therefore, receptors such as dap12 and ig - like receptors associated with fcr, initially characterized in myeloid cells and in natural killer lymphocytes, are also involved in rank induced osteoclastogenesis . Tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells . For example, cd80/cd86 blocks oc generation by binding to ctla4, an inhibitory molecule of the monocyte induced t lymphocyte costimulation, which is highly expressed on treg surface . Cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components, such as type i collagen . In addition to osteoclastic bone resorption, cathepsin k is also implicated in dendritic cell activation through tlr 9 . Moreover, cathepsin k supports the secretion of il-6 and il-23, inflammatory cytokines involved in the production of th17 cells, which in turn promote osteoclastic bone resorption . Eph receptors and their associated ligands, expressed by cells found within the bone marrow microenvironment, including ob and oc, are implicated in the regulation of physiological and pathological bone remodeling, but also are central in many other different cellular processes including, in addition to immune regulation, angiogenesis, neuronal development and neoplastic metastatization . Osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . Since sema4d also regulates a variety of immune functions, such as antigen presentation, b lymphocyte activation and chemotaxis of monocytes, it could be regarded as an osteoimmunological mediator . The matrix glycoprotein osteopontin (opn), produced by different types of cells, including immune cells, oc, endothelial and epithelial cells, increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44, essential for cell migration, and by directly enhancing oc attachment to bone extracellular matrix (ecm), required for ocp activation . As a consequence of bone resorption, more opn is further released from the ecm into the bone microenvironment and into the blood, thus amplifying local and systemic osteoclastogenesis . Rankl and cd40l expressed on t cells, apc, stromal cells and ob, activate the cognate receptors rank and cd40 in ocp and ob, respectively . Cd40/cd40l signaling promotes macrophage activation and differentiation, antibody isotype switching, and the development of b cell memory . Cd40l also increases the commitment of msc to the osteoblastic lineage . Through cd80/86 signaling in ocp boh these conditions are mimicked by continuous pth infusion (cpth), whereas daily or intermittent pth injection (ipth), therapeutically employed in several osteoporotic conditions, stimulates bone formation . Pth binds its receptors (ppr) on stromal cells, ob, and ocy but also on t cells and macrophages . The catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells . The pth anabolic effect is mediated by wnt signaling activation: pth increases -catenin levels in ob, promotes lrp6 signaling and decreases the production of sclerostin . Cpth stimulates bone cells and immune cells to release growth factors and cytokines, including il-6 and tnf-, which induce th17 cell differentiation and the production of il-17, that plays a pivotal role in the pth induced bone loss . Tnf- in turn stimulates oc formation and activity via multiple mechanisms, including increased rankl production . Moreover, tnf- upregulates the expression of cd40 in ob and stromal cells, increasing their sensitivity to cpth and suppressing opg . Bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth . Therapy with teriparatide, a form of ipth treatment, increases the bone marrow levels of wnt10. Bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 . Inflammation results in disturbances in the immunoskeletal interface, i.e. The convergence of cells and cytokines that regulate both the immunity that the bone, causing osteoporosis . The inflammatory cytokines tnf-, il-1, il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption . An excessive or abnormal immune activation can induce osteoporosis, as for example in autoimmune diseases, infections and also in senile and postmenopausal osteoporosis . All these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . However, in addition to osteoclastogenic cytokines, there are also cytokines which counteract bone resorption and exert osteoblastogenic properties, resulting in a complex bone remodeling cytokine network . Each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act, the maturation stage of target cells and/or the influence of other cytokines . For example, the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling, by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl . However, in postmenopausal osteoporosis, inflammation or infections, the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression . In fact, ifn- is a powerful stimulator of class ii major histocompatibility complex (mhcii) antigen expression on apc, with consequent increased stimulation of t cells through their antigen specific receptor (tcr), inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [35 - 40]. A cascade of cytokines drives ocp homing, differentiation and activation . Circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells, in relation to the different cytokines implicated . The activation of cd8 t cells by oc induces il-2, il-6, il-10 and ifn- production . Ocp can also enhance the expression of the suppressor of cytokine signaling (socs). Treg, whose main marker is the transcription factor foxp3, balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4, which in turn inhibits oc activity . Endothelial cells, activated by il-1 and tnf-, attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules (cam), such as icam-1 and cd44 . These cd14 + monocytoid cells, under the stimulation of rankl, become activated bone resorbing oc . Resident tissue macrophages of bone, termed osteal macrophages, are predominantly located adjacent to ob . Osteal macrophages play diverse roles in skeletal homeostasis, their specific functions depending on the macrophage subset considered . A central function of macrophages is their phagocytic ability . In particular, efferocytosis (phagocytosis of apoptotic cells) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis . Finally, not only the immune system regulates bone remodeling, but also the bone is able to influence the immune system, actively interacting with immune cells . The same bone cells would then be able to influence or even also perform many immune functions, such as cytokine production and antigen presentation [46 - 48]. In this sense, the bone would be regarded as a sort of expanded immune system . Cytokines secreted by bone cells drive naive t cell differentiation into several lineages, leading to expansion of mature t cell populations that further regulate bone homeostasis . Oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 (oc - induced regulatory cd8 t cells). In turn, these cd8 + treg cells suppress bone resorption, decrease inflammatory / osteoclastogenic cytokine production, and stimulate bone formation, creating a regulatory loop: oc and rankl induce treg, and then treg blunt osteoclastic bone resorption . Osteocalcin, expressed on mature bone cells, regulates the production of thymic - seeding t lymphoid progenitors . They secrete a variety of proinflammatory and immunosuppressive factors . A subset of mesenchimal cells expressing osterix, a marker of bone precursors, regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 (igf-1) production . Skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8, ifn-, gm - csf and mif . They inhibit b cell proliferation, differentiation, and antibody production, and can also directly inhibit t cell function, rendering them anergic or shifting their phenotype to that of functional regulatory cells . Msc induce macrophages to switch from classically activated proinflammatory (m1) to alternatively activated anti - inflam - matory (m2) phenotype, and inhibit mast cell degranulation attenuating allergic reactions . Msc express active tlr, through which they sense bone microenvironment, recognizing exogenous (bacterial products) and endogenous (heat shock proteins, rna) danger signals . The common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules . Both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling . Inflammation and bone turnover share the same mediators, such as cytokines and transcription factors . Various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk . T lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone (pth), in physiological and pathological conditions . During inflammatory diseases or in conditions characterized by low - grade systemic inflammation, such as menopause and aging, oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes . However, bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells . Two mechanisms are involved: the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors, and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage . The final effect of t lymphocytes on bone depends on their activation state and their specific phenotype . The prevailing bone marrow t cells are activated central memory cd8 + lymphocytes, secreting relatively high levels of effector cytokines, mainy tnf-. These cells are abundant in postmenopausal women with osteoporotic fractures . T helper (th17) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis, rheumatoid arthritis, periodontal disease, and inflammatory bowel disease . Th17 cells induce osteoclastogenesis by secreting interleukin (il)-17, rankl, tnf-, il-1, and il-6, along with low levels of ifn-. Moreover, il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc . Treg exert anti - osteo - clastogenic activity by producing suppressor cytokines, including il-4, il-10, and tgf- . For example it is interesting to note how even the cells of the immune sytem, mainly activated t and b lymphocytes and dendritic cells, express rankl . Moreover, ran - kl, the principal osteoclastogenic mediator, stimulates bone resorption through the nfatc1, which is also a crucial factor in the immune system regulation . Rankl, initially regarded as activator of apc by t lymphocytes, also plays an important role in the generation of treg, which suppress the development of cd8 + lymphocytes into cytotoxic cells . The expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus . Thus, depending on the context in which it acts, rankl can stimulate or suppress immune reactions . Other examples of shared receptor signals are the immunoglobulin (ig)-like receptors which amplify the nfatc1 signal . The toll like receptors (tlr), stimulated by pathogen associated molecular patterns (pamp), utilize traf6 in their cascade signaling . Tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells, leading to bone resorption stimulation . Their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases . The osteoclast associated receptor (oscar), which belongs to the ig - like receptor family, mediates interactions between ob and oc and is also involved in the regulation of both the adaptive and innate immunity . It associates with the adaptor molecule fcr subunit, which harbors an immunoreceptor tyrosine - based activation motif (itam) critical for calcium signaling activation in the immune system . Also, the itam - harboring adaptor dap12 plays a role in oc differentiation and function . Therefore, receptors such as dap12 and ig - like receptors associated with fcr, initially characterized in myeloid cells and in natural killer lymphocytes, are also involved in rank induced osteoclastogenesis . Tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells . For example, cd80/cd86 blocks oc generation by binding to ctla4, an inhibitory molecule of the monocyte induced t lymphocyte costimulation, which is highly expressed on treg surface . Cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components, such as type i collagen . In addition to osteoclastic bone resorption, cathepsin k is also implicated in dendritic cell activation through tlr 9 . Moreover, cathepsin k supports the secretion of il-6 and il-23, inflammatory cytokines involved in the production of th17 cells, which in turn promote osteoclastic bone resorption . Eph receptors and their associated ligands, expressed by cells found within the bone marrow microenvironment, including ob and oc, are implicated in the regulation of physiological and pathological bone remodeling, but also are central in many other different cellular processes including, in addition to immune regulation, angiogenesis, neuronal development and neoplastic metastatization . Osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . Since sema4d also regulates a variety of immune functions, such as antigen presentation, b lymphocyte activation and chemotaxis of monocytes, it could be regarded as an osteoimmunological mediator . The matrix glycoprotein osteopontin (opn), produced by different types of cells, including immune cells, oc, endothelial and epithelial cells, increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44, essential for cell migration, and by directly enhancing oc attachment to bone extracellular matrix (ecm), required for ocp activation . As a consequence of bone resorption, more opn is further released from the ecm into the bone microenvironment and into the blood, thus amplifying local and systemic osteoclastogenesis . Rankl and cd40l expressed on t cells, apc, stromal cells and ob, activate the cognate receptors rank and cd40 in ocp and ob, respectively . Cd40/cd40l signaling promotes macrophage activation and differentiation, antibody isotype switching, and the development of b cell memory . Cd40l also increases the commitment of msc to the osteoblastic lineage . Through cd80/86 signaling in ocp boh these conditions are mimicked by continuous pth infusion (cpth), whereas daily or intermittent pth injection (ipth), therapeutically employed in several osteoporotic conditions, stimulates bone formation . Pth binds its receptors (ppr) on stromal cells, ob, and ocy but also on t cells and macrophages . The catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells . The pth anabolic effect is mediated by wnt signaling activation: pth increases -catenin levels in ob, promotes lrp6 signaling and decreases the production of sclerostin . Cpth stimulates bone cells and immune cells to release growth factors and cytokines, including il-6 and tnf-, which induce th17 cell differentiation and the production of il-17, that plays a pivotal role in the pth induced bone loss . Tnf- in turn stimulates oc formation and activity via multiple mechanisms, including increased rankl production . Moreover, tnf- upregulates the expression of cd40 in ob and stromal cells, increasing their sensitivity to cpth and suppressing opg . Bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth . Therapy with teriparatide, a form of ipth treatment, increases the bone marrow levels of wnt10. Bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 . Inflammation results in disturbances in the immunoskeletal interface, i.e. The convergence of cells and cytokines that regulate both the immunity that the bone, causing osteoporosis . The inflammatory cytokines tnf-, il-1, il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption . An excessive or abnormal immune activation can induce osteoporosis, as for example in autoimmune diseases, infections and also in senile and postmenopausal osteoporosis . All these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . However, in addition to osteoclastogenic cytokines, there are also cytokines which counteract bone resorption and exert osteoblastogenic properties, resulting in a complex bone remodeling cytokine network . Each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act, the maturation stage of target cells and/or the influence of other cytokines . For example, the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling, by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl . However, in postmenopausal osteoporosis, inflammation or infections, the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression . In fact, ifn- is a powerful stimulator of class ii major histocompatibility complex (mhcii) antigen expression on apc, with consequent increased stimulation of t cells through their antigen specific receptor (tcr), inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [35 - 40]. A cascade of cytokines drives ocp homing, differentiation and activation . Circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells, in relation to the different cytokines implicated . The activation of cd8 t cells by oc induces il-2, il-6, il-10 and ifn- production . Ocp can also enhance the expression of the suppressor of cytokine signaling (socs). Treg, whose main marker is the transcription factor foxp3, balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4, which in turn inhibits oc activity . Endothelial cells, activated by il-1 and tnf-, attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules (cam), such as icam-1 and cd44 . These cd14 + monocytoid cells, under the stimulation of rankl, become activated bone resorbing oc . Resident tissue macrophages of bone, termed osteal macrophages, are predominantly located adjacent to ob . Osteal macrophages play diverse roles in skeletal homeostasis, their specific functions depending on the macrophage subset considered . A central function of macrophages is their phagocytic ability . In particular, efferocytosis (phagocytosis of apoptotic cells) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis . Finally, not only the immune system regulates bone remodeling, but also the bone is able to influence the immune system, actively interacting with immune cells . The same bone cells would then be able to influence or even also perform many immune functions, such as cytokine production and antigen presentation [46 - 48]. In this sense, the bone would be regarded as a sort of expanded immune system . Cytokines secreted by bone cells drive naive t cell differentiation into several lineages, leading to expansion of mature t cell populations that further regulate bone homeostasis . Oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 (oc - induced regulatory cd8 t cells). In turn, these cd8 + treg cells suppress bone resorption, decrease inflammatory / osteoclastogenic cytokine production, and stimulate bone formation, creating a regulatory loop: oc and rankl induce treg, and then treg blunt osteoclastic bone resorption . Osteocalcin, expressed on mature bone cells, regulates the production of thymic - seeding t lymphoid progenitors . A subset of mesenchimal cells expressing osterix, a marker of bone precursors, regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 (igf-1) production . Skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8, ifn-, gm - csf and mif . They inhibit b cell proliferation, differentiation, and antibody production, and can also directly inhibit t cell function, rendering them anergic or shifting their phenotype to that of functional regulatory cells . Msc induce macrophages to switch from classically activated proinflammatory (m1) to alternatively activated anti - inflam - matory (m2) phenotype, and inhibit mast cell degranulation attenuating allergic reactions . Msc express active tlr, through which they sense bone microenvironment, recognizing exogenous (bacterial products) and endogenous (heat shock proteins, rna) danger signals . The common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules . Osteoporosis is a systemic disease of the skeleton, whose main features are loss of bone mass, bone mineral density (bmd) decrease and disruption of bone microarchitecture, so the skeleton becomes fragile, exposing patients to increased risk of fractures . Aside from senile and postmenopausal osteoporosis, the first recognized types of primary osteoporosis, many other causes of secondary osteoporosis have been subsequently recognized, for example vitamin d and calcium deficits, lack of sun exposure, immobility, drugs such as cortisone, malabsorption syndromes, endocrine and dismetabolic diseases such as diabetes, disthyroidisms, hypercortisolism, and so on . Only later, clinical and experimental findings evidenced a close connection between osteoporosis and immune mediated inflammatory conditions, for example, rheumatoid arthritis (ra) and acquired immune deficiency syndrome (aids), and a common inflammatory background has been finally discovered as pathogenetic factor even in conditions of major osteoporotic risk, such as old age and estrogen deficiency [57 - 59]. More recently, other unpredictable pathological conditions, such as obesity, are coming out as potential osteoporotic risk factors . Even in these cases, the main pathogenetic mechanism leading to bone tissue alteration seems to be inflammation . From this point of view osteoporosis could be therefore regarded as an immune mediated disease in which immune activation, through the induction of cytokine production and inflammation, leads to a remodeling of oc and ob activity and dysregulation of bone turnover with consequent increased bone resorption and osteoporosis . Paradigmatic examples of the link between inflammation and osteoporosis are inflammatory arthritis, mainly ra (fig . 1). Ra is an autoimmune disease that is characterized by inflammation of the synovial joint, leading to severe structural damage and bone destruction . An increased bone resorption is the main pathogenetic mechanism in both disease progression leading to juxta - articular bone erosions and irreversible joint damage and systemic osteoporosis . Bisphosphonates, drugs used for some time in the therapy of osteoporosis, are potent inhibitors of oc activity both in the primitive and secondary osteoporosis, such as that associated with autoimmune diseases . A decreased bmd in the spine and hip and higher prevalence of osteoporosis have been described in ra patients . In early untreated ra, bmd is related to longer symptom duration, the presence of rheumatoid factor (rf) and cyclic citrulinated peptide antibodies (anti - ccp), disease activity score, and the presence and progression of joint damage [1, 19]. Monoclonal antibodies (moab) against various proinflammatory cytokines and their receptors, such as tnf-, are useful in preventing and/or reversing bone erosions as well as systemic osteoporosis . Autoimmune reactions induce rankl expression and subsequent osteoclastogenesis . That the rank/ rankl / opg pathway is central to the osteoporosis pathogenesis is confirmed by the elevated antiresorptive capacity of denosumab, an anti - rankl moab, utilized in osteoporosis therapy . Activated immune cells at sites of inflammation produce a wide spectrum of proinflammatory and osteoclastogenic cytokines, resulting in bone erosions, osteitis, and peri - inflammatory and systemic bone loss . Local peri - inflammatory bone loss and osteitis occur early and precede and predict erosive bone destruction in ra . Moreover, peri - inflammatory bone formation is impaired, resulting in non - healing of erosions, and this allows a local vicious circle of inflammation between synovitis, osteitis, and local bone loss . Rankl is highly expressed in the ra synovium, and inflammation - mediated bone damage is largely attributable to its abnormally high expression . In addition to activated t lymphocytes and macrophages, a pivotal role in inducing bone erosions is also played by rankl - expressing b cells, as highlighted by the observation of the therapeutic effect of anti - cd20 antibody in ra . Cd4+t - cells, especially th17 cells, play a prominent role, particularly in the initiation of systemic immune response in ra . The interaction between immune and mesenchymal cells in joints, including synovial fibroblasts, which are characterized by hyperproliferative and hyperactive properties in response to an inflammatory environment, is of paramount importance in rheumatoid inflammation . In fig . Thus, in the affected joints, hyperplasia of the synovial membrane is characterized by both hyperproliferation of synovial fibroblasts and massive infiltration of inflammatory immune cells, including cd4 + t cells and innate immune cells . Autoimmune diseases, including arthritis, often result from an imbalance between treg cells and th17 cells . The th17 cells derived from foxp3 + t cells in ra comprise a novel th17 cell subset with a distinct pattern of gene expression and arthritogenic properties . The fate of plastic foxp3 + t cells may be a key determinant of the treg / th17 balance that is critically involved in the self - tolerance and autoimmunity . Mesenchymal cells are a determinant of the development of ra that links the systemic immune response and the local disorder in the joints . Mesenchymal cells contribute to the th17 mediated chronic inflammation by promoting the migration of th17 cells to the inflamed joint and concomitant increase in il-17 production . Thus, the interaction of immune and mesenchymal cells plays a key role in both the chronic inflammation and bone destruction in ra . In particular, pathogenetic autoreactive immune cells migrate into joints and activate the mesenchymal cells resident in joint, such as synovial fibroblasts . Moreover, since soluble inhibitors of the wnt pathway, such as dkk-1, produced by synovial fibroblasts, are upregulated by tnf-, antibodies against dkk1 could be able to both promote bone formation and prevent bone erosions in ra . Osteoporosis has long been regarded as simply the consequence of the menopausal estrogen decline . Os- the progression of articular erosions is clearly driven by proinflammatory and osteoclastogenic cytokines produced by immune cells in the inflamed joint . Both activated lymphocytes and macrophages stimulate osteoclast differentiation by producing proinflammatory mediators . Activated lymphocytes of the synovial pannus overexpress rankl and tnf- that stimulate bone marrow cd11b+ ocp to proliferate and enter the bloodstream where they themselves produce inflammatory factors amplifying inflammation . Activated macrophages in the inflamed joints produce various chemokines which drive the localization of periarticular bone ocp . The stimulation of osteoclastogenesis induced by the high concentrations of rankl and tnf- results in bone resorption . These cells do not produce antiosteoclastogenic cytokines, such as ifn- or il4, while expressing high levels of rankl and secrete il-17, that in turn stimulates ob, synoviocytes and fibroblasts to express rankl, and induces macrophages to produce proinflammatory cytokines, such as tnf- and il-6 in the synovium amplifying local inflammation . Il-17 also induces the synthesis of enzymes capable of degrading the bone matrix such as matrix metalloproteinases (mmp). These effects are balanced by treg that inhibit bone destruction by suppressing oc formation through both cell - cell contact and the secretion of inhibitory cytokines such as tgf-, il-4 and il-10 . Teoimmunology, suggesting that immune cells take part in the bone changes typical of menopause, has led to a shift in the concept of osteoporosis, that is currently considered an inflammatory condition . Post - menopausal osteoporosis is a clear example of the mutual influences between immune system, bone and endocrine system . Like many other hormones, in addition to specific target organs (breast and reproductive system), estrogens have their receptors also on immune cells and bone, as well as on bone marrow precursors . (3) shows the effects of estrogen deficiency on cells and molecules involved in bone metabolism . Menopausal estrogen decline leads to proliferation and activation of t cells by increasing mhcii expression on monocytes and apc function and by inhibiting t cell apoptosis . These effects leads to the expansion of activated t lymphocytes resulting in hyperproduction of inflammatory cytokines and chronic oc stimulation which is responsible for bone loss and increased fracture risk . Maintenance of inflammatory reactions leading to bone resorption and skeletal fragility is also present during senescence and inflammaging (fig . 4), that is the condition of chronic inflammation characterizing aging, as the result of the immune system s ability to cope with stressors . Inflammaging is now considered the background of a broad range of age - related diseases with an inflammatory pathogenesis . Many of the biological mechanisms implicated in the aging process, such as cell senescence, proinflammatory immune profile, apoptosis and metabolism imbalance, are also implicated in bone remodeling . Also in the absence of overt inflammatory diseases, the heightened catabolic signals induced by inflammation enhance apoptosis of ob and muscle cells, causing both osteoporosis and sarcopenia . During aging, the lifelong exposure to oxidative stress and chronic antigenic load leads to the loss of the regulatory process which counteracts t cell activation induced bone resorption . In aged people, lipid oxidation mediated by ros oxidized polyunsaturated fatty acids induce the association of ppar with -catenin and promote its degradation . Oxidized lipids also potentiate oxidative stress, enhance ob apoptosis and inhibit bmp-2 induced ob differentiation . Antioxidant agents seem to have some action on bone remodeling: resveratrol decreases nf - k activation induced by rankl and oc differentiation and also promotes osteogenesis in msc via the sirt1/foxo3 axis stimulation . During senescence, besides the impaired treg function, the number of effector memory cells is increased . These are senescent cells with proinflammatory properties, secreting several inflammatory cytokines able to influence bone remodeling . Interestingly, this immunological pattern (accumulation of activated cells and memory / effector lymphocytes secreting proinflammatory cytokines) characterizing immunosenescence, is also peculiar of other immunological conditions associated with osteoporosis, such as ra, aids, chronic viral infections, etc . . The already complex cross - talk between bone and immune system is further expanding as they emerge new mediators involved . Interestingly, the scenario of osteoimmunolgy is increasingly including interconnections with other organs and systems, therefore influencing several homeostatic functions in addition to bone turnover and immunity, such as hemopoiesis, metabolic and neuro - endocrine functions . The crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell (hsc) function and throughout the hemopoiesis . Hsc occupy specific locations in the bone marrow microenvironment, commonly referred to as niches, comprising multiple cell types able to regulate hsc proliferation and differentiation . Hematopoietic and skeletal stem cell homeostasis are closely related: msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis, including kit ligand and stromal - derived factor, but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis, whose cognate receptors are highly expressed by skeletal stem cells . Interestingly, these latter exhibit receptors to circulating hormones, such as leptin and thyroid - stymulating hormone, suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system . Primitive mesenchymal progenitors are more critical for hsc function, whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors . Bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis . The estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation, in particular ifn-, m - csf, tnf-, il-1, il-6 and il-7, in addition to prostaglandins (pge) and reactive oxygen species (ros), which play a driving role in the development of osteoporosis . Of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets, cd3 + cd56 + t lymphocytes, major producers of tnf-, and b220 + igm - b lymphocyte precursors, that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines . The enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women . The same ifn-, which in some situations could be protective against osteoporosis, during estrogen deficiency has a prevailing osteoclastogenic stimulating action . The estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone . The age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells, in addition to an impaired treg function . A peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines, mainly tnf-, il-1, il-6 and il-17 . These cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle . Both the increased transcriptional activity of nf - kb due to genotoxic, inflammatory, and oxidative stresses in aging, and the chronic p53 activation induced by the age - related progressive loss of telomere length, result in impaired ob proliferation and osteoporosis development . Cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment . Hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors, which surprisingly do not take part in the regulation of immunity and hemopoiesis . Ob, together with msc, are crucial components of the niche of growth of hsc . Ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential . The wnt antagonist secreted frizzled - related protein 1 (sfrp1) is involved in osteoblastic mediated hsc regulation; through its regulation of ob, pth controls the hematopoietic niche function, involving crosstalk with wnt signaling . The upregulation of notch ligand protein jagged-1 (jag1) expression in ob and the increased notch signaling are involved in increasing hsc numbers; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche, inducing quiescence of these cells . Osteopontin, an ob - secreted protein, participates in hsc location and is a negative regulator of their proliferation . Also, ocy derived from ob that become embedded within the bone matrix, are involved in the complex regulation of hemopoiesis through the osteoimmune interface: mainly by the production of sclerostin, they appear to have an inhibitory effect on hsc support . Even oc, as well as activated endothelial cells, are likely involved in the mobilization of hsc by cytokine induced cam expression . Being the only cells capable of bone resorption, in addition to allowing the renewal of the skeleton, they also open the space in the bone marrow for hematopoietic cells . Moreover, osteoclastic bone resorption releases calcium, which attracts and retains calcium sensing receptor expressing hsc at the endosteal region . Macrophages play diverse roles in the bone and marrow . At the sites of bone remodeling, a subset of hsc is located in close proximity to megakaryocytes in the bone marrow, where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors, such as platelet derived growth factor (pdgf)-, to promote hsc proliferation . The bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases, including both hematologic malignancies and metastases of solid tumors, mainly breast and prostatic cancer . In multiple myeloma, an osteolytic hematological malignancy characterized by an important skeletal involvement, neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob . In addition to osteolytic factors, myeloma cells produce dkk1, which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . On the other hand, the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma, perpetuating a vicious circle of mutual reinforcement . A promising new field of interest in this regard is the osteo - immune - oncology . There is in fact a close relationship between immune regulation of bone turnover and tumor growth . Often, cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . Tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix, fueling a vicious circle of impaired bone turnover and tumor proliferation (fig . Not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth, as previously believed, but sometimes some patterns of immune activation can be facilitators of the development of tumors . Then the block of rankl, in addition to inhibiting bone resorption, also decreases tumor growth, enhances apoptosis of malignant cells and diminishes proneoplastic inflammation . Osteal macrophages, as well as macrophages in other tissues, have a role in tumor progression, supporting cancers which preferentially metastasize to the skeleton, in particular breast and prostate cancer . Osteal macrophages expressing cd68, a phagocytic capacity marker of cells infiltrating metastatic lesions, could facilitate tumor establishment and growth . Tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2, which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . As for bone, adipose tissue is a kind of immune tissue and produces immunoregulatory factors . Adipocytes and ob derive from the same msc, as well as visceral adipose tissue macrophages and oc derive from the same hsc . Shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . By action of peroxisome proliferative activated receptor gamma (ppar), the principal regulator of adipogenesis, the msc differentiates into adipocytes rather than into ob . In contrast, the expression of runt - related transcription factor 2 (runx2), associated with ob differentiation, and osterix, an ob zinc finger mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal . Not surprisingly, ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes, also increase the risk of osteoporotic fractures . Leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior, where it promotes satiety and prevents weight gain . Leptin can also directly signal through its receptor expressed on immunocytes, where it induces tnf- and il-6 production by monocytes, chemokines by macrophages, and th1 cytokines from polarized cd4 + t cells . Adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance . It can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . Tnf- and il-6 inhibit adiponectin production in adipocytes . Adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob . Fat tissue, mainly visceral fat tissue, may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf-, which stimulate oc activity through the regulation of the rankl / rank / opg pathway . Leptin and adiponectin act on the bone through different signaling pathways with contrasting effects (table 2). It promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling . Therefore, leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . Various cell populations within the fat tissue can exacerbate the development of the chronic, low - grade inflammation associated with obesity and metabolic dysfunction . White adipocytes store lipid as triglycerides within unilocular droplets, and are responsive to various stimuli, such as insulin . Brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated . Beige adipocytes, dispersed within white adipose tissue, can dissipate heat, similarly to classical brown adipocytes, when exposed to cold temperatures or after prolonged highfat diet feeding . Visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity, have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity . Conversely, in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines . Notwithstanding epidemiological evidence indicates that an increase in body mass index (bmi) is related to increased bone mass, probably due to the effects of the mechanical load of the body weight on the skeleton, not always obesity, and mainly the increase in visceral fat mass, has a positive effect on bone . Obesity is associated with increased leptin and decreased adiponectin serum levels . Moreover, in the visceral adipose tissue there are activated macrophages producing cytokines . In obese subjects, especially with central obesity, in which the visceral fat is increased, there is a significant increase of several markers of inflammation such as c reactive protein (crp), il-1, il-6 and tnf-, that can alter the quality of the bone, making it more fragile . Therefore, these new clinical and experimental evidences definitively connect obesity and other related pathological conditions, such as metabolic syndrome, nonalcoholic fatty liver disease (nafld) and diabetes, to impaired bone health and fragility fractures [90 - 93]. In conclusion, it is currently emerging that adipose tissue, liver, bone and immune system modulate each other through a complex network of interconnected signals . Both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines, sex hormones, redox balance, ppar and liver x receptors (lxr). Osteocalcin, an ob secreted bone matrix noncollagen protein, takes part in calcium metabolism and in bone deposition, as well as in energy homeostasis and glucose metabolism . Fetuin - a, produced by the liver, is involved in the regulation of bone metabolism and insulin action, vascular calcification, neurodegenerative diseases and cancer cell proliferative signaling . Ob lineage cells express receptors for adiponectin, leptin, angiotensin ii, insulin and insulin - like growth factor-1, able to influence rank / rankl / opg signaling pathway . Interestingly, these hormones are implicated in the pathogenesis of type - ii diabetes, obesity and hypertension, all of which are risk factors for metabolic syndrome . The development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products (age) and receptor of age (rage). Age are the end products of glucose, as well as other sugars, proteins, lipids, and nucleic acids via a non - enzymatic glycosylation reaction, able to bind to multiple membrane receptor proteins, including rage . Age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . In particular, age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis . The binding of age to organic bone matrix may also increase the fragility of bones . Autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions . However, excessive autophagy is harmful to cells and leads to damage or massive death of cells . Autophagy deficiency increases oxidative stress levels in ob, decreases bone mineralization and induces ran - kl secretion . It is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . In turn sympathetic nerves produce catecholamines, which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings . Hsc express catecholaminergic receptors, suggesting that they are able to directly respond to signals from the sympathetic nervous system . Adrenergic signaling reduces cxcl12 expression in the bm . Affecting maintenance of hsc and the differential lineage commitment . An association between major depression and osteoporosis has been recognized [96 - 99]. The prevalence of low bmd is higher in people with depression than the general population . Interestingly, patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression, who also have a lower bmd compared with healthy controls [100, 101]. Neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal (hpa) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone (crh). Leptin is expressed in the hypothalamus and pituitary gland, where it modulates corticotropin - releasing hormone and acth secretion, probably acting in an autocrine - paracrine manner . It inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla, activating the sympathoadrenal axis . The neuromodulator serotonin or 5-hydroxy - tryptamine (5ht), which is an important vasoactive mediator of allergic reactions, in addition, it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling, resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . On the other hand, 5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . Moreover, serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb, which regulates many genes involved in circadian rhythms in different tissues (period 1,2,3 and clock genes). There are two anatomically and functionally distinct pools of serotonin: the first one, synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system, where it functions as neurotransmitter; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1, regulated by the low - density lipoprotein receptor (ldlr)-related protein-5 (lrp5). The circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine, where it stimulates peristalsis in response to the meal . The peripherally produced serotonin acts as a hormone inhibiting bone formation, whereas serotonin produced in the brain functions as a neurotransmitter, enhancing bone formation and limiting bone resorption . The neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin, in an integrated homeostatic network with fat tissue and metabolism (fig . A portion of 5ht is also produced by the mammary gland, where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects . In the course of pregnancy, lactation and menopause, the local serotonin production increases, contributing to the increased bone resorption typical of these phases of the woman's life . Small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier, have recently been proposed for the treatment of osteoporosis . In alzheimer s disease (ad), a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid peptide (a) deposits, neurofibrillary tangles, chronic inflammation, and neuronal loss, increased bone fracture rates and reduced bmd are commonly observed, suggesting common denominators between both disorders . Amyloid precursor protein (app) is transmembrane protein involved in ad pathogenesis: app gene mutations characterize early - onset ad, and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production . Rage, acting as an app / a binding partner, is therefore implicated in the pathogenesis of both ad and osteoporosis . The role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin, the s100 family, and the high mobility group box 1(hmgb1). In particular, hmgb1 is a proin - flammatory cytokine released from activated macro - phages, that promotes rankl - induced oc differentiation in a rage - dependent manner . Shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth . The release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation . In addition to the hyperproduction of proinflammatory cytokines, rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . The upregulation of rankl, particularly in myeloma and breast cancer, promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . In this context, bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia . For example, bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well . The block of rankl by the monoclonal antibody denosumab, in addition to inhibiting bone resorption, is also useful in reducing tumor growth, in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia . Its predominant effect on bone is through the central nervous system: by stimulating specific receptors (lepr) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus, which interact each other, it increases central sympathetic activity . Inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors (2-ar) expressed on ob, suppressing their differentiation and osteocalcin production . Serotonin is synthesized in serotonergic neurons of the central nervous system, exerting various functions in the brain; it is also synthesized in the gut, mediating different peripheral functions . It acts on bone cells using three different receptors: through 5ht1b receptors, it negatively regulates bone mass, while it enhances bone formation through 5ht2b and 5ht2c receptors . The crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell (hsc) function and throughout the hemopoiesis . Hsc occupy specific locations in the bone marrow microenvironment, commonly referred to as niches, comprising multiple cell types able to regulate hsc proliferation and differentiation . Hematopoietic and skeletal stem cell homeostasis are closely related: msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis, including kit ligand and stromal - derived factor, but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis, whose cognate receptors are highly expressed by skeletal stem cells . Interestingly, these latter exhibit receptors to circulating hormones, such as leptin and thyroid - stymulating hormone, suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system . Primitive mesenchymal progenitors are more critical for hsc function, whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors . Bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis . The estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation, in particular ifn-, m - csf, tnf-, il-1, il-6 and il-7, in addition to prostaglandins (pge) and reactive oxygen species (ros), which play a driving role in the development of osteoporosis . Of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets, cd3 + cd56 + t lymphocytes, major producers of tnf-, and b220 + igm - b lymphocyte precursors, that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines . The enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women . The same ifn-, which in some situations could be protective against osteoporosis, during estrogen deficiency has a prevailing osteoclastogenic stimulating action . The estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone . The age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells, in addition to an impaired treg function . A peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines, mainly tnf-, il-1, il-6 and il-17 . These cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle . Both the increased transcriptional activity of nf - kb due to genotoxic, inflammatory, and oxidative stresses in aging, and the chronic p53 activation induced by the age - related progressive loss of telomere length, result in impaired ob proliferation and osteoporosis development . Cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment . Hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors, which surprisingly do not take part in the regulation of immunity and hemopoiesis . Ob, together with msc, are crucial components of the niche of growth of hsc . Ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential . The wnt antagonist secreted frizzled - related protein 1 (sfrp1) is involved in osteoblastic mediated hsc regulation; through its regulation of ob, pth controls the hematopoietic niche function, involving crosstalk with wnt signaling . The upregulation of notch ligand protein jagged-1 (jag1) expression in ob and the increased notch signaling are involved in increasing hsc numbers; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche, inducing quiescence of these cells . Osteopontin, an ob - secreted protein, participates in hsc location and is a negative regulator of their proliferation . Also, ocy derived from ob that become embedded within the bone matrix, are involved in the complex regulation of hemopoiesis through the osteoimmune interface: mainly by the production of sclerostin, they appear to have an inhibitory effect on hsc support . Even oc, as well as activated endothelial cells, are likely involved in the mobilization of hsc by cytokine induced cam expression . Being the only cells capable of bone resorption, in addition to allowing the renewal of the skeleton, they also open the space in the bone marrow for hematopoietic cells . Moreover, osteoclastic bone resorption releases calcium, which attracts and retains calcium sensing receptor expressing hsc at the endosteal region . Macrophages play diverse roles in the bone and marrow . At the sites of bone remodeling, a subset of hsc is located in close proximity to megakaryocytes in the bone marrow, where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors, such as platelet derived growth factor (pdgf)-, to promote hsc proliferation . The bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases, including both hematologic malignancies and metastases of solid tumors, mainly breast and prostatic cancer . In multiple myeloma, an osteolytic hematological malignancy characterized by an important skeletal involvement, neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob . In addition to osteolytic factors, myeloma cells produce dkk1, which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . On the other hand, the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma, perpetuating a vicious circle of mutual reinforcement . A promising new field of interest in this regard is the osteo - immune - oncology . There is in fact a close relationship between immune regulation of bone turnover and tumor growth . Often, cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . Tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix, fueling a vicious circle of impaired bone turnover and tumor proliferation (fig . 5). In skeletal metastases, not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth, as previously believed, but sometimes some patterns of immune activation can be facilitators of the development of tumors . Then the block of rankl, in addition to inhibiting bone resorption, also decreases tumor growth, enhances apoptosis of malignant cells and diminishes proneoplastic inflammation . Osteal macrophages, as well as macrophages in other tissues, have a role in tumor progression, supporting cancers which preferentially metastasize to the skeleton, in particular breast and prostate cancer . Osteal macrophages expressing cd68, a phagocytic capacity marker of cells infiltrating metastatic lesions, could facilitate tumor establishment and growth . Tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2, which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . As for bone, adipose tissue is a kind of immune tissue and produces immunoregulatory factors . Adipocytes and ob derive from the same msc, as well as visceral adipose tissue macrophages and oc derive from the same hsc . Shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . By action of peroxisome proliferative activated receptor gamma (ppar), the principal regulator of adipogenesis, the msc differentiates into adipocytes rather than into ob . In contrast, the expression of runt - related transcription factor 2 (runx2), associated with ob differentiation, and osterix, an ob zinc finger containing transcription factor, shift the equilibrium towards the osteoblastogenesis . Mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal . Not surprisingly, ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes, also increase the risk of osteoporotic fractures . Leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior, where it promotes satiety and prevents weight gain . Leptin can also directly signal through its receptor expressed on immunocytes, where it induces tnf- and il-6 production by monocytes, chemokines by macrophages, and th1 cytokines from polarized cd4 + t cells . Adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance . It can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . Tnf- and il-6 inhibit adiponectin production in adipocytes . Adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob . Fat tissue, mainly visceral fat tissue, may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf-, which stimulate oc activity through the regulation of the rankl / rank / opg pathway . Leptin and adiponectin act on the bone through different signaling pathways with contrasting effects (table 2). It promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling . Therefore, leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . Various cell populations within the fat tissue can exacerbate the development of the chronic, low - grade inflammation associated with obesity and metabolic dysfunction . White adipocytes store lipid as triglycerides within unilocular droplets, and are responsive to various stimuli, such as insulin . Brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated . Beige adipocytes, dispersed within white adipose tissue, can dissipate heat, similarly to classical brown adipocytes, when exposed to cold temperatures or after prolonged highfat diet feeding . Visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity, have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity . Conversely, in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines . Notwithstanding epidemiological evidence indicates that an increase in body mass index (bmi) is related to increased bone mass, probably due to the effects of the mechanical load of the body weight on the skeleton, not always obesity, and mainly the increase in visceral fat mass, has a positive effect on bone . Obesity is associated with increased leptin and decreased adiponectin serum levels . Moreover, in the visceral adipose tissue there are activated macrophages producing cytokines . In obese subjects, especially with central obesity, in which the visceral fat is increased, there is a significant increase of several markers of inflammation such as c reactive protein (crp), il-1, il-6 and tnf-, that can alter the quality of the bone, making it more fragile . Therefore, these new clinical and experimental evidences definitively connect obesity and other related pathological conditions, such as metabolic syndrome, nonalcoholic fatty liver disease (nafld) and diabetes, to impaired bone health and fragility fractures [90 - 93]. In conclusion, it is currently emerging that adipose tissue, liver, bone and immune system modulate each other through a complex network of interconnected signals . Both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines, sex hormones, redox balance, ppar and liver x receptors (lxr). Osteocalcin, an ob secreted bone matrix noncollagen protein, takes part in calcium metabolism and in bone deposition, as well as in energy homeostasis and glucose metabolism . Fetuin - a, produced by the liver, is involved in the regulation of bone metabolism and insulin action, vascular calcification, neurodegenerative diseases and cancer cell proliferative signaling . Ob lineage cells express receptors for adiponectin, leptin, angiotensin ii, insulin and insulin - like growth factor-1, able to influence rank / rankl / opg signaling pathway . Interestingly, these hormones are implicated in the pathogenesis of type - ii diabetes, obesity and hypertension, all of which are risk factors for metabolic syndrome . The development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products (age) and receptor of age (rage). Age are the end products of glucose, as well as other sugars, proteins, lipids, and nucleic acids via a non - enzymatic glycosylation reaction, able to bind to multiple membrane receptor proteins, including rage . Age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . In particular, age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis . The binding of age to organic bone matrix may also increase the fragility of bones . Autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions . However, excessive autophagy is harmful to cells and leads to damage or massive death of cells . Autophagy deficiency increases oxidative stress levels in ob, decreases bone mineralization and induces ran - kl secretion . It is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . In turn sympathetic nerves produce catecholamines, which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings . Hsc express catecholaminergic receptors, suggesting that they are able to directly respond to signals from the sympathetic nervous system . Adrenergic signaling reduces cxcl12 expression in the bm . Affecting maintenance of hsc and the differential lineage commitment . An association between major depression and osteoporosis has been recognized [96 - 99]. The prevalence of low bmd is higher in people with depression than the general population . Interestingly, patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression, who also have a lower bmd compared with healthy controls [100, 101]. Neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal (hpa) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone (crh). Leptin is expressed in the hypothalamus and pituitary gland, where it modulates corticotropin - releasing hormone and acth secretion, probably acting in an autocrine - paracrine manner . It inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla, activating the sympathoadrenal axis . The neuromodulator serotonin or 5-hydroxy - tryptamine (5ht), which is an important vasoactive mediator of allergic reactions, is also likely involved in interactions between the central nervous and immune systems . In addition, it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling, resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . On the other hand, 5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . Moreover, serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb, which regulates many genes involved in circadian rhythms in different tissues (period 1,2,3 and clock genes). There are two anatomically and functionally distinct pools of serotonin: the first one, synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system, where it functions as neurotransmitter; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1, regulated by the low - density lipoprotein receptor (ldlr)-related protein-5 (lrp5). The circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine, where it stimulates peristalsis in response to the meal . The peripherally produced serotonin acts as a hormone inhibiting bone formation, whereas serotonin produced in the brain functions as a neurotransmitter, enhancing bone formation and limiting bone resorption . The neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin, in an integrated homeostatic network with fat tissue and metabolism (fig . A portion of 5ht is also produced by the mammary gland, where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects . In the course of pregnancy, lactation and menopause, the local serotonin production increases, contributing to the increased bone resorption typical of these phases of the woman's life . Small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier, have recently been proposed for the treatment of osteoporosis . In alzheimer s disease (ad), a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid peptide (a) deposits, neurofibrillary tangles, chronic inflammation, and neuronal loss, increased bone fracture rates and reduced bmd are commonly observed, suggesting common denominators between both disorders . Amyloid precursor protein (app) is transmembrane protein involved in ad pathogenesis: app gene mutations characterize early - onset ad, and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production . Rage, acting as an app / a binding partner, is therefore implicated in the pathogenesis of both ad and osteoporosis . The role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin, the s100 family, and the high mobility group box 1(hmgb1). In particular, hmgb1 is a proin - flammatory cytokine released from activated macro - phages, that promotes rankl - induced oc differentiation in a rage - dependent manner . Shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth . The release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation . In addition to the hyperproduction of proinflammatory cytokines, rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . The upregulation of rankl, particularly in myeloma and breast cancer, promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . In this context, bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia . For example, bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well . The block of rankl by the monoclonal antibody denosumab, in addition to inhibiting bone resorption, is also useful in reducing tumor growth, in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia . Its predominant effect on bone is through the central nervous system: by stimulating specific receptors (lepr) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus, which interact each other, it increases central sympathetic activity . Inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors (2-ar) expressed on ob, suppressing their differentiation and osteocalcin production . Serotonin is synthesized in serotonergic neurons of the central nervous system, exerting various functions in the brain; it is also synthesized in the gut, mediating different peripheral functions . It acts on bone cells using three different receptors: through 5ht1b receptors, it negatively regulates bone mass, while it enhances bone formation through 5ht2b and 5ht2c receptors . Immune system and skeletal system interact with each other both in physiological and pathological conditions . Considerable progress has been made in clarifying the crosstalk between bone and immune system that occurs in a complex and dynamic bone microenvironment . A central role in this crosstalk osteoimmunology therefore represents a new approach to studying osteoporosis that in the past was not considered an inflammatory condition . Moreover, the discoveries of the existence of the immunoskeletal interface has also highlighted interesting repercussions for a wide range of pathological conditions beyond osteoporosis, including infections, autoimmune diseases, and neoplasia, in which same pathogenetic pathways are shared . Osteoporosis could be therefore considered as a systemic model of integrated signaling pathways and cytokines working in a cooperative fashion . Further important research horizons are opened with the extension of the discoveries of osteoimmunology and the disclosure of the immunoskeletal interface, whose practical implications may provide novel therapies for diseases other than osteoporosis, by targeting specific transcription factors, cytokines and their receptors . The correct understanding and decoding of the complex language through which immune system and bone communicate, although still in its dawn, is the essential requirement for the identification of such potentially useful therapeutic targets for both osteoporosis and other correlated inflammatory conditions, which share same mediators and signaling pathways.
Lymphangioleiomyomatosis (lam) is a rare idiopathic disease that exclusively occurs in women of childbearing age, and this disease is characterized by the proliferation of abnormal smooth muscle cells in the lungs and along the thoracic and abdominal lymphatics (1 - 3). The disease primarily affects the lungs in the majority of cases, but extrapulmonary lam occasionally occurs with or without subsequent involvement of the lungs . Lam involvement of the uterus is extremely rare and there have been only a few such reported cases (2 - 7), and the imaging findings of uterine lam have only been briefly mentioned in two reports (2, 5). In this article, we report the cross - sectional imaging findings of extensive lam involving the uterus and pelvic cavity in a 29-year - old woman with tuberous sclerosis complex (tsc) and we propose that certain imaging features and the clinical history may suggest the diagnosis . To the best of our knowledge, this is the first report of the imaging findings of lam involvement of female pelvic organs . When she was ten years old, she was diagnosed with tsc based on her history of seizure and mental retardation . Five years previously, she underwent a pelvic magnetic resonance imaging (mri) study for the evaluation of menometrorrhagia and dysmenorrhea . The mr images showed several small (less than 3 cm) intramural and subserosal hemorrhagic lesions in the uterus and hematometra (fig . Adenomyotic cysts and uterine leiomyomas with red degeneration were considered in the differential diagnosis based on the imaging at that time . Endometrial drainage was performed for the hematometra, but no further treatment such as hormonal therapy was done . For the current visit, her serum ca-125 level was 204.01 u / ml, the ca 19 - 9 level was 50.11 u / ml and the hemoglobin level was 8.7 g / dl . 1c) showed multiple, large, lobulated, thick - walled cystic masses involving the uterus and the entire pelvic cavity, and these masses extended to the lower abdomen . The attenuation of the cystic masses was higher than that of simple fluid, which suggested hemorrhagic contents . Both kidneys were enlarged by multiple, various sized masses with attenuations matching fat and soft tissue, which are findings compatible with angiomyolipomas . Pelvic mr imaging showed huge, irregularly - shaped, cystic masses involving the uterus and parauterine pelvic cavity, and these masses were predominantly hyperintense on the fat - saturated t1-weighted image (fig . 1d) and they were heterogeneously hypointense and hyperintense on the t2-weighted image (fig . These cystic masses were thought to originate from the uterine myometrium on the sagittal t2-weighted image (fig . Multiple loculated fluid collections with fluid - fluid levels were seen in the cul - de - sac . On the contrast - enhanced t1-weighted image markedly enlarged lam involving the uterus with extension to the pelvic cavity was suggested as the most probable diagnosis based on the follow - up imaging and clinical findings . The chest ct showed numerous, well - defined, thin - walled cysts that were diffusely distributed throughout the lungs (fig . 1 g). Because the cysts were regularly round in shape and there was no associated nodular lesion, pulmonary lam was the suggested diagnosis, but she had no pulmonary symptoms . Brain mr imaging showed multiple cortical and subcortical tubers as well as several subependymal nodules in the bilateral lateral ventricles, and this represented tuberous sclerosis (fig . The intraoperative findings revealed that the uterus was enlarged and distorted by multiple subserosal and intramural hemorrhagic cystic masses . The cystic masses also involved both adnexae, the pelvic side wall and the omentum . There were severe adhesive changes between the cystic lesions and the sigmoid colon and bladder . Microscopic examination revealed that the tumor was composed of atypical smooth muscle cells (lam cells) arranged in short fascicles around dilated lymphatics and a ramifying network of endothelium - lined spaces (fig . Immunohistochemical staining showed that the tumor cells were diffusely positive for smooth muscle actin and they were strongly multifocally positive for human melanin black-45 (hmb-45) (fig . The patient's hospital course was uneventful and she was discharged on the tenth postoperative day . Lam results from the proliferation of abnormal - appearing smooth muscle cells in the lymph vessels, which causes dilatation and obstruction in the lymph vessels and this results in cystic collections of chylous material (1). These smooth muscle cells are classified in the family of perivascular epithelioid cells (pec), which is a cell type that is constantly present in a group of tumors, including lam, sugar tumors of the lung and pancreas, renal angiomyolipomas and clear cell myomelanocytic tumor of the falciform ligament (8, 9). These so - called " pecoma " all express hmb-45 (8). Lam occurs in about 30% of the women with tuberous sclerosis complex (tsc), which is an autosomal dominant multisystem neurocutaneous disorder of highly variable penetrance, and it is characterized by hamartomas, seizure and mental retardation (1). Due to the striking similarities in the pathological processes between the lam and tsc, lam has been considered a forme fruste of tsc, and lam is classified as a tsc - associated or sporadic form (5, 10). Lymphangioleiomyomatosis predominantly affects the lung parenchyma and this is characterized by pulmonary cysts seen on ct . The extrapulmonary manifestation of lam is uncommon and this is mainly located in the retroperitoneum, pelvic cavity and the posterior mediastinum along the lymphatic channels (1 - 7). In a large study of 80 patients with pulmonary lam (11), the ct imaging findings of retroperitoneal lam were described as a low - attenuating (3 - 25 hounsfield unit [hu]), multilobulated mass, and the ultrasound findings were reported as a cystic mass with a thick echogenic rind . Uterine involvement of lam is extremely rare, and to the best of our knowledge, only eight cases of pathologically proven uterine lam have currently been reported (2 - 7). Six cases of uterine lam occurred in patients with the stigmata of tsc, and two patients did not have stigmata of tsc . Most uterine lams were microscopic and they are incidentally found in patients undergoing evaluation for extrauterine disease (2). Menorrhagia and/or pelvic pain have been reported in half of the cases, the same as in our case (2 - 7). The imaging findings of uterine lam were briefly mentioned in two gynaecological reports (2, 5). (2) reported that uterine lam simulated high - stage endometrial stromal sarcoma, and the ct finding of uterine lam was described as a large uterine mass, which was thought to be a fibroid . However, the surgery revealed multiple subserosal and intramural hemorrhagic nodules ranging in size from 2.5 to 4.0 cm and bloody ascites due to tumor perforation . (5) recently reported on a 5.5-cm hypervascular tumor between the uterus and the right ovary that showed low signal intensity on the t1-weighted mr image and intermediate signal intensity on the t2-weighted mr image . This mri finding is quite different from that of retroperitoneal lam and it is rather similar to that of uterine leiomyoma . The pathological examination in that case revealed multiple intramural leiomyomas and several fragments of irregular soft masses composed of hmb 45 positive lam cells . The most remarkable aspects of our case are the initial involvement of lam in the uterus that gradually grew into the pelvic cavity and the intratumoral bleeding . Intratumoral bleeding may be caused by overdistention and rupture of the cysts . Although the ct findings showed highly attenuating cystic masses involving the uterus and pelvic cavity, the mr imaging confirmed the hemorrhagic cystic nature of the masses by their signal intensity and the mr imaging exactly localized the masses . These imaging findings are consistent with the known imaging findings of retroperitoneal lam . However, if a patient is without a clinical history of tsc, then uterine intramural and subserosal leiomyomas with hemorrhagic and cystic degeneration, adenomyotic cysts and/or endometrial cysts, and malignant uterine and/or ovarian tumors with hemorrhage should be considered in the differential diagnosis . In summary, we report here on the extraordinary radiological findings of lam involving the uterus and pelvic cavity in a young woman with tsc . Although uterine lam is rare, radiologists should consider the possibility of this disease when they see multiple cystic uterine or parauterine masses with or without internal hemorrhage in a patient with a history of tsc or pulmonary lam.
Ts is a key enzyme in the de novo synthesis of dtmp, an essential precursor of dna, which catalyses the methylation of dump to dtmp . The critical role of ts in the nucleotide metabolism has made it an important target of a variety of chemotherapeutic agents including 5-fu, 5-fu prodrugs as capecitabine, and novel folate - based ts inhibitor such as raltitrexed and pemetrexed, used in the treatment of colorectal and other solid tumours . Resistance to fluoropyrimidines and other ts inhibitors may occur through a variety of mechanisms including elevated intracellular ts levels resulting from increases in ts transcription and translation . In crc patients the ts intratumoural expression may predict for the sensitivity to 5-fu and other ts inhibitor - based chemotherapies [5, 6] and may also be an important prognostic marker . High ts expression in early stage crc patients seems to predict for poorer overall survival in both chemotherapy - treated and untreated patients following surgery . Also, metastatic crc patients with high ts levels are unlikely to respond to infusional treatment with 5-fu, whereas patients with low ts levels have higher than average response rate [8, 9]. In a previous study, however, we demonstrated that higher ts levels are favourable prognostic factors for disease free and overall survival . To date the real value of the ts level as prognostic factor is doubtful and some authors are in disagreement about it . The ts gene, containing 7 coding exons, is located in chromosome 18q, for which a high percentage of monosomic loss has been reported to be cell - cycle dependent, although some recent evidence points are more oriented towards proliferation - dependence . It can be predicted that mutations in the ts gene might result in a modification of its structure and then in its ability to interact with the fluoropyrimidines . This prediction has been born out in a variety of studies [1215]. S. h. berger and f. g. berger reported that the human colon tumour cell line hct116 produces a variant structural form of ts, in addition to the common form found in all colon cell lines tested . Among the ts overproduction derivatives of hct116, cells overexposing the novel forms are more resistant to fdurd, compared with cells overexpressing the normal form so, an association between drug response and altered ts structure could suggest that the novel ts form, which is encoded by a variant structural ts gene, confers relative resistance to fdurd; this was supported by preliminary kinetic data indicating that this novel form has a reduced affinity for fdump . This variant form presents a replacement of an evolutionary conserved tyrosine by a histidine at residue 33 of the ts polypeptide [17, 18]; this mutation represents the only difference between the two ts forms and must account for the structural and functional differences between them . In an our previous paper we performed the analysis of the ts structure in patients bearing crc to try to demonstrate the presence of that specific mutation, but we did not find it in any patient . Here, we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer and previously untreated and try to find relationship between any hypothetical ts variant form with the 5-fu treatment . We performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes' stages (a, b, and c) and histological grade but we did not find any change in the ts - dna structure . Ts structure was assessed in a series of 68 patients who underwent surgery for primary colorectal carcinoma confirmed histologically and previously untreated . The following exclusion criteria were applied: history of other neoplasias apart of crc and death occurring within 30 days following surgery and due to postoperative complications . The tumours were staged and graded according dukes' system and their clinicopathological features were summarised in tables 1, 2, and 3 . The samples at surgery were divided in two parts: one was frozen at 80 until analysis, and the other portion was embedded in paraffin to confirm histologically the absence of contamination by normal and necrotic tissue and lymphocytes . Genomic dna was extracted from tumour and mucosa samples with a commercial kit purchased from qiagen (kjvenlo, the netherlands) and stored at 20. the ts gene of tumour and mucosa samples was amplified in seven different pcr reactions using dna primers of 1620 bases in length placed in the adjacent intronic regions of exons 1, 2, 3, 4, 5, 6, and 7 and listed in table 4 . Amplification was performed using a perkin elmer model 2400 thermal cycler (boston usa). Reaction mixture included 5 l of 10% dimethylsulfoxide, 5 l of 10 buffer, 1.5 l of 1.5 mm mgcl2, 1 l of 10 mm dntps, 200500 ng of genomic dna, 200 pmol of both upstream and downstream primers, and 1 units of taq dna polymerase, in a final volume of 50 l . Amplification was run for 35 cycles with each cycle consisting in a denaturation step at 95c for 1 minutes, a primer annealing step at 51c for 1 minute, and an extention step at 72c for 2 minutes . Pcr was terminated by incubation at 72c for 10 minutes . The length of the amplification products ranged from 180 to 310 bases . Dna samples, generated with independently repeated pcr products, were sequenced with the sequitherm excel ii dna sequencing kit (epicenter, madison, wi) on a li - cor 4000 (mwg - biotech, ebersberg, germany) sequencer . The enzyme structure obtained by sequencing the ts genomic dna of each sample was performed in 68 patients with operable crc, untreated with previous chemotherapy . Primary tumours and corresponding colonic mucosa were obtained from each patient and the ts genomic structure was performed on each of them, after histological control . 7 patients had g1 histological grade, 36 had g2, and 25 had g3 histological grade; 10 patients had dukes' a tumours, 27 patients had dukes' b, and 31 patients had dukes c cancers . The median age of patients was 67 years; 30 patients were male and 38 female . In all the tumour samples evaluated we did not find any ts - dna variant structure in tumour: all a, b, and c dukes' patients showed stable ts - dna, and also in the germline genome no ts - dna variants have been observed . The structure of the macromolecular target of a cytotoxic drug is a critical determinant of cellular sensitivity to that drug . Recently, ts has become the subject of several studies aimed to elucidate a possible clinical role of ts detection either as determinant of drug resistance or as prognostic marker of the disease and predictive factor of the treatment . Many studies are currently examining the real value of ts expression levels as a prognostic factor, but in the mean time significant uncertainty prevails; whatever its prognostic influence, to date no studies have been able to establish a cut - off all of these in vitro studies have promising implications in the study of the role of ts in clinical practice . Moreover, the unclear meaning of levels not only as a prognostic indicator but also as marker predictive of resistance could be due to mechanisms different from over expression, as mutation . In fact some authors focused their attention on the ts structure with a double aim: development of a new classes of ts - inhibitors with a different mechanism of action and generation of ts mutants to develop gene - therapy strategies . Berger and coworkers showed that a single naturally occurring change, a tyr to his replacement, in the primary structure of the ts molecule confers relative resistance to the ts - directed antimetabolite fdurd in hct 116 cells . The tyr to his mutation is the only difference between the altered form of ts and the normal form and, therefore, must be responsible for the diminished effectiveness of the enzyme as a drug target . The data in those studies, together with previously published experiments, strongly favour the notion that the tyr to his' mutation is responsible for the relative fdurd resistance of cell line hct 116 [15, 16]. However, as noted earlier, it is quite possible that factors in addition to this mutation contribute to the phenotype of hct116 and other colon cell lines . The frequency of the tyr33 to his33 mutation in the normal and in the pathological human populations is unknown . The altered ts may exist as a polymorphism in humans; alternatively, it may have spontaneously arisen during tumorigenesis or during establishment of the cell lines in culture . Distinguishing among these possibilities will be of great utility in assessing whether variant forms of ts identified in cultured cell lines are segregating in the human population and have an impact upon clinical response to 5-fluorouracil therapy in cancer patients . On the basis of this knowledge we hoped to see if in human crc samples it could be possible to find the same tyr33 to his33 substitution in the ts structure and eventually to correlate this finding with some clinicopathological parameters such as age and sex, tumour size and location, histological grade, dukes' stage, 5-fu and raltitrexed treatment, and disease free and overall survivals, to see if that point mutation could be considered a reliable marker of drug resistance and of prognosis . In those samples we did not find that point mutation at the codon 33 . In this study we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer (crc) and previously untreated and try to find a relationship between any hypothetical ts variant form with the 5-fu treatment and prognosis . We performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes' stages (a, b, and c) and histological grade, but we did not find any mutation in the ts - dna structure . The conclusions that could be drown are that in dukes' a, b, and c crcs there are no changes in the ts - dna gene structure and that the evaluation of the ts expression is the main crc prognostic and drug response marker . What remains is to evaluate the ts gene structure of the d metastatic dukes' crcs: in these tumours it might be possible to find ts - dna structural changes related with their higher genomic instability and this fact could give an explanation of the 5-fu drug resistance and worse prognosis.
Colorectal cancer is one of the most common cancers, representing the second (males; 9%) and third (females; 8%) most prevalent cancer in the usa (1), and the third overall leading cause of cancer - related death in the western world (2). The past several decades have seen a dramatic increase in the incidence of colorectal cancer also in japan (3); in 2008, age - standardized incidence rates in males and females in japan were 41.7 and 22.8 cases per 100 000, with corresponding death rates of 15.2 and 8.9 per 100 000, respectively (4). The introduction of monoclonal antibody - based therapy targeting epidermal growth factor receptor (egfr) has provided new alternative treatment options for various types of malignancy, including colorectal cancer (58). Panitumumab is a high - affinity, fully human igg2 monoclonal antibody specific to the extracellular domain of egfr (5,9). The clinical efficacy and tolerability of panitumumab have been well established in large, randomized, controlled clinical trials as monotherapy (1013) or in combination with cytotoxic agents (1417), in the first-, second- and third - line, or later, settings for patients with unresectable, advanced or recurrent colorectal cancer with wild - type kirsten rat sarcoma-2 virus oncogene (kras). Panitumumab was first approved in the usa (september 2006) and european union (eu) (december 2007) as monotherapy and for use in combination therapy in the eu (november 2011). In japan, panitumumab was approved (april 2010) for the treatment of wild - type kras gene unresectable, advanced and recurrent colorectal cancer as monotherapy, and for use in combination therapy in the first-, second- and third - line, or later, settings based on global clinical trials (1012,1417). The approval of panitumumab in japan for use in combination therapy represents the first such approval in the world . However, because the number of japanese individuals enrolled in the global clinical trials was relatively limited (13), the japanese ministry of health, labour and welfare required the market authorization holders to conduct a post - marketing surveillance study, called all cases surveillance (18), as a condition of its approval to evaluate the safety and effectiveness of all japanese patients treated with panitumumab . The main purpose of this study was to collect detailed information, particularly with regard to safety issues, on all panitumumab - treated patients; additionally, by evaluating a large number of patients, the study aimed to identify rare adverse drug reactions (adrs), which are often undetectable in smaller clinical trials . This post - marketing surveillance study was planned to include all patients treated with panitumumab from the start date (15 june 2010) of its launch in japan . To promote the appropriate use and evaluate safety information, a vectibix appropriate use committee, a vectibix safety evaluation committee and a vectibix interstitial lung disease (ild) review subcommittee all reported ild - like events were assessed individually by an ild review subcommittee, comprising external experts in the field of radiology, pulmonology and medical oncology . Evaluation of ild - like events was performed using computed tomography (ct) and x - ray imaging, and by assessment of clinical information . Completion of a specific registration form (fig . 1), which reported patient characteristics and treatment information, was mandatory before panitumumab treatment was initiated . The registration period of this post - marketing surveillance was june 2010 to november 2010 . Patients were carefully observed, and information during their clinical courses for 10 months (42 weeks), or until discontinuation of panitumumab for any reason, was collected through the pre - specified case report forms (crfs). At the time of registration, a recommendation letter to stop initiating treatment with panitumumab was sent from the vectibix appropriate use committee to each attending doctor if at least one item on the dark gray zone of the registration form was checked (fig . 1). Subsequently, the following information relating to clinical course was collected using crfs: (1) patient background including prior chemotherapy and reason for its discontinuation / termination; (2) administration of panitumumab including its dose and date, premedication to prevent infusion reaction, concurrent medications and combined therapy; (3) laboratory tests including serum magnesium, calcium and potassium; (4) adverse events including time to onset, grade according to national cancer institute common terminology criteria for adverse events (ctcae) ver . 4.0, treatment, causality of panitumumab and recovery and (5) outcomes including disease progression, survival or death . Figure 1.patient registration form excerpt . An adr was defined as an adverse event for which a causal relationship with panitumumab could not be ruled out . Adrs were coded using medical dictionary for regulatory activities (meddra) version 15.0 . With regard to toxicity, skin disorders, ild, infusion reaction, electrolyte abnormalities and cardiac disorders were monitored with special interest . The effectiveness of panitumumab was assessed by overall survival, calculated using the kaplan meier method . Overall survival time was defined as the time from the date of first administration of panitumumab to the date of death (regardless of the cause of death) or censored on the last date of survival (date of confirmation of survival as recorded in the crf or, if there was no information regarding the date of confirmation of survival, the last date of panitumumab administration was used for calculation purposes). This survey was initiated in june 2010 and the data lock point of the final analysis was set at 12 september 2012 . During the registration period from june to november 2010, a total of 3091 patients were registered (completion of mandatory patient registration forms) from 1031 clinical institutes and departments in japan . Of the 3091 registered patients, the crfs of five registered patients were not obtained despite repeated requests to physicians . Of these, 3085 patients were eligible for safety analysis; 1 patient was excluded from the safety analysis as no information was available regarding drug administration (fig . 2). Figure 2.patient registration in the panitumumab japanese post - marketing surveillance study (safety analysis set). Patient registration in the panitumumab japanese post - marketing surveillance study (safety analysis set). While a kras test was attempted for all registered patients, it was not determined for 79 patients (2.6%) mainly due to the condition of the tissues . Despite a recommendation letter to stop, panitumumab was administered to three patients (0.1%) with mutant kras at each physician's discretion . The majority of patients (91.4%) had an eastern cooperative oncology group (ecog) performance status (ps) of 0 - 1 . Although the ecog ps of 3080 patients was reported to be 02 at registration, 5 patients with ps 3 were registered despite requests for reassessment of administration . The general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab . Therefore, patients with ps 3 (0.7%) or 4 (0.1%) were included in this surveillance . There were no other registered patients who met at least one item in the dark gray zone of the registration form (fig . 1). Table 1.patient demographics and baseline characteristicsall patients (n = 3085)baseline characteristicnumber%gender male196563.7 female112036.3age (years) <65152449.4 6574105834.3 7550316.3 median (range) (years)65.0 (1890)kras status wild300397.3 mutant30.1 not determinable792.6primary tumor type (duplicate counting) colon186060.3 resected162152.5 unresected2397.8 rectal124440.3 resected105434.2 unresected1896.1 colorectal3085100.0 resected266186.3 unresected42313.7treatment line first - line31010.1 second - line54317.6 third - line or later223272.4ecog performance status 0187760.9 194230.5 22417.8 3220.7 430.1past treatment regimens no1735.6 yes (duplicate counting)291194.4 folfox243979.1 folfiri190761.8 bevacizumab211368.5 cetuximab91729.7kras, kirsten rat sarcoma-2 virus oncogene; ecog, eastern cooperative oncology group; folfox, 5-fluorouracil, leucovorin, oxaliplatin; folfiri, 5-fluorouracil, leucovorin, irinotecan.mainly due to the condition of tissue samples; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease, excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration . Although 3080 patients were reported to be ps 02, the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . Patient demographics and baseline characteristics kras, kirsten rat sarcoma-2 virus oncogene; ecog, eastern cooperative oncology group; folfox, 5-fluorouracil, leucovorin, oxaliplatin; folfiri, 5-fluorouracil, leucovorin, irinotecan . Mainly due to the condition of tissue samples; reasonable attempts were made to determine the kras status for all patients . Treatment for metastatic or recurrent disease, excluding postoperative adjuvant chemotherapy from counting treatment lines . Specified ps immediately before panitumumab administration . Although 3080 patients were reported to be ps 02, the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . Panitumumab was used in the third - line, or later, setting for metastatic disease in 72.4% of patients, and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients, such as folfox (79.1%), folfiri (61.8%) and bevacizumab (monoclonal antibody, vascular endothelial growth factor - specific angiogenesis inhibitor; 68.5%); 29.7% of patients had received cetuximab (monoclonal antibody, egfr antagonist) (duplicate counting). Figure 3 shows treatment regimens administered and the status of administration (safety analysis set). At the start of panitumumab administration, 1254 (40.7%) patients received monotherapy and 1831 (59.4%) patients received combination chemotherapy . Combination therapy consisted (duplicate counting) of folfox4 (3.6%), mfolfox6 (15.1%), folfiri (33.9%), 5fu / l - lv (2.0%), cpt-11 (9.0%), s-1 + cpt-11 (1.3%) or other chemotherapy (3.1%). Of the 310 patients receiving first - line treatment, the majority (86.5%) received combination therapy, of which 193 patients received concomitant folfox (62.3%). With regard to the use of panitumumab monotherapy in the first - line setting, primary physicians did not select combination chemotherapy considering various patient factors, such as age and general condition, because there is no limitation in the indication of panitumumab for combination or monotherapy in japan . Combination therapy was administered in 394 of 543 (72.6%) patients as second - line treatment, of whom 276 (50.8%) received folfiri . Almost half (47.6%) of the 2232 patients receiving third - line, or later, treatment were in the monotherapy group . The median period of treatment with panitumumab was 113.0 days (range: 1559 days), with 14.6% of patients receiving panitumumab for> 10 months . Panitumumab was discontinued in 2592 (84.0%) patients in the safety analysis set (n = 3085). The main reasons for discontinuation (with some duplicate counting) were as follows: disease progression in 1903 (61.7%) patients, of which 1484 patients (78.0%) had confirmed disease progression by diagnostic imaging; occurrence of adverse events in 431 (14.0%) patients; patient refusal (190 patients; 6.2%); lack of patient hospital visits (30 patients; 1.0%) and other reasons (185 patients; 6.0%). The overall incidence of adrs in all grades was 84.1% (2595/3085 patients) (table 2). The most common classification, according to the meddra system organ class (soc), was skin and subcutaneous tissue disorders' (76.6%; 2364/3085) (table 2), followed by infections and infestations' [(25.0%; 771/3085), which included paronychia (23.7%; 731/3085)], gastrointestinal disorders' (20.8%; 642/3085) and metabolism and nutrition disorders' (17.9%; 552/3085). Table 2overall incidence of adverse drug reactions (adrs) by system organ class (soc)any gradegrade 3n%n%all adrs259584.179725.8 skin and subcutaneous tissue disorders236476.639212.7 infections and infestations77125.01605.2 gastrointestinal disorders64220.81264.1 metabolism and nutrition disorders55217.91494.8 investigations2046.6742.4 general disorders and administration site conditions1113.6290.9 eye disorders832.780.3 nervous system disorders682.2180.6 respiratory, thoracic and mediastinal disorders632.0280.9 injury, poisoning and procedural complications521.750.2 blood and lymphatic system disorders331.1240.8 hepatobiliary disorders190.640.1 renal and urinary disorders120.470.2 musculoskeletal and connective tissue disorders100.320.1 vascular disorders90.320.1 cardiac disorders70.210.03 overall incidence of adverse drug reactions (adrs) by system organ class (soc) the incidence of adrs in the monotherapy group (n = 1254) was 80.1% (1004/1254); 19.7% of these were grade 3 and 5.4% were classified as serious cases . In patients receiving combination therapy (n = 1831), 86.9% reported adrs; 30.0% of these were grade 3 and 7.9% were serious cases . There were no major differences in the incidence of adrs by combined regimens in the combination therapy group: folfox (overall: 86.7%, grade 3: 29.3% and serious cases: 9.1%) and folfiri (87.4, 29.8 and 8.1%, respectively). A summary of safety data from the post - marketing surveillance study and panitumumab clinical trials (19) is presented in table 3 . Table 3.summary of safety datapost - marketing survey in japanp - mab monotherapy (n = 1254)combination therapy (n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders (soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders (soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy (n = 1052: kras wt)p - mab + folfox4 (n = 322: kras wt)p - mab + folfiri (n = 302: kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders (soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders (soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab, panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos: 20050216, 20040192, 20020408, 20030194, 20030167, 20030250, 20025405, 20030138, 20040116, 20030251, 20020375.trial no: 2005203.trial no: 2005181 . Summary of safety data based on the evaluation of the ild review subcommittee . Including grade-2 serious cases . Trial nos: 20050216, 20040192, 20020408, 20030194, 20030167, 20030250, 20025405, 20030138, 20040116, 20030251, 20020375 . The overall incidence rate of major skin disorders' (which includes all related events) was 78.4% (grade 3, 14.7%). Dermatitis acneiform occurred in 69.9% of patients (grade 3, 10.5%), paronychia in 24.2% (grade 3, 4.3%), dry skin in 21.7% (grade 3, 2.1%) and pruritus in 4.8% (grade 3, 0.4%). The median time from the first day of treatment to the onset of each of the major skin disorders was as follows: 15 days for dermatitis acneiform, 43 days for paronychia, 29 days for dry skin and 21 days for pruritus . The prevalence of skin disorders was similar with panitumumab monotherapy (74.7%; 937/1254) and panitumumab plus chemotherapy (80.9%; 1482/1831). Serum magnesium, calcium and potassium were checked at least once during treatment in 46.2% (1425/3085), 65.4% (2016/3085) and 92.5% (2855/3085) of the patients, respectively . The incidence rate of electrolyte abnormalities was 19.3% (596/3085), with that of grade 3 cases being 4.8% (149/3085). The respective incidence rates of hypomagnesemia, hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% (520/1425), 6.7% (136/2016) and 2.3% (67/2855). The incidence rate of hypomagnesemia in the overall population was 16.9% (520/3085), with that of grade 3 hypomagnesemia being 4.0% (123/3085). The median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases: 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not . The incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration . Of 105 patients who experienced both hypomagnesemia and hypocalcemia, 42 patients had the same grade of hypomagnesemia and hypocalcemia; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia, respectively . Of 30 patients with grade 3 hypomagnesemia and hypocalcemia, 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities: qtc prolongation (3 cases), rhabdomyolysis, paralysis, tetany and convulsion (1 case of each). Infusion reaction occurred in 1.5% (47/3085) of patients, with an incidence rate of grade 3 or serious cases of 0.2% (6/3085). Infusion reaction occurred at initial administration in 61.7% (29/47) of patients and in 14.9% (7/47) of patients at second administration, while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients (with the latest infusion reaction occurring at the 21st administration). Panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab, following premedication and at reduced speed of infusion in almost all cases . Two of these patients (2.9%) experienced grade 1 infusion reaction, while infusion reaction occurred in 9/847 (1.1%) patients without a history of infusion reaction to cetuximab administration . Overall, the incidence rate of ild was 1.3% (39/3085): 1.3% in monotherapy and 1.3% in combination therapy, resulting in a mortality rate of 0.6% (20/3085). The time to onset of ild varied (table 4). As determined by ct imaging, diffuse alveolar damage (dad) was observed in 46.2% (18/39) of patients and 15 of the 18 dad cases were fatal . The remaining ild cases were diagnosed as hypersensitivity pneumonia (9 cases) or organizing pneumonia (8 cases), with 4 unevaluable cases . Table 4.time to onset of interstitial lung disease (ild)time (months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20) time to onset of interstitial lung disease (ild) the incidence of cardiac disorders was 0.2% (7/3085), with death due to heart failure (grade 5) reported in one patient . In this patient, treated with panitumumab and folfiri, pancytopenia (not related to panitumumab) appeared on day 23, followed by pneumonia (day 28) and heart failure (day 32). In total, 1135 deaths were recorded, and most of them were related to progression of colorectal cancer . However, in 25 (2.2%) patients, a relationship to panitumumab treatment could not be ruled out . Among these, the most common cause of death was ild (20 patients, according to the evaluation of the ild subcommittee). Meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line, or later, setting . Figure 4.overall survival (n= 1062) in patients receiving third - line, or later, therapy with panitumumab monotherapy . Overall survival (n= 1062) in patients receiving third - line, or later, therapy with panitumumab monotherapy . The median overall survival time (95% confidence interval) was 10.3 months (9.011.8 months), with 56.6% censored cases . During the registration period from june to november 2010, a total of 3091 patients were registered (completion of mandatory patient registration forms) from 1031 clinical institutes and departments in japan . Of the 3091 registered patients, the crfs of five registered patients were not obtained despite repeated requests to physicians . Of these, 3085 patients were eligible for safety analysis; 1 patient was excluded from the safety analysis as no information was available regarding drug administration (fig . 2). Figure 2.patient registration in the panitumumab japanese post - marketing surveillance study (safety analysis set). Patient registration in the panitumumab japanese post - marketing surveillance study (safety analysis set). While a kras test was attempted for all registered patients, it was not determined for 79 patients (2.6%) mainly due to the condition of the tissues . Despite a recommendation letter to stop, panitumumab was administered to three patients (0.1%) with mutant kras at each physician's discretion . The majority of patients (91.4%) had an eastern cooperative oncology group (ecog) performance status (ps) of 0 - 1 . Although the ecog ps of 3080 patients was reported to be 02 at registration, 5 patients with ps 3 were registered despite requests for reassessment of administration . The general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab . Therefore, patients with ps 3 (0.7%) or 4 (0.1%) were included in this surveillance . There were no other registered patients who met at least one item in the dark gray zone of the registration form (fig . 1). Table 1.patient demographics and baseline characteristicsall patients (n = 3085)baseline characteristicnumber%gender male196563.7 female112036.3age (years) <65152449.4 6574105834.3 7550316.3 median (range) (years)65.0 (1890)kras status wild300397.3 mutant30.1 not determinable792.6primary tumor type (duplicate counting) colon186060.3 resected162152.5 unresected2397.8 rectal124440.3 resected105434.2 unresected1896.1 colorectal3085100.0 resected266186.3 unresected42313.7treatment line first - line31010.1 second - line54317.6 third - line or later223272.4ecog performance status 0187760.9 194230.5 22417.8 3220.7 430.1past treatment regimens no1735.6 yes (duplicate counting)291194.4 folfox243979.1 folfiri190761.8 bevacizumab211368.5 cetuximab91729.7kras, kirsten rat sarcoma-2 virus oncogene; ecog, eastern cooperative oncology group; folfox, 5-fluorouracil, leucovorin, oxaliplatin; folfiri, 5-fluorouracil, leucovorin, irinotecan.mainly due to the condition of tissue samples; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease, excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration . Although 3080 patients were reported to be ps 02, the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . Patient demographics and baseline characteristics kras, kirsten rat sarcoma-2 virus oncogene; ecog, eastern cooperative oncology group; folfox, 5-fluorouracil, leucovorin, oxaliplatin; folfiri, 5-fluorouracil, leucovorin, irinotecan . Mainly due to the condition of tissue samples; reasonable attempts were made to determine the kras status for all patients . Treatment for metastatic or recurrent disease, excluding postoperative adjuvant chemotherapy from counting treatment lines . Specified ps immediately before panitumumab administration . Although 3080 patients were reported to be ps 02, the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients . Panitumumab was used in the third - line, or later, setting for metastatic disease in 72.4% of patients, and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients, such as folfox (79.1%), folfiri (61.8%) and bevacizumab (monoclonal antibody, vascular endothelial growth factor - specific angiogenesis inhibitor; 68.5%); 29.7% of patients had received cetuximab (monoclonal antibody, egfr antagonist) (duplicate counting). Figure 3 shows treatment regimens administered and the status of administration (safety analysis set). At the start of panitumumab administration, 1254 (40.7%) patients received monotherapy and 1831 (59.4%) patients received combination chemotherapy . Combination therapy consisted (duplicate counting) of folfox4 (3.6%), mfolfox6 (15.1%), folfiri (33.9%), 5fu / l - lv (2.0%), cpt-11 (9.0%), s-1 + cpt-11 (1.3%) or other chemotherapy (3.1%). Of the 310 patients receiving first - line treatment, the majority (86.5%) received combination therapy, of which 193 patients received concomitant folfox (62.3%). With regard to the use of panitumumab monotherapy in the first - line setting, primary physicians did not select combination chemotherapy considering various patient factors, such as age and general condition, because there is no limitation in the indication of panitumumab for combination or monotherapy in japan . Combination therapy was administered in 394 of 543 (72.6%) patients as second - line treatment, of whom 276 (50.8%) received folfiri . Almost half (47.6%) of the 2232 patients receiving third - line, or later, treatment were in the monotherapy group . The median period of treatment with panitumumab was 113.0 days (range: 1559 days), with 14.6% of patients receiving panitumumab for> 10 months . Panitumumab was discontinued in 2592 (84.0%) patients in the safety analysis set (n = 3085). The main reasons for discontinuation (with some duplicate counting) were as follows: disease progression in 1903 (61.7%) patients, of which 1484 patients (78.0%) had confirmed disease progression by diagnostic imaging; occurrence of adverse events in 431 (14.0%) patients; patient refusal (190 patients; 6.2%); lack of patient hospital visits (30 patients; 1.0%) and other reasons (185 patients; 6.0%). The overall incidence of adrs in all grades was 84.1% (2595/3085 patients) (table 2). The most common classification, according to the meddra system organ class (soc), was skin and subcutaneous tissue disorders' (76.6%; 2364/3085) (table 2), followed by infections and infestations' [(25.0%; 771/3085), which included paronychia (23.7%; 731/3085)], gastrointestinal disorders' (20.8%; 642/3085) and metabolism and nutrition disorders' (17.9%; 552/3085). Table 2overall incidence of adverse drug reactions (adrs) by system organ class (soc)any gradegrade 3n%n%all adrs259584.179725.8 skin and subcutaneous tissue disorders236476.639212.7 infections and infestations77125.01605.2 gastrointestinal disorders64220.81264.1 metabolism and nutrition disorders55217.91494.8 investigations2046.6742.4 general disorders and administration site conditions1113.6290.9 eye disorders832.780.3 nervous system disorders682.2180.6 respiratory, thoracic and mediastinal disorders632.0280.9 injury, poisoning and procedural complications521.750.2 blood and lymphatic system disorders331.1240.8 hepatobiliary disorders190.640.1 renal and urinary disorders120.470.2 musculoskeletal and connective tissue disorders100.320.1 vascular disorders90.320.1 cardiac disorders70.210.03 overall incidence of adverse drug reactions (adrs) by system organ class (soc) the incidence of adrs in the monotherapy group (n = 1254) was 80.1% (1004/1254); 19.7% of these were grade 3 and 5.4% were classified as serious cases . In patients receiving combination therapy (n = 1831), 86.9% reported adrs; 30.0% of these were grade 3 and 7.9% were serious cases . There were no major differences in the incidence of adrs by combined regimens in the combination therapy group: folfox (overall: 86.7%, grade 3: 29.3% and serious cases: 9.1%) and folfiri (87.4, 29.8 and 8.1%, respectively). A summary of safety data from the post - marketing surveillance study and panitumumab clinical trials (19) is presented in table 3 . Table 3.summary of safety datapost - marketing survey in japanp - mab monotherapy (n = 1254)combination therapy (n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders (soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders (soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy (n = 1052: kras wt)p - mab + folfox4 (n = 322: kras wt)p - mab + folfiri (n = 302: kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders (soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders (soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab, panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos: 20050216, 20040192, 20020408, 20030194, 20030167, 20030250, 20025405, 20030138, 20040116, 20030251, 20020375.trial no: 2005203.trial summary of safety data based on the evaluation of the ild review subcommittee . Including grade-2 serious cases . Trial nos: 20050216, 20040192, 20020408, 20030194, 20030167, 20030250, 20025405, 20030138, 20040116, 20030251, 20020375 . The overall incidence rate of major skin disorders' (which includes all related events) was 78.4% (grade 3, 14.7%). Dermatitis acneiform occurred in 69.9% of patients (grade 3, 10.5%), paronychia in 24.2% (grade 3, 4.3%), dry skin in 21.7% (grade 3, 2.1%) and pruritus in 4.8% (grade 3, 0.4%). The median time from the first day of treatment to the onset of each of the major skin disorders was as follows: 15 days for dermatitis acneiform, 43 days for paronychia, 29 days for dry skin and 21 days for pruritus . The prevalence of skin disorders was similar with panitumumab monotherapy (74.7%; 937/1254) and panitumumab plus chemotherapy (80.9%; 1482/1831). Serum magnesium, calcium and potassium were checked at least once during treatment in 46.2% (1425/3085), 65.4% (2016/3085) and 92.5% (2855/3085) of the patients, respectively . The incidence rate of electrolyte abnormalities was 19.3% (596/3085), with that of grade 3 cases being 4.8% (149/3085). The respective incidence rates of hypomagnesemia, hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% (520/1425), 6.7% (136/2016) and 2.3% (67/2855). The incidence rate of hypomagnesemia in the overall population was 16.9% (520/3085), with that of grade 3 hypomagnesemia being 4.0% (123/3085). The median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases: 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not . The incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration . Of 105 patients who experienced both hypomagnesemia and hypocalcemia, 42 patients had the same grade of hypomagnesemia and hypocalcemia; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia, respectively . Of 30 patients with grade 3 hypomagnesemia and hypocalcemia, 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities: qtc prolongation (3 cases), rhabdomyolysis, paralysis, tetany and convulsion (1 case of each). Infusion reaction occurred in 1.5% (47/3085) of patients, with an incidence rate of grade 3 or serious cases of 0.2% (6/3085). Infusion reaction occurred at initial administration in 61.7% (29/47) of patients and in 14.9% (7/47) of patients at second administration, while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients (with the latest infusion reaction occurring at the 21st administration). Panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab, following premedication and at reduced speed of infusion in almost all cases . Two of these patients (2.9%) experienced grade 1 infusion reaction, while infusion reaction occurred in 9/847 (1.1%) patients without a history of infusion reaction to cetuximab administration . Overall, the incidence rate of ild was 1.3% (39/3085): 1.3% in monotherapy and 1.3% in combination therapy, resulting in a mortality rate of 0.6% (20/3085). The time to onset of ild varied (table 4). As determined by ct imaging, diffuse alveolar damage (dad) was observed in 46.2% (18/39) of patients and 15 of the 18 dad cases were fatal . The remaining ild cases were diagnosed as hypersensitivity pneumonia (9 cases) or organizing pneumonia (8 cases), with 4 unevaluable cases . Table 4.time to onset of interstitial lung disease (ild)time (months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20) time to onset of interstitial lung disease (ild) the incidence of cardiac disorders was 0.2% (7/3085), with death due to heart failure (grade 5) reported in one patient . In this patient, treated with panitumumab and folfiri, pancytopenia (not related to panitumumab) appeared on day 23, followed by pneumonia (day 28) and heart failure (day 32). In total, 1135 deaths were recorded, and most of them were related to progression of colorectal cancer . However, in 25 (2.2%) patients, a relationship to panitumumab treatment could not be ruled out . Among these, the most common cause of death was ild (20 patients, according to the evaluation of the ild subcommittee). The overall incidence rate of major skin disorders' (which includes all related events) was 78.4% (grade 3, 14.7%). Dermatitis acneiform occurred in 69.9% of patients (grade 3, 10.5%), paronychia in 24.2% (grade 3, 4.3%), dry skin in 21.7% (grade 3, 2.1%) and pruritus in 4.8% (grade 3, 0.4%). The median time from the first day of treatment to the onset of each of the major skin disorders was as follows: 15 days for dermatitis acneiform, 43 days for paronychia, 29 days for dry skin and 21 days for pruritus . The prevalence of skin disorders was similar with panitumumab monotherapy (74.7%; 937/1254) and panitumumab plus chemotherapy (80.9%; 1482/1831). Serum magnesium, calcium and potassium were checked at least once during treatment in 46.2% (1425/3085), 65.4% (2016/3085) and 92.5% (2855/3085) of the patients, respectively . The incidence rate of electrolyte abnormalities was 19.3% (596/3085), with that of grade 3 cases being 4.8% (149/3085). The respective incidence rates of hypomagnesemia, hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% (520/1425), 6.7% (136/2016) and 2.3% (67/2855). The incidence rate of hypomagnesemia in the overall population was 16.9% (520/3085), with that of grade 3 hypomagnesemia being 4.0% (123/3085). The median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases: 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not . The incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration . Of 105 patients who experienced both hypomagnesemia and hypocalcemia, 42 patients had the same grade of hypomagnesemia and hypocalcemia; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia, respectively . Of 30 patients with grade 3 hypomagnesemia and hypocalcemia, 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities: qtc prolongation (3 cases), rhabdomyolysis, paralysis, tetany and convulsion (1 case of each). Infusion reaction occurred in 1.5% (47/3085) of patients, with an incidence rate of grade 3 or serious cases of 0.2% (6/3085). No grade 4 cases, or deaths due to infusion reaction, infusion reaction occurred at initial administration in 61.7% (29/47) of patients and in 14.9% (7/47) of patients at second administration, while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients (with the latest infusion reaction occurring at the 21st administration). Panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab, following premedication and at reduced speed of infusion in almost all cases . Two of these patients (2.9%) experienced grade 1 infusion reaction, while infusion reaction occurred in 9/847 (1.1%) patients without a history of infusion reaction to cetuximab administration . Overall, the incidence rate of ild was 1.3% (39/3085): 1.3% in monotherapy and 1.3% in combination therapy, resulting in a mortality rate of 0.6% (20/3085). Of the total ild cases, 51.3% (20/39) the time to onset of ild varied (table 4). As determined by ct imaging, diffuse alveolar damage (dad) was observed in 46.2% (18/39) of patients and 15 of the 18 dad cases were fatal . The remaining ild cases were diagnosed as hypersensitivity pneumonia (9 cases) or organizing pneumonia (8 cases), with 4 unevaluable cases . Table 4.time to onset of interstitial lung disease (ild)time (months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20) time to onset of interstitial lung disease (ild) the incidence of cardiac disorders was 0.2% (7/3085), with death due to heart failure (grade 5) reported in one patient . In this patient, treated with panitumumab and folfiri, pancytopenia (not related to panitumumab) appeared on day 23, followed by pneumonia (day 28) and heart failure (day 32). In total, 1135 deaths were recorded, and most of them were related to progression of colorectal cancer . However, in 25 (2.2%) patients, a relationship to panitumumab treatment could not be ruled out . Among these, the most common cause of death was ild (20 patients, according to the evaluation of the ild subcommittee). Meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line, or later, setting . Figure 4.overall survival (n= 1062) in patients receiving third - line, or later, therapy with panitumumab monotherapy . Overall survival (n= 1062) in patients receiving third - line, or later, therapy with panitumumab monotherapy . The median overall survival time (95% confidence interval) was 10.3 months (9.011.8 months), with 56.6% censored cases . The main objective of the current post - marketing surveillance study was to evaluate the safety of panitumumab in japanese patients with unresectable colorectal cancer (and with the majority of patients possessing wild - type kras tumors) in clinical practice . The overall incidence of adrs was 84.1%, with skin disorders representing the most common classification (78.4%). Skin toxicity associated with panitumumab (and other egfr antibodies) is well known (20). Indeed, the development of skin disorders, particularly rash, is considered to be a predictive factor for the efficacy of egfr inhibitors (21,22). As reported previously with egfr inhibitors (22,23), dermatitis acneiform (69.9%) was the most frequent skin disorder reported in the current study, occurring about 2 weeks after the initiation of panitumumab . In the current study, although major skin disorders such as dermatitis acneiform, paronychia, dry skin and pruritus occurred in 78.4% of patients, 93% of the patients who experienced skin disorders were able to continue panitumumab therapy . The egfr - signaling pathway potentially plays a pivotal role in regulating magnesium homeostasis, and, indeed, hypomagnesemia / magnesium has been shown in patients with colorectal cancer receiving egfr - targeting antibodies (24). In the current study, 46.2% of patients were evaluated for serum magnesium, and grade 3 or higher hypomagnesemia occurred in 4.0% of patients, with a shorter median time to onset in patients treated with cetuximab immediately prior to panitumumab (39.5 and 71 days, respectively), indicating that the total duration of exposure to an egfr inhibitor is an important factor . From these findings, it is considered that the longer - term administration of anti - egfr treatment worsens the severity of hypomagnesemia (24). However, in the present study, the incidence of hypomagnesemia and hypocalcemia peaked 3 and 4 months after the start of panitumumab administration, respectively . Moreover, the incidence of hypomagnesemia observed in the current post - marketing surveillance study is similar to that reported in a phase ii clinical trial of panitumumab in japanese patients with metastatic colorectal cancer (13)., almost all patients with prior experience of infusion reaction to cetuximab could receive panitumumab safely following premedication and with a reduced speed of infusion . Some reports have shown that panitumumab can be administered successfully in patients after a severe infusion reaction to cetuximab (2529). On the other hand, post - marketing fatalities due to infusion reaction with panitumumab have been reported in patients with a history of severe cetuximab - related infusion reaction (30). Therefore, administration of panitumumab to patients with a history of infusion reaction to cetuximab is not currently recommended . Pulmonary toxicity including drug - induced ild represents an important adverse reaction that is associated with egfr - targeted cancer therapy (31), with the potential to be fatal in some patients (32). We prospectively established an ild subcommittee consisting of experts to accurately evaluate the incidence of ild . Furthermore, in order to avoid panitumumab treatment, at the time of registration, a recommendation letter was issued to reconsider treatment with panitumumab if a patient had a history or complication of ild (fig . 1). In the current post - marketing surveillance study of panitumumab, the overall incidence rate of ild was 1.3%, with a mortality rate of 0.6%.the incidence of ild was similar to that reported in a post - marketing survey of cetuximab in japan (1.2%) (23). The ild subcommittee carried out a full evaluation of ild with panitumumab, concluding that the onset of ild can occur at any time after the start of panitumumab administration and that there were no findings specific to panitumumab, either clinically or in ct imaging . The dad type of ild occurred in some cases, as with cetuximab and the egfr tyrosine kinase inhibitors gefitinib and erlotinib, and may be fatal . At present, no major differences in the incidence of ild or in the rate of death due to ild were found in comparison with cetuximab (23). Thus, ild is one of the particular concerns with the use of panitumumab as well as other anti - egfr drugs . With regard to all panitumumab - related adrs, there were no differences between its use as monotherapy or in combination therapy with cytotoxic agents, and the profile, incidences, onset and outcomes of these adrs were very similar to those reported in previous clinical trials . In contrast to the combination setting with chemotherapy regimens, the monotherapy setting represents the efficacy of panitumumab in clinical practice more accurately; therefore, patients treated with panitumumab monotherapy as a third - line or later therapy were selected for analysis of the effectiveness of panitumumab . The effectiveness of panitumumab, reported previously in clinical trials, was also confirmed in the current post - marketing surveillance study . In a pivotal, phase iii, randomized, controlled study (15), the median overall survival time in the panitumumab group (wild - type kras) was 8.1 months (6.39.4 months) (11); data obtained from the present surveillance study exhibit a median overall survival of 10.3 months (56.6% censored cases). Whilst acknowledging that post - marketing surveillance studies have some inevitable limitations (e.g. The lack of a control group) that differ from those in well - controlled, prospective clinical trials, it is recognized that post - marketing surveillance studies provide a information based on the general population (33). These factors result in greater external validity for post - marketing surveillance studies compared with well - controlled, randomized clinical trials (33). Despite these limitations, this large - scale, non - interventional study has some advantages . This study also provides health - care professionals with valuable information on the safety and effectiveness of panitumumab based on real - world data . In conclusion, the current post - marketing surveillance study in japanese patients with unresectable colorectal cancer confirmed the safety profile and effectiveness of panitumumab that has been reported previously in clinical trials . It is considered that the benefit / risk balance for the use of panitumumab in patients with unresectable colorectal cancer remains favorable also in japan . This work was supported by takeda pharmaceutical company limited . Funding to pay the open access publication charges for this article was provided by takeda pharmaceutical company . Has received a consultancy fee from takeda, speaker honoraria from takeda, taiho and yakult, and research funding from takeda, bristol - myers squibb, merck serono and chugai . Has received consultancy fees from taiho, chugai, takeda and merck serono, speaker honoraria from taiho, chugai, yakult, bristol - myers squibb, merck serono, kyowa hakko kirin, takeda, kaken, johnson & johnson, toray, aska and bayer, a manuscript fee from chugai, and a donation from taiho, chugai, yakult, bristol - myers squibb, merck serono, kyowa hakko kirin, takeda, daiichi - sankyo, ono, eisai, kaken and tori . Y.k . Has received a consultancy fee, speaker honoraria and non - specific research support from takeda . K.m . Has received a consultancy fee from takeda, speaker honoraria from takeda, taiho, yakult, chugai, merck serono and bristol - myers squibb, and research funding from taiho, pfizer and merck serono . T.h . Has received a consultancy fee from takeda and speaker honoraria from takeda, chugai, taiho, pfizer, yakult and daiichi - sankyo . T.y . Has received a consulting fee from takeda, speaker honoraria from chugai, takeda and merck serono, and research grants from taiho, daiichi - sankyo, lily, pfizer and yakult.
Nevus lipomatosus cutaneous superficialis (nlcs) was first described by hoffman and zuhrelle in 1921.1 it s a rare idiopathic hamartomatous benign condition characterized by the presence of an ectopic mature adipose tissue within the dermis . It is classified into two clinical variants: the classical form, usually composed of multiple and grouped skin - colored, pedunculated and cerebriform nodules that often coalesce to form a plaque and a second more rare form, presenting as a solitary dome - shaped sessile papule or nodule.24 we report here eight cases of nlcs in order to assess the epidemiologic, clinical, and pathological features, as well as the management of this uncommon neoplasm . This study was a retrospective case series including all patients with histopathologically documented nlcs, who attended the dermatology department of charles nicolle hospital in tunisia during the last 14 years; between january 1997 and december 2010 . For each patient, we recorded the following data: age, sex, duration of the lesions, their localizations, number, and size as well as their management and evolution . Ethical approval and informed consent eight patients with nlcs were observed, three males and five females, aged between 7 and 41 years . In patients number 4, 5, 7, and 8, lesions had begun during childhood (at 1, 4, 5, and 6 years respectively). For the other four patients (number 1, 2, 3, and 6) lesions had begun in the third to fourth decade of life . Clinical presentation was that of the classical form in seven patients and of the solitary form in one patient (patient number 3). In four patients it was one of the limbs which had the lesion, and in the other four patients it was the trunk (figure 1: patient 8; figure 2: patient 5). Soft, skin - colored voluminous tumor (10 cm x 7 cm) of the left buttock composed of multiple nodules with a cerebriform surface . Histopathological features were typical of nlcs showing ectopic mature adipose tissue in the dermis (figure 3). Table 1 summarizes the main epidemiologic, clinical and pathological features, and management of nlcs in all eight patients . Table 1epidemiologic, clinical and pathological features, and management of nevus lipomatosus cutaneous superficialis (nlcs) in all eight patients.patientsexage (years)durationlocationclinical aspecthistopathologytreatment1m381 yearleft buttockplaque of several soft skin - colored nodules of 1 cm to 2 cmmature adipose tissue in reticular dermis without connection to the hypodermissurgical resection2f383 monthsrigth thighmultiple soft yellowish papules coalescing into plaquesectopic mature adipocytes in reticular dermisabstention3f391 yearleft shoulderone soft skin - colored nodule of 1 cmlobular proliferation of fat tissue in the dermissurgical resection4m76 yearsright shoulderlarge (6 cm x 2 cm) soft skin - colored verrucous plaque composed of nodules and papulesectopic mature adipocytes in reticular dermissurgical resection5m2420 yearslumbar area1 cm to 3 cm - grouped soft skin - colored papulesepidermal acanthosis, ectopic mature adipocytes in papillary dermissurgical resection6f3210 yearsthighmultiple soft skin - colored papules coalescing into a 5 cm plaqueepidermal acanthosis; hyperkeratosis; dermal proliferation of mature adipocytessurgical resection7f149 yearsflanklarge (6 cm x 2 cm) soft skin - colored plaque composed of nodulesthinned epidermis, ectopic mature adipose tissue in reticular dermissurgical resection8f4135 yearsleft buttocksoft, skin - colored voluminous tumor (10 cm x 7 cm) composed of multiple nodules with a cerebriform surfacenormal epidermis, mature fat cells in reticular dermis without hypodermis connectionsurgical resectionf = female; m = male . Epidemiologic, clinical and pathological features, and management of nevus lipomatosus cutaneous superficialis (nlcs) in all eight patients . Nlcs is more commonly present from birth, but can appear later in life; there is neither sex predilection nor familial trend in this disorder.1 in this study, there were five cases in which the lesion appeared in the first three decades, and in three cases nlcs appeared after 35 years . Two clinical patterns of nlcs exist; a multiple form or classical type, and the solitary form . In the classical type, lesions are congenital or they develop usually during the first two to three decades of life.5,6,7 it consists of multiple papules, skin colored or yellowish, coalescing into plaques with zosteriform, linear or segmental distribution . The lesions are slow - growing, with a smooth or cerebriform surface, and can reach a large size if left untreated . The largest size reported so far has been 40 cm x 28 cm.8 the most common sites are the pelvic girdle, the lower trunk, the gluteal region, and the thigh . The second clinical pattern of nlcs is a solitary papule or nodule mimicking skin tag,8 usually appearing later than the classical form, during the third to sixth decades of life.3,6,9 it can occur at any site, including unusual ones like the scalp, the eyelid, the nose, and the clitoris.1,1013 solitary form was also called pedonculated lipofibroma by mehregan et al . Because of its distinctive clinicopathologic features in comparison with the multiple form.9,14 among the presented cases, there were seven cases with the classical form and one case with the solitary form (case 3), whilst in another tunisian study the solitary type was predominant: 11 cases out of 13.15 nlcs have an indolent and asymptomatic course . Recently, a case of ulnar nerve entrapment accompanied by numbness and straining in the forearm, caused by nlcs, was reported and was treated by partial excision for symptomatic recovery.16 occasionally, nlcs may ulcerate after external trauma or ischemia . In two previously reported cases, surface of nevus was studded with multiple open comedons,2,17 and foul - smelling discharge may be associated.2 nlcs has been reported in association with other cutaneous disorders; follicular papules and hypertrophic pilo - sebaceous units,18 angiokeratoma of fordyce,4 scattered leukoderma, caf - au - lait macules,2 and hemangioma.4 in our study, no associated skin abnormalities were present . To our knowledge, there are no systemic abnormalities or malignant transformation described with nlcs . Nlcs should be clinically differenciated from nevus sebaceous, neurofibroma, lymphangioma, focal dermal hypoplasia, cylindroma, trichoepithelioma, and angiolipoma . Histopathological evaluation is required for diagnosis showing ectopic mature adipocytes, intermingled with collagen bundles, and proliferating around the periadnexial adventitial dermis and the perivascular area . Mature fat cells proliferate in reticular dermis and may extend to the papillary dermis.2,6,7 in the most characteristic feature of nlcs, there is usually no connection to the subcutaneous fat tissue; for some authors this condition is necessary to establish diagnosis of nlcs.6 several studies documented increased vascularity in the subpapillary and papillary dermis; epidermal changes have been reported, like mild to moderate acanthosis, basket weave hyperkeratosis, increased basal pigmentation, and focal elongation of rete pegs.3 adnexal structures may be reduced,3 and abnormal folliculosebaceous structures were reported in some cases such as sebaceous trichofolliculoma, folliculosebaceous cystic hamartoma, and dermoid cysts.7,19 in our study, diagnosis of nlcs was based on characteristic clinical aspects and confirmed by typical histopathological features . The genetic background of nlcs is still unknown, only one cytogenetic study was reported recently showing a 2p24 deletion.20 the pathogenesis of nlcs remains unknown . Several theories have been proposed: hoffman and zuhrelle postulated that fat deposition in the dermis is secondary to degenerative changes (metaplasia) in the connective tissue.3 other authors hypothesized that adipocytes originate from the pericytes of dermal vessels.2,21 for others, fat cells represented a true nevus that resulted from the focal heterotopic development of adipose tissue.22 treatment is usually not necessary unless for cosmetic reasons . The treatment of choice is surgical excision, which is curative, and post surgical recurrence is rare . Patients unwilling for surgery may undergo cryotherapy which yields partial but satisfactory results.8 recently, a case of classic nlcs successfully treated with co2 laser was reported with no recurrence during a follow - up period of 12 months.23 in this retrospective study, we did nt investigate chromosomal abnormalities to identify the genetic biomarkers of this congenital pathology, since cytogenetic analysis was reported in only one previous study.20 physicians should be aware of this rare condition because early recognition enables more conservative resection of the tumor and less invasive reconstruction of the defect . Nlcs is a rare skin malformation; we report herein a large case series of eight patients where the classical form is predominant . Our study may give additional data about epidemiological and clinical pattern of this hamartoma . Further studies are needed, especially prospective studies, by using chromosomal analysis to reach conclusions about the role of the genetic abnormalities in the development of this hamartoma.
The use of chemotherapy in the treatment of cancer has opened new possibilities for improvement of the quality of life of cancer patients . Despite its success, treatment with some of the most effective anticancer drugs, the mechanism of cisplatin (cis - diamminedichloroplatinum (ii) or cddp) induced nephrotoxicity has gradually been elucidated . Studies have shown an increase in lipid peroxides in the renal tissue of cddp - administered animals, a decrease in reduced glutathione levels, and the induction of metallothionein, an antioxidant . These changes have been considered to result from the generation of reactive oxygen species (ros). Studies using chemiluminescence or electron spin resonance (esr) have shown that cddp generates oh radical [5, 6]. Nephrotoxicity involves kidney damage or dysfunction arising from direct or indirect exposure to drugs and industrial or environmental chemicals . Cisplatin ((cis - diamminedichloroplatinum (ii) or cddp)), an anti - neoplastic drug have been reported to induce nephrotoxicity . The kidney which is the major route of cisplatin excretion also accumulates it to a greater degree than other organs [6, 8]. Oxidative stress, inflammation, and apoptosis are some of the mechanisms already established to explain cisplatin induced acute kidney injury . A number of strategies have been proposed for the prevention / management of cisplatin induced nephrotoxicity, since there is no specific treatment, with the use of some synthetic drugs which have been popular . However, these drugs have some associated risks and side - effects, hence, the need for natural alternatives of plant origin (plant foods / extracts) with little or no side - effect . Plants have limitless ability to synthesize aromatic substances such as polyphenols, mainly flavonoids, and phenolic acids, which exhibit antioxidant properties due to their hydrogen - donating and metal - chelating capacities . Polyphenols are secondary metabolites of plants and are widely distributed in plant - derived foods, such as cereals, legumes, nuts, vegetables, and fruits, and in beverages such as green or black tea, and fruit juice . Tannic and gallic acids are two commonly phenolic acids that are structurally related . Tannic acid, a naturally occurring plant polyphenol, is composed of a central glucose molecule derivatized at its hydroxyl groups with one or more galloyl residues, whereas gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is widely distributed in green tea, red wine and grapes, witch hazel, sumac, oak bark, and other plants . Considerable amounts of experimental data on the antioxidant activity of both tannic acid and gallic acids with emphasis on structure - function antioxidant activity have been reported . Also, several authors have demonstrated that tannic acid and other polyphenols have antimutagenic and anticarcinogenic activities [1316]. Extensive studies have been carried out on the protective effect of cotreatment and posttreatment of phenolic acids against cisplatin induced nephrotoxicity . Hence, this study was carried out to investigate and compare the protective effect of administration of both tannic and gallic acids on normal and cisplatin induced nephrotoxicity in rats . Male albino rats weighing 110185 g used for this experiment were purchased from a private animal colony, ikere - ekiti metropolis . The rats were maintained at 25c on a 12 hour light / dark cycle with free access to food and water . They were acclimatized under these conditions for two weeks prior to the commencement of the experiments . The experimental study was approved by the institutional animal ethical committee of the university of ado - ekiti, nigeria . Chemicals such as tannic acid, gallic acid, oxidized and reduced glutathione, hydrogen peroxide (h2o2), dithionitrobenzene (dtnb), thiobarbituric acid (tba), and adrenaline were purchased from sigma chemical co. (st . Ethanol, acetic acid, h2so4, sodium carbonate, sodium citrate, sodium azide (nan3), sodium chloride, potassium dichromate, tris - hcl buffer, sodium dodecyl sulphate (sds), and ascorbic acid were sourced from bdh chemicals ltd . May - grnwald, giemsa, and hematoxylin and eosin (h&e) stains were purchased from hi - media labs, mumbai . All the kits used for bioassay were sourced from randox laboratories ltd ., crumlin, county antrim, uk . Except stated otherwise, all other chemicals and reagents were of analytical grades and the water was glass - distilled . After two (2) weeks of acclimatization, 80 male rats were randomly divided into eight (8) groups of ten animals each: group i served as a normal control and received saline (0.85 w / v%) orally for 7 consecutive days and on the 7th day, 1 hr after receiving the oral saline dose, the rats received a single i.p . Group ii served as toxicant group and received saline (0.85%) orally for 7 consecutive days . Ga was orally administered at two doses, 20 and 40 mg / kg body weight (bwt), to groups iii and iv, respectively, for 7 consecutive days . Also, ta was orally administered at two doses, 20 and 40 mg / kg body weight (bwt), to groups vi and vii, respectively, for 7 consecutive days . On the 7th day of pretreatment, a single i.p . Dose of cisplatin (7.5 mg / kg bwt) after oral treatment of ga and ta was given to the animals in groups ii, iii, iv, vi, and vii . Groups v and viii received only higher dose (40 mg / kg) of ga and ta orally for 7 consecutive days, respectively, and on the 7th day 1 hr ga and ta treatment received a single i.p . Injection of saline (0.85%) to ensure that groups v and viii received only the higher dose of ga and ta; this was done in order to test that the higher dose did not produce any kind of toxic effects . The doses of ta and ga used were actually selected on the basis of preliminary dose escalation studies to determine the minimum dose of ta and ga required to produce an observable effect (data not shown). Uric acid, urea, and creatinine were determined using commercially available kits (randox laboratories uk). Tissue malondialdehyde (mda) content was determined as described by ohkawa et al . . Tissue antioxidant parameters were also determined; superoxide dismutase (sod) by the method of alia et al ., catalase (cat) by the method of sinha, reduced glutathione (gsh) by the method of ellman, and glutathione peroxidase (gpx) by the method of rotruck et al . . At the end of the experiment, whole blood of the sacrificed rats were collected into edta bottles and centrifuged at 800 g for 10 min to separate the plasma . The plasma was then decanted into plain sample bottle and stored in a refrigerator prior to analysis . The rat's tissues (kidney) were rapidly isolated, placed on ice, and weighed . The tissue was rinsed in cold (0.9% w / v) normal saline (1: 3, w / v) and subsequently homogenized in sodium phosphate buffer (ph 7.4) with (1: 5 bt w / v) using mortar and pestle as homogenizer and the homogenates were centrifuged at 4,000 g . The uric acid concentration was determined using colorimetric method as described by collin and diehl and morin and prox . Briefly, 20 l of distilled water was added to 20 l of the sample which was mixed with 1 ml of hepes reagent (50 mm phosphate buffer, 4 mm 3,5-chloro-2-hydroxybenzenesulfonic acid) and enzyme reagent (0.25 mm 4-aminophenazone, peroxidise, and uricase). Thereafter, the mixture was incubated for 5 min at 37c and the absorbance at 520 nm was taken against reagent blank within 30 min . The urea concentration was determined using colorimetric method as described by searcy et al . . Briefly, 10 l of sample was added to 0.1 ml of sodium nitroprusside 6 mm sodium nitroprusside, 1 g / l urease) after which the mixture was incubated for 10 min at 37c . 2.5 ml of 120 mm diluted phenol and 2.5 ml of 27 mm sodium hypochlorite solution containing 0.14 n sodium hydroxide which was then added to the reaction mixture . Thereafter, the mixture was incubated for 15 min at 37c and the absorbance at 546 nm was taken against reagent blank within 8 hours . The creatinine concentration was determined using colorimetric alkaline picrate method as described by jaffe method . Briefly, 50 l of distilled water was added to 2 ml of working reagent (35 mm picric acid and 0.32 m sodium hydroxide) before 50 l of sample was added . The absorbance at 492 nm was taken twice, firstly after 30 sec and secondly after 2 min . The creatinine concentration was subsequently calculated against the standard, using change in the sample absorbance (absorbance). Arginase activity was determined by the measurement of urea produced by the reaction of ehrlich's reagent according to the modified method of kaysen and strecker . The reaction mixture contained 1.0 mm tris - hcl buffer, 1.0 mm mncl2 (ph 9.5), 0.1 m arginase solution and 500 ml of the enzyme preparation . The mixture was incubated for 10 mins at 37c . The reaction was terminated by the addition of 2.5 ml ehrlich reagent (2.0 g of p - dimethylaminobenzaldehyde in 20.0 ml of concentrated hydrochloric acid and made up to 100 ml with distilled water). Superoxide dismutase (sod) was determined by the method of alia et al . . 50 l of supernatant was treated with 1000 l of 50 mm carbonate buffer (ph 10.2) and 17 l of adrenaline (0.06 mg / ml). The absorbance was read at 480 nm in spectrophotometer for 2 minutes at 15-second intervals . Sod activity was expressed as ui per 100 g protein (480 = 4.02 mmcm). The reaction mixture (1.5 ml) contained 1.0 ml of 0.01 m phosphate buffer (ph 7.0), 0.1 ml of tissue homogenate, and 0.4 ml of 2 m h2o2 . The reaction was stopped by the addition of 2.0 ml of dichromate - acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1: 3 ratio). Then, the absorbance was read at 620 nm: cat activity was expressed as moles of h2o2 consumed / min / g protein . . 1 ml of supernatant was treated with 500 l of ellman's reagent (19.8 mg of 5,5dithiobisnitrobenzoic acid in 100 ml of 0.1% sodium citrate) and 3.0 ml of 0.2 m phosphate buffer (ph 8.0). The activity of glutathione peroxidase (gpx) was assayed by the method of rotruck et al . . The reaction mixture containing 0.2 ml of edta (0.8 mm, ph 7.0), 0.4 ml of phosphate buffer (10 mm), and 0.2 ml of tissue homogenate was incubated with 0.1 m of h2o2 and 0.2 ml of glutathione for 10 min . The activity of gpx was expressed as l mol glutathione oxidized / min / g protein . The lipid peroxidation assay was carried out using the modified method of ohkawa et al . . Briefly, 300 l of tissue homogenate, 300 l of 8.1% sds (sodium dodecyl sulphate), 500 l of acetic acid / hcl (ph = 3.4), and tba (thiobarbituric acid) were added, and the mixture was incubated at 100c for 1 thereafter, the thiobarbituric acid reactive species (tbars) produced was measured at 532 nm and calculated as malondialdehyde (mda) equivalent . One - way analysis of variance was used to analyze the results and duncan multiple tests were used for the post hoc analysis . Statistical package for social science (spss) 10.0 for windows was used for the analysis . As evident from table 1, administration of a single dose of cisplatin (7.5 mg / kg bwt) caused a significant increase in the biomarkers of renal function (creatinine, urea, and uric acid) when compared with the control group that received only saline (0.85% w / v). However, pretreatment with gallic and tannic acids orally at two doses, 20 and 40 mg / kg body weight, respectively, shows a significant (p <0.05) improvement of renal function due to a decrease in the creatinine, urea, and uric acid levels when compared with the induced group (group 2). Also, administration of a single i.p dose of cisplatin (7.5 mg / kg bwt) caused a significant decrease in the biomarkers of oxidative stress [superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and reduced glutathione (gsh)] when compared with the control group that received only saline (0.85% w / v) (table 2). However, pretreatment with gallic and tannic acids orally at two doses, 20 and 40 mg / kg body weight, respectively, shows a significant (p <0.05) improvement in the body's antioxidant status by an increase in the activities of superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and reduced glutathione (gsh) when compared with the induced group (table 2). Likewise, there was a significant (p <0.05) increase in the malondialdehyde (mda) content in rat kidney administered a single i.p dose of cisplatin (7.5 mg / kg bwt). However, both pretreatment with gallic and tannic acids orally at two doses, 20 and 40 mg / kg body weight, respectively, shows a significant (p <0.05) reduction of mda content in rat kidney when compared with the induced group (figure 1). Figure 2 revealed that administration of a single i.p dose of cisplatin (7.5 mg / kg bwt) caused a significant (p <0.05) increase in arginase activity when compared with the control group that received only saline (0.85% w / v). However, pretreatment with gallic and tannic acids orally at two doses, 20 and 40 mg / kg body weight, respectively, shows a significant (p <0.05) decrease in arginase activity when compared with the induced group (group 2). Photomicrographs of kidney sections from various treatment groups are shown in figures 3, 4, and 5 . Histopathological examination of sections from rat kidney administered a single i.p dose of cisplatin (7.5 mg / kg bwt) shows severe and generalized tubular epithelial cell necrosis associated with diffuse tubular lumina when compared with the control without cisplatin . However, pretreatment with gallic and tannic acids orally at two doses, 20 and 40 mg / kg body weight, respectively, shows a marked improvement on kidney damage . About 25% of most commonly used drugs in intensive care units (icus) are potentially nephrotoxic and are recognized as considerable health and economic burden worldwide . Among them, cisplatin, when used in cancer chemotherapy, induces renal impairment and acute renal failure by induction of reactive oxygen species, tubulointerstitial inflammation, and apoptosis . Although various studies have been reported on the benefits of several agents in cisplatin induced renal toxicity, the basis of nephroprotection remains elusive . This makes the search for strategies to prevent nephrotoxicity constitute an active area of investigation . Hence, it is reasonable to assume that a reinforcement of antioxidant defense of renal tissue by exogenous antioxidant such as phenolic acids should be a strategy to protect the kidney from the oxidative damage . In the present study, we compare the protective effect of administration of gallic and tannic acids (two commonly phenolic acids that are structurally related) of the same dosage against normal and cisplatin induced renal injury in rats for the first time by attenuating renal oxidative stress . The elevations of key kidney function biomarkers such as creatinine, uric acid, and urea have been suggested to be indicative of reduced renal functions [30, 31]. Thus, estimation of plasma creatinine and uric acid has been employed as key test to assess kidney function [30, 31]. In the present study, the observed elevation in plasma creatinine, urea, and uric acid levels in induced rats this finding is consistent with that reported by earlier studies [32, 33]. However, the restoration of the plasma creatinine, urea, and uric acid level in rats treated with both gallic and tannic acids (table 1) suggests that both phenolic acids have the ability to prevent kidney damage and protect the kidney against nephrotoxicity . This, however, may be a function of their antioxidant properties and ability to inhibit arginase activity (figure 2). Cisplatin increased arginase activity in the rat's kidney, while the pretreatment with both gallic and tannic acids at two doses (20 and 40 mg / kg bwt), respectively, for 7 days resulted in a decrease in kidney arginase activity (figure 2). Arginase is a hydrolytic enzyme responsible for the conversion of l - arginine to l - ornithine and urea . Ornithine is an important biosynthetic precursor of polyamines which have been implicated to facilitate cell proliferation in certain cancer cells . Another important enzyme, endothelium nitric oxide synthase (enos), competes with arginase for the same substrate, arginine . The no produced plays an important role in both regulating renal hemodynamics and modulating inflammatory and proliferating response to various stimuli . Therefore, inhibition of arginase activity slows the progression of renal failure in renal ablation . The increase in the kidney and plasma mda content (figure 1) in the induced rats suggests lipid peroxidation . This agreed with earlier studies where administration of cisplatin caused inflammation and lipid peroxidation [6, 7]. This could be as a result of increased hydrogen peroxide concentration produced in the kidney due to the depletion of antioxidant enzymes: superoxide dismutase (sod), reduced glutathione (gsh), and catalase (cat) activity (table 2). This also is consistent with earlier study where depletion of sod, cat, and gpx in rats resulted in increased mda concentration due to lipid peroxidation . The depletion of these antioxidants suggests that cisplatin induced nephrotoxicity could be a result of oxidative stress or suppression of the antioxidant enzymes, as previously reported by earlier studies [3537]. However, the reduced kidney and plasma mda content (figure 1) and restoration of sod, gsh, and cat activities (table 2) in pretreated rats suggest an improvement in the in vivo antioxidant status, which may be a function of the antioxidant properties of the phenolic acids (gallic and tannic acids) therefore, the observed decrease in the kidney gsh level (table 2) in the induced rats suggests cisplatin induced nephrotoxicity, which is associated with a drastic reduction in kidney gsh content . This finding is consistent with earlier studies where gsh depletion was suggested to be due to the interaction of cisplatin with the molecules contain sulfhydryl groups [39, 40]. However, restoration of gsh levels in the pretreated rats suggests the antioxidant and nephroprotective properties of the phenolic acids (table 2). Furthermore, histopathology study revealed normal glomerulus and tubules with intact renal architecture in normal (figure 3(a)), gallic (figure 5(a)), and tannic acid (figure 5(b)) group without cisplatin injection . Degenerated tubular structures with vacuolization and loss of architecture were seen in cisplatin induced group (figure 3(b)). Pretreatment with both gallic and tannic acids at two doses (20 and 40 mg / kg bwt), respectively, for 7 days resulted in excellent protection against nephrotoxicity induced by cisplatin and showed predominant normal kidney morphology (figures 4(a), 4(b), 4(c), and 4(d)). The results of the present study revealed that oxidative stress and apoptosis / necrosis play an important role in pathogenesis of cisplatin nephrotoxicity . Gallic and tannic acids, two important pharmacologically active phenolic compounds, reduced cisplatin induced functional and histological renal damage . Furthermore, they suppressed the generation of ros, lipid peroxidation, and oxidative stress in kidney tissues . These results indicated that both gallic and tannic acids exhibit nephroprotective effect and the possible mechanism of action by which they exert this effect could be due to their antioxidant properties and inhibition of arginase activity . However, tannic acid exhibited better nephroprotective potential than gallic acid which may be due to the glycosidation with a glucose moiety.
Cavernous haemangioma is the most common primary orbital tumour in adults, accounting for 6% of all orbital lesions [13]. Orbital schwannomas are benign peripheral nerve sheath tumours and constitute 1% of all orbital tumours [1, 3]. Orbital cavernous haemangiomas and schwannomas present as slow progressive enlargement and the most common presenting sign and symptom is painless proptosis [48]. Although both orbital cavernous haemangiomas and schwannomas share many similar clinical and radiological characteristics [58], it is important to distinguish between the tumours because they demonstrate important different features and the treatments of choice for the two are different [3, 79]. Magnetic resonance imaging, especially dynamic mr imaging, plays a crucial role in the differentiation of orbital cavernous haemangiomas and schwannomas [7, 8, 1014]. The spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is considered the most important finding distinguishing the two tumours [1014]. However, the literature on mr imaging findings in the differentiation of these two tumours is based on small patient populations . Consequently, this study aims to assess mr imaging findings distinguishing orbital cavernous haemangiomas from schwannomas and to elaborate on specific differences between the two tumours . Between june 2004 and february 2009, 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol . Mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients . Most patients presented with painless insidious proptosis . Some patients demonstrated a limitation of extraocular motion, diplopia and strabismus . Informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data . Magnetic resonance imaging was performed on a general electric (ge) signa 1.5-t mr imaging system (ge healthcare, milwaukee, wi, usa) with an eight - channel head coil . Mr imaging was performed with t1-weighted (repetition time / echo time, 400/15 ms) and t2-weighted (3,000/120) fast spin - echo images . A 16-cm field of view, a 3-mm - thick section at 0 interval, and a 288 256 matrix with two signals acquired were used . Dynamic contrast - enhanced (dce) mr imaging was performed by using a fast spoiled gradient recalled (fspgr) sequence with 8.4/4 ms, a flip angle of 15, one excitation, a matrix of 256 160, a field of view of 220 220 mm, and a section thickness of 3.2 mm at 0 interval . A power injector (medrad, indianola, pa, usa) with an injection flow rate of 2 ml / the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1 mmol of gadopentetate dimeglumine (magnevist, schering, berlin, germany) per kilogram . Each mr sequence yielded 12 anatomical slices in 13 s; this was repeated 12 times with an interval between each sequence of 12 s. total imaging time was 288 s (4 min 48 s). Dce mr imaging was evaluated by using a ge aw 4.2 workstation (ge healthcare). A region of interest (roi) was drawn manually on the dynamic images for assessment of the enhancement kinetics . We chose the area that demonstrated the greatest degree of early enhancement so that the time intensity curve (tic) could be generated . The area of the roi typically was 8 mm . The contrast index (ci) was calculated from: ci = [(signal intensity post - contrast) (signal intensity pre - contrast)]/(signal intensity pre - contrast). The tics were analysed qualitatively as wash - out, persistent or plateau - shaped curves . Magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist (15 years experience). Location, configuration, margins, signal intensity, homogeneity and enhancement pattern of the tumour were assessed . The location was defined as anterior orbit (anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery), posterior orbit (posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery), or both anterior and posterior orbit; extraconal or intraconal space; superior or inferior orbit; and nasal or temporal orbit . On contrast - enhanced mr imaging including dce mr imaging, the spread pattern of the contrast enhancement, enhancement pattern, ci and type of tic were evaluated . The spread pattern of the contrast enhancement was divided into two groups: one starting from a small point or portion and the other starting from a wide area . Progressive enhancement means that on early imaging after the injection, one small point or portion of enhancement was initially noted, then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement (fig . 1). Other patterns of enhancement included gradually heterogeneous enhancement and gradually homogeneous enhancement . Fig . B the tumour appeared hyperintense (arrowhead) relative to muscle on axial t2-weighted imaging . C e dynamic mr images obtained at 25, 75 and 225 s, respectively, after administration of gadopentetate dimeglumine, showed enhancement starting from one point of peripheral portion of the tumour (arrowhead). F post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions (arrowhead) a 54-year - old woman with a cavernous haemangioma . B the tumour appeared hyperintense (arrowhead) relative to muscle on axial t2-weighted imaging . C e dynamic mr images obtained at 25, 75 and 225 s, respectively, after administration of gadopentetate dimeglumine, showed enhancement starting from one point of peripheral portion of the tumour (arrowhead). F post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions (arrowhead) frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests . Fisher s exact test was performed when the sample size in the subgroups was deemed too small . Between june 2004 and february 2009, 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol . Mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients . Most patients presented with painless insidious proptosis . Some patients demonstrated a limitation of extraocular motion, diplopia and strabismus . Informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data . Magnetic resonance imaging was performed on a general electric (ge) signa 1.5-t mr imaging system (ge healthcare, milwaukee, wi, usa) with an eight - channel head coil . Mr imaging was performed with t1-weighted (repetition time / echo time, 400/15 ms) and t2-weighted (3,000/120) fast spin - echo images . A 16-cm field of view, a 3-mm - thick section at 0 interval, and a 288 256 matrix with two signals acquired were used . Dynamic contrast - enhanced (dce) mr imaging was performed by using a fast spoiled gradient recalled (fspgr) sequence with 8.4/4 ms, a flip angle of 15, one excitation, a matrix of 256 160, a field of view of 220 220 mm, and a section thickness of 3.2 mm at 0 interval . A power injector (medrad, indianola, pa, usa) with an injection flow rate of 2 ml / s was used . The acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1 mmol of gadopentetate dimeglumine (magnevist, schering, berlin, germany) per kilogram . Each mr sequence yielded 12 anatomical slices in 13 s; this was repeated 12 times with an interval between each sequence of 12 s. total imaging time was 288 s (4 min 48 s). Dce mr imaging was evaluated by using a ge aw 4.2 workstation (ge healthcare). A region of interest (roi) was drawn manually on the dynamic images for assessment of the enhancement kinetics . We chose the area that demonstrated the greatest degree of early enhancement so that the time intensity curve (tic) could be generated . The contrast index (ci) was calculated from: ci = [(signal intensity post - contrast) (signal intensity pre - contrast)]/(signal intensity pre - contrast). The tics were analysed qualitatively as wash - out, persistent or plateau - shaped curves . Magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist (15 years experience). Location, configuration, margins, signal intensity, homogeneity and enhancement pattern of the tumour were assessed . The location was defined as anterior orbit (anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery), posterior orbit (posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery), or both anterior and posterior orbit; extraconal or intraconal space; superior or inferior orbit; and nasal or temporal orbit . On contrast - enhanced mr imaging including dce mr imaging, the spread pattern of the contrast enhancement, enhancement pattern, ci and type of tic were evaluated . The spread pattern of the contrast enhancement was divided into two groups: one starting from a small point or portion and the other starting from a wide area . Progressive enhancement means that on early imaging after the injection, one small point or portion of enhancement was initially noted, then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement (fig . 1). B the tumour appeared hyperintense (arrowhead) relative to muscle on axial t2-weighted imaging . C e dynamic mr images obtained at 25, 75 and 225 s, respectively, after administration of gadopentetate dimeglumine, showed enhancement starting from one point of peripheral portion of the tumour (arrowhead). F post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions (arrowhead) a 54-year - old woman with a cavernous haemangioma . B the tumour appeared hyperintense (arrowhead) relative to muscle on axial t2-weighted imaging . C e dynamic mr images obtained at 25, 75 and 225 s, respectively, after administration of gadopentetate dimeglumine, showed enhancement starting from one point of peripheral portion of the tumour (arrowhead). F post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions (arrowhead) frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests . Fisher s exact test was performed when the sample size in the subgroups was deemed too small . Tables 1 and 2 describe the frequency distribution of non - enhanced mr imaging features (table 1) and contrast - enhanced mr imaging features (table 2). There was a significant difference between cavernous haemangiomas and schwannomas regarding the location (including anterior orbit, posterior orbit, or both anterior and posterior orbit; extraconal or intraconal space), configuration and margins of the mass, signal intensity and homogeneity on t1- and t2-weighted imaging, the spread pattern of the contrast enhancement, enhancement pattern and type of time intensity curve (tic) (p <0.05) (figs . 1, 2, 3). B axial t2-weighted imaging revealed that the tumour was isointense relative to muscle (arrowhead). C e dynamic mr images obtained at 25, 75 and 225 s, respectively, after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour (arrowhead). F axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour (arrowhead)fig . A obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour (arrow) and slight hypointensity of the posterior portion (arrowhead) relative to muscle . B axial t2-weighted imaging showed isointensity of the anterior portion of the tumour (arrow) and hyperintensity of the posterior portion (arrowhead) relative to muscle . C obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour (arrow) and no enhancement of the posterior portion (arrowhead)table 1frequency distribution of non - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no . Of patients594316anterior or posterior orbit0.004 anterior4779.63990.8850 posterior610.224.6425 both610.224.6425extraconal or intraconal space0.049 intraconal4474.63581.4956.2 extraconal1525.4818.6743.8nasal or temporal orbit0.250 nasal2644.11739.5956.2 temporal3355.92660.5743.8superior or inferior orbit0.937 superior2949.22148.8850 inferior3050.82251.2850configuration0.015 round610.249.3212.6 ovoid5084.73990.71168.7 irregular35.100318.7margin0.531 smooth4474.63376.71168.7 lobulated1525.41023.3531.3t1 signal intensity<0.001 hypointense711.900743.7 isointense5288.143100956.3t1 homogeneity<0.001 homogeneous5084.743100743.7 heterogeneous915.300956.3t2 signal intensity0.001 isointense58.500531.3 hyperintense5491.5431001168.7t2 homogeneity<0.001 homogeneous467843100318.7 heterogeneous1322001381.3signal intensity was compared with that of extraocular muscletable 2frequency distribution of contrast - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no . Of patients594316spread pattern of contrast enhancement<0.001 starting from a small point or portion4372.94310000 starting from a wide area1627.10016100enhancement pattern<0.001 homogeneous or heterogeneous1627.10016100 progressive4372.94310000ci1.8 0.61.4 0.30.003tic type<0.001 persistent1220.337956.2 plateau - shaped2440.71944.2531.3 wash - out23392148.8212.50 a 42-year - old man with a schwannoma . B axial t2-weighted imaging revealed that the tumour was isointense relative to muscle (arrowhead). C e dynamic mr images obtained at 25, 75 and 225 s, respectively, after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour (arrowhead). F axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour (arrowhead) a 47-year - old woman with schwannoma . A obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour (arrow) and slight hypointensity of the posterior portion (arrowhead) relative to muscle . B axial t2-weighted imaging showed isointensity of the anterior portion of the tumour (arrow) and hyperintensity of the posterior portion (arrowhead) relative to muscle . C obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour (arrow) and no enhancement of the posterior portion (arrowhead) frequency distribution of non - enhanced mr imaging features signal intensity was compared with that of extraocular muscle frequency distribution of contrast - enhanced mr imaging features orbital cavernous haemangiomas were always a well - defined, ovoid, intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging . Schwannomas often demonstrated a heterogeneously isointense or mildly hyperintense mass in the intraconal or extraconal space . After gadolinium administration, all 43 cavernous haemangiomas showed enhancement starting from a small point or portion and contrast media filling up the tumour later, i.e. Progressive enhancement (fig . 1). Compared with the cavernous haemangiomas, all 16 schwannomas showed enhancement starting from a wide area and heterogeneous or homogeneous enhancement later (fig . 2). Orbital cavernous haemangiomas, the most common primary orbital tumour in adults, demonstrate a predilection to affect middle - aged women (6070%), with a mean age of 43 to 48 years and range of 18 to 72 years based on several large series [4, 7, 9]. The most common presenting sign and symptom of the orbital cavernous haemangioma is painless proptosis . In the past, orbital cavernous haemangiomas were invariably resected because they could not be conclusively discerned from other tumours clinically or radiologically . However, orbital cavernous haemangiomas have recently been classified as type 3 low - flow arteriovenous malformations with a direct arterial in - flow and venous out - flow mechanism (arterial low - flow type) rather than as true neoplasms or proliferating hamartomas, and, to our knowledge, there is no evidence of malignant transformation in the literature . Moreover, ct and mr imaging, especially mr imaging, have evolved to deliver high sensitivity and specificity in diagnosing orbital cavernous haemangiomas and distinguishing cavernous haemangiomas from other tumours, which have played a vital role in accurate diagnosis and correct treatment planning [5, 7, 12, 14, 16]. Therefore, recent literature supports observation as an alternative to surgical excision for asymptomatic orbital cavernous haemangiomas and surgery can be withheld until vision is threatened or cosmetic appearance resulting from proptosis is unacceptable [3, 9]. Orbital schwannomas, constituting 1% of all orbital tumours, are benign peripheral nerve sheath tumours originating purely from schwann cells and usually seen in young to middle - aged adults and rarely in children . Clinical findings of orbital schwannomas are generally similar to those of cavernous haemangiomas, but they are somewhat more likely to exhibit progressive growth and symptoms . Thus, surgical excision is the treatment of choice for orbital schwannomas, different from orbital cavernous haemangiomas [3, 79]. Our results demonstrate that, in presence of a well - defined, ovoid, intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging, one small point or portion of enhancement initially noted after administration of contrast medium and progressive enhancement on later enhancement images, favours the diagnosis of cavernous haemangioma instead of schwannoma . Most orbital cavernous haemangiomas are identified in the intraconal space, predominantly in the lateral aspect of the intraconal space [7, 9]. In our series, only 8 of the 43 orbital cavernous haemangiomas were in the extraconal space . However, orbital schwannomas more commonly arise from sensory nerves in the orbit, specifically the supraorbital, supratrochlear and lacrimal nerves, and thus are usually outside the orbital muscle cone . The nasociliary nerve is the only division of the ophthalmic nerve within the annular tendon . Therefore, orbital schwannomas can be seen as an intraconal or extraconal mass . In our series, the frequency of the schwannoma being located in the extraconal or intraconal space is almost the same . Orbital cavernous haemangiomas appear isointense to muscle on t1-weighted imaging and hyperintense to muscle on t2-weighted imaging [5, 7]. Although non - contrast mr imaging findings are non - specific, they may offer some advantage in the differentiation of orbital cavernous haemangiomas by demonstrating marked hyperintensity signal to muscle with internal septations on t2-weighted imaging, a hypointense circumferential rim corresponding to the fibrous pseudocapsule, and chemical shift artefact in the frequency - encoded direction secondary to high water content within the lesion surrounded by orbital fat [5, 7, 17]. Orbital schwannoma is isointense with respect to the extraocular muscle on t1-weighted images and mildly hyperintense on t2-weighted images [6, 18]. Orbital schwannomas may undergo cavitary change, which appears as a cystic mass with straw - coloured fluid on gross pathological examination and looks homogeneously hypointense to muscle on t1-weighted images and hyperintense on t2-weighted images [8, 19]. Consequently, markedly homogeneous hyperintensity signal on t2-weighted imaging is one of the reliable findings distinguishing the two tumours . Variable degrees of contrast enhancement observed in orbital cavernous haemangiomas were reported, probably related to variable imaging times after contrast medium infusion . However, multiphase dynamic contrast ct and mri demonstrated virtually pathognomonic imaging features of orbital cavernous haemangiomas [5, 7, 17]. On early imaging after the injection, one small point or portion of enhancement is initially noted because of its low - flow arterial supply, and the slow progressive accumulation of contrast medium continues within the dilated vascular spaces filling in centrally during the late venous phase . Subsequently, homogeneous contrast enhancement occurs and persists in the delayed equilibrium phases [7, 1216]. This enhancement pattern is referred to as a progressive enhancement pattern in the literature [10, 11, 13]. Several multiphase dynamic contrast mr imaging protocols have been reported for studying orbital cavernous haemangiomas [7, 11, 12, 14]. Compared with orbital cavernous haemangiomas, no progressive enhancement pattern was found in orbital schwannomas [12, 18]. Therefore, our results and the literature showed that the progressive enhancement pattern will be the most reliable finding in distinguishing orbital cavernous haemangiomas from schwannomas . Some reports in the literature indicated that orbital cavernous haemangiomas showed an initial central point or portion enhancement after administration of gadopentetate dimeglumine [7, 1214]. However, our results show that enhancement starts from one peripheral point or portion of the tumour in most orbital cavernous haemangiomas . The initial enhancement point represents the connecting point of feeding vessels to the lesion, which is useful for estimating the connecting point of the feeding vessels . An unexpected result is that nearly half of orbital cavernous haemangiomas demonstrate a wash - out tic type frequently seen in the malignant tumour rather than a plateau - shaped or persistent tic type . This may be because contrast medium fills one portion of the tumour during an early phase after administration of contrast medium and contrast medium diffuses into adjacent spaces through the foramen of the fibrous interstitium between spaces at a later phase [1214]. Thus it results in a decrease in concentration of contrast media in the portion of the tumour filled by contrast media during the early phase . There is a significant difference in tic type between orbital cavernous haemangiomas and schwannomas in contrast to a previous report that there is no significant difference in tic type between the two tumours . This is probably because we chose an roi covering the greatest degree of early enhancement rather than the whole tumour . Recent literature on dynamic mr imaging in the breast, soft tissue and orbital masses indicates that an roi covering the greatest degree of early enhancement of the mass reflects haemodynamic alterations of the tumour better than an roi covering the whole tumour [10, 21, 22]. The multiphase dynamic contrast mr imaging findings of orbital cavernous haemangiomas have been described in the literature [1014]. However, our results provided additional information on dynamic contrast - enhanced mr imaging findings of orbital cavernous haemangiomas . First, short imaging time and interval of every phase on dynamic contrast mr imaging may accurately show one point or small portion of enhancement of the mass on early phase images after the injection of contrast medium and allow characterization of specific spread pattern of the contrast enhancement in small orbital cavernous haemangiomas, which can differentiate small orbital cavernous haemangiomas from small schwannomas and other tumours . Second, tic type of orbital cavernous haemangioma is significantly different from that of schwannoma . Previous literature showed small haemangiomas could not be differentiated from haemangiopericytomas as their enhancement appears early and strongly and calcification could be noticed in cavernous haemangiomas, contributing to the differential diagnosis [5, 7]. However, calcification is occasionally seen in cavernous haemangiomas and plays a limited role in the differential diagnosis . Our results revealed that strong enhancement starting from a wide area and rapid wash - out on dce mr imaging were seen in haemangiopericytomas that might be helpful in the differential diagnosis . Capillary haemangiomas are a different sort of haemangiomatous tumour that are more common in children, often regress and have a different imaging appearance [5, 7]. Cavernous haemangiomas and schwannomas have different mr imaging features including the location, configuration and margins of the mass, signal intensity and homogeneity on t1- and t2-weighted imaging, the spread pattern of contrast enhancement, the enhancement pattern and tic type, which could be helpful in the differentiation of the two tumours . The spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is the most reliable finding distinguishing cavernous haemangiomas and schwannomas . Markedly homogeneous hyperintensity signal on t2-weighted imaging is another reliable finding distinguishing the two tumours . Mr imaging, especially dynamic contrast - enhanced mr imaging, plays a vital role in the differential diagnosis of cavernous haemangiomas and schwannomas.
Gingival enlargement is a common observation in clinical practice that may occur as a result of a response to varied stimuli and/or interactions with the host and the environment . These lesions could be either localized to certain aspect of the oral cavity or generalized affecting larger areas . The possible reasons for this condition may be related to plaque, hormonal imbalances, or systemic - induced manifestation . These excessive gingival distensions may adversely affect speech, mastication, tooth eruption, esthetics along with major hindrance to the maintenance of routine oral hygiene . Such cases should be treated in a methodical manner, involving a detailed medical history followed by conventional nonsurgical therapy . However, in order to maintain successful therapeutic outcome, it is crucial to create patient awareness and motivation, along with a timely recall visits . This case series describes three different conditions of localized gingival overgrowths and its management with prime emphasis on the importance of patient compliance . Neoplasms are characterized by progressive autonomous growth that can be either a benign or a malignant course, whereas nonneoplastic lesions are usually inflammatory or represent a reaction to some kind of irritation or low - grade injury . Most of these lesions have similar clinical findings such as sessile or pedunculated nodule located at the interdental papilla with color variations from pale pink to erythematous . Pyogenic granuloma (pg) may occur in all ages and is predominant in the second decade of life in females . It is a common nonneoplastic, exophytic vascular growth often associated with the history of trauma or chronic irritation . The present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year . On detailed conversation with the patient, he revealed an occasional habit of smoking since 2 years with no contributory medical history . Clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine, measuring 12 mm 15 mm with an interproximal lingual extension [figure 1a and b]. The color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft . In general, early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent, whereas mature lesions tend to become firm due to the presence of more collagen network . There was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity . An intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 . Hence, based on the location and clinical findings, a provisional diagnosis of pg was assigned . A treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation . Oral hygiene instructions were given and the use of chlorhexidine mouthwash (0.2% clohex, dr . Hyderabad, telangana, india) twice a day for 1 week was advised . At the follow - up interval, . A differential diagnosis of pg, peripheral fibroma, and hyperplastic gingival overgrowth was suggested . Hence, an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence . The surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days . Case 1 (a) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43, (b) lingual extension of overgrowth, (c) histopathological picture in confirmation with pyogenic granuloma, and (d) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification . The stained section showed parakeratinized stratified squamous epithelium, containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion . The underlying connective tissue stroma was delicate to dense and composed of fibroblasts, fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells, which is considered a hallmark of pg . There was moderate, focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [figure 1c]. The patient was recalled after a week and healing was found to be satisfactory [figure 1d]. Correlating the clinical findings with the histological features, a diagnosis of pg the patient is still on follow - up since 6 months with no evidence of recurrence . However, literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias . Peripheral ossifying fibroma (pof) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . A common gingival growth, it is typically seen on the interdental papilla and is believed to comprise about females are more commonly affected, and anterior maxilla is the most prevalent location of involvement . The etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque, calculus, microorganisms, masticatory forces, ill - fitting dentures, and poor - quality restorations . Following the elimination of causative factors, excision of the lesion is the choice of treatment . The present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back . Intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [figure 2a and b]. A tentative diagnosis of fibroma was made and patient was informed about the treatment plan . The patient was advised on the importance of oral care, which included brushing technique and prescription of chlorhexidine mouthwash (0.2% clohex, dr . India) was advised twice daily so as to prevent further disease progression and was recalled after a week . The results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . At some areas, stroma appeared to be dense with ossifying areas assembled as bony trabeculae [figure 2c]. Thus, based on clinical and histopathological findings, the final diagnosis was determined as pof . Case 2 (a) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27, (b) palatal extension of overgrowth, (c) histopathological picture in confirmation with peripheral ossifying fibroma, and (d) follow - up at 2 years as reported in literature, the average time interval for recurrence is within a 12 months period . However, in this present case, no recurrence was seen even at 2 years follow - up [figure 2d]. Gingival enlargement is a known adverse effect of calcium channel blockers, especially the dihydropyridine group . It is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . Among the calcium channel blockers, gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine; however, the incidence with amlodipine is rare . In the present case, a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums, which had been progressively increasing in size over the previous 6 months . The disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene . The patient's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years . Intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [figure 3a]. The enlargement was pink in color, nodular, and slightly edematous with no surface ulcerations . A prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm 15 mm, soft on palpation, and bleeding on provocation present [figure 3b]. Case 3 (a) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors, (b) isolated overgrowth wrt 42 and 43, (c) 4 months post nonsurgical therapy, (d) residual isolated overgrowth wrt 42 and 43, and (e) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor . Patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits . She was referred to a physician for drug substitution who replaced amlodipine with atenolol, a beta - blocker . A full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling . The patient had revealed satisfactory results and the gingival tissues had shown marked regression [figure 3c]. The sessile growth revealed a reduction in size to approximately 7 mm 9 mm [figure 3d]. Differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement, combined gingival enlargement, peripheral fibroma, peripheral giant cell granuloma, pg, and peripheral fibroma . Hence, an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis . The nodular lesion along with surrounding tissues was excised for a complete histologic analysis [figure 3e]. The lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [figure 3e (i)], elongated rete pegs extending deep into connective tissue [figure 3e (ii)] that exhibited densely arranged collagen fibers and new blood vessels [figure 3e (iii)] suggestive of drug - induced gingival enlargement . The other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [figure 3e (iv)]. Connective tissue stroma was edematous, composed of delicate to dense collagen fibers, proliferating blood vessels, and markedly infiltrated by acute and chronic inflammatory cells [figure 3e (v)] that are seen in pg . Thus, the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg . Previously, a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported; however, there has not been any report of amlodipine - induced gingival enlargement associated with pg, to the author's knowledge . Pyogenic granuloma (pg) may occur in all ages and is predominant in the second decade of life in females . It is a common nonneoplastic, exophytic vascular growth often associated with the history of trauma or chronic irritation . The present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year . On detailed conversation with the patient, he revealed an occasional habit of smoking since 2 years with no contributory medical history . Clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine, measuring 12 mm 15 mm with an interproximal lingual extension [figure 1a and b]. The color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft . In general, early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent, whereas mature lesions tend to become firm due to the presence of more collagen network . There was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity . An intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 . Hence, based on the location and clinical findings, a provisional diagnosis of pg was assigned . A treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation . Oral hygiene instructions were given and the use of chlorhexidine mouthwash (0.2% clohex, dr . Hyderabad, telangana, india) twice a day for 1 week was advised . At the follow - up interval, . A differential diagnosis of pg, peripheral fibroma, and hyperplastic gingival overgrowth was suggested . Hence, an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence . The surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days . Case 1 (a) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43, (b) lingual extension of overgrowth, (c) histopathological picture in confirmation with pyogenic granuloma, and (d) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification . The stained section showed parakeratinized stratified squamous epithelium, containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion . The underlying connective tissue stroma was delicate to dense and composed of fibroblasts, fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells, which is considered a hallmark of pg . There was moderate, focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [figure 1c]. The patient was recalled after a week and healing was found to be satisfactory [figure 1d]. Correlating the clinical findings with the histological features, a diagnosis of pg the patient is still on follow - up since 6 months with no evidence of recurrence . However, literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias . Peripheral ossifying fibroma (pof) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . A common gingival growth, it is typically seen on the interdental papilla and is believed to comprise about females are more commonly affected, and anterior maxilla is the most prevalent location of involvement . The etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque, calculus, microorganisms, masticatory forces, ill - fitting dentures, and poor - quality restorations . Following the elimination of causative factors, excision of the lesion is the choice of treatment . The present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back . Intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [figure 2a and b]. A tentative diagnosis of fibroma was made and patient was informed about the treatment plan . The patient was advised on the importance of oral care, which included brushing technique and prescription of chlorhexidine mouthwash (0.2% clohex, dr . India) was advised twice daily so as to prevent further disease progression and was recalled after a week . The results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . At some areas, stroma appeared to be dense with ossifying areas assembled as bony trabeculae [figure 2c]. Thus, based on clinical and histopathological findings, the final diagnosis was determined as pof . Case 2 (a) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27, (b) palatal extension of overgrowth, (c) histopathological picture in confirmation with peripheral ossifying fibroma, and (d) follow - up at 2 years as reported in literature, the average time interval for recurrence is within a 12 months period . However, in this present case, no recurrence was seen even at 2 years follow - up [figure 2d]. Gingival enlargement is a known adverse effect of calcium channel blockers, especially the dihydropyridine group . It is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . Among the calcium channel blockers, gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine; however, the incidence with amlodipine is rare . In the present case, a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums, which had been progressively increasing in size over the previous 6 months . The disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene . The patient's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years . Intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [figure 3a]. The enlargement was pink in color, nodular, and slightly edematous with no surface ulcerations . A prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm 15 mm, soft on palpation, and bleeding on provocation present [figure 3b]. Case 3 (a) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors, (b) isolated overgrowth wrt 42 and 43, (c) 4 months post nonsurgical therapy, (d) residual isolated overgrowth wrt 42 and 43, and (e) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor . A provisional diagnosis of combined gingival enlargement patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits . She was referred to a physician for drug substitution who replaced amlodipine with atenolol, a beta - blocker . A full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling . The patient had revealed satisfactory results and the gingival tissues had shown marked regression [figure 3c]. The sessile growth revealed a reduction in size to approximately 7 mm 9 mm [figure 3d]. Differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement, combined gingival enlargement, peripheral fibroma, peripheral giant cell granuloma, pg, and peripheral fibroma . Hence, an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis . The nodular lesion along with surrounding tissues was excised for a complete histologic analysis [figure 3e]. The lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [figure 3e (i)], elongated rete pegs extending deep into connective tissue [figure 3e (ii)] that exhibited densely arranged collagen fibers and new blood vessels [figure 3e (iii)] suggestive of drug - induced gingival enlargement . The other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [figure 3e (iv)]. Connective tissue stroma was edematous, composed of delicate to dense collagen fibers, proliferating blood vessels, and markedly infiltrated by acute and chronic inflammatory cells [figure 3e (v)] that are seen in pg . Thus, the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg . Previously, a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported; however, there has not been any report of amlodipine - induced gingival enlargement associated with pg, to the author's knowledge . It is possible to misdiagnose reactive lesions arising from the gingiva . A detailed case history to determine the etiology and histopathological examination is essential for an accurate diagnosis and for proper management . Clinical differential diagnosis for localized gingival overgrowths includes fibroma, peripheral giant cell granuloma, pg, peripheral odontogenic fibroma, and peripheral ossifying fibroma . A relatively healthy oral environment provided by the dentist and maintained by the patient will reduce local microflora that will help in eliminating the major focus of infection . A critical factor that assists successful therapeutic outcome the patient should be placed on a regular dental visit, in which a complete description of the existing dental condition should be informed . Creating awareness and educating patients regarding the influence of poor oral hygiene and habits to these gingival lesions are essential to avoid further recurrence.
Successful management and treatment of periodontal disease depends upon simple medical procedures such as health education, prophylaxis, scaling and root - planing, gingivectomy, gingival grafts, and different types of gingival flaps . Periodontal pocket refers to the pathological increase in the depth of gingival sulcus, and is one of the main clinical features of periodontal disease . Elimination of pathological changes of the pocket wall is among the objectives of surgical treatment for periodontal pockets, so that a stable and sustainable situation can be achieved . Currently, several flap techniques have been applied for the treatment of pocket, among which modified widman flap is worth mentioning . This technique facilitates the use of equipment, in which pocket lining is removed with no effect on reducing the pocket depth . Dental treatments are accompanied by patients hemodynamic changes; for instance, increase in blood pressure and heart rate during dental treatment are influenced by various factors such as physical and physiological stress, painful stimuli, and activity of catecholamines present in local anesthetic solutions . Heart rate, which is indicative of ventricular contraction, changes in different conditions, including pain, anxiety, stress, heart diseases, metabolic and endocrine disorders and heart muscle diseases, and is evaluated by peripheral pulse measurement or the electrocardiogram and pulse oximetry . For the measurement of blood pressure, different methods are applied, from which riva - rocci, korotokoff, dinamap, and finometer techniques can be enumerated . Due to the risk of hypoxia in oral surgeries, pulse oximetry pulse oximeter is a reliable and sensitive apparatus in detecting small amounts of blood oxygen saturation, and in all patients, spo2 (spo2) normal percentage is not less than 95% . A dental patient is exposed to stressors, such as physiological responses to emotional factors and/or pain . Pain and anxiety, are important stimuli for the secretion of endogenous adrenaline, which plays a significant role in cardiovascular responses during dental treatment . Local anesthetics mainly affect a limited area; however, they will be absorbed from the injection site and exert general effects, especially on cardiovascular or central nervous system . It is obvious that more the concentration of epinephrine, the better the control of bleeding, but there will be more cardiovascular changes . In spite of the effect of local anesthetics on vital signs, their accurate application is able to dramatically reduce stress . Given the above bodies of evidence and the importance of monitoring patients hemodynamic changes during oral surgery, the present study has been conducted to investigate these alterations during periodontal surgery outside the operating room with local anesthesia . This practical clinical trial study was carried out on patients with gingival disease referred to the clinic of dental faculty of babol university of medical sciences, and underwent modified widman flap surgery on anterior maxillary region in periodontology department . The equipments used in the study included (1) finger pulse oximeter (soor afarinesh company), and (2) automatic sphygmomanometer (omron company) according to the following inclusion criteria, 50 patients ranging from 30 to 50 years participated in this study . In all patients, changes in heart rate, blood pressure, and also pulse oximetry were examined during five steps; the first step before injecting the anesthetic, the second step after the anesthetic injection and before the incision, the third step after the incision, the forth step after debridement, and the fifth step following stitching and the end of the operation . It should be noted that all the operations were performed by one surgeon, and all patients received two anesthetic cartridges containing lidocaine (2%) and epinephrine (1:80000) (darou pakhsh company), using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa, in anterior 1/6 region and incisive canal; no additional anesthetic cartridge was used during surgery, and those in need of re - injection were excluded from the study . Spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient's finger during surgery . Having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . Having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period . Data were analyzed by spss statistical software using anova repeated measure test, and p <0.05 was considered as statistically significant level . According to the following inclusion criteria, 50 patients ranging from 30 to 50 years participated in this study . In all patients, changes in heart rate, blood pressure, and also pulse oximetry were examined during five steps; the first step before injecting the anesthetic, the second step after the anesthetic injection and before the incision, the third step after the incision, the forth step after debridement, and the fifth step following stitching and the end of the operation . It should be noted that all the operations were performed by one surgeon, and all patients received two anesthetic cartridges containing lidocaine (2%) and epinephrine (1:80000) (darou pakhsh company), using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa, in anterior 1/6 region and incisive canal; no additional anesthetic cartridge was used during surgery, and those in need of re - injection were excluded from the study . Spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient's finger during surgery . Having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . Having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period . Data were analyzed by spss statistical software using anova repeated measure test, and p <0.05 was considered as statistically significant level . The present research has been implemented on 50 patients referring to periodontology department of dental faculty for periodontal surgery . Among the study participants with the mean age of 42.58 7.14 years, ranging from 30 to 55 years old, there were 22 men and 28 women . In all cases, systolic and diastolic blood pressure, heart rate, and spo2 were evaluated during five stages; the first step before injecting the anesthetic, the second step after the anesthetic injection and before the incision, the third step after the incision, the forth step after debridement, and the fifth step following stitching and the end of the operation . The mean systolic blood pressure was highest in the second stage and lowest in the first one . In the five steps studied, there was a significant difference between the first step and the second (p <0.0001) and the third (p = 0.001), the second step and the first (p = 0.001) and the fourth and the fifth (p <0.0001), the third step and the first (p = 0.001) and the fourth (p = 0.010), the fourth step and the second (p <0.0001) and the third (p = 0.010), and also the fifth step and the second (p <0.0001) [table 1 and figure 1]. Comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery the maximum and the minimum diastolic blood pressures were, respectively, obtained in the second and the first stage . Statistical analysis showed significant difference between the first stage and the second (p = 0.001), the second stage and the first (p = 0.001) and the third (p <0.0001) and the fourth (p = 0.003), the third stage and the second (p <0.0001), and the fourth step and the second (p = 0.003); however, no meaningful relationship was found between the fifth step and the other stages [table 2 and figure 2]. Comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery according to statistical analysis, the maximum and the minimum heart rate was respectively observed in the second and the fifth stage . Significant differences were found between the first step and the second and the third (p <0.0001), the second and the third steps and the other stages, the fourth step and the second and the third (p <0.0001) and the fifth (p = 0.001), and also the fifth step with the second and the third (p <0.0001) and the fourth (p = 0.001) steps [table 3 and figure 3]. Comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery similarly, based on statistical analysis, no significant difference has been achieved in the mean spo2 during the five stages studied (p> 0.05) [table 4 and figure 4]. Comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery the mean systolic blood pressure was highest in the second stage and lowest in the first one . In the five steps studied, there was a significant difference between the first step and the second (p <0.0001) and the third (p = 0.001), the second step and the first (p = 0.001) and the fourth and the fifth (p <0.0001), the third step and the first (p = 0.001) and the fourth (p = 0.010), the fourth step and the second (p <0.0001) and the third (p = 0.010), and also the fifth step and the second (p <0.0001) [table 1 and figure 1]. Comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery the maximum and the minimum diastolic blood pressures were, respectively, obtained in the second and the first stage . Statistical analysis showed significant difference between the first stage and the second (p = 0.001), the second stage and the first (p = 0.001) and the third (p <0.0001) and the fourth (p = 0.003), the third stage and the second (p <0.0001), and the fourth step and the second (p = 0.003); however, no meaningful relationship was found between the fifth step and the other stages [table 2 and figure 2]. Comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery according to statistical analysis, the maximum and the minimum heart rate was respectively observed in the second and the fifth stage . Significant differences were found between the first step and the second and the third (p <0.0001), the second and the third steps and the other stages, the fourth step and the second and the third (p <0.0001) and the fifth (p = 0.001), and also the fifth step with the second and the third (p <0.0001) and the fourth (p = 0.001) steps [table 3 and figure 3]. Comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery similarly, based on statistical analysis, no significant difference has been achieved in the mean spo2 during the five stages studied (p> 0.05) [table 4 and figure 4]. Comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery hemodynamic changes during dental treatment have long been one of the main concerns among practitioners and researchers . Increase in blood pressure and heart rate during surgery is inevitable; however, the effect of sympathetic nervous system is not well - identified on this augmentation . Since the use of local anesthesia does not merely contribute to increased concentration of plasma noradrenaline without dental treatment, blood pressure response appears to be depending on dental treatment process itself . As mentioned earlier, elevated blood pressure during oral surgery is attributed to the sympathetic nervous system . Due to possessing vasoconstrictors and anesthetic components, adrenaline is used as a vasoconstrictor along with lidocaine to increase the amount and duration of anesthesia . Moreover, many researchers have pointed to changes probably effective on normal patients physiological stability . Sedative drugs and local anesthetics can be used to control the autonomic nervous system responses and help to reduce the cardiovascular alterations . In addition, different people react differently to anxiety and pain, which could be significantly influential on their hemodynamic alterations . In the present study, blood pressure and heart rate were remarkably increased after injection of anesthesia (p <0.05). According to figures 1, 2, and 3, a meaningful difference has been found in the second step and following the anesthetic injection containing lidocaine and epinephrine (1:80000) (p <0.0001) in systolic and diastolic blood pressure and heart rate, on which the result was almost predictable . This can be associated with hemodynamic effects of adrenaline, as well as alpha - adrenergic effects to peripheral vasoconstriction and beta - adrenergic effects to increased cardiac contraction . Furthermore, patient's physiological stress before surgery and also painful stimulus following injection might lead to increased sympathetic activity and subsequently blood pressure and heart rate . Thus, application of anxiety - reducing protocols such as psychological sedatives during treatment and minimizing patient's waiting time can greatly control endogenous secretion of adrenaline . The study figures present a reduction in systolic and diastolic blood pressure following the second step and during the third, fourth, and fifth steps, indicating the elimination of stimulatory effect of adrenaline on blood pressure and heart rate and showing insignificant impact of other surgical steps on vital signs in cases of no anesthetics injection . Furthermore, over time and acceptance of treatment processes by patient, the initial anxiety will be diminished and the person will reach to a relatively stable cardiovascular balance . Since the peak effects of local anesthetics and alpha and beta epinephrine impact elimination occur in the third and the fourth steps, decrement in heart rate and blood pressure seems to be obvious in these stages . According to figure 2, diastolic blood pressure has been slightly elevated in the fifth step, after stitching at the end of the operation, which is probably due to feeling of pain by patient and decrease in the effect of anesthetic . In a study by matsumura et al . Investigating changes in blood pressure and heart rate during dental surgery, the results revealed an increase in both parameters after anesthesia with lidocaine (2%) and epinephrine (1:80000), which is compatible to findings of the present research . In faraco survey on the effect of anesthetics containing lidocaine and epinephrine on cardiovascular changes during dental implant surgery, blood pressure was evaluated using the automated noninvasive oscillometric technique during ten steps; the results showed no significant difference in blood pressure and heart rate variability, which is not in accordance to the findings achieved in this research . In another study by gedik et al . On blood pressure, heart rate and temperature variability during periodontal surgery, patients were divided into four groups according to types of surgery, and blood pressure and heart rate were measured after anesthetic injection and the end of the operation; blood pressure and heart rate were decreased in patients undergoing gingivectomy and periodontal flap, which is in consistence with the results obtained in the present research . However, no changes in heart rate and blood pressure have been observed following frenectomy and curettage, indicating that the difficulty in treatment process is an effective factor in blood pressure . As shown in figure 4, no statistically significant difference has been found in the mean spo2, measured by pulse oximetry, in different stages (p> 0.05). In other words, conventional periodontal surgery with technical and safety observations in an investigation by kaviani and birang, nausea, vomiting, decreased or increased blood pressure and other hypoxia complications following the use of nitrous oxide inhalation sedation during periodontal treatment have only been observed in 1 out of 32 cases, which was probably due to patient restlessness and movement during treatment . Have addressed to pulse oximetry during oral surgery with and without intravenous sedation and found out a significant reduction in arterial blood oxygen levels in the group receiving midazolam . This study illustrates remarkable sedative effect of midazolam on anxious patient prior to the surgery; nonetheless, this effect does not sustain during the operation . As mentioned, findings of various studies in terms of hemodynamic changes following dental treatments are contradictory, and such discrepancies could be related to different research approaches and different local anesthetics applied . Different surgical stages will be accompanied by diverse effects on hemodynamic variability and pulse oximetry . According to the present study, blood pressure and heart rate are significantly increased after anesthetic injection and are decreased in the following stages by the elimination of adrenaline stimulatory effect . Moreover, no significant alteration has been observed in spo2 during the operational steps in this research.
Chronic kidney disease (ckd) is defined as a structural or functional kidney abnormality lasting for 3 or more months . The global prevalence of ckd is estimated to be more than 10%, and ckd has emerged as a public health problem . Adverse outcomes of ckd such as kidney failure, cardiovascular disease (cvd), and premature death can be prevented or delayed when treatment is initiated in the early stages of disease . As the earlier stages are often asymptomatic, ckd is usually detected during laboratory evaluation of comorbid conditions . Glomerular filtration rate (gfr) is considered the index of kidney function, and for convenience is usually estimated using equations instead of by direct measurement . The modification of diet in renal disease (mdrd) study equation, which was replaced by the chronic kidney disease epidemiology collaboration 2009 equations (ckd - epi equations), has been used to estimate gfr . However, this tended to underestimate the measured gfr and had less precision, especially at a gfr of 60 ml / min/1.73 m or more . The ckd - epi equations were reported as having less bias, improved precision, and greater accuracy than the mdrd study equation at a higher gfr of 60 ml / min/1.73 m or more . The ckd - epi equations led to a lower estimation of ckd prevalence, primarily due to a lower prevalence of gfr stage 3, compared to the mdrd study equation . Persistently increased protein excretion has usually been considered to be a marker of kidney damage, and screening with urine dipsticks has been acceptable for detecting proteinuria . Recently, urinary albumin measurement was recommended as a marker of kidney damage, both for its standardization and also because albumin is the most important protein lost in the urine in most ckd . The albumin - to - creatinine ratio (acr) is the most preferred value among urinary albumin measurements . Kidney disease: improving global outcomes (kdigo) has updated guidelines for defining, diagnosing, staging, and managing ckd since the establishment of the first uniform definitions and staging system for ckd in 2002 . More objective criteria, such as the ckd - epi equations for estimating gfr and classification of albuminuria, have been incorporated . Moreover, the etiology of ckd was included in the definition; hypertension and diabetes mellitus (dm) are systemic diseases known to cause ckd . Consequently, kdigo guidelines provide relatively simple and precise methods for early diagnosis of ckd, and tend to yield fewer false positives, thereby avoiding incorrect diagnosis . The number of patients treated with renal replacement therapy (rrt) including dialysis and transplantation has increased dramatically in korea, from 28,046 in 2000 to 75,042 in 2013 . The prevalence of rrt was 1,446 patients per million population (pmp), with an incidence of 234 pmp in 2013 . The cause of kidney failure was estimated to be dm in 48.0%, hypertension in 19.7%, and chronic glomerulonephritis in 8.3% of cases . The previously reported ckd prevalence calculated from the mdrd study equation the ckd prevalence calculated from the mdrd study equation alone was 8.8% in 2005 and 7.2% in 2007 in korea national health and nutrition examination survey (knhanes) subjects aged 20; the prevalence calculated using the mdrd study equation with the dipstick proteinuria method has tended to decrease since 2001 in men and since 2005 in women among knhanes i to iv (1998 to 2009) subjects aged 20 . Since there are no reports on ckd prevalence using the ckd - epi equations to estimate gfr and acr to measure albuminuria for koreans, a new analysis of ckd prevalence is necessary . This study was therefore designed to investigate the prevalence of ckd in the korean population using knhanes samples, based on the definition and classification by kdigo 2012 guidelines, as well as the impact of changes through a comparison of the ckd - epi and mdrd study equations for the estimation of gfr, and the associations between ckd and major risk factors, including dm, hypertension, age, and gender . This retrospective, population - based study was performed using data acquired by the knhanes v from 2010 to 2012 . Knhanes v was a cross - sectional, nationally representative survey with a multistage, stratified, and probability sampling design based on sex, age, and geographical area using household registries . Knhanes, composed of a health questionnaire survey, physical examination, and nutrition survey, has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention (kcdc). The study protocol was approved by the institutional review board of kcdc (2010 - 02con-21-c), and written informed consents were obtained from all subjects or their guardians . Knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 . The inclusion criteria were (1) adults aged 19, and (2) available serum creatinine (scr), urine albumin, and urine creatinine test results . Individuals aged 19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 . The health examination was performed in a mobile health examination center . Was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association . Three readings were obtained via mercury sphygmomanometer (baumanometer, baum, copiague, ny, usa), and the mean of the second and third reading was reported as the final bp for individual participants . Body mass index (bmi) was calculated as body weight divided by the square of the height, and expressed in units of kg / m . Blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast . A random urine sample (first urine in the morning, if possible) was obtained in a conical container . The urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day (neodin medical institute, seoul, korea). Serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako (wako, osaka, japan) following the jaffe method; fasting glucose concentration (fasting blood glucose, fbg) by an enzymatic method using pureauto s glu (sekisui, osaka, japan); total cholesterol, high density lipoprotein cholesterol (hdl - c), and triglycerides by an enzymatic method; and urine albumin by turbidimetric assay using albumin (roche, mannheim, germany), all performed using a hitachi automatic analyzer 7600 (hitachi high - technologies co., tokyo, japan), which was calibrated using calibrator for automated systems (roche diagnostics, indianapolis, in, usa). Glycated hemoglobin (a1c) was assayed by high performance liquid chromatography (hlc-723g7 a1c, tosoh, tokyo, japan). Urine chemistry was tested by test strip (urysis 2400 cassette, roche) using urisys 2400 (roche). Low density lipoprotein cholesterol (ldl - c) was computed using the friedewald formula when triglycerides were less than 400 mg / dl . Ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr . Gfr was estimated using ckd - epi equations, taking into account gender and serum creatinine . Individuals with gfr <60 ml / min/1.73 m or acr 30 mg / g were classified as ckd patients . Hypertension was defined as having a systolic blood pressure (sbp) of 140 mmhg or a diastolic blood pressure (dbp) of 90 mmhg, or having received a physician s diagnosis . Dm was defined as having a1c 6.5%, fbg of 126 mg / dl, or having received a physician s diagnosis . Dyslipidemia was defined as having total cholesterol 240 mg / dl, ldl - c 160 mg / dl, hdl - c <40 mg / dl, triglycerides 200 mg / dl, or having received a physician s diagnosis . Analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations . The study population was separated as a subpopulation, and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias . In addition, weight adjustments were applied to the user - missing values to prevent skews in sampling design . The demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables . The mean and the 95% confidence interval (ci) were presented for data on continuous variables, whereas unweighted count with weighted percentage was presented for data on categorical variables . The complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories, and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage . A subpopulation was added to strata when performing the complex samples chi - square test . Associations of ckd, gfr, and acr categories with various risk factors, including gender, age, bmi, dm, hypertension, and dyslipidemia were expressed as an odds ratio (or), which was calculated using complex samples multivariate logistic regression analyses . This retrospective, population - based study was performed using data acquired by the knhanes v from 2010 to 2012 . Knhanes v was a cross - sectional, nationally representative survey with a multistage, stratified, and probability sampling design based on sex, age, and geographical area using household registries . Knhanes, composed of a health questionnaire survey, physical examination, and nutrition survey, has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention (kcdc). The study protocol was approved by the institutional review board of kcdc (2010 - 02con-21-c), and written informed consents were obtained from all subjects or their guardians . Knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 . The inclusion criteria were (1) adults aged 19, and (2) available serum creatinine (scr), urine albumin, and urine creatinine test results . Individuals aged 19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 . Blood pressure (bp) was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association . Three readings were obtained via mercury sphygmomanometer (baumanometer, baum, copiague, ny, usa), and the mean of the second and third reading was reported as the final bp for individual participants . Body mass index (bmi) was calculated as body weight divided by the square of the height, and expressed in units of kg / m . Blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast . A random urine sample (first urine in the morning, if possible) the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day (neodin medical institute, seoul, korea). Serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako (wako, osaka, japan) following the jaffe method; fasting glucose concentration (fasting blood glucose, fbg) by an enzymatic method using pureauto s glu (sekisui, osaka, japan); total cholesterol, high density lipoprotein cholesterol (hdl - c), and triglycerides by an enzymatic method; and urine albumin by turbidimetric assay using albumin (roche, mannheim, germany), all performed using a hitachi automatic analyzer 7600 (hitachi high - technologies co., tokyo, japan), which was calibrated using calibrator for automated systems (roche diagnostics, indianapolis, in, usa). Glycated hemoglobin (a1c) was assayed by high performance liquid chromatography (hlc-723g7 a1c, tosoh, tokyo, japan). Urine chemistry was tested by test strip (urysis 2400 cassette, roche) using urisys 2400 (roche). Low density lipoprotein cholesterol (ldl - c) was computed using the friedewald formula when triglycerides were less than 400 mg / dl . Ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr . Gfr was estimated using ckd - epi equations, taking into account gender and serum creatinine . Individuals with gfr <60 ml / min/1.73 m or acr 30 mg / g were classified as ckd patients . Hypertension was defined as having a systolic blood pressure (sbp) of 140 mmhg or a diastolic blood pressure (dbp) of 90 mmhg, or having received a physician s diagnosis . Dm was defined as having a1c 6.5%, fbg of 126 mg / dl, or having received a physician s diagnosis . Dyslipidemia was defined as having total cholesterol 240 mg / dl, ldl - c 160 mg / dl, hdl - c <40 mg / dl, triglycerides 200 mg / dl, or having received a physician s diagnosis . Analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations . The study population was separated as a subpopulation, and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias . In addition, weight adjustments were applied to the user - missing values to prevent skews in sampling design . The demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables . The mean and the 95% confidence interval (ci) were presented for data on continuous variables, whereas unweighted count with weighted percentage was presented for data on categorical variables . The complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories, and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage . A subpopulation was added to strata when performing the complex samples chi - square test . Associations of ckd, gfr, and acr categories with various risk factors, including gender, age, bmi, dm, hypertension, and dyslipidemia were expressed as an odds ratio (or), which was calculated using complex samples multivariate logistic regression analyses . All statistical analyses were performed using ibm spss version 21.0 (ibm corp ., armonk, ny, usa). The statistical significance level was set at p <0.05 . Among the total sample size of 16,576 from 2011 and 2012, 64.7% satisfied the inclusion criteria (10,636 individuals: 5,388 and 5,248 from 2011 and 2012, respectively) as shown in fig . The composition of neither the total adults nor the study population was statistically different between 2011 and 2012 (p = 0.232 and p = 0.087, respectively). The study population comprised 81.8% of total adults (81.0% and 82.7% from 2011 and 2012, respectively, the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable, except for hdl - c, ldl - c, and prevalence of dyslipidemia (table 1). The percentage of men was 52.6%, and the mean age was 45.8 years (95% ci, 45.2 to 46.3). Mean serum creatinine was 0.86 mg / dl (95% ci, 0.85 to 0.86), with an estimated gfr of 96 ml / min/1.73 m (95% ci, 96 to 97). Mean acr was 18 mg / g (95% ci, 15 to 21), with urine albumin 22 g / ml (95% ci, 19 to 26) and urine creatinine 165 mg / dl (95% ci, 163 to 168). Mean sbp was 118 mmhg (95% ci, 117 to 119), and dbp 76 mmhg (95% ci, 76 to 77). Mean fbg was 97 mg / dl (95% ci, 97 to 98), and a1c 5.7% (95% ci, 5.67 to 5.71). Mean total cholesterol, hdl - c, ldl - c, and triglycerides were 190 mg / dl (95% ci, 188 to 191), 49 mg / dl (95% ci, 49 to 50), 114 mg / dl (95% ci, 113 to 115), and 136 mg / dl (95% ci, 133 to 139), respectively . The ckd prevalence in korean adults was estimated as 7.9% (7.8% in 2011 and 8.0% in 2012, p = 0.770) (table 1). The prevalence of hypertension, dm, and dyslipidemia was 27.8% (27.2% in 2011 and 28.4% in 2012, p = 0.380), 9.3% (9.7% in 2011 and 9.0% in 2012, p = 0.306), and 32.8% (34.6% in 2011 and 31.0% in 2012, p = 0.004), respectively . The distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 . The prevalence of low, moderately increased, high, and very high ckd risk prognosis was 92.0%, 6.3%, 1.1%, and 0.6%, respectively . The prevalence of gfr <60 ml / min/1.73 m was 2.2%, whereas acr 30 mg / g was 6.5% (a2 of 5.6% and a3 of 0.9%). The prevalence of acr 30 mg / g in individuals with gfr 60 ml / min/1.73 the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig . The prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation, and g2 was 29.8% and 38.7%, respectively . The prevalence of gfr <60 ml / min/1.73 m by the mdrd study equation was 2.0%, which was significantly less than the 2.2% by ckd - epi equations (p <0.001). The prevalence of at least 1 + proteinuria in a1, expected to be fully negative, was 0.1% whereas or proteinuria in a2 and a3, expected to be fully positive, were 5.3% and 0.3%, respectively . Some interactions between demographic covariates, such as gender, age, and bmi, were present . The risk of bmi 25 for men was higher than for women (or, 1.296; 95% ci, 1.164 to 1.443; p <0.001). The study population was stratified into six groups by age in 10-year increments: 19 to 28, 29 to 38, 39 to 48, 49 to 58, 59 to 68, and 68 . The ors of increasing age (using the age group of 19 to 28 as a reference) to bmi 25 were 1.731 (95% ci, 1.400 to 2.139), 2.150 (95% ci, 1.757 to 2.632), 1.689 (95% ci, 1.376 to 2.074), 2.092 (95% ci, 1.690 to 2.590), and 1.554 (95% ci, 1.255 to 1.924; p = 0.001), respectively . In addition, the composition of men significantly decreased in the 49 age groups; ors of men to increasing age (using the age group of 19 to 28 as a reference) were 0.931 (95% ci, 0.784 to 1.105), 0.894 (95% ci, 0.750 to 1.065), 0.735 (95% ci, 0.625 to 0.864), 0.691 (95% ci, 0.578 to 0.825), and 0.505 (95% ci, 0.421 to 0.607; p = 0.001), respectively . Thus, the association analysis was performed separately for each gender . Because ckd is defined by both gfr and albuminuria, the associations between the risk factors and either decreased gfr or increased acr were also studied . The frequency of women among ckd patients was 54.3%, which was significantly higher than that for men (p <0.001). Among individuals with acr 30 mg / g, women accounted for 55.4% (p <0.001); however, women constituted 48.7% of gfr <60 ml / min/1.73 m (p = 0.683). The ors of gender to ckd, acr 30 mg / g, and gfr <60 ml / min/1.73 m for men were 1.354 (95% ci, 1.162 to 1.577; p <0.001), and 1.057 (95% ci, 0.811 to 1.376; p = 0.683), respectively . The prevalence of bmi 25, ckd, gfr <60 ml / min/1.73 m, acr 30 mg / g, dm, hypertension, and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 . The ors of covariates to the prevalence of ckd, gfr <60 ml / min/1.73 m, and acr 30 mg / g, after adjusting for age, bmi, hypertension, dm, and dyslipidemia in men and women are listed in table 6 . Hypertensive patients showed a higher risk of ckd than the normotensive population (or, 3.394 for men; or, 2.935 for women; both p <0.0001). The ors of hypertension to cause albuminuria were 3.351 (p <0.001) for men and 3.067 (p <0.001) for women, and to decrease gfr, 3.436 (p <0.001) for men and 1.904 (p = 0.006) for women . Diabetic patients showed a higher risk of ckd than the nondiabetic population (or, 3.145 for men; or, 1.983 for women; both p <0.001). The ors of dm to cause albuminuria were 3.144 (p <0.001) for men and 2.022 (p <0.001) for women, and to decrease gfr 1.965 (p = 0.004) for men and 1.764 (p = 0.013) for women . Dyslipidemia showed a significant association with ckd in women (or, 1.324; p = 0.023), but not in men (or, 1.175; p = 0.243). Increasing age and obesity (bmi 25) showed a positive correlation with ckd . Among the total sample size of 16,576 from 2011 and 2012, 64.7% satisfied the inclusion criteria (10,636 individuals: 5,388 and 5,248 from 2011 and 2012, respectively) as shown in fig . The composition of neither the total adults nor the study population was statistically different between 2011 and 2012 (p = 0.232 and p = 0.087, respectively). The study population comprised 81.8% of total adults (81.0% and 82.7% from 2011 and 2012, respectively, the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable, except for hdl - c, ldl - c, and prevalence of dyslipidemia (table 1). The percentage of men was 52.6%, and the mean age was 45.8 years (95% ci, 45.2 to 46.3). Mean serum creatinine was 0.86 mg / dl (95% ci, 0.85 to 0.86), with an estimated gfr of 96 ml / min/1.73 m (95% ci, 96 to 97). Mean acr was 18 mg / g (95% ci, 15 to 21), with urine albumin 22 g / ml (95% ci, 19 to 26) and urine creatinine 165 mg / dl (95% ci, 163 to 168). Mean sbp was 118 mmhg (95% ci, 117 to 119), and dbp 76 mmhg (95% ci, 76 to 77). Mean fbg was 97 mg / dl (95% ci, 97 to 98), and a1c 5.7% (95% ci, 5.67 to 5.71). Mean total cholesterol, hdl - c, ldl - c, and triglycerides were 190 mg / dl (95% ci, 188 to 191), 49 mg / dl (95% ci, 49 to 50), 114 mg / dl (95% ci, 113 to 115), and 136 mg / dl (95% ci, 133 to 139), respectively . The ckd prevalence in korean adults was estimated as 7.9% (7.8% in 2011 and 8.0% in 2012, p = 0.770) (table 1). The prevalence of hypertension, dm, and dyslipidemia was 27.8% (27.2% in 2011 and 28.4% in 2012, p = 0.380), 9.3% (9.7% in 2011 and 9.0% in 2012, p = 0.306), and 32.8% (34.6% in 2011 and 31.0% in 2012, p = 0.004), respectively . The distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 . The prevalence of low, moderately increased, high, and very high ckd risk prognosis was 92.0%, 6.3%, 1.1%, and 0.6%, respectively . The prevalence of gfr <60 ml / min/1.73 m was 2.2%, whereas acr 30 mg / g was 6.5% (a2 of 5.6% and a3 of 0.9%). The prevalence of acr 30 mg / g in individuals with gfr 60 ml / min/1.73 the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig . 2 . The prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation, and g2 was 29.8% and 38.7%, respectively . The prevalence of gfr <60 ml / min/1.73 m by the mdrd study equation was 2.0%, which was significantly less than the 2.2% by ckd - epi equations (p <0.001). The prevalence of at least 1 + proteinuria in a1, expected to be fully negative, was 0.1% whereas or proteinuria in a2 and a3, expected to be fully positive, were 5.3% and 0.3%, respectively . Some interactions between demographic covariates, such as gender, age, and bmi, were present . The risk of bmi 25 for men was higher than for women (or, 1.296; 95% ci, 1.164 to 1.443; p <0.001). The study population was stratified into six groups by age in 10-year increments: 19 to 28, 29 to 38, 39 to 48, 49 to 58, 59 to 68, and 68 . The ors of increasing age (using the age group of 19 to 28 as a reference) to bmi 25 were 1.731 (95% ci, 1.400 to 2.139), 2.150 (95% ci, 1.757 to 2.632), 1.689 (95% ci, 1.376 to 2.074), 2.092 (95% ci, 1.690 to 2.590), and 1.554 (95% ci, 1.255 to 1.924; p = 0.001), respectively . In addition, the composition of men significantly decreased in the 49 age groups; ors of men to increasing age (using the age group of 19 to 28 as a reference) were 0.931 (95% ci, 0.784 to 1.105), 0.894 (95% ci, 0.750 to 1.065), 0.735 (95% ci, 0.625 to 0.864), 0.691 (95% ci, 0.578 to 0.825), and 0.505 (95% ci, 0.421 to 0.607; p = 0.001), respectively . Thus, the association analysis was performed separately for each gender . Because ckd is defined by both gfr and albuminuria, the associations between the risk factors and either decreased gfr or increased acr were also studied . The frequency of women among ckd patients was 54.3%, which was significantly higher than that for men (p <0.001). Among individuals with acr 30 mg / g, women accounted for 55.4% (p <0.001); however, women constituted 48.7% of gfr <60 ml / min/1.73 m (p = 0.683). The ors of gender to ckd, acr 30 mg / g, and gfr <60 ml / min/1.73 m for men were 1.354 (95% ci, 1.162 to 1.577; p <0.001), 1.416 (95% ci, 1.193 to 1.682; p <0.001), and 1.057 (95% ci, 0.811 to 1.376; p = 0.683), respectively . The prevalence of bmi 25, ckd, gfr <60 ml / min/1.73 m, acr 30 mg / g, dm, hypertension, and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 . The ors of covariates to the prevalence of ckd, gfr <60 ml / min/1.73 m, and acr 30 mg / g, after adjusting for age, bmi, hypertension, dm, and dyslipidemia in men and women are listed in table 6 . Hypertensive patients showed a higher risk of ckd than the normotensive population (or, 3.394 for men; or, 2.935 for women; both p <0.0001). The ors of hypertension to cause albuminuria were 3.351 (p <0.001) for men and 3.067 (p <0.001) for women, and to decrease gfr, 3.436 (p <0.001) for men and 1.904 (p = 0.006) for women . Diabetic patients showed a higher risk of ckd than the nondiabetic population (or, 3.145 for men; or, 1.983 for women; both p <0.001). The ors of dm to cause albuminuria were 3.144 (p <0.001) for men and 2.022 (p <0.001) for women, and to decrease gfr 1.965 (p = 0.004) for men and 1.764 (p = 0.013) for women . Dyslipidemia showed a significant association with ckd in women (or, 1.324; p = 0.023), but not in men (or, 1.175; p = 0.243). Increasing age and obesity (bmi 25) showed a positive correlation with ckd . This study is the first to report ckd prevalence using gfr estimated by using ckd - epi equations together with acr for albuminuria in the korean population . The prevalence of ckd was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged 19, and the ckd risk prognosis was reported in table 2 . The ckd risk prognosis from moderately increased to very high reported in this study was lower than that in the usa . Gfr and albuminuria are independent and complementary predictors of important clinical outcomes, including ckd progression, end - stage renal disease, acute kidney injury, cardiovascular mortality, and all - cause mortality [16 - 19]. The frequencies of gfr <60 ml / min/1.73 m showed sharp increases starting from around age 50 for men and 60 for women, whereas those of acr 30 mg / g increased relatively gradually, starting from earlier ages, as shown in table 4 and 5 . The rate of decline of measured gfr was reported to double in kidney transplantation donors aged> 45 years . The prevalence of estimated gfr <60 ml / min/1.73 m using ckd - epi equations and the mdrd study equation was 2.2% and 2.0% in this study, but 2.6% and 3.2%, respectively in the previous study . The statistical method used in the current study was complex sample analysis incorporating sampling weight, in contrast to the previous study which did not consider weight in the analysis . The frequencies of gfr stage g1 and g2 by ckd - epi equations were 67.7% and 29.8%, whereas those by the mdrd study equation were 59.4% and 38.7%, respectively, in this study . These numbers are comparable to the previous study, considering the different analytical methods; the frequencies of gfr stage g1 and g2 by ckd - epi equations were 64.5% and 28.9%, whereas those by the mdrd study equation were 47.6% and 49.2%, respectively . These results confirm the tendency to underestimate gfr by the mdrd study equation when gfr is 60 ml / min/1.73 m or greater . The previous report of lower prevalence of gfr stage g3 using ckd - epi equations than with the mdrd study equation was not observed in this study . Among the population of the previous study, the frequencies of g3 by ckd - epi equations and the mdrd study equation (33.2% and 37.2%, respectively) were far greater than those among the korean population (2.1% and 1.9%, respectively). The prevalence of acr 30 mg / g in the entire study population, and acr 30 mg / g in individuals with gfr 60 ml / min/1.73 the prevalence of acr 30 mg / g in the entire study population and acr 30 mg / g in individuals with gfr 60 ml / min/1.73 m aged 35 years was 10.2% and 8.7%, respectively, in the previous korean population . Besides the different age criteria, the populations of the previous and current study are quite different; the previous study was designed by the researcher group considering age, gender, and city factors not using the knhanes sample data . Although the absolute values are different, the important point in common is that a large number of people have albuminuria before gfr decreases to <60 ml / min/1.73 m (85.3% and 87.7% of the albuminuria cases did not have decreased gfr in the previous and current study, respectively). As shown in table 3, test strips did not detect albuminuria in 81.5% of cases . At present, laboratory tests for albuminuria are not routinely performed during regular physical examinations in korea . The prevalence of ckd in men and women was 6.9% and 9.1%, respectively (p <0.001). Besides gender itself, age distribution, frequency of obesity (bmi 25), and concurrent chronic diseases, including dm, hypertension, and dyslipidemia, were different according to gender . Thus, the risk of ckd is higher in women than men (or, 1.354; p <0.001), and the difference seems to be caused by the susceptibility to albuminuria (or, 1.416; p <0.001) rather than decreased gfr (or, 1.057; p = 0.683). When gfr was estimated using the mdrd study equation, the increased risks of gfr <60 ml / min/1.73 m in women were observed in previous studies: or, 3.16 (p <0.001) using knhanes iii and iv-1; frequencies of 3.4%, 9.7%, 10.2%, and 4.6% in women, and 1.0%, 5.4%, 3.1%, and 2.6% were seen in men through knhanes i - iv . There seemed to be no difference in acr according to gender in the previous study (10.1% and 10.3% in women and men, respectively). A similar observation was reported for proteinuria by gender (frequencies of 3.3%, 2.3%, 1.4%, and 1.9% in women, and 3.1%, 3.0%, 2.8%, and 2.2% in men through knhanes direct comparison is difficult due to the difference in the designs of study populations and laboratory test methods using dipstick proteinuria and acr . Increasing age and obesity (bmi 25) correlated significantly with ckd, as shown in table 6, which agreed with previous studies [11 - 13]. Hypertension and dm are both major risk factors and consequences of ckd [21 - 23]. Concurrent chronic diseases such as dm and hypertension increased the risk of ckd consistently [11 - 13]. Diabetic patients are susceptible to glomerular disease and hypertensive patients to renal vascular disease . Since the prevalence of dm was higher in men than women, with the opposite for prevalence of ckd, the or of dm to ckd in men was higher than women, as shown in table 6 . Moreover, dm was correlated with increased acr rather than decreased gfr in this study . Since increased acr appeared at an earlier age in women than men, and decreased gfr was preceded by increased acr, the difference between ors to gfr <60 ml / min/1.73 m and acr 30 mg / g in hypertensive women was greater than hypertensive men . On the other hand, limited availability of direct ldl - c measurement, unstandardized laboratory assay methods for hdl - c, and lack of a uniform definition of dyslipidemia between the studies made it difficult to evaluate their associations . The major limitation of this study is the lack of the evaluation for chronicity assessment and use of single measurements for laboratory tests due to the cross - sectional data characteristics . According to the definition of ckd by the kdigo guideline the rationale for defining chronicity is to differentiate ckd from acute kidney diseases, which may require different interventions and have different etiologies and outcomes . In addition, there is still the discrepancy between measured and estimated gfr using ckd - epi equations . Gfr estimation using equations is convenient, but measurement of gfr should be considered to diagnose a specific patient . Finally, association analysis could not provide conclusive causal relationships due to the study characteristics . However, the representative features of the knhanes sample data cannot be overlooked . In conclusion, the prevalence of ckd using estimated gfr by the ckd - epi equation and acr for albuminuria measurement was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged 19 . The prevalence of albuminuria (acr 30 mg / g) before decline in gfr (gfr 60 ml / min/1.73 m) was 5.7% . Despite kdigo guidelines to prevent or delay progression of ckd in its early stages, implementation remains difficult due to practical limitations . The failure to recognize ckd in its early stage may result in complications such as premature rrt . Thus, urinalysis to detect albumin should be strongly recommended for patients with higher risk of ckd and cvd . As there is a consensus about the risk of ckd with dm and hypertension, regular albuminuria testing for at least these patients to detect kidney disease at an early stage 1 . The prevalence of chronic kidney disease (ckd) was estimated to be 7.9% in the korea national health and nutrition examination survey samples of 2011 and 2012 in korean adults aged 19 . Early identification of ckd paired with appropriate management and earlier referral to specialty kidney services results in economic and clinical benefits . The majority of ckd patients have albuminuria prior to a decrease in glomerular filtration rate . 4 . Regular laboratory tests for albuminuria in the high - risk group, especially for hypertensive or diabetes mellitus patients, should contribute to early detection of ckd.
While it is true that not all patients are able to dialyse themselves at home, it is a sad fact that many are never given the chance, despite the fact that patient choice has never been more discussed as an answer to almost any health care problem . Although studies suggest that renal physicians and nurses regard home dialysis as the best treatment, it is in decline in most european countries, yet in - centre hospital - based dialysis is the most expensive and most disempowering option available . The practical barriers to home therapy vary from unit to unit but need to be identified and overcome if patients are to benefit from self - management . However, it can be difficult to convince both staff and patients if they have no direct experience or training in this aspect of renal replacement therapy . Patient pathway to (relative) freedom a step which can be made much easier if staff are familiar with the stages of change model and have a basic understanding of motivational interviewing . Pre - emptive living donor transplantation should always be promoted as the first - line treatment for kidney failure . However, where that is not possible, patients must receive timely, adequate and unbiased information and advice regarding the complete array of dialysis options available, including home - based peritoneal dialysis (pd) and haemodialysis (hd). Interestingly, a comparison of survival in canadian patients treated with nocturnal hd or deceased donor kidney transplantation showed no difference between the two treatments, suggesting that this intensive dialysis modality may be a bridge to transplantation, or even a suitable alternative, in the absence of an available living donor . Traditionally around the world, medical and nursing staff, sometimes in conjunction with a social worker, will . A clear example of this was evident in the 1990s, when the uk had a large pd population simply because space in hospital hd facilities was limited at that time, and home hd had shrunk to the extent that most uk units no longer had viable programmes . Since that time, in - centre hd facilities in the uk have expanded enormously, such that in some areas supply exceeds demand and the pd population has approximately halved in number . Therefore, many patients currently are not able to choose home hd and patient selection presumably does not generally occur . Staff are unlikely to be able to advocate a therapy of which they have no experience and are even less likely to advocate one which they perceive to be unavailable . Therefore, health service managers and commissioners also have an enabling role and must embrace the value of self - care by providing the practical resources required . Understandably, a patient may never be adequately informed about home hd or assisted automated peritoneal dialysis if these modalities are not operating in the unit concerned . Many patients will assume that they are not suitable for a home therapy if it is not offered to them, without realizing why it has not been offered . Ideally, every patient should learn about every dialysis modality, accepting that it is probably pointless to discuss pd with a patient who has previously had major abdominal surgery . If this were done, then some patients would inevitably demand the creation of home services where none currently exists, and in one or two uk units, this has already happened . Experience within our own service suggests that where a dialysis unit enables and actively encourages self - management, patients will tend to select themselves, and if well motivated may overcome significant difficulties, such as needle phobia, housing problems and literacy issues, to exceed the expectations or predictions of even quite experienced dialysis staff . Patients then become advocates themselves and can provide other patients with the necessary motivation to consider a home treatment, such that they approach staff, rather than vice versa . Interestingly, the general consensus among nephrologists in canada, usa and uk in 2006 was that 45% of patients are suitable for a home therapy, and if you question the staff in your local hd unit, very few would wish themselves to be dialysed in - centre if the need arose . Despite this, the reality is that pd numbers are in decline and home hd is virtually non - existent in many areas . Selection to medical and nursing staff simply does not work and that many capable patients are not finding their way through there is much in our health care culture that expects care and treatment to be administered by caring and expert professionals, and this expectation is present in the providers of health care as much as it is in the recipients . There are of course individual differences in both staff and patients willingness to embrace self - care, but if we assume that cultures and individual preferences are not fixed, then we may find ways to move people along the self - care continuum . Often, patients contemplating the need for renal replacement therapy are understandably and predictably hoping that dialysis will be done for them and will probably shy away from the thought of doing it at home. Clearly, it is easier and quicker to point them in the direction of in - centre hd than to embark on a difficult and time - consuming discussion around self - care home dialysis and training . To undertake this task, the staff must first be themselves totally convinced that home treatment is in the patient s best interest if not, the patient will rapidly detect any hint of uncertainty and further discussion is probably futile . This also means that all members of staff with whom the patient comes into contact must be equally capable of espousing some or all of the benefits of home therapy, from nurse, to doctor, to social worker, to dietician and others . In other words, ethos must be that home therapy is not only viable but preferable and beneficial for those who are able to pursue it . For a unit s staff to be able to talk to patients with confidence requires direct experience of home dialysis, but obviously in many units which do not have a full range of home therapies, this may initially be impossible . Under these circumstances, collaboration with another unit may enable staff to gain the training and experience required to be able to talk with authority and instill confidence in the patient . Visiting patients in their home environment is an essential part of training for both medical and nursing staff . It is known that patients who start dialysis after adequate preparation tend to select a home therapy [5, 6]. This is encouraging, but in some instances, education can default to telling the patient a series of facts, and once the full list of facts is ticked off, the patient is expected to make a choice . Yet, we all know from our own educational experiences that this approach alone is insufficient . On the face of it, it seems plausible that suitable patients would accept the benefits of a home therapy, once they have been educated as to the reasons for the recommendation, and staff may be bemused when they do not do so . This aspect of everyday human behaviour has been most extensively studied in the field of smoking cessation, where it is recognized that someone who has accepted the need to quit is much more likely to be successful than someone who has not, despite both being aware of the future health benefits . The stages of change model suggests that, for most people, a change in behaviour occurs gradually, with the patient moving from being uninterested, unaware or unwilling to make a change (pre - contemplation), to considering a change (contemplation), to deciding and preparing to make a change (preparation then action). Therefore, before a patient is able to begin to engage in discussion about any dialysis therapy, they must have reached a point of acceptance that dialysis is necessary . If they are not at this point, then any attempt at education will be largely futile, especially if it is simply repeated when it may be counter productive and alienating . Even once they have accepted, the need to change a patient may still not be able to engage in the process of change due to real and significant hindrances, such as needle phobia, general anxiety, low mood or fatigue . 5the goal for chronic kidney disease patients at the pre - contemplation stage is to begin to think about the likely 5need for dialysis in the future and where the dialysis should take place . The task for physicians is to empathetically engage the patient in contemplating this change to their life . During this stage, patients may appear argumentative, hopeless or in denial, and the natural tendency is for physicians to try to convince them with more facts, which usually engenders resistance . Figure 1 describes the stages of change which anyone confronting a life changing event, such as the need to start dialysis, must pass through in order to come to terms with their new situation . Asking the patient to indicate which rung of they ladder they feel they are on can be a helpful first step . Bringing a patient to the stage of taking action in a defined timescale is of great importance since progressive renal disease will not wait, and it is clear that those who start dialysis in an unplanned fashion fare less well . On the other hand, a patient who has reached the action stage may be able to select themselves for either home or hospital therapy as they begin to understand what is, and is not, desirable and possible given their specific circumstances . The uk national institute for health and clinical excellence (nice) produced guidance on home compared to hospital hd for patients with end - stage renal failure in 2002 . Suitable for home hd should be offered the choice of having hd in the home or in a renal unit . The definition of practical characteristics of patients suitable for home hd according to nice guidance 2002 although these guidelines are entirely clear and sensible, the manchester experience is that the definition of enthusiastic and determined patients who might initially have been deemed unsuitable are found to be not only capable but successful home dialysers . Indeed, the current feeling is that very few physical disabilities preclude home treatment if the patient is keen to proceed, and level of enthusiasm is a more reliable guide . Once a patient has arrived at the point of choosing a home therapy, the pathway to their first dialysis at home must be as smooth and pot - hole free as possible . This requires some resources and infrastructure to be already in place, and if they are not, then all but the most determined patient is likely to be put off . Many patients commitment at this stage is fragile and if the path is too rough or steep, or not well signposted, they may understandably decide to turn back . Therefore, from this point on, communication, planning and action must be slick and efficient in order to build the patient s confidence . The experience and confidence of the staff is crucial so that even if the patient is unsure of the process, they feel fully able to trust the team around them and to know who to turn to for reassurance or answers . It requires staff to engage with patients in conversations about what is important to them and what they value in more general terms (e.g. Freedom, independence, safety, quality and quantity of life) and about what the complex hindrances to self - care may be so that an individualized response to these sorts of questions can move patients along the self - care continuum . When faced with the spectre of dialysis treatment, most of us would not naturally opt for a home therapy . Traditional methods of patient selection have not prevented a steady decline in home therapies over the past two decades, yet most dialysis unit staff say they would opt for a home therapy for themselves . The stages of change model coupled with basic motivational interviewing techniques can enable staff and patients to engage with each other in a more meaningful way such that many patients will be better able to understand the benefits of self - management and treatment at home . Practical infrastructure barriers must also be tackled in order to smooth the pathway to home dialysis.
The epstein barr virus (ebv) was described by michael anthony epstein and yvonne barr, who isolated ebv virus particles from endemic burkitt lymphoma specimens . Ebv is a double stranded dna virus of the herpes virus family and is the most common virus affecting humans . The infection occurs through oral transmission via saliva . By the age of 40, over 90% of the world population has experienced an ebv infection . Ebv also plays a pathogenic role, mainly in the development of several t cell and b cell lymphoproliferations (see below) but is also associated with different types of rare epithelial and mesenchymal neoplasms . Ebv is mainly a b - lymphotropic and epitheliotropic virus and rarely infects t cells and nk cells . The life cycle of ebv is composed of a lytic state and a latency state, the latter allowing lifelong persistence of the virus in the host . The replication cycle of ebv is composed of three phases: (i) entry into either epithelial cells or naive b cells after oropharyngeal transmission, (ii) lytic replication, and (iii) latency . Once a cell has become infected, the viral capsid dissolves and the dna is transported into the cell nucleus . In epithelial cells, the entry of ebv into the cell is usually directly followed by the lytic replication of the virus, which leads to production of infectious virions using the virus s own replication machinery . Ebv persists in b cells throughout the differentiation from naive b cells to germinal center cells to long - lived memory b cells . In memory b cells, dependent on the expression of specific ebv - associated proteins and rnas, three different viral latency types have been described, which can be reproduced to a certain extent by immunohistochemical markers (table 1).table 1ebv - latency types and expressed antigenslatency typeebv - associated antigens0/ieber, ebna1ii (default program)eber, ebna1, lmp1, lmp2biii (growth program)eber, ebna1 - 6, lmp1, lmp2a + blyticall lytic antigens (e.g., zebra) ebv - latency types and expressed antigens most humans become infected with ebv during childhood, when the infection usually proceeds asymptomatically or with mild symptoms that are undistinguishable from other viral infections . If the infection occurs in young adults and adolescents, it can manifest as mononucleosis (glandular fever). Ebv tonsillitis differs from unspecific tonsillitis related to other causes in its histologically detectable polymorphic lymphoid infiltration with abundant plasmablasts and occasional hodgkin - like cells expanding in the interfollicular area of the tonsils . In affected lymph nodes, an extension of the interfollicular areas with a similarly polymorphic infiltration to that in tonsils can be observed . Within the peripheral blood and bone marrow, cd8 positive lymphoblasts (pfeiffer cells) might be observed . The histomorphological picture of primary ebv infection shares histomorphological features with other ebv - driven lymphoproliferative diseases (lpd, table 2). Therefore, the definite diagnosis of infectious mononucleosis is based on the following features: (i) young patient age, (ii) sole or main manifestation in the tonsils, (iii) absence of immunosuppression in the patient, and most importantly (iv) a serological test indicating ebv primary infection (positive igm titer).table 2spectrum of polymorphic ebv driven lpd with variable neoplastic potential spectrum of polymorphic ebv driven lpd with variable neoplastic potential following the primary infection, patients become asymptomatic virus carriers with a very limited number of ebv - positive b cells . A tissue correlate of this phenomenon is the occasional presence of single small ebv - encoded rna (eber)-positive b cells, so called bystander cells, within lymph nodes, mucosa - associated lymphatic tissue, or lungs . Rare complications of primary ebv infection, such as the chronic active ebv (caebv) disease and/or ebv - associated hemophagocytic syndrome have recently been reviewed in this journal . Since the vast majority of humans have been infected by ebv in childhood and adolescence, ebv - associated lpds in adults usually arise from reactivation of the virus from ebv - infected memory cells . Such reactivation is generally considered to consist in an escape from the normal immunosurveillance and preferentially occurs at sites representing the physiological reservoir of ebv - infected b cells, such as the lymphatic tissues and mucosa . In these patients, age - related decline in immunocompetence is assumed to be the cause of ebv - lpd [4, 5]. However, the diagnostic pathologist should stress that a clinical work - up of the immune status is warranted once an ebv reactivation is detected . Ebv reactivation in adults occurs primarily in the form of ebv - lpd presenting with a polymorphic histological and clinical picture . Dependent on the clinical scenario and the preferential site of involvement, different types of polymorphic ebv - lpd can be distinguished . They are outlined in table 2 and include polymorphic posttransplantation lymphoproliferations (ptld), ebv - positive mucocutaneous ulceration (ebvmcu), and lymphomatoid granulomatosis . Depending on the clinical scenario and the extent of tissue involvement, these conditions cause variable clinical symptoms . Polymorphic ebv - lpd regress in most patients if immunocompetence can be reestablished, e.g., by withholding immunosuppressive therapy . Nevertheless, a subgroup of polymorphic ebv - lpd progress to more aggressive diseases, usually accompanied by a transformation to the morphology of a fully developed lymphoma (designated as monomorphic polymorphic ebv - associated lymphoproliferations share histomorphological and immunophenotypical features, which are certainly not specific but should alert the histopathologist to an ebv - driven disease when analyzing lymphatic proliferations . These features include the following: tissue necrosisepithelial ulcerationangiotropic vasculitis - like lymphoid infiltratespolymorphic picture rich in large cells, hodgkin - like cells, plasmablasts and plasma cellsintermingled large t cells (often cd8 positive)b blasts, with weak cd20 expression and expression of cd30 epithelial ulceration angiotropic vasculitis - like lymphoid infiltrates polymorphic picture rich in large cells, hodgkin - like cells, plasmablasts and plasma cells intermingled large t cells (often cd8 positive) b blasts, with weak cd20 expression and expression of cd30 if the clinical scenario (immunosuppression) or the histological features are suggestive of an ebv - lpd, testing by eber in situ hybridization should be initiated (see below). When the diagnosis of an ebv - positive polymorphic lpd has been confirmed by detection of ebv in the tissue, the nomenclature (classification) of the disease is dependent on (i) the clinical context and (ii) the involved site (table 2). Ebv - lpd histologically resembling a lymphomatoid granulomatosis might occur after organ transplantation, in which case the disorder is named according to both the clinical scenario and the histopathological picture as ebv - positive polymorphic ptld under the histopathological picture of a lymphomatoid granulomatosis . The most important diagnostic message from the pathologist to the treating clinican should be (i) the ebv association of the lesion and (ii) the clear distinction of polymorphic ebv - lpd from fully developed lymphomas.fig . A and d illustrates the features of a primary ebv infection in the oral mucosa of a child . As examples of ebv reactivation, an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia (b and e) and a lymphomatoid granulomatosis in the lung of a patient (c and f) are shown . As shared features in all lesions necrosis (arrow) and angiotropism (arrow head) f 400 histomorphology of ebv - associated lymphoproliferative diseases . A and d illustrates the features of a primary ebv infection in the oral mucosa of a child . As examples of ebv reactivation, an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia (b and e) and a lymphomatoid granulomatosis in the lung of a patient (c and f) are shown . As shared features in all lesions necrosis (arrow) and angiotropism (arrow head) are indicated . Ebv - positive lymphomas occur in immunocompetent patients, but are much more frequent in immunocompromised patients . In the latter patient group, the latency type of ebv detectable in the lymphoma cells frequently but not exclusively indicates higher viral activity with expression of more ebv encoded proteins (latency type 3) and sometimes active viral replication . Lymphomas occurring in immunocompetent patients, such as hodgkin lymphoma, usually display latency types 0/1 or 2 with a more restricted pattern of ebv - associated proteins and no viral replication (table 3). The diagnostic criteria for these lymphomas have been described elsewhere and do not necessarily require the detection of ebv because of the presence of ebv in the lymphoma cells . In rare cases, detection of ebv is a mandatory diagnostic feature (e.g., in ebv - positive lpd of childhood). Nevertheless, in certain lymphoma entities, e.g., nasal and extranasal nk t cell lymphoma, angioimmunoblastic t cell lymphoma, primary effusion lymphoma, and plasmablastic lymphoma, ebv association remains an important clue towards the correct diagnosis . Of further relevance might be the identification of ebv - positive diffuse large b cell lymphoma (dlbcl) of the elderly . Histological features that are useful for identifying dlbcl candidates for screening for ebv have been described as angiotropic growth, necrosis, and polymorphic, plasmablastic or hodgkin - like differentiation and expression of cd30 .table 3ebv - positive lymphomas and latency types and their association with immunocompromised statelineageimmunocompetence of patientsentitylatency typeb cellcompetentclassical hodgkin lymphoma2endemic burkitt lymphoma0/1sporadic burkitt lymphoma0/1ebv + dlbcl of the elderlyvariableebv + dlbcl associated with chronic inflammation (pyothorax lymphoma)predominantly 3compromisedprimary effusion lymphoma0/1plasmablastic lymphoma0/1lymphomatoid granulomatosis grade 3 and dlbcl arising from the former3monomorphic ptld variablelymphomas associated with hiv infection variablelymphoproliferative disease associated with primary immune disorders variableother iatrogenic immunodeficiency - associated lymphoproliferative disorders 3 t cellcompetentangioimmunoblastic t cell lymphoma0/1 or 2extranodal nk / t cell lymphoma2compromisedebv - positive t cell lymphoproliferative disease of childhood and young adults variable diseases under this category are further specified according to histopathology the immunodeficiency in many of these young patients is postulated but not always objectifiable dlbcl diffuse large b cell lymphoma, ptld posttransplant lymphoproliferative disease ebv - positive lymphomas and latency types and their association with immunocompromised state diseases under this category are further specified according to histopathology the immunodeficiency in many of these young patients is postulated but not always objectifiable dlbcl diffuse large b cell lymphoma, ptld posttransplant lymphoproliferative disease the diagnostic criteria for ebv - positive lymphomas are described in the current who classification . However, distinguishing polymorphic ebv - lpd and a fully developed ebv - positive lymphoma can be challenging . The most important feature, large sheets of ebv - positive blasts and absence of a polymorphic cellular picture, should raise suspicion of an ebv - positive lymphoma, especially a dlbcl . A specifically difficult task is the differentiation of a polymorphic ebv - lpd from a fully developed classical hodgkin lymphoma, because the latter may be composed of a similarly polymorphic cellular infiltrate to that of the polymorphic ebv - lpd . Pathologists should be aware of the fact that proteins expressed in hodgkin lymphoma, such as cd15, can also be observed in polymorphic ebv - lpd, like ebvmcu, and therefore the final diagnosis will depend not only on the histopathological features but also on the clinical context, including the site of involvement . For example, hodgkin lymphomas rarely involve epithelial barrier organs, but ebvmcu by definition always do (table 2). In our experience, clonality testing is not helpful in differentiating ebv - positive lymphoma from polymorphic lymphoproliferations, as viral - driven lpd can also result in a clonal expansion of t and b cells . Testing for ebna2 expression is useful in some cases, since expression of ebna2 strongly argues against hodgkin lymphoma (table 2).table 4differential diagnostic of polymorphic ebv - positive lymphoproliferative disease (lpd) compared to hodgkin lymphomafeaturehodgkin lymphomapolymorphic ebv + lpdhistomorphologymixed cellularity infiltration with hodgkin (like) cells, necrosispredominant epitheloid cells and eosinophilspredominant plasmablasts, plasmacells, angiotropismimmunophenotype of large cellscd30> cd20cd20> cd30expression of ebv antigens eber + cells equal to lmp1 + cells eber + large cells only ebna2 negative eber + cells more than lmp1 + cells eber + small and large cells ebna2 + /clinical scenariopredominant immunocompetentpredominant immunocompromisedsite mediastinum nodal>> extranodal extranodal>> nodal mucosa differential diagnostic of polymorphic ebv - positive lymphoproliferative disease (lpd) compared to hodgkin lymphoma the question as to which specimen should be tested for ebv depends on the histopathological findings on the one hand and on the clinical context on the other hand . Table 3 summarizes our criteria and favored method of testing for ebv, fig . If pathologists are confronted with lymphoid tissue histologically resembling inflammatory (reactive) tissue in immunocompetent patients, the decision whether or not to test for ebv is primarily based on histological features (table 3). In immunocompromised patients, the method of testing should be eber in situ hybridization, which is most sensitive for detecting ebv, especially in lesions that cannot necessarily be expected to express other viral antigens such as lmp1 (table 3). Since indolent (low grade) b cell lymphomas are virtually never associated with ebv, testing cannot be recommended . For other entities, which are listed in tables 3 and 5, testing should be part of the standard diagnostic work - up . Immunohistochemical staining for lmp1 is sufficient for hodgkin lymphoma, since these lymphomas express the latency type 2 . However, other lymphomas listed in table 5, such as diffuse large b cell lymphomas, require testing by eber because lmp1 is frequently not detectable.fig . 2an ebv - associated mucocutaneous ulcer in the colonic mucosa (a hematoxilin and eosin). Eber usually labels the majority of ebv - infected cells, whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells . D 400table 5recommendations for selecting tissue specimen and methods for ebv testinglymphomanodal and extranodal lymphatic tissue not presenting overt lymphoma (polymorphic lpd)histopathological featuresclinical contexttesting by ebertesting by ebertesting by eberb cell lymphomas endemic burkitt lymhoma hodgkin - like large cells and hodgkin or reed - sternberg cells inherited immunodeficiency plasmablastic lymphoma primary effusion lymphoma posttransplantation pyothorax associated lymphoma dlbcl with histological features suspicious for ebv association necrosis drug induced immunocompromised state angiotropism of blastic cells any monomorphic ptld lymphomatoid granulomatosis plasmablastic infiltrate hiv infectiont cell lymphomas ailt extranodal nk / t cell lymphoma ebv - positive t cell lymphoproliferative disease of childhood and young adultstesting by eber or lmp hodgkin lymphomatesting by eber, lmp1 and ebna2 differential diagnosis between hodgkin lymphoma and ptld ptld posttransplantation lymphoproliferative disease, aitl angioimmunoblastic t cell lymphoma an ebv - associated mucocutaneous ulcer in the colonic mucosa (a hematoxilin and eosin). Eber usually labels the majority of ebv - infected cells, whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells . Original magnification a d 400 recommendations for selecting tissue specimen and methods for ebv testing ptld posttransplantation lymphoproliferative disease, aitl angioimmunoblastic t cell lymphoma although epidemiologic data are lacking, ebv - lpd distinct from overt lymphoma will be increasingly recognized . This is due on the one hand to the aging of many populations, with an increase in age - associated ebv reactivation . On the other hand, widespread access to commercial assays and automatization leads to an increasing use of in situ hybridization for eber by diagnostic pathologists . Additionally, newly recognized subgroups of lymphomas, such as the ebv - positive dlbcl of the elderly, require the detection of ebv in more lymphoma entities as part of the diagnostic process.
Echinococcosis or cystic hydatid disease is an endemic parasitic disease in human populations in iran and some parts of the world . It is caused by larvae stage of echinococcus granulosus (1). In the life cycle of e. granulosus, clinical treatment of cysts includes albendazole (abz) or mebendazole (mbz) therapy in combination with either surgical resection (2). The role of enzymes in living organism is clear and remarkable and the worms are seriously dependent on these activities . Parasite components such as enzymes have specific biological functions, which are necessary for parasite survival and are supposed to have an important role in host - parasite interactions and disease progress (3). Parasite s enzymes are attractive purposes that are be explored for the development of diagnostic method and vaccines . They mediate processes like tissue invasion, feeding, evasion (escape the immune system) of host immune response etc . Glutathione - s - transferase (gst) is an enzyme, which has a significant role in the detoxification of parasite metabolites (endogeous), host metabolites (xenobiotics) and drugs through their conjugation to glutathione (5). Gst activity in e. granulosus has been described in the cytosolic portion of protoscoleces obtained from sheep cysts and activated by pre - treatment of protoscoleces with gst inducers (6). Alkaline phosphates (alp) is an enzyme that plays an important role in dampening host immune responses and also plays a role in feeding parasites (7). Most phosphatases have been found in the absorption system of cestodes, excretory system in trematodes and intestinal cells of the nematode (8). Proteolytic enzymes of parasites have been given more attention than other enzymes, because they play a vital role in parasite survival and are involved in many fundamental physiologic processes (3). The activities of protease described in e. granulosus (9), has been detected in hydatid cyst fluid, cyst wall and in protoscoleces . This enzyme is responsible for breakdown of proteins in all living tissues in order to be used by the cells (10). In addition to their known role in the catabolism, they have a part in protein processing in evasion from immune system, leaving the cyst, molting of the parasites and in diagnosis, especially cysteine proteases as serological markers . Proteases have generally been identified as potential drug targets in parasites (11, 12). The purpose of this study was to determine the effects of abz and mbz on the activity of the gst, alp and proteases in the protoscoleces of hydatid cyst and to evaluate their inhibitory effects on enzyme activity . Protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran (center of iran). Protoscoleces were allowed to settle in a 50 ml falcon tube, and then washed several times in phosphate - buffered saline (pbs ph, 7.2). Viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays (13). Mbz used in this study was obtained from rouzdarou pharmaceutical company (iran) and abz was purchased from tolide daruhai dami iran company . No: k4111 - 500), 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each] containing 500l protoscoleces and 1 g / ml abz and/ mbz [stock solution 1 mg / ml of dimethyl sulphoxide (dmso)] were considered as test groups and 10 culture medium [five culture containing 500 l protoscoleces with 0.6 l dmso, and five culture medium without dmso] regarded as control groups and were incubated at 37 c in 5% co2 (14). Dmso, and five culture medium the culture supernatants containing the parasite e / s products were collected at time interval, every 12 h. at the end of each time interval, the entire 1 ml of culture medium (culture supernatants) was removed and replaced with the same volume of fresh medium (13). The precipitates were discarded and the total protein and enzyme activities were measured as follows: the concentrations of total proteins of e / s samples were measured by bradford method, which involves reacting the e / s samples with a dye that binds to protein . To measure the protein concentration, standard solutions (bovine serum albumin) and e / s products were prepared and the bradford reagent (100 mg coomassie brilliant blue g-250, 50 ml 95% ethanol, 100 ml 85% phosphoric acid) was added . The absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature . A standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated (15, 16). In order to measure the activity of gst in e / s samples, reagent stock including potassium phosphate buffer 0.1 m, 100 mm reduced glutathione (gsh) and 100 mm 1-chloro-2,4- dinitrobenzene (cdnb) substrates were prepared in a microtube . To each test, from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well . The same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min . Finally, total gst activity (u / ml), of samples was calculated . To calculate the specific activity of gst enzyme, the rate of enzyme activity was divided by the mg protein concentration (17). Eight hundred l of buffer reagent (r1) and 200 l of substrate reagent (r2) were poured into cuvette and mixed . Then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure . Sasein solution 0.65% (6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm, ph7.5) prepared and incubated at 37 c for 5 min, and then e / s samples were added to test tubes and were incubated 37 c for 10 min . The reaction was stopped using trichloroacetic acid (110 mm tca, prepared by diluting a 6.1n stock 1:55 with purified water). E / s products were added to control tubes simultaneously and incubated for 30 min at 37 c, and then centrifuged for 5 min at 14000 g at 25 c . The supernatant was poured into the test tube; 1cc sodium carbonate solution (500 mm sodium carbonate solution, prepared using 53 mg / ml of anhydrous sodium carbonate in purified water) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c . Finally, tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm . Solution l - tyrosin 1.1 mm, dw, na2co3, phenol) and reported as units /ml enzyme (16). Folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color . In order to determine the statistically significant difference between protein concentrations, gst, alp and protease activities of e / s samples of treated and control groups, t - test was used . A duplicate set of samples were taken for each test and at the end, their average was taken in to account . Protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran (center of iran). Protoscoleces were allowed to settle in a 50 ml falcon tube, and then washed several times in phosphate - buffered saline (pbs ph, 7.2). Viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays (13). Mbz used in this study was obtained from rouzdarou pharmaceutical company (iran) and abz was purchased from tolide daruhai dami iran company . No: k4111 - 500), 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each] containing 500l protoscoleces and 1 g / ml abz and/ mbz [stock solution 1 mg / ml of dimethyl sulphoxide (dmso)] were considered as test groups and 10 culture medium [five culture containing 500 l protoscoleces with 0.6 l dmso, and five culture medium without dmso] regarded as control groups and were incubated at 37 c in 5% co2 (14). The culture supernatants containing the parasite e / s products were collected at time interval, every 12 h. at the end of each time interval, the entire 1 ml of culture medium (culture supernatants) was removed and replaced with the same volume of fresh medium (13). The precipitates were discarded and the total protein and enzyme activities were measured as follows: the concentrations of total proteins of e / s samples were measured by bradford method, which involves reacting the e / s samples with a dye that binds to protein . To measure the protein concentration, standard solutions (bovine serum albumin) and e / s products were prepared and the bradford reagent (100 mg coomassie brilliant blue g-250, 50 ml 95% ethanol, 100 ml 85% phosphoric acid) was added . The absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature . A standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated (15, 16). In order to measure the activity of gst in e / s samples, reagent stock including potassium phosphate buffer 0.1 m, 100 mm reduced glutathione (gsh) and 100 mm 1-chloro-2,4- dinitrobenzene (cdnb) substrates were prepared in a microtube . To each test, from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well . The same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min . Finally, total gst activity (u / ml), of samples was calculated . To calculate the specific activity of gst enzyme, the rate of enzyme activity was divided by the mg protein concentration (17). Eight hundred l of buffer reagent (r1) and 200 l of substrate reagent (r2) were poured into cuvette and mixed . Then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure . Sasein solution 0.65% (6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm, ph7.5) prepared and incubated at 37 c for 5 min, and then e / s samples were added to test tubes and were incubated 37 c for 10 min . The reaction was stopped using trichloroacetic acid (110 mm tca, prepared by diluting a 6.1n stock 1:55 with purified water). E / s products were added to control tubes simultaneously and incubated for 30 min at 37 c, and then centrifuged for 5 min at 14000 g at 25 c . The supernatant was poured into the test tube; 1cc sodium carbonate solution (500 mm sodium carbonate solution, prepared using 53 mg / ml of anhydrous sodium carbonate in purified water) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c . Finally, tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm . Solution l - tyrosin 1.1 mm, dw, na2co3, phenol) and reported as units /ml enzyme (16). Folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color . In order to determine the statistically significant difference between protein concentrations, gst, alp and protease activities of e / s samples of treated and control groups, t - test was used . A duplicate set of samples were taken for each test and at the end, their average was taken in to account . The mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml, respectively . The mean protein concentration in the control group was 6.06 g / ml . Protein concentration of e / s products of protoscoleces treated and control groups p<0.05; between control and treated groups p>0.05; between treated groups the results of gst, alp and protease activity are shown in table 2 . Gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml . Alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml, receptively . Alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml . Protease specific activity in abz and mbz treated protoscoleces e / s products was not detected, while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml . Gst, alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005, t = test or sample n / d = not detected gst activity = od / min0.0096total voluesample voluedilution factor alp activity = od 2764 protease activity=(umole tyrosine equivalents released)(total volume of assay)volume of enzymetime of assayvolume of filtratespecific activity = units / ml enzymemg solid statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group (p<0.05). A significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz (p<0.05). Meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz (p>0.05) the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml, respectively . The mean protein concentration in the control group was 6.06 g / ml . Protein concentration of e / s products of protoscoleces treated and control groups p<0.05; between control and treated groups p>0.05; between treated groups gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml . Alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml, receptively . Alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml . Protease specific activity in abz and mbz treated protoscoleces e / s products was not detected, while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml . Gst, alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005, t = test or sample n / d = not detected gst activity = od / min0.0096total voluesample voluedilution factor alp activity = od 2764 protease activity=(umole tyrosine equivalents released)(total volume of assay)volume of enzymetime of assayvolume of filtratespecific activity = units / ml enzymemg solid statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group (p<0.05). A significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz (p<0.05). Meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz (p>0.05) glutathione transferases (gst) are multifunctional enzyme present in both animal and plant kingdoms . The enzymes are regarded as parts of the phase ii detoxification system that catalyse glutathione (gsh) conjugation of a multitude of exogenous and endogenous toxic compounds (18). Gst is one of the major detoxification system component found in helminthes, particularly at high levels in cestodes and digeneas . In the tissue extracts of parasites gst activity gst activity exists in e / s products and in the surface of digenea worms such as schistosoma, fasciola and hookworm necator . Gst in e / s products act as an anti - inflammatory agent and neutral lipid peroxidation products in the mucous membranes (20, 21). Researches related to chemotherapy and immunotherapy, have identified glutathione of parasitic worms independently as a potential target for treatment (22). These acts include the digestion of host s tissue in order to provide food for parasites, preventing blood coagulation, facilitating entry into the host s immune system, and disrupting it (23). Alp is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins (24). The phosphatases have been found frequently in the structure of absorption like tegument of cestodes, intestinal cell of the nematodes and excretory system of trematodes . While alkaline phosphatases have been found in adult cestode, the larval forms have a predominance of acid phosphates . This change in these enzymes may be associated with the growth and development of the reproductive system in adult worms (8). Alp as protein plays a role in immune evasion by generating the potent immunosuppressant adenosine . Since adenosine is also an important nutrient, alp may also play a role in parasite feeding (7). Several studies have been carried out on characterization of hydatid cyst protoscoleces e / s products (25, 26). In the present study, the effect of abz and mbz on protein content and some enzymatic activities of e / s products were examined . Protein concentration in treated groups e / s products were less than that of the control group e / s sample . Abz and mbz cause degenerative changes in intestinal cells of the worm by binding to the colchicine - sensitive site of tubulin, therefore blocks its polymerization into microtubules and inhibits the cell proliferation in metaphase stage (27, 28). Based on these findings we can expect the decreasing of total protein in treated parasite culture media, which correlates our study that shows an average protein of test group is less than control group . In this study, a significant decrease observed in the activity of gst, alp and protease enzymes of the e / s products of treated groups in comparison with the control group . The inhibition of enzymes activity induced by mbz and abz might damage the defensive system, metabolism and nutrition of the parasite . The inhibition of gst activity induced by anti - helmintics may contribute to passive the detoxification mechanism (29). The abz and mbz cause reduction in protease enzymes activity, and effect on the parasite activities including assault, migration, feeding, and particularly survival in the host . Since protease enzymes are proteins, the reduction of their activity could be attributed to interference of benzimidazole with protein synthesis (30). Alp is an important enzyme of helminthes parasites associated with absorption and/or digestion of food materials . It has a role in modulating the host immune response (7, 31). Higher concentrations of alp found in the some areas of intestine and sub - cuticular layers of the worm associated with protein transport processes (32). Various commercial drugs (anthelmintic) and chemical compounds are shown to bring about changes in the activity of alp in various soft - bodied helminth parasites like cestodes (33). When taenia solium cysts was treated with praziquantel and abz, alp secretion in the culture medium based on the results obtained in this study, the reduction or inhibition of the enzymes (gst and alp) caused by abz and mbz could lead to alteration of metabolism in helminthes parasites . Therefore, the study indicates that both anti hydatid benzimidazoles (abz and mbz) have an inhibitory effect on the activity of gst, alp and protease enzymes in e / s products of e. granulosus protoscoleces in vitro . Protein concentration in e / s products of mbz groups was slightly higher than that of the abz groups but this difference was not significant . The results showed that the effect of abz on the activity of gst and alp is more than mbz and this may be due to the different structures of these drugs . In addition, the above - mentioned results might account for at least in part, difference in antihydatid mechanism of the both benzimidazole (5). Both drugs have an inhibitory effect on the activity of gst, alp and protease enzymes . The results of the two drugs indicated that abz is more effective on the enzymes activities (gst and alp) as compared with mbz . This may by attrirbuted tothe different structures of the two drug and might account for at least in part, differencein antihydatid mechanism of these benzimidazole derivation.
Anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus (hiv), anoreceptive intercourse, chronic inflammatory bowel disease . An established association exists with human papillomavirus (hpv) infection and premalignant intra - epithelial dysplasia . Phased - array mri is recommended as the preferred imaging modality for regional staging . An uncommon malignancy in the general population, squamocellular anal carcinoma (scac) accounts for approximately 1% of all gastrointestinal neoplasms and less than 5% of anorectal tumours . In past decades, scac was usually diagnosed at a relatively advanced age with a significant female predominance, and believed to be an indolent disease secondary to chronic irritation . In recent years, similarly to uterine cervix dysplastic changes, oncogenic human papillomavirus (hpv) has been detected in the vast majority (up to 90%) of invasive scacs, and linked to the development of low- and high - grade premalignant anal intra - epithelial neoplasms (ain), particularly with high - risk or multiple hpv serotypes infection [1, 2]. Furthermore, the incidence of scac is steadily increasing, particularly in patients with risk factors such as human immunodeficiency virus (hiv) infection, history of anoreceptive intercourse, coexistent cervical dysplasia or cancer, immunosuppression, inflammatory bowel diseases (ibd) and cigarette smoking . Currently, at least half of scacs occur in relatively young (4060 years) hiv - positive individuals, most often men who have sex with men (msm) [35]. The surgical anus is about 4 cm long from the anorectal junction to the perianal skin on the external anal margin (verge). The internal anal sphincter consisting of smooth muscle is separated from the external, striated muscle sphincter by the fatty intersphincteric space . Along with the puborectalis and levator ani muscles, the external anal sphincter forms the sphincter complex . Located approximately halfway along the anus, the dentate line marks the transition from the squamous epithelium to the intestinal mucosa . Thus, histologically scacs can be either keratinising or non - keratinising according to their origin below or above the dentate line, although with similar biological behaviour [6, 7]. Anal margin and anal canal scac originating distal to the dentate line drain to the inguinal and femoral lymph nodes . When the primary tumour arises above the dentate line, regional lymph nodes include the inguinal, internal iliac and perirectal nodes, whereas the external, common iliac and para - aortic nodes are considered non - regional [68]. Because of its anatomical location, in most cases scac is diagnosed clinically in patients with rectal bleeding, pain, discharge or palpable masses . Alternatively, lesions may be detected during follow - up of high - risk individuals . Following physical examination including digital rectal and vaginal examination, ano - proctoscopy and biopsy, imaging is required to evaluate the local extent of the lesion, lymph node involvement, possible invasion of adjacent organs and distant metastases [6, 9]. Imaging the anal canal and perianal structures may prove technically challenging to perform and interpret . In past years, trans - anal ultrasound (trus) and magnetic resonance imaging (mri) techniques allowed an accurate assessment of tumour size and depth of mural invasion [7, 10, 11]. Unfortunately, in patients with anal lesions, positioning of endoanal sonography probes and mri coils is hampered by pain and stricture . Trans - anal imaging combines an excellent spatial detail with a limited field - of - view that prevents panoramic assessment of entire ischiorectal spaces and of regional lymph nodes . Furthermore, trus has limited specificity for differentiation of residual tumour versus post - treatment fibrosis [1113]. Currently, mri performed using external phased - array coils on high - magnetic - field scanners is the imaging modality of choice to investigate the anal region . Significant advantages of mri include its native multiplanar capability, superior soft - tissue differentiation, biological non - invasiveness and optimal safety profile of gadolinium - based contrast agents . Acquisition protocols heavily rely on high - resolution t2-weighted sequences along three planes, with coronal and axial scans planned slightly oblique, respectively parallel and perpendicular to the long axis of the anal canal . Despite the increased tumour conspicuity provided by background fat suppression, short - tau inversion recovery (stir) sequences are less useful because of limited spatial detail and difficulty to delineate anatomic landmarks . At our centre, t1-weighted sequences including fat suppression in at least one plane are routinely acquired following standard - dose intravenous gadolinium contrast, to allow detection of lesion enhancement . Conversely, other authors discourage post - contrast mri acquisitions by stating that enhanced images do not offer additional information to the high soft tissue contrast intrinsic to t2-weighted imaging [7, 10, 1315]. Although with limited contrast resolution compared with mri, volumetric multidetector computed tomography (mdct) acquisitions including image reformations along arbitrary planes allow visualisation of anorectal abnormalities in their cranio - caudal extent with relationship to key anatomical landmarks such as the sphincter complex [1618]. Although the dentate line is not directly recognisable, its position can be inferred as it corresponds approximately to the upper portion of external sphincter muscles . Sensitivity of trus and mri for the identification of scac has been reported to approach 90100%, with high concordance regarding tumour size, although on a limited number of patients, and more precise results with ultrasound for smaller, superficial tumours . Neoplastic tissue in the anal canal has low - to - intermediate t1 signal intensity and positive enhancement after intravenous gadolinium contrast . On t2-weighted and stir sequences, untreated neoplasms display intermediate signal intensity, lower to that of normal ischioanal fat and almost always superior to the internal reference standard represented by uninvolved anal sphincters and gluteal muscles (figs . 1, 2). Although with limited sensitivity compared with mri, on ct images scac may be detected as solid, enhancing nodules or masses within the anus (figs . 2, 3). Axial (a) and sagittal (b) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle, intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image (c), consistent with t1 tumour (arrowheads)fig axial t2-weighted (a), post - contrast fat - suppressed axial (b) and coronal (c) t1-weighted images, and corresponding enhanced image from body ct (d) show a 5.5-cm long (t3) enhancing tumour with infiltration of the left ischioanal fatty space (arrowheads)fig . Post - contrast axial (a) and coronal reformatted (b) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule . Subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scacfig . 4an elderly, 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri . Sagittal t2- (a) and axial t1-weighted (b) images show 5-cm long solid, inhomogeneous neoplastic tissue () extending from the anus to encase the proximal urethra (note catheter in place) a 40-year - old msm with bioptic diagnosis of scac . Axial (a) and sagittal (b) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle, intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image (c), consistent with t1 tumour (arrowheads) a 62-year - old female with biopsy - proven scac . Axial t2-weighted (a), post - contrast fat - suppressed axial (b) and coronal (c) t1-weighted images, and corresponding enhanced image from body ct (d) show a 5.5-cm long (t3) enhancing tumour with infiltration of the left ischioanal fatty space (arrowheads) a 57-year - old woman undergoing abdomino - pelvic mdct for unrelated reasons . Post - contrast axial (a) and coronal reformatted (b) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule . Subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scac an elderly, 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri . Sagittal t2- (a) and axial t1-weighted (b) images show 5-cm long solid, inhomogeneous neoplastic tissue () extending from the anus to encase the proximal urethra (note catheter in place) staging is essential for both prognostic information and correct therapeutic planning, and is performed according to the uicc / ajcc tumour - node - metastasis (tnm) system (table 1), including local lesion extent, lymph node status and distant metastatic spread . In 2010 the european society for medical oncology (esmo) recommended mri as the primary imaging modality to accurately stage scac, taking into account the maximum tumour diameter, possible invasion of adjacent organs and nodal involvement [6, 13].table 1tumour - node - metastasis (tnm) staging of anal carcinoma according to lesion site of originanal canalanal marginprimary tumour (t)txprimary tumour cannot be assessedt0no evidence of primary tumourt1tumour 2 cm in greatest dimensiont2tumour 25 cm in greatest dimensiont3tumour> 5 cm in greatest dimensiont4tumour of any size invading adjacent organ(s) e.g. Vagina, urethra, bladdertumour invading deeper structures (skeletal muscle, cartilage)regional lymph nodes (n)nxregional lymph nodes cannot be assessedn0no regional lymph node metastasisn1metastasis in perirectal lymph node(s)regional lymph node metastasisn2metastasis in unilateral internal iliac and/or inguinal lymph node(s)n3metastasis in internal iliac and perirectal lymph nodes and/or bilateral internal iliac and/or bilateral inguinal lymph nodesdistant metastasis (m)m0no distant metastasism1distant metastasis tumour - node - metastasis (tnm) staging of anal carcinoma according to lesion site of origin particular care should be applied to choosing the longest lesion diameter on t2-weighted images, since correct t parameter staging relies on this measure being below 2 cm, over 5 cm or intermediate (fig . 1) mri demonstrates good correlation with physical findings concerning t stage, whereas infiltration of adjacent organs and sometimes tumour size are clinically underestimated . Extramural neoplastic spread may involve the sphincter complex muscles (external sphincter, levator ani and puborectalis) and most commonly occurs towards the anterior urogenital triangle with possible vaginal, urethral or bladder involvement . Sometimes, the tumour may also extend laterally with invasion of the ischioanal fossa (fig . 2), superiorly to the rectum and mesorectal compartment, or inferiorly to the skin and subcutaneous planes of the perianal region . In such instances, t2-hyperintense solid tissue is seen infiltrating the even more hyperintense fat in the ischiorectal (fig . 2) and subcutaneous spaces, encasing the lower signal intensity skeletal muscles, or isointense structures such as the vagina (fig . 5), prostate and urethra (fig . Notably, radiologists should remember that anal canal carcinoma directly invading the rectal wall, perianal skin, subcutaneous or the sphincter muscle does not imply assessing the tumour stage as t4 . For anal margin scac, a t4 lesion is defined by invasion of deeper structures such as the skeletal muscle or cartilage [9, 10, 13].fig . Axial t2-weighted (a) and post - contrast fat - suppressed axial t1-weighted (b) images show inhomogeneous anal tissue invading the left aspect of the vagina (arrowheads), with internal non - enhancing necrosis and peripheral enhancement . Biopsy diagnosed scac with superimposed infection a 32-year - old hiv - positive woman with clinical diagnosis of anovaginal fistula . Axial t2-weighted (a) and post - contrast fat - suppressed axial t1-weighted (b) images show inhomogeneous anal tissue invading the left aspect of the vagina (arrowheads), with internal non - enhancing necrosis and peripheral enhancement . Biopsy diagnosed scac with superimposed infection the incidence of regional nodal involvement increases with primary tumour size . Lymph node metastases may be present (in 25% of cases) even with superficial (up to t2) scacs, and are unreliably assessed clinically . Nodal staging evaluation relies on the distance from the primary tumour rather than on the number of involved nodes . Mri is highly helpful to assess lymph node metastatic involvement, although the mere size criterion is far from accurate and associated with both false - positive and false - negative results . Short - axis threshold values of 8 mm, 5 mm and 10 mm have been suggested for pelvic, perirectal and inguinal lymph nodes, respectively . Additional helpful features to increase specificity include loss of the normal bean - shaped morphology and fatty hilum, internal t1 and t2 signal heterogeneity with central necrosis, and inhomogeneous enhancement (figs . 6a 65-year - old woman with history of previously treated small scac 3 years earlier . Axial t2- (a) and post - contrast t1-weighted (b) images show roundish 1-cm left inguinal node (arrowheads) with internal fluid - like necrosis and inhomogeneous enhancement, confirmed by ultrasound (c) as hypoechoic with loss of normal nodal structure . Surgical exeresis (postoperative status as seen in d, follow - up mri) confirmed metastatic node from scacfig . Axial t2-weighted (a, b) and post - contrast fat - suppressed t1-weighted (c) images show inguinal nodal metastases, larger on left side (arrowheads) plus bilateral enhancing perirectal adenopathies (arrows) a 65-year - old woman with history of previously treated small scac 3 years earlier . Axial t2- (a) and post - contrast t1-weighted (b) images show roundish 1-cm left inguinal node (arrowheads) with internal fluid - like necrosis and inhomogeneous enhancement, confirmed by ultrasound (c) as hypoechoic with loss of normal nodal structure . Surgical exeresis (postoperative status as seen in d, follow - up mri) confirmed metastatic node from scac the same patient as in fig . 2 . Axial t2-weighted (a, b) and post - contrast fat - suppressed t1-weighted (c) images show inguinal nodal metastases, larger on left side (arrowheads) plus bilateral enhancing perirectal adenopathies (arrows) as suggested by ecco guidelines, search for distant spread is usually performed by means of contrast - enhanced body mdct, with conventional imaging appearances of liver and lung metastases . Dissemination is very uncommon (less than 5% of patients at initial diagnosis, and is usually encountered in association with post - treatment recurrence . Alternatively, in immunocompetent patients f - fluorodeoxygluocose positron emission tomography (fdg - pet / ct) has high specificity for nodal and visceral dissemination . At diagnosis, fdg - pet / ct may alter staging of anal scac in 20% of patients, leading to inclusion of involved pelvic or inguinal lymph nodes in the radiation field [6, 7]. In our experience, not infrequently anal tumours coexist with inflammatory conditions such as proctitis and abscesses . In such instances, mdct and mri provide confident detection of perirectal inflammatory changes and purulent collections that are differentiated from solid neoplastic tissue, thus allowing a correct therapeutic choice including surgical drainage as necessary (figs . 5, 8, 9, 10). Resolution of associated inflammatory changes during treatment is easily monitored by cross - sectional imaging (figs . 8a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass . Axial (a) and coronal reformatted (b) ct images show moderately heterogeneous tissue () in its entire longitudinal extent from the anorectal junction to below the anal verge, associated with large necrotic iliac adenopathies ( in c). Axial images (b, c) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal (arrowheads) associated with abscess collections with mixed gas - fluid content () and fistulas crossing the ischioanal space . Surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . After surgical drainage and subsequent chemo - radiotherapy, mri (c) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracksfig . Staging mri confirms left - sided anal thickening (arrowheads) with abnormal t2 signal intensity (a) and strong contrast enhancement (b). Incidentally, two abscess collections with necrotic content and peripheral enhancement are seen ventrally, connected to the anal canal by a fistulous track (arrow in c). Subtotal regression of changes after treatment is seen on follow - up mri (d) a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass . Axial (a) and coronal reformatted (b) ct images show moderately heterogeneous tissue () in its entire longitudinal extent from the anorectal junction to below the anal verge, associated with large necrotic iliac adenopathies (* in c). Biopsy confirmed superinfected scac a 53-year - old man with purulent drainage and clinical diagnosis of perianal inflammation . Axial images (b, c) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal (arrowheads) associated with abscess collections with mixed gas - fluid content () and fistulas crossing the ischioanal space . Surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . After surgical drainage and subsequent chemo - radiotherapy, mri (c) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracks a 39-year - old hiv - infected man with biopsy - proven scac . Staging mri confirms left - sided anal thickening (arrowheads) with abnormal t2 signal intensity (a) and strong contrast enhancement (b). Incidentally, two abscess collections with necrotic content and peripheral enhancement are seen ventrally, connected to the anal canal by a fistulous track (arrow in c). Subtotal regression of changes after treatment is seen on follow - up mri (d) furthermore, cross - sectional imaging particularly with mri also proves useful to differentiate anal carcinoma from other causes of local pain and perineal masses, such as pilonidal sinus diseases, gartner duct or bartolini gland cysts, tailgut cysts, uncommon soft - tissue neoplasms, urethral cancer, lymphoma or metastases [14, 22]. In the past, scac was treated with abdomino - perineal resection and permanent colostomy . Currently, anal margin and small canal tumours without evidence of nodal spread may be successfully excised . In all other cases, the standard treatment includes radiation combined with mitomycin - c plus infusional 5-fu chemotherapy, yielding an 80% 5-year survival rate with preservation of sphincter function . Advanced t3/t4 tumours have worse outcomes with a 4068% 3-year disease - free survival . Salvage surgery with abdomino - perineal resection is reserved for persistent or recurrent tumours [3, 6, 2325]. After radio - chemotherapy, imaging follow - up with mri represents a useful complement to clinical evaluation in the assessment of therapeutic response . Shortly after treatment completion, interpretation of mri is usually challenging due to the superimposition of inflammatory changes resulting from radiotherapy . Performed at least 68 weeks after treatment completion, mri provides confident, reproducible assessment of post - treatment modifications . Findings indicative of a positive response include size reduction and diminished t2 signal intensity of the treated tumour and associated adenopathies (figs . The appearance of t2-hypointense signal is consistent with fibrosis, although it does not allow excluding minor residual neoplastic foci for sure (fig . Stability in size and signal intensity of any residual abnormality visible at mri in the site of the treated lesion 1 year after therapy has been reported to be strongly associated with a favourable outcome . Locoregional and/or distant recurrence occurs in up to 35% of treated patients, and is strongly associated with advanced (t3t4) stage and nodal involvement at presentation [7, 20]. Persistent and locally recurrent tumours often display an aggressive behaviour, with possible extensive invasion of the adjacent organs and pelvic bony structures, and a tendency for lymphatic dissemination (figs . Initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal (a) and contrast enhancement (b) (arrowheads). Complete disappearance of the lesion is observed on axial t2 (c) and post - contrast fat - suppressed t1-weighted (d) images following chemo - radiotherapyfig . Mri staging including axial (a) and coronal (b) t2-weighted images detect a solid, 3-cm eccentric anal mass (arrowhead) consistent with t2 tumour, associated with right inguinal adenopathies with analogous signal features . After chemo - radiotherapy, mri follow - up (c, d) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathiesfig . Initially, mdct (a) shows left - sided thickening with involvement of the external sphincter (arrowheads). After chemo - radiotherapy, lesion regression is observed on mri with appearance of t2-hypointense fibrosis () and hyperintense nodule (arrow) consistent with residual tumourfig . 14an elderly, 85-year - old lady with biopsy - proven scac and multiple comorbidities . Initial mri (a, b) shows showed circumferential anal wall thickening with abnormal solid signal (arrowheads) measuring 6 cm in length, plus a suspicious centimetric left inguinal lymph node (arrow in a), findings consistent with t3n2 lesion . After reduced chemo - radiotherapy, follow - up mri (c, d) 4 months later disclosed progression and partial necrosis of both primary tumour (arrowhead) and inguinal adenopathy (arrow), plus appearance of an exophytic tissue mass protruding from the external anal orifice (* in d) an 83-year - old man with known scac . Initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal (a) and contrast enhancement (b) (arrowheads). Complete disappearance of the lesion is observed on axial t2 (c) and post - contrast fat - suppressed t1-weighted (d) images following chemo - radiotherapy a 46-year - old female patient with biopsy - proven scac . Mri staging including axial (a) and coronal (b) t2-weighted images detect a solid, 3-cm eccentric anal mass (arrowhead) consistent with t2 tumour, associated with right inguinal adenopathies with analogous signal features . After chemo - radiotherapy, mri follow - up (c, d) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathies a 46-year - old hiv - infected man with biopsy - proven scac . Initially, mdct (a) shows left - sided thickening with involvement of the external sphincter (arrowheads). After chemo - radiotherapy, lesion regression is observed on mri with appearance of t2-hypointense fibrosis () and hyperintense nodule (arrow) consistent with residual tumour an elderly, 85-year - old lady with biopsy - proven scac and multiple comorbidities . Initial mri (a, b) shows showed circumferential anal wall thickening with abnormal solid signal (arrowheads) measuring 6 cm in length, plus a suspicious centimetric left inguinal lymph node (arrow in a), findings consistent with t3n2 lesion . After reduced chemo - radiotherapy, follow - up mri (c, d) 4 months later disclosed progression and partial necrosis of both primary tumour (arrowhead) and inguinal adenopathy (arrow), plus appearance of an exophytic tissue mass protruding from the external anal orifice ( in d) after the introduction of highly active anti - retroviral treatment (haart), people with hiv infection or acquired immunodeficiency syndrome (aids) gained a greatly improved life expectancy with better immune conditions, at the price of an increased tendency to develop tumours . Although it is considered a non - aids - defining malignancy, currently scac ranks third (8.2%) among neoplasms observed in hiv / aids populations, with a substantially higher incidence in msms and in long - standing infected people [4, 26]. Dysplastic intraepithelial lesions are highly prevalent in hiv - infected people, and hiv represents a marker for coinfection with other sexually transmitted diseases such as hpv . Since the risk of developing scac increases with the total time elapsed with cd4 + count below 200 cells/l, it has been hypothesised that hiv - related immune suppression acts as a cofactor to hpv in the development of anal dysplasia and progression to overt carcinoma [1, 3, 4]. Screening procedures including high - resolution anoscopy and cytology smears are increasingly adopted at hiv care centres, to allow detection of scac precursors and early - stage tumours amenable to limited excision and topical therapies . Prevention should limit the occurrence of advanced stages at diagnosis in the future [4, 2629]. A high prevalence (at least 30%) of anorectal complaints is characteristic of hiv - positive patients, particularly those practicing anoreceptive intercourse . Differential diagnosis encompasses a wide spectrum of abnormalities, including non - specific anal diseases such as haemorrhoids, fissures, fistulas and abscesses, along with venereal infections, viral ulcers, and a non - negligible (7%) rate of neoplasms such as scac, lymphoma and kaposi s sarcoma . Therefore, when performing cross - sectional imaging procedures on hiv - infected patients, even for unrelated complaints, special attention should be paid to the anal region, with a focus on the possible identification of solid, enhancing tissue consistent with tumour that indicates need for biopsy (figs . 5, 8, 10, 13) [21, 30, 31]. Despite initial discouraging reports, in the haart era hiv - positive patients are likewise treated with standard chemo - radiation regimens, reaching satisfactory results in terms of local control and survival rates, although with increased toxicity and frequent local recurrences [4, 5, 26, 27, 32]. Some literature reports have highlighted the increased risk of anus and lower rectum carcinomas associated with long - standing, severe perianal fistulising crohn s disease (cd) (fig ., scac reaches a 14% proportion among all colorectal cancers, which is ten - times higher than the usual figure . Furthermore, cd patients develop anorectal carcinomas at a younger age (20 years earlier) than the general population . According to the hypothesised pathogenesis, fistulas probably allow hpv an easier access to the epithelial layers, and chronic mucosal regeneration may ultimately lead to neoplastic changes . However, anal tumours may occasionally develop in patients with ulcerative colitis (uc)-related (fig . 15a 40-year - old male patient with long - standing perianal crohn s disease, being treated with seton . Contrast - enhanced mdct images (a, b, c in cranio - caudal order) show right - sided levator ani abscess (), extensive perianal fistulisation occupying the ischioanal space (arrow), and marked solid - appearing circumferential anorectal thickening (arrowheads). Initial multiplanar mdct (a, b) identified enhancing anal thickening (arrowheads) with right - sided vaginal infiltration and solid tissue () extending to reach the internal obturator muscle . After biopsy confirmation of scac and surgical debulking with colostomy, repeat mdct (c, d) detected enlarging neoplastic residue (). Shortly after chemo - radiotherapy, mri (e) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour (thin arrows). Mri follow - up (e) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence (arrow). After negative clinical reassessment and pet findings, this post - treatment finding remained stable on further mri studies (not shown) a 40-year - old male patient with long - standing perianal crohn s disease, being treated with seton . Contrast - enhanced mdct images (a, b, c in cranio - caudal order) show right - sided levator ani abscess (), extensive perianal fistulisation occupying the ischioanal space (arrow), and marked solid - appearing circumferential anorectal thickening (arrowheads). Abdomino - perineal resection for scac was performed a 39-year - old woman with history of ulcerative colitis and perianal inflammation . Initial multiplanar mdct (a, b) identified enhancing anal thickening (arrowheads) with right - sided vaginal infiltration and solid tissue () extending to reach the internal obturator muscle . After biopsy confirmation of scac and surgical debulking with colostomy, repeat mdct (c, d) detected enlarging neoplastic residue (). Shortly after chemo - radiotherapy, mri (e) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour (thin arrows). Mri follow - up (e) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence (arrow). After negative clinical reassessment and pet findings, this post - treatment finding remained stable on further mri studies (not shown) diagnosis is often unsuspected or delayed because of pre - existent, unspecific complaints and clinical assessment is hampered by complex inflammation with stricture and local pain . As a result, ibd - associated anal cancers are often advanced at presentation, may require extensive surgery plus chemotherapy and radiotherapy, and are associated with a severe prognosis . Therefore, patients with early - onset or long - standing perianal cd should undergo clinical and imaging surveillance, particularly when new or changed symptoms develop . Radiologists should be aware of the increased risk for anorectal cancer in middle - aged ibd patients, and clearly report any solid tissue as suspicious for neoplasm and suggest biopsy (figs . The established association with hpv infection and premalignant intra - epithelial dysplastic changes provides insight into the pathogenesis of hiv- and ibd - related anal cancers, and the possibility of prevention or early diagnosis through screening of high - risk individuals [1, 2]. State - of - the - art cross - sectional imaging with high - resolution mri using external phased - array coils and multiplanar mdct allow detailed, comprehensive visualisation of abnormalities involving the anus and perineal region . Currently, mri represents the modality of choice for primary regional staging of scac, assessment of complications, of therapeutic response following chemo - radiotherapy, and of possible recurrences [9, 10, 13].
Black yeasts from the family herpotrichiellaceae (order chaetothyriales) are fungi with a remarkable dual ecology . On the one hand, they have a unique ability to adapt to extreme environments (exposure to toxic chemicals, high temperature, scarcity of nutrients, acidic, and/or dry conditions), while on the other hand, they exhibit a significant human pathogenic potential . Unlike common opportunistic fungi, herpotrichiellaceous black yeasts frequently cause infections in individuals without known underlying disease and only occasionally in immunocompromised patients . Also, more often than any other fungal group, these organisms have been reported from environments that are rich in aromatic compounds . The first evidence on such phenomenon arose from the frequent isolation of black yeasts from wood treated with creosote, while their occurrence on untreated wood was comparatively low, indicating that accumulation of aromatic compounds might promote the growth of these fungi . The concurrence of these two ecological traits in a single species has been observed for exophiala dermatitidis, isolated abundantly not only on tropical creosoted railway ties but also from clinical cases of severe mycoses [26, 27]. It has been hypothesized that wild berries constitute a natural niche of e. dermatitidis, which might then be ingested by birds and humans and sporadically resulting in mycoses . Deposition of feces on creosoted railway ties subsequently leads to the massive enrichment of this fungus . The related species exophiala bergeri, exophiala heteromorpha, exophiala oligosperma, and exophiala xenobiotica have also been reported as opportunistic fungi causing infections that are generally less serious and from creosoted wood [57, 11, 27]. However, the connection between route of infection and natural occurrence is less evident with these species . The tendency of herpotrichiellaceous fungi toward aromatic metabolism has been confirmed by their recurrent isolation from biofilters treating vapors of volatile aromatic hydrocarbons . Besides hydrocarbon exposure, environmental conditions in biofilters are characterized by a relative low water activity and acidification of the filter bed, which might lead to biomass inhibition problems . In this respect, fungal colonization in air biofilters has generally been related to an improved bioreactor performance . Detailed metabolic studies on fungi from gas biofilters have demonstrated their capacity to assimilate alkylbenzene hydrocarbons as the sole source of carbon and energy [3, 20, 29], which appears to be quite an uncommon metabolic feature in the eukaryotes . Molecular phylogenetic characterization of these biofilter fungi has shown a predominant affiliation to the genera exophiala and cladophialophora (fam . Herpotrichiellaceae), particularly to the species exophiala lecanii - corni, e. oligosperma, and the recently described e. xenobiotica, cladophialophora saturnica, and cladophialophora inmunda [1, 6]. Despite the medical and environmental relevance of herpotrichiellaceous black yeasts, little is understood on their biodiversity and natural occurrence . In order to address questions on niche shifts and environmental prevalence in relation to virulence factors and routes of transmission, the aim of the present work is to apply a specific isolation method based on the enrichment of black yeasts by simulating in batch solid state - like cultures the environmental conditions that are found in gas biofilters for the treatment of volatile aromatic hydrocarbons . Different environmental samples related to the life cycle of e. dermatitidis were used as inocula . The list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table 1 . These samples were collected around utrecht (the netherlands) and concerned wild berries from different plants, guano - rich soil, as well as samples from creosote - treated oak railway ties, which might contain both fecal pollution and contamination with aromatic hydrocarbons . Environmental samples were collected with sterile lab tools, placed in plastic bags and plates, then stored at 4c, and processed in the laboratory within 7 to 14 days . The same locations had previously been sampled for black yeasts, but without enrichment on volatile aromatic hydrocarbons . Table 1samples and sampling sites in the utrecht area (the netherlands) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates (wgs84)a1oak railway tie, outside railsnear station, hollandsche rading521041.95 n, 51045.96 ea2oak railway tie, between railsnear station, hollandsche rading521041.95 n, 51045.96 ea3oak railway tie, outside railsforest area, hilversum521220.68 n, 5119.66 ea4oak railway tie, between railsforest area, hilversum521220.68 n, 5119.66 ea5concrete railway tie, between railsforest area, hilversum521220.68 n, 5119.66 ec1berry, sorbus aucupariaroadside, de bilt52 717.18 n, 5 948.13 ec2berry, sorbus aucuparialight forest, voordaansepad, groenekan52 749.19 n, 5 931.42 ec3berry, sorbus aucuparialight hedge, oostveensepad, maartensdijk52 833.70 n, 5 951.98 ec4berry, sorbus aucuparialapersveld park, hilversum521254.74 n, 51110.25 ec5berry, viburnum opuluslight hedge, oostveensepad, maartensdijk52 833.70 n, 5 951.98 ec6berry, crataegus monogynahedge, vuursche pad, hollandsche rading521010.52 n, 51051.99 ed1guano - rich soil of jackdaw and starlinglapersveld park, hilversum, roosting under thuja521254.74 n, 51110.25 ed2guano - rich soil covered with hedera sp.lapersveld park, hilversum, roosting under thuja521254.74 n, 51110.25 ed3guano - tic soil of jackdaw and starlinglapersveld park, hilversum, roosting under thuja521254.74 n, 51110.25 ed4fresh goose feceslapersveld park, hilversum521254.74 n, 51110.25 ed5old goose feceslapersveld park, hilversum521254.74 n, 51110.25 e samples and sampling sites in the utrecht area (the netherlands) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source . Serum flasks of 100 ml were filled with approximately 25 ml of perlite granules, saturated with mineral medium . Each environmental sample (13 g) was washed with phosphate - buffered saline, and the suspension (40 ml) was used to inoculate four different batches (10 ml each batch). As a control, these suspensions were also plated on sabouraud s glucose agar (sga) amended with antibiotics in order to prevent bacterial growth . The inoculated flasks were then closed with a cotton wool plug covered with aluminum foil and placed inside four desiccators where they were exposed, respectively, to a gaseous phase of benzene, toluene, xylene, or naphthalene . This gas phase was generated by placing 10 ml of a 5% (v / v) solution of the aromatic substrate in dibutyl - phthalate . A solution of 140 g l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9, and the whole set was incubated at 30c for at least 3 months . After this time, the perlite granules in each flask were washed with 5060 ml sterile water . One milliliter of 1-, 10-, or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c . Colony growth was observed daily, and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters . A sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi . Dna was extracted using an ultra clean microbial dna isolation kit (mobio, carlsbad, ca 92010, usa) according to the manufacturer s instructions . The internal transcribed spacer (its) regions and the small subunit (its1 - 5.8s - its2) of the rrna genes were amplified by using the primer set its1 (5-tcc gta ggt gaa cct gcg g-3) and its4 (5-tcc tcc gct tat tga tat gc-3). Pcr reactions were performed on a gene amp pcr system 9700 (applied biosystems, foster city, ca, usa) in 50 l volumes containing 25 ng of template dna, 5 l reaction buffer (0.1 m tris hcl, ph 8.0, 0.5 m kcl, 15 mm mgcl2, 0.1% gelatine, 1% triton x-100), 0.2 mm of each dntp and 2.0 u taq dna polymerase (itk diagnostics, leiden, the netherlands). Amplification was performed with cycles of 2 min at 94c for primary denaturation, followed by 35 cycles at 94c (45 s), 52c (30 s) and 72c (120 s), with a final 7-min extension step at 72c . Amplicons were purified using gfx pcr dna and gel band purification kit (ge healthcare, ltd ., buckinghamshire, uk). Sequence pcr was performed as follows: 95c for 1 min, followed by 30 cycles consisting of 95c for 10 s, 50c for 5 s, and 60c for 2 min . Reactions were purified with sephadex g-50 fine (ge healthcare bio - sciences ab, uppsala, sweden), and sequencing was done on an abi 3730xl automatic sequencer (applied biosystems). Sequence data obtained in this study were adjusted using the seqman of lasergene software (dnastar inc ., madison, wisconsin, usa). For the phylogenetic assignment, the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases (ncbi, usa). Phylogenetic analyses were conducted using mega version 4 (center for evolutionary functional genomics, the biodesign institute, usa). The list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table 1 . These samples were collected around utrecht (the netherlands) and concerned wild berries from different plants, guano - rich soil, as well as samples from creosote - treated oak railway ties, which might contain both fecal pollution and contamination with aromatic hydrocarbons . Environmental samples were collected with sterile lab tools, placed in plastic bags and plates, then stored at 4c, and processed in the laboratory within 7 to 14 days . The same locations had previously been sampled for black yeasts, but without enrichment on volatile aromatic hydrocarbons . Table 1samples and sampling sites in the utrecht area (the netherlands) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates (wgs84)a1oak railway tie, outside railsnear station, hollandsche rading521041.95 n, 51045.96 ea2oak railway tie, between railsnear station, hollandsche rading521041.95 n, 51045.96 ea3oak railway tie, outside railsforest area, hilversum521220.68 n, 5119.66 ea4oak railway tie, between railsforest area, hilversum521220.68 n, 5119.66 ea5concrete railway tie, between railsforest area, hilversum521220.68 n, 5119.66 ec1berry, sorbus aucupariaroadside, de bilt52 717.18 n, 5 948.13 ec2berry, sorbus aucuparialight forest, voordaansepad, groenekan52 749.19 n, 5 931.42 ec3berry, sorbus aucuparialight hedge, oostveensepad, maartensdijk52 833.70 n, 5 951.98 ec4berry, sorbus aucuparialapersveld park, hilversum521254.74 n, 51110.25 ec5berry, viburnum opuluslight hedge, oostveensepad, maartensdijk52 833.70 n, 5 951.98 ec6berry, crataegus monogynahedge, vuursche pad, hollandsche rading521010.52 n, 51051.99 ed1guano - rich soil of jackdaw and starlinglapersveld park, hilversum, roosting under thuja521254.74 n, 51110.25 ed2guano - rich soil covered with hedera sp.lapersveld park, hilversum, roosting under thuja521254.74 n, 51110.25 ed3guano - tic soil of jackdaw and starlinglapersveld park, hilversum, roosting under thuja521254.74 n, 51110.25 ed4fresh goose feceslapersveld park, hilversum521254.74 n, 51110.25 ed5old goose feceslapersveld park, hilversum521254.74 n, 51110.25 e samples and sampling sites in the utrecht area (the netherlands) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source . Serum flasks of 100 ml were filled with approximately 25 ml of perlite granules, saturated with mineral medium . Each environmental sample (13 g) was washed with phosphate - buffered saline, and the suspension (40 ml) was used to inoculate four different batches (10 ml each batch). As a control, these suspensions were also plated on sabouraud s glucose agar (sga) amended with antibiotics in order to prevent bacterial growth . The inoculated flasks were then closed with a cotton wool plug covered with aluminum foil and placed inside four desiccators where they were exposed, respectively, to a gaseous phase of benzene, toluene, xylene, or naphthalene . This gas phase was generated by placing 10 ml of a 5% (v / v) solution of the aromatic substrate in dibutyl - phthalate . A solution of 140 g l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9, and the whole set was incubated at 30c for at least 3 months . After this time, one milliliter of 1-, 10-, or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c . Colony growth was observed daily, and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters . A sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi . Dna was extracted using an ultra clean microbial dna isolation kit (mobio, carlsbad, ca 92010, usa) according to the manufacturer s instructions . The internal transcribed spacer (its) regions and the small subunit (its1 - 5.8s - its2) of the rrna genes were amplified by using the primer set its1 (5-tcc gta ggt gaa cct gcg g-3) and its4 (5-tcc tcc gct tat tga tat gc-3). Pcr reactions were performed on a gene amp pcr system 9700 (applied biosystems, foster city, ca, usa) in 50 l volumes containing 25 ng of template dna, 5 l reaction buffer (0.1 m tris hcl, ph 8.0, 0.5 m kcl, 15 mm mgcl2, 0.1% gelatine, 1% triton x-100), 0.2 mm of each dntp and 2.0 u taq dna polymerase (itk diagnostics, leiden, the netherlands). Amplification was performed with cycles of 2 min at 94c for primary denaturation, followed by 35 cycles at 94c (45 s), 52c (30 s) and 72c (120 s), with a final 7-min extension step at 72c . Amplicons were purified using gfx pcr dna and gel band purification kit (ge healthcare, ltd ., buckinghamshire, uk). Sequence pcr was performed as follows: 95c for 1 min, followed by 30 cycles consisting of 95c for 10 s, 50c for 5 s, and 60c for 2 min . Reactions were purified with sephadex g-50 fine (ge healthcare bio - sciences ab, uppsala, sweden), and sequencing was done on an abi 3730xl automatic sequencer (applied biosystems). Sequence data obtained in this study were adjusted using the seqman of lasergene software (dnastar inc ., madison, wisconsin, usa). For the phylogenetic assignment, the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases (ncbi, usa). Phylogenetic analyses were conducted using mega version 4 (center for evolutionary functional genomics, the biodesign institute, usa). A total of 71 fungal strains were isolated upon enrichment of environmental samples on atmospheres of different volatile aromatic hydrocarbons (table 2). Of those, a single mold (aspergillus fumigatus) other than black yeasts was isolated from berries of sorbus aucuparia (sample c1). The highest isolation rates were achieved with samples taken from creosoted wooden railway ties, particularly from those arising from the tie section located outside the rails and using toluene as the incubation substrate (39 isolates, samples a1 and a3). These samples were the only ones that yielded isolates when xylene or benzene was applied as enrichment substrates (17 and 10 strains, respectively), but not a single fungus was isolated when naphthalene was supplied as the sole carbon source . Though in lower numbers, black yeasts were also isolated from the tie section located between the rails (14 strains, samples a2 and a4). Besides creosote, these samples were likely to contain fecal and mineral oil contamination as well . Table 2number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9, under an atmosphere rich in specific volatile aromatic hydrocarbonssample codebenzenetoluenexylenenaphthalenetotal strainsa10411015a203003a31086024a40110011c105005c207007d206006total strains104317071one isolated strain within this treatment was identified as aspergillus fumigatus; this was the only non - black yeast strain from the whole obtained strain collection number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9, under an atmosphere rich in specific volatile aromatic hydrocarbons one isolated strain within this treatment was identified as aspergillus fumigatus; this was the only non - black yeast strain from the whole obtained strain collection from the natural sampled environments, black yeasts were isolated in two out of four berries from s. aucuparia (12 isolates, samples c1 and c2), but no fungi were obtained from berry samples of viburnum opulus and crataegus monogyna (samples c5 and c6, respectively). Soil mixed with bird feces (guano) also yielded melanized fungi in one out of five tested samples (six isolates, sample d2). No black yeasts were isolated when suspensions of the samples described above (table 1) were directly plated onto sga plates, without the enrichment step on volatile aromatic hydrocarbons, while several common and heavily sporulating fungal species were encountered . Phylogenetic analysis of aligned its1 - 5.8s - its2 rrna sequences from the obtained strain collection showed that the isolates were affiliated to five major groups (fig . 1). Sequences from reference strains deposited at the cbs collection (utrecht, the netherlands) that were related to the isolates in terms of their sequence homology and/or ecophysiology were also included in the analysis . Those encompassed the type strains of e. xenobiotica, e. bergeri, exophiala spinifera, e. oligosperma, e. heteromorpha, e. dermatitidis, e. lecanii - corni, and phialophora sessilis, as well as from those of the cladophialophora species that have been so far related to the metabolism of aromatic hydrocarbons: cladophialophora immunda, c. saturnica, and cladophialophora psammophila sp . Nov ., the latter species being in the process of description (badali et al ., submitted). Strains of e. xenobiotica, e. bergeri, and e. dermatitidis that have previously been isolated from creosoted railway ties were also used . Figure 1neighbor joining phylogenetic tree (kimura 2-parameter model) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study, in relation to the isolation sample and enrichment substrate . Sequences from relevant reference type strains (bold characters) and from other isolates obtained previously from creosoted wood and related environments (underlined characters) were also added [26, 27]. For phylogenetically unassigned groups, sequences from close genbank matches were also included in the analysis (genbank sequence codes are given between square brackets) neighbor joining phylogenetic tree (kimura 2-parameter model) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study, in relation to the isolation sample and enrichment substrate . Sequences from relevant reference type strains (bold characters) and from other isolates obtained previously from creosoted wood and related environments (underlined characters) were also added [26, 27]. For phylogenetically unassigned groups, sequences from close genbank matches were also included in the analysis (genbank sequence codes are given between square brackets) sequence comparisons against those from reference strains revealed that most of the isolates were similar to exophiala xenobiotica (32 strains). The majority was highly homologous (99%) to the ex - type strain, and some were slightly deviating (97% homology). A second group of isolates (nine strains) was identified as belonging to e. bergeri on the basis of a 99% sequence homology to the ex - type strain . The cluster encompassing e. bergeri also included a major group (24 strains), the sequence of which deviated significantly from the ex - type strain (94% sequence homology), so that its affiliation to e. bergeri might be put into question . Blast searches on this latter group into the genbank genomic database revealed a close match (98% homology) with an unidentified exophiala strain isolated from a rock surface . A minor e. bergeri sibling group was composed of two strains for which no known sequence match was found . A third group (three strains) did not match any known reference type strain, but genbank searches revealed a high sequence homology (98%) to the sequence of an uncultivated fungus from a municipal composting plant . Except for a single isolate (dh18150) obtained from a s. aucuparia berry (sample c1), the e. xenobiotica - related strains were isolated exclusively from creosoted oak tie sections located outside the rails (samples a1 and a3). Within this group, three strains were obtained under benzene enrichment, while the remaining 29 strains were isolated with toluene or xylene as enrichment substrates . Besides e. xenobiotica, sample a3 also yielded five isolates upon benzene enrichment, which were identified as e. bergeri . The related between - the - rails section from that same tie (sample a4) and the guano - rich soil (sample d2) yielded 1 and 3 strains of e. bergeri, respectively, using toluene as the enrichment substrate . The occurrence of black yeasts in the human - dominated environment has previously been underestimated due to the application of routine microbial isolation methods . Under common laboratory culture conditions, numerous protocols for the selective isolation of black yeasts have been developed during the last decades (an overview is presented in table 3), which have yielded new data on the presence of these fungi in a wide range of artificial environments, sometimes with no obvious counterpart in nature . Several methods are based on osmotolerant and oligotrophic abilities of target fungi . With these regimens, the isolated black yeasts are mainly osmotolerant members of the saprophytic order dothideales, although the rock - inhabiting fungi appear to be an area of overlap between the dothideales and the chaetothyriales . Applying enrichment methods based on the inoculation of rodents, exposure to aromatic hydrocarbons, assimilation of rare sugars, and/or incubation at high temperature, most isolated black yeast - like fungi are members of the chaetothyriales . It should be noted though that neither of these enrichment conditions might represent the natural niche for these fungi, and they are therefore classified with difficulty in any of the known ecological categories . Table 3overview of selective methods used for environmental isolation of black yeasts and related melanized fungi, with approximate resultsmethodprevalent genusorderecologyreferenceanimal baitexophiala, fonsecaea, cladophialophorachaetothyrialesopportunists[2, 8]erythritolexophialachaetothyrialesopportunistsmineral oilexophiala, fonsecaeachaetothyrialesopportunistsraulin 40cexophialachaetothyrialesopportunistsneedleconiosporiumchaetothyrialesrock fungialkylbenzene vaporscladophialophora, exophialachaetothyrialesxenobioticsacidicexophialachaetothyrialesacidophileshortaeadothidealescrushconiosporiumchaetothyrialesrock fungihormonemadothidealeslow strengthexophialachaetothyrialesoligotrophssarcinomycesdothidealesrock fungicadophoraleotialesoligotrophshigh saltaureobasidium, hortaeadothidealeshalophiles[31, 32]suspendcryomyces, friedmanniomycesdothidealespsychrophilesethanolbaudoiniadothidealesethanophiles overview of selective methods used for environmental isolation of black yeasts and related melanized fungi, with approximate results the present study demonstrates that enrichment on inert solid media incubated under a controlled atmosphere rich on volatile aromatic hydrocarbons compounds displays an extraordinary selectiveness toward chaetothyrialian fungi from the herpotrichiellaceae family . For example, under hydrocarbon atmospheres, the berry samples were free from aureobasidium pullulans, which is a member of the dothideales, otherwise an extremely common component of honeydew mycobiota and slightly osmotic surfaces of fruits and berries . This species was encountered abundantly along with many contaminants on sga control plates without alkylbenzene enrichment . The presence of heavily sporulating airborne fungi, frequently found in soil and litter, was strongly reduced by alkylbenzenes and limited to a single strain of a. fumigatus . Interestingly, the dothidiaceous species amorphoteca resinae, known as creosote fungus for its common isolation from creosoted wood, was found neither in this study nor in previous fungal isolation surveys from creosoted wood [26, 27]. Consistent and relatively abundant isolation of herpotrichiellaceous black yeasts from unpolluted environmental samples, such as wild berries, only after addition of volatile aromatic hydrocarbons strongly suggests that these substances play an essential role in the ecology and competitive ability of those fungi . In particular, the utilization of aromatic compounds as carbon and energy source might explain the key factor determining their success in anthropized environments . With advanced analytical techniques, it has been shown that volatile aromatic hydrocarbons that traditionally were associated with environmental pollution are in fact ubiquitously present in nature, though at very low concentrations . Toluene, for example, is produced biologically in different natural environments including maturing berries . On the other hand, as extremophilic and slow - growing microorganisms, incubation on a support that is relatively dry and poor in nutrients is also a determinant factor for the enrichment of black yeasts, when compared to traditional liquid enrichment cultures . Regarding both the presence of aromatic compounds and exposure to fluctuating and/or extreme environmental conditions, wood treated with creosote can be regarded as a paradigmatic environmental niche for black yeasts . Creosote is a distillate derived entirely from tar, which is rich in a wide variety of polycyclic aromatic hydrocarbons, phenols, and cresols, and has widely been used as wood preservative against microbial decay . Due to its carcinogenic character, the use of creosote has been banned in the european union . Yet, creosoted wood utilities are still widely present in the open environment, such as telephone poles, railway cross ties, switch ties, and bridge timbers . Also, creosote is an important soil contaminant in former wood creosoting plants . In a previous study, e. dermatitidis was isolated from wild berries and massively from creosoted railway ties in thailand when using the pre - incubation in raulin s solution at low ph and incubated at 40c, this protocol being quite selective for e. dermatitidis . It was hypothesized that the life cycle of this species involved the occurrence on wild berries, ingestion and passage through the human intestinal tract, and feces deposition on railway ties . Subsequent enrichment on creosoted wood might then be related not only to the oligotrophy, thermotolerance (particularly under tropical conditions), acidotolerance, and moderate osmotolerance of e. dermatitidis but also to the presence of aromatic compounds . The temperate wild berry sampling locations that were the subject of our study (table 1) had previously been analyzed for the occurrence of black yeasts [18, 26]. Collected berries were homogenized, diluted, and used for spread plates or were subjected to the raulin s pre - incubation protocol . In these earlier studies involving several hundreds of samples, only a single strain of e. dermatitidis the lack of this fungus in temperate climate conditions suggests that e. dermatitidis has an origin in the tropics . This hypothesis is further supported by the isolation of e. dermatitidis from creosoted railway ties in brazil, using the mineral oil - flotation protocol [15, 24]. From the present enrichment program on volatile alkylbenzenes, it appears that exophiala species, though other than the thermophilic e. dermatitidis, are regularly present on substrates that are characteristic from the latter species (berries, bird feces, and creosoted railway ties). It is known to be associated with mild cutaneous infections in humans and is also commonly found in habitats rich in xenobiotics such as hydrocarbon - polluted soil and gas biofilters for the biodegradation of btex compounds [6, 24, 26]., it was regarded to be an extremely rare opportunist, although a recent study showed that it was underdiagnosed due to a general lack of application of sequencing for identification . Using the selective oil - flotation method, e. bergeri was isolated from samples of wood treated or non - treated with creosote preservatives . A distinct cluster was identified within the e. bergeri group, which might belong to a yet - undescribed species . Strains from this group were predominantly isolated from the section between the rails of sampled wooden ties, which were partly contaminated by machine oil and human feces . The remaining strains of e. xenobiotica and e. bergeri senso stricto were obtained from the section outside the rails, where this contaminating effect was less marked . Three identical strains were isolated from guano - rich soil covered with hedera by enrichment under a toluene atmosphere . No close sequence homology against reference strains was found for these fungi, indicating that a yet - undescribed species is concerned . The phylogenetic affiliation of the undescribed strains will be reported in detail in a subsequent study . The extremophilic nature of black yeast, in combination with their capacity to metabolize aromatic pollutants, makes them ideal candidates for specific bioremediation purposes . Prospects on the biotechnological application of these fungi until recently have been hampered by potential biohazard, but, as phylogenetic data on these organisms accumulates, it seems that severe pathogens and hydrocarbon - associated species are consistently well - separated siblings (de hoog, unpublished). Indeed, enrichment on volatile aromatic hydrocarbons in gas biofilters and similar systems thus far has never yielded pathogenic species of the higher fungal biohazard risk categories, such as cladophialophora bantiana or e. dermatitidis . This suggests that a certain degree of speciation between pathogenic and aromatic hydrocarbon metabolizing species has occurred in evolutionary terms . Yet, more studies are needed to emphasize the importance of selective methods to isolate black yeasts and acquire more understanding of their ecological niches . The application of the enrichment method used in the present paper contributes to the isolation of fungi that have a potential for bioremediation of sites polluted with monoaromatic hydrocarbons.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.