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Osteoporotic vertebral compression fractures are a major cause of morbidity and disability among the elderly . Percutaneous vertebroplasty is a procedure used to treat a painful compression fracture in the absence of a neurologic deficit . Percutaneous vertebroplasty is a relatively safe and effective treatment for osteoporotic compression fractures.2) after vertebroplasty, progressive decrease of augmented vertebral body height is noted . Recollapse of augmented vertebrae may also occur with reappearance of pain.6) when this occurs, aggravation of kyphotic change may be noted . Gradual vertebral body height decrease and kyphotic change may occur even without the reappearance of severe pain . Recollapse of augmented vertebrae may induce severe kyphotic deformity.22) the purpose of this study was to evaluate the risk factors for gradual vertebral body height decrease in augmented vertebrae after vertebroplasty . From january 2012 to december 2013, we performed 186 level vertebroplasties for compression fractures in 160 patients . We selected 44 patients, with a first occurrence of a single vertebral compression fracture at the thoracolumbar junction (from t11 to l2). We analyzed sex, age, bone mineral density, body mass index (bmi), sagittal cobb angle, volume of injected cement, level of compression fracture, presence of cement leakage into the disc space and presence of air cleft within the vertebral body on admission (figure 1). Refracture was defined as recurrent fracture of the same cemented vertebra as on initial admission with severe back pain during follow - up, for which repeat vertebroplasty was performed . Our selected patients did not complain of recurrent severe pain and did not undergo repeat vertebroplasty . In that cases, we defined recollapse or gradual vertebral height decrease in our study . Chicago, il, usa). The chi - square test (pearson) was used to compare the groups . The average follow - up period was 16.5 months (range, 13 - 29 months). The fractured vertebrae, included 1 at t11, 14 at t12, 20 at l1, and 9 at l2 (table 1). Height decrease of augmented vertebrae occurred in 10 of 26 men (38.5%) and 12 of 18 women (66.7%). The height decrease was more common in women, but the difference was not statistically significant (p=0.066). Fourteen were in a group aged less than 75 years, and 30 were aged 75 years or over (average age, 76.1 years). They height decrease of augmented vertebrae occurred at the same rate in the two groups (50.0%). We defined severe osteoporosis as a t - score less than -3.5, which was seen in 29 patients . Height decrease was less common in the severe osteoporosis group (46.7% vs. 51.7%, p=0.750). Height decrease occurred more commonly (55.6% vs. 46.2%, p=0.540) in this group . Height decrease of augmented vertebrae was more common in the thoracic spine than in the lumbar spine (p=0.340). When cement leaked into the disc space, height decrease was more common (66.7% vs. 41.4%,p=0.112). When the volume of injected cement was greater than 4cc, height decrease was more common (75.0% vs. 35.7%). Air clefts within the vertebral body on admission were visualized by plain radiography or computed tomography . When an air cleft was present, height decrease wa more common (83.3% vs. 37.5%, p=0.007). The gradual height decrease of augmented vertebrae increased the sagittal cobb angle during follow - up with statistical significance (table 2). Vertebral body height often increases during vertebroplasty, although the clinical significance of restoring height is unknown.7) kyphoplasty and vertebroplasty showed similar reduction in pain and disability, with similar safety and complication rates.4) vertebroplasty was not associated with increased refracture rates in different vertebrae.2123) however, other report showed refracture of different vertebrae in the vertebroplasty group occurred much sooner than that in a conservative treatment group.21) recollapse of augmented vertebrae is not relatively common . In our study, vertebral body height gradually decreased during the follow - up period in about half of the patients, without reappearance of severe back pain . Air clefts and greater height restoration of fractured vertebrae were risk factors for recollapse.610) air clefts within the vertebrae were also prone to vertebral height decrease in our study . Non - cemented - endplate - contact was another important predisposing factor for recollapse.10) preoperative kyphotic change also influenced recollapse of cemented vertebrae.8) cement augmentation restores the strength of treated vertebrae, but leads to an increased endplate bulge and altered load transfer in adjacent vertebrae.16) cemented vertebrae had a lower elastic modulus, but there was no significant effect on the frequency or severity of an induced fracture within the vertebrae.15) pneumaticos et al.18) reported that there is no difference in the compressive load of failure between augmented and non - augmented fractured vertebrae . These mechanisms may induce recollapse of augmented vertebrae as well as non - augmented fractured vertebrae . Recollapse was more common with use of calcium phosphate.17) the mechanism of recollapse after vertebroplasty may be thermal necrosis19) and mismatch of elastic modulus, stiffness, and strength at the cement - bone interface.11) the optimal volume of cement to inject into fractured vertebrae remains controversial . Vertebral augmentation with appropriate amounts of bone cement does not lead to stress peaks under the endplate.1) bone cement increases subsidence in the posterior regions of the treated endplate and the anterior region of the superior caudal endplate.14) greater height restoration and solid lump filling cement are risk factors for refracture of cemented vertebrae.313) greater volume of injected cement contributed to frequent subsequent fracture.12) cement volume was related to recollapse of fractured vertebrae as well as subsequent fracture.9) but, other studies showed that there was no correlation between injected cement volume and subsequent fracture.520) our study suggested that more than 4 cc of injected cement may induce a gradual vertebral height decrease after vertebroplasty . Larger cement volume may induce more thermal necrosis and mismatch at cement - bone interface . A limitation of our study is that follow - up periods were relatively short and did not define the relationship between gradual vertebral height decrease and the clinical significance . So, further studies will be needed about clinical significance of gradual vertebral height decrease . More than 4 cc of injected cement and air cleft within the fractured vertebrae on admission induced gradual height decrease of augmented vertebrae . Thus, longer follow - up is needed in such cases, even when patients do not complain of the reappearance of severe back pain.
A 41-year - old woman prompted a visit to an optometrist due to blurred vision in june 2013, and was diagnosed to have retinal hemorrhage . Complete blood count revealed marked leukocytosis (326 k/l), neutrophilia (25%, 64.6 k/l) with a prominent myelocyte peak (27.5% myelocytes), basophilia (4.0%, 12.9 k/l), and mild normocytic anemia (hemoglobin 10.4 g / dl). Bone marrow study revealed hyperplastic myeloid series (myeloid / erythroid ratio of 8.1/1) with only 1.2% blasts . Further both karyotype and fish analysis revealed presence of philadelphia chromosome in nearly all cells (fig . 1a and b) based on these findings patient was diagnosed with chronic myeloid leukemia (cml), chronic phase . She was started on imatinib 400 mg daily and hydroxyurea . Despite achieving hematological remission three months after diagnosis, patient continued to have persistent cytogenetic disease as detected on follow up fish assays with 32% cells showing t (9; 22). In june 2014, patient was switched to nilotinib, as she appeared to have imatinib resistant disease, although molecular testing for abl kinase mutational analysis was negative . In july 2015 cbc done at that time revealed a normal white cell count of 9.1 k/l with 17.5% circulating blasts, (fig . 1c) and hemoglobin of 7.5 g / dl . Bone marrow aspiration and biopsy was performed and the specimen was also submitted for flow cytometry and cytogenetic analysis . Prominent erythroid hyperplasia was noted on the bone marrow aspirate smears with 69.5% erythroid precursors, several of which showed dysplastic features (fig . Although only 9% blasts were counted on the bone marrow differential count, flow cytometry revealed 14% cells in the blast gate (fig . 1 g) mostly expressing myeloid markers cd117, cd33, stem cell marker cd34 and with partial aberrant expression of lymphoid marker cd7 . Although no solid sheets of blasts were seen, cd34 highlighted scattered blasts throughout the biopsy section with a variable distribution estimated at approximately 1015% (fig . Based on the presence of marked erythroid hyperplasia (69.5%) and 9% bone marrow blasts the diagnostic criteria for acute erythroid / myeloid leukemia were met and a diagnosis of blast transformation of underlying cml to acute erythroid / myeloid leukemia was made . Patient received 7 + 3 induction chemotherapy with idarubicin and cytoxan . Despite lowering of her she further developed subarachnoid hemorrhage, and septicemia and subsequently died six weeks after her hospitalization . Chronic myelogenous leukemia (cml) is a clonal disorder involving the pluripotent stem cell and is consistently associated with the bcr - abl1 fusion gene located on the philadelphia chromosome . The disease typically evolves in 3 distinct clinical stages: chronic and accelerated phases and blast crisis . In approximately 70% of cases, the blast lineage is myeloid, of which granulocytic and monocytic blasts are more common . Erythroid blast phase of cml is relatively rare and a literature review suggests that the incidence ranges from 0% to 10% . Acute erythroid leukemia is a rare subtype (<5%) of acute myeloid leukemia that may arise de novo or from transformation of an underlying myelodysplastic syndrome . It is further subdivided into two subtypes namely: acute erythroleukemia and pure erythroid leukemia . Unlike pure erythroid leukemia in which the erythroid series is mostly comprised of proerythroblasts and basophilic erythroblasts, in acute erythroleukemia (erythroid / myeloid) all maturation stages of the erythroid precursors are present (comprise> 50% of the entire nucleated cell population), may frequently show a shift to immaturity, are dyspoietic and myeloblasts comprise greater than 20% of non - erythroid cells . Chronic myelogenous leukemia with erythroid crisis is a rare entity with variable reported incidence rates . Based on our review of literature we came across very few reported cases of transformation of underlying cml to acute erythroid leukemia,,,,, . We also searched our institutional database for all cases of cml that transformed to acute leukemia over the course of last twenty years and did not find any other cml patient with erythroid blast crisis . Whether the criteria listed for diagnosis of erythroleukemia should be applied in cml erythroid blast phase is poorly defined . Some studies have considered the percentage of normoblasts below 50% as criteria for erythroblast phase but not erythroleukemia . Although acute erythroid leukemia is far less common than cml erythroblast crisis, a few cases of philadelphia - positive acute erythroid leukemia have been reported . Studies have also suggested that erythroid blast phase is not independent of cml chronic phase . Bcr - abl fusion product in the normoblasts of cml, which provides concrete evidence confirming erythroid leukemia rather than a hyperplastic process . In our case at the time of disease progression in 2015 we were able to demonstrate presence of both bcr - abl fusion and monosomy 7 in majority of the bone marrow cells that on morphology were mostly erythroid precursors by fish assays . Although the 9; 22 translocation was seen at the time of diagnosis, the anomalies of chromosome 7 and 3 were newly acquired in 2015, indicating karyotype evolution and disease progression . In the blastic phase of cml, several additional chromosome aberrations in addition to the philadelphia chromosome have been reported in 7580% of patients,, . Complex rearrangements are widely dominant in acute erythroleukemia with clonal abnormalities mostly involving chromosomes 5 and 7 followed by 8, 16 and 21 . Ph - positive acute erythroid leukemia represents an even less common occurrence than erythroid blast phase cml . It is difficult to distinguish the erythroblast phase of cml from a ph - positive acute leukemia . Although complex karyotype and presence of multiple chromosomal abnormalities is fairly common in all cases of acute erythroleukemia, very few cases of ph - positive erythroleukemia have been reported . Blast phase of cml is often associated with a complex karyotype, including trisomy 8 and 19, double ph chromosomes, and isochromosome i (17q), . The who classification does not specifically address the issue of erythroid hyperplasia in patients with cml or erythroid blast phase of cml . We feel due to presence of more than 50% erythroid precursors and increased myeloblasts (greater than 20% of the non - erythroid cells) our case meets the who diagnostic criteria for acute erythroleukemia (erythroid / myeloid). The criteria for diagnosing acute erythroleukemia arising from an underlying cml have not been firmly established, partly due to the rare occurrence of this phenomenon . Chronic myelogenous leukemia blast crisis is highly refractory to standard induction chemotherapy, with a response rate of less than 2030%, . In patient's with imatinib resistant disease dastanib and nilotinib can help achieve hematological response however neither drug has been reported to be entirely effective in achieving complete cytogenetic remission or for treatment of blast crisis . Further acute erythroid leukemia has an aggressive clinical course mostly with an adverse clinical outcome.
This article presents statistics on health care utilization, prices, expenses, employment, and work hours, as well as on national economic activity . Some of these statistics are based on sample surveys conducted monthly or quarterly by government agencies or private organizations and are available 1 to 3 months after the completion of the period . They provide the first glimpse at changes occurring within the general economy and the health care sector . The accompanying tables report quarterly statistics for 1992, and the calendar year aggregation of quarterly information for the past 3 to 10 years ., this calculation permits analysis of quarterly data to focus on the direction and magnitude of changes, without interference introduced by seasonal fluctuations . In the national health accounts, indicators such as these play an important role in the estimation of the latest historical year of health care expenditures . Information that is more comprehensive tends to lag behind the close of a calendar year by 9 to 12 months or more . Therefore, we rely extensively on indicators such as these to anticipate and predict changes in health care sector expenditures for the most recent year . Other indicators help to identify specific reasons (e.g., increases in price inflation or declines in utilization) for expenditure change . In the following sections we will discuss the sources of this information, and then describe how it can be used to predict trends in health care expenditure and the share of national economic activity that is consumed by health care purchases . Since 1963, the american hospital association (aha), in cooperation with member hospitals, has collected data on the operation of community hospitals through its national hospital panel survey . Community hospitals, which comprised more than 80 percent of all hospital facilities in the united states in 1991, include all non - federal, short - term general, and other special hospitals open to the public . They exclude hospital units of institutions; psychiatric facilities; tuberculosis, other respiratory, and chronic disease hospitals; institutions for the mentally retarded; and alcoholism and chemical dependency hospitals . The sample is designed to produce estimates of community hospital indicators by bed size and region (american hospital association, 1963 - 92). In tables 1 and 2, statistics covering expenses, utilization, beds, and personnel depict trends in the operation of community hospitals annually since 1983 and quarterly for 1992 . Figures 1 and 2 show annual changes in various measures of hospital utilization for 1981 - 92 . For purposes of national health expenditures (nhe), survey statistics on revenues (not shown on table 1) are analyzed in estimating the growth in the largest component of health care costs community hospital expenditures . This one segment of nhe accounted for 33 percent of all health spending in 1991 (letsch et al ., 1992). The survey also identifies important factors influencing expenditure growth patterns, such as changes in the number of beds in operation, number of admissions, length of stay, use of outpatient facilities, and number of surgeries . The u.s . Bureau of labor statistics (bls) collects monthly information on employment for all workers, and earnings and work hours for non - supervisory workers in a sample of 350,000 establishments . Data are collected through cooperative agreements with state agencies that also use this information to create state and local area statistics . The survey is designed to collect industry - specific information on wage and salary jobs in non - agricultural industries . It excludes statistics on self - employed persons and on those employed in the military (u.s . Department of labor, 1991). Approximately 5 percent of the population hold more than one job at any point in time . (other surveys that are household based, such as the current population survey [cps], also record employment . In the cps, however, each person's employment status is counted only once, as either employed, unemployed, or not in the labor force .) Once each year, monthly establishment - based employment statistics are adjusted to benchmarks created from annual establishment census information, resulting in revisions to previously published employment estimates . Tables 3 and 4 and figure 3 present statistics on employment, average hourly earnings, and average weekly hours in private (non - government) health service establishments . Similar statistics for the all private non - agricultural sector, included on these tables, provide a basis for comparing the economy as a whole with the health sector in employment, earnings, and work hours . Table 5 summarizes business activity in the health sector and the overall economy by measuring change in the implied non - supervisory work hours and payroll . Implied work hours are the product of the number of non - supervisory employees and average weekly hours . Implied non - supervisory payrolls figure 4 shows annual changes in non - supervisory payroll for 1983 - 92 . For purposes of nhe, changes in work hours by industry combined with changes in prices (discussed in a later section) can be used to gauge the direction and magnitude of expenditure change in specific industries . We use these composite indicators in the estimation of growth in physician and dental expenditures for the most recent period . We study the historical relationship of changes in this indicator to changes in expenditures, and estimate this relationship for the most recent period . Bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services . Tables 6 and 7 and figure 5 present information on the consumer price index (cpi) for all urban consumers that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states . (the more restrictive wage - earner cpi gauges prices faced by wage earners and clerical workers . The index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . For most items, the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products . Those shares or weights remain constant in all years, even though consumption patterns of the household may change over time . Cpis for health care goods and services depict price changes for out - of - pocket expenditures made by consumers directly . The composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items . For example, the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out - of - pocket by consumers; the remaining 97 percent of the costs of hospital care paid by private health insurers, medicare, medicaid, and other payers are not weighted into the cpi for medical care . In addition, some medical care sector indexes measure changes in list or charged prices, rather than the prices actually received by providers after discounts are deducted . In several health care areas, received or transaction prices are difficult to capture, although bls is making advances in this area . In the nhe, a combination of cpis for selected medical care items, an input price index for nursing homes, and the bls cpi for hospital and related services adjusted by hcfa to provide transaction price changes are used as measures of inflation for the health industry . The indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index (letsch, 1993). In 1979, hcfa developed the prospective payment system (pps) hospital input price index which was designed to measure the pure price changes associated with expenditure changes for hospital services . In the early 1980s, the skilled nursing facility (snf) and home health agency (hha) input price indexes, often referred to as market baskets, were developed to price a consistent set of goods and services over time . They have played an important role in helping to set payment percent increases and in understanding the contribution of input price increases to growing health expenditures . The input price indexes, or market baskets, are laspeyres or fixed - weight indexes that are constructed in two steps . For example, for the pps hospital input price index, the base period is 1987 . Cost categories, such as food, fuel, and labor, are identified and their 1987 expenditure amounts determined . The proportion or share of total expenditures included in specific spending categories the purpose of the price proxy is to measure the rate of price increase of the goods or services in that expenditure category . The price proxy index for each spending category is multiplied by the expenditure weight for the category . The sum of these products (weights multiplied by the price index) over all cost categories yields the composite input price index for any given time period, usually a fiscal year or a calendar year . The percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider . The input price indexes are estimated on a historical basis and forecasted out several years . The hcfa - chosen price proxies are forecasted under contract with data resources, inc./mcgraw - hill (dri). Following every calendar year quarter, in march, june, september, and december, dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions . Some of the data in tables 813 are forecasted and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received . The methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps, hha, and snf cost limits . A description of the current structure of the pps input price index was published september 4, 1990 (federal register, 1990). The most recent pps update for payment rates was published in the september 1,1992 (federal register, 1992a). The latest hha regulatory input price index was published july 1,1992 (federal register, 1992b), and the latest snf input price index was published october 7,1992 (federal register, 1992c). Periodically, the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased . Each revision allows for new base weights, a new base year, and changes to certain price variables used for price proxies . Each input price index is presented in two tables: the first is a percent - change table, and the second provides the actual index numbers from which the percentages were computed . Bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services . Tables 6 and 7 and figure 5 present information on the consumer price index (cpi) for all urban consumers that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states . (the more restrictive wage - earner cpi gauges prices faced by wage earners and clerical workers . The index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . For most items, the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products . Those shares or weights remain constant in all years, even though consumption patterns of the household may change over time . Cpis for health care goods and services depict price changes for out - of - pocket expenditures made by consumers directly . The composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items . For example, the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out - of - pocket by consumers; the remaining 97 percent of the costs of hospital care paid by private health insurers, medicare, medicaid, and other payers are not weighted into the cpi for medical care . In addition, some medical care sector indexes measure changes in list or charged prices, rather than the prices actually received by providers after discounts are deducted . In several health care areas, received or transaction prices are difficult to capture, although bls is making advances in this area . In the nhe, a combination of cpis for selected medical care items, an input price index for nursing homes, and the bls cpi for hospital and related services adjusted by hcfa to provide transaction price changes are used as measures of inflation for the health industry . The indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index (letsch, 1993). In 1979, hcfa developed the prospective payment system (pps) hospital input price index which was designed to measure the pure price changes associated with expenditure changes for hospital services . In the early 1980s, the skilled nursing facility (snf) and home health agency (hha) input price indexes, often referred to as market baskets, were developed to price a consistent set of goods and services over time . They have played an important role in helping to set payment percent increases and in understanding the contribution of input price increases to growing health expenditures . The input price indexes, or market baskets, are laspeyres or fixed - weight indexes that are constructed in two steps . For example, for the pps hospital input price index, the base period is 1987 . Cost categories, such as food, fuel, and labor, are identified and their 1987 expenditure amounts determined . The purpose of the price proxy is to measure the rate of price increase of the goods or services in that expenditure category . The price proxy index for each spending category is multiplied by the expenditure weight for the category . The sum of these products (weights multiplied by the price index) over all cost categories yields the composite input price index for any given time period, usually a fiscal year or a calendar year . The percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider . The input price indexes are estimated on a historical basis and forecasted out several years . The hcfa - chosen price proxies are forecasted under contract with data resources, inc./mcgraw - hill (dri). Following every calendar year quarter, in march, june, september, and december, dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions . Some of the data in tables 813 are forecasted and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received . The methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps, hha, and snf cost limits . A description of the current structure of the pps input price index was published september 4, 1990 (federal register, 1990). The most recent pps update for payment rates was published in the september 1,1992 (federal register, 1992a). The latest hha regulatory input price index was published july 1,1992 (federal register, 1992b), and the latest snf input price index was published october 7,1992 (federal register, 1992c). Periodically, the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased . Each revision allows for new base weights, a new base year, and changes to certain price variables used for price proxies . Each input price index is presented in two tables: the first is a percent - change table, and the second provides the actual index numbers from which the percentages were computed . National economic indicators provide a context for understanding health specific indicators, and how change in the health sector relates to change in the economy as a whole . Tables 14 and 15 and figure 6 present national indicators of output, employment, and inflation . Economy as the value of output produced within the geographic boundaries of the united states by u.s . Or foreign citizens or companies . Real gdp removes the effects of prices from the valuation of goods and services produced, so that the growth of real gdp reflects changes in the physical output of the economy (u.s . Department of commerce, 1992). Indicators can be used to predict the share of gdp allocated to health care prior to the availability of more complete health expenditure data . Growth rates for five major components of national health expenditures (nhe) can be estimated using growth rates of selected statistics shown in tables 110 . The major components are hospital care, physician services, dental services, drugs and other non - durable medical products, and nursing home care . During the past decade an expected range for the share of gdp consumed by health care can be determined from the five estimated components . In 1992, most currently available indicators show some degree of deceleration in growth when compared with growth in 1991 . First, the aha panel survey reports that growth in community hospital expenses (table 2) and revenues were slower in 1992 than in 1991 by 0.6 and 0.9 percentage points respectively, which suggests growth in hospital expenditures will decelerate . A preliminary measure of growth in expenditures for physician and dental services is the product of growth in bls - reported work hours (table 5) and cpis (table 7). For physician services, a slight deceleration in growth is observed in 1992, as the product of growth in physician work hours and prices was 10.0 percent in 1992, compared with 10.5 percent in 1991 . For dental services, a somewhat larger deceleration occurred from 1991 to 1992, as the product of growth in dental work hours and prices was 10.1 percent in 1991 and 8.6 percent in 1992 . Growth in expenditures for retail purchases of drugs and other medical non - durables has been close to growth of the cpi for prescription drugs . In 1992, the cpi showed prices for prescription drugs grew 7.6 percent, a deceleration compared with the growth of 9.9 percent in 1991 (table 7). Trends in expenditures for nursing home care are approximated by the product of growth in bls work hours in nursing and personal care facilities (table 5) and in the snf input price index (table 10). In 1992, growth in this measure (8.2 percent) was 1.9 percentage points slower than in 1991, when the growth rate was 10.1 percent . A preliminary 1992 estimate for the five major components of nhe is calculated by applying the predicted growth rates for 1992 to the 1991 nhe estimates for those sectors . From 1987 to 1991, these five components have fallen as a share of total nhe, decreasing from 78.2 percent in 1987 to 76.5 percent in 1991 . By continuing this trend, a five - component share of nhe for 1992 is developed . An estimate of nhe for 1992 is then made by dividing the sum of the five estimated components for 1992 by the 1992 five - component ratio . Given this method, growth in nhe from 1991 to 1992 is likely to be 1 - 2 percentage points slower than the 11.4-percent growth experienced 1 year earlier . Although deceleration is expected in 1992, nhe growth will still likely be double the growth in gdp, which increased at 4.8 percent . Given these estimates, a range for health expenditures as a percent of gdp can be determined . In 1991, health expenditures accounted for 13.2 percent of gdp, up from 12.2 percent in 1990 . Slow growth in gdp2.8 percent (table 15) rather than accelerating health care spending, was largely responsible for the unprecedented 1.0 percentage point increase . In 1992 deceleration in health care indicator growth, combined with an acceleration in gdp growth, will likely result in this more moderate increase in the share of the nation's resources devoted to health . However, this 0.6 - 0.8 percentage point increase is still higher than the average 0.3 percentage point annual increase experienced in the 1980s . Indicators can be used to predict the share of gdp allocated to health care prior to the availability of more complete health expenditure data . Growth rates for five major components of national health expenditures (nhe) can be estimated using growth rates of selected statistics shown in tables 110 . The major components are hospital care, physician services, dental services, drugs and other non - durable medical products, and nursing home care . During the past decade, an expected range for the share of gdp consumed by health care can be determined from the five estimated components . In 1992, most currently available indicators show some degree of deceleration in growth when compared with growth in 1991 . First, the aha panel survey reports that growth in community hospital expenses (table 2) and revenues were slower in 1992 than in 1991 by 0.6 and 0.9 percentage points respectively, which suggests growth in hospital expenditures will decelerate . A preliminary measure of growth in expenditures for physician and dental services is the product of growth in bls - reported work hours (table 5) and cpis (table 7). For physician services, a slight deceleration in growth is observed in 1992, as the product of growth in physician work hours and prices was 10.0 percent in 1992, compared with 10.5 percent in 1991 . For dental services, a somewhat larger deceleration occurred from 1991 to 1992, as the product of growth in dental work hours and prices was 10.1 percent in 1991 and 8.6 percent in 1992 . Growth in expenditures for retail purchases of drugs and other medical non - durables has been close to growth of the cpi for prescription drugs . In 1992, the cpi showed prices for prescription drugs grew 7.6 percent, a deceleration compared with the growth of 9.9 percent in 1991 (table 7). Trends in expenditures for nursing home care are approximated by the product of growth in bls work hours in nursing and personal care facilities (table 5) and in the snf input price index (table 10). In 1992, growth in this measure (8.2 percent) was 1.9 percentage points slower than in 1991, when the growth rate was 10.1 percent . A preliminary 1992 estimate for the five major components of nhe is calculated by applying the predicted growth rates for 1992 to the 1991 nhe estimates for those sectors . From 1987 to 1991, these five components have fallen as a share of total nhe, decreasing from 78.2 percent in 1987 to 76.5 percent in 1991 . By continuing this trend, a five - component share of nhe for 1992 is developed . An estimate of nhe for 1992 is then made by dividing the sum of the five estimated components for 1992 by the 1992 five - component ratio . Given this method, growth in nhe from 1991 to 1992 is likely to be 1 - 2 percentage points slower than the 11.4-percent growth experienced 1 year earlier . Although deceleration is expected in 1992, nhe growth will still likely be double the growth in gdp, which increased at 4.8 percent . Given these estimates, a range for health expenditures as a percent of gdp can be determined . In 1991, health expenditures accounted for 13.2 percent of gdp, up from 12.2 percent in 1990 . Slow growth in gdp2.8 percent (table 15) rather than accelerating health care spending, was largely responsible for the unprecedented 1.0 percentage point increase . In 1992 deceleration in health care indicator growth, combined with an acceleration in gdp growth, will likely result in this more moderate increase in the share of the nation's resources devoted to health . However, this 0.6 - 0.8 percentage point increase is still higher than the average 0.3 percentage point annual increase experienced in the 1980s.
Electrical impedance tomography (eit) [1, 2] reconstructs the spatial distribution of electrical conductivity or resistivity of a closed conducting domain () from the surface potentials developed by a constant current injection through the surface electrodes surrounding the domain to be imaged . Before carrying out the practical measurements on patients, it is advised to test an eit system with a tissue mimicking model of known properties called practical phantoms [410]. Hence, phantoms are often required to assess the performance of eit systems for their validation, calibration, and comparison purposes . Two - dimensional (2d) eit (2d - eit) assumes that the electrical current flows in a 2d space which is actually three - dimensional inside real volume conductors . Hence, the development of a perfect 2d practical phantom is a great challenge as the real electrodes always have a definite surface area, and hence the injected current signal cannot be confined in a 2d plane in bathing solution . Researchers have developed a number of practical phantoms which are three - dimensional objects, and those phantoms are designed and developed, generally, for their own eit systems . Practical phantoms containing electrolyte (or other conducting medium) [410] are three - dimensional in shape and hence they will have some data error due to the three dimensional current conduction . Also, the phantoms containing electrolytes (e.g., nacl solution or saline) [5, 7, 8] are difficult to transport and are prone to errors since the evaporation of the water gives rise to changes in conductivity . In addition, temperature variations have a marked effect on the conductivity because the temperature coefficient is large . Therefore, the practical phantoms will have a poor stability and a gradually increasing data error over time . Network or mesh phantoms [12, 13] are compact, more stable, rugged, portable, easy to move, consistent over time, and less temperature dependent . But these phantoms need a huge number of identical electronic components properly designed in a mesh mimicking the conductivity distribution of a practical biological tissue . Furthermore, for a large tissue structure, a mesh phantom requires a huge number of very precision components . The reproduction of these kinds of phantoms having different properties is often time - consuming . The option for changing the position and property of an inhomogeneity is limited by the phantom structure and the number of elements in mesh phantom but the practical phantoms allow us to put several types of object in different positions in the bathing solution, but they produce several errors contributing to the poor signal to noise ratio (snr) in boundary data . Reconstructed image quality in impedance tomography depends on the errors associated with practical phantom, electronic hardware, and inverse solver performance . Image quality is largely affected by the practical phantom design parameters such as phantom geometry, electrode geometry, electrode materials, and the nature and behavior of the inhomogeneity and bathing solution . Snr is also reduced by the error contributed by current injector, data acquisition system, and signal conditioner circuits . In practical phantoms, the voltage data developed by a three - dimensional current conduction are collected form surface electrodes connected to an analog instrumentation . Therefore, it is quite confusing to identify the source of the errors responsible for poor image quality in a 2d - eit system . In order to overcome the difficulties and limitations of practical and mesh phantoms, a matlab - based boundary data simulator (bds) bds is an absolute 2d data simulator which is required to generate the errorless 2d boundary data to study and modify the inverse solver of a 2d eit system . As the bds is a computer program, it is free from the instrumentation errors and allows us to generate voltage profile with different types of phantom geometry, inhomogeneity and background conductivity profile, and inhomogeneity geometry (shape, size, and position). Moreover, it is absolutely stable, compact, easy to use, and easy to handle and modify for further development . Boundary data for different phantom geometries are generated in bds, and resistivity images are reconstructed in standard reconstruction algorithm . Bds is studied to conform its suitability to use for boundary data generation with different phantom configurations which are required to assess the eit inverse solvers . Eit image reconstruction is a nonlinear inverse problem in which the electrical conductivity distribution of a closed domain () in a volume conductor is reconstructed from the surface potential data developed at the boundary () by injecting a constant current signal . A low frequency and low magnitude constant sinusoidal current is injected through an array of electrodes attached to the boundary, and the boundary potentials are measured using a data acquisition system . The voltage data collected from surface electrodes are then used by an image reconstruction algorithm which reconstructs the conductivity distribution of the domain under test (dut). The reconstruction algorithm computes the boundary potential for a known current injection and known conductivity values and tries to compute the conductivity distribution for which the difference between the measured boundary potential (vm) and the calculated (vc) is minimum . The reconstruction algorithm is developed with two parts: forward solver (fs) [5, 1517] and inverse solver (is) [1517]. Forward solver calculates the boundary potential data for a known current injection and known conductivity values . Inverse solver computes the conductivity distribution for which the boundary voltage difference (v = vm vc) becomes minimum . The dut will have the distinct conductivity values at each points defined by their corresponding coordinates (x, y). Due to a constant current injection, a potential profile is developed within dut, and its potential profile without any internal energy sources depends on the conductivity profile . Hence, a relationship, called eit governing equation, between the electrical conductivity () of the points within the dut and their corresponding potential values () can be established . The governing equation in eit [1, 2] can be derived from the maxwell's equation and can be represented as (1)=0 . To calculate the domain potential developed for a constant current injected to the dut with a known conductivity distribution, the above equation is essentially to be solved . As the eit governing equation is a nonlinear partial differential equation, the direct or analytical technique fails to solve it . Therefore, to calculate the domain potential, the equation is solved by developing a mathematical model called forward model which is derived from (1) using a numerical technique like finite element method (fem). The eit governing equation has an infinite number of solutions, and hence the fem formulation of the eit technique is essentially required to be provided by some boundary conditions [1820] to restrict its solutions space . The boundary conditions are imposed into the fem formulation of eit by specifying the value of certain parameters (voltage or current). The parameters defining the boundary conditions may be either the potentials at the surface or the current density crossing the boundary or mixed conditions . The boundary conditions, in which the parameters are the potential at the surface, are called the dirichlet boundary conditions and are represented as [1, 5, 19, 20](2a)=i, where i = 1, the boundary conditions, in which the parameters are current density crossing the boundary, are known as the neumann boundary conditions [1, 5, 19, 20] which are given by (2b)n={+ion the source electrodeion the sink electrode0otherwise, where is the boundary, and n is the outward unit normal vector on an electrode surface . In eit, the fem technique is used to derive the forward model from the governing equation in the form of a matrix equation establishing the relationship between the injected current and the developed potential within a dut . The relationship can be assumed as the transfer function of the system which is mathematically represented as a matrix called global stiffness matrix (gsm) or transformation matrix constructed with the elemental conductivities () and nodal coordinates (x, y). In eit, fem discretizes the dut by a finite element mesh containing finite number of elements of defined geometry and finite number of node . Fem applied on the governing equation to derive the forward model of a dut in the form of a matrix equation using the and nodal coordinates . In the eit forward model, the relationship established between the current injection matrix [c] (matrix of the applied signal) and the nodal potential matrix [] (matrix of the developed signal) through the transformation matrix [k()] is mathematically represented as (3)[]=[k()]1[c]. Now, in fem formulation in eit, when the current matrices [c] and [k()] are known, and the nodal potential matrix [] is unknown, the forward model or the mathematical problem is termed as the forward problem . The procedure of calculating the [] by solving the forward problem (3) with known [k()] and known [c] is termed as forward solution . In eit, the forward solver first computes the potential distribution with the assumed initial conductivity distribution (0) with a known constant current simulation, and then the inverse solver reconstructs the conductivity distribution from the measured boundary potential data for a same constant current injection through surface electrodes . The eit reconstruction algorithm tries to mathematically find the elemental conductivity values (conductivity distribution) for which the difference between the estimated nodal potentials (vc) computed in the fs and the potentials measured (vm) on the surface electrodes (for a same current injection values) becomes minimum . The inverse solver of the eit reconstruction algorithm is developed with a mathematical minimization algorithm (mma) [1922] such as gauss - newton - based mathematical minimization algorithm (gn - mma). In gn - mma, the conductivity update vector ([]) is calculated and the boundary data mismatch vector (v = vm vc) is minimized by an iteration technique like the modified newton - raphson iteration technique (nrit) [1922]. The [] matrix is the desired variation in the elemental conductivity values in [] matrix for which the forward solver calculates the boundary potentials more similar to the measured value in next iteration using nrit . Therefore, the algorithm starts with an initial elemental conductivity vector ([0]), and it is then updated to ([1] = [0] + []) in the next iteration . Using this [1], fs calculates a new potential distribution in dut and a new voltage mismatch vector [v1] is thus obtained and compared with the previous voltage mismatch vector [v0]. If the v1 is not found as the minimum, the iteration process is continued till the kth iteration using the conductivity update vector ([k]) developed by gn - mma . Using, nrit the [] matrix is iteratively updated to [k+1] = [k]+[k] and repetitively tries to find out the minimum value of [v]. Hence, in the eit inverse solver, it is understood that the desired elemental conductivity matrix is obtained by a minimization algorithm (mma) which is composed of gauss - newton method and newton - raphson iteration in which the technique iteratively tries to find out an optimum conductivity distribution [k] for which the voltage mismatch vector is minimized [v]. At a particular iteration in this mma, the elemental conductivity matrix is calculated when the current matrices [c] and [] or [v = vm vc] are known . Thus, when the current matrices [c] and [] are known, and the elemental conductivity matrix [] is unknown, the model or the problem is called the inverse problem . The procedure of calculating the [] or [] using with known [v] and the known [c] is termed as inverse solution . Electrical conductivity imaging is a highly nonlinear and ill - posed inverse problem [1922]. In eit, a minimization algorithm is used to obtain an optimized elemental conductivity value [] for which the voltage mismatch vector [v] becomes minimum . In the image reconstruction process, the minimization algorithm [17, 18] first defines an objective function (s) from the computational predicted data [vc] and the experimental measured data [vm] and runs iteratively to minimize it . Generally, in the eit image reconstruction algorithm, the inverse solver searches for a least square solution of the minimized object the function (s) using by a gauss - newton method and the nrit - based iterative approximation techniques . If f is a function mapping a t - dimensional (t is the number of element in the fem mesh) impedance distribution into a set of m (number of the experimental measurement data ([vm]) available) approximate measured voltages, then the gauss - newton - method - based minimization algorithm [1926] tries to find a least square solution of the minimized object function (s) [1926] which is defined as: (4)s=12\|\|vmf\|\|2=12(vmf)t(vmf). Now, differentiating (4) with respect to the conductivity, it reduces to (5)s=[f]t[vmf]=jtv, where the matrix j = f is known as jacobin matrix [1922], which may be calculated by a method as described in [19, 22] or by the adjoint method represented by (6) (6)j=sdd, where s is the forward solution for a particular source location, and d is the forward solution for the adjoint source location (source at the detector location and detector at the source location). Differentiating (5) with respect to again, the equation reduces to (7)s=[f]t[f][f]t[vmf]. By gauss - newton method, thus, the conductivity update vector is given by (9)=[[f]t[f][h]tv]1jtv, where the higher - order term h = [f] is known as the hessian matrix . In (9) by neglecting h, the update conductivity vector reduces to (10)=[[f]t[f]]1jt[v]. In general, using nrit method, the conductivity update vector expressed as in (10) can be represented for kth iteration (where k is a positive integer) as (11)k=[[jk]t[jk]]1[jk]t[vk], where [vk] and [jk] are the voltage mismatch matrix and jacobian matrix, respectively . The [f]matrix in (11) is always ill conditioned [1924], and hence small measurement errors will make the solution of (11) changes greatly . In order to make the system well posed, the regularization method [1926] is incorporated into the reconstruction algorithm by redefining the object function [1926] with regularization parameters as (12)sr=12\|\|vmf\|\|2 + 12\|\|g\|\|2, where sr is the constrained least - square error of the regularized reconstructions, g is the regularization operator, and (the positive scalar) is called the regularization coefficient [1926] (13)sr=12(vmf)t(vmf)+12(g)t(g). Differentiating the inject function in (12) with respect to the elemental conductivity: the following relations are obtained (14)sr=(f)t(vmf)+(g)t(g),(15)sr=(f)t(f)(f)t(vmf)+gtg . Now, using gauss - newton- (gn-) method - based minimization process, the conductivity update vector [] is obtained as (16)=srsr=(f)t(vmf)(g)t(g)(f)t(f)(f)t(vmf)+gtg . Neglecting the hessian matrix in (15) (17)=srsr=(f)t(vmf)(g)t(g)(f)t(f)+gtg . Replacing f by j and gg by i (identity matrix) (21) reduces to (18)=jt(vmf)ijtj+i, where the matrix j = f is the jacobin as stated earlier . Thus, the conductivity update vector ([]) is found as (19)=(jtj+i)1(jt(vmf)i). Sometimes, the last term (i) is neglected, and the conductivity update vector [] is calculated as (20)=(jtj+i)1jt(vmf). In general, the eit image reconstruction algorithm provides a solution of the conductivity distribution of the dut for the kth iteration as (21)k+1=k+((jtj+i)1(jt(vmf)i))k . The eit algorithm starts with the solution of fp obtained from the eit governing equation, and the [vc] is calculated for a known current injection matrix [c] and an initial guess (known or assumed) conductivity matrix [0]. The voltage mismatch matrix [v] is estimated, and then it is used to calculate the conductivity update matrix [] using gn - mma and is added to the initial conductivity matrix ([o]) to update it to a new conductivity matrix [1 = o +] using nrit . New update matrix [1] is used in forward solver to obtain a new calculated boundary data matrix [vc1] which provides a new voltage mismatch matrix [v1]. Therefore, the nrit algorithms iteratively calculate the [] using gn - mma to find out an optimized [] matrix for which the [v] reaches its minimum value . Thus, the eit reconstruction algorithm is found to work in the following sequences:(1)forward solver calculates the boundary potential matrix [vc] for a known current injection matrix [c] and an initial guess (known) conductivity matrix [0],(2)measured voltage data matrix [vm] is compared with [vc] to estimate the [v] as [v = vc vm],(3)jacobian (j) is computed,(4)conductivity update vector [] is calculated by gauss - newton - based minimization algorithm,(5)[o] matrix is updated to a new conductivity matrix [1 = o +] by adding [] to [] using newton - raphson iteration technique (nrit),(6)new update matrix [1] is used in forward solver to calculate the new voltage mismatch matrix [v1],(7)check whether the [v1] is minimum or not or compare the [v] with a specified error limit () if provided,(8)stop the algorithm if v condition is achieved, otherwise repeat the steps 1 to 7 until the specified stopping criteria (v) is achieved . Forward solver calculates the boundary potential matrix [vc] for a known current injection matrix [c] and an initial guess (known) conductivity matrix [0], measured voltage data matrix [vm] is compared with [vc] to estimate the [v] as [v = vc vm], jacobian (j) is computed, conductivity update vector [] is calculated by gauss - newton - based minimization algorithm, [o] matrix is updated to a new conductivity matrix [1 = o +] by adding [] to [] using newton - raphson iteration technique (nrit), new update matrix [1] is used in forward solver to calculate the new voltage mismatch matrix [v1], check whether the [v1] is minimum or not or compare the [v] with a specified error limit () if provided, stop the algorithm if v condition is achieved, otherwise repeat the steps 1 to 7 until the specified stopping criteria (v) is achieved . A two - dimensional boundary data simulator (bds) is developed in matlab r2010a using finite element method (fem) to generate accurate boundary data for studying the eit reconstruction algorithms . The matlab - based bds is developed as an absolute 2d data simulator for eit image reconstruction studies, and it is used suitably to generate the errorless 2d boundary data to study and modify the inverse solver of a 2d eit system . As bds is developed in a computer software, it is found free from errors produced by the eit instrumentation and phantom . Bds also allows us to generate boundary potential data for different type of phantom geometry, inhomogeneity geometry (shape, size, and position), inhomogeneity conductivity profiles, and background conductivity profiles . Moreover, it is developed as a compact, absolutely stable, and easy to use and handle for eit studies . It is developed in such a way that it can be modified for further modifications . Bds is developed with matlab - based computer program consisting of four - part imaging domain simulator (ids), eit model developer (emd), current injection simulator (cis), and boundary data calculator (bdc). Imaging domain simulator (ids) in bds simulates a domain with inhomogeneity with their corresponding conductivity distributions . Eit model developer (emd) derives a mathematical model of the forward solver by applying fem on the governing equation of the dut in the form of a matrix equation . Current injection simulator (cis) simulates a constant current injection through the definite points at the domain boundary with neighbouring current injection protocol [1, 2, 2830]. The boundary data calculator (bdc) solves the governing equation by solving the forward model and calculates the potentials at all electrodes at the domain boundary . Imaging domain simulator (ids) first defines a dut with a desired area (ad) defined by a required diameter and defined with a particular coordinate system . Imaging domain simulator applies the fem to discretize the domain with a 2d finite element mesh containing finite element of triangular elements (t) and finite number of nodes (n). In ids, a circular domain () to be imaged is defined with a required radius (rp) using the cartesian coordinate system (figure 1(a)), and the domain is discretized with a finite element (fe) mesh (figure 1(b)). The mesh is symmetrically composed of the first - order triangular elements with linear shape functions [18, 31]. The fe mesh is generated with the pdetool of matlab r2010a in such a way that it can be refined further to increase the number of elements as per the requirement . All the coordinates and parameters assigned to the finite elements and the nodes are stored in corresponding matrices . Boundary nodes are identified, and the sixteen nodes among the boundary nodes are assigned as the electrodes called the electrode nodes . Inside the domain one (or more) smaller region (regions) is (are) defined as the inhomogeneity (inhomogeneities) positioned at a particular place . The center point (p) of the inhomogeneity with the required shape and size is positioned inside the phantom domain by defining its center with a polar coordinate (r,) as shown in figure 1(a). Single or multiple inhomogeneities are defined with their desired areas (ai) inside the dut, and elements within the inhomogeneity and the background are identified . The background area is defined as the area of the domain surrounding the inhomogeneity (ab = ad ai), and the elements within the background area (ab) are identified . The elements within the inhomogeneity are assigned with a particular conductivity called inhomogeneity conductivity, (i) while the rest of the elements are assigned with a different conductivity called background conductivity (b) as shown in figure 1(b). The assigned conductivity values of all the elements are assumed to be featured at their corresponding centroids . Eit model developer (emd) develops the mathematical model of the forward solver by applying fem on the governing equation and derive the forward model of a dut in the form of a matrix equation (3) using the elemental conductivities and nodal coordinates . The emd establishes a relationship between the current injection matrix, [c] (matrix of the applied signal), and the nodal potential matrix, [] (matrix of the developed signal), through the transformation matrix [k()] which is mathematically represented by (3). The global stiffness matrix [k()] in eit is actually an admittance matrix that is formed using the nodal coordinates of all the elements with their corresponding conductivities . Thus, the [k()] inforward model represents the transfer function of the eit system obtained from the governing equation by fem formulation . The current injection simulator (cis) is used to simulate a constant current injection through the sixteen nodes called simulated electrodes (se) on the domain boundary with neighbouring current injection protocol . The cis works in a for loop to execute all the projections [1, 28, 30, 32] of current injection process . In bds, a constant current injection is simulated into the dut surrounded by the sixteen simulated current electrodes (sei) with all the possible combination of sei pairs, and the potential data are calculated on all the electrodes called voltage electrodes (sev) in bdc . The current injection through a particular current electrode pair (say sei1 and sei2) and corresponding voltage data collection from all the possible voltage electrodes (sev1, sev2, sev3, sev4, sev15, sev16, sev7, sev8, sev9, sev10, sev11, sev12, sev13, sev14, sev15 and sev16) is known as a simulated current projection (scp). Hence, in an n - electrode eit system, there will be n - different current projections each of which will inject current through a particular current electrode pair and collect m voltage (differential / grounded) data where m may be either equal to n or less than n depending on the eit data collection strategy called the current pattern [1, 28, 30, 32]. Therefore, a complete scan (containing all the current projections) conducted on the dut yields n m voltage data . As the bds is studied for sixteen electrode system, the cis runs for sixteen times and provides sixteen current projections (scpv1, scpv2, scpv3, scpv4, scpv15, scpv16, scpv7, scpv8, scpv9, scpv10, scpv11, scpv12, scpv13, scpv14, scpv15, and scpv16). Therefore, a complete data collection procedure (called a complete scan) in the bds collects m voltage data from the voltage electrodes or voltage electrode pairs in all the sixteen current projections and computes 16 m voltage data . Boundary data calculator (bdc) calculates the potentials (developed for a constant current injection by cis) at all electrode points (electrode nodes) at the domain boundary in each current projection for a particular current pattern . The current injection matrix is formed in cis using the neumann type boundary conditions, and the potential matrix is calculated from (3) using the matrix inversion technique working on l - u factorization process . The bds is developed to run in an another for loop for m times to calculate the m electrode potentials from voltage electrodes or voltage electrode pairs at each of the steps of the loop . This second for loop runs within the first for loop for m times and collects m voltage data for each step of first for loop and hence collects 16 m voltage data as first for loop runs for sixteen times . Moreover, as the eit reconstruction process needs a complete scan, the bds runs in each current projection and computes sixteen electrode potentials at each projection . The domain potential is calculated from the forward model (3), and the potential values of all the nodes are stored in a nodal potential matrix [33, 34] denoted by [mnp]. Boundary potential data are separated from [mnp] and stored in a different matrix called boundary potential matrix [mbp]. The electrode potential data are extracted from the nodal potential matrix [mnp] and are stored in a separate matrix called electrode potential matrix [mep]. In sixteen electrode eit system, the [mep] is formed as a column matrix and contains the 16 m electrode potentials (differential or grounded) obtained for all the projections . In neighbouring or adjacent current injection method, first reported by brown and segar, the current is applied through two neighbouring or adjacent electrodes, and the differential voltages is measured successively from all other adjacent electrode pairs excluding the pairs containing one or both of the current electrodes . For a sixteen electrode eit system with domain under test surrounded by equally spaced sixteen electrodes (e1, e2, e3, e4, e5, e6, e7, e8, e9, e10, e11, e12, e13, e14, e15, and e16), the neighbouring method injects current through the current electrode pairs for sixteen current projections (figure 2), and the differential voltages are measured across the voltage electrode pairs using four electrode method in each projection . As shown in figure 2(a) in the first current projection (p1) of adjacent method, the current is injected through electrode 1 (e1) and electrode 2 (e2), and the thirteen differential voltage data (v1, v2, v3,, v13) are measured successively between the thirteen electrode pairs e3-e4, e4-e5,, and e15-e16, respectively (figure 2(a)). As reported by brown and segar, in neighbouring current injection method, the current density within the dut is found highest between the current electrodes (e1 and e2 for p1); the current density then decreases rapidly as a function of distance . Similarly, in current projection 2 (p2), the current signal is injected through electrodes 2 (e2) and 3 (e3), and an another set of thirteen differential voltage data (v1, v2, v3,, v13) are collected between the thirteen electrode pairs e4-e5, e5-e6,, e16-e1, and so on . Lastly, in the current projection 16 (p16), the last set of thirteen differential voltage data (v1, v2, v3,, v13) are collected between the thirteen - electrode pairs e2-e3, e3-e4,, and e14-e15 by injecting the current through the electrodes e16 and e1 . Thus, the neighbouring current injection method in a sixteen electrode eit system data collection procedure consists of sixteen current projections (p1, p2, p3,, p15, and p16), and each of the current projection yields thirteen differential voltage data (v1, v2, v3,, v13). Therefore, a complete data collection scan with the neighbouring current injection method in a sixteen electrode eit system yields 16 13 = 208 voltage measurements . Though in neighbouring method, eit boundary data are not collected across the electrode pairs containing one or two current electrode for contact impedance problem, but sometimes it is advantageous to collect the boundary data from all the electrodes including the current electrodes to obtain the greatest sensitivity to the resistivity changes in the domain as reported by cheng et al . . In the present study, the boundary potentials are calculated at all the electrodes (figure 2(b)) with respect to a virtual ground point selected within the dut . Hence, in a complete data collection scan, the potentials on all the electrodes are collected in all the sixteen current projection and are stored in [mep]. Therefore, the [mep] is found as a column matrix containing 16 16 voltage data all collected with respect to the virtual ground point of the dut . Hence, in the present study, with neighbouring current injection method, the [mep] is found as a 256 1 matrix containing 256 electrode potentials . In the present study, 1 ma current injection is simulated through the electrodes of the simulated domain containing sixteen nodal electrodes using adjacent or neighboring current injection protocol (figure 2(b)). The potentials on all the sixteen electrodes are calculated using boundary data calculator (bdc) for all the current projections, and the electrode potential matrix [mep] is used as the calculate boundary potential matrix [vc] to reconstruct the conductivity distribution of dut . The bds is designed in such a way that a huge number of voltage data sets can be generated using different types of phantoms with their different design parameters . Boundary potential data [vc] are generated for different type of phantom configurations, and the boundary data have been tested with electrical impedance tomography and diffuse optical tomography reconstruction software (eidors) [37, 38] for 2d - eit . A large number of data sets are generated by changing the values of one or more phantom parameters like: phantom diameter (d = 2rp), inhomogeneity radius (ri), inhomogeneity geometry (shape, size, and position), inhomogeneity number (ni), bathing solution conductivity (b), and inhomogeneity conductivity (i). 1 ma current injection is simulated to the domain boundary, and data generation in bds and image reconstruction in eidors are studied for different inhomogeneity geometries in dut . Reconstruction is also studied for different iterations and for multiple inhomogeneity reconstruction to evaluate the bds . Image reconstruction quality in eit depends on the boundary data accuracy which is dependent on the geometric accuracy of the inhomogeneity developed in bds . Dimensional accuracy of the inhomogeneity depends on the number of finite elements in the fe mesh or mesh refinement number (nmr) as shown in figure 3 . As the nmr increases, the number of elements in the fe mesh is increased, and hence the geometric accuracy of the inhomogeneity increases which gives more accurate boundary data and better image reconstruction (figure 3). But the bds with a highly refined mesh needs a high pc memory and large computation time . In this paper, the mesh refinement is found suitable as nmr = 4 as per the configuration of the pc (2.4 ghz/1.5 gbram/ p - iv) used . It is observed that the fe mesh with nmr = 4 (containing 2048 elements and 1089 nodes) gives almost an accurate geometry (figure 3) to the desired inhomogeneity and generates a reconstructible data set in less than 10 seconds . Eidors reconstructs the resistivity images from the bds data sets using regularized image reconstruction technique . Results show that the resistivity or conductivity can be successfully reconstructed from the boundary data generated by our bds using a circular domain (rp = 75 mm) with a circular inhomogeneity (r = 37.5 mm, ri = 25 mm, = 45, i = 0.005 s / m, and b = 0.21 s / m) in the 9th iteration (figure 4). It is also observed that the reconstructed shape of the inhomogeneity is similar to that of the original one (figure 4(a)), and the reconstructed conductivity profile in figure 4(b) is almost similar to that of the original object in figure 4(a). Iteration studies shows that in different reconstruction steps called iterations (figure 5), the reconstructed images become more localized from iteration to iteration and the reconstruction errors (appeared by the red color at phantom periphery) are gradually reduced (figure 5). It is observed that the resistivity is successfully reconstructed from the boundary data in the 9th iteration (figures 5(i) and 5(j)), though the shape of all the reconstructed images in 9th12th iterations is almost similar to that of the original one (shown by dotted circles in figure 5). As the reconstructed resistivity profile similar to that of the original is obtained only in the 9th iteration, the 9th iteration is taken as the optimum reconstruction . In 13th and 14th the optimum iteration number depends on the data accuracy and reconstruction algorithm, and hence the bds can be used to generate the boundary data sets required for assessing the inverse solver in eit . Voltage data are also generated for a domain (rp = 75 mm) with the circular inhomogeneities (ri = 25 mm, i = 0.005, s / m, and b = 0.21 s / m) positioned at different places using the bds (figure 6). It is observed that the reconstructed image is more circular for an inhomogeneity positioned at the phantom centre where r = 0 and = 0 (figure 6(a)). On the other hand, for r 0, that is, for the inhomogeneities near domain boundary (figure 6(b)), reconstructed images are not perfectly circular because of the comparatively less accurate shape of the original object obtained for r 0 . For a less number of mesh refinements, the geometry of the original side objects is not exactly circular itself (figure 4), and hence the corresponding boundary data have lower accuracy . An fe mesh with large nmr can easily produce an accurate geometry for the boundary objects (objects near domain boundary) with proper shape, which gives a boundary data without geometric error and automatically improves the image shape . Boundary data sets are also generated with a circular domain (rp = 75 mm and b = 0.21 s / m) with a circular inhomogeneity (i = 0.005 s / m) with different diameters (2ri) and all positioned at the phantom center (r = 0). Results show that for the domain discretized with nmr = 4, the data sets, generated with a diameter larger than 13.3% of the phantom diameter, are accurate enough (figures 7(a)7(f)) to reconstruct the resistivity images in eidors-2d . It is clearly observed that for nmr = 4, the triangular elements within the inhomogeneity with smaller ri are unable to shape themselves into a proper circle (figure 7(g)). Hence, the data obtained for the inhomogeneity with a diameter of 20 mm has low accuracy (figure 7(g)), and hence the resistivity image (figure 7(h)) is found with low resolution showed and some reconstruction error (appeared in the red color at phantom periphery). Increasing the fe elements in bds, the boundary data error can be minimized, and the improved resistivity image can be achieved even for smaller inhomogeneities with a diameter less than 13.3% of rp . Boundary potential data are also generated for domains (rp = 75 mm) containing multiple circular inhomogeneities (ri = 25 mm, r = 37.5 mm, i = 0.005 s / m, and b = 0.21 s / m) placed at different positions inside the domain (figure 8). Figure 8(a) shows a domain with two circular inhomogeneities (180 apart from each other) which are placed at a central distance (r) of 37.5 mm . Similarly, another domain with three circular inhomogeneities (120 apart from each other) placed inside the phantom domain is shown in figure 8(c). All the inhomogeneities in both the domains are positioned at a central distance (r) of 37.5 mm . 1 ma current is simulated with the neighbouring current pattern, and the boundary data are collected for resistivity reconstruction . It is noticed that the resistivity images (figures 8(b) and 8(d)) of inhomogeneities in both the domains are reconstructed successfully . Results show that the boundary data simulator can be efficiently used to generate boundary potential data for a huge number of phantom configurations in less than 10 seconds . Bds is software - based virtual eit phantom, and hence it has a number of advantages over the practical and mesh phantoms . The literatures [3941] presenting the phantom simulations are limited, and they only discuss the software phantoms developed for their own systems . Bds is a software - based versatile boundary data simulator which generates boundary data suitable for studying the reconstruction algorithm required for several eit systems, and hence it is better suited for assessing the performance of the inverse solver of 2d electrical impedance tomography . A matlab boundary data simulator (bds) is developed for studying the resistivity reconstruction in inverse solvers of 2d - eit . Bds is developed with four parts: imaging domain simulator (ids), eit model developer (emd), current injection simulator (cis), and boundary data calculator (bdc). Imaging domain simulator (ids) simulates a domain with single or multiple inhomogeneities of different geometries defined with their corresponding conductivity distributions, whereas the eit model developer (emd) derives a forward model using fem to solve the governing equation of the dut . Current injection simulator (cis) simulates a constant current injection through the simulated electrodes positioned at the domain boundary with the neighbouring current injection protocol . The boundary data calculator (bdc) solves the forward model to solve the governing equation and calculates the potentials at all the simulated electrodes . Boundary data are generated with different type of domains simulated in bds by changing its input parameters . Resistivity images are reconstructed from the boundary data using standard eit reconstruction software called eidors, and the bds is evaluated . It is observed that the bds with fe mesh with 2048 elements can simulate an inhomogeneity of desired geometry with suitable accuracy . The bds with 2048 elements suitably generates the boundary data for simulated domains containing the objects with different geometries which are found efficient for image reconstruction in eidors . Results also show that the conductivity or resistivity profiles of the domains simulated in bds are successfully reconstructed from their corresponding boundary data generated for different type of single and multiple inhomogeneities . By changing the inhomogeneity position, diameter, and number in bds, multiple inhomogeneity imaging shows that the bds suitably generates boundary data with the desired accuracy, and the boundary data are found efficient for resistivity reconstruction in eidors . Results also show that for the simulated domains discretized with nmr = 4, the boundary data sets generated for circular inhomogeneity with a diameter larger than 13.3% of the phantom diameter are accurate enough to reconstruct the resistivity images in eidors . Increasing the fe elements in bds, the boundary data error can further be minimized, and the improved resistivity image reconstruction can be obtained even for smaller inhomogeneities . Hence, it is concluded that the bds generated a number of boundary data sets which can suitably be used for inverse solver assessment in eit.
The number of older adults affected by dementia has been rising significantly in japan and a number of other countries1, 2 . Mild cognitive impairment (mci)3,4,5,6 is considered to be a preclinical stage of dementia . Studies have revealed that approximately 10% to 15% of people with mci progressed to dementia annually7,8,9 . In order to reduce the number of people who experience cognitive dysfunction, it is important to delay, or even reverse, the progression of mci to dementia . While aging is regarded as the greatest risk factor for cognitive dysfunction10, several studies have indicated that habitual exercise and physical activity may have a beneficial effect on the prevention of dementia11,12,13,14,15 . A wide variety of exercises, such as walking, have been recommended for older adults in efforts to maintain and promote health . Aquatic exercises in particular are recommended for older adults with obesity and osteoarthritis16,17,18 . One such aquatic exercise regimen is synchronized swimming, which is suitable for older adults, as the swimmer may stand on the floor of a shallow pool19 . While a number of studies have indicated that land - based exercise exerts a beneficial effect on cognitive function in older adults11,12,13,14, no studies to date have examined the effect of synchronized swimming on cognitive function in older adults . The study participants were recruited from among 23 women practicing synchronized swimming - exercise at a sport club in fukushima - ku, osaka city, japan (ss group). All participants provided written informed consent, after having gained a full understanding of the present study . The study was approved by the ethics committee of the osaka university of health and sport sciences (approval number 15 - 21). Synchronized swimming was performed in a shallow pool from 1.2 to 1.3 meters in depth and the participants were allowed to touch the bottom of the pool with their hands and feet during the performance . Participants who had participated in the olympic games or the domestic competitions for seniors were excluded . Participants practiced synchronized swimming twice a week for 90 minutes under the supervision of an instructor, who had been a member of the japanese synchronized swimming team . The ages of participants in the ss group ranged from 49 to 85 years (mean age: 69.8 11.6 years), and the duration of synchronized swimming experience ranged from 1 to 39 years (mean year: 23.9 12.6 years). Control participants were recruited age - matching women with exercise custom other than synchronized swimming . They were 36 individuals participating in a health promotion program at our university in 2015, and provided written informed consent as controls (age range: 4977 years; mean age: 67.0 6.0 years). Among the participants in the control group, 16 participants exercised three or four times a week, 17 exercised once or twice a week, and 3 exercised one to three times a month . Cognitive functions was compared between the two groups using the japanese version of the montreal cognitive assessment (moca - j)20 . The moca is a brief cognitive test containing test items related to memory, language, executive function, working memory (attention), visuospatial recognition, abstract thinking, and orientation21 . The sensitivity and specificity of the moca - j in screening for mci has been demonstrated20 . The assessment takes only approximately 10 minutes to complete, and a number of studies have demonstrated the effectiveness of moca - j as a screening tool for mci21,22,23 . Total scores of less than 26 points may be indicative of mild cognitive impairment . Information on the presence of neurological disease, psychiatric disease, alcoholism, and hospitalization was collected via an interview . Unpaired t - tests were used to compare results between the ss group and control group when data were normally distributed, while u tests were performed when the data were not normally distributed . Two - sided p values of <0.05 were considered to indicate a statistically significant difference . No significant differences in education level were observed between the control and ss groups (12.1 2.0 and 12.2 1.8 years, respectively). In addition, no significant differences in total moca - j scores were observed between the two groups (22.2 3.6 and 23.2 3.1, respectively). Twenty - nine participants in the control group and 17 in the ss group scored below 26 on the moca - j and were suspected of having mci (table 1table 1.comparison of moca - j between the control group and the ss groupscorecontrol group (n=36)ss group (n=23)moca - j<2629 (80.6)17 (73.9)267 (19.4)6 (26.1)no significant differences are shown between the two groups . Moca - j: japanese version of montreal cognitive assessment; ss: synchronized swimming). With regard to moca - j sub - scores, significant differences in delayed recall (p=0.005) however, no significant differences were observed with regard to measures of but not in visuospatial / executive function, naming, attention, language, abstraction, or orientation (table 2table 2.comparison of moca - j sub - score between the control group and the ss groupcontrol group (n=36)ss group (n=23)visuospatial / executive function (5)3.5 1.03.4 1.0/5naming (3)2.9 0.43.0 0.0attention (6)4.9 1.14.8 1.0language (2)1.6 0.91.7 0.8abstraction (2)1.1 0.80.9 0.7delayed recall * (5)1.8 1.73.0 1.4orientation (6)5.8 0.45.6 0.7total scores22.2 3.623.2 3.1values are shown as the mean sd . * moca - j: japanese version of montreal cognitive assessment; ss: synchronized swimming values are shown as the mean sd . * p<0.005 for the difference between the two groups . No significant differences in years of education or total moca - j scores were observed between the ss group and the control group in the present study . The results of total moca - j scores in the present study align with those of a previous report involving 1,552 participants (mean age: 72 years; mini - mental state examination score> 24 points) living in sasaguri, fukuoka prefecture, in which mean the moca - j score was 22.4 3.024 . However, in the comparison of moca - j sub - score in the present study, delayed recall scores, which are considered indicative of recent memory function, were significantly higher in the ss group than in the control group . Suzuki et al.25 conducted an exercise - based intervention program that included exercise, calculation, word - chain game, and multiple tasks using a ladder diagram program (90 minutes two times per week for 6 months) with participants aged 65 years or older experiencing mci . Significant differences in mini - mental state examination, wechsler memory scale - logical memory i, word fluency test - category, and word fluency test - letter scores were observed following this intervention . In synchronized swimming, participants must swim and act in a coordinated manner with other swimmers while performing to music . In other words, the swimmers are required to perform multiple coordinated tasks in an aquatic setting to be more complex than those of the multicomponent exercise program developed by suzuki et al25 . Moreover, it has been reported that group music intervention delayed the deterioration of cognitive function, particularly short - term recall among aging adults with mild and moderate dementia26 . Performing to music in synchronized swimming might be one of the reason that delayed recall scores were significantly higher in the ss group than in the control group . Voelcker - rehage et al.22 reported that not only physical fitness, as indexed by cardiovascular fitness and muscular strength, but also aspects of motor fitness, such as movement speed, balance, motor coordination, and flexibility exhibit associations with executive control and perceptual speed . They further demonstrated physical and motor fitness to be differentially related to various cognitive processes using functional brain magnetic resonance imaging . Given that most synchronized swimmers exhibit good cardiopulmonary function, muscle strength, and flexibility27, it is also possible that enhanced physical fitness may exert direct positive effect on cognitive function . According to a report by suzuki et al.28, 12 months of multicomponent exercise in particular, positive effects were observed for general cognitive function, immediate memory, and language ability . We therefore consider that synchronized swimming exerted beneficial effects on cognitive function in the participant who experienced synchronized swimming for one year . In conclusion, the results of the present study suggest that synchronized swimming may improve cognitive function in older adults, particularly with regard to recent memory . No relevant disclosures.
Ig genes in b lymphocytes undergo three types of genetic alterations during their development, i.e., v(d)j recombination, somatic hypermutation (shm), and class switch recombination (csr). V(d)j recombination takes place in developing b lymphocyte precursors and its biochemical mechanism is well characterized (1). Both events occur in activated mature b lymphocytes such as germinal center cells, but outcomes of the events are apparently very different . In shm, mostly point mutations are introduced in ig variable (v) region genes, giving rise to ig with high affinity (2). Dna cleavages are shown to be introduced in the v region during shm (36). On the other hand, in csr, two switch (s) regions located 5 to heavy - chain constant (ch) region genes are cleaved and a large dna fragment between the cleavages is excised out from the chromosome to bring in a downstream ig ch region gene to the proximity of a rearranged v gene (7). In addition, neither shm nor csr is prerequisite of the other (8, 9). Therefore, it is striking that a defect of aid, a putative rna editing enzyme, virtually abolishes both shm and csr without affecting germinal center formation (10, 11). To explain this unexpected finding, we have proposed a model that aid edits a precursor mrna to synthesize an endonuclease essential for generating dna cleavages in both shm and csr reactions (12). However, it remains to be tested whether aid edits separate pre - mrnas for csr and shm, and thus is involved in different steps in the two genetic events . In the present study we provide the evidence that hypermutation takes place in the unrearranged ig s region under the condition that induces csr but not shm in the v region gene . The results imply that csr and hypermutation may be mediated, at least in part, by the same molecular machinery . Wild - type (wt) and aid mice on (cba c57bl/6) c57bl/6 back ground were maintained in our animal facility and used 28 mo of age . Spleen b cells were purified by depleting cd43 cells with the magnetic cell sorting system (macs; miltenyi biotec). After 58 d cultivation, live cells were harvested and high molecular weight nuclear dna was extracted with sds / proteinase k lysis, followed by phenol / chloroform extraction . In some experiments, switched igg cells were enriched (6890%) or depleted (1.56%) by macs with combination of biotinylated anti - igg1 and anti igg3 antibodies (bd pharmingen) and streptavidin microbeads (miltenyi biotec). Mutation frequencies in each population were determined by sequencing 10,391 and 10,328 bp, respectively, as described in table i. induction of hypermutation in the s region upon csr stimulation dna was extracted from purified spleen b cells cultured with or without lps and il-4 . The s region (1.1 kb) composed of the 5 and 3 subregions (0.5 and 0.6 kb, respectively) and vj558-jh4 downstream region (1.2 kb but 0.5 kb of the jh4 3 flanking sequence was examined) were amplified, subcloned, and then sequenced . The switch efficiency to igg1 and igg3 in the lps / il-4 cultured wt cells was 2956% . Statistic tests were done with sas version 2000 software (sas institute inc . ). Pcr were performed with the primers shown below using pyrobest dna polymerase (takara) that has the 3 exonuclease activity and high fidelity . After purification, the pcr fragments were digested with ecori or spei and ligated into pbluescript vector . The ligation mixture was used for transformation and the library was plated without preculturing to avoid amplification of sister clones . No more than 21 clones were sequenced from a single pcr reaction for the s. clonality of the v region clones were checked by their cdr3 sequences . Nucleotide sequences were determined with abi prism 3100 genetic analyser (perkinelmer). The s region germline sequence of cba and c57bl/6 were determined and compared . 6 and 5 bp polymorphic differences were found in the s and jh4 downstream regions of our interest, respectively, and excluded from mutations . Primers used for s pcr are: 5-ggaattcattccacacaaagactctggacc-3; 5-ggaattccagtccagtgtaggcagtaga-3 (a and d in fig . 1 a, respectively) with 30 cycles of 94c for 30 s, 62c for 30 s, 72c for 1 min . Primers used for sequencing in addition to common primers for pbluescript are: 5-ggaattcgtaaggagggacccaggctaag-3; 5-ggaattcttccagaatcccaggattgcc-3 (b and c in fig . The 3 subregions of nonswitched alleles were amplified by nested pcr as follows: the first step, 20 cycles of 98c for 10 s, 68c for 7 min, with the primer a and 3-agcccatgctagctcagcctcacataa-5 (3 of the s core); the second step, 15 cycles of 98c for 10 s, 68c for 80 s, with the b and d primers . For vj558-jh4 downstream region pcr, primers described (13) were used with 35 cycles of 98c for 10 s, 68c for 80 s. distribution of mutations in the s region . (a) a total of 58 point mutations and 6 deletions shown in table i (closed and hatched symbols) and table ii (open symbols) are mapped by triangles and rectangles, respectively, on the s region (bar). Position from the transcription start site (arrow) is indicated by a scale below . Lengths (bp) of deletions (a f) are: a, one; b, 17; c, 30; d, 52; e, 7; f, 19 . The s nucleotide sequence of the c57bl/6 mouse tail dna is shown by upper case letters . Sequences that are predicted to form s / l structures are underlined (references 12 and 34). Conditions used for the s / l prediction were: the ionic conditions (mm), [na] = 150, [mg] = 0.5; the folding temperature, 37c; the maximum distance between paired bases, 25 . Recombinant retrovirus constructs (pmx - aid - ires - gfp) to express aid or aid, inactive mutant of aid, and preparation and infection of retroviruses were described before (14). Wild - type (wt) and aid mice on (cba c57bl/6) c57bl/6 back ground were maintained in our animal facility and used 28 mo of age . Spleen b cells were purified by depleting cd43 cells with the magnetic cell sorting system (macs; miltenyi biotec). After 58 d cultivation, live cells were harvested and high molecular weight nuclear dna was extracted with sds / proteinase k lysis, followed by phenol / chloroform extraction . In some experiments, switched igg cells were enriched (6890%) or depleted (1.56%) by macs with combination of biotinylated anti - igg1 and anti igg3 antibodies (bd pharmingen) and streptavidin microbeads (miltenyi biotec). Mutation frequencies in each population were determined by sequencing 10,391 and 10,328 bp, respectively, as described in table i. induction of hypermutation in the s region upon csr stimulation dna was extracted from purified spleen b cells cultured with or without lps and il-4 . The s region (1.1 kb) composed of the 5 and 3 subregions (0.5 and 0.6 kb, respectively) and vj558-jh4 downstream region (1.2 kb but 0.5 kb of the jh4 3 flanking sequence was examined) were amplified, subcloned, and then sequenced . The switch efficiency to igg1 and igg3 in the lps / il-4 cultured wt cells was 2956% . Statistic tests were done with sas version 2000 software (sas institute inc . ). Pcr were performed with the primers shown below using pyrobest dna polymerase (takara) that has the 3 exonuclease activity and high fidelity . After purification, the pcr fragments were digested with ecori or spei and ligated into pbluescript vector . The ligation mixture was used for transformation and the library was plated without preculturing to avoid amplification of sister clones . No more than 21 clones were sequenced from a single pcr reaction for the s. clonality of the v region clones were checked by their cdr3 sequences . Nucleotide sequences were determined with abi prism 3100 genetic analyser (perkinelmer). The s region germline sequence of cba and c57bl/6 were determined and compared . 6 and 5 bp polymorphic differences were found in the s and jh4 downstream regions of our interest, respectively, and excluded from mutations . Primers used for s pcr are: 5-ggaattcattccacacaaagactctggacc-3; 5-ggaattccagtccagtgtaggcagtaga-3 (a and d in fig . 1 a, respectively) with 30 cycles of 94c for 30 s, 62c for 30 s, 72c for 1 min . Primers used for sequencing in addition to common primers for pbluescript are: 5-ggaattcgtaaggagggacccaggctaag-3; 5-ggaattcttccagaatcccaggattgcc-3 (b and c in fig . The 3 subregions of nonswitched alleles were amplified by nested pcr as follows: the first step, 20 cycles of 98c for 10 s, 68c for 7 min, with the primer a and 3-agcccatgctagctcagcctcacataa-5 (3 of the s core); the second step, 15 cycles of 98c for 10 s, 68c for 80 s, with the b and d primers . For vj558-jh4 downstream region pcr, primers described (13) were used with 35 cycles of 98c for 10 s, 68c for 80 s. distribution of mutations in the s region . (a) a total of 58 point mutations and 6 deletions shown in table i (closed and hatched symbols) and table ii (open symbols) are mapped by triangles and rectangles, respectively, on the s region (bar). Position from the transcription start site (arrow) is indicated by a scale below . Lengths (bp) of deletions (a f) are: a, one; b, 17; c, 30; d, 52; e, 7; f, 19 . The s nucleotide sequence of the c57bl/6 mouse tail dna is shown by upper case letters . Sequences that are predicted to form s / l structures are underlined (references 12 and 34). Conditions used for the s / l prediction were: the ionic conditions (mm), [na] = 150, [mg] = 0.5; the folding temperature, 37c; the maximum distance between paired bases, 25 . Recombinant retrovirus constructs (pmx - aid - ires - gfp) to express aid or aid, inactive mutant of aid, and preparation and infection of retroviruses were described before (14). We reasoned that csr target s regions might receive extensive sequence alterations like shm even without actual csr upon stimulation of b cells if shm and csr share a common mechanism for dna cleavage . To assess this possibility, we examined dna sequences of the s region in splenic b lymphocytes stimulated with lps and il-4 . To avoid pcr artifacts due to highly g rich repetitive sequences in the s core region (15), we chose to analyze the upstream flanking region to the s core sequence (hereafter called the s region), in which practically frequent csr can take place (16, 17). We also analyzed such s regions that are located on nonswitched alleles, using nested pcr . We found a significant number of mutations accumulating in the s region of spleen b cells stimulated with lps and il-4 but not of unstimulated b cells (table i). The mutations are located mainly in the 3 subregion of the s region (fig . The mutation frequency observed was 4.5 10/bp and the fraction of mutated clones reached 21.6% of sequenced clones . These mutations in the s region are independent of csr because (a) the mutation frequency in s regions on nonswitched alleles is as high as that of total s (table i), (b) the mutation frequency was not significantly changed between switched and unswitched b cells (4.8 10/bp and 2.9 10/bp, respectively; p = 0.58, fisher's exact test), and (c) no csr junctions were included in the s region sequenced although frequent mutations are found in the proximity (most often within 10 bp) of csr junctions (18, 19). Furthermore, no mutations were found in a non - ig gene, c - myc (total 9,430 bp sequences of 26 clones), excluding the possibility of nonspecific genomewide hypermutation due to dna damage and repair . Most importantly, these mutations are not a part of shm in the v region because lps and il-4 stimulation could not induce hypermutation in vj558-jh4 downstream regions (table i). The vj558 family is shown to constitute the major vh population in c57bl mice and vj558-jh4 downstream regions are known to accumulate mutations in in vivo activated b cells (13). The absence of shm induction by lps and il-4 stimulation in vitro is consistent with the previous reports (2022). A few heavily mutated clones exist in both before and after the stimulation in wt samples, which are likely due to memory cells . Direct comparison of the mutation rate in the s region to that in the v region is not straightforward because unlike shm this mutation frequency represents unselected clones in in vitro primary cultures . Nonetheless, the v region of a human b lymphoma cell line, ramos spontaneously accumulates 2.3 10/bp mutations during 2 wk (3), the frequency of which is slightly higher than but comparable to the present data . 1 a), which is in agreement with previous reports that internal deletions of s (s and s1) regions can occur upon csr induction (23, 24). These data indicate that csr stimulation of b cells induces wide spread cleavage in s dna, which can cause point mutations as well as deletions . To determine if the hypermutation in the s region also depends on aid, we analyzed spleen b cells from aid mice (10) in parallel with those of wt mice . Altogether the 109.5 kb sequences of the s region were mutation free in aid b cells (table i). We conclude that the recombination - uncoupled hypermutation in the s region is mediated by the function of aid . To exclude the possibility that an apparent induction of the hypermutation in the s region represents the outgrowth of the population already with mutations, most likely memory b cells, we transfected aid primary b cells with aid - expressing retroviruses . As aid b cells have no background mutations, population changes in the cell culture would not affect the result . Splenic b cells that had been stimulated with lps and il-4 1 d before virus infection were harvested for analysis 5 d after infection . 29% of infected aid cells, which were distinguished by green fluorescent protein (gfp) coexpressed bicistronically with aid, switched to igg whereas only a background level of igg cells was found in uninfected cells (fig . The switch efficiency of rescued aid cells is comparable to that (29.5%) of wt cells without aid virus infection . By contrast, a control virus carrying a deletion mutant aid (aid) did not rescue csr ability of aid cells . Purified spleen b cells from aid (reference 10) and wt mice were stimulated with lps (25 g / ml) and il-4 (75 /ml). On day 1, the culture were split into three, and two of them were infected with either aid or aid expressing virus . On day 6, cells were harvested and stained with anti - b220, anti - igg1, anti - igg3 (bd pharmingen), and propidium iodide (pi). B220 and pi - negative cells were electronically gated and their igg and gfp expressions are shown . We then analyzed the sequence of the s region in aid b cells infected with aid virus . Virus - uninfected cells were removed by enrichment of igg cells or gfp cells by cell sorting . Clearly, only aid - virus transfected cells mutated their s region while aid - virus infected cells did not (table ii). The mutation frequencies of igg and gfp cells were 1.2 10 and 1.3 10 per bp, respectively, which are almost the same as the frequency in wt cells infected with aid virus (1.2 10/bp). Almost all mutations are found in the 3 subregion in consistence with virus non - infected wt cells (table i, fig ., practically no mutations were observed in vj558-jh4 downstream regions in aid or wt b cells infected with aid virus (table ii). These results indicate that the mutations of the s region are introduced de novo upon csr stimulation without shm in the v region, and absolutely dependent on aid, implicating that both recombination and hypermutation in s regions may be catalyzed by a certain common reaction regulated by aid . Retrovirus - mediated aid transfection rescues the hypermutation phenotype in aid b cells cells were infected and selected as indicated . The percentage of virus - infected cells within the selected populations was measured by expression of gfp coexpressed with aid . Data were obtained and presented as in table i. p = 0.031 (fisher's exact test). If so, the s hypermutation may be biased to stem and loop (s / l) structures as shown for csr cleavage sites (12, 18, 25). Therefore, we mapped the hypermutation sites to the predicted s / l structures on both strands of the s region, and found significant bias of the mutation to the s / l structures (table iii, fig . 1 b). Note that the majority of mutations coupled with csr are located in the proximity (within 10 bp) of the csr junctions but do not necessarily coincide with cleavage sites (18). This is because mutations are probably introduced during error prone dna synthesis to repair cleavages . Accordingly, the present level of the coincidence between s mutations and s / l structures well supports the assumption that both csr and s mutations may be mediated by an enzyme recognizing s / l structures . Mutation bias to rgyw / wrcy and s / l structure mutations in the 3 subregion were analyzed for association with sequence and structural motifs . Mutations which associate or do not associate with the rgyw / wrcy motif were counted as (+) and (), respectively . S / l structure was predicted by a computer program that is developed by zuker as described and shown in fig . 1 b. the two strands were analyzed separately for association with the s / l structure and the results are combined . S region hypermutation shares important features with v region shm: aid dependency, high frequency point mutations with occasional deletions, and mutations biased to transition (64%) and to the rgyw / wrcy motif (r, purine; y, pyrimidine; w, a / t; table iii) (2). It is therefore reasonable to assume that either a similar mechanism activated by aid or aid itself is responsible for hypermutations in the s as well as v region . It has been shown that the defect in the non - homologous end - joining (nhej) system affects csr but not shm (2629), and that aid mice show no obvious abnormality in the nhej system (10), indicating that csr and shm differ in the repair mechanism . These results taken together suggest that a common step of csr and shm, most likely at dna cleavage rather than dna repair, may be regulated by aid . It is well established that csr and shm are independent events in b cells (8, 9). In fact, under the present conditions both csr and hypermutation in s regions occur concurrently without shm in the v region . There must be another level of regulation to distinguish the targets of the aid - activated system . As aid deficiency does not affect germline transcription and nhej repair, aid is likely to be involved in cleavage of the s region during csr (10). This conclusion is confirmed by the recent finding that accumulation of h2ax and nbs1 at double strand breakages in the igh locus during csr is dependent on aid (30). The present finding suggests that csr and shm are likely to be mediated by the same enzyme . It is therefore less likely that aid edits separate pre - mrnas for csr and shm . These results taken together argue for, but do not prove, the hypothesis that aid edits a precursor mrna to synthesize an endonuclease essential for generating dna cleavage in both shm and csr reactions (12). Alternatively, aid itself may have dna attaching activity although we consider it less likely because (a) aid does not bind to double - stranded or single - stranded s sequences (data not shown) and (b) aid is not associated with the s core region by chromatin immunoprecipitation assay of csr - induced b cell chromatin (data not shown). We have further proposed that the endonuclease introduces nicks by recognizing secondary structures such as stem and s / l in s and v regions, which are formed transiently by transcription - promoted strand separation (12). Nicking in the v region will be repaired by exonuclease and error - prone dna synthesis (3133), followed by mismatch repair and ligation . By contrast, in s regions frequent nicking generates staggered cleavages (18) which are also repaired by exonuclease and error - prone dna synthesis, followed by the nhej repair system (2628).
In this issue of critical care, kao and colleagues consider whether open lung biopsy (olbx) can assist in the management of patients with acute respiratory distress syndrome (ards). Clinical outcome in ards remains poor despite substantial advances in our understanding of the biology of this syndrome . Although limiting transpulmonary pressure can clearly prevent worsening of ards, no other major therapeutic advances with proven benefit have occurred in this area . Progress has been limited potentially due to the heterogeneous phenotypes that are known to underlie the american european consensus definition of this disease . Thus, methods to improve diagnostic specificity are likely to be helpful in making progress . Olbx has been used for years as a method of defining the underlying pathology in patients with lung disease . While its role has become established in the setting of interstitial lung disease, its utility and safety are more controversial in critically ill patients . Proponents of olbx argue that knowledge of underlying etiology can be helpful in defining the best course of treatment . In addition, the risk of biopsy in experienced hands is fairly low if adequate precautions are taken . Opponents of olbx cite the lack of specific therapies for underlying etiologies of ards and believe that defining the underlying mechanism of injury is largely academic . A similar discussion has taken place in the interstitial lung disease arena, where some advocate the demonstration of usual interstitial pneumonitis among patients with idiopathic pulmonary fibrosis, whereas others believe that a therapeutic trial of steroids in the majority of patients is justifiable until new therapeutic strategies emerge . The work by kao and colleagues supports the existing literature that open lung biopsy is fairly safe and frequently revealing in the context of ards . First, the authors corroborate prior reports that the underlying pathology in clinical ards is often a pattern other than diffuse alveolar damage or fibro - proliferation . Of note, this and prior studies were retrospective analyses making the generalizability of these findings difficult to define . Without knowing the total number of ards cases potentially eligible for biopsy, we have no easy way to know how common the observed abnormalities would be in an unselected ards population . Second, the authors found minimal morbidity attributable to the surgical procedures that their patients underwent . These data support the existing literature that, in experienced hands, olbx can be safely performed in carefully chosen patients . The risk of bronchopleural fistula was fairly low in the present study, which may reflect the use of protective mechanical ventilation . We have recently observed that high pressures measured at the airway opening are strongly predictive of prolonged bronchopleural fistula risk following lung biopsy in ards . Thus, attention to mechanical ventilator settings may be one factor that led to the low risk of this procedure . Nearly 75% of patients had changes made in their therapeutic management due to findings from olbx . Whether these changes were helpful to the patient is not entirely clear due to the lack of a control group however, at least 14 patients (11 with infections, 1 with hypersensitivity pneumonitis, and 2 with pulmonary edema) had a disorder found for which accepted therapies exist . Interestingly, the most common change in management recorded in response to olbx results was the institution of glucocorticoid therapy . The role of glucocorticoid therapy in ards has been controversial, with some smaller studies showing benefits whereas other larger studies demonstrated no important benefit . A number of critiques have emerged after the recently published new england journal of medicine trial examining the role of steroids in persistent ards, leading some to speculate that, despite the negative results of that trial, some ards patients may still benefit from anti - inflammatory therapy . In this recent study, more than 95% of patients were excluded prior to enrollment, leading to results that may not be generalizable to the overall ards population . The most common reason for exclusion was glucocorticoid therapy, yielding the possibility that the best candidates for steroid therapy (from both an efficacy and safety perspective) were excluded from the study . In addition, the frequent use of paralytics (in up to 50% of steroid treated participants) and marked hyperglycemia (mean values in excess of 200 mg / dl) may have contributed to avoidable complications of steroid therapy . Thus, the frequent re - intubations and neuromyopathies that occurred in this recent study may have offset the potential benefits of steroid therapy . Regardless, the stratification of patients likely to benefit from steroid therapy, while avoiding the potential morbidity of pharmacological therapies and other intensive care unit measures (including mechanical ventilation) is likely to be a successful strategy . Future studies that aggressively limit the side effects of steroids and that examine treatment response stratified by olbx findings may demonstrate subgroups of patients that derive important benefit from this therapy . In the future, biomarkers that could be defined either in the serum or by bronchoalveolar lavage would be preferable to olbx to stratify the likelihood of benefit from steroid therapy . Such biomarkers may help define the underlying pathobiology and so become a surrogate for olbx in assessing the steroid responsiveness of the disease . Another class of biomarkers that may prove useful in the management of ards patients would be ones that provided information on the intrinsic steroid responsiveness of the patient . The search for genetic polymorphisms that predict individual responsiveness to steroid therapies is well underway in other conditions such as asthma and ulcerative colitis . Both types of biomarkers would aid treatment decisions by better defining subgroups most likely to benefit from steroid therapy . Thus, further work is clearly needed to determine whether individualized therapy will improve outcome in various subgroups of ards patients.
Tuberculosis of oral cavity is a rare entity . Even before antitubercular chemotherapy era, oral lesions occurred less commonly in patients with pulmonary tuberculosis . A 55-year - old labourer was referred to the outpatient department of our centre as a case of suspected gingivobu - ccal carcinoma for further evaluation and management . He had a non - healing ulcer in the oral cavity for one and a half months, which was associated with pain . He had been well until six weeks earlier when he noticed a lesion in his oral cavity . He visited the local doctor for this and was given symptomatic treatment and vitamin supplements . On enquiry, he had no systemic symptoms of chronic disease like fever, weight loss, and loss of appetite or weight . He also had no history of tuberculosis or contact with a tuberculosis patient . On clinical examination, the patient had an ulcer in the right lower gingivobuccal sulcus of size about 21 cm and was covered with slough . There was also mild fullness and soft tissue induration in the cheek below the lip . The patient's height was 167 cm; weight, 62 kg; blood pressure, 124/76 mm hg; and pulse, 74 beats per minute . On further investigation, haemoglobin was 13.9 gm%, total white blood cell count was 6,400 cells / dl, and erythrocyte sedimentation rate was 13 mm at the end of one hour . These are usually accompanied by associated history like a stressor event or the presence of a sharp tooth, etc, pointing to diagnosis . . The most common cause of a non - healing ulcerative lesion in the oral cavity is malignancy . The history is usually that of a rapidly growing lesion that does not respond well to any form of management . In most cases it is also usually accompanied by weight loss and features of chronic disease . In a developing country every year, approximately 2.2 million people develop tuberculosis, of which about one million are new - smear positive highly infectious cases and about 5 lakh people die of tuberculosis every year . This should be considered in the differential diagnosis, particularly in a non - healing lesion that does not respond to the usual therapy . Actinomycosis is a rare, chronic, spreading, suppurative, granulomatous and fibrosing infection characterised by the formation of multiple abscess, draining sinuses and the release of characteristic sulphur granules . Peak incidence occurs between 15 and 30 years, and males are more frequently infected than females . The majority of examples of the clinical disease are cervicofacial (55%), with only 20% occurring in an abdo - minopelvic form and 15% as a thoracopulmonic form . . Are anaerobic or aerotolerant (facultatively anaerobic), non - sporulating, gram - positive bacteria that tend to form branching rods and filaments and have a fermentative type of carbohydrate metabolism . Are not regarded as virulent human pathogens and are best considered as opportunistic pathogens, as they are normally present in healthy individuals as commensals of the mouth cavity and the upper respiratory tract . Their presence in the oral and respiratory specimens does not necessarily signify clinical disease and might often not be reported . Are polymicrobial . The copathogens are most commonly colonisers of the respective organ systems involved . They act synergistically by inhibiting host - defense mechanisms or reducing the oxygen tension in the affected tissue, which promotes the growth of actinomyces spp . Syphilis is an infectious disease caused by a spirochete called treponema pallidum (tp), which has a tropism for several organs and tissues in the body, causing complex clinical manifestations . Transmission of syphilis occurs mostly through sexual intercourse and sites of inoculations are usually genitals, though it can also be extragenital . It is characterised by several different dermatological lesions, involving both skin and mucous membrane . In the first stage of the disease (primary syphilis), oral chancres syphilitic chancre is a solitary, painless, indurated, hard in consistency, reddish ulcer, accompanied by regional lymphadenopathy, which is localised at the site of tp inoculation and usually resolves after approximately one month without scarring . In secondary syphilis, the oral manifestations are characteristic lesions, which are multiple mucous patches that are slightly raised and covered by grayish, white pseudomembranes and surrounded by erythema . Oral lesions are often painful and snail track ulcers result when multiple mucous patches become confluent . The diagnosis is confirmed by the specific serology and the demonstration of tp in the lesion . Tp has a slender, coiled morphology and when examined by dark - field microscopy, it was seen moving in a drifting rotary motion (corkscrew). Naat is better than dark - field microscopic examination in the case of oral lesions because of the possible presence of saprophytic tp in the mouth . Chest x - ray revealed an ill - defined patchy opacity and reticulo - nodular opacities in the upper and mid zone of both lungs [figure 2]. Sputum was positive for the presence of acid - fast bacilli 1 + in two consecutive samples . These are usually accompanied by associated history like a stressor event or the presence of a sharp tooth, etc, pointing to diagnosis . . The most common cause of a non - healing ulcerative lesion in the oral cavity is malignancy . The history is usually that of a rapidly growing lesion that does not respond well to any form of management . In most cases it is also usually accompanied by weight loss and features of chronic disease . In a developing country every year, approximately 2.2 million people develop tuberculosis, of which about one million are new - smear positive highly infectious cases and about 5 lakh people die of tuberculosis every year . This should be considered in the differential diagnosis, particularly in a non - healing lesion that does not respond to the usual therapy . Actinomycosis is a rare, chronic, spreading, suppurative, granulomatous and fibrosing infection characterised by the formation of multiple abscess, draining sinuses and the release of characteristic sulphur granules . Peak incidence occurs between 15 and 30 years, and males are more frequently infected than females . The majority of examples of the clinical disease are cervicofacial (55%), with only 20% occurring in an abdo - minopelvic form and 15% as a thoracopulmonic form . Are anaerobic or aerotolerant (facultatively anaerobic), non - sporulating, gram - positive bacteria that tend to form branching rods and filaments and have a fermentative type of carbohydrate metabolism . Are not regarded as virulent human pathogens and are best considered as opportunistic pathogens, as they are normally present in healthy individuals as commensals of the mouth cavity and the upper respiratory tract . Their presence in the oral and respiratory specimens does not necessarily signify clinical disease and might often not be reported . They act synergistically by inhibiting host - defense mechanisms or reducing the oxygen tension in the affected tissue, which promotes the growth of actinomyces spp . Syphilis is an infectious disease caused by a spirochete called treponema pallidum (tp), which has a tropism for several organs and tissues in the body, causing complex clinical manifestations . Transmission of syphilis occurs mostly through sexual intercourse and sites of inoculations are usually genitals, though it can also be extragenital . It is characterised by several different dermatological lesions, involving both skin and mucous membrane . In the first stage of the disease (primary syphilis), oral chancres syphilitic chancre is a solitary, painless, indurated, hard in consistency, reddish ulcer, accompanied by regional lymphadenopathy, which is localised at the site of tp inoculation and usually resolves after approximately one month without scarring . In secondary syphilis, the oral manifestations are characteristic lesions, which are multiple mucous patches that are slightly raised and covered by grayish, white pseudomembranes and surrounded by erythema . Oral lesions are often painful and snail track ulcers result when multiple mucous patches become confluent . The diagnosis is confirmed by the specific serology and the demonstration of tp in the lesion . Tp has a slender, coiled morphology and when examined by dark - field microscopy, it was seen moving in a drifting rotary motion (corkscrew). Naat is better than dark - field microscopic examination in the case of oral lesions because of the possible presence of saprophytic tp in the mouth . Chest x - ray revealed an ill - defined patchy opacity and reticulo - nodular opacities in the upper and mid zone of both lungs [figure 2]. Sputum was positive for the presence of acid - fast bacilli 1 + in two consecutive samples . Primary tuberculosis of the oral cavity is rare, as it occurs in young individuals and is associated with cervical lymphadenopathy. [258] secondary tuberculosis of the oral cavity usually occurs following pulmonary tuberculosis . It is more common than the primary variety and occurs more often in old age. [257] tuberculosis of the oral cavity is rare because the intact mucosa is resistant to tuberculous infection and saliva has a protective effect . Other possible causes for the relative rarity of oral tuberculosis include the presence of saprophytes in the oral cavity and resistance of stratified muscles to bacterial invasion . There are reports in which dentists with pulmonary tuberculosis have infected patients during dental manipulation . Nasolabial infection occurred in a physician who administered mouth - to - mouth resuscitation to a tuberculosis patient . Other sites include the gingiva, soft palate, lip, buccal mucosa, floor of the mouth, and gingivobuccal sulcus . Oral tuberculosis may present as single or multiple, painful or painless ulcers with irregular border . It can also present as nodules, fissures, vesicles, or peri - apical granulomas . The oral tuberculous ulcers have some characteristic clinical features including chronicity, hyperemic changes, and multiple or satellite lesions, which lead to the suspicion of a tuberculous lesion . Oral tuberculosis occurs in 0.4 - 1.5% of patients with pulmonary tuberculosis . At present, with improved community health and anti - tubercular medication, the incidence of tuberculosis has decreased . Oral tuberculosis is not considered in the differential diagnosis of patients with oral ulcer by many clinicians; hence, it is missed or the treatment is delayed . The purpose of this paper is to alert clinicians to consider oral tuberculosis in patients with non - healing oral ulcer . On consultation with the physician, anti - tubercular therapy was initiated with isoniazid (10 mg / kg of body weight), rifampicin (10 - 20 mg / kg of body weight), and pyrazinamide (10 - 20 mg / kg of body weight) and ethambutol for two months, followed by isoniazid and rifampicin for the following four months . During the period, the patient was instructed not to undergo any surgical procedure within the oral cavity and was warned of transmitting the disease to others . Tuberculous ulcer of the oral cavity secondary to pulmonary tuberculosis . From the department of head and neck oncology, tata memorial hospital, mumbai.
Malaria has been man's problem since the 14th century (the era of the black death in europe), and now in the 21st century, its effect has again reached an alarming proportion . Malaria remains a major threat to health and economic development of local communities and nations [2, 3]. Almost half of the world's population is at risk of the disease [46]. Africa south of the sahara records about 90% of the world's all malaria deaths for two main reasons . The first is that infections are caused by plasmodium falciparum (p. f.) (the most dangerous of the four human malaria parasites) and the second is because the mosquito anopheles gambiae (the most effective malaria vector and also the most difficult to control) is most widespread in africa . Malaria infection during pregnancy is a major public health problem in tropical and sub - tropical regions throughout the world . Children under 5 of years of age and pregnant women are recognised by the world health organization as the vulnerable groups to malaria infection . The disease causes about 300500 million clinical cases and 1.5 to 2.7 million deaths worldwide each year (who in) even though there is a downward trend in these figures . The prevalence of malaria in africa is high with an estimated average prevalence of p. f. of 63% in west africa and 39% in eastern and southern africa . Reports of outpatient visits due to malaria for some africa countries such as malawi, ur tanzania, uganda, and zambia vary from 18% in 1985 to 46% in 2000 while hospital admission due to malaria in these countries varies from 20% in 1985 to 60% in 2000 according to rbm . Child mortality due to malaria seems to have been higher in eastern / southern africa countries than in west african countries between 1990 and 1998 . Malaria accounts for 63% of the diseases reported in healthcare facilities across the six geopolitical zones of nigeria, while it accounts for 11% of maternal deaths and its prevalence among pregnant women was put at insecticide - treated nets (itns) are known to be highly effective in the reduction of morbidity and mortality in malaria rbm . In recent times, their usage in the african continent has been vigorously scaled up for coverage and in the introduction of long - lasting insecticide nets (llins). Itns have been scaled up in many african countries among which are tanzania, zambia, kenya, mali, and malawi (rbm). In these countries, itns are usually distributed freely to parents of children and pregnant women (the vulnerable groups) while, in some countries, nigeria inclusive, they are made available at retail shops and/or are supplied at subsidised prices or under the voucher scheme [12, 13]. Rbm programme is put in place to assist endemic countries to conduct national needs assessments from which strategic plans could be developed . It also intended to serve as a forum to match country's plans with international donors . In view of the complications of malaria in pregnancy, especially in this period of drug resistant malaria parasites, roll back malaria (rbm) programme was launched in 1998 with at least three new or refined tools to combat the disease . The tools are artemisinin - based combination therapies (acts), insecticide - treated nets (itns), and intermittent preventive treatment (ipt) for pregnant women . Besides, other rbm measures used for malaria control are irs, larval control, quick diagnosis, administration of artemisinin - based combination therapies (acts) (freely given in some countries for all age groups), and adoption of chloroquine, mefloquine, and sulphadoxine - pyrimethamine (sp) (fansidar) and primaquine . In spite of the efforts galvanised to combat malaria in africa, there is still a high burden of malaria of 20%40% with an average of 30% of all outpatient visits to the clinics in all african countries where malaria is endemic (rbm). In many of these countries, the report indicated that between 20% and 50% of all hospital admissions are due to malaria . Reports (rbm) indicate that less than 40% of malaria morbidity and mortality occur in formal health facilities which constitute a small fraction of the total burden but many of the reports do not include nongovernmental facilities like the faith clinics . By 2005, it was expected that 60% of pregnant women in nigeria would sleep under an itn and 60% of pregnant women would receive ipt using sulphadoxine - pyrimethamine (sp) at least twice during anc . These targets were to gradually rise to a value between 75 and 80% by 2010 and coverage was sustained thereafter . A national malaria situation survey conducted in 2000 reported by indicated that preventive health behaviour in malaria in terms of net use among pregnant women in nigeria seems to be generally low in all the six geopolitical zones . Also, reported a generally poor usage of nets among all categories of people in nigeria with only 20% of households owning nets, some of which are not necessarily itns, whereas observed a higher coverage of the usage of nets in urban areas . The need to further confirm current state of usage of rbm tools among this vulnerable group and the influence of location in this regard was crucial . In view of this, the present study had the impetus to investigate the acceptability and utilisation of rbm tools, their influencing factors, and the influence of location on the behaviour outcomes among pregnant women who access government and faith clinics in oyo state, nigeria, which seem to be usually sidelined by most health programmes initiated even when a sizeable number of pregnant women patronise them . What is the level of acceptance of intermittent preventive treatment (ipt) of malaria in pregnancy and itns by pregnant women who attend different health facilities in oyo state?is there a difference in acceptance of ipt and itns by pregnant women in terms of the health facilities accessed and location?to what extent are ipt and itns utilised by pregnant women who access different health facilities in oyo state?is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location?is there a relationship between acceptance and utilisation of ipt and itns by these pregnant women?what are the factors influencing (i) acceptance and (ii) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state? What is the level of acceptance of intermittent preventive treatment (ipt) of malaria in pregnancy and itns by pregnant women who attend different health facilities in oyo state? Is there a difference in acceptance of ipt and itns by pregnant women in terms of the health facilities accessed and location? To what extent are ipt and itns utilised by pregnant women who access different health facilities in oyo state? Is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location? Is there a relationship between acceptance and utilisation of ipt and itns by these pregnant women? What are the factors influencing (i) acceptance and (ii) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state? The study adopted the survey and causal comparative types of research as none of the variables were manipulated; they were studied as they occurred and inferences made on the basis of the findings obtained . The levels of acceptance and utilisation of sulphadoxine - pyrimethamine (fansidar), the drug of choice for intermittent preventive treatment (ipt) in pregnancy, and insecticide - treated nets (itns) between government clinics and faith clinics or mission homes were compared . Oyo state is located in the southwest geopolitical zone of nigeria and is one of the malaria endemic areas of the country . The disease afflicts children under five and pregnant women two of the most vulnerable groups resulting in high morbidity and mortality in the state . For instance, data from puts the trend of malaria prevalence in the state from a total of 358,780 cases in 2006 to 403,468 in 2009 . The study population consisted of all pregnant women in oyo state, nigeria, who were attending antenatal (anc) clinics at both the primary healthcare centres (phc) and faith clinics, respectively, in 2009 - 2010 when the study was conducted . The 33 local government areas in the state were stratified into urban and rural composition and purposive sampling technique was employed to select three local government areas, respectively, from both urban and rural locations to take care of the location of the faith clinics / mission homes which were dominant in urban locations . All the phc clinics and mission homes (faith clinics) in the selected local government areas were used for the study, while purposive sampling technique was used to select a total of 650 pregnant women but 582 who accessed government clinics and 50 who accessed faith clinics in these lgas who had complete information on them formed the final sample . A structured questionnaire that was valid and reliable pregnant women acceptance and utilisation questionnaire (pwauq, = 0.81)used for data collection was administered once to the participants . Pwauq consisted of background information about the pregnant women and their spouses' occupations and educational levels, locations, and number of pregnancies and their outcomes among others . The instruments on acceptance of rbm tools, utilisation of itns, and factors influencing acceptance and utilisation were closed - ended questions administered once to the participants . Data collection was carried out by the researchers with the assistance of fourteen trained assistants (six rbm programme managers in the six lgas and eight others trained for the purpose of data collection). The researchers explained the purpose of the research work to all pregnant women that were present including the health workers at each phc or faith clinic or mission home visited . Pregnant women in rural locations who had low educational qualification filled their questionnaire through the assistants who interpreted the contents to them in the local language yoruba without influencing their responses . The data obtained were coded and analysed using the statistical package for the social sciences (spss) version 20 . Positive responses were coded with one (1) while negative responses were coded with zero (0). Percentages were computed for the responses relating to levels of acceptance and utilisation of rbm tools . Inferential statistics such as t - test and analysis of variance (anova) were computed to determine significant levels of acceptance and utilisation of rbm tools between pregnant women who accessed government and faith - based health facilities and those in urban and rural locations . Pearson product moment correction was used to analyse the relationship between acceptance and utilisation of rbm tools . Multiple regressions were computed to establish the factors that influenced the acceptance and utilisation of the rbm tools . What is the level of acceptance of intermittent preventive treatment (ipt) of malaria in pregnancy and itns by pregnant women attending different health facilities in oyo state? What is the level of acceptance of intermittent preventive treatment (ipt) of malaria in pregnancy and itns by pregnant women attending different health facilities in oyo state? Pregnant women who accessed government clinics (49.8%) tended to accept itns even though 72% of them claimed to like sleeping under a mosquito net, while 52.2% claimed they a total of 28% and 30% of pregnant women who accessed faith clinics reported that they accepted these rbm tools, respectively, though 72% of them indicated they like sleeping under a mosquito net and 64% react to anti - malarial drugs (table 1). (2)is there a difference in the acceptance of ipt and itns by pregnant women in terms of the health facility accessed and location? Is there a difference in the acceptance of ipt and itns by pregnant women in terms of the health facility accessed and location? There was a significant difference in acceptance of ipt and itns by pregnant women in the two health facilities in table 2, t(630) = 3.51, p 0.05, and t(630) = 4.15, p 0.05, respectively . Pregnant women in government clinics with m = 4.74, sd = 1.08 and m = 4.66, sd = 1.15 seem to accept ipt and itns compared to their counterparts in faith clinics, mean = 4.16, sd = 1.59; m = 3.96, sd = 0.93 . The effect sizes (cohen's d) of 0.14 or 14% and 0.16 or 16%, respectively, were obtained for the variance in acceptance in the two health facilities . Significant influence of location was observed on the extent to which pregnant women in government health facility only accepted ipt (table 3). Pregnant women in rural locations with mean 4.92, sd = 1.00 were better in the acceptance of ipt than those in the urban areas, mean 4.55, sd = 1.13, t(580) = 4.12, p 0.05, with an effect size (cohen's d) of 0.17 or 17% . The influence of location on acceptance of itns was not significant, t(580) = 0.58, p 0.05 . Faith clinics were not found in rural locations. (3) to what extent are ipt and itns utilised by pregnant women attending different health facilities in oyo state? To what extent are ipt and itns utilised by pregnant women attending different health facilities in oyo state? Pregnant women who accessed government clinics (60.8%) utilised itns in spite of the claim that 62.9% reported sleeping under an itn before their current pregnancy and 73.2% claimed it is comfortable sleeping under an itn . Among pregnant women who accessed faith clinics, 18% utilise itns even though 48% claimed they find sleeping under an itn comfortable . More women (66.8%) in government clinics than their counterparts in faith clinics (38.0%) used ipt to the expected two doses . As many as 79% of the pregnant women in government clinics and 62% in faith clinics indicated their willingness to use ipt in their subsequent pregnancies (table 4). (4) is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location? Is the utilisation of ipt and itns by pregnant women a function of the health facility accessed and location? A significant difference in the utilisation of ipt and itns as presented in table 5 was observed between these women, t(630) = 5.81, p 0.05, and t(630) = 3.99, p 0.05 . Pregnant women in government clinics utilised ipt and itns more than those who accessed faith clinics . The effect sizes (cohen's d) of 0.23 or 23% and 0.16 or 16%, respectively, were obtained for the variance in the utilisation of ipt and itns, respectively . A significant difference in the utilisation of ipt by the pregnant women who accessed government clinics by location, t(580) = 641, p 0.05, was observed with an effect size of 0.26 or 26% but the itns utilisation was not significant, t(580) = 1.170, p> 0.05, with an effect size of 0.05 or 4.86% (table 6). Faith clinics were not found in rural locations. (5) is there a relationship between acceptance and utilisation of rbm tools by these pregnant women? Is there a relationship between acceptance and utilisation of rbm tools by these pregnant women? As presented in table 7, the utilisation of rbm tools which was found to be significantly related with the acceptance of rbm tools among pregnant women in the state government clinics (r = 0.398, n = 582, p 0.05; r = 0.16) and pregnant women in faith clinics (r = 0.379, p 0.05; r = 0.14). This explained 16% and 14% of the variation between acceptance and utilisation among these women, respectively. (6) what are the factors influencing (i) acceptance and (ii) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state? What are the factors influencing (i) acceptance and (ii) utilisation of ipt and itns among pregnant women who access these health facilities in oyo state? The multiple regression model summary of the nine influencing variables (table 8) significantly explained and predicted the acceptance of rbm tools among pregnant women in government clinics (f(9,572) = 6.320, p 0.05) but not in faith clinics . The variables accounted for 8% of variance in the pregnant women's acceptance of rbm tools whereas other variables not investigated accounted for the remaining 92% . With respect to the t - test results in relation to the significant multiple regression coefficient obtained, the nine variables significantly predicted the acceptance of rbm tools through number of pregnancies (t = 5.172, = 0.217). Four other variables, educational qualification of the pregnant woman (t = 2.871, = 0.120), marital status (t = 2.928, = 0.117), age of pregnant woman (t = 2.805; = 0.115), and husband's occupation (t = 0.2212, = 0.093), also contributed significantly but inversely to the acceptance of rbm tools (table 8). The multiple regression of the nine influencing independent variables of the utilisation significantly explained and predicted the utilisation of rbm tools among pregnant women in government clinics (f(9,572) = 3.607, p 0.05) but not in faith clinics (table 9). The nine variables accounted for 3.9% variance in the utilisation of rbm tool whereas other variables not investigated accounted for the remaining 96.1% . Concerning the t - test results in relation to the significant effect of the multiple regression coefficient obtained, the nine variables significantly predicted the utilisation of rbm tools by pregnant women in government clinics through the number of pregnancies had (t = 2.818, = 0.121). Marital status and educational qualification of the pregnant women also contributed significantly but inversely to the utilisation of rbm tools among these women (t = 3.248, = 0.132 and t = 2.496, = 0.106, resp . ). The findings with respect to the acceptance of rbm tools (ipt and itns) by pregnant women indicated that pregnant women who accessed government clinics accepted these rbm tools better than those who accessed faith clinics . Between 50% and 72% of the pregnant women who accessed government clinics accepted ipt and itns whereas between 30% and 72% of pregnant women who accessed faith clinics accepted these rbm tools . The overall acceptance by these women (even when pregnant women who accessed faith clinics claimed they engage in dual registration) could be as a result of adequate enlightenment on the knowledge of rbm programme provided to the pregnant women in government clinics through health education at anc visits, which is a regular practice of primary healthcare in the clinics . The targets of over 82% in the use of rbm tools by pregnant women were set by the african heads of state to be achieved by year 2010 . The observed trend in this study the level of education of an individual generally dichotomises one into an ignorant or informed person . According to, health education helps people understand health and how their behaviour may affect their health . Since such education encourages people to make their own choices for a healthy life, it will ensure the success of the prevention, treatment, and control of malaria programme put in place in order to reduce to the barest minimum maternal and child morbidity and mortality, in the state . Other findings in this study indicate that health facility accessed by the pregnant women significantly influenced acceptance: government clinics: t(630) = 3.51, p 0.05; faith clinics: t(630) = 4.15, p 0.05 . Acceptance was also location based as women in the rural locations significantly accepted ipt in pregnancy compared to those in urban locations (t = 4.12, p 0.05) but location did not significantly influence the acceptance of itns for this group of women . This implied that rbm programme in oyo state is a healthcare focused programme geared towards the rural people through the primary healthcare . Thus, the rural dwellers see rbm programme as their own programme which they embrace wholeheartedly more than the urban dwellers . Personal experiences of the researchers show that dwellers in the urban areas take malaria for granted and probably see it as a common disease occurrence . Findings indicate a modest level of the utilisation of ipt and itns by the pregnant women . More of the pregnant women in government clinics (between 58% and 73%) utilised ipt and 67.2% used itns whereas between 18% and 60% and 38.0% of those who accessed faith clinics used these tools, respectively . The observed difference could be attributed to the religious beliefs and practices of the women in faith clinics who do not believe in the use of drugs even though they engage in dual registration . The study of finds support in respect of this finding . Although researchers like revealed that factors such as availability of nets, cost of nets, and inadequate information on where to obtain the nets and the attitude of users towards the use of net had great influence on the poor and improper utilisation of nets (itns), the findings of this present study seem to have indicated a considerable improvement in the utilisation of itns among the pregnant women . Findings differed markedly from the findings of that reported less than 10% usage of nets among pregnant women and children in uganda but are in consonance with the study which reported 58% usage of itns among women attending anc and delivery units in burkina faso and that reported a variation between 32% and 69% usage of itns among pregnant women in six african countries, nigeria inclusive . Findings equally find support in the study of which found that itns use in niger improved after a nationwide integrated campaign . The influence of location on the utilisation of rbm tools by the pregnant women in this study was important as pregnant women in rural locations who accessed government clinics utilised ipt compared to those in urban areas, significant at p 0.05 . In this regard, findings in nigeria [22, 23] which corroborated are in support of the fact that the use of nets was very variable among urban and rural communities in oyo and ekiti states, respectively . A national malaria situation survey conducted in 2000 reported by also indicated that net use among pregnant women in nigeria seems to be generally low in all the six geopolitical zones . It also indicated a generally poor usage of nets among all categories of people in nigeria with only 20% of households owning nets, some of which are not necessarily itns, whereas observed a higher coverage of the usage of nets in urban areas . There is therefore need to further reach pregnant women in urban locations to improve their attitude to ipt and itns utilisation as well as those who access faith clinics . In addition, a positive but low correlation seems to exist between acceptance and utilisation of ipt and itns among pregnant women who accessed government clinics (r = 0.398) accounting for 16% variance in utilisation compared to those who accessed faith clinics (r = 0.379) accounting for 14% variance in utilisation . This implied that, all things being equal, high acceptance implies reliable utilisation of ipt and other rbm programme packages . The acceptability of and receptiveness to rbm tools by some of the pregnant women could have facilitated their utilisation of the tools with ease . The implication of this finding is that any effective public enlightenment and sensitisation carried out on rbm programme and tools will make the right impact on reducing the menace of malaria on pregnant women irrespective of the health facility accessed . From the findings in this study, pregnant women in the state have a high acceptance of rbm tools . It appears, however, that the acceptance of these tools by pregnant women who accessed government clinics depended on factors such as number of pregnancies had (= 0.217) which significantly and positively influenced these women's acceptance of these tools, but educational qualification of the pregnant woman (= 0.120), marital status (= 0.117), age of the pregnant women (= 0.115), and husband's occupation (= 0.093), though significant, inversely influenced acceptance . What these findings imply is that as the age increased or educational qualification and marital status or husbands' occupations of these women improved, their acceptance of rbm tools became low . On the issue of the utilisation of the tools by pregnant women who accessed government clinics, the findings revealed that utilisation was influenced by such factors as the number of pregnancies had (= 0.121) which contributed significantly and positively to utilisation and marital status (= 0.132) and educational qualification of the pregnant women (= 0.106) which contributed significantly and inversely to the utilisation of rbm tools among these women . These variables did not influence both the acceptance and utilisation of pregnant women in faith clinics . Three variables . Number of pregnancies, educational qualification of the pregnant women, and marital status which were observed to significantly and consistently influence the acceptance and utilisation of rbm tools in this study seem to be contiguously related to one another and portend strong indications that the observed finding about these women has grave implications for their purchasing powers . Precisely, decisions about spending money are predominantly and traditionally made by men in many african cultures . Also, a large number of the pregnant women who participated in the study were housewives who were with no adequate means of livelihood of their own and who had to depend on their husbands' incomes to be able to purchase the nets and the drugs . Some of the women may, therefore, not be able to make independent financial decisions since they do not have much income of their own and as such, their acceptance and utilisation of these tools could have been influenced not only by the number of pregnancies had, but also by marital status and husbands' occupations . Thus, the spouse and the nature of occupation are central to the women's responses to these tools . Findings also corroborate pulford, herzel, bryant, siba, and mueller in who reported that lack of affordability is an important barrier to ownership of itns . With this kind of findings, the attainment of the two main objectives of rbm programme which are reducing the burden of malaria particularly in the two most vulnerable groups (pregnant women and children under 5 years of age) and contributing to the positive health and socio - economic development of the nation is feasible and possible, all things being equal . Among the pregnant women who accessed faith clinics, the findings indicated that all the nine variables did not predict their acceptance and utilisation of the rbm tools primarily because these women are not necessarily inclined to accept or utilise these tools due to their religious beliefs and practices . The study established the current status of the acceptance and utilisation of rbm programme tools (itns and sp in pregnancy) among pregnant women who accessed two models of healthcare facilities in oyo state from 2001 to 2009, an initiative implemented with the purpose of stemming of malaria - related morbidity and mortality among these pregnant women and their unborn children . Though the acceptance and utilisation of rbm tools were higher among pregnant women who accessed government phc clinics and in rural areas than among pregnant women who accessed faith clinics and in urban areas, these were far from meeting the set targets . Pregnant women's marital status and husbands' occupations significantly and statistically influenced acceptance while husbands' occupations influenced the utilisation of rbm tools implying that spouses' statuses play significant roles in achieving set targets . We recommend that intensive effort should be made by healthcare providers to encourage especially more of the educated urban women to utilise rbm programme tools, especially the itns . Rbm programme should not focus only on the utilisation of rbm tools but the scope should be expanded to include information dissemination on behavioural change towards malaria and its consequences . Malaria consumables such as insecticide - treated nets (itns) and medications such as sulphadoxine - pyrimethamine should be made available at the various health centres and given freely . They should also be available at the open chemist stores at cheap and affordable prices to the average person.
Recent clinical and experimental studies have indicated that the long - term effects of severe inflammatory events often include the suppression of immune system functions . The decrease of th1/th2 ratio is one of the major characteristics of immunosuppression in sepsis . It is reported that membrane adhesive protein annexin - a1 (anxa1) and transcription factor gata-3, which were both decreased in sepsis patients [2, 3], play important roles in the th1/th2 shift . Many researchers have studied anxa1 and gata-3, respectively, but the relationship between anxa1 and gata-3 is still unknown . Exploring the interactions between anxa1 and gata-3 may provide clues to understand the immunosuppression and improve the treatment effects of sepsis patients . As an anti - inflammatory protein, anxa1 plays a homeostatic role in the innate immune system through mediating immune cells, such as neutrophils and macrophages . Endogenous anxa1 markedly reduced leukocyte adhesion to postcapillary venules through formyl peptide receptor (fpr) pathway . Furthermore, anxa1 promotes inflammatory cell apoptosis associated with transient rise of intracellular calcium and caspase-3 activation . Moreover, anxa1 has been recently identified as one of the eat me signals on apoptotic cells to be recognized and ingested by phagocytes . Studies on the expression of anxa1 in human and mouse leukocytes have shown that this protein is also expressed at higher levels in cells of the adaptive immune response, such as t and b lymphocytes [810]. Further research indicates that anxa1 increases t cells activation and favors their differentiation to th1 cells by modulating t cell receptor (tcr) signaling . In addition, analysis of the inflammatory response of anxa1 mice has demonstrated an exquisite role of anxa1 in modulation of tcr signaling by the fpr family . These findings suggest a potential role of anxa1/fprl-1 pathway in the adaptive immune response . Upon antigen stimulation of their tcr by antigen presenting cells, nave cd4 t cells can differentiate to at least two different types of t helpers, th1 and th2 cells, which were documented to be involved in adaptive immunity . The transcription factor gata-3 is selectively upregulated during th2 differentiation in vitro [13, 14]. Gata-3 is important not only for the transactivation of th2 cytokine genes but also for the suppression of th1 development . . In vivo experiment from anxa1 mice exhibited that anxa1-deficient t cells expressed th2 skewing . However, there is not enough evidence to support the interrelation between gata-3 and anxa1 . In our previous studies, the expressions of anxa1 and gata-3 were both decreased in the burnt mouse with sepsis . The purpose of this study is to investigate whether anxa1 and gata-3 interact with each other to influence the immune response in t lymphocyte, as well as exploring the possible molecular mechanisms involved . Our results show that overexpressed anxa1 (or gata-3) represses the expression of gata-3 (or anxa1), while knockdown of anxa1 (or gata-3) increases the gata-3 (or anxa1) expression . Further studies indicate that anxa1 regulates gata-3 expression through anxa1/fprl-1/erk and pkb / akt signaling pathways, and gata-3 mediates anxa1 transcription activity by binding to anxa1 promoter . Thus this study, together with our previous observations of anxa1, suggests that the anxa1/fprl-1 axis and gata-3 are potential therapeutic targets of the th1/th2-mediated immunological suppression in sepsis . Anti - mouse cd3e (clone 145 - 2c11) and anti - mouse cd28 (clone 37.51) were purchased from bd bioscience (san jose, ca). Murine il-2, il-4, ifn, il-12, anti - il-4 (clone 11b11), and anti - ifn (clone xmg1.2) were purchased from ebioscience (wembley, united kingdom). Anti - mouse gata-3, total and phosphorylated erk1/2, and akt were purchased from cell signaling (danvers, ma). Anti - t - box transcription factor (t - bet / tbx21) was purchased from abcam (cambridge, uk), anti - fprl-1 purchased from novus (colorado, usa), and anti - anxa1 purchased from proteintech (manchester, uk). Unless otherwise specified, all the other reagents were from sigma - aldrich (st . Louis, mo). Km male mice with weight of 3550 g were obtained from department of experimental animals, central south university (changsha, china). Animal work has been performed according to the approval of the ethics of xiangya hospital, central south university . Mice were anesthetized with sodium pentobarbital [4050 mg / kg intraperitoneal (i.p . )]. Their spleen was teased apart to make a single - cell suspension and stained with apc - conjugated anti - cd4 . The isolated naive cd4 t cells (> 95%, 1 10/6 wells) were cultured in rpmi 1640 (gibco, grand island, ny) complete medium . The culture conditions of t cells are as follows: th0: anti - cd3/cd28 (5 g / ml) and il-2 (50 u / ml); th1: il-12 (2 ng / ml), il-2 (50 u / ml), and anti- il-4 (20 g / ml) and th2: il-4 (1000 u / ml), il-2 (50 u / ml), and anti - ifn (10 g / ml). The sequences for anxa1 and gata-3 overexpression were artificially synthesized and cloned into the plv (exp) puro - ires - egfp vector (cyagen, guangzhou, china). Interference lentiviral (plent ix1 puro - egfp vector) against anxa1 and gata-3 was constructed . The above constructed lentivirus was stably transfected into the t cells with an optimal multiplicity of infection (moi) of 100 tu / ml after stimulated with anti - cd3/cd28 (5 g / ml) for 24 h. upregulated (downregulated) expressions of anxa1 and gata-3 were confirmed by qrt - pcr and western blot (see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/1701059). The shrna sequences with the maximum of more than 75% knockdown efficiency were selected for further stable anxa1 and gata-3 silencing in t cells . The most efficient anxa1 shrna sequence of three was constructed as follows: (shanxa1 - 3) 5-gagatctggccaaagacataa-3; (anti - shanxa1 - 3) 5-ttatgtctttggccagatct-3. The most efficient gata-3 shrna sequence of three was constructed as follows: (shgata-3 - 2) 5-gctcagtatccgctgacggaa-3; (anti - shgata-3 - 2) 5-ttccgtcagcggatactgagc-3. Th0 cells (1 10/ml), obtained after infection with upanxa1 and culturing in th0 conditions with complete rpmi medium for seven days, were stimulated with phorbol 12-myristate 13-acetate(pma) 10 ng / ml and ionomycin 1 g / ml for 4 h in 6-well plates to produce cytokine . The ifn and il-4 levels were measured by elisa kits purchased from multi - sciences (china). Qrt - pcr was performed to determine the expression levels of gata-3, t - bet, and anxa1 . Total rna extraction was performed using simply p total rna extraction kit (bioflux, europe). The cdna synthesis was performed using all - in - one first - strand cdna synthesis kit (genecopoeia, maryland, usa). Cdna was used to set up a quantitative real - time pcr (qpcr) reaction using the all - in - one qpcr mix (genecopoeia). The expression levels of gata-3, anxa1, and t - bet were normalized to actin . The primer sequences were as follows: mouse gata-3 forward primer: 5-gctggatggcggcaaag-3, mouse gata-3 reverse primer: 5-gtgggcgggaaggtgaa-3, mouse anxa1 forward primer: 5-aaggtggtcctgggtcagc-3, mouse anxa1 reverse primer: 5-tgagcattggtcctcttggt-3, mouse tbx21/t - bet forward primer: 5-atgttcccattcctgtccttca-3, mouse tbx21/t - bet reverse primer: 5-aaatgaaacttcctggcgcatc-3, mouse actin forward primer: 5-catcctgcgtctggacctgg-3, and mouse actin reverse primer: 5-taatgtcacgcacgatttcc-3. All protocols were carried out according to the manufacturer's instructions . 30 g protein was separated by sds - page, transferred onto pvdf membrane, and hybridized with a primary antibody followed by a horseradish - conjugated secondary antibody . The cells permeabilized with 0.1% triton x-100 were incubated with 2% bovine serum albumin for 30 min and followed by the primary antibodies against gata-3 and anxa1 at 4c overnight . The slides of cells were subsequently incubated with the corresponding alexa fluor 488-conjugated secondary antibodies for 1 h at room temperature . The gata-3 penter (nm-001002295) and control penter plasmids were obtained from vigene biosciences (rockville, md, usa). The pgl3 enhancer vector and prl - tk vector were obtained from promega (madison, wi, usa). Anxa1 (nm00200.2) promoter region 2000 bp base - pairs and firefly luciferase reporter gene were built in pgl-3 enhancer vector and the renilla luciferase reporter gene was built in prl - tk vector . The renilla luciferase plasmid was used in all the experiment to normalize the efficiency of the transfection . Briefly, on the day of transfection, 5 10 cells were plated in 100 l in each well of a 96-well pate prior to incubation with vigenefection (vigene, rockville, md) complexes according to the manufacturer's instruction using a total of 0.3 g dna and 0.9 l vigenefection . At 48 to 72 hours after transfection, cells were lysed in 20 l reporter lysis buffer (promega, madison, wi) for 15 minutes on ice . Add the 100 l luciferase assay reagent ii (promega) and record the firefly luciferase activity measurement . And then add the 100 l stop & glo reagent (promega) and read the renilla luciferase activity measurement . At least three independent experiments were performed and the data were presented as mean sem . All data were presented as mean sem and analyzed by student's t - test . To explore the expression of anxa1 and/or gata-3 of the th0, th1, and th2 cells, immunofluorescence was carried out to confirm that anxa1 and gata-3 expression were located in cell nucleus and cytoplasm (figure 1). To determine the roles of overexpressed endogenous anxa1 in the balance between gata-3 and t - bet expression, two major transcriptional switches in th1/th2 differentiation, we examined the differentiation of t cells, which is driven by the strength of overexpressed anxa1 in th0 conditions . Naive t cells from mouse were infected with upanxa1 viruses and cultured in th0 medium for seven days, followed by being exposed to pma (10 ng / ml) and ionomycin (1 g / ml) for 4 h to stimulate the production of cytokines in th1/th2 . Upanxa1 significantly increased the expression of ifn and reduced il-4 expression in t cells, compared with those infected with up - control (p <0.01, figure 2). Anxa1-silenced t cells exhibited decreased production of ifn and increased approximately 50% higher production of il-4, compared with t cells infected with down - control (p <0.01, figure 2). These results demonstrated that anxa1 induced th0/th1 differentiation, whereas cells infected with silenced anxa1 promoted th0/th2 differentiation . The important roles of gata-3/t - bet in influencing differentiation of t cell lineage into th1 or th2 effector cells were well accepted [19, 20]. As shown in figure 3, the results indicated that anxa1-silenced t cells expressed higher levels of gata-3 and low level of t - bet, compared with the control and upanxa1 groups (p <0.01). Considering that erk and pkb / akt are two major downstream transcription factors of fprl-1, we further investigated the role of fprl-1 signaling pathways in the regulation of anxa1 on gata-3 . The result showed that anxa1 significantly phosphorylated erk and pkb / akt, while knockdown of anxa1 decreased erk and akt activation . The expression levels of frrl-1 in all groups, regardless of anxa1 status, were not significantly different (figure 4). To investigate the effect of gata-3 on anxa1 expression in cd4 t cells, cd4 cells with different gata-3 expression levels were used . As shown in figures 5(a) and 5(b), overexpressed gata-3 inhibited anxa1 protein and mrna expression, while silenced gata-3 increased the expression of anxa1 . As a transcription factor, gata-3 mediates anxa1 expression through anxa1 promoter . Anxa1 promoter area about 2000 bp was found in the eukaryotic promoter database and ucsc genome bioinformatics . The jaspar database was used to predict the binding domain of anxa1 promoter with gata-3 . The result suggested that gata-3 could combine anxa1 promoter agataaga (start-281 end-274) area on the level of nucleic acid sequences . The area the same as zinc finger of gata-3 closet to the c terminus binds to the consensus dna sequence area (a / t)gata(a / g). To verify this hypothesis, anxa1 promoter area (2000 bp) and luciferase reporter gene were built in pgl-3 enhancer vector and renilla luciferase reporter gene was built in prl - tk . Three plasmids gata-3 penter, anxa1 promoter pgl3, and prl - tk were transfected in 293 t cell . As shown in figure 5(c), the luciferase activity was significantly increased, compared with up - controls when cotransfected with upgata-3 . These results demonstrate that gata-3 binds to anxa1 promoter to repress its expression in 293 t cells . Excessive inflammatory responses including the release of a cytokine storm and the activation of a large number of t cells are characteristics of early stage sepsis . In spite of their ability to kill pathogens, they are equally effective in inducing cell and tissue damage . On one hand, the cytokine storm could be protective by activating the host anti - inflammatory proresolving response . On the other hand, during sepsis, the cytokine storm induces excessive activation induced t cell death (aicd); the cause of aicd is an aberration in the cell cycle program . Enhanced anti - inflammatory response, characterized by a large number of t cells apoptosis and decreased responsiveness to antigen / mitogen, leads to the immunosuppression . More patients die in the immunosuppression phase where the risk of a secondary infection increases . Anti - inflammatory therapy can improve the survival rate of patients in hyperinflammatory status, while it may be worse if applied during the sepsis - induced immunosuppression . The conventional understanding of immune responses of post - sepsis involves a shift away from th1 responses towards th2 responses . Promoting a low ratio of th1/th2 immune responses to th1 skewing might be a treatment of immunosuppression . Studies have shown that anxa1 is an endogenous anti - inflammatory factor in the innate immune system [6, 29]. Increasing evidences suggest that anxa1 plays an important role in the regulation of the adaptive immune response . Consistent with above studies, we found that overexpressed anxa1 induced the differentiation of th1/th2 to th1 skewing with increased ifn and reduced il-4 cytokines expression . In the process of th1/th2 differentiation, another important factor is gata-3 . As a specific transcription factor of th2, gata-3 activates tcr signaling in pre - t cells and promotes the cd4 t cell lineage differentiation after positive selection . Many researches and our previous studies have found that the anxa1 plasma levels were decreased in the rabbit and patients with severe sepsis [2, 32]. Our previous animal model observed that anxa1 and gata-3 both present the same downtrend in mouse with severe sepsis [18, 33]; this phenomenon may be related with a large number of t cells apoptosis . The major finding of this study is the discovery of a novel role of transcription factor gata-3 regulation on anxa1 in the adaptive immune response and the molecular mechanisms of mutual regulation contributing to immunosuppression in inflammatory processes . Our results indicated that expressions of anxa1 and gata-3 are both located in th0, th1, and th2 cells nucleus and cytoplasm . Oliani et al . Also found that anxa1 was expressed in the eosinophil cytosol and nucleus . Further study manifested that overexpressed endogenous anxa1 exerted an effect on decline of gata-3 expression and increase of t - bet expression, which may be one reason of anxa1 inducing the differentiation of th1/th2 to th1 skewing . One viewpoint deems that anxa1 modulates th1/th2 differentiation by promoting t cell activation [4, 30]. But another study finds t cell - expressed anxa1 inhibits t cell activation in the adaptive immune response . Our view is that t cells differentiate into various subtypes according to environmental signals, and anxa1 modulates t cell differentiation through modulating their transcription factors . It is known that anxa1 is one endogenous ligand of fprl-1, which belongs to the members of the g protein - coupled receptor family . Exogenous anxa1 protein and its n - terminal peptide ac2 - 26 were demonstrated to activate erk in vitro and in vitro . It is reported that erk suppresses many t cell differentiation related transcription factors, including gata-3 and foxp3 in the t cells [33, 38]. Interestingly, some studies show that erk - mapk cascade controls gata-3 stability through the ubiquitin / proteasome - dependent pathway . These studies further verify the important roles of activated erk - mapk cascade in the gata-3 phosphorylation and its ubiquitin - mediated degradation through the 26 s proteasome . So erk - mapk is essential for gata-3 activation and stability to avoid gata-3 excessive expression in stimulated t cells . Meanwhile, erk pathway is also essential for t cell proliferation and th1 skewing by inducing the tbx21 expression . Akt is a gata-3 phosphorylase kinase and the activation of akt1 induces derepression of tbx21 and ifn expression in th2 cells . Akt phosphorylates gata-3 and induces the dissociation of hdac2 from the gata-3 complex, resulting in a failure in the repression of ifn expression in th2 cell . These findings highlight a pivotal role of posttranscriptional modification of gata-3, which is a kind of negative feedback regulation mechanism of th2 differentiation to avoid excessive differentiation to th2 cell . Therefore, it is proposed that the enhanced anxa1 regulates gata-3 expression to induce th1/th2 shift by erk and pkb / akt to influence the inflammatory response and balance the early proinflammatory function . Another interesting discovery in this study is that the gata-3 also downregulated anxa1 expression in cd4 t cells . Further research indicated that gata-3 exerted a transcriptional - level control by combining with the promoter of anxa1 in 293 t cells . This experimental conclusion needs to be verified in cd4 t cells and determined further by electrophoretic mobility shift assay or chromatin immunoprecipitation . In the early sepsis stage, tcr trigger gata-3 upregulation via pi3k - mtor dependent pathways . Moreover, gata-3 is suggested to mediate notch signaling that is important for promoting t cell development and proliferation . In our studies, if we downregulate gata-3 expression, the level of anxa1 expression increased . On one hand, it can remit th1/th2 toward to th2 skewing to prevent immunosuppression . On the other hand, as an anti - inflammatory protein, anxa1 can balance the early proinflammatory function . In the immunosuppression stage gata-3 can strengthen the tcr activation to improve adaptive immune and decreased anxa1 can reduce anti - inflammatory response at a certain extent for better care of patients . In conclusion, our results suggest that gata-3 binds to anxa1 promoter area to inhibit anxa1 expression and functions . Anxa1-mediated gata-3 and t - bet might favor th1 differentiation through erk and akt pathway, reversing tendency characterized by th2 skewing of immunosuppression . Based on the th1/th2 mediation, our discoveries might provide a potential treatment of immunosuppression after sepsis.
Achaete - scute homolog 1 basic helix - loop - helix chromodomain helicase dna binding protein 7 central nervous system gamma - aminobutyric acid histone acetyl transferase long interspersed nuclear element 1 neuroepithelial precursor nucleosome remodelling and deacetylase oligodendrocyte precursor cell silencing mediator for retinoid and thyroid receptors nuclear receptor corepressors a look into the pubmed database reveals that sox2 is one of the most studied transcription factors of recent years . In 2013 alone, there are about 750 publications mentioning sox2 in title or abstract . After all, sox2 is intimately linked in the scientific consciousness to stem cells, both embryonic and neural, where it is key to induction and maintenance of pluripotency . It is also one of the 4 classical reprogramming factors that are necessary for the generation of induced pluripotent stem cells . Sox2 is originally expressed in all blastomeres, and later in all cells of the inner cell mass and the epiblast . High sox2 expression results both in the suppression of mesendodermal differentiation, and the induction of a neuroectodermal fate . In the early neuroectoderm, sox2 is expressed in all nep (neuroepithelial precursor) cells often together with its close relatives sox1 and sox3 . These 3 related proteins constitute the soxb1 group, and jointly ensure expansion and maintenance of nep cells . Expression of sox2 remains associated with nep cells throughout development of the early neuroectoderm into the cns (central nervous system), and is even maintained in the adult where sox2 is found in adult neural stem cells in the subventricular zone of the lateral ventricle walls and in the subgranular zone of the dentate gyrus . . One of the mechanisms by which this is achieved, is suppression of premature neurogenesis . In accord, soxb1 proteins have been shown to counteract the activity of proneural bhlh (basic helix - loop - helix) proteins such as ascl1 and ngn1 in the early neural tube, and to repress neuronal gene expression . At first glance soxb1 proteins therefore appear to function prominently as repressors . Indeed, such functions have been described for sox2 in studies on the transcriptional regulation of the neurod1 transcription factor and line-1 retrotransposons which both exert neurogenic activity . It has been postulated that sox2 represses neurod1 and line-1 expression by direct binding to the corresponding regulatory regions and recruitment of transcriptional co - repressors such as hdac1 (histone deacetylase1) so that wnt/-catenin - dependent activation of transcription cannot take place . While such function as a classical transcriptional repressor exists for a subset of its target genes and is thus biologically relevant, there is ample evidence that soxb1 proteins when acting as transcription factors this is already suggested by standard in vitro tests for transcriptional activity, which readily identify a transactivation domain in the proteins carboxyterminal half, but fail to detect repressor functions . Results from in ovo electroporation experiments in the early chicken neural tube also point to a predominantly transactivating function . In this type of experiment, sox2 function is mimicked by a chimeric protein in which the aminoterminal half of sox2 (which includes its dna - binding hmg - domain) is combined with a constitutively active heterologous transactivation domain from the herpesviral vp16 protein . Analogous combination of the aminoterminal half of sox2 with the strong repressor domain of the drosophila engrailed protein in contrast changed properties completely and reversed sox2 from an anti - neurogenic into a neurogenic factor as the resulting chimeric protein forced nep cell cycle exit and induced premature expression of neuronal markers . This argues that suppression of neurogenesis is mostly indirect and involves the induction of factors, that then act as inhibitors of neurogenic proteins . What these factors are has not yet been analyzed in detail . Sox2 exerts its diverse effects on gene expression during cns development by influencing epigenetics, transcription and micrornas . It regulates histone modifying enzymes and chromatin remodellers as a pre - patterning factor, and at the same time acts as an activating (or repressing) transcription factor to influence transcription of pluripotency and differentiation genes . Additionally, it impacts on neural and glial gene expression through modulation of micrornas . Sox2 exerts its diverse effects on gene expression during cns development by influencing epigenetics, transcription and micrornas . It regulates histone modifying enzymes and chromatin remodellers as a pre - patterning factor, and at the same time acts as an activating (or repressing) transcription factor to influence transcription of pluripotency and differentiation genes . In addition to its role as transcription factor, sox2 has epigenetic, chromatin - associated functions (fig . Genome - wide chromatin immunoprecipitation studies suggest that sox2 functions as a pre - patterning or pioneer factor (for review see ref . Such factors have the capacity to preselect and bind regulatory regions and thereby configure the local chromatin in a way that the corresponding genes are kept in a poised state . As such they are not yet actively transcribed, but have the potential to be easily activated in ensuing developmental stages, provided certain preconditions are met . In the context of neurogenesis, this means that sox2 not only binds to regulatory regions of genes whose products are required for realization and maintenance of nep cell properties . It is also bound to regulatory regions of many neuronal differentiation genes, and thereby ensures that these genes can later be activated . For this to happen, sox2 is down - regulated and replaced on these regulatory regions by other factors, in particular the soxc proteins sox4 and sox11, which then cooperate with proneural and other neurogenic factors to drive neurogenesis . This role as pioneer factor is supported by results from recent proteomic analyses, that find sox2 associated with many different histone modifying and chromatin remodelling complexes and proteins including nurd, smrt / ncor, swi / snf, chd7, hats and hdacs . This aspect of the overall function of sox2 is equally important as its classical transcription factor function . Although impossible to separate, one might argue in a somewhat simplistic manner that the role as transcription factor is most obvious for maintenance of nep cells and suppression of premature neuronal differentiation, whereas its role as pre - patterning / pioneer factor defines developmental potential and thus guarantees pluripotency . During development, oligodendroglia develop through the opc (oligodendrocyte precursor cell) stage into mature oligodendrocytes, the myelinating glia of the cns . We noted that expression of sox2 and sox3 is maintained after nep cell specification into opc, and is downregulated in the oligodendroglial lineage not earlier than during terminal differentiation to mature myelinating oligodendrocytes . By analogy to neuronal development we initially assumed that soxb1 proteins would ensure opc maintenance and suppress differentiation into oligodendrocytes . Neither deletion of sox2 alone nor in combination with sox3 interfered with proper proliferation or generation of opc in normal numbers . Thus, the role of sox2 and the related sox3 during oligodendrogenesis differs dramatically from the one during neurogenesis with soxb1 proteins being involved in oligodendroglial differentiation events rather than the maintenance of progenitor characteristics . Despite this clear - cut overall difference, it should be noted that differentiation functions have also been detected or proposed in late developmental phases of certain subtypes of retinal or gabaergic telencephalic neurons . This argues that it is not a complete black - and - white between neurons and glia . Our mechanistic studies indicated that sox2 is bound to regulatory elements of myelin genes and that soxb1 proteins can act as moderate transactivators of glial differentiation genes (fig . However, activation potential appears much less than that of other activators of myelin gene expression such as sox10 and olig2 . Considering that expression of the latter is essentially unaltered in the combined absence of sox2 and sox3, it appears unlikely that the differentiation defect observed in soxb1-deficient oligodendroglia is attributable to the lack of direct activation of differentiation genes by sox2 and sox3 . Rather we found evidence for an alternative mode of action that involved a microrna (fig ., sox2 counteracts expression of mir145, which targets pro - differentiation factors such as the transcription factor myrf (myelin gene regulatory factor) or the mediator subunit med-12 that is equally required for terminal differentiation of oligodendrocytes . In the absence of sox2, terminal differentiation of oligodendrocytes and hence myelination are reduced at least in part because mir145 is de - repressed and inhibits expression of myrf and other factors that favor myelination . Our studies thus identify sox2-dependent modulation of microrna expression as another important facet of its action . Intriguingly, we also found evidence for a repressive influence of mir145 on sox2 expression, similar to what has been described in glioblastoma cells . These findings argue that a reciprocal negative feedback - loop may exist in developing oligodendroglia between the 2 regulators . The overall impression from current studies therefore is that in addition to its role as a transcriptional activator sox2 primarily functions as a pre - patterning / pioneer factor during neurogenesis and as a microrna regulator during oligodendrogenesis (fig . Thus, it is legitimate to ask whether sox2 function during the 2 processes is indeed mechanistically as different as the evoked effect . Although there is no definite answer to this question, we like to suggest that this is not the case . We rather assume that sox2 relies on all its different modes of action in both processes . As already mentioned, we have found sox2 on regulatory regions of myelin genes in our study . While this is compatible with a role of sox2 as a weak activator of myelin gene expression, it could equally be interpreted as the result of a pre - patterning function in which sox2 preselects those regulatory regions in opc that have to be activated later on in differentiating oligodendrocytes following the exchange of sox2 on these regions by sox10 . Thus, it seems perfectly plausible that sox2 also functions as a pre - patterning / pioneer factor in oligodendroglial differentiation . On the other side, a first functional link between sox2 and a microrna was recently detected during in vitro neurogenesis . Sox2 was found to activate expression of lin28, which is a potent inhibitor of expression of the let-7 microrna family . Let-7i in turn inhibits neuronal differentiation, by targeting among others the 2 proneural genes ascl1 and ngn1 . These findings provide support for the assumption that at least some of the long known repressive effects of sox2 in nep cells on neurogenesis may be mediated by micrornas that are under direct or indirect control of sox2 . In our opinion, sox2 is a true central regulator of gene expression because it acts not only at the transcriptional level, but also pretranscriptionally as a chromatin - associated pioneer factor and posttranscriptionally as a microrna modulator . Its mechanistic versatility is not only basis for its central role in many regulatory networks, but also endows it with a tremendous flexibility that allows it to acquire different tasks in diverse developmental setups including maintenance of the precursor state, suppression of neurogenesis and promotion of glial differentiation.
Hypoplasia is defined as a quantitative defect of enamel visually and is histomorphologically identified as an external defect involving the surface of the enamel and associated with reduced thickness of enamel . The cervical and the incisal borders of the defect have a rounded appearance due to the prisms in the non - affected enamel being bent, which may be attributed to a change in the prism direction . The macro and microscopical appearances suggest that only some specific ameloblasts have ceased to form enamel, whereas others are partly or completely able to fulfil their task . Unlike other abnormalities which affect a vast number of teeth, turner's hypoplasia usually affects only one tooth in the mouth and it is referred to as a turner's tooth . If turner's hypoplasia is found on a canine or a premolar, the most likely cause is an infection that was present when the primary tooth was still in the mouth . Most likely, the primary tooth was heavily decayed and an area of inflamed tissues around the root of the tooth affected the development of the permanent tooth . The appearance of the abnormality will depend on the severity and longevity of the infection . If turner's hypoplasia is found in the anterior area of the mouth, the most likely cause is a traumatic injury to a primary tooth . The traumatized tooth, which is usually a maxillary central incisor, is pushed into the developing tooth underneath it and consequently affects the formation of enamel . Because of the location of the permanent tooth's developing tooth bud in relation to the primary tooth, the most likely affected area on the permanent tooth is the facial surface . Type i hypoplasia: enamel discoloration due to hypoplasia type ii hypoplasia: abnormal coalescence due to hypoplasia type iii hypoplasia: some parts of enamel missing due to hypoplasia type iv hypoplasia: a combination of previous three types of hypoplasia . Both dentitions could be affected by enamel hypoplasia; however, the incidence is more severe in permanent dentition . The characteristics of clinical enamel hypoplasia include unfavorable esthetics, higher dentin sensitivity, malocclusion and dental caries susceptibility . The treatment challenge in this type of injury is to promote a complete oral rehabilitation in both esthetics and function . We have come across few cases of unattended hypoplastic teeth which had turned non - vital without any carious insult or trauma . A 7-year - old female patient reported to the department of pedodontics and preventive dentistry with chief complaint of painful discolored right upper front tooth for the past 2months . On clinical examination, the maxillary right lateral incisor showed yellowish brown discoloration [figure 1] with type iv enamel hypoplasia (enamel discoloration, abnormal coalescence, some parts of enamel missing). Patient had a history of trauma at the age of 3 years, followed by exfoliation of 52 . Intraoral periapical radiograph (iopa) showed an open apex with no other abnormalities [figure 2]. A test cavity was prepared in 12 and the tooth had no response, indicating non - vitality . Intraoral photograph of 12 with type iv turner's hypoplasia intraoral periapical radiograph of 12 with open apex a 20-year - old male patient reported with a complaint of painful discolored left upper front tooth with deposits on it . Patient had history of trauma to his upper front primary teeth at the age of 2years . On clinical examination, a horizontal groove with brown discoloration around the cervical region of 21 was observed [figure 3], resembling circular enamel hypoplasia (type iv). Iopa radiograph showed loss of enamel around the cervical region of the tooth with no other abnormalities [figure 4]. A test cavity was prepared in 21 and the tooth had no response, indicating non - vitality . The treatment plan included root canal therapy followed by jacket crown . Clinical photograph of circular enamel hypoplasia intraoral periapical radiograph of 21 revealing loss of enamel around cervical region an 8-year - old male patient reported with chief complaint of discolored lower front two teeth for the past 4months . Patient gave a history of loss of lower primary incisors due to trauma at the age of 2years . Clinical examination showed yellowish brown discoloration with type iv enamel hypoplasia [figure 5]. A test cavity was prepared in 31 and 41 and the tooth had no response, indicating non - vitality . Clinical photograph of 31, 41 with type iv turner's hypoplasia intraoral periapical radiograph of 31, 41 with open apex a 20-year - old female patient reported with a complaint of painful discolored lower right back tooth for past 3months . Patient gave a history of retained, long - standing, untreated carious primary tooth . On clinical examination of 45, the tooth was tender on percussion and showed yellowish brown discoloration with typeiv enamel hypoplasia [figure 7]. A 7-year - old female patient reported to the department of pedodontics and preventive dentistry with chief complaint of painful discolored right upper front tooth for the past 2months . On clinical examination, the maxillary right lateral incisor showed yellowish brown discoloration [figure 1] with type iv enamel hypoplasia (enamel discoloration, abnormal coalescence, some parts of enamel missing). Patient had a history of trauma at the age of 3 years, followed by exfoliation of 52 . Intraoral periapical radiograph (iopa) showed an open apex with no other abnormalities [figure 2]. A test cavity was prepared in 12 and the tooth had no response, indicating non - vitality . Intraoral photograph of 12 with type iv turner's hypoplasia intraoral periapical radiograph of 12 with open apex a 20-year - old male patient reported with a complaint of painful discolored left upper front tooth with deposits on it . Patient had history of trauma to his upper front primary teeth at the age of 2years . On clinical examination, a horizontal groove with brown discoloration around the cervical region of 21 was observed [figure 3], resembling circular enamel hypoplasia (type iv). Iopa radiograph showed loss of enamel around the cervical region of the tooth with no other abnormalities [figure 4]. A test cavity was prepared in 21 and the tooth had no response, indicating non - vitality . The treatment plan included root canal therapy followed by jacket crown . Clinical photograph of circular enamel hypoplasia intraoral periapical radiograph of 21 revealing loss of enamel around cervical region an 8-year - old male patient reported with chief complaint of discolored lower front two teeth for the past 4months . Patient gave a history of loss of lower primary incisors due to trauma at the age of 2years . Clinical examination showed yellowish brown discoloration with type iv enamel hypoplasia [figure 5]. A test cavity was prepared in 31 and 41 and the tooth had no response, indicating non - vitality . . Clinical photograph of 31, 41 with type iv turner's hypoplasia intraoral periapical radiograph of 31, 41 with open apex a 20-year - old female patient reported with a complaint of painful discolored lower right back tooth for past 3months . Patient gave a history of retained, long - standing, untreated carious primary tooth . On clinical examination of 45, the tooth was tender on percussion and showed yellowish brown discoloration with typeiv enamel hypoplasia [figure 7]. Hypoplasia is a disturbance that occurs at the time when teeth are developing and is associated with macroscopic enamel defects . Traumatic injuries to the primary dentition are very common, affecting from 4 - 30% of all children . The effect of trauma is more pronounced if it occurs prior to third year of life . The assessment of trauma in primary dentition seems to be very important because of the presence of sequelae in the permanent dentition . Diana ribeiro et al reported from their longitudinal study of 8years that discolorations of enamel and/or enamel hypoplasia (46.08%) were the most prevalent sequelae on permanent dentition due to traumatic injury . In the four cases we reported here, all patients had a history of trauma / infection in their deciduous dentition below the age of 3 years . In the present case reports, all patients had type - iv enamel hypoplasia (enamel discoloration, abnormal coalescence, some parts of enamel missing). The brown discoloration occurs due to disturbances in ameloblastic layer, leading to defective matrix formation caused by traumatic injuries, but the stretched inner enamel epithelium continues to induce the differentiation of new odontoblasts and hence the dentine formation is not affected . In one of our cases, severe pulpal infection of deciduous teeth results in excessive osteolysis of inter - radicular bone and early exposure of the succedaneous tooth before adequate root length formation . These structurally defective teeth are not only weak but also provide favorable area for colonization of bacteria . The hypoplastic permanent teeth are seven times more sensitive to carious attack compared to those without hypoplasia . Vahid golpaygani and mehrdad et al reported that, the rate of dental caries among hypoplastic teeth was much higher than the normal teeth . In type - iv enamel hypoplasia, the incidence of dental caries was significantly increased . In our cases, the hypoplastic tooth had turned non - vital without any history of trauma or any carious insult . The tooth would have turned non - vital because of the defective enamel and open dentinal tubules which act as a nidus for bacterial entry into the pulp space, thereby leading to pulp necrosis . The base of the enamel hypoplasia in primary tooth under polarized light microscope showed no sign of normal aprismatic surface layer, revealing a rough surface . Also, a more porous zone was discerned, indicating a pore volume distribution of <5% . Nina sabel et al reported that the porous enamel (which constitutes a pathway for bacteria and other stimuli that may affect the pulp), leads to the formation of reparative dentin . It should be stressed that the parents tend to forget, or overlook history of minor injuries which happened years before . Delayed treatment on affected deciduous and permanent teeth can lead to pulp pathosis and its sequelae . The aim of treatment protocol should be to preserve the vitality of the tooth and prevent further enamel destruction rather than esthetical consideration . Fortunately, enamel hypoplasia can usually be managed by restoring the affected enamel (bonding a tooth - coloured material to the tooth in order to protect it from further wear), following proper oral hygiene methods . Topical fluoride application is effective in reducing dentin sensitivity and caries attack . In most cases, the need for close periodic examination and early detection of all possible developmental defects in the permanent dentition and the importance on preventive measures should be stressed for maintaining the vitality of the tooth . Since information on the microstructural level of enamel hypoplasia is still limited, further studies to be conducted to better understand the mechanisms behind non - vitality.
Several studies suggest that postoperative atrial fibrillation (poaf) is associated with an increased duration of hospitalization, early and long term morbidity and mortality [2, 3]. Off - pump coronary artery by - pass (cabg) grafting (op - cabg) has been hypothesized to decrease incidence of poaf [4, 5]; however contrasting results has been reported . Aim of present prospective investigation was to evaluate the incidence of poaf in patients undergoing isolated surgical revascularization in a tertiary heart surgery centre . Patients with poaf were followed for an average period of 2 years to assess the recurrence rate of the arrhythmia and its prognostic role on early and late risk of stroke and mortality . Finally, the role of cardiopulmonary by - pass (cpb) surgical revascularization (cpb - cabg) and op - cabg on poaf was evaluated . Study population . Among 822 patients who underwent heart surgery between jan 1 2009 and dec 31 2009 in a tertiary heart surgery centre, 229 patients in sinus rhythm on hospital admission (179 males, 50 females) underwent isolated cabg (138 - op - cabg, 91 - cpb - cabg). Patients with atrial fibrillation, hyperthyroidism or scheduled for maze procedure were excluded from the study . In patients undergoing isolated cabg, bipolar maze procedure was usually planned for subjects with persistent or frequent episodes of paroxysmal atrial fibrillation . Finally, patients with more than mild valvular disease and creatinine clearace <30 ml / min were excluded . Echocardiographic evaluation was performed within 48 hours before surgery using a sequoia acuson instrument (siemens medical solution, mount view, ca, usa). Clinical and echocardiographic characteristics of patients included in the study mean (standard deviation). * bleeding requiring transfusion at least 2 units of packed red blood cells or surgical revision . * * hemodynamic deterioration with the need to infuse amines . Op = off pump; cpb = cardipulonary - by pass; cabg = coronary artery by - pass; sd= standard deviation . In table 2 clinical diagnosis, indications for surgery (elective, urgency / emergency), the number of diseased vessels and graft performed in the groups under investigation are reported . Op = off pump; cabg = coronary artery by - pass; cad = coronary artery disease; acs = acute coronary syndrome; stemi = elevated st acute myocardial infarction; lmc = left main coronary; lima = left internal mammary artery; rima = right internal mammary artery; saph = saphene vein . Thirty clinical and echocardiographic variables were considered to evaluate a relationship with occurrence of poaf . After surgery all patients were continuously monitored electrocardiography (ecg), blood pressure, non - invasive oxygen saturation for at least the first 48 hours . Transient electric stimulation through epicardic wires was used for severe bradycardia or atrio - ventricular (av) block until restoration of heart rhythm . All symptomatic arrhythmic episodes or asymptomatic atrial fibrillation lasting more than 15 minutes at ecg monitoring were considered as poaf and included in the analysis . Patients who did not recover sinus rhythm (sr) within 30 minutes were usually treated with intravenous amiodarone (300 mg in 1 hour followed by 900 mg/24 h e.v . Electrical cardioversion was considered when sinus rhythm was not restored within 24 hours after the beginning of pharmacological treatment . Perioperative complications including bleeding needing transfusion of at least 2 units of packed red blood cells and/or surgical revision, severe hypotension requiring amines (norepinephrine, epinephrine, dobutamine or dopamine), and new onset av block or severe bradycardia requiring electrical stimulation were recorded . Postoperative pericardial inflammation was diagnosed in the presence of pericardial rubs and/or ecg or echocardiogram signs of pericardial involvement . In the end, the study was approved by the ethic committee of our institution and all participants gave their informed consent . Holter monitoring was performed every 3 months during the first year and thereafter every 6 months . Primary end point of the study was the evaluation of recurrence of atrial fibrillation and related hospitalization; secondary end points were all cause hospitalization and mortality . Finally we evaluated the role of surgical technique (cpb - cabg vs op - cabg) on poaf . Statistical analysis . Data were described as mean and standard deviation (sd) for continuous variables and as number and percent for categorical variables . Preoperative and operative patient characteristics were compared according to the occurrence of postoperative af by means of the student t test or fisher exact test for continuous and categorical variables, respectively, or finally by anova . In - hospital outcomes . Overall incidence of poaf during hospitalization was 24.4% (56 of 229 patients), 38 males and 18 females . Patients who developed af after surgery were older than ones in stable sinus rhythm (70.5 vs 64.9 years, p=0.005). Poaf was not related to clinical indication to surgery (elective vs urgency / emergency), number of diseased vessels or graft performed (table 3). Comparison of clinical, echocardiographic characteristics and conduits used for grafting between patients with and without poaf according to surgical technique . * bleeding requiring transfusion at least 2 units of packed red blood cells or surgical revision . * * hemodynamic deterioration with the need to infuse amines . Poaf = postoperative atrial fibrillation; op = off pump; cpb = cardipulonary - by pas; copd = chronic obstructive pulmonary disease; cad = coronary artery disease; acs = acute coronary syndrome; stemi = elevated st acute myocardial infarction; lmc= left main coronary; lima = left internal mammary artery, rima = right internal mammary artery, saph = saphen vein; sd = stabdard deviation . Poaf left ventricular ejection fraction was not significantly lower than in sinus rhythm group (49% vs 51%). The use of beta - blockers, angiotensin converting enzyme (ace) inhibitors / angiotensin a1 receptor (at1) blockers and statins did not influence the prevalence of postoperative af . Patients with af did not show a different prevalence of intra - aortic balloon pump use or treatment with vasopressors or inotropic drugs after surgery . Transient electric stimulation after surgery was needed in 2 patients with poaf and in 3 who did not develop arrhythmias . Multivariate analysis revealed that only antero - posterior left atrium diameter was associated with an increased risk of poaf (odds ratio = 1.15; 95% confidence interval (ci) [1.02, 1.30], p<0.001) (table 4). Od = odds ratio; ci = confidence interval; la = left atrium; ace = angiotensin - converting - enzyme; lv = left ventricle; bp = blood pressure . The frequency of poaf was not statistically different between patients undergoing op - cabg and those undergoing cardiopulmonary by - pass (24.6% for op - cabg vs 24.1% for cpb - cabg .) Table 3 reports the relative number of elective in comparison to urgency / emergency procedures, and the number of grafts conduits employed in the two groups . Patients with poaf undergoing op - cabg were on average 7 years older than cbp - cabg (74.3 vs 67 years, p<0.001). Length of hospitalization was on average 2 days longer in patients with poaf after cpb - cabg . The 56 patients with postoperative atrial fibrillation were followed - up for a median of 685 days . Age was not significant different in those patients (average age 72.5 vs 71.3 years). Among those patients, preoperative left ventricular ejection fraction was not different in patients with af in comparison to patients without recurrence (48% vs 49%), while mean left atrium anterior - posterior diameter was respectively 42 mm and 40 mm . On average ultimately, in the last patient sinus rhythm could not be restored . At the end of follow up incidence of atrial arrhythmias after cardiac surgery has been reported to range from 10 to 65% . According to a large multi - centre study, poaf after cabg occurs in near 30% . Several factors, including type of surgical procedure, patient demographics, criteria used for diagnosis and methods of ecg monitoring, may account for the wide range of poaf incidence reported in literature . Dispersion in atrial refractoriness induces multiple local re - entry wavelets; therefore, atrial fibrillation may be induced by several factors . Among these: trauma from surgical dissection and manipulation, myocardial ischemic damage, an exaggerated local inflammatory response with or without pericarditis, an elevation in atrial pressure from post operative ventricular stunning a chemical stimulation due to postoperative support with catecholamine and other inotropic agents, a reflex sympathetic activation from volume loss, anemia or pain, parasympathetic activation, fever from atelectasis or infection, hypoglycaemia, metabolic and electrolyte imbalance, fluid overload, prolonged post operative electrical stimulation . Cardiopulmonary by - pass related hemodynamic changes may induce intraoperative atrial ischemia that has been hypothesized to play a role in the development of poaf . . Reported at 6 months after surgery a significantly higher mortality in af patients compared with patients without af (9.4% vs 4.2%). . Showed in 6475 patients undergoing first isolated cabg that poaf was associated with at increased risk of death (odds ratio = 1.5; 95% ci [1.3, 1.8]). In this study, cumulative survival rate at 1 and 4 years was 87% and 74% in poaf patients versus 94% and 87% for non - af population . In more than 8500 isolated cabg patients a significantly increased risk of death was observed among those who developed postoperative af compared with those who did not (odds ratio = 1.2; 95% ci, 1.1 to 1.3). Patients affected by postoperative af had an increased 1-year mortality (4.6% versus 2.0%), and af was confirmed to independently predict late mortality (hazard ratio, 1.7; 95% ci [1.2, 2.5]). Results from present investigation do not support an association of poaf with an increase of late mortality in patients undergoing surgical revascularization . Only one patient died at 2 years follow - up and not for cardiovascular cause . Restoration of sinus rhythm in all patients during hospitalization and low recurrence rate may have significantly decreased the risk of mortality, in particular due to stroke or complications of oral anticoagulant treatment . Decreased risk of ischemic damage in beating heart cabg initial favourable results [6, 7] has not been confirmed by other authors [4, 12]. Siebert et al during the postoperative intensive care unit stay reported a 9.8% rate of poaf in patients after cpb - cabg, 10.2% after op - cabg, and 21% after cabg combined with valve replacement . A recent meta - analysis suggested a decreased incidence of af in op - cabg although overall mortality was not affected . A not significant difference in the incidence of poaf between the two techniques was reported by several other studies [14, 15]. Two randomized, controlled trials and one large scale concurrent cohort study addressed the issue of beating - heart cabg . Ascione et al found a significantly lower rate of postoperative af in the op - cabg group (11.0%) than in the cbp - pump cabg group (45.0%) in 200 patients who had been randomized to undergo cabg either with or without cpb . A significant difference in postoperative af favouring the op - cabg group (21.2%) compared to the on - pump cabg group (6.3%) in contrast, in 281 patients randomized to cabg with or without cpb, van dijk et al reported no difference in the rate of postoperative af . In patients undergoing emergency revascularization for acute coronary syndromes off - pump surgery vs cpb surgery was performed in patients with more severe clinical conditions: op - cabg patients were more frequently in cardiogenic shock, had an impaired renal function, a log euroscore> 20 or a left ventricular ejection fraction <30% . Overall survival and event rate, however, were similar at 5 five year follow - up . Postoperative af occurred in 30.2% patients undergoing cpb - cabg vs 29.3% in op - cabg surgery while the incidence of stroke was two - fold in the former group (6.7 vs 2.5%, p <0.035). Noteworthy the incidence of af was two folds in patients with cardiogenic shock undergoing cpb versus op surgery (62.5 vs 39.8%), with a significant increase in the number of stroke (33.3 vs 9.6%, p<0.009). 0.901 ng / ml at icu admission has been identified as cut - off value for prediction of af in patients undergoing elective cabg . Patients with serum tni> of 0.901 ng / ml showed an 11.5 times increased risk for the onset of af after elective cabg . In present investigation no significant relation was found between tni serum concentration after surgery and the risk of af (odds ratio = 0,96 - 95% ci [0,85, 1,08], p=0.17). In our experience, the incidence of poaf resulted not significantly different in patients undergoing op - cabg in comparison to cpb - cabg . The two groups were comparable for severity of coronary disease, number of grafts performed and clinical presentation (urgent versus elective surgery). However, mean age of patients undergoing op - cabg was on average 7 years older in comparison to patients treated with cpb - cabg . The present study was limited by its observational nature, by a relative short follow - up period (2 years), and by the low number of patients investigated . Otherwise the short time of enrolment (1 year), the similar characteristics of the two groups, with the cited exception of age, and the limited number of operators decreased the risk of non homogeneity of the population under investigation . The low number of recurrences occurred, similarly to other studies, may be related to potential bias due to the reliance on self - reporting for follow - up cardiac rhythm data . Although scheduled holter monitoring in our investigation did not reveal paroxysmal episodes of atrial fibrillation, only continuous monitoring systems may provide definitive data . Similarly, the use of questionnaires and clinical examination during follow - up may not accurately identify paroxysmal episodes of af and may potentially have underestimated the incidence of the arrhythmia recurrences . Despite the reported limitations, our study does not support the hypothesis that postoperative af is associated with an increased late mortality, rate of stroke or rehospitalization . Restoration of sinus rhythm before hospital discharge may have significantly limited the negative prognostic effects of post operative atrial fibrillation . At present, the guidelines of the american college of chest physician state that op - cabg cannot be recommended to decrease post operative af because of conflicting results resulted from randomized controlled trials or large - scale concurrent cohort studies.
From may 1 through july 30, 2007, a total of 29 cases of locally acquired cyclospora infection were reported in british columbia (figure 1; table 1). An initial investigation was conducted around the 6 laboratory - confirmed case - patients reported in the last 2 weeks of may and the first week of june (phase 1). No common exposure was reported, and case reports subsided . During the last week of june, case reports resumed, and phase 2 of the investigation was initiated . A total of 19 confirmed and 4 probable cases were identified with symptom onsets during june 28july 20, 2008 . Average time from symptom onset to positive laboratory result was 17 days (range 631 days). Confirmed and probable cases of cyclosporiasis (n = 29), by date of onset, british columbia, canada, may august 2007 . * laboratory - confirmed cases were reported to public health by medical diagnostic laboratories and specimens forwarded for confirmation to the public health reference laboratory . Oocysts included shape (spherical), size (810 m in diameter), oocyst wall (well - defined), internal contents with refractile globules, autofluorescence, and modified acid - fast or safranin staining (1). During phase 2, a total of 17 confirmed case - patients were interviewed with hypothesis - generating questionnaires about items eaten in the 2 weeks before symptom onset . The instrument included questions about restaurant history with meal details; grocery stores frequented; and yes / no questions about> 70 fruits and vegetables, 8 herbs, and 16 mixed foods (e.g., salsa, pesto) previously implicated in outbreaks of foodborne disease . Frequently reported foods were compared with population controls from canadian (waterloo, ontario) and american (oregon; us foodborne diseases active surveillance network [foodnet]) published food consumption surveys (35). Although such measurements may be limited by the timing of questionnaire administration and the recall period considered, they can be useful comparators during the hypothesis - generating stages of an investigation . By the end of phase 2, strawberries, cilantro, and sweet basil garlic and red peppers also were commonly eaten by case - patients; however, population comparisons were unavailable . Eighty - eight percent of case - patients reported having eaten romaine lettuce; 85% of controls in the waterloo survey (4,5) had eaten lettuce of any type, and romaine lettuce consumption was much less commonly reported in the foodnet survey (3) (table 2). A formal case control study was considered premature in the early stages of phase 2 because no strong hypothesis emerged from early interviews and comparisons to population controls . We further explored the plausibility of various hypotheses through a combination of methods described below that allowed room for additional hypotheses to emerge or existing hypotheses to strengthen as cases accrued . * case - patients were 17 persons with laboratory - confirmed cases interviewed during phase 2 (june 24july 21, 2007). Us foodnet, foodborne diseases active surveillance network; na, not applicable . Detailed questionnaires asked whether foods were eaten in a restaurant or were store - bought and about type of packaging and method of preparation (because c. cayetanensis is heat - sensitive) (6). We reinterviewed early case - patients using the second questionnaire and interviewed later case - patients using both questionnaires . In phase 1, garlic eaten at restaurants by all 4 persons with confirmed infections were traced back to different suppliers; only 1 case - patient ate raw garlic in a restaurant . Three case - patients also reported eating cooked garlic at home; cooking would have inactivated the pathogen . Early and proactive collaboration with cfia involved a general assessment of the country of origin and distribution patterns for frequently eaten foods . According to cfia records, romaine lettuce and red peppers sold during the exposure period were not imported from a known cyclospora - endemic country and were widely distributed in canada and the united states . Comm . ). Because interviews, population control comparisons, and product distribution limited suspected foods to strawberries, cilantro, and basil, we began preliminary traceback of all 3 suspected items . Environmental health officers and regional cfia staff interviewed grocery store owners, restaurant managers, and distributors to trace produce to its supplier . Local strawberries eaten by case - patients from 3 small markets were traced back to 2 local farms in geographically separate regions of british columbia . Cilantro eaten by case - patients was traced to 2 suppliers; both supplied home - grown rather than imported produce . Of 14 case - patients with confirmed basil exposures, 4 (57%) ate only organic basil supplied by distributor a. additionally, 4 (29%) reported multiple basil exposures, including exposure to organic basil from distributor a (figure 2). In british columbia, organic basil enjoys a smaller market share than the conventional product . Traceback of basil eaten by persons with confirmed cyclosporiasis (n = 14), british columbia, canada, may august 2007 . In phase 2, 12 (71%) of 17 case - patients reported shopping at grocery c. records of any grocery store purchases for the households of 8 consenting case - patients were obtained through grocery c s savings card program; other case - patients were not cardholders . All purchase histories were requested for 1 month before symptom onset to account for the typical incubation period plus product shelf life . Records from 3 (38%) case - patients showed purchases of the same organic basil supplied by distributor a. two case - patients had bought organic basil on the same day at the same location . Of the remaining 5 case - patients who recalled purchasing organic basil but whose consumer card records did not confirm it, 2 had not used their cards for large portions of the incubation period . We collected supplier information for organic basil during a visit to the distribution warehouse and local farm site of distributor a. the remaining 2 (14%) case - patients with basil exposure previously unlinked to distributor a were confirmed through trace - forward from distributor a. the first had eaten organic basil at a smaller market supplied by distributor a under another trade name . The second had eaten conventional basil from a grocery store supplied by distributor a. distributor a confirmed using organic basil to supplement conventional basil shipments when supply was low . Late summer outbreaks of cyclosporiasis in british columbia are unusual; distributor a confirmed that imported product was used throughout the summer in 2007 because of a poor local growing season . All case - patients in phase 2 who recalled basil exposure (82%) could have been exposed to organic basil from distributor a. once this common vehicle was identified, cfia conducted a full traceback of organic basil by using formal documentation including invoices, shipment numbers, and airway bills . The suspected imported basil was no longer available for testing . Using distributor a invoices, we identified a specific shipment of organic basil imported from 1 of 2 mexican supplier farms, and cfia notified mexican authorities . The mexican farm was located in a region previously linked to cyclosporiasis outbreaks (r. cardinal, cfia, pers . Detailed interviews, modified traceback of several suspected items, and information about product distribution and market share led to organic basil as a primary hypothesis . Food regulators could pinpoint a specific shipment and trace it to its origin because consumer cards provided the exact purchase dates for basil that case - patients could not recall . Overall, the approach used in this investigation increased the work load typically requested of team members during foodborne outbreaks . However, this combination of investigative methods successfully identified a single vehicle during a community cyclosporiasis outbreak where a common menu was not available.
One normally needs to maintain postural control while performing one or more other concurrent tasks such as walking while talking . An individual s attention resources and information processing capacity are presumably limited, and must be shared among all the tasks concurrently being performed1 . Therefore, when two tasks are being performed simultaneously, performance of one or both can be impaired, if together they require attention that exceeds an individual s capacity2 . Postural control, control of the body s position, orientation and balance, is often regarded as an automatic or reflex - controlled task with minimal attentional demands . Performance of the postural control system declines with age due to deterioration of the visual, proprioceptive and vestibular systems, slower nerve conduction velocity and reduced musculature4 . Studies have shown that the attentional demands of balance control increase with age in compensation, even as general attention resources decrease5, 6 . Consequently, older adults are more likely to have insufficient attention resources for effective postural control in dual - task situations, which would affect their dual - task performance . Reported that older adults have significantly higher values of sway area and center of pressure velocity in older adults during dual - tasks when compared with young adults7 . This suggests why the risk of balance loss, and hence falling, increases among older adults8 . Older adults dual - task performance may also be influenced by other factors, such as task difficulty, postural threat, stimulus modalities or response modes . In tasks with increasing difficulty, such as walking backward, an increased cognitive demand occurs and the concurrent execution of a cognitive task may be compromised9 . Falls are a common problem for the aged . Among community - dwelling adults aged 65 or above impact on the hip is likely in a backward or sideways fall11, and direct impact on the hip in a fall from standing height can easily be forceful enough to cause a hip fracture in older adults12 . Besides physical impact, other possible consequences include psychological problems, a reduction in subsequent activity or even social isolation due to fear of falling13 . Many falls occur during transitional movements which require an older person to step backward . Backward walking is associated with reduced velocity and stride length, and increased gait variability, and these parameters are associated with a greater fall risk in older adults14 . Examples include pulling open a door, backing to sit on a chair, or responding to a forward perturbation . These are common situations that older adults encounter in daily life, but the effects of aging on postural control when stepping backward have not been well - studied . Therefore, this study was designed to examine (i) whether there are differences between young and older adults in cognitive performance and postural control after stepping back, and (ii) to what extent dual - tasking affects cognitive performance and postural control . The hypothesis was that older adults would have poorer cognitive performance and postural stability after stepping back, especially while dual - tasking . A total of fifty - eight subjects, including thirty young adults and twenty - eight older adults, participated in this cross - sectional study . The older subjects, community - dwelling adults aged 65 or above, were recruited from elderly centers in hong kong . The subjects were all independent in their daily living activities, able to walk unaided, and native cantonese speakers . Candidates were excluded if they had a neurological disorder, myocardial infarction or heart failure, uncontrolled diabetes or hypertension, deformity of the lower limbs, a history of musculoskeletal injury to either lower limb in the previous 12 months, or cognitive or hearing impairment . The validated cantonese version of the mini - mental state examination was used to screen the older subjects for cognitive impairment15 . Candidates with scores less than 24 were regarded as impaired and excluded from the study16 . Subjects who reported dizziness on the day of the assessment or failed to follow the verbal commands ethics approval was obtained from the ethics committee of the hong kong polytechnic university . Written informed consent was obtained from all of the participants prior to their participation . Each participant performed three tasks: an auditory stroop test and a stepping - backward task with and without a concurrent auditory stroop test . The classic stroop test requires subjects to name the color in which a word is printed where the word itself is the name of a different color . Subjects react more quickly and accurately to congruent stimuli (when the word corresponds to the color of the printing) than to incongruent stimuli (where the word and the print color differ). Cantonese is a tonal language where meaning depends both on the phoneme and the tone in which it is pronounced . Two cantonese words meaning high and low were used in this test, and they were pronounced at either a high or low pitch, generating four combinations of pitch and meaning . The subjects were required to respond to the voice as quickly as possible by pressing the right thumb switch when a high pitch voice was heard or pressing the left one for low pitch, disregarding the meaning of the word as pronounced . For the test, each participant had four familiarization trials (one for each combination) before sixteen test trials (four trials per combination in a random order). The reaction time (the time between the sound and the moment when the switch was pressed) and error rate (percentage of wrong responses) were recorded . The stepping - backward task started with the subject standing barefoot on a force plate (model or6 - 51000, advanced mechanical technologies inc ., newton, ma, usa) with the feet shoulder - width apart, looking at a fixed visual target placed two meters from the center of the force plate . The participants were required to make a step backward from one force plate onto another in response to a verbal command . The dominant leg, which was defined as the leg used to kick a ball, was used to step back18 . Meanwhile, before landing, the subject was required to press one of the thumb switches (without an auditory stroop test). This served to control for any possible perturbation of postural control arising from the action of pressing a thumb switch . After stepping back the subject was required to maintain a single - leg stance on the dominant leg for 10 seconds and keep looking at the fixed visual target . A prolonged phase of double - leg stance (with the non - dominant leg stays in the front force plate) or a shortened single - leg stance of the dominant leg occurs when postural control deteriorates . Thus, the duration of the double - leg stance and the sway parameters during the single - leg stance were recorded and compared between the older and young participants . A familiarization trial was given before the four test trials, and rests were allowed if necessary . The center of pressure (cop) data collected during the first 5 seconds of the single - leg stance were analyzed to quantify postural control, since the average single - leg stance duration of older adults is normally about 6 to 7 seconds19 . Two body - sway measures: (i) the total sway path of the cop and (ii) the sway area covered by the maximum sway excursion, were used to evaluate the postural control performance after stepping back . The data were recorded at a sampling frequency of 1,000 hz and then re - sampled at 120 hz for analysis using matlab 7.1 software (the math - works inc ., natick, usa). When performing stepping - backward with a concurrent cognitive task, subjects were requested to respond to the auditory stroop test as quickly and accurately as possible while simultaneously performing the stepping - backward task . The auditory stroop test was triggered either when the subjects lifted their dominant leg (detected by a pressure sensor placed underneath their heels), or stepped back onto the force plate . They were instructed that the auditory stroop test could be triggered at any time during stepping back or the single - leg stance . Version 20.0 of the commercially available statistical package for the social sciences (spss) software (ibm corp . The average ages, heights and weights of the young and older subjects were compared using the independent t - test . The test was used to compare the gender distribution of the two groups . Repeated measures multivariate analysis of variance (manova) was used to compare the results of the auditory stroop tests, the stepping - back task and the dual - task performances between the two groups . Univariate tests were conducted for each of the measures when an overall statistically significant difference was found . The confidence level for statistical significance the baseline characteristics of the two groups are shown in table 1table 1.participants characteristicsvariablesyoung adult group (n=30)older adult group (n=28)age (years)21.3 1.2 72.2 5.7height (cm)163.9 7.3154.8 6.4weight (kg) 54.8 8.8 59.1 8.6female, n (%) 22 (73.3) 21 (75.0)mean standard deviation . * significant difference between the young and older adults groups .. the two groups differed significantly in terms of age and height (both p<0.001), but there was no significant difference in their gender distribution or weights . A significant overall (group task) difference (f=24.835; p<0.001) in the auditory stroop test was found by repeated measures manova . Univariate analysis demonstrated a significant (group task) difference in reaction times (p<0.001), but not in error rates (p=0.064) (table 2table 2.auditory stroop test results during single- and dual - taskingyoung adult group (n=30)older adult group (n=28)single taskdual task% change (%) single taskdual task% change (%) reaction time (s)0.62 0.100.69 0.15 + 11*1.03 0.32 1.81 0.46+76 * error rate (%) 4.6 5.38.9 12.0 + 9515.2 11.3 * 27.2 21.0+80 * significant difference between the young and older adults groups . * * significant difference between single- and dual - tasking . ). The independent t - test showed there were significant between - group differences in average reaction times and error rates of both the single- and dual - tasking (p<0.001). The paired t - test showed that, when comparing dual - tasking with single - tasking, both the young and older subjects had significantly increased average reaction times (p=0.002 and p<0.001, respectively), but that the older adults had significantly higher average error rates (p=0.001) but this was not evident among the young adults (p=0.1). * significant difference between the young and older adults groups . * * significant difference between single- and dual - tasking . Since the average height of the two groups was significantly different, height was treated as a covariate in the statistical analysis . In addition, the double - leg stance durations from the dominant leg landing to the lifting of the non - dominant leg were significantly longer for the older subjects in both single- and dual - tasking (both p<0.001) (table 3table 3.center of pressure excursions during single- and dual - taskingyoung adult group (n=30)older adult group (n=28)single taskdual task% change (%) single taskdual task% change (%) cop path (mm)92.3 66.1101.8 71.0 + 10273.1 54.2 * 286.0 60.5+5cop area (mm)474.8 343.8679.5 565.5 + 431,081.5 444.9 1,180.0 574.2+9double - leg stance duration (ms) 444.5 125.4459.4 108.2 + 3771.1 287.4 705.2 201.4*9mean standard deviation . The younger subjects were simply nimbler and that would be expected to affect subsequent postural performance . Therefore, the double - leg stance durations were also treated as covariates in the statistical analysis . Repeated measures manova showed no significant group task difference in either average path length or the area subtended by cop peregrination during the first 5 seconds of the single - leg stance (p>0.05). The independent t - test revealed there were significant between - group differences in terms of both total sway path and total sway area for both the single- and dual - tasking . But the paired t - test showed that neither the young nor the older adults had a significant difference in total sway path of the cop or the sway area in the comparison of single- versus dual - tasking (all p>0.05). This study investigated the postural control and cognitive performance of older adults in both single- and dual - tasking . Compared with young adults, older adults showed significantly longer reaction times, higher error rates, longer total sway paths and larger total sway areas when stepping backward with and without a concurrent cognitive task . When dual - tasking, both groups had significantly longer reaction times than when single - tasking, but only older adults showed significantly higher error rates . These results suggest that older adults tend to prioritize postural control over cognition when performing a dual - task . For cognitive performance, the results showed that the older adults had a significantly longer average reaction time, and a higher error rate in the auditory stroop test than the young adults . This was as expected because of the well - understood decline with age in the central processing capacity of the brain20 . When an additional postural task was included, both groups demonstrated increased average reaction times . It was hypothesized that if two tasks were performed simultaneously and exceeded the processing capacity, the performance of one or both tasks would be degraded2 . The competition for attention resources generated in this experimental protocol was sufficient that the cognitive performance of both groups was affected in the dual - tasking condition . These results are comparable to those of a study published by our research group in which the participants stepped down from a 19-cm platform with and without an accompanying auditory stroop test18 . While the cognitive task was the same, the postural task was different from the present study . Nevertheless, those results showed that older adults had significantly longer reaction times and higher error rates in dual - tasking than single - tasking, as well as when compared with young adults in both conditions . However, the present study found young adults had slower reaction times in dual - tasking than in single - tasking . This was probably due to the task sequence randomization adopted in the present study, which might have minimized the practice effect on the cognitive task results . Related to this, lajoie and co - workers examined the effect on auditory reaction time of the maintenance of static posture and gait using eight young and eight older adults . They also found that the reaction times of both groups were fastest during sitting, and concluded that both young and older adults required more cognitive resources for standing and walking than for sitting21 . Bergamin and colleagues7 investigated the effects of different secondary tasks, namely a brooks spatial - memory task (visual construct), counting backwards aloud test (verbal), and mental arithmetic task (cognitive) on a static postural stability task . They found that the verbal task influenced the performance of the postural task most and they ascribed this finding to either vocalization and/or cognitive demands . The adoption of the auditory stroop test in this study, with the pressing of the thumb switch arbitrarily during stepping backward (single - task), eliminated the influence of a secondary physical task on the postural control task . Compared with the younger subjects, the older ones showed significantly longer average sway paths and larger average sway areas in both single- and dual - tasking . This result is in agreement with our previous study on stepping down, which found that older adults have larger body sway than young adults in both single and dual tasks18 . Possible reasons for the increased postural sway include the age - related decline in the speed of torque development in the lower extremities22, delayed onset of postural muscle activation23, and a diminished capacity to detect and integrate somatosensory information24 . All of these may contribute to a greater sway amplitude and a larger sway area . In contrast, the younger subjects faster ankle torque development and quicker postural muscle responses should have helped them restore balance more quickly, leading to better postural performance25 . The older subjects had similar performance in terms of both postural control measures, but their cognitive performance declined significantly in dual - tasking when compared with single - tasking, showing that they were prioritizing postural control . Hypothesis suggested by shumway - cook which proposes that older adults allocate resources to postural control at the expense of cognitive performance in dual - tasking conditions26 . Task prioritization tends to be more obvious when the motor task involves a risk of falling, especially for frail older people with balance impairments for whom a fall could have critical survival significance9 . Backward stepping may be considered as an alternative to stepping down, which is presumably a more challenging postural task18, for assessing the balance ability of older adults and/or identifying those at greater risk of falling . The postural task may be more suitable for frail subjects, such as patients with parkinson disease, who often have backward walking deficits which might lead to backward falls . It has also been suggested that assessing backward walking may be illustrative of the degree of basal ganglia impairment27 . However, more balance - related data should be collected before a cut - off point for differentiation can be definitively set . The dual - task paradigm is more demanding of attention, so it might be a more sensitive test than the single - task protocol for testing postural control28 . Since this was a cross - sectional study, no causality can be attributed . Also, most of the young subjects were university students . The generalizability of the results of the cognitive assessments might be affected due to this limited variety in educational level . Only the auditory stroop test was used in this study . Further studies are needed to examine the motor and cognitive costs when different cognitive tasks are performed during stepping backward . No electromyographic data was collected, so the muscle recruitment strategies adopted by subjects during stepping back remain to be explored . This could be included in future investigations to search for differences in the strategies employed by different subject groups . Future studies might fruitfully compare older fallers with similar non - fallers to find out if there is any difference in postural control performance between them during single- and dual - task activities . It has been suggested that exercise training is beneficial for the static and dynamic balance of older adults with multiple disease conditions performing single and dual tasks29 . However, insufficient evidence has been found to verify these benefits in healthy older adults30 . Therefore, one possible direction for future study is to evaluate intervention trials which may increase the performance of postural control and selective attention while walking backward . High quality studies should compare different exercise modalities, and study the optimal dose of exercise needed to improve dual - task performance and reduce the risk of fall . In conclusion, young adults had better postural control and superior cognitive performance in both single- and dual - tasking as they showed significantly shorter reaction times and lower error rates in the auditory stroop test, as well as shorter total sway paths and smaller total sway areas in the postural task . Although neither group showed a significant difference in postural control between single- and dual - tasking, they had significantly longer reaction times during dual - tasking, indicating older adults tend to prioritize postural control over cognitive performance when dual - tasking.
Investigar si la precisin de las mediciones de la presin intraocular (pio), utilizando la tonometra de rebote sobre las lentes de contacto (lc) desechables de hidrogel (etafilcon a), se ve afectada por la potencia positiva de dichas lentes . El grupo experimental incluy a 26 sujetos, (8 varones, 18 mujeres). Se realiz la medicin de la pio en los ojos derechos de los sujetos, de modo aleatorio, utilizando un tonmetro de rebote (icare). Las lc tenan potencias de + 2,00 d y + 6,00 d. se realizaron mediciones el valor de la pio obtenido con ambas lc fue considerablemente menor al valor sin lc (t del test; p <0,001), aunque no se hall una diferencia significativa entre las dos potencias de las lentes . La tonometra de rebote sobre las lc positivas de hidrogel origina un cierto grado de subestimacin del pio . Este resultado no sufri variacin entre las dos lentes positivas utilizadas en el experimento, a pesar de la gran diferencia de potencia, y por tanto del espesor de las lentes . Los optometristas deberan de tener en cuenta estos resultados en a la hora de medir el pio con un tonmetro de rebote, con lentes de contacto de mayor potencia . Primary open angle glaucoma is a potentially blinding condition . Raised intraocular pressure (iop) is an important risk factor for the development and progression of optic nerve damage in glaucoma, and is the target of both medical and surgical treatment being currently the only treatable risk factor . Measuring iop over a soft contact lens (cl) can be very useful for several reasons . These include avoiding topical anaesthesia, minimizing trauma in conditions of corneal pathology, whenever there is a need to undertake tonometry several times, when the corneal surface is extremely irregular and finally to allow iop measurement without removing the cls . Villani was probably the first to use a soft contact lens with contact tonometry having the aim of avoiding pharmacological anaesthesia . Several studies showed that measurement of iop can be performed over soft contact lenses using the goldmann tonometer,5, 6, 7, 8, 9, 10, 11, 12 the mackay marg tonometer, the tono - pen,14, 15, 16, 17 the gas pneumotonometer,16, 17 the non - contact tonometer18, 19, 20, 21, 22, 23, 24, 25, 26 and the dynamic contour tonometer.11, 27, 28, 29 in 2005 a new handheld device became available to measure iop, the icare rebound tonometer, having the advantage over other instruments that no topical anaesthesia is required . A light magnetized small, disposable probe, characterized by a round plastic tip, is launched towards the eye using a solenoid . The return rate of the probe after it touches the cornea permits information about iop.30, 31 the results are reproducible and reasonably accurate.32, 33 several investigators34, 35, 36, 37, 38, 39, 40 have evaluated the icare tonometer compared with other tonometry devices, showing a reasonable overall correlation and concordance between the iop obtained with the goldmann or pascal types . It has been shown that with the rebound tonometer it is possible to measure iop over soft cls, either hydrogel or silicone hydrogel, with good clinical accuracy.39, 40 however it has been found that the type of material and the power of the cl can cause an underestimation of iop or overestimation.25, 42 the aim of this study was to verify this effect not only for a + 2.00 d cl but also for a higher positive + 6.00 d cl . Other corneal parameters such as thickness and curvature were evaluated to investigate their influence on the measurement . Twenty - six subjects (8 male and 18 female), age range from 21.2 to 48.7 years (mean 28.8; sd 8.9 years), were enrolled in the study . Inclusion criteria were normal corneas (no corneal scarring, corneal pathology or prior corneal surgery), assessed by slit lamp examination and videokeratoscopy, and corneal astigmatism of not more than 2.50 d. contact lens wearers were enrolled only if they had taken their lenses out for 12 h before the experiment . All subjects had been informed about the experiment in detail and had signed the consent document in compliance with the declaration of helsinki before the experiment . All tonometric measurements were carried out with a rebound tonometer (icare; finland oy). Two different spherical powers were used: + 2.00 d and + 6.00 d. to evaluate the effect of power on the measurement of iop, a repeated measurements design was used . Four measures of iop were taken on the right eye of each subject . The first measurement (rt1) and the last measurement (rt4) were taken without cls . The second and third measurements were performed over the two different powers of the cls . In order to prevent a possible effect on iop of the repetition of the measurement or the insertion and removal of cls, each subject was assigned randomly to one of two different sequences (table 2). In order to control accommodation, that might influence the measurement of iop during experiments,45, 46 the left eye (corrected with cls for any hyperopic defect) viewed a distance target (6/24 or 0.6 logmar). One investigator assigned each subject randomly to one experimental condition and fitted all cls to each subject . A second investigator, experienced in rebound tonometry, performed all iop measurements on each subject for all conditions in order to reduce between - observer bias . A third investigator checked the position of the rebound tonometer probe on the cornea during the measurement . This control was performed because it has been demonstrated that the location of the tonometer on the cornea can affect the measurement of iop47, 48 even though a recent study showed that the rebound tonometer appears insensitive to misalignments . After the measurement the third investigator read the measure on the display of the tonometer . Thus the measurements were repeated up to the moment the third investigator had two valid readings . The number of measures required to achieve two valid readings was recorded . To reduce between observer and fitter bias all measurements were taken between 1.00 and 3.00 pm in order to minimize the effect of diurnal variation of iop on the results . Before the iop measurements, a corneal topographic map as well as a pachymetric map of each cornea was taken by a scheimpflug camera system (sirius acquiring system; cso, florence, italy) in order to evaluate a possible effect of corneal thickness and curvature on tonometric measurement . Smirnov test was used to evaluate the results for a normal distribution of iop, and corneal parameters data . All the statistical processing used to analyze the comparison between the measurements with and without cls was performed using a value for the latter (rt) that was the mean of the first (rt1) and last measurements (rt4). The strength of the relationship between iop measurements without cls and with the two powers of cls was evaluated using a correlation analysis (r of pearson). Altman plot was used to assess the difference in iop reading with and without the two powers of cls as a function of iop value . A student's paired t test for repeated measurement was applied in order to evaluate the differences between the measurements obtained without cls and with each positive cl . Considering the sample size, the statistical powers of the significant comparisons of paired t test + 2 and rt and + 6 and rt were 0.987 and 0.965 respectively . Any possible relationship between corneal parameters (thickness, curvature, asphericity) and the measurement of iop over positive cls was evaluated using a correlation analysis (r of pearson) between every corneal parameter measure and the difference in iop measurement with and without the cls . Twenty - six subjects (8 male and 18 female), age range from 21.2 to 48.7 years (mean 28.8; sd 8.9 years), were enrolled in the study . Inclusion criteria were normal corneas (no corneal scarring, corneal pathology or prior corneal surgery), assessed by slit lamp examination and videokeratoscopy, and corneal astigmatism of not more than 2.50 d. contact lens wearers were enrolled only if they had taken their lenses out for 12 h before the experiment . All subjects had been informed about the experiment in detail and had signed the consent document in compliance with the declaration of helsinki before the experiment . All tonometric measurements were carried out with a rebound tonometer (icare; finland oy). The cls used were bi - weekly replacement hydrogel (acuvue 2). Two different spherical powers were used: + 2.00 d and + 6.00 d. to evaluate the effect of power on the measurement of iop, a repeated measurements design was used . Four measures of iop were taken on the right eye of each subject . The first measurement (rt1) and the last measurement (rt4) the second and third measurements were performed over the two different powers of the cls . In order to prevent a possible effect on iop of the repetition of the measurement or the insertion and removal of cls, each subject was assigned randomly to one of two different sequences (table 2). In order to control accommodation, that might influence the measurement of iop during experiments,45, 46 the left eye (corrected with cls for any hyperopic defect) viewed a distance target (6/24 or 0.6 logmar). One investigator assigned each subject randomly to one experimental condition and fitted all cls to each subject . A second investigator, experienced in rebound tonometry, performed all iop measurements on each subject for all conditions in order to reduce between - observer bias . A third investigator checked the position of the rebound tonometer probe on the cornea during the measurement . This control was performed because it has been demonstrated that the location of the tonometer on the cornea can affect the measurement of iop47, 48 even though a recent study showed that the rebound tonometer appears insensitive to misalignments . After the measurement the third investigator read the measure on the display of the tonometer . Thus the measurements were repeated up to the moment the third investigator had two valid readings . The number of measures required to achieve two valid readings was recorded . To reduce between observer and fitter bias all measurements were taken between 1.00 and 3.00 pm in order to minimize the effect of diurnal variation of iop on the results . Before the iop measurements, a corneal topographic map as well as a pachymetric map of each cornea was taken by a scheimpflug camera system (sirius acquiring system; cso, florence, italy) in order to evaluate a possible effect of corneal thickness and curvature on tonometric measurement . Data were analyzed using statistica (statsoft inc ., tulsa, ok, usa) v.6.0 for windows . Smirnov test was used to evaluate the results for a normal distribution of iop, and corneal parameters data . All the statistical processing used to analyze the comparison between the measurements with and without cls was performed using a value for the latter (rt) that was the mean of the first (rt1) and last measurements (rt4). The strength of the relationship between iop measurements without cls and with the two powers of cls was evaluated using a correlation analysis (r of pearson). Altman plot was used to assess the difference in iop reading with and without the two powers of cls as a function of iop value . A student's paired t test for repeated measurement was applied in order to evaluate the differences between the measurements obtained without cls and with each positive cl . Considering the sample size, the statistical powers of the significant comparisons of paired t test + 2 and rt and + 6 and rt were 0.987 and 0.965 respectively . Any possible relationship between corneal parameters (thickness, curvature, asphericity) and the measurement of iop over positive cls was evaluated using a correlation analysis (r of pearson) between every corneal parameter measure and the difference in iop measurement with and without the cls . Mean corneal astigmatism of the subjects right eyes was 0.73 0.37 d (range 0.15/1.60 d). Twenty right eyes had with the rule astigmatism (steepest corneal meridian 90 20), three had against the rule astigmatism (steepest corneal meridian 180 20) and three had oblique astigmatism (steepest meridian between 21 and 69 or 111 and 159). The right eye central corneal thickness and the corneal thickness at the pupil centre was 540 32 m and 542 32 m respectively . Mean spherical equivalent refraction of the subjects right eyes was 2.10 2.28 d (range 0.75/7.13 d). Mean of intraocular pressure without cls and with + 2.00 d and + 6.00 d was 19.0 4.1 mmhg (range: 9.027.5), 17.6 4.6 mmhg (range: 10.525.0) and 17.8 4.1 mmhg (range: 10.824.8) respectively (fig . The correlations between iop measurements without cls and with the positive cls were> 0.9 (p <0.05 in all cases). Altman plots for the comparison between the measurement with + 2.00 d cl and without cl (rt) and the measurement with + 6.00 d cl and without cl respectively are shown in figure 2, figure 3 . 2) shows that there is no proportional bias: no significant trend was detected for differences between + 2.00 d and rt measurements as a function of their mean value (r = 0.30, p = 0.141). 3) for the + 6.00 d and rt measurements gave a similar result, mean value (r = 0.006, p = 0.98). Table 3 gives the paired - samples t - test between the measurements with and without cls . All the comparisons between the measurements without cls and the measurements with the positive cls were significant . The comparison between measures obtained with the + 2.00 d and + 6.00 d cls was not significant . In order to evaluate if corneal parameters such as thickness, curvature or asphericity were affecting the difference in iop measurement with and without cls, a coefficient of correlation was calculated . None of these parameters correlated with the difference between iop values obtained with + 2.00 d and rt or + 6.00 d and rt . Iop measurement with a rebound tonometer over positive hydrogel cls provides statistically significant lower values than the measurement without cls . The decrease is not proportional to the increase in refractive power of the cl . Despite the fact the difference is statistically significant, from a clinical point of view this difference is minimal because it is almost at the cut off value of more than 1.5 mmhg that is considered relevant . Although the rebound tonometer is among the most recent instruments used today, it is gaining a relative acceptance especially for the simplicity of the procedure and that topical anaesthesia and fluorescein are not required . The results are reproducible and reasonably accurate.32, 33 furthermore it can be used in challenging patients such as children51, 52 or the disabled . It has been shown that with the rebound tonometer it is possible to measure iop over soft cls, either hydrogel (etafilcon a) or silicone hydrogel (senofilcon a), with good accuracy . In this study the underestimation of iop was greater for power + 2.00 d compared to cls of negative power . Our results appear to conflict with previous studies where the tonometry measurement was taken using conventional applanation tonometers, especially the air puff type, where positive soft cls contribute to an increase in the value of iop.7, 20, 21, 22, 23, 54, 55, 56 the results from tonometry can be influenced by the characteristics of the patient's cornea such as corneal thickness57, 58 corneal curvature and corneal biomechanical factors . True iop will be overestimated in eyes with thick corneas, a steep corneal curvature and high corneal hysteresis . However, most researchers have considered the effect of tonometry based on applanation principles . Regarding the rebound tonometer, chui et al ., have found that the result is affected by biomechanical corneal properties but not corneal thickness . Jorge et al ., found that although corneal thickness can play a role in rebound tonometry, individual physiological variations of biomechanical corneal properties such as the elastic and viscoelastic responses, may be more relevant factors . In a recent cross - sectional study, the effect of plano power lotrafilcon a contact lenses in situ on iop measurement from three portable tonometers, including icare, a statistically significant overestimation of 1.00 mmhg was found between iop measurements obtained with the rebound tonometer with and without cls . This result could be attributed to the higher modulus of lotrafilcon a which should offer more resistance to the deformation than cls in senofilcon a (used in the previous study), and etafilcon a (used in the previous and present studies). A similar increment in iop was found by anton et al ., when measuring iop with a rebound tonometer over a silicone hydrogel soft cl (balafilcon a) characterized by a water content of 36%, back vertex power of plano and a central thickness of 0.07 mm . The decrement in iop found in the present study could be attributed to low resistance to deformation produced by etafilcon a a hydrogel characterized by high water content . In this study, we have had the opportunity to compare the effect on iop induced by two cls having positive powers, and significantly different thickness . The presence of the same trend in the change of iop induced by the two cls leads us to believe, in accordance with chui et al ., that the thickness at the centre of the cornea, or cornea together with the cl, does not affect the value of iop assessed with a rebound tonometer . In conclusion, the measurement of iop while positive power cls having a water content of 58%, such as those in etafilcon a, are worn, tends to give lower values than those obtained without cls . Eye care practitioners should keep this in mind when analyzing iop values or remove positive cls before performing rebound tonometry measurements.
Interacting with other human beings is a basic element of daily life, yet not a trivial challenge . Many joint actions between adults are highly sophisticated, but are performed with apparent ease (knoblich and jordan 2003; sebanz et al . 2006). Cooking a meal with friends, lifting a heavy bag together, or dancing with others are only a few of numerous examples for joint actions . In the early years of life, however, children still have difficulties coordinating their actions with those of others (brownell et al . The question arises as to which mechanisms underlie the development of joint action capabilities . For adults, it has been shown that a key factor for successful joint action is the involvement of the motor system of the brain . Motor - related brain activity is not only observed during execution of one s own actions but is also important for predicting and incorporating a partner s actions (see bekkering et al . Recent findings by kourtis and colleagues indicate that the motor system in adults is more strongly activated when they predict actions of a joint action partner as compared to those of an individual actor . For adults, being engaged in a joint action thus has an effect on the involvement of their own motor system when observing the actions of another person (kourtis et al . An interesting possibility to investigate the underlying neurocognitive mechanisms of action observation during joint action is to look at early stages of joint action in development . To date, it is unclear whether similar effects of motor system involvement can be observed when young children are engaged in a joint action . Therefore, the aim of the current study was to investigate young children s brain activity around the age that they begin to incorporate others actions into their own action performance more successfully (cf . More specifically, we examined whether being engaged in a joint action modulates the involvement of young children s own motor system when observing the actions of another person, and if so, how this motor involvement is associated with their joint action performance . Early forms of joint action such as handing over a toy can be observed already in infancy (carpenter 2009). However, infants cooperation with adults often requires extensive scaffolding by the adult action partner (see e.g., warneken and tomasello 2007). Despite infants motivation to act jointly with another person (see carpenter 2009, for a review), the nature of their cooperation attempts is still not entirely mutual . It is during the second and third year of life that joint actions with peers and adults become more reciprocal and sophisticated (brownell et al ., young children can cooperate successfully in simple interactive games like letting a ball bounce on a little trampoline by holding and moving the frame of the trampoline jointly (warneken et al . Children aged 2 years and older reliably solve simple cooperation tasks with peers (brownell and carriger 1990) and show improvement in monitoring their partner s actions and integrating them into their own action performance (brownell et al ., children have been shown to coordinate their actions in a sequential button - pressing task as accurately with an adult partner as when acting on their own (meyer et al ., children reliably succeed in joint actions that involve complementary roles for the two action partners (ashley and tomasello 1998), something which appears to be difficult for younger children (hunnius et al ., the ability to successfully cooperate with others is a skill that develops gradually during early childhood . From a neurocognitive perspective, successful joint actions require the brain to connect observed actions of others with their own motor system in order to adapt own actions accordingly . First developmental studies on action execution and action perception in young children reveal mechanisms of motor system involvement comparable to those found in adults (see e.g., hari and kujala 2009; rizzolatti and craighero 2004, for adult studies): already during infancy, motor - related brain activity has been found for action execution as well as for the observation of another person s actions (lepage and thoret 2006; marshall et al . 2010; nystrm 2008; shimada and hiraki 2006; southgate et al . Motor involvement during one s own actions and during the observation of others actions can be studied by measuring oscillatory activity in the eeg signal . In both children and adults, motor activation has been associated with a power reduction in the mu- and beta - frequency bands above motor areas (caetano et al . A recent eeg study investigated 14-month - old infants brain activity while they were pressing a button on their own or observing an adult doing the same (marshall et al . Decreased power was found in the infant equivalent of the mu - frequency range over central electrode sites . This is in line with previous findings of reduction in mu - power during action execution and observation in adults (caetano et al . Moreover, the involvement of the motor system during action observation was shown to be modulated by children s action experience (van elk et al . More motor involvement indicated by less mu- and beta - power over motor areas was found during observation of an action that children had more experience with . Until now, developmental brain imaging studies have focused on execution and observation of individual actions . However, how young children s brain activity is modulated in the context of real - time joint action is still unexplored . Previous research in adults reveals that the involvement in a joint action has an effect on the neurocognitive processes associated with the other person s actions (see bekkering et al . For instance, monitoring a person s action in a cooperative context showed an early component of error - related brain activation that was absent when the other person acted in a competitive context (koban et al . 2010). In a recent set of experiments, kourtis and colleagues (2010) investigated the involvement of the motor system when observing a joint action partner or an uninvolved actor . In one of their experiments, they created a social context in which two action partners were facing each other, while an individual actor was sitting next to them . In a go / no - go paradigm, motor - related brain activity was assessed using eeg . A stronger decrease in beta - power and changes in motor - related potentials, which reflected stronger anticipatory motor activation, were found for one s own action partner than for the individual actor (kourtis et al . Hence, the mere involvement in a joint activity with another person modulated the observer s motor activation when observing the partner s actions as compared to observing the actions of a person not involved in the joint action . As indicated by previous research, the involvement of one s own motor system plays a crucial role in understanding and predicting others actions (see sebanz and knoblich 2009, for a review). When acting jointly, it is especially important to be able to understand and predict the other person s actions so that it is possible to constantly adjust one s own actions to those of the partner . Therefore, the activation of the motor system, which is thought to support action understanding and action prediction (de lange et al . 2008; iacoboni et al . 2005; rizzolatti and sinigaglia 2010; southgate et al . 2010), is of special relevance in joint actions . Still, little is known about the role of the motor system in early joint action development . The current study is the first to investigate motor involvement in young children who are engaged in a joint action with another person . In particular, we were interested in whether motor - related brain activity while observing another person s actions is modulated by being involved in a common joint action . Furthermore, the aim of this study was to shed light on the link between the activation of young children s motor system during action observation and their joint action performance . In order to investigate young children s motor involvement in joint action, we simultaneously assessed 3-year - olds brain activity and their performance in a joint action task . The experimental setup consisted of a simple computerized button - pressing game in which a cartoon figure had to be moved up a ladder by pushing two buttons alternately (cf . The game was played in different conditions that varied the children s involvement in the joint action . In the joint action condition, children were playing together with an adult partner, taking turns to push the two buttons . In the joint action observation condition, the children watched the same adult play the game together with a third adult actor . To determine modulations in the child s motor system activation, we compared children s brain response during their partner s actions in the two conditions . Finally, we correlated the outcome of the eeg analysis with children s joint action performance to examine the relation between their motor system involvement for the partner s actions and their own action performance . The final sample consisted of seven 3-year - old children (5 boys) with a mean age of 36.7 months (sd = .99). Seventeen of these 29 participants were excluded due to lack of or bad eeg recording traces resulting from insufficient time (i.e., limited by the little participants patience) to lower impedances in the preparation phase . Another twelve participants were excluded, either due to a lack of at least 8 movement- and artifact - free trials per condition (n = 10) or due to experimental errors (n = 2). The high dropout rate in the current experiment is consistent with other developmental studies assessing electrophysiological recordings (cf . 2007; southgate et al . 2010). During a sequential joint action game, we recorded brain activity and performance accuracy of the 3-year - old children . Figure 1 illustrates the experimental setup that consisted of a simple computerized button - pressing game proved to be suitable for children of this age in a previous study (meyer et al . 2010). In this game, a cartoon figure of a frog could be moved up a ladder by alternately pushing two buttons . As can be seen in fig . 1, the visual stimuli were presented on a wide - screen that was tilted to increase the height of the presented ladder and thereby the number of steps required to reach the top . In total, the ladder consisted of 42 steps that were shown on the screen . At the top of the ladder, there was a target location for the frog represented by a cartoon figure of a pig, the frog s friend on a cloud . In front of the screen, we placed two custom - made buttons to control the game and a board with the contours of two hands indicating starting and resting positions of the hands . The two buttons were interconnected via a tilt mechanism such that pushing one button down caused the other button to move up . More precisely, a right button press triggered the frog to move up using its right leg and pressing the left button moved up the left leg of the frog, so that alternating left button presses also elicited a short beep tone (60 ms duration) in order to keep the child s interest and attention . With each button press, eeg markers were sent such that button presses could be traced back in the eeg recordings . 1the experimental setup of the joint button - pressing game . In front of a tilted wide - screen, we positioned two chess - clock buttons and resting positions marked by hand contours . By pressing the two buttons alternately, a cartoon figure could be moved up a ladder on the screen the experimental setup of the joint button - pressing game . In front of a tilted wide - screen, we positioned two chess - clock buttons and resting positions marked by hand contours . By pressing the two buttons alternately, a cartoon figure could be moved up a ladder on the screen each participant was involved in three different conditions of the game: a joint action condition, a joint action observation condition, and an individual action condition . The focus of this paper is on children s motor - related brain activity during action observation with respect to joint actions . We were interested in whether the motor system of the 3-year - olds was activated more strongly while observing others actions when involved in a joint action as compared to watching two people acting jointly without being involved . The same person acted both as the child s joint action partner and together with a third actor in the joint action observation condition . In the current study, we concentrate on the results of the joint action and the joint action observation condition.1 in the joint action condition, we instructed the children to push the right button with their right hand in turns with their adult action partner (actor1) who pushed the left button with her left hand . More specifically, the button - pressing action was supposed to start with the hand on the resting position, which was marked by drawings of hand contours on a board in front of the buttons (see fig . 1). Starting from this position, the action was executed by pushing the respective button and ended when the hand was placed back on the resting position . We thereby aimed to prevent children from leaving their hands on the button throughout the joint play and introduced a standardized action pattern that was comparable across conditions . During the measurement, the children sat on their parent s lap on a chair to the right of actor1 . In the joint action observation condition, the children watched two adults (actor1 and actor2) playing the same game together . While actor1 and actor2 were playing jointly, actor2 sat between the child and actor1 such that the child would have to move only minimally to the right . For all children, the children were not explicitly instructed where to look during the game, but in subsequent steps, only data of trials were included during which children looked at the experimenter, the buttons, or the screen (see eeg data analysis section). Video recordings of the entire measurement session were made and aligned with the experimental events on the screen, and children s eeg and button presses were recorded . For demonstration purposes, the joint action observation condition always preceded the joint action condition . Depending on the attention span of the children, we additionally included another run of the joint action observation condition after the joint action condition . Six out of the seven participants therefore watched actor1 and actor2 play both before and after they played together with actor1 . Before pooling together the data of the observation condition from the two time points, we tested for order effects . To make sure that children s motor activation did not differ significantly between the two time points, we compared activity during action observation of actor1 s button press (t = 450 to 0 ms). Since no differences in mu- and beta - power were found between data collected before and after the children had played themselves (for details, see eeg data analysis), the data of the joint action observation condition were subsequently pooled . Electrophysiological recordings were conducted using child - sized eeg caps with 30 electrode sites on the scalp . The ag / agcl active electrodes were placed in an acticap (brain products, munich), arranged in the 1020 system, and referenced to electrode fcz over the central midline . The signal was amplified using a 32-channel brainamp dc eeg amplifier, band - pass filtered (.1125 hz), and digitized at 500 hz . We strived to keep all impedances below 60 k. we analyzed the data using fieldtrip, an open source matlab (version 7.0, themathworks, inc .) Toolbox developed at the donders institute for brain, cognition and behaviour (http://www.ru.nl/neuroimaging/fieldtrip). The eeg data were locked to the button press of the first experimenter (actor1) and determined 450 ms before and 450 ms after the button was pressed . During this time, the children were observing actor1 s actions and the effect on the screen when actor1 was either their joint action partner (joint action condition) or the joint action partner of actor2 (joint action observation condition). By including exclusively data from actor1, we kept the comparison between the two conditions constant . To examine the involvement of the motor system in these two conditions, we focused on electrodes c3 and c4 over motor cortices . As mentioned in the introduction, power decrease in the mu(711 hz)- and beta(1721 hz)-frequency range over motor areas is associated with motor activation (cf . Hari 2006) and thus is the focus of the current analysis . On the basis of the video recordings of the measurement session, trials were rejected if children moved their hands or did not pay attention to the game (i.e., when they looked at neither the experimenter, nor the buttons, nor the screen) during the critical period of the experimenter s action (i.e., the hand movement toward the button). Since it was an interactive game in which we relied on children s spontaneous behavior, many trials had to be excluded due to children moving during the window of interest (t = 450 to 0 ms). Participants with less than 8 trials per condition were excluded from the analyses (see participants). We visually inspected the remaining trials to exclude eeg artifacts (such as noisy channels or eye blinks). As a result, on average, 15 trials remained for the joint action condition (range 836) and 35 trials for the joint action observation condition (range 1860). A dft filter2 was used to remove line noise from the data, and for each trial, we took out the offset by subtracting the mean signal of the entire trial . We then calculated time - resolved spectral power estimates using the fourier transform in combination with a hanning taper . For this, we used a 300-ms sliding time window that was advanced in steps of 50 ms . We obtained separate tfrs for the joint action condition and the joint action observation condition . To contrast children s brain response in these two conditions, we computed the normalized difference per time frequency sample between the two conditions ([tfr actor1 as joint partner tfr actor1 as partner of actor2]/[tfr actor1 as joint partner + tfr actor1 as partner of actor2]) (cf . The eeg data were locked to the button press of actor1, which is denoted as zero . Hence, children observed actor1 moving her hand toward the button from about 450 ms to 0 . At zero, the button press of actor1 made the frog on the screen move upward . In the period of 0450 ms, children were preparing to press the button themselves in the joint action condition, while it was actor2 s turn in the joint action observation condition . At the same time, actor1 was placing her hand back on the resting position in front of the button.fig . 2a time - resolved normalized difference in power at electrode site c3 . Power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . At time 0, actor1 pushed the button that moved up a cartoon figure on the screen . Before time 0, actor1 moved her hand toward the button . After time 0, actor1 moved her hand back to the resting position, while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . Frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography (see methods and results). B correlation between the individual beta - power difference and the percentage of errors children made during the joint game . C topography of the normalized beta - power difference, including the data points marked by the white box . Power differences are displayed on seven electrodes (only electrodes were used that were sufficiently noise - free for all seven children) a time - resolved normalized difference in power at electrode site c3 . Power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . At time 0, actor1 pushed the button that moved up a cartoon figure on the screen . After time 0, actor1 moved her hand back to the resting position, while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . White boxes indicate the time frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography (see methods and results). B correlation between the individual beta - power difference and the percentage of errors children made during the joint game . C topography of the normalized beta - power difference, including the data points marked by the white box . Power differences are displayed on seven electrodes (only electrodes were used that were sufficiently noise - free for all seven children) in the statistical evaluation of the electrophysiological data, we determined whether power estimates during observation of the goal - directed action of actor1 differed significantly between conditions . We used the window of 450 ms prior to the button press until the button press (t = 450 to 0 ms) and the frequency bands of 711 hz (mu) and 1721 hz (beta). 2) were then averaged over the respective frequency range and time window . By means of one - sample t tests analogous to this analysis, we evaluated the data of the joint action observation condition collected before and after the joint action condition (with t = 450 to 0 ms; mu: 711 hz; beta: 1721 hz). Due to the relatively small sample size of 7 participants, it might be argued that effects in the mu- and beta - power could be driven by extreme outliers . To exclude this possibility and to provide an overview of the strength of the observed effect in both mu- and beta - power, we ran complementary analyses on an individual participant level . For this purpose, we estimated the power in the mu- and beta - frequency ranges as described earlier for each individual trial per condition and participant . To determine the average power in the two conditions for each participant, we averaged power estimates of mu - power of all trials per participant over the time window of 450 to 0 ms . Using the same approach, we obtained average power values in the beta - frequency range for the two conditions per child . Subsequently, we evaluated the difference in power between the two conditions (separately for mu- and beta - power) on an individual participant basis using independent - sample t tests with trials as units of observation . Additionally to the general power difference on a group and individual level, we were interested in how time - locked these differences were to the actions of the joint action partner . In other words, we examined whether an increase in motor activation (reflected by less power in the mu- and beta - band) was specifically locked to actor1 s actions or rather reflected a general activation of the children s motor system . To test this, we compared the normalized difference in the mu- and beta - frequency range for the two conditions before and after actor1 pressed the button . If the children s motor system was generally more activated in the joint action condition, we would expect no difference between these two time periods of actor1 s action . However, if the motor activation was time - locked to the actions of children s joint partner, we would expect the power differences to be different before and after actor1 s button press . Therefore, we also calculated the average normalized difference of mu - and beta - power for the time period after actor1 s button press (t = 0450 ms). To evaluate the statistical difference between the two time periods, we used paired - sample t tests with time period (before vs. after actor1 s button press) as an independent factor, one for testing differences in the mu- and one for differences in the beta - frequency range . To further evaluate the relation of mu- and beta - power during observation of joint action with regard to children s own joint action performance, we correlated the eeg results with the behavioral button press data . On the basis of the significant power differences between conditions, we chose representative time frequency windows (see white boxes in fig . 2). Frequency windows because they represent strongest continuous effect windows time - locked to the button press of actor1 . The normalized difference in power of the respective frequency and time was then averaged to obtain a representative effect value . We subsequently correlated those effect values with the percentage of errors children made when acting jointly . The percentage of errors indicates how often the 3-year - olds pushed their button when it was not their turn . The percentage was computed as number of incorrect button presses of the child (i.e., button presses when it was actor1 s turn to press) divided by the total number of times the child pushed the button when playing the joint game . We used a pearson correlation across participants to test the relation between the effect value of each specified window (see white boxes in fig . 2) and the percentage of errors . The final sample consisted of seven 3-year - old children (5 boys) with a mean age of 36.7 months (sd = .99). Seventeen of these 29 participants were excluded due to lack of or bad eeg recording traces resulting from insufficient time (i.e., limited by the little participants patience) to lower impedances in the preparation phase . Another twelve participants were excluded, either due to a lack of at least 8 movement- and artifact - free trials per condition (n = 10) or due to experimental errors (n = 2). The high dropout rate in the current experiment is consistent with other developmental studies assessing electrophysiological recordings (cf . During a sequential joint action game, we recorded brain activity and performance accuracy of the 3-year - old children . Figure 1 illustrates the experimental setup that consisted of a simple computerized button - pressing game proved to be suitable for children of this age in a previous study (meyer et al . 2010). In this game, a cartoon figure of a frog could be moved up a ladder by alternately pushing two buttons . As can be seen in fig . 1, the visual stimuli were presented on a wide - screen that was tilted to increase the height of the presented ladder and thereby the number of steps required to reach the top . In total, the ladder consisted of 42 steps that were shown on the screen . At the top of the ladder, there was a target location for the frog represented by a cartoon figure of a pig, the frog s friend on a cloud . In front of the screen, we placed two custom - made buttons to control the game and a board with the contours of two hands indicating starting and resting positions of the hands . The two buttons were interconnected via a tilt mechanism such that pushing one button down caused the other button to move up . More precisely, a right button press triggered the frog to move up using its right leg and pressing the left button moved up the left leg of the frog, so that alternating left button presses also elicited a short beep tone (60 ms duration) in order to keep the child s interest and attention . With each button press, eeg markers were sent such that button presses could be traced back in the eeg recordings . 1the experimental setup of the joint button - pressing game . In front of a tilted wide - screen, we positioned two chess - clock buttons and resting positions marked by hand contours . By pressing the two buttons alternately, a cartoon figure could be moved up a ladder on the screen the experimental setup of the joint button - pressing game . In front of a tilted wide - screen, we positioned two chess - clock buttons and resting positions marked by hand contours . By pressing the two buttons alternately, a cartoon figure could be moved up a ladder on the screen each participant was involved in three different conditions of the game: a joint action condition, a joint action observation condition, and an individual action condition . The focus of this paper is on children s motor - related brain activity during action observation with respect to joint actions . We were interested in whether the motor system of the 3-year - olds was activated more strongly while observing others actions when involved in a joint action as compared to watching two people acting jointly without being involved . The same person acted both as the child s joint action partner and together with a third actor in the joint action observation condition . In the current study, we concentrate on the results of the joint action and the joint action observation condition.1 in the joint action condition, we instructed the children to push the right button with their right hand in turns with their adult action partner (actor1) who pushed the left button with her left hand . More specifically, the button - pressing action was supposed to start with the hand on the resting position, which was marked by drawings of hand contours on a board in front of the buttons (see fig . 1). Starting from this position, the action was executed by pushing the respective button and ended when the hand was placed back on the resting position . We thereby aimed to prevent children from leaving their hands on the button throughout the joint play and introduced a standardized action pattern that was comparable across conditions . During the measurement, the children sat on their parent s lap on a chair to the right of actor1 . In the joint action observation condition, the children watched two adults (actor1 and actor2) playing the same game together . While actor1 and actor2 were playing jointly, actor2 sat between the child and actor1 such that the child would have to move only minimally to the right . For all children, the same first experimenter (actor1) acted as their joint action partner . The children were not explicitly instructed where to look during the game, but in subsequent steps, only data of trials were included during which children looked at the experimenter, the buttons, or the screen (see eeg data analysis section). Video recordings of the entire measurement session were made and aligned with the experimental events on the screen, and children s eeg and button presses were recorded . For demonstration purposes, depending on the attention span of the children, we additionally included another run of the joint action observation condition after the joint action condition . Six out of the seven participants therefore watched actor1 and actor2 play both before and after they played together with actor1 . Before pooling together the data of the observation condition from the two time points, we tested for order effects . To make sure that children s motor activation did not differ significantly between the two time points, we compared activity during action observation of actor1 s button press (t = 450 to 0 ms). Since no differences in mu- and beta - power were found between data collected before and after the children had played themselves (for details, see eeg data analysis), the data of the joint action observation condition were subsequently pooled . Electrophysiological recordings were conducted using child - sized eeg caps with 30 electrode sites on the scalp . The ag / agcl active electrodes were placed in an acticap (brain products, munich), arranged in the 1020 system, and referenced to electrode fcz over the central midline . The signal was amplified using a 32-channel brainamp dc eeg amplifier, band - pass filtered (.1125 hz), and digitized at 500 hz . We analyzed the data using fieldtrip, an open source matlab (version 7.0, themathworks, inc .) Toolbox developed at the donders institute for brain, cognition and behaviour (http://www.ru.nl/neuroimaging/fieldtrip). The eeg data were locked to the button press of the first experimenter (actor1) and determined 450 ms before and 450 ms after the button was pressed . During this time, the children were observing actor1 s actions and the effect on the screen when actor1 was either their joint action partner (joint action condition) or the joint action partner of actor2 (joint action observation condition). By including exclusively data from actor1, we kept the comparison between the two conditions constant . To examine the involvement of the motor system in these two conditions, we focused on electrodes c3 and c4 over motor cortices . As mentioned in the introduction, power decrease in the mu(711 hz)- and beta(1721 hz)-frequency range over motor areas is associated with motor activation (cf . Hari 2006) and thus is the focus of the current analysis . On the basis of the video recordings of the measurement session, trials were rejected if children moved their hands or did not pay attention to the game (i.e., when they looked at neither the experimenter, nor the buttons, nor the screen) during the critical period of the experimenter s action (i.e., the hand movement toward the button). Since it was an interactive game in which we relied on children s spontaneous behavior, many trials had to be excluded due to children moving during the window of interest (t = 450 to 0 ms). Participants with less than 8 trials per condition were excluded from the analyses (see participants). We visually inspected the remaining trials to exclude eeg artifacts (such as noisy channels or eye blinks). As a result, on average, 15 trials remained for the joint action condition (range 836) and 35 trials for the joint action observation condition (range 1860). A dft filter2 was used to remove line noise from the data, and for each trial, we took out the offset by subtracting the mean signal of the entire trial . We then calculated time - resolved spectral power estimates using the fourier transform in combination with a hanning taper . For this, we used a 300-ms sliding time window that was advanced in steps of 50 ms . We obtained separate tfrs for the joint action condition and the joint action observation condition . To contrast children s brain response in these two conditions, we computed the normalized difference per time frequency sample between the two conditions ([tfr actor1 as joint partner tfr actor1 as partner of actor2]/[tfr actor1 as joint partner + tfr actor1 as partner of actor2]) (cf . The eeg data were locked to the button press of actor1, which is denoted as zero . Hence, children observed actor1 moving her hand toward the button from about 450 ms to 0 . At zero, the button press of actor1 made the frog on the screen move upward . In the period of 0450 ms, children were preparing to press the button themselves in the joint action condition, while it was actor2 s turn in the joint action observation condition . At the same time, actor1 was placing her hand back on the resting position in front of the button.fig . Power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . At time 0, actor1 pushed the button that moved up a cartoon figure on the screen . Before time 0, actor1 moved her hand toward the button . After time 0, actor1 moved her hand back to the resting position, while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . Frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography (see methods and results). B correlation between the individual beta - power difference and the percentage of errors children made during the joint game . C topography of the normalized beta - power difference, including the data points marked by the white box . Power differences are displayed on seven electrodes (only electrodes were used that were sufficiently noise - free for all seven children) a time - resolved normalized difference in power at electrode site c3 . Power differences represent the contrast between the observation of actor1 s actions when children were involved in the joint action and when they were not involved . At time 0, actor1 pushed the button that moved up a cartoon figure on the screen . Before time 0, actor1 moved her hand toward the button . After time 0, actor1 moved her hand back to the resting position, while it is the child s next turn in the joint action condition and actor2 s next turn in the joint action observation condition . Frequency windows of the effects for which we evaluated the correlation with joint action performance and the topography (see methods and results). B correlation between the individual beta - power difference and the percentage of errors children made during the joint game . C topography of the normalized beta - power difference, including the data points marked by the white box . Power differences are displayed on seven electrodes (only electrodes were used that were sufficiently noise - free for all seven children) in the statistical evaluation of the electrophysiological data, we determined whether power estimates during observation of the goal - directed action of actor1 differed significantly between conditions . We used the window of 450 ms prior to the button press until the button press (t = 450 to 0 ms) and the frequency bands of 711 hz (mu) and 1721 hz (beta). 2) were then averaged over the respective frequency range and time window . By means of one - sample t tests analogous to this analysis, we evaluated the data of the joint action observation condition collected before and after the joint action condition (with t = 450 to 0 ms; mu: 711 hz; beta: 1721 hz). Due to the relatively small sample size of 7 participants, it might be argued that effects in the mu- and beta - power could be driven by extreme outliers . To exclude this possibility and to provide an overview of the strength of the observed effect in both mu- and beta - power, we ran complementary analyses on an individual participant level . For this purpose, we estimated the power in the mu- and beta - frequency ranges as described earlier for each individual trial per condition and participant . To determine the average power in the two conditions for each participant, we averaged power estimates of mu - power of all trials per participant over the time window of 450 to 0 ms . Using the same approach, we obtained average power values in the beta - frequency range for the two conditions per child . Subsequently, we evaluated the difference in power between the two conditions (separately for mu- and beta - power) on an individual participant basis using independent - sample t tests with trials as units of observation . Additionally to the general power difference on a group and individual level, we were interested in how time - locked these differences were to the actions of the joint action partner . In other words, we examined whether an increase in motor activation (reflected by less power in the mu- and beta - band) was specifically locked to actor1 s actions or rather reflected a general activation of the children s motor system . To test this, we compared the normalized difference in the mu- and beta - frequency range for the two conditions before and after actor1 pressed the button . If the children s motor system was generally more activated in the joint action condition, we would expect no difference between these two time periods of actor1 s action . However, if the motor activation was time - locked to the actions of children s joint partner, we would expect the power differences to be different before and after actor1 s button press . Therefore, we also calculated the average normalized difference of mu - and beta - power for the time period after actor1 s button press (t = 0450 ms). To evaluate the statistical difference between the two time periods, we used paired - sample t tests with time period (before vs. after actor1 s button press) as an independent factor, one for testing differences in the mu- and one for differences in the beta - frequency range . To further evaluate the relation of mu- and beta - power during observation of joint action with regard to children s own joint action performance, we correlated the eeg results with the behavioral button press data . On the basis of the significant power differences between conditions, we chose representative time frequency windows (see white boxes in fig . 2). Frequency windows because they represent strongest continuous effect windows time - locked to the button press of actor1 . The normalized difference in power of the respective frequency and time was then averaged to obtain a representative effect value . We subsequently correlated those effect values with the percentage of errors children made when acting jointly . The percentage of errors indicates how often the 3-year - olds pushed their button when it was not their turn . The percentage was computed as number of incorrect button presses of the child (i.e., button presses when it was actor1 s turn to press) divided by the total number of times the child pushed the button when playing the joint game . We used a pearson correlation across participants to test the relation between the effect value of each specified window (see white boxes in fig . 2) and the percentage of errors . To examine whether young children s involvement in joint action modulates their motor activation for others actions, we focused the eeg analysis on power differences in frequency bands (mu: 711 hz, beta: 1721 hz) and electrode sites (c3, c4) associated with motor activation in the brain (cf . We were mainly interested in the contrast between children s brain activity while observing the actions of their joint action partner (joint action condition) and of the same person acting as the joint action partner of a third person (joint action observation condition). Figure 2 illustrates the difference in activity when children were observing actor1 acting as their own joint action partner and as the joint action partner of another person (actor2). More specifically, the figure shows the normalized difference in power estimated for frequencies 530 hz at electrode c3 . Since results for electrode c4 did not show any significant difference in either of the frequency bands for the two conditions, the subsequent results only include data of electrode c3 (see topography in fig . 2). Data of the joint action observation condition obtained before and after the joint action condition did not differ significantly (all p>.05). Therefore, further reported results include pooled data of the joint action condition . Cold colors in the tfr of fig . 2 represent less power for observing actor1 as their own joint action partner, whereas warm colors represent more power for observing actor1 as their joint action partner . The difference between the conditions is most pronounced in two frequency bands, namely around 10 hz and around 18 hz . In both bands, there is less power when children are observing their own joint action partner than when observing actor1 playing together with a third person (actor2). Previous studies have associated less power in these frequency ranges with more motor activation (cf . Pfurtscheller and lopes da silva 1999). Consequently, the current findings of decreased power indicate more motor involvement in the joint action condition compared with the joint action observation condition . For statistical evaluation, we analyzed the power differences in the time before the button was pushed down . In both the mu- and the beta - frequency range, the normalized difference was significantly different from zero (mu, t(6) = 3.49, p = .013, r = .81; beta, t(6) = 5.06, this indicates that the 3-year - olds showed significantly more motor involvement when observing their own joint partner acting compared with observing the same person in joint action with another person . Figure 3a and b show the resulting average power for each participant separately for the two conditions (joint action observation condition: represented in blue; joint action condition: represented in green). 3a . The same data pattern, namely lower average power for the joint action condition compared with the joint action observation condition, can be seen in six out of seven participants . Using single - subject statistics, this tendency in mu - power differences reaches significance in two participants (participant 1: t(35.976) = 2.39, p = .000, r = .37; participant 2: t(55) = 2.07, p = .043, r = .26). All seven participants exhibit the tendency of more attenuated power when observing actor1 in the joint action condition than in the joint action observation condition, and this difference reaches significance in three out of seven participants using single - subject statistics (participant 2: t(52.525) = 3.29, p = .002, r = .41; participant 4: t(52.592) = 3.55, p = .001, r = .43; and participant 6: t(24) = 2.12, p = .044, r = .39).fig . 3power averaged over the time window of 450 to 0 ms in the a mu(711 hz)- and b beta(1721 hz)-frequency range displayed as a function of condition (joint action observation; joint action) on an individual participant level . Vertical black lines represent standard errors of the means power averaged over the time window of 450 to 0 ms in the a mu(711 hz)- and b beta(1721 hz)-frequency range displayed as a function of condition (joint action observation; joint action) on an individual participant level . Vertical black lines represent standard errors of the means shifting the focus of the analysis back to the grand average, fig . 2 shows that the enhanced motor activation in the mu - frequency range seems to be persistent throughout the entire time window (900 ms), whereas the beta - band effect appears to occur time - locked to actor1 s button press . We tested the time - specificity of the effect by comparing the time period of reaching toward the button (t = 450 to 0 ms) with a time period of the same duration after the button had been pressed (t = 0450 ms). Comparing the two time periods within the mu - frequency range did not show significant differences between the two time windows of observation in the grand average, t(6) = .35, p = .737, r = .14 . In contrast to this, the beta - power difference was more pronounced during the goal - directed action of actor1 than after actor1 had pressed the button, t(6) = 3.52, p = .013, r = .82 . Thus, while activity in the beta - frequency range appears to be related specifically to the timing of the joint action partner s button press, the effect in the mu - range might be indicative of a general involvement of motor activation throughout the joint play compared with the mere observation of two people playing . Finally, we examined the relationship between the mu- and beta - effects and children s joint action performance . To determine whether the enhanced motor activation in the two bands was related to how well children acted together with actor1, we correlated the effects in the frequency bands with children s performance during joint play . For this purpose, the percentage of errors served as an indicator of performance quality . On average, the children pushed their own button about 10% of the times during the joint play (range 030.6%) when it was actually the turn of actor1 . Based on the tfr effects (i.e., the difference between observing actor1 in the joint action condition and the joint action observation condition) illustrated in fig . 2, we selected time frequency windows that represent the strongest difference time - locked to actor1 s button press . Results revealed a significant correlation between the effect in the beta - frequency range and the percentage of errors in children s joint action performance, r = .83, p = .021 . No significant correlation was found between the effect in the mu - frequency range and performance, r = .465, p>.5 . The present study is one of the first to explore the role of the motor system in young children s involvement in a naturalistic joint action . As hypothesized, involvement in joint action modulated activity in the motor system of 3-year - old children when observing the actions of another person . The results of the eeg analysis show significantly less power in the mu(711 hz)- and beta(1721 hz)-frequency range over motor areas when children observed actions of their own joint action partner as compared to when they observed the actions of the same person playing with someone else . Since power decrease in these frequency bands is acknowledged to be associated with activation of the motor system, the current findings indicate enhanced motor system activation during action observation when the 3-year - old children were involved in the joint action . This data pattern was consistently observed over individual participants in both the mu- and beta - frequency range . Moreover, the power decrease in the beta - frequency range appears to be time - locked to the action of children s joint action partner . After the button press of the joint action partner, power differences in this frequency band vanished . In contrast, power differences in the mu - band continued even when the partner moved her hand away from the button after having pushed it . To address possible relations between motor system involvement of the children and their joint action performance, we correlated the effects in both frequency bands with children s performance accuracy . The effect in the beta - frequency range correlated significantly with the percentage of errors children made during the joint action . This points to a negative relation between children s motor system involvement when observing their partner s actions and the amount of errors they made during the joint action: the less the child s motor system was activated during observation of their joint action partner, the more errors (i.e., erroneous button presses) the child performed . The effect in the mu - frequency range, however, turned out to be not significantly correlated to the children s joint action performance . In general, motor activation during action observation is thought to facilitate the understanding and prediction of others actions (cattaneo et al . 2007; de lange et al . 2008; iacoboni et al . 2005; rizzolatti and sinigaglia 2010; southgate et al . 2010; stapel et al . 2010). During joint actions, it is particularly important to predict the timing and type of action to be performed by the action partner . Anticipating what our joint action partner will do next facilitates our coordination with the other person and allows for a successful interaction (see sebanz and knoblich 2009, for a review). The current findings of enhanced motor activation for observing the own partner s actions as well as the correlation between motor system involvement and children s joint action performance might reflect children s predictive processing . However, it should be mentioned that the final sample size of the current experiment is rather small such that future research would be needed to further establish the observed effects . Further research is also required to make causal inferences on the function of the motor system for children s prediction of their partner s actions . Given children s motor system involvement and its link to joint action performance, the question arises of how exactly the motor system is involved in joint actions . Does the motor activation reflect a precise spatial and temporal simulation of the other s actions? With respect to the topography, enhancement in the motor system was found in an electrode over the left hemisphere (c3) while children were observing left - hand movements . In adults, activation of left motor areas is associated with performance of right - side movements (cf ., it therefore seems unlikely that the children simulated the actions of their partner on an effector level, which is in line with results of an action observation study with adults using meg (kilner et al . One might speculate that while observing the action partner the motor system of the 3-year - olds represented actions of the right hand, which was also the effector they needed for their own actions . All in all, however, the spatial resolution of the current findings is not sufficient to answer this question properly and further research is required to allow conclusive interpretations . Although the extent to which spatial aspects of the other s actions are integrated in children s motor system remains speculative, the findings show a clear integration of the action partner s timing as indicated by beta - power modulation . Integrating temporal information about the partner s actions into one s own motor system points thereby to a certain purpose enhanced motor system involvement might serve, namely facilitating action prediction in joint actions . There may be various reasons why we found different patterns of effects for mu- compared with beta - power . One explanation for finding beta to be more time - locked to the partner s actions might simply be the nature of beta, which is a faster rhythm than mu . Oscillations in a higher frequency range might be more effective and flexible in adapting to events for instance by recovering faster (pineda 2005). In fact, a recent study by van ede and colleagues showed that modulations of beta - oscillations during somatosensory anticipation were deployed with more temporal specificity than mu - oscillations (van ede et al . The continuous suppression of mu - power throughout the action partner s turn might reflect a general activation of the motor system bridging the time that it is not the child s turn by keeping their motor system alert . Alternative explanations might refer to differences in the function of these two rhythms as indicated by previous research . Modulations in these frequency bands might be related to the type of action or the context in which actions are performed . While beta - power during action execution and observation in adults has been shown to be modulated by the correctness of actions (koelewijn et al . 2008), in 12-month - old infants mu - suppression has been reported to differ depending on whether the infants were observing ordinary or extraordinary actions (stapel et al . More importantly, in adults, anticipatory suppression of the beta- but not mu - power was found to be stronger when observing actions of a partner than when observing actions of an individual actor (kourtis et al . In accordance with this, our findings point to different modulations of mu- and beta - frequency range activity with regard to observing others actions depending on whether one is involved in a joint action game or merely observing others playing jointly . We have interpreted decreased power in the mu- and beta - frequency band as reflecting the activation of motor - related areas . However, the precise neural origin of modulations in the mu- and beta - frequency bands remains a matter of debate with some evidence suggesting origins in primary motor and premotor areas, whereas others suggest more posterior (e.g., somatosensory) areas (caetano et al . 2007; pineda 2005; salmelin and hari 1994; stancak and pfurtscheller 1996; van ede et al . 2010 although the precise neural sources of decrease in mu- and beta - power in the scalp - recorded eeg remain to be determined, studies in both children and adults consistently show that the execution and observation of actions is accompanied by power decreases in the mu- and beta - frequency bands (caetano et al . 2010; muthukumaraswamy and johnson 2004; nystrm 2008; van elk et al . 2008). In line with these findings, the present study clearly shows that the observation of an action resulted in a decrease in mu- and beta - power, which was stronger when the 3-year - olds were engaged in joint action with the observed person as compared to when they were not . Being engaged in a joint action appears to result in stronger motor involvement during observation of others actions . What exactly makes a joint action situation so different from merely observing two people act together? Which factors might play a role in eliciting stronger motor involvement for others actions when involved in a joint action? It can be speculated that motivational and attentional factors play a role here . Being involved in a joint action implies that the actions of the partner gain relevance for one s own subsequent actions . In line with this, previous research has shown that different aspects of social relevance in the relation between actor and observer, such as the identity of the actor or eye contact between actor and observer, modulate motor activation during action observation (see frith and frith 2010, for a review). Developmental research has shown that 3-year - olds, but not younger children, monitor their peer s actions in a selective manner (see gauvain 2001, for a review). More precisely, 3-year - olds direct their attention to their peer s attempts to solve a task, while children below the age of three pay social attention in general to their peers without a special focus on their task - solving activities (see gauvain 2001, for a review). An increased attentional focus on other s task performance might thus have elicited enhanced motor involvement during the joint action . However, the extent to which attentional and motivational factors contribute to the modulation in children s motor involvement when acting jointly remains to be clarified . Moreover, in joint action, different aspects of an observed action (such as the timing) might serve as relevant cues for adapting their own action . The question arises whether in an individual action context similar nonsocial cues would subserve the same purpose and result in the same neural response . Since this question cannot be answered by the current experimental design, further investigations contrasting social and nonsocial situations are needed . How do the current findings of 3-year - olds brain activity relate to the development of young children s joint action performance? A recent developmental study by grfenhain and colleagues revealed that it is around the same age that children understand the obligations and commitments they have toward a joint action partner (grfenhain et al . At this age, children were also found to interact successfully when the joint action requires more complex interactions of the action partners (ashley and tomasello 1998). This indicates changes in children s responsiveness to the joint action partner occurring around the age of 3 years . We have previously investigated 3-year - olds joint action coordination in a behavioral study with a comparable joint task as used in the current experiment (meyer et al ., we found that 3-year - old children made less errors when acting with an adult action partner than two - and - a - half - year - olds, while both age groups performed on a similar level when playing bimanually on their own (meyer et al . 2010). Assessing children s brain activity during a similar task and at the age when children begin to establish well - coordinated joint actions revealed that their motor system involvement during action observation was related to their joint action performance . More activation in the motor system during action observation was thereby associated with fewer errors when playing jointly . This suggests that involvement of the motor system in observing the joint action partner might play a crucial role for the development of successful joint action performance . Together, the results show an enhanced motor activation as indicated by decreased mu- and beta - power during action observation when the 3-year - olds were involved in a joint action game with the observed actor . While power differences in the beta - range show time - locked motor activation for the partner s actions, differences in mu - power rather indicate a more general involvement of the motor system in a joint action task . Furthermore, the results show that the stronger the time - locked effect in beta - power, the fewer errors children made when acting jointly . This study is one of the first to investigate the neurocognitive mechanisms underlying joint action in young children . The present findings suggest that already in early childhood, others actions are integrated differentially in the motor system depending on whether or not children are engaged in a joint action . This context - specific involvement of the motor system might have important consequences for developing success in joint action.
To report an unusual case of multifocal bacterial keratitis that despite success - ful treatment caused chronic ocular hypertension . A 67-year - old woman with unilateral multifocal keratitis and no previous ocular pathology was admitted to our hospital . Corneal scrapings and conjunctival samples were obtained for culture and the patient received intensive therapy with fortified vancomycin and tobramycin eye drops . The cultures demonstrated two strains of staphylococcus epidermidis, one resistant to ciprofloxacin and both sensitive to vancomycin . Treatment was effective and gradually discontinued after total cessation of the inflammatory activity . During the follow - up period, the patient developed late and persistent ocular hypertension of unknown etiology, in absence of any detectable inflammation or complication, and received permanent antiglaucoma therapy . Patients with multifocal bacterial keratitis may need intraocular pressure monitoring, even after complete infection healing . Staphylococcus is a common gram - positive bacterium causing keratitis that tends to present with a focal and well - defined white or yellow - white infiltrate . During the infection, corneal inflammation can lead to neovascularization and scarring, while internal ocular inflammation can cause synechia formation, elevated intraocular pressure and cataract [1, 2]. Several staphylococcal strains have been reported resistant to topical quinolone therapy [3, 4]. We report an unusual case of quinolone - resistant multifocal staphylococcal keratitis without a history of previous ocular pathology, which caused chronic intraocular pressure elevation despite successful keratitis treatment and complete resolution of external and internal ocular inflammation . A 67-year - old woman presented in the emergency section of our department with gradual onset of redness, tearing, photophobia and a 4-day history of blurred vision in her right eye . There was no history of contact lens wear, previous ocular disease, trauma or surgery . Examination of the right eye revealed visual acuity of counting fingers, intense conjunctival injection, mild corneal edema and three corneal ulcers of different extension (15 mm in diameter). A moderate aqueous flare was noted in the anterior chamber without presence of hypopyon or pupillary synechiae (fig . 1). Intraocular pressure and fundus were normal, and there were no pathological findings in her left eye . Corneal scrapings and conjunctival samples were obtained from the right eye and sent to the laboratory for culture . The patient was hospitalized and received empirical treatment with fortified vancomycin + tobramycin (voncon, lilly + tobrex, alcon) eye drops every 30 min the first day, every hour the second day and afterwards every 2 h. topical cyclopentolate (cyclogyl, alcon) 3 times daily was also administered . The cultures demonstrated the presence of two strains of coagulase - negative staphylococcus epidermidis, one resistant to ciprofloxacin and both sensitive to vancomycin . One week after treatment implementation, the patient reported partial relief from her initial symptoms . A considerable reduction of the extension of corneal ulcers was noted, though aqueous flare and intraocular pressure increased (32 mm hg) and corneal neovascularization appeared at the limbus adjacent to the corneal ulcers . Treatment was modified to topical vancomycin, dexamethasone (maxidex, alcon) and cyclopentolate initially 3 times daily, with a progressive tapering scheme during the following 3 weeks . After the 3-week - period, the corneal ulcers were completely healed; there was no conjunctival injection, no visible aqueous flare but only a mild scarring at the superior limbus (fig . Two months later, a persistent ocular hypertension (2628 mm hg) of unknown etiology and without any external or internal ocular inflammation was noted in the right eye at three consecutive visits . A goldmann contact lens was used to perform a 360 gonioscopy that did not reveal any pathological findings . Intraocular pressure was normal in the left eye and corneal pachymetry was normal in both eyes . Topical brinzolamide was restarted and follow - up was continued monthly . For the next 6 months, intraocular pressure in the right eye fluctuated within normal limits and no inflammatory recurrence was observed . Occasionally, staphylococcal keratitis can present with predominantly multifocal epithelial infiltration, especially in the setting of hydrophilic contact lens wear or after laser in situ keratomileusis . Multiple foci of abscesses resemble fungal satellite lesions [1, 5]. In the present case, furthermore, we did not detect any predisposing factors for chronic intraocular pressure elevation, such as history of glaucoma or ocular hypertension, trauma, relapsing inflammation and anterior or posterior synechiae . The existence of aqueous flare in the anterior chamber could explain the temporary intraocular pressure rise diagnosed in the second week, though after total cessation of the inflammatory activity the intraocular pressure returned to normal values as expected . Steroid - induced ocular hypertension does not seem to be relevant since steroid treatment was of limited duration, low dosed and gradually discontinued . A possible explanation for the late and persistent intraocular pressure elevation may be a permanent damage of the trabecular meshwork caused by staphylococcal toxins . Despite normal appearance of the iridocorneal angle on gonioscopy, invisible microscopic changes could not be excluded . The resistance of one s. epidermidis strain to ciprofloxacin is in accordance with previous reports and indicates that microbial culture is necessary in cases of extensive bacterial corneal ulcers [3, 4]. Furthermore, it also shows that an initial empirical treatment with a combination of fortified topical antibiotics may be superior to topical quinolone monotherapy in these cases . However, further evidence is required to support the above hypothesis since there are studies showing equivalence of fluoroquinolone monotherapy to fortified medications in microbial keratitis [6, 7]. Unfortunately, it was not possible for our laboratory to test the sensitivity of the ciprofloxacin - resistant strain to newer - generation quinolones, which could have had better activity against these gram - positive organisms . In conclusion, the present case demonstrates that in patients with multifocal keratitis, differential diagnosis between fungal and bacterial infection is critical for successful treatment . Besides treatment, patient follow - up and intraocular pressure monitoring may be needed, even after complete infection healing and resolution of inflammatory signs.
Due to their bioactive properties, some vegetables or their extracts have been applied to a large number of human activities (e.g. Food preservation) since ancient times (medina et al ., 2006). In particular, a wide range of plant extracts showed certain levels of inhibition against bacterial growth . In most cases, the antibacterial activity is attributed to the presence of phenolic compounds that are metabolites involved in the resistance against parasites (servili et al . Olives, virgin olive oil and its secondary products such as olive mill wastewater showed a high level of phenolic compounds . The glucoside oleuropein is the main phenolic compound (secoiridoids) in fruit; moreover in olive products the molecules of its hydrolysis exert a stronger antimicrobial activity (medina et al ., 2006). However, most part of these compounds is lost in wastewater during the olive oil extraction process . It was recognized that the presence of phenolic compounds reduces the microbial degradability of oil vegetation waters (vws) leading to pollution problems (capasso et al ., 1995; tafesh, et al ., 2011; saadi et al ., 2007) however, vws could be considered as additional resources for the virgin olive oil (voo) industry . In fact several phenolic compounds occurring in vw, having the same chemical characteristics of voo phenols, possess many biological properties that include antioxidant activity and, for this reason, may be used in food industry as natural preservatives or bio - active ingredients (servili et al ., 2011b). The aim of the study was to assess the in vitro bactericidal effect of purified phenols extracted from oil vegetation water (peow) obtained by membrane filtration techniques from vw on several food - borne strains (spoilage bacteria, food - borne pathogens and starter cultures). The assessment of bactericidal activity could define some threshold doses for a further application on real food models . In figure 1 the protocol for the separation of the phenols extracted from oil vegetation water is reported . A panel of 18 food - borne strains was investigated for the minimum bactericidal concentration (mbc; table 1) in a microtiter assay . Several two - fold dilutions of the peow (figure 1) were performed in a 20% ethanol / water solution (12.0; 6.0; 3.0; 1.5; 0.75; 0.375 mg / ml), considering a 65% of total phenolic content on the initial extract (carraro et al ., 2014). Each well was added with 50 l of peow and 200 l of bacterial suspension in a 96 well plate with a final bacterial concentration of 10 ufc / ml . For each plate 3 wells without bacteria were performed according peow dilution (negative control). Moreover, for each strain 3 wells were added with 200 l of bacterial suspension and 50 l of a 20% ethanol / water solution; an additional positive control was performed for each strain using 250 l of bacterial suspension . Microtiter plates were incubated at specific temperature / time for each strains and the mbc was evaluated on agar plate by spreading 10 l of each suspension . The dose that kills the bacteria was recorded and the mbc was defined as the level that did not allowed the survival of all replicates . A visual examination of well bottoms was also applied in order to have a visible growth and define the minimum inhibitory concentration (mic). Table 1 reported the mbc value for the food - borne bacteria tested . Among pathogenic strains, s. aureus and l. monocytogens showed the lowest level of resistance to peow (mbc=1.53 mg / ml). For gram positive strains, some events of heteroresistance were observed and a stringent evaluation of mbc was based on the highest level that did not allowed the growth on all replicates . On the opposite, in gram negative strains (e.g s. typhimurium and pseudomonas spp .) The mbcs ranged between 6 to 12 mg / ml with the exception of escherichia coli o:157 h7 (3 mg / ml). Starter cultures were dramatically reduced in growth (e.g. S. xylosus; mbc 0.75 - 1.5 mg / ml). Pediococcus pentosaceus seemed the more resistant species among actic acid bacteria (lab), the strain was able to survive at higher concentrations of peow (mbc 12 mg / ml). The ethanol / water solution did not affect the survival of any tested strains, the results were in agreement with the previously observation on e. coli (carraro et al ., 2014). The final concentration of ethanol was around 1/4 of the initial solution (5%), this level was ineffective to kill the pre - inoculum of the tested bacteria . The mic is the lowest concentration of an antimicrobial that inhibit the growth of a microorganism . The visual examination has resulted in some misinterpretation due to the turbidity of the extract after the incubation, especially at higher peow concentrations (6 - 12 mg / ml); for this reason the evaluation of mic is not reported . As reported by carraro et al . (2014) the composition of the extract showed that oleuropein - aglycone di - aldehyde was the major secoiridoid constituent (471.7 1.9 mg / g). Others chemical compounds are hydroxytyrosol (72.7 0.6 mg / g), tyrosol (17.8 0.1 mg / g) and verbascoside (83.6 1.0 mg / g). The in vitro tests suggested an interesting bactericidal effect of peow, especially on gram positive food - borne pathogens . Other studies reported that s. aureus and l. monocytogenes showed a higher sensitivity to phenols derived from several olive matrices (e.g olive oil, olive leaf and purified compounds) (medina et al ., 2006; pereira et al ., 2007). Moreover, thafesh et al . (2011) suggested that the use of a combination of polyphenols extracted by olive mill wastewater is effective against several human pathogens . The present results confirmed the potential of peow, though the bioactivity could be related to the content of certain specific phenolic compounds (thafesh et al ., 2011). This is probably due to the formation of a brown - coloured quinonic form due to the oxidative degradation of phenols . The present results suggested two thresholds of bactericidal effect on gram positive food - borne pathogens . However, other pertinent considerations need to be considered when these products are added to the food (e.g organoleptic traits, diffusion, bonding with some food constituents). Taking into account mbc results, the lab and s. xylosus are among the most sensitive bacteria . In a functional milk beverage fortified with phenolic compounds still, the level of inclusion was limited (100 and 200 mg / l). In other fermented products, the supplementation of higher levels of peow, as a natural ingredient, could reduce the performance or the quality of ripening . For further applications, a deep screening of starter cultures is required in order to select the species able to grow in presence of phenol during fermentation . The mbc suggested that one strain of pediococcus pentosaceus was able to grow at 12 mg / ml: this species could be applied to some fermented food (e.g salami). This work is the first step before the use of these substances on the food models (e.g. Challenge test and storage test) for the further application of this extract as an ingredient . The two thresholds proposed for the pathogenic food - borne bacteria need to be considered together with other pertinent food aspects (e.g. Organoleptic traits, antioxidant effects).
Pemphigus vulgaris (pv), the most prevalent type of pemphigus, is a life - threatening autoimmune bullous disease characterized by an autoantibody predominant target epitopes to desmoglein 3 (dsg3), a desmosomal cell adhesion glycoprotein [14]. It can be considered as a chronic organ - specific disorder because the autoimmune injury which leads to the formation of intraepidermal blisters and acantholysis is confined to the skin and mucosa . A number of studies about pv, using patient samples or animal models, have been previously reported . However, despite recent advancements, the pathogenesis of pv to date remains to be fully elucidated . Autoreactive t cells are thought to play a central role in the pathogenesis of pv [1, 57]. Recently, emerging findings put the spotlight on the contribution of a newly discovered subset of interleukin-17 (il-17) producing t helper (th) cells accordingly named th17 cells to autoimmune states [812]. Furthermore, over the past few years, increasing evidence suggested that the development and maintenance of th17 cells have been linked to interleukin-23 (il-23), a key initiating cytokine in the development of autoimmunity . It was reported that il-23 was mainly secreted by macrophages and dendritic cells (dcs) and il-23 promoted the expansion of the novel th17 population [1315]. As a result, a crucial role was proposed for the il-23/il-17 axis in mediating tissue inflammation and autoimmunity recently, such as psoriasis [16, 17]. However, despite the current evidences indicating that il-17 may play an important role in pv [18, 19], few reports have explored the crucial role of the il-23/il-17 axis in the immunopathogenesis of pv . We thus hypothesized that the il-23/il-17 axis will also be functionally involved in the development and maintenance of pv . In this study, we examined the immunoexpression of il-23 and il-17 in the lesional biopsy specimens from 10 cases of pv, comparing the results with those of pf patients and normal control skins from 6 healthy individuals, and evaluated the correlation between il-23 + cells and il-17 + cells; moreover, the sources of il-23 were also evaluated . In this descriptive - analytical study, the subjects were 10 (3 men and 7 women) unrelated patients with pv and 3 (1 man and 2 women) patients with pemphigus foliaceus (pf) diagnosed by clinical and immunohistochemical criteria . The lesional biopsy specimens of the patients were obtained before treatment during the active phase for the purpose of evaluating the acute state . The normal control skins (eyelid skin) were taken from 6 (1 man and 5 women) healthy people who were selected randomly . This study was approved by our local ethics committee, and written informed consent was obtained from all participants . Immunofluorescence staining of cryosections from those specimens, both the pv patients and the healthy individuals, was performed with the following primary antibodies: rabbit anti - human il-17 pab (santa cruz), goat anti - human il-23 pab (santa cruz), and mouse anti - human cd163 mab (santa cruz). For negative control preparations, the first antibodies were replaced with mouse f(ab)2 igg (abcam), an irrelevant isotype control . The second antibodies are alexa fluor 488 goat anti - mouse igg (invitrogen), alexa fluor 555 rabbit anti - goat igg (invitrogen), and alexa fluor 555 goat anti - rabbit igg (invitrogen). The counts of il-17 + cells, possibly th17 cells (il-17), il-23 + cells (il-23), and cd163 + cells, possibly macrophages (cd163), in three sections were quantitatively evaluated . The counts of positive staining cells were initially reported in the form of descriptive statistics . Unpaired t - test was utilized for comparison of il-17 and il-23 between the three groups and the significance of the correlation was assessed by pearson test . The numbers of il-23 + cells and il-17 + cells were significantly increased in pv lesions (figures 1(a)1(d) and figures 2(a)2(d)), compared to healthy controls (figures 1(e)1(h); figures 2(e)2(h); figure 3, p <0.05). The counts of il-23 + cells in each 200x field of view in pv lesions and normal control skin were 73.70 5.315 and 29.50 4.448, respectively (figure 3(a), p <0.05), while the numbers of il-17 + cells in each 200x field of view in pv lesions and normal control skin were 46.60 5.673 and 9.50 3.354, respectively (figure 3(b), p <0.05). Besides, the il-23 + staining was overlapped with the cd163 + macrophages (figures 1(d) and 1(h)), which indicated that the il-23 was secreted by macrophages . As the il-23 + staining was overlapped with the cd163 + macrophages (figures 1(d) and 1(h)), we adopted the number of cd163 + cells instead of il-23 + cells' counts for the correlation analysis (figure 2). Based on pearson test, the correlation between il-23 + cells and il-17 + cells in pv lesions was significant (r = 0.7546; p <0.05) (figure 4). The counts of il-23 + cells and il-17 + cells in each 200x field of view in pf lesions were 42.67 5.812 and 21.33 4.485, respectively (see supplementary figure in the supplementary material available online at http://dx.doi.org/10.1155/2014/463928), where we found a statistically significant decrease in number compared to pv (p <0.05). But no statistically significant differences of these numbers between pf and the control group were identified . In the current research, we examined the lesions and found that both il-23 and il-17 were overexpressed in pv patients, compared to the healthy controls and pf patients . More importantly, the results showed a correlation between il-23 + cells and il-17 + cells . Finally, this study demonstrated that the il-23 was secreted by a cell population in dermis expressing cd163, which was considered as a surface marker of the macrophages . In a previous experimental study conducted by arakawa et al ., the authors presented the possibility of th17 (il-17 + cells) that played an important role in the pathogenesis of pv . They quantified th17 cells in lesional biopsy specimens from pv patients and found a significantly higher expression of il-17 + cells compared to controls, which was also confirmed by our results . However, the importance of il-23 was not investigated in arakawa's study . To our knowledge il-23 was clearly not required for the initial induction of il-17 production in naive t cells either in vitro or in vivo . However, production of il-17 by memory effector cells was clearly enhanced in the presence of il-23, and it was shown that il-23 maintained expression of il-17 in activated th17 cells . Since its discovery, il-23/il-17 axis has been linked to the pathogenesis of various autoimmunity disorders, such as psoriasis and systemic lupus erythematosus [16, 2325]. However, a limited number of reports have explored the crucial role of the il-23/il-17 axis in the immunopathogenesis of pv . Taken together, this provided us with the basis for a rising interest in the il-23/il-17 axis in pv . Our results showed overexpression of il-23 and il-17 in the lesion of pv patients and a correlation between il-23 + cells and il-17 + cells, which suggested that the il-23/il-17 axis probably played an important role in the immunopathogenesis of pv . The first issue which should be considered is which type of immunopathology il-23/il-17 axis may show in pv ., il-23/il-17 axis may not be a cause but a result of the disease; in other words, it may possibly appear in a protective response to maintain epithelial homoeostasis . Traditionally, il-23 was found to be expressed mainly by macrophages and dendritic cells (dcs) in dermis [21, 26]. In an earlier study, cd163 was recommend to be considered as an alternative marker to identify dermal macrophages for its more specific and more useful in flow cytometry applications . Our findings that the il-23 + staining was overlapped with the cd163 + macrophages in all specimens indicated that the il-23 in dermis was expressed mainly by the cd163 + macrophages . In other words, macrophages, which have been confirmed to play an important role in the pathogenesis of psoriasis, may be also involved in the pathogenesis of pv . In summary, the present study suggested the importance of the il-23/il-17 axis in the development of pv, which provided us with some clues for the elucidation of the pathogenesis of pv . The reason why more il-17 + cells and il-23 + cells were present in lesional specimens from pv than those from pf is not certain . Actually, the accurate role of il-23/il-17 axis in the pathogenesis of pv is still unresolved in the present study . Additionally, the current research had some limitations that should be noted, such as the following: (i) the results were descriptive mainly and lacked some mechanistic studies, (ii) as a retrospective study, the serum levels of il-23 and il-17 were not tested, and a correlation between il-23/il-17 and disease activity or antibody titers was not investigated, and (iii) the relatively small number of patients was employed in this study . Prospectively, further study of the il-23/il-17 pathway in the pathogenesis of pv in mice model may be encouraged to further validate our hypothesis . Additionally, we hypothesized that targeting the il-23/th17 pathway maybe a highly effective therapeutic approach in the treatment of pv . Future studies are required in order to better explore this pathway as a potential therapeutic target . In conclusion, our present study provides evidence that the expression of il-23 and il-17 was elevated and correlated in pv patients, which suggested the crucial role of the il-23/il-17 axis in the development of pv and provided us with some clues for the elucidation of the pathogenesis of pv.
Dendritic cells (dcs) have been identified as a key component in manipulating and stimulating the immune system . Activated dcs are potent antigen presenting cells that express both major histocompatibility complex (mhc) class i and ii molecules (signal 1) and costimulatory molecules (signal 2) and secrete immune modulating cytokines (signal 3) resulting in activation of t lymphocytes . Depending on the cytokine environment, dcs may elicit either a th (t helper) 1 or th2 cd4 t - cell response . For tumor immunotherapy, induction of a th1 t - cell response is pivotal, and secretion of il-12 (interleukin 12) by dcs is of critical importance for differentiation of naive t cells into th1 cells . Furthermore, il-12 stimulates the production of interferon - gamma (ifn-) and tumor necrosis factor - alpha (tnf-) from t cells and natural killer cells . In contrast, th2 responses, associated with cytokines il-4, il-5, il-6, and il-10, suppress th1 activity and may anergize effector t cells to tumor antigens . One of these strategies involves fusing dcs with tumor cells using electrical currents in a method called electrofusion, hence combining the antigen presenting properties of dcs with the full repertoire of antigens present within a tumor cell in order to stimulate effector t cells [6, 7]. While dc - tumor hybrids alone are insufficient to elicit significant immune responses in vivo and are critically dependent upon exogenously administered 3rd signal adjuvants, murine studies using dc - tumor hybrids for vaccination given concomitantly with an adjuvant third signal, such as il-12, ox-40-, 4 - 1bb - monoclonal antibody, or toll - like receptor agonists, showed regression of tumor metastases after a single vaccination in several tumor types including melanoma, breast, sarcoma, and squamous cell carcinoma [811]. However, systemic delivery of 3rd signal along with a dc - tumor fusion vaccine is clinically problematic due to 3rd signal toxicity and/or availability . Therefore, a better understanding of the mechanisms affecting the dependence of dc - tumor fusions on 3rd signal adjuvants is of paramount importance for optimizing this immunotherapeutic approach . In this study, we show that production of the th1 skewing cytokine il-12 was dramatically downregulated in dc - tumor fusion cells . In addition, gene products that are involved in signaling pathways including nfb (nuclear factor kappa - light - chain - enhancer of activated b - cells), pi3k / akt / mtor (phosphatidylinositol 3-kinase / akt, protein kinase b / mammalian target of rapamycin), wnt (wingless - related integration site), and mapk (mitogen - activated protein kinase) were differentially expressed in fusion cells . Inhibitor studies revealed that interruption of the canonical wnt pathway did not affect il-12 production by dc - tumor fusion cells and that inhibition of mek (mitogen extracellular signal - regulated kinase) only increased il-12 production marginally . In contrast, il-12 production could significantly be enhanced by treatment of dc - tumor hybrids with inhibitors of the pi3k and mtor . Given the critical role of the pi3k / akt / mtor signaling pathway in cancer biology and the immunostimulatory effect of pi3k / akt / mtor inhibitors on dc - tumor hybrids, combination therapy may represent a promising and novel cancer vaccine with enhanced clinical impact . Animals were housed in a specific pathogen - free environment at the animal facility of the durham veteran affairs medical center . All mice used in this study were cared for in accordance with the guide for humane care and use of laboratory animals published by the national institutes of health . All the animal experimental protocols were approved by the duke university medical center institutional animal care and use committee . D5lacz is a -galactosidase expressing derivative of the b16 f10.9 melanoma cell line and has been shown to be poorly immunogenic . Cells were cultured in complete media (cm) composed of rpmi 1640 media supplemented with 10% fetal bovine serum, 2 mm l - glutamine, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 100 u / ml penicillin, 100 g / ml streptomycin, 0.5 g / ml fungizone, 50 g / ml gentamicin, and 5 10 m 2-mercaptoethanol (invitrogen, carlsbad, ca). These cells were maintained at 37c with 5% co2, harvested following a short incubation period with 0.05% trypsin with edta, and irradiated at 100 gy prior to use . B- and t - lymphocytes were depleted using antibody - coated magnetic beads (dynal biotech, carlsbad, ca). The dc - enriched cell fraction was then cultured in cm supplemented with 10 ng / ml gm - csf and 10 ng / ml il-4 (peprotech, rocky hill, nj) at a concentration of 0.5 10 cells / ml at 37c with 5% co2 . On day 6, cells were harvested, resuspended in fresh cm + gm - csf / il-4 media at 1 10 cells / ml, and incubated at 37c with 5% co2 for 24 hours . Then, lps (lipopolysaccharide, 100 ng / ml, sigma - aldrich, saint louis, mo) was added to stimulate dc maturation . After 24 hours after 24 hours, dcs were stained intracellularly with cfse prior to use (molecular probes, eugene, or). Irradiated tumor cells and cfse stained dc were mixed in a 1: 1 ratio and washed in prefusion media, followed by resuspension in fusion media at a concentration of 20 10 cells / ml . For electrofusion, the pulse generator (model ecm 2001 generator, btx instruments, san diego, ca) was used . Cells were exposed to two consecutive, independent electrical currents: (1) a low voltage alternating current of 120 v / cm for 10 seconds to achieve alignment and chain formation, and (2) a high voltage direct current of 1100 v / cm for 25 microseconds to cause a reversible breakdown of cell membranes . The multinucleated hybrid cells were allowed to stand for at least 5 minutes before incubation in culture media overnight at 37c with 5% co2 . To separate unfused tumor cells (t) from t - t hybrids and unfused dcs from dc - dc hybrids facs sorting by size on forward scatter (fsc) and side scatter (ssc) dc - t hybrids were purified using a combination of mechanical and facs sorting techniques, based on their plastic adherence characteristics as well as cfse staining . Therefore, after electrofusion and overnight culture, the nonadherent cell population representing unfused dcs and dc - dc hybrids was discarded . Facs was then performed only on the adherent cell population containing unfused tumor cells, t - t hybrids, and dc - t hybrids . Since only dcs were stained with cfse, facs sorting was used to separate cfse positive cells from the cfse negative populations (unfused tumor cells and t - t hybrids). All cell samples were analyzed using the facs aria ii (bd biosciences, san jose, ca). 24 hours after electrofusion, total rna was isolated using the rneasy plus mini kit protocol (qiagen, valencia, ca). The cdna template was synthesized from 0.51.0 ug of total rna using the rt first strand kit protocol (sabiosciences, frederick, md). Each template was then combined with rt sybr green qpcr master mix (sa biosciences) and aliquoted into a 96-well mouse common cytokine plate array (sa biosciences). The pcr cycling program was 95c for 10 minutes, followed by 40 cycles of 95c for 15 seconds, and then 60c for 1 minute on a stratagene mx3005p qpcr machine . Briefly, all threshold values (ct) reported as greater than 35 indicated no detectable gene expression . Genomic dna (gdna) contamination was detected if the gdna control ct value was below 35 . A reverse transcription control (rtc) detected impurities in the rna sample that affect the reverse transcription of the template and was considered positive if the ct was greater than 5 . Gene expression associated with th1 (ifn-, il-2, il-12p40, il-15, il-18, and tnf-) and th2 (il-4, il-10, il-13, and il-25) immune responses was analyzed . Total rna was isolated from tumor cells, dcs, and dc - t fusion cells using the rneasy plus mini kit protocol (qiagen, valencia, ca). Rna isolation for tumor and dendritic cells was done in triplicate . For the dc - t fusion cells, triplicate samples of d5lacz tumor cells, dcs, and dc - t fusion cells were each run through a microarray chip (affymetrix) by the duke dna microarray core facility . Multiway anova was performed and fold change was determined to select target genes that were differentially expressed between fusion cells and dcs, or fusions cells and tumors cells, respectively . Top differentially expressed genes were selected with p value cutoff of 0.01 based on anova test and fold change cutoff of> 5 . Hierarchical clustering was performed on differentially expressed genes based on average linkage with pearson's dissimilarity . The murine il-4 elisa kit (ebioscience, san diego, ca) and the murine il-12p70 elisa kit (bd biosciences, san jose, ca) were used according to the manual provided by the manufacturer . To determine cytokine secretion by dcs or dt - tumor fusion cells, 2 10 cells in 1 ml of aimv media (invitrogen, carlsbad, ca) were incubated in the presence of 100 ng / ml of lps for 24 hours at 37c, 5% co2 . Where indicated, lps stimulation was performed in the presence of the following inhibitors (purchased from sigma - aldrich, saint louis, mo): u0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) is a highly selective inhibitor of both mek1 and mek2 and was used at a concentration of 100 nm, jw 74 (4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4h-1,2,4-triazol-3-yl]-pyridine) an inhibitor of the canonical wnt pathway was used at a concentration of 10 m, rapamycin (23,27-epoxy-3h - pyrido[2,1-c]oxaazacyclohentriacontine) forms a complex with fkbp12 (fk506 binding protein 12) that binds to and inhibits mtor which was used at 0.5 m, and wortmannin which inhibits the pi3k / akt signal transduction cascade was used at 100 nm . Experiments were performed in duplicate and error bars represent the sem (standard error of the mean). In a first set of experiments, d5lacz tumor - tumor (t - t) cell hybrids, dc - dc hybrids, and dc - t hybrids were generated by electrofusion . Fusion cells were purified by facs and rna isolated from hybrid cells was analyzed by quantitative real - time pcr (qpcr) for expression levels of mrnas encoding the th1 cytokines ifn-, tnf-, il-2, il-12p40 (the -subunit of bioactive il-12p70), il-15, and il-18 or the th2 cytokines il-4, il-10, il-13, and il-25 . Figure 1 shows the results of our qpcr analyses . Comparison of dc - t hybrids with dc - dc fusion cells (white bars) reveals that cytokines associated with a th1 response including il-12p40 and il-15 were downregulated by more than 100- and 15-fold, respectively . In contrast, the th2 cytokine il-4 was dramatically upregulated by 115-fold . Among all cytokines analyzed, only tnf- and il-12p40 exhibited higher expression levels in dc - t fusion cells when compared to t - t fusions (figure 1, black bars). In another series of experiments, the th1 and th2 cytokine expression profile of cells exposed to electrofusion was compared to unexposed cells . However, no significant changes in cytokine gene expression between tumor cells and t - t fusion cells or dcs and dc - dc fusion cells were observed (data not shown). For this reason, we focused on the comparison of gene expression levels between dc - t hybrid cells and dcs in the subsequent analyses presented in this study . We next sought to determine changes in the expression of genes that may negatively impact the immunologic properties of dc - t fusion cells . In order to do so, rnas were isolated from facs - isolated dc - t hybrids cells, dcs, or d5lacz tumor cells, and microarray assays were performed . Consistent with our qpcr data, expression of il12p40 and il-15 by dc - t fusion cells was markedly downregulated (13.2- and 8-fold) when compared to dcs, albeit to a lesser degree than observed in pcr analyses (figure 2(a)). Also, il-4 was upregulated 59.4-fold in dc - t fusions . The proinflammatory cytokines il-1 and il-1 were downregulated 5.5- and 8.2-fold, respectively, while tgf3 was upregulated 8.8-fold . Furthermore, we observed a downregulation of receptors for colony - stimulating factor (csfr1), tnf- (tnfr2), and il-7 (il-7r). In contrast the receptors for tweak (tnf - like weak inducer of apoptosis, tweakr) and for il-17 (il-17rc) were upregulated 9.5- and 6.5-fold . While overexpression of il-17rc has been implicated in bcl-2- and bcl - xl - independent protection of cancer cell lines from tnf-induced apoptosis, tweakr signaling has been shown to enhance the expression of nfb (nuclear factor kappa - light - chain - enhancer of activated b - cells)-regulated genes including il-6, il-8, rantes, and icam-1 (cd54). However, upregulation of none of these gene products was observed in our study (figures 2(a), 2(c), and 3(a)). In addition to cytokine gene and cytokine receptor expression, there were also significant changes in the expression level of gene products that are involved in cytokine signaling (figure 2(b)). Expression of tgfi (transforming growth factor beta - induced), a protein that is induced by tgf and that acts to inhibit cell adhesion, was downregulated 14.1-fold . Downregulation of this gene product was unexpected given that tgf3 was upregulated in dc - t hybrids (figure 2(a)) and implies that the tgf-signaling pathway may not be hyperactive in dc - t hybrid cells . There were no differences in expression levels of tgf-receptors between dcs and dc - t hybrid cells . However, nedd4l (neural precursor cell expressed developmentally downregulated gene 4-like) was upregulated 13.8-fold in dc - t hybrid cells . Nedd4l negatively regulates tgf signaling by ubiquitination - mediated degradation of tgf- receptor 1 and receptor - regulated smad2 (mothers against decapentaplegic homolog 2). As such, it is reasonable to assume that neddl4 overexpression suppressed transcriptional activity induced by tgf. Expression of il-1ra, the interleukin-1 receptor antagonist, which modulates a variety of il-1 related immune and inflammatory responses, was downregulated 7.5-fold . Moreover, expression of irfs (interferon regulatory factors) 4, 7, and 9, which are involved in transcriptional regulation of type i interferon genes, interferon signaling, and hence the janus kinase- (jak-) signal transducer and activator of transcription (stat) pathway, was downregulated 7-, 6-, and 10.5-fold, respectively . Also, jak-2 and stat-4, known to be involved in il-12 receptor signaling, are downregulated 6.5- and 11.3-fold . Last, three gene products that are associated with nf-b signaling were found to be downregulated in dc - t hybrid cells, namely, relb (reticuloendotheliosis viral oncogene homolog b, 5.3-fold), traf-1 (tnf receptor associated factor-1, 11.3-fold), and the nf-b inhibitor ib (nuclear factor of kappa light polypeptide gene enhancer in b - cells inhibitor alpha, 7.3-fold). Relb is known to form heterodimers with nf-b p50 or p52, and traf-1 forms a heterodimeric complex with traf2, which is required for tnf--mediated activation of mapk8/jnk (jun kinase) and nf-b . On the other hand, these results are somewhat contradictory and argue that nf-b activity in dc - tumor fusion cells is not regulated at the transcriptional level . As shown in figure 2(c), expression of chemokines or their receptors which are involved in chemotaxis of neutrophils, monocytes, dcs, t cells, and nk cells were generally downregulated in dc - tumor fusions, with the exception of cxcl-10 (ip-10, interferon - gamma - induced protein 10). Surprisingly, even chemokines involved in chemotaxis of th2 cells and regulatory t cells (ccl-17 and ccl-22) were downregulated while ip-10 which is implicated in the induction of th1 responses and chemotaxis of th1 cells was significantly upregulated [23, 24]. We therefore hypothesize that the chemokine expression profile of dc - tumor hybrids does not have a major impact on the th - polarizing capacity of dc - tumor hybrid cells . It has been demonstrated that the expression of matrix metalloproteinases mt-1 (mmp-14) and mmp-9 is a major contributing factor to the migratory capacity of dcs to lymph nodes through the degradation of extracellular matrix components . In this context, mmp-9 activity is of particular importance since it cleaves collagen iv, a major component of basement membranes . Furthermore, it has been shown that the balance of mmp-9 and timp (tissue inhibitor of mmps) expression is crucial for dc migration in vivo . Our data reveal that timp-2 was upregulated 11.8-fold in dc - t fusion cells, while mmp-9 is downregulated 7-fold (figure 2(d)). As such, these results suggest that the migratory capacity of dc - t hybrids toward lymph - node derived chemokines, namely, ccl-19 and ccl-21, may be impaired . As shown in figure 3(a), expression of genes involved in antigen presentation in the context of mhc classes i and ii or cd1d was downregulated 5.7-, 16.5-, and 6-fold in fusion cells . Furthermore, the expression of all well - established costimulatory molecules, including cd40, cd54, cd80, cd83, cd86, 4 - 1bb, gitr (glucocorticoid - induced tnfr - related protein), ox40l, and slam (signaling lymphocytic activation molecule), was downregulated in dc - tumor fusion cells . These data explain to some degree why targeting of costimulatory molecules with agonistic antibodies can enhance the potency of dc - tumor fusion - based vaccines, as has been described previously . Last, expression of pd - l2 (programmed death ligand 2), an inhibitory immune checkpoint molecule, was suppressed 7.8-fold in dc - fusion cells . No differences in pd - l1 expression between dcs and dc - t hybrid cells were observed . The development of melanocytes is highly dependent on the action of the microphthalmia - associated transcription factor (mitf) which has been shown to regulate a broad variety of genes, whose functions range from pigment production to cell - cycle regulation, migration, and survival . Mitf was upregulated in dc - tumor fusion cells (figure 3(b)). Concomitantly, also mitf - regulated mrnas encoding melanoma antigens, including tyr (tyrosinase), trp-1 and trp-2 (tyrosinase - related protein), gp100 (silver), melan, melanophilin, m - cam (melanoma cell adhesion molecule), and matp (membrane - associated transporter protein also known as solute carrier family 45 member 2 (slc45a2) or melanoma antigen aim1), were also highly upregulated . Moreover, expression of mitf - regulated mcr1 (melanocortin 1 receptor), trpm1 (transient receptor potential cation channel subfamily m member 1), gpr143 (g protein - coupled receptor 143), and mbp (myelin basic protein) was highly upregulated in fusion cells . Expression of mbp by melanoma cells is somewhat surprising, but it has been shown that b16f10 cells undergo differentiation to a myelinating glial phenotype characterized by induction of the transcriptional activity of the mbp promoter . Last, osteonectin (secreted protein acidic and rich in cysteine (sparc)), which has been implicated in metastasis of melanoma to the lungs, and plagl1 (pleomorphic adenoma gene - like 1), a potential tumor suppressor gene, were also overexpressed in dc - tumor fusions . These results suggest that the entire antigenic repertoire of melanoma cells is indeed strongly expressed in dc - tumor hybrid cells, as has been hypothesized . Next, we analyzed expression levels of genes that are involved in signaling pathways known to be aberrantly regulated in cancer cells . The transcription factor tcf7l1 (transcription factor 7-like 1) which is activated by -catenin and thus mediated wnt signaling was upregulated 6.2-fold (figure 4(a)). Also, expression of frzb (frisbee), a wnt - binding protein and competitor for the cell - surface receptor frizzled, and expression of wntless (g protein - coupled receptor 177), another receptor for wnt proteins, were increased 12.2- and 61.3-fold . Furthermore, target genes of the canonical wnt pathway, wisp-1 (wnt1-inducible - signaling pathway protein 1) and nrcam (neuronal cell adhesion molecule), were upregulated 13.7- and 148.7-fold, indicating activation of the wnt pathway in dc - tumor fusion cells . Expression of the fk506 binding proteins fkbp4, fkbp6, and fkbp9, immunophilins known to interact with mtor, was upregulated in dc - tumor fusions 5.3-, 6.7-, and 28-fold (figure 4(b)). Furthermore, expression of pik3r1 (phosphatidylinositol 3-kinase regulatory subunit alpha, p85) was downregulated 6.5-fold, which may indicate aberrant activity of pi3k in dc - tumor fusion cells . Nedd4 directly binds to and poly - ubiquitinates pten (phosphatase and tensin homolog), targeting it for proteasomal degradation . Therefore, posttranslational suppression of its expression level may lead to hyperactivation of the pi3k / akt signaling pathway . The lps - inducible mitogen - activated protein kinase 12 (mapk12), also known as extracellular signal - regulated kinase 6 (erk6) or p38-, was upregulated 11.7-fold in dc - tumor fusions (figure 4(c)). In contrast, the lps - inducible gadd45 (growth arrest and dna damage - inducible 45) was downregulated 10.8-fold . Gadd45 is an nf-b target gene which, in combination with mekk4, activates p38mapk . Furthermore, mitogen - activated protein kinase kinase kinase 14 (map3k14) also known as nf - kappa - b - inducing kinase was downregulated 7.25-fold in fusion cells . Lastly, expression of c - jun amino - terminal kinase interacting protein 1 (mapk8ip1), a negative regulator of mapk8 (c - jun amino - terminal kinase), was upregulated 6.9-fold in dc - tumor hybrid cells . Lipid mediators such as prostaglandins have been implicated in tumor - mediated immunosuppression [37, 38]. As presented in figure 4(d), several genes involved in eicosanoid biosynthesis and signaling were differentially expressed in dc - tumor fusion cells . Additionally, cyclooxygenase-2 was downregulated 16.2-fold and the cysteinyl - leukotriene c4 synthase (ltc4s) was downregulated 12.1-fold . In contrast, phospholipase a2 (pla2) and prostaglandin d2 synthase (pgds) were upregulated 47.2- and 61.3-fold, respectively . We next sought to determine whether inhibition of signaling pathways, for which inhibitors are available clinically, could restore secretion of bioactive il-12p70 by dc - tumor fusion cells . We chose wortmannin as an inhibitor of pi3k upstream of akt (pkb), u0126 as an inhibitor of mek1 and mek2, rapamycin as an inhibitor of mtor, and jw74 as an inhibitor of the canonical wnt pathway . Admittedly, our data provide several lines of evidence that nf-b - signaling is impaired in dc - tumor fusion cells, but, even though nf-b - inhibitors are starting to emerge in the clinic, it would obviously not make sense to administer nf-b - agonists to cancer patients . Dcs and dc - tumor fusion cells were stimulated with lps in the presence or absence of inhibitors as indicated in figure 5(a) and supernatants were analyzed for il-12p70 secretion by elisa . As expected, dc - tumor fusions did not produce il-12p70 in response, while dcs responded to lps stimulation . U0126 led to a modest increase of il-12p70 by both dc - tumor fusions and dcs . Inhibition of pi3k with wortmannin and inhibition of mtor with rapamycin increased secretion of il12-p70 significantly (11 - 13-fold). Inhibition of the canonical wnt pathway with jw74 did not have any impact on il-12p70 production by dcs or dc - tumor fusion cells . We next asked whether combined inhibition of pi3k and of mtor could further enhance il-12p70 secretion by dc - tumor fusion cells . As shown in figure 5(b), combining wortmannin and rapamycin to inhibit pi3k and mtor did not significantly enhance il-12p70 secretion by dc - tumor hybrid cells, hence excluding a synergistic or additive effect of these inhibitors . This study is the first to investigate the mechanisms responsible for the dependence of dc - tumor hybrid vaccines on exogenously provided 3rd signal adjuvants . These include the induction of apoptosis of immune cells via expression of fas ligand, trail (tnf - related apoptosis - inducing ligand) [39, 40], or pd - l1 and pd - l2 (programmed death ligand). Furthermore, induction of tolerance through cytokines such as tgf-, il-6, and il-10 or lipid mediators [37, 38] has been described . Lastly, activation of the mapk pathway by melanoma cells has been described as a mechanism to inhibit il-12 production by dcs in a paracrine manner . Our results do not provide evidence for overexpression of apoptosis - inducing ligands by dc - tumor cell hybrids, nor did we observe an enhanced production of tolerance - inducing cytokine il-6 or il-10 by these cells . Tgf-3 was upregulated 8.8-fold in dc - tumor fusions, but the observed downregulation of the tgf-induced protein in combination with upregulation of nedd4l argues against a major impact of this cytokine on fusion cells . Surprisingly, despite a profound upregulation of mrna encoding il-4 in dc - tumor hybrids, there was no evidence of il-4 signaling in these cells . We did not observe any upregulation of target genes of the il-4 receptor i or ii, including socs-1, il-4 receptor, ccl11 (eotaxin 1), or fc receptor ii . In addition, there were no changes in expression of gene products that are components of the il-4 receptors, namely, il-4 receptor, il13 receptor, and cd25 . Even though the expression levels of phospholipases a2, which release arachidonic acid from phospholipids, and of prostaglandin d2 synthase were upregulated in dc - tumor fusions, expression of cyclooxygenase, which catalyzes the downstream conversion of arachidonic acid into eicosanoids, was downregulated . Furthermore, while pgd-2 has been shown to upregulate cd80 and to downregulate ip-10 in lps - matured dcs, the exact opposite was observed in our experiments (figures 2(c) and 3(a)). Accordingly, we conclude that pgd-2 may not be the main culprit for the dramatic downregulation of il-12 production in dc - tumor hybrids . However, it has been described that, in the spontaneous b16f10 melanoma cell line, expression of p16ink4a (inhibitor of cdk4a), which inhibits cell - cycle progression by inactivating cyclin - dependent kinases, and of p19arf (alternate reading frame tumor suppressor), which causes mdm2 (mouse double minute 2 homologue) induced translational silencing and p53 degradation, is lost and that there is no evidence of activation of the mapk - signaling pathway in this cell line . It is therefore highly unlikely that the mapk - signaling pathway would be a major contributor to the loss of immunostimulatory capacity of dc - tumor hybrid cells . Nevertheless, our data reveal that treatment of dc - tumor hybrid cells with mek inhibitor u0126 led to a modest increase in il-12 secretion . This however might be a result of the previously published observation that treatment with u0126 can result in a slight but significant inhibition of p70 (s6 ribosomal protein kinase) activation, a downstream target of akt . Our results further indicate that molecules that are involved in wnt signaling, including tcf7l1, frzb, and wntless, were upregulated in dc - tumor fusions . Additionally, wisp-1 and nrcam, targets of the canonical wnt pathway, were upregulated 12- and 148.7-fold, respectively . In the canonical wnt pathway, activation of wnt receptors leads to stabilization and import of -catenin into the nucleus where -catenin associates with t - cell factor / lymphoid enhancer factor (tcf / lef) and activates target genes . However, treatment of dc - tumor fusions with jw74, a specific inhibitor of the canonical wnt pathway, had no impact on il-12 secretion by these cells . On the other hand, it is conceivable that the mtor pathway was activated through the noncanonical wnt / ca pathway, wnt - dependent activation of pka (protein kinase a) and creb (camp response element - binding protein), or mtor activation via wnt - mediated inhibition of glycogen synthase kinase 3 . Alternatively, we cannot exclude that the pi3k / akt / mtor pathway was activated independent of wnt signaling . The pi3k / akt / mtor pathway has been shown to play a critical role in cell proliferation, survival, and metastasis of cancer cells, and we observed that inhibition of the pi3k / akt and inhibition of the mtor pathway enhanced the immune - stimulatory capacity of dc - tumor fusions through induction of bioactive il-12p70 secretion . The fact that combined inhibition of pi3k and mtor signaling did not further improve il-12p70 secretion by dc - tumor fusions may indicate that inhibition acted on the same signaling pathway, likely to involve p70 as has been described previously . In sum, we conclude that combining pi3k / akt / mtor inhibition with dc - melanoma fusion cell - based cancer vaccination appears to be a promising strategy and warrants further studies in vitro and in animal models . Ultimately, this research may lead to the development of improved dc - fusion - based cancer vaccines with enhanced clinical impact.
Marks (2015) review and analysis significantly advances the understanding of the obesity epidemic by (a) identifying the factors that could be at the origin of weight gain and clarifying how they contribute to the obesity epidemic; (b) highlighting the distinction between factors that contribute to initial weight gain, as well as to the processes involved in the circle of discontent (cod); (c) describing the psychological and health problems that result from weight gain and obesity and (d) proposing prevention strategies . Although we applaud the article s advancement in the field, we nevertheless see some issues that may benefit from a different perspective . Our differences with marks largely revolve around his conceptualization of the motivational processes underlying eating regulation and the factors that could lead to successful, versus unsuccessful self - regulation . In general, we believe that marks model focuses almost entirely on environmental factors that may derail the internal process of homeostatic regulation, resulting in the development of obesity . Consequently, the strategies proposed to circumvent the obesity epidemic are aimed at legislations, public policies and facilitating conditions (see also gearhardt et al ., 2012; pomeranz and brownell, 2012). The notion that behaviour should be regulated by homeostasis is nearly a truism; people should be motivated to eat when they are hungry and stop when they are satiated . Unfortunately, many people find it challenging to regulate their eating behaviours and, more so, to sustain this over a long period of time . Why is this? Marks (2015) proposes that over - consumption of high - caloric, low - nutrient foods with low satiating power explains why people initially gain weight, and that the resulting body dissatisfaction and negative affect lead people to consume even more high - density foods and beverages . Once people reach that stage, they gain more weight, become more dissatisfied with their bodies and feel even worse about themselves . Consequently, they attempt to control their weight through different means, which aggravates the problem and leads to more weight gain and, eventually, obesity . Although we agree with marks that a considerable amount of research supports these processes and the association between the variables included in the cod, this view could be problematic for several reasons: (a) it does not explain why some people exposed to the same conditions (e.g. Abundance of unhealthy foods, negative life events) do not gain weight and become obese; (b) it suggests that the individual has a limited capacity to self - regulate; (c) it devotes very little attention to the psychological resources needed for long - term maintenance and (d) it emphasizes strategies to prevent obesity that do not consider individuals as active agents of their own behaviours . We propose that an important aspect of fighting obesity is determining the psychological factors that explain why some individuals may be less susceptible to the processes described in marks theory and how this relates to their motivation to regulate their eating behaviours (patrick and williams, 2012; pelletier et al . We believe that people have the potential to play an active role in the regulation of their eating behaviours, above and beyond what was proposed in marks model . For instance, individuals can plan their eating behaviours on a daily basis by preparing grocery lists and planning daily or weekly meals (otis and pelletier, 2008). They can also engage in activities that are conducive to the achievement of their goals (e.g. To be healthy) (pelletier and dion, 2007), that are integrated with other goals and values (e.g. Improving nutrition for family members) or that are intrinsically enjoyable (e.g. Making meals special family time) (pelletier et al ., 2004). In agreement with self - determination theory (sdt; deci and ryan, 2000), people differ in their reasons for engaging in their nutritional choices and these reasons correspond to the level of autonomy they experience in that life domain . When someone has a more autonomous motivation orientation for a behaviour, they engage in that behaviour for the pleasure, interest and satisfaction derived from the behaviour itself; because it is consistent with other values in their self - system; and it is congruent with their values and goals (e.g. Eating a plant - based diet because you view yourself as a healthy individual that is environmentally conscious). When someone has a more controlling motivation orientation for a behaviour, they perform that behaviour because of self - imposed pressures such as guilt or anxiety (ryan and connell, 1989) or they want to achieve a reward or avoid a punishment (e.g. Eating behaviours engaged in to avoid feeling ashamed for not eating healthy). People who have an autonomous orientation, versus a controlled orientation, assume greater responsibility for their actions because they have personally endorsed their course . Autonomously motivated behaviours are better maintained because they are either inherently enjoyable or are well internalized into the person s sense of self (ryan and deci, 2006). In relation to eating, autonomously motivated eating behaviours lead to better regulation of eating and weight management (pelletier et al ., 2004) and more sustained regulation over time (guertin et al ., 2015). In sum, we believe that the form of regulation portrayed by marks (2015) corresponds to controlled regulations and although the model could explain why some people fail to regulate their eating behaviours, it falls short when trying to explain why some people succeed and, most importantly, how this pattern could be prevented and even reversed . Like several other researchers before him (dittmar, 2005; levine and harrison, 2004; polivy and herman, 2004), marks (2015) suggests that body image, sociocultural pressures about body image and the internalization of the thin - ideal represent risk factors for body dissatisfaction and lead to several eating - related problems (levine and piran, 2004; stice, 2002). Pelletier and dion (2007) have examined how sdt could contribute to our understanding of the associations between these risks factors by examining how motivation at two different levels (life in general and in the context of eating) could explain why some people differ in their responses to sociocultural pressures and messages related to body image . Their results suggest that an autonomous motivation orientation at the general level can help people protect against pressures related to body image and endorsement of society s beliefs about thinness and obesity and has a direct influence on their motivation towards eating behaviour . Specifically, autonomous motivation at the general level is positively associated with autonomous motivation for healthy eating behaviour, which leads to increased healthy eating, and negatively associated with controlled motivation of eating behaviours, which is associated with dysfunctional and unhealthy eating . Overall, these results suggest that a general autonomous motivation orientation may serve as a buffer against sociocultural pressures and messages of thinness and promote autonomous motivation towards healthy eating, which is in accordance with one s own integrated values, instead of a response to external controlling forces . As a contrast, it appears that body dissatisfaction resulting from pressures about body image and endorsement of society s beliefs about thinness and obesity may be more closely associated with controlled motivation towards eating behaviours, which may explain its relation with eating pathology . Thus, the more people are autonomous in their life in general and, as well as towards their eating, the less likely they are to perceive sociocultural messages about body image as a source of pressure, but instead, as information that they are free to use or dismiss . Although we agree with the four prevention strategies proposed by marks (2015), we fail to see how they could lead to behaviour change and, more importantly, to sustained self - motivation for health behaviours . Environmental changes such as those proposed by marks may be slow to implement, can be very expensive and could be stalled by industries with competing interests (pomeranz and brownell, 2012). Therefore, it might be important to develop strategies that emphasize self - regulatory processes that help individuals become active agents in their own behaviours in the pursuit of healthy and sustained eating behaviour changes . A critical point is that both individual - based and population - based initiatives to fight obesity should be guided by information campaigns to promote healthy eating which apply sound theory - based motivational principles . For instance, providing people with strategic messages is often perceived as an important step to motivate people to change a specific behaviour . One systematic approach that could be used to facilitate behaviour change consists in tailoring persuasive messages in function of the processes that underlie behaviour change (i.e. Detecting a problem, deciding on a course of action and implementing a behaviour) and framing persuasive messages in terms of whether they serve autonomous (health, well - being, personal growth) or controlling (physical appearance and appealing to others) goals . Regarding message framing, research suggests that when a goal is framed as a function of autonomous motives, relative to controlling motives, it should lead to more engagement in an activity, more persistence over time and its effects should generalize to health - related behaviours (pelletier and sharp, 2008). In summary, although we commend marks for the contributions to the obesity literature, we highlight three points that should be considered when attempting to explain and propose solutions for the obesity epidemic . Greater emphasis should be placed on the role the individual plays in regulating their own eating behaviours, with less responsibility attributed to external sources, in order to facilitate feelings of self - control and accountability . A substantial base of research has indicated that there are multiple forms of motivation that differ in their degree of internalization and result in very different outcomes, with autonomous motives leading to desirable outcomes, and controlled or amotivated motives resulting in less desirable consequences . Instead of simply de - valorizing the thin - ideal, messages about eating should be designed to highlight autonomous goals and motives (e.g. Pleasure, health, personal development or family time) in order to facilitate sustained changes in eating behaviours.
A 55-year - old man was admitted to the general ward at a gyeongsang national university hospital with an obstructive hydrocephalus visualized by brain magnetic resonance imaging . A ventriculoperitoneal shunt had been placed 2 years earlier due to an obstructive hydrocephalus, but the physical examination was within the normal limits with the exception of an upward gaze disturbance . The laboratory studies including a complete blood count, electrocardiogram, liver and renal function tests were within the normal limits . The patient was premedicated with midazolam (2.5 mg) and glycopyrrolate (0.2 mg). Anesthesia was induced with 250 mg sodium thiopental and 50 mg rocuronium while he received oxygen (5 l / min) through a face mask . Fiberoptic orotracheal intubation was then accomplished using a reinforced endotracheal tube (internal diameter: 7.5 mm). Anesthesia was maintained with nitrous oxide in oxygen (50%: 50%) and 6 vol% desflurane . The intraoperative end - tidal carbon dioxide tension was maintained within 30 - 35 mmhg . He received irrigation with approximately 3,000 ml normal saline at room temperature while introducing the neuroendoscope (minop neuroendoscopy system, aesculap, center valley, pennsylvania, usa) with a shaft diameter of 2.7 mm, a maximum infusion rate of 1,000 ml / min and a maximum pumping pressure of 500 mmhg . During the surgical procedure, irrigation fluid was naturally drained and the neuroendoscopic intracranial pressure was not measured because monitoring equipment was unavailable . Two hours after beginning the procedure, his systolic blood pressure and heart rate increased from 100 mmhg to 170 mmhg and from 45 - 90 beats / min to 100 - 110 beats / min, respectively . Intravenous hydralazine (10 mg) and esmolol (10 mg) was administered to control the increased blood pressure and heart rate . However, the increased heart rate persisted from 2 hours after beginning the procedure to the end of the operation . Otherwise, the operation and anesthesia were uneventful . In order to rule out a postoperative hemorrhage, brain computerized tomogram taken immediately after the endoscopic third ventriculostomy revealed the external ventricular drainage catheter and pneumocephalus on the left frontal convexity . Although he showed mild confusion, the patient was extubated after confirming adequate voluntary respiration . Four hours after surgery, the respiratory rate increased to 50 - 66/min and he showed seizure - like movements . Although administering intravenous diazepam (10 mg), haloperidol (3 mg) and orfil (300 mg) to treat the seizure - like movement, mild seizure - like activity such as myoclonal jerk persisted for 2 hours and was treated successfully with intravenous administration of another 10 mg diazepam used for endotracheal intubation . Arterial blood gas analysis at that time revealed acute respiratory alkalosis (table 1). Orotracheal intubation using a direct laryngoscope was then performed with the intravenous administration of 10 mg diazepam, and controlled mechanical ventilation (assist - control mechanical ventilation mode: tidal volume; 500 ml, respiratory rate; 16/min, inspired oxygen fraction; 40%) was applied for 15 hours (table 1). The complication rate for endoscopic third ventriculostomy ranges from 6% to 8%, and is similar to the expected infection rate of a shunt . The complications of an endoscopic third ventriculostomy include cerebrospinal fluid leak, pneumocephalus, ventriculitis, subdural hematoma, injury to the periventricular structures (hypothalamus, basal ganglia and brain stem), bradycardia, asystole, minor bleeding and hypokalemia . The etiology of respiratory alkalosis includes hypoxia, parenchymal lung disease, bronchial asthma, drug, mechanical ventilation, central nervous system disorder, metabolic causes and hyperventilation . The floor of the third ventricle is not a membrane but a part of the hypothalamus . Massive or high - speed irrigation with normal saline has been associated with a hypothalamic dysfunction . As the neuroendoscope used in this patient was not equipped with device for a critical setting of infusion rate and pumping pressure, an unchecked maximum infusion rate (1,000 ml / min) and pumping pressure (500 mmhg) which produce a transient increase in pressure inside neuroendoscope may cause a transient injury of periventricular st ructures such as hypothalamus . A> 30 mmhg pressure inside the endoscope is associated with postoperative morbidity, particularly unexpected delayed recovery . First, considering the above reports, hypothalamic dysfunction caused by an unrecognized increased intracranial pressure due to the high - speed irrigation of normal saline during an endoscopic third ventriculostomy may contribute to the hyperventilation observed in the recovery room . A critical setting of infusion rate and pumping pressure during an endoscopic third ventriculostomy might help reduce pressure transferring to periventricular structures . Therefore, we recommend that the neuroendoscopic intracranial pressure be monitored to prevent the complications associated with hypothalamic dysfunctions . Second, this hyperventilation may be partially associated with csf acidosis induced by normal saline irrigation used during the procedure . The ph of the cerebrospinal fluid is 7.31, whereas that of normal saline is 6.1 . A total of 3,000 ml normal saline used during the endoscopic third ventriculostomy would fill the enlarged lateral and third ventricles with normal saline instead of csf . After fenestrating the floor of the third ventricle, acidic normal saline in the enlarged lateral and third ventricles drained into the interpuduncular cisterna filled with csf, which might have led to a more acidic csf compared to normal csf . Central lactate production of a meningeal tumor can also cause csf acidosis, which induces hyperventilation and respiratory alkalosis . The capacity of the male elderly lateral and third ventricle is 35.19 20.35 and 2.63 0.96 ml, respectively . Our findings and those of previous reports [8 - 10] suggest that the ph in the csf and arterial blood should be measured simultaneously to confirm the respiratory alkalosis caused by normal saline irrigation - induced csf acidosis . The causes of peripheral hyperventilation, which include hypoxia, parenchymal lung disease, medication, mechanical ventilation, central nervous system disorder and metabolic disturbance, were excluded by a physical examination and laboratory tests . In conclusion, central neurogenic hyperventilation was developed in a patient receiving high - speed irrigation and large amount of normal saline (maximum infusion rate of 1000 ml / min and maximum pumping pressure of 500 mmhg) during an endoscopic third ventriculostomy, which may be ascribed to a transient hypothalamic dysfunction and/or acidic csf.
Stevens johnson syndrome (sjs) is thought to be a hypersensitivity complex affecting the skin and the mucous membranes . A new anticonvulsant, oxcarbazepine, which is structurally related to carbamazepine (cbz), was introduced for use in patients with epilepsy . Drug hypersensitivity reactions can occur with most drugs, although the frequency, severity, and clinical manifestations vary . Drug hypersensitivity can be defined as an inappropriate immune response leading to tissue damage from an otherwise nontoxic agent . Drug hypersensitivity reactions to benzodiazepines often fall into the category of type b (or bizarre) adverse drug reactions, according to the classification proposed by rawlins and thompson . The incidence of hypersensitivity varies according to the drug, the disease being treated, and the ethnicity of the patient . Drug response (including drug hypersensitivity) is a multifactorial and multigenic process, dependent on a complex interaction between multiple genes and the environment [figure 1]. Each gene contributes to the risk of developing the hypersensitivity reaction, but each individual gene is neither necessary nor sufficient by itself to cause the reaction . A female patient of 38 years with a history of drug allergy was complaining of fever, sore throat, and fatigue, at the time of admission . She was administered oxcarbazepine for the management of right partial bronchial seizure due to left parasagittal mass lesion following which she developed papular rashes all over the body and diagnosed as sjs . In this case, during the first week, we used 600 mg / day oxcarbazepine for seizure control, and then increased the dose after 3 days to 900 mg / day . After 10 days of treatment, ulcers and other lesions begin to appear in the mouth and lips along with the genital and anal regions . Ulcers in the mouth are usually extremely painful and reduce the patient's ability to eat or drink . High fever and multiple maculopapular rashes were found over the patient's face and neck initially on the 12 day after taking oxcarbazepine . She was brought to our emergency department and admitted under the presumed diagnosis of sjs . Laboratory investigations showed leukocytosis (wbc, 14660/l; reference value, 4,000 - 10,000/l), and elevated c - reactive protein (59.30 g / ml; reference range, 0 - 5 g / ml). After obtaining informed consent, we carried out genotyping and took photos of the patient . There was marked liquefactive degeneration in the lower half of the epidermis with some dyskeratotic keratinocytes . The dermis showed predominant cd8 + lymphohistiocytic infiltration around the blood vessels and scanty eosinophils . After corticosteroids (iv dexamethasone 1.0 mg / kg body weight) and antihistamine treatment for 10 days, the patient improved and was discharged from the hospital . Anticonvulsant hypersensitivity syndrome is a potentially fatal drug reaction with cutaneous and systemic reactions (incidence, 1 in 1000 to 1 in 10,000 exposures) to the arene oxide - producing anticonvulsants phenytoin, cbz, and phenobarbital sodium . Cbz and its derivatives are widely used anticonvulsants that can cause rashes in up to 10% of patients, and in occasional cases, this may be the precursor to the development of a hypersensitivity syndrome characterized by systemic manifestations such as fever and eosinophilia . The diagnosis of sjs is based on clinical manifestations with acute onset of rapidly expanding targetoid erythematous macules, necrosis and detachment of the epidermis along with erythema, erosions, and crusting of two or more mucosal surfaces . Our patient had skin targetoid erythematous rashes and mucosa involvement 2 weeks after starting oxcarbazepine treatment . During these 2 weeks the skin pathology finding revealed lymphohistiocytic infiltration around the blood vessels and scanty eosinophils, which was consistent with sjs . The patients usually develop a hypersensitivity reaction between 2 and 12 weeks after starting medicine . It has been postulated that metabolites and not the parent drug are the causal agents . One of the first reports showed that hla - b*1502 was present in 100% of cbz - induced patients with sjs but in only 3% of patients tolerating cbz and in 9% of the general population . Other studies have confirmed these results in han chinese and in the thai population . In this case, we used a dose of 600 mg / day and then titrated it to 900 mg / day . It has been reported that higher daily doses of drugs are associated with an increased risk of sjs than lower doses, which is the case for allopurinol . However, lam et al . Found that the early use of short - term systemic steroids for 35 days lacked any significant side effects and did not increase mortality or morbidity . No sequels were found during 3 months of follow - up . Compared with other categories of drugs, such as antibiotics and nsaids, antiepileptic therapies are associated with a high incidence of sjs and toxic epidermal necrolysis (ten). Case reports by chen et al . Suggest that identification of such genetic factors is important, not only to realize the prospect of developing preventive strategies but also to learn about the mechanisms of these reactions, which may ultimately lead to other preventive strategies through better drug design and to better treatment strategies for patients who develop the reactions.
This sticky, greenish - brown product has different compositions depending on the location of the bees and what trees and flowers they have access to . Propolis from turkey or egypt will not have the same chemical properties as propolis from europe or brazil . This is because it is very difficult for researchers to come to general conclusions about its health benefits . Caffeic acid phenethyl ester (cape) [figure 1] is one of important compounds found in propolis that has antiviral, antioxidant, anti - inflammatory, antiproliferative, antitumor, and immunomodulatory effects . This marvelous compound has been used to prevent oxidative stress - based deterioration in cells / tissues / organs in both cell culture and experimental animals . Lately, the protection of cape on central and peripheral nervous system as well as a reproductive system have been extensively reviewed [3 - 5]. It has extensively been used to treat a broad of malignancies including hodgkin s and non - hodgkin s lymphoma, burkitt s lymphoma, chronic lymphocytic leukemia, ewing s sarcoma, breast cancer, testicular cancer, etc . It has toxic effects in almost every system in the human body including the heart, liver, and kidney of which mostly due to its structural properties prone to induce oxidative stress in vitro and in vivo . The chemical illustration of caffeic acid phenethyl ester this study aimed to collect data and compare the protective effects of propolis and cape against cp - induced injury in animals ., ameliorative effect of propolis against cp - induced toxicity in mice was studied by el - naggar et al . . It throws light on the side effects of a common anticancer agent, cp, used in the treatment of various malignancies and possible remedies to prevent that type of side effects in vital organs such as liver and kidney . Uysal et al . Conducted an experimental animal study to determine protective role of cape on cp - induced hemorrhagic cystitis (hc). While cp - induced hc lead to increase in superoxide dismutase, catalase, and malondialdehyde activities / levels, cape significantly reduced these parameters showing the protective effects . In addition to this biochemical effects, cape also ameliorates edema, hemorrhage, inflammation, and mucosal ulceration of cp - induced hc . We published a review article about toxicities of some therapeutic compounds and the protective effect of cape on chemotherapy- and radiotherapy - induced toxicity . We have shown that cape has protective effects on oxidative stress - induced toxicities by doxorubicin (nephrotoxicity), cisplatin (nephrotoxicity, ototoxicity, and hepatotoxicity) [10 - 13], and bleomycin (lung fibrosis). Currently, there is no medically recommended dose for propolis, since the mixture of propolis is subjected to change depending on its source . The most successful medical application field of propolis is beauty and skin care, especially in acne vulgaris because of its antibacterial, antiviral, antifungal, and anti - inflammatory properties . Despite the fact that both water and ethanolic extractions of propolis have been used in the in vivo and in vitro experiments, water - soluble extracts of propolis exhibit higher antioxidant and inhibitory activities as compared ethanolic extract in vitro . In this perspective, even though the extraction method selection is dependent on the authors desire, it would be expected for authors to study propolis for their experiments comparatively by selecting propolis extracted by both extraction methods . Cape is the most potent antioxidant agent of propolis mixture having free radical scavenging activity and potent inhibition of nf-b . So, the protective antioxidant effect of ethanol extract of propolis on organs depends mostly on cape rather than other polyphenolic compounds such as flavonoids, phenolic acids, and their esters [figure 2]. Cape was shown to completely block the production of reactive oxygen species in human neutrophils and in the xanthine / xanthine oxidase systems at 10 m concentration by its competent antioxidant capacity . Indeed, cape has a regulatory effect on antioxidant enzyme activities such as catalase, superoxide dismutase, and glutathione peroxidase [figure 2]. Proposed mechanism of how cyclophosphamide - induced oxidative stress is blocked by antioxidant enzymes in several parts of hepatocytes and renal cells and how cape shows its protective effects against oxidative stress . Aas: amino acids, cape: caffeic acid phenethyl ester, cat: catalase, fe: ferrous iron, gpx: glutathione peroxidase, gsh: reduced glutathione, gr: glutathione reductase, gssg: oxidized glutathione, h2o: water, nadp: oxidized nicotinamide adenine dinucleotide phosphate, nadph: reduced nicotinamide adenine dinucleotide phosphate, o2: molecular oxygen, o2: superoxide anion radical, oh: hydroxyl ion, oh: hydroxyl radical, onoo: peroxynitrite, no: nitric oxide, nos: nitric oxide synthase, pufa: polyunsaturated fatty acid, sod: superoxide dismutase it has been shown that cape application to the rats modifies the enzyme activity of cytochrome p450 (cyp) isoforms involved in the activation of diethylnitrosamine such as cyp1a1/2 and cyp2b12 . Furthermore, treatment with cape of carbon tetrachloride - induced hepatotoxicity in mice blocks cyp2e1-mediated ccl4 bioactivation and protects against fas / fasl - mediated apoptosis . It will be very interesting to see the effect of cape on cyp2b6, which constitutes 3 - 6% of total hepatic cyp content and metabolizes several pharmaceuticals including cp . To achieve this, further studies on the every single bioactive constituent of propolis such as cape and some other polyphenols are necessary to identify interactions mediating their biological effects on cyp2b6, since there are roughly 150 different polyphenolic compounds within propolis . As a conclusion, studying propolis to prevent cp - induced oxidative stress in animals has several limitations since the proposed effect cannot be specified to one or several molecules within the mixture . In that case, every single bioactive constituent of propolis needs to be studied to show the source of real effects and the molecular mechanisms of this effects.
Langerhans cell histiocytosis (lch) refers to a clonal, neoplastic expansion of langerhans cells . Stimulation by tumor necrosis factor and granulocyte - monocyte colony - stimulating factor has been implicated in the pathogenesis (as has human herpesvirus 6). It most commonly affects children between the age of 1 and 4, although cases have been described in all age groups; the incidence in pediatrics has been estimated at 25 per million / year . It is exceptional for lch to present with brain involvement as the sole presenting symptom . This complication is reported as occurring in 14% of patients with multisystem disease, typically 520 years from the initial diagnosis . He developed depression, anxiety and agitation, which led to his retirement from construction work . Shortly thereafter, he developed episodes described as hyperventilation, lasting from seconds to minutes, mostly in the morning, which were followed by confusion and a severe pounding headache affecting the whole head, both of which lasted for hours . One year prior to presentation, his family described personality changes (it was hard for them to characterize this further) and worsening confusion, particularly over the preceding 3 months . Three months before presentation, he developed weakness of the right leg, which had been slowly progressing since . Two weeks prior, it became so bad that he needed help walking . At this time, he also noticed weakness of the right arm and slurring of speech, which led him to seek medical help . He had been diagnosed with peripheral arterial disease (claudication in his left leg) 4 months prior to presentation, and paroxysmal atrial fibrillation 1 month prior his initial review of systems revealed that he was anorexic and had lost 30 lb over the last 2 years . He also complained of a loss of libido and an occasional shortness of breath when walking . On initial exam, he was unable to sustain upward gaze; vertical saccades were followed by a slow downward drift, then a compensatory saccade . He had a weakness of the right side of his face, sparing the forehead . He was weak throughout, with strength of 4/5 on his right side in an upper motor neuron pattern with signs of spasticity; babinski's reflex was present on this side . His initial complete blood count, comprehensive metabolic panel, serum protein electrophoresis and folate were normal apart from a white cell count of 13 10/l (95% neutrophils) and platelets of 570 10/l . His sodium was initially normal, but, during his admission, it was generally in the range of 145160 mmol / l . Mg / l (<10), and the erythrocyte sedimentation rate 60 mm / h (<20). He was hypothyroid with a thyroid - stimulating hormone (tsh) level of 5.6 iu / ml (<4.9) and a free t4 of 0.6 ng / dl (> 0.7). He had hypogonadism (presumably secondary) with total testosterone of <20 ng / dl (> 200). A lumbar puncture was done shortly after admission, showing 4 red cells, 9 white cells (56% lymphocytes, 43% macrophages), and a protein level of 45 mg / dl (<35). Oligoclonal bands were absent and cytology showed no signs of malignancy . Shortly after admission, a complete serum paraneoplastic panel was sent; this was negative . The antibodies reported were: anti - neuronal nuclear types 13, anti - glial nuclear type 1, purkinje cell types 12 and type tr, amphiphysin, crmp-5-igg, striatal, t / q and n - type calcium channels, ach receptor - binding, ach receptor ganglionic and neuronal voltage - gated k channels . Mri is notable for the presence of t2-hyperintense lesions in the left frontal lobe, the right mesial temporal lobe and the brainstem, which were minimally enhanced . An mr perfusion scan of the brain showed no significant increase in relative blood volume or relative blood flow in these lesions . A ct of the thorax, abdomen and pelvis showed 5 small lung nodules (maximum diameter 6 mm) and a pneumatocele in the left lower lobe . As there was nothing further on history, exam or initial investigations to suggest cancer anywhere else, a pet / ct was not performed . Immunohistochemistry shows the lesion to be cd1a- and s100-positive, which is characteristic for histiocytosis . Initially, we were unsure of the cause of his right temporal lesion . Given the rarity of lch with onset in the central nervous system (cns) in adults, the possibility of another tumor was raised, and so a second biopsy was performed . In retrospect, it would have been better to avoid performing this, as the diagnosis was already clear and his other lesions were attributable to lch . Furthermore, the procedure precipitated status epilepticus and led to preventable complications which ultimately contributed to his death . This illustrates mild edema, hypercellularity and reactive changes, all of which are consistent with the edge of an inflammatory process . Septic shock (likely due to ventilator - associated pneumonia) and a pulmonary embolism complicated his course thereafter . Histiocytosis affecting his skull was thought to be in remission as a result of the treatment, as there was no sign of new or enlarging lytic lesions . After discharge from the icu, he showed signs of critical illness myopathy / neuropathy . There was widespread muscle atrophy and generalized weakness . He was unable to stand without support and his tracheostomy remained in place . Given the suspicion of paraneoplastic brainstem inflammation, he was started on intravenous immune globulin (ivig) 400 mg / kg 5 days . Two weeks after the initial treatment, the brainstem showed some improvement in the degree of flair hyperintensity (fig . An mri 1 month after a second course of ivig showed these changes to be stable overall, with some increase in pontine enhancement (fig . He then developed recurrent pneumonia and septic shock, again requiring an icu admission . Following this, his family elected a palliative approach to care and he died 6 months after his initial presentation . Our patient's episodes of hyperventilation followed by confusion are typical for seizures involving the right mesial temporal lobe; the similarity to panic attacks has previously been identified in a case series . The development of status epilepticus following biopsy of this region lends support to this being an epileptogenic focus . In his cerebrospinal fluid (csf), the finding of a high proportion of macrophages is striking . We were unable to find literature specifically addressing the significance of this finding; classically it is said to be associated with meningitis due to fungi or tuberculosis . None of the other case reports we studied, including one with langerhans cells in the csf, reported this finding . One series has shown elevations of neurofilament protein light chain, glial fibrillary acid protein and total tau protein in the csf of patients with lch affecting the brain . These advise the use of cladribine for patients such as ours with multisystem and cns involvement (cytarabine is thought be a reasonable alternative). Myelosuppression is the main dose - limiting toxicity; this has best been studied in the setting of hairy cell leukemia where neutropenia was reported in 70%, anemia (hb <8 g / dl) in 35% and thrombocytopenia in 10% of patients . The most recent and thorough description of the neuropathology of lch comes from a series of 12 cases from grois et al . In 2005 the authors divide the pathology into 3 categories: (1) the typical circumscribed granulomas found in other organs; (2) infiltrating granulomas with profound t - cell dominated inflammation and severe neurodegeneration; (3) neurodegenerative lesions lacking langerhans cells, typically affecting the brainstem and cerebellum and resembling paraneoplastic encephalitis . It appears that the temporal and frontal lesions in our patient were of the second type; it is probable that the biopsy came from the neurodegenerative part of the lesion rather than that containing langerhans cells . The mri appearances of the brainstem in our patient are typical of the third type . The presence of inflammation without langerhans cells has led to the view that these lesions are paraneoplastic in origin and that the neurodegeneration seen is thus autoimmune . The langerhans cells are thought to take up normal neuronal antigens, provoking an immune response to these; alternatively, the immune system may target antigens shared by langerhans cells and microglia . If an antigen is involved, the pathology reported by grois et al . The antigens responsible for this phenomenon have not been identified and thus it was to be expected that our patient's paraneoplastic panel was negative . Using multiple immunosuppressants langerhans cells in their normal form are thought to have a lifespan of months, and lch is characteristically a slowly progressive disorder . By contrast, cytotoxic t - cells typically have a half - life of 2448 h when activated . When used in the treatment of leukemia and lymphoma, it is typically given every 4 weeks . Thus, the initial doses of cladribine would have been expected to lead to the death of a majority of neoplastic histiocytes while the t - cell - mediated inflammation would have been expected to resume within 34 weeks . Ivig would not have been expected to affect the viability of langerhans cells, but should have reduced t - cell activity for 34 weeks . It leads to a selective decrease in t - cells overall and an increase in regulatory t - cells . In children with lch, ivig has been reported to contribute to a stabilization of inflammation in 4/5 patients treated and to enable long - term remission in 1 case [11, 12]. It has been suggested that ivig be given prophylactically to prevent the development of cns complications of lch, particularly diabetes insipidus, which tends to be irreversible . Alternative agents reported to treat the paraneoplastic inflammation in lch include vincristine / cytarabine and all - trans - retinoic acid . The use of steroids in our patient would not have been expected to have had long - lasting effects . Bearing the above in mind, as well as the results of treatment in other t - cell - mediated paraneoplastic conditions, strong consideration should be given to agents that primarily target t - cells: steroids, ivig, plasma exchange and mycophenolate mofetil or cyclophosphamide . Other paraneoplastic disorders recognized in patients with lch include cancer - associated retinopathy and spinocerebellar degeneration . Brain imaging should always be undertaken in an older patient with episodes suggestive of seizures, diabetes insipidus of central origin or symptoms suggestive of pituitary dysfunction . Once a diagnosis of lch is made, a brain biopsy is not essential if the mri appearances are typical for the inflammatory processes that accompany this condition . Once lch appears stable, prolonged treatment may be needed to prevent recurrence of inflammation . Ivig appears to be a reasonable adjunct to cladribine, particularly in a case such as this where cytopenias or infections limit the use of conventional chemotherapy.
Congenital heart disease (chd) is one of the most common birth defects, with an incidence of nine out of every 1,000 live births.1,2 critical chd (cchd) is defined as cardiac lesions that require surgery or cardiac catheterization within the first month (or within the first year by different definitions) of life to prevent death or severe end - organ damage.2 although infant mortality has decreased over the past 3 decades for children with all forms of chd, many children are still diagnosed too late to avoid significant morbidity or death.35 delayed diagnosis of cchd is unfortunately all too common, with up to 25% of infants with these defects being missed in newborns when identification is based on clinical symptoms or signs of heart disease even in settings with routine prenatal sonograms.3,68 approximately 40% of these infants with missed diagnoses at birth present in cardiogenic shock at a medical facility and 5% are diagnosed at autopsy.4,5,9 studies in europe and the us have suggested that newborn screening with pulse oximetry testing prior to discharge from the nursery can decrease the number of missed diagnoses by 30%.2,10 in 2011, pulse oximetry screening for cchd was added to the recommended uniform screening panel by the health and human services secretary.11 in the subsequent years, many states have implemented their own protocols to comply with this recommendation.1215 pulse oximetry has been the mainstay procedure for indirectly detecting hypoxemia in medically ill patients since the 1980s.16,17 the screening of cchd by pulse oximetry involves taking advantage of its ability to detect clinical and more importantly subclinical levels of hypoxemia that should raise suspicion for a cchd . However, pulse oximetry does not readily offer information about decreased stroke volume, which is another physiologic feature of several cchds that could be detectable during the neonatal transition . In this review, we highlight the relevant principles and limitations of pulse oximetry in the context of detecting cchd . The beer lambert bouguer law of physics describes the attenuation of light to the properties of materials through which the light is traveling.16 oxygenated blood absorbs red light at a wavelength of 660 nm, and deoxygenated blood absorbs light in the infrared spectrum at 940 nm.16 computation of oxygen saturation is achieved with the use of calibration algorithms, based on the amount of signals from nonpulsatile (venous, capillary, bone, and skin) and pulsatile arterial blood flow, in the red and infrared wavelengths mentioned earlier . A microprocessor removes the continuous signal from the nonpulsatile tissues and vessels, which leaves the pulsatile signal from the arteries to be displayed as a plethysmographic wave form on the pulse oximeter monitor.16 safe use of pulse oximetry requires knowledge of its limitations . There are multiple sources of potential artifacts, which are particularly relevant for neonates and can cause false readings . These include motion artifacts, poor perfusion and cold skin at the site of measurement, irregular rhythms, ambient light, phototherapy or electromagnetic interference, skin pigmentation and jaundice, inappropriate probe positioning (penumbra effect), venous pulsation, intravenous dyes, and presence of abnormal hemoglobin molecules.1618 pulse oximeters recommended for screening for chd should report functional oxygen saturation (referring to the hemoglobin that is capable of transporting oxygen), be motion tolerant, be validated for low perfusion states, and have a 2% root - mean - square accuracy.11 of note, a pulse oximeter s performance is optimized for oxygen saturations (spo2) in the range of 80%100% . The development of the modern pulse oximeter is based on healthy, fit adult individuals who were exposed to different degrees of subambient oxygen with their spo2 being kept between 80% and 100% . Therefore, any spo2 <80% is extrapolated by a computer program.19,20 most new pulse oximeters are able to detect motion and label it as artifact or perform calculations quickly in a way that renders them motion tolerant . The pulse oximeter uses an algorithm to average readings over a period of time . For the most accurate measurement, the average is taken over a shorter period of time, which also increases the delay of the reading and decreases the speed of computation by the machine . For a pulse oximetry measurement to be accurate, the peripheral tissue needs an adequate pulse volume and pressure . In situations such as septic shock, where the extremities are cool and have low perfusion, the pulse oximeter may not reliably assess the oxygen saturation.19,20 competing light sources, such as other machinery, fluorescent lighting, and even cell phones, can overload the semiconductor sensor . In pediatrics, a phenomenon called the penumbra effect occurs.18 pulse oximeters may over - or under - read the spo2 in infants and children because of the small size of their fingers, or the other areas where the pulse oximeter probe is placed, and the different light paths for each wavelength through the peripheral tissue . Specific probes for infants and children have been created and should be used to prevent these false measurements . Finally, pulse oximetry should not be used in certain circumstances in which the hemoglobin molecule is not completely saturated with oxygen, such as with carbon monoxide poisoning or when methemoglobinemia occurs after administration of certain medications such as antimalarial drugs, nitrates, nitrites, or dyes such as methylene blue . In these instances, pulse oximetry is used in all aspects of newborn care, including resuscitation of newborns in the delivery room to routine monitoring in the operating room . A study by levesque et al21 in 2000 described the normal range of oxygen saturations in term newborns in the first days of life . These investigators evaluated normal oximetry values at sea level, from admission to the newborn nursery to discharge . They also evaluated variables such as sex, gestational age at birth, birth weight, mode of delivery, apgar scores, pre- or postductal site of measurement, and status of the infant at the time of measurement (sleeping, quiet, and crying). Pulse oximetry measurements were taken upon admission to the newborn nursery, at 24 hours of life, and at discharge . The overall oxygenation saturation was 97.2%1.6% (95% ci 97.1%97.2%).21 they compared the mean values at each time period, which showed a slight increase in oxygen saturation over time for the right hand (preductal) and right foot (postductal). Rising from statistical significance were only two variables, postnatal age and activity of the infant . Postnatal age was found to be statistically significant (+ 0.17% per 24-hour interval from admission to discharge, p=0.0001) but not clinically relevant.21 infant s activity was statistically significant with values obtained while the infant was crying, fussy, or awake being lower than the values obtained while sleeping.21 de wahl granelli et al10,22 systematically measured the oxygen saturation in 40,000 newborns and compared the values obtained in children with cchd vs otherwise healthy newborns . In addition, these investigators showed that two of the three infants with cchd were missed by physical examination alone.22 addition of pulse oximetry screening raised the diagnosis rate to 82% . Several other studies mostly from europe revealed similar findings.12,2328 these were summarized in a systematic review and meta - analysis of pulse oximetry screening for cchd in the newborn nursery, which included 13 studies with 229,421 infants.28 sensitivity of pulse oximetry was 76.5% (95% ci 67.783.5) and specificity was 99.9% (95% ci 99.799.9) for the detection of chd, with the average false - positive rate for these infants being 0.14% (95% ci 0.160.33).28 with the average pulse oximetry value being 97.2% during the first days of life for all newborns, pulse oximetry is an excellent tool to evaluate subclinical hypoxemia, that occurs during transitioning physiology of certain chd, such as transposition of the great arteries, truncus arteriosus communis, hypoplastic left heart syndrome, total anomalous pulmonary venous connection, tricuspid atresia, tetralogy of fallot, and pulmonary atresia . These lesions are usually associated with hypoxemia in the newborn period and can cause significant morbidity and mortality if the diagnosis is delayed . In september 2010, the secretary s advisory committee on heritable disorders in newborns and children (sachdnc) considered these seven lesions as primary targets for pulse oximetry screening in the newborn period on the basis of advice from a technical expert panel.2 in 2011, the sachdnc, in collaboration with the american academy of pediatrics, the american college of cardiology foundation, and the american heart association, convened a work group to outline implementation strategies for pulse oximetry screening in newborns for chd . After reviewing data from existing large studies in sweden and the uk,22,25 the work group proposed a screening protocol based on results of measurements from the right hand (preductal) and either foot (postductal).11 according to the sachdnc protocol, an infant would have a positive (failing) screen if at 24 hours of life: 1) a pulse oximeter reading was <90% in either the right hand or either foot . 3) a persistent> 3% difference in the right hand and either foot measurement on three measurements each separated by 1 hour . An infant who had 95% in either extremity with 3% difference in the pre- and postductal oxygen saturation would have a negative screen and no further work - up is needed . Many states that have initiated pulse oximetry screening have adapted this protocol or a variation there of.12,13,15 kochilas et al14 demonstrated that the sachdnc protocol was the most efficient protocol with the fewest false - positive pulse oximetry screens in the newborn period . Data from various studies suggest that this protocol is adequate to detect clinical and subclinical hypoxemia that is associated with the seven primary target lesions based on their almost universal association with at least mild hypoxemia in the neonatal period, when the fetal circulation transitions to postnatal circulation . The protocol should also be effective, although to a lesser degree, to detect the hypoxemia associated with five additional congenital heart lesions that are considered as secondary targets.6,9 these lesions are frequently associated with at least some degree of neonatal hypoxemia and include proximal aortic arch anomalies (such as interrupted aortic arch or aortic atresia), coarctation of the aorta (coa) with patent ductus arteriosus, ebstein s anomaly, double outlet right ventricle, and single ventricle lesions . There are additional lesions that are possibly screenable in the neonatal period with the same protocol based on their anatomy and potential for intracardiac or ductal - level shunting . These include aortic stenosis with a patent ductus arteriosus, severe pulmonary stenosis, and complete common atrioventricular canal . Finally, there is a category of cardiac lesions that will not have hypoxemia in the newborn period and can be classified as not screenable and include left - sided obstructive lesions such as coa without patent ductus arteriosus and aortic stenosis without a patent ductus arteriosus, ebstein s anomaly without interatrial shunting from right - to - left, and all other lesions that cause left - to - right shunting and valve anomalies that were not included in the previous categories29 (table 1). Although not all chds cause hypoxemia, these types of lesions can still lead to serious complications if not detected early enough to avoid end - organ damage . Among them, the most frequent category is comprised of some form of left - sided obstructive lesions for which additional diagnostic strategies may soon become clinically available . One of the most promising ones is the peripheral perfusion index (ppi), which is based on the analysis of the pulse oximetry signal and is displayed on some newer generation pulse oximeters . More specifically, this technique uses the ratio between the pulsatile and nonpulsatile component of the pulse oximetry signal to detect changes in the relative amount of arterial perfusion in the examined site.29,30 as measured, the ppi may be useful in detecting reduction in the arterial circulation at the monitoring site and can be used to detect decreased perfusion in settings of left heart obstructive lesions either globally (ie, aortic stenosis) or regionally (coa). Important challenges for this technique remain the wide and highly skewed distribution of ppi values in the normal population and sensitivity to environmental factors such as skin temperature . Preliminary work with ppi in neonates has established reference values for newborns with cutoff values of <0.70 for possibly impaired peripheral perfusion and <0.50 definite hypoperfusion.31 however, further studies with children with various cardiac lesions are needed before incorporating ppi in the screening process for cchd . Few studies have shown the cost - effectiveness of pulse oximetry screening in the newborn nursery . Two studies published by peterson et al32 estimated that routine screening of newborns would identify an additional 1,189 infants with cchd at their respective birth hospitals that would not have been diagnosed prior to discharge . In addition, they estimated that 20 infant lives would be saved each year by screening and a cost of $40,385 per life - year will be gained.32 the estimated cost for pulse oximetry screening in the newborn nurseries in new jersey was $14.09 per newborn, with supplies and labor dividing this cost almost equally.33 peterson et al33 extrapolated that the estimated average cost to screen all newborns in the us regardless of which level of nursery they were in was $13.50 per newborn . In the scientific statement from the american heart association and the american academy of pediatrics, mahle et al evaluated the statistical analysis of pulse oximetry screening with data from ten different studies . Analysis on the studies with infants who were evaluated after 24 hours of age showed 18 false positives, along with seven false negatives and 51,063 true negatives . With these data, the sensitivity of pulse oximetry was 69.9% and the specificity was 99.9%, with a negative predictive value of 99.9% and a positive predictive value of 47%.2 pulse oximetry is an adequate screening tool, with few false positives and a high negative predictive value . As far as we know at the time of writing this article, there are no sufficient data available on the burden to the health care system with the increase in infant echocardiograms completed due to failed pulse oximetry screening in the nursery . Each type of chd affects cardiopulmonary circulation differently, and pulse oximetry is a good screening tool to detect lesions that cause hypoxemia in the first few days of life . Chd with impaired perfusion rather than oxygenation will, though, remain undetected by pulse oximetry . The addition of ppi, a derivative of the quantitative analysis of the pulsatile vs nonpulsatile signal of the pulse oximetry, is a promising technique to cover the diagnostic gaps for this population.
In the last decade we have witnessed a dramatic increase both in the proportion and absolute number of bacterial pathogens presenting multidrug resistance to antibacterial agents . Organizations such as the us centers for disease control and prevention (cdc), the european centre for disease prevention and control (ecdc) and the world health organization (who) are considering infections caused by multidrug - resistant (mdr) bacteria as an emergent global disease and a major public health problem . The emergence of resistant microorganisms, either by mutations or the acquisition of mobile genetic elements carrying resistance genes, may take place irrespective of the presence of antibacterial agents . It is the exposure to these drugs what provides the necessary selective pressure for the rise and spread of resistant pathogens . Therefore, the driving force behind the increasing rates of resistance can ultimately be found in the abuse and misuse of antibacterial agents, whether used in patients and livestock or released into the environment . Antimicrobial resistance has become a global health threat that will require the coordinated action of many different stakeholders to tackle antibiotic resistance at its very root . The aim of the meeting organized jointly by b - debate (bio - cat) and the barcelona institute for global health (isglobal) in partnership with the european society of clinical microbiology and infectious diseases (escmid) and the spanish network for research in infectious diseases (reipi) was to generate debate among the main stakeholders (i.e. Policy makers, public health authorities, regulatory agencies, pharmaceutical companies and the scientific community at large) and come up with a coordinated set of strategies to fight antimicrobial resistance in a multifaceted approach . The meeting focused on three major areas: antimicrobial resistance in animals and the food chain; in the environment and the community; and within the healthcare setting . The widespread use of antimicrobial agents in animals and the food chain constitutes an important source of antimicrobial resistance, although the impact of such use on human health remains controversial . Massive amounts of antibiotics have been used as growth promoters as well as for prophylaxis and the treatment of infections among farm animals and in aquaculture, increasing the selective pressure on both commensal and pathogenic microorganisms that can spread to humans through direct contact and via the food chain or indirectly from the environmental pollution of farm effluents . Interventions to limit the emergence and spread of resistant bacteria in the animal setting may include the following: (a) banning antibiotic use as growth promoters and limiting its use for other nontherapeutic applications, (b) reducing the dissemination of mdr bacteria through the food chain by improving farm biosecurity and developing alternative treatment strategies and increasing hygienic conditions and practices along the food chain, (c) developing education programs, mainly directed at veterinarians, farmers, and food handlers and (d) linking surveillance systems on antibiotic resistance established for humans and animals . The european food safety authority (efsa) is playing an essential role in detecting emerging risks in the area of mdr bacteria within the food industry . Several proposals have been made by efsa for the harmonization of monitoring and reporting of resistant bacteria, such as: (a) agreeing a comprehensive set of antibacterial agents to be included in the monitoring plans, (b) reinforcing antimicrobial resistance monitoring in sentinel bacteria, (c) conducting active monitoring programs in healthy animals based on randomized sampling plans and (d) harmonizing of epidemiological values . Antibiotics that have become critical for human health should be clearly identified and their use restricted to humans only in order to avoid cross - resistance . In this respect, the who has established a list of essential antimicrobial agents for human use to be avoided in nonhuman interventions . Compliance with the who recommendations, however, is neither mandatory nor regulated . Who has also initiated different collaborative programs to tackle foodborne antimicrobial resistance through the promotion of national coordination and integrated surveillance, prevention and control of antibiotic resistance in the food chain and the improvement of awareness on antibiotic use and risk of resistance among veterinarians and the food industry . Antimicrobial resistance in the community has steadily been increasing during the last decades, especially regarding resistance to quinolones, carbapenems and third - generation cephalosporins . The latter relates to the increased prevalence of extended - spectrum -lactamases among enterobacteriaceae and with the spread of high - risk clones such as escherichia coli st131 . Surveillance studies of antimicrobial resistance and antibiotic consumption have drawn attention to this phenomenon and should be used to drive political campaigns to contain resistance . The task of the cdc / ecdc is crucial in identifying, assessing and communicating current and emerging human health threats on antimicrobial resistance . The latest ecdc report on antimicrobial resistance surveillance in europe (http://www.ecdc.europa.eu/en/publications/publications/antimicrobial-resistance-surveillance-europe-2012.pdf) showed that methicillin - resistant staphylococcus aureus prevalence is stabilizing or even decreasing in some countries, while resistance to third - generation cephalosporins in particular and multidrug resistance (three or four antibacterial agents) in general continues to show a sharp and widespread increase in e. coli and klebsiella pneumoniae . Currently, antimicrobial consumption data from the european union and countries belonging to the european economic area / european free trade association are expressed as a number of defined daily doses (ddd) per 1000 inhabitants and per day . Complementary to ddd, the number of packages per 1000 inhabitants and per day are also reported, depending on the availability of data on packages from the national surveillance networks . Information on packages is deemed to improve the understanding and interpretation of differences in the levels and trends of antimicrobial consumption observed between and within countries, as the ddd system cannot take into account changes in package content . In addition, a drug resistance index that aggregates information about antibiotic resistance and antibiotic used into a single composite measure has also been proposed . Such drug resistance index, similar to the way the dow jones is used in economics, would allow the continuous quantitation of antibiotic effectiveness overtime in particular geographic areas . As stated above, antibiotic abuse has greatly contributed to speed up the development of antibiotic resistance, and in this regard human medicine has played a key role . Inappropriate prescribing (whether caused by obsolete guidelines or pharmaceutical pressures), over - the - counter antibiotic availability and self - medication reflect a general lack of awareness on the global threat that antibiotic resistance poses to our society . Educational programs on the rational use of antibiotics addressed to primary care physicians, drug vendors and the community in general must be enforced to ease the pressure on prescribers and reduce antibiotic consumption . Similarly, the prescription of delayed receipts conditioned to the remission or worsening of clinical symptoms might also contribute to such reduction . Additional measures should include up - to - date local antibiotic prescribing guidelines, active reporting on antibiotic prescribing and consumption and the enforcement of local surveillance programs on antibiotic resistance . The implementation of such measures, however, needs substantial legislation amendments and increased funding, which depend on a strong commitment by policy makers at both national and international scales . Of particular note is the use of antibiotics in low - income countries, where there are additional factors contributing to the emergence of resistance, including (a) less potent activity of some antibacterial agents (including counterfeit drugs), (b) over - the - counter availability, with insufficient dosages, (c) lack of diagnostic laboratories and (d) poor level of sanitation, facilitating the familiar and community spread of resistant organisms . The excessive use of antimicrobial agents to treat both humans and animals has also caused the accumulation of these compounds in the environment, and the impact of such accumulation on the emergence of antibiotic resistance should not be underrated . Antibacterial agents have several routes of entry into the environment, such as sewage from the community or hospitals through manure and water bodies . The accumulation of antibacterial agents further selects resistant microorganisms, turning the environment into a gigantic reservoir for antibiotic resistance genes that feeds on the constant and increasing environmental pollution . Wastewater treatment plants have become a hot spot for horizontal gene transfer and the coselection of genetic determinants providing resistance to antibiotics, pollutants, heavy metals, biocides, disinfectants, or detergents . The current legislation on water quality mainly focuses on the presence of indicator microorganisms but does not address the antibiotic concentrations of sewages and treatment plants . Strategies to mitigate the risks of environmental exposure should be aimed at improving industrial systems for sanitation and decontamination of hospital sewage water . The concurrence of high antibiotic consumption, critically ill patients and a permanent influx of pathogenic species within the healthcare setting nurtures the development of resistance and provides an ideal scenario for the dissemination of resistant microorganisms and horizontal transfer of resistance genes . The degree of antimicrobial resistance in these settings depends on intrinsic factors related to the particular idiosyncrasies of each centre as well as on external factors such as the influx of resistant pathogens that originate in the community . Intrinsic differences in resistance rates between hospitals can be attributed to use of individual rooms vs. two or three bedrooms or open units, staffing, antibiotic stewardship, environmental cleaning, adherence to hand hygiene precautions and infection control programs . Antimicrobial consumption also shows huge interhospital differences that might be partially explained by case mix as well as by differences in the flow of patients carrying resistance bacteria that are transferred from other healthcare facilities . In order to minimize the unwanted consequences of antimicrobial use, implementation of antimicrobial stewardship programs ideally, antimicrobial stewardship should be designed to include the following: (a) passive educational measures (antibiotic guidelines, educational sessions), (b) active interventions (clinical rounds, prospective audits, reassessment of antibiotic perspectives), (c) restrictive measures (limiting antibiotics on the hospital formulary, reporting of susceptibility by the microbiology laboratory, antibiotic order form, preauthorization), and (d) supplemental measures (computer - assisted management programs, multidisciplinary stewardship teams, consultancy services). In addition, infection control measures should be planned according to the microorganisms of interest, case mix of patients and whether endemic or epidemic . Risk assessment in target pathogens needs to consider (a) the pathogen itself, as there are different dosing strategies for different species, (b) time of exposure the duration of treatment should be kept for as short as possible, (c) drug exposure related to risk in an inverse u relationship, (d) patterns of drug exposure, (e) inoculum size, with different dosing for high - load (pneumonia) and low - load infections (surgically treated complicated skin and soft tissue infection) and (f) combination therapy to suppress resistance . We use breakpoints to categorize microorganisms as susceptible (treatable with the agent in question) and resistant (not treatable with the agent) to guide therapy . At the international level, breakpoints are officially determined by two main institutions: the clinical and laboratory standards institute (clsi) in the united states, which is a not - for - profit membership organization sponsored by the industry and the medical community; and the european committee on antimicrobial susceptibility testing (eucast), a joint effort among escmid, ecdc, and the european medicines agency (ema). Breakpoints are reviewed at regular intervals because changes in doses, administration modes and indications for therapy occur and new resistance mechanisms are discovered . Differences among breakpoints suggested by clsi or eucast are subtle but significant for certain antimicrobial classes, and such differences reflect the use of divergent criteria as well as different conflicts of interest . In addition, there is also a plethora of likewise national organizations providing breakpoints and guidelines (i.e. Bsac, ca - sfm, crg, din, nwga, srga, among others) that all together contribute to a very poor standardization of procedures . An international harmonization of breakpoints is clearly needed, and in order to achieve it, either all countries must adopt the standards of one of the two main committees (clsi or eucast), or a novel international breakpoint committee must be created that embraces all of the above . The rapid detection of resistance mechanisms plays an important epidemiological role in surveillance studies to evaluate the potential dissemination of resistance genes . From the therapeutic point of view, however, this information does not exclude the presence of other mechanisms of resistance affecting the same antibacterial agent . Hence, its presence has a high predictive value for resistance but not for susceptibility . The prompt identification of the antimicrobial susceptibility of a microorganism, on the other hand, ensures the administration of the correct treatment and reduces the need for broad - spectrum drugs, limiting the emergence of antimicrobial resistance . Molecular methods have shortened the time to detect specific resistance mechanisms and the development of next generation sequencing technologies has increased the number of sequenced bacterial genomes at an exponential rate . A better understanding of the molecular basis of antimicrobial resistance has facilitated the development of bioinformatic tools to identify antibiotic resistance genes in bacterial genomes, such as arg - annot, resfinder, and the card database . In addition, mass spectrometry techniques have proven extremely useful in the rapid identification of bacterial species and their use in the detection of resistance profiles to certain classes of antibiotics has provided excellent results . Similarly, advanced applications of nanoparticles and bacterial microencapsulation to clinical are very promising and might be fully developed in the years to come . The development of novel therapeutic strategies, however, seems to have reached a dead end . Despite the urgent need to find new antibacterial products, many pharmaceutical companies have abandoned antibiotic drug discovery programs . Several factors have contributed to this situation: (a) difficulty predicting the development of resistance adding risk to research and development (r&d) investment, (b) significant scientific bottlenecks, (c) complex and divergent regulatory requirement and (d) the challenge of the commercial model there is high investment but low returns compared to alternative r&d investment . Relaunching antimicrobial drug discovery and development should be a global priority, and some initiatives have been suggested to encourage investment by the pharmaceutical industry, such as extending the period of exclusivity for certain antibiotics (i.e. The gain (generating antibiotics incentives now) act) and amending regulatory requirements to accelerate access to antibiotics for serious infections for which there are few, if any, alternatives . Promoting research and development of novel antimicrobial drugs needs to address the issue of the challenging commercial model and come up with strategies to reconcile public health needs with an attractive economic model for the pharmaceutical industry . Initiatives such as the 10 20 initiative, promoted by the infectious diseases society of america (idsa), that attempts to produce ten new systemic antibiotics by the year 2020, or the european innovative medicine initiative (http://www.imi.europa.eu), supporting collaborative research projects between the pharmaceutical industry and the academia, combine public and private funding to invigorate antimicrobial drug research . One of these research projects to tackle antimicrobial resistance is combacte (combatting bacterial resistance in europe), which aims to give antibiotic drug development a much - needed boost by pioneering new ways of designing and implementing efficient clinical trials for novel antibiotics . Fortunately, there are also a handful of small- and medium - size companies as well as multiple research groups that are investing in antimicrobial drug discovery, although such initiatives will eventually have to rely on larger corporations to proceed through the expensive phase 2/3 of clinical development . In this regard, efforts are being made by ema to relax the regulations for clinical trials, a measure thought to cheapen and accelerate the release of new drugs to the market . Table 1 summarizes the different strategies that are being used to discover and develop drugs to fight bacteria . The current global threat of antimicrobial resistance and the urgent need to control it and to find new antibacterial products has prompted the different stakeholders to take action in integrating research and public health, and in maintaining and promoting the national and international antimicrobial resistance research community . The joint programming initiative on antimicrobial resistance is a collaborative research initiative supported by 18 european countries plus canada as a response to this threat . It has defined a strategic research agenda under the assumption that only a collaborative effort will provide the necessary critical mass and scientific expertise to answer the most important and urgent research questions related to antimicrobial resistance . Other actions promoted by nonprofit entities such as the world alliance against antibiotic resistance (waaar, france), antibiotic action (uk), react (sweden) and the antibiotic resistance initiative (isglobal, spain), among others, are also playing an important role in this process . To summarize, the following measures can be taken to prevent the emergence and spread of antibiotic resistance worldwide: (a) rational use of antibiotics in all settings, (b) implementation of infection control measures in healthcare settings, (c) development of strategies to mitigate the risks of environmental exposure, (d) development of rapid diagnostic tests, (e) promotion of research on antibacterial resistance prevention and surveillance, (f) promotion of research and development of novel antimicrobial strategies and antibacterial agents and (g) improved general awareness of antibiotic use and risk of increasing resistance . The flow of resistance and the main interventions that are needed to prevent the threat of antibiotic resistance at specific key points are summarized in fig . 1 . The factors that have led us to the current situation and the measures proposed to circumvent it are no novelty to the scientific community, and much has been written about it . Implementing those measures will demand a concerted action of all stakeholders involved (policy makers, public health authorities, regulatory agencies, pharmaceutical companies and the scientific community at large), but above all, it will require strong regulatory modifications and a great deal of investment . The treatment of patients infected with drug - resistant pathogens is much more expensive as a result of longer hospitalization times and the use of more expensive last - resort drugs . The annual economic burden associated with the treatment of antibiotic - resistant infections has been estimated to be between $21,000 and $34,000 million in the united states alone, and around 1500 million in europe, which includes the economic impact associated with the number of days of lost productivity, estimated to be approximately 450 million each year in europe . A global and coordinated initiative to tackle antibiotic resistance will be needed to persuade the general population and policy makers of the advantages, both medical and economic, of combating the threat of antimicrobial resistance.
The world health organization (who) considers lymphatic filariasis (lf) the second leading cause of physical disability worldwide [1, 2]. Of the 1.4 billion people who lives in filaria endemic areas in the 73 endemic countries, 120 million people are currently infected . Globally, there are 15 million people with lymphoedema and 25 million men with urogenital swelling, principally scrotal hydrocele . The chronic attacks in the form of acute filarial adenolymphangitis caused by the death of adult filarial worms and acute dermatolymphangioadenitis (adla) due to secondary infection are common in lymphoedema . Repeated acute episodes spur the progression from lymphoedema to elephantiasis and have greater short term disability . A regimen of rigorous foot care practices with skin hygiene and simple self - help measures, such as limb elevation, exercise, use of topical antibiotics, and antifungals, aimed at minimizing episodes of acute dermatolymphangioadenitis (adla) attacks and lymph stasis is the model recommended by the world health organization for the management of filarial lymphoedema [6, 7]. People with higher grades of lymphoedema and hydrocele had more severe psychosocial problems than physical ones . They are at higher risk of depression . A study from sri lanka reported individuals suffering from chronic lymphoedema were depressed (8.5%), felt shy (33.3%), had fear of lymphoedema (7.3%), and perceived a major problem affecting their lives (61.8%). The disease is prevalent in the rural areas of the country predominantly affecting the poorer sector of the community . The gravity of the problem lies in 60 out of 75 districts being endemic and more than 25 million individuals are at risk . Prevalence rates above 20% were found in 11 districts (with the highest rate of 40%), those of 619% were found in 15 districts, and 0.15% were in 7 districts . The government of nepal is committed to eliminating lf by 2020 initiating the mass drug administration (mda) in the parsa district in 2003, which was later expanded to 46 districts in 2011 . This programme is focused on interrupting parasite transmission by employing annual, community - wide mass drug administration [13, 14]. Information and insights on health seeking behaviors and self - care practices of lymphoedema in nepal remain meager . The objective of this qualitative paper is to explore specific health beliefs, health - seeking behaviors, and self - care practices of people with lymphoedema in nepal for designing socially acceptable and culturally compatible prevention and morbidity management strategies . An exploratory study was carried out in the three of sixty lf endemic districts in nepal from july to september, 2013 . The selected districts were dhading (salyantar village development committee (vdc)), kapilvastu (maharajgunj vdc), and kailali (pahalbanpur and malakheti vdcs). This study is a part of the larger study conducted on parasitological and sociocultural aspects of lymphatic filariasis in nepal . The cases of lymphoedema were mapped with the help of female community health volunteers and health workers in the study sites . A semistructured interview schedule was prepared after considering previous studies as works of references [1517] and authors' experiences in the field . The developed tool was then reviewed by a panel of experts from the department of community medicine and public health at the institute of medicine, kathmandu, nepal . Questions were translated into nepali, pretested on a sample of lymphoedema patients in the salyantar vdc of the dhading district, and later modified to correspond to the cultural setting of the study site . Qualitative study techniques were used [18, 19]. In - depth interviews (idis) were conducted by research assistants who spoke the local language fluently . At the end of the interview, all patients were also educated on lymphoedema management practices recommended by the world health organization . Each interview lasted 4560 minutes and was tape recorded and later translated into english . Over 200 pages of typed verbatim transcripts data were systematically examined for emerging codes and patterns and were divided into a priori themes: health beliefs, health seeking behaviors, and self - care practices for data analysis . This was followed by indexing, charting, mapping, and interpretation (figure 1). This study obtained ethical clearance from the institutional review board at the institute of medicine, tribhuvan university, nepal . This word is used to signify someone who has swollen legs that resemble the legs of an elephant . While hattipaile was the most commonly used word in dhading and kailali districts, godfuluwa was used in awadhi - speaking communities in kapilvastu . We did not find any local terminology used to signify lymphoedema of hands in kapilvastu and kailai districts . However, in the dhading district, people used hatfuluwa and compound terminologies like hatma hattipaile bhako . Knobs, lesions, and skin folds in the body, right leg swelling, left leg swelling, swelling of both the legs, and swelling of single or both arms were the major signs found . Hydrocele was found to be a well - known condition and a major health problem in the study communities . Patients and other key informants were asked what they believed to be the cause of lymphoedema . Respondents reported that the cause of disease was related to their past work and physical activity, insect / mosquito bites, and bodily abnormality . Although people say that the mosquito bite causes it, i believe it is due to the defect of my internal body . I also suffer from fever, swelling and tingling of nerves and once i got unconscious for 2 - 3 days due to a high grade fever . Although people say that the mosquito bite causes it, i believe it is due to the defect of my internal body . I also suffer from fever, swelling and tingling of nerves and once i got unconscious for 2 - 3 days due to a high grade fever . Others associated diseases with itching, wounds and infection, massage / contact, inheritance, excessive sexual activity, dirty blood, trauma, trapped gas, and witchcraft were reported by respondents to be the causes of lymphoedema . A few of the respondents reported that prevention could be achieved by taking diethyl carbamazine (dec), using a mosquito - nets and chemical spray . I get a fever and swelling which becomes worse during the cold . Respondents were asked what kind of services they had taken for the treatment of lymphoedema . Most of the respondents said that they used traditional and home - based care in the first episodes of lymphoedema . Faith / beliefs such as dhami / jhakri, avoidance of food, astrologists and pandits, and home - based treatments such as hot water and sponging had been used . A man who had sought care from traditional healers said, for treatment, at first i approached dhami / jhakri and even performed naag puja (a hindu ritual worshipping snake god). Later i went to amppipal hospital (local hospital) where lymphoedema was diagnosed . Following this, i took medicine but did not get cured . For treatment, at first i approached dhami / jhakri and even performed naag puja (a hindu ritual worshipping snake god). Later i went to amppipal hospital (local hospital) where lymphoedema was diagnosed . Following this traditional medical remedies such as applying neem (azadirachta indica) leaves and leeches over the swollen area were reported . I preferred home remedies and, therefore, took garlic and cow urine followed by hot sponging but nothing cured me . I preferred home remedies and, therefore, took garlic and cow urine followed by hot sponging but nothing cured me . Most of the respondents mentioned that they had frequently used the analgesics, dec, diuretics, nsaids, vitamins tablets, antifungal creams, antibacterial creams, and antibiotics bought from medical shops on prescription from health practitioners . There were many hydrocele patients aspiring to the treatment for hydrocele but actually did not have hydrocelectomy . However, most pricked the hydrocele with sharp objects to drain out the fluid . The high cost of surgery and lack of money and fear of death, as well as impotence and/or sterility, were reported as the reasons why they did not have hydrocelectomy . I went to delhi (india) where lymphoedema was diagnosed and i took medicine, but still i am not cured . Now, i don't have money for operation (hydrocelectomy). I went to delhi (india) where lymphoedema was diagnosed and i took medicine, but still i am not cured . Respondents reported various psychological problems such as anxiety, worry and stress, depression, low self - esteem, feeling weak, and fear of being abandoned . I am afraid that my husband will leave me to marry other women . Due to this disease, i got married to a poor family females were worried more about transmitting the disease to children compared to their counter parts . More males reported painful sexual intercourse and psychological problems . However, none had taken treatment or counseling for the management of the problem . A man having a lymphoedema in legs and only a quarter of the study population was aware and a few of them had been practicing standard foot care practices like soap water washing, elevation and exercise of the affected limb, nail hygiene, trauma reduction and care for the entry lesion, antiseptics, and use of footwear on a regular basis . The majority of the respondents had been practicing washing the affected limbs only . Among the six foot care measures asked, they did not have knowledge of foot care practices; patients said when asked about why they did not practice such practices . Application of medicinal bandages, herbal preparations, and leeches on the swollen area was reported . In rare cases, patients used herbal preparations orally, or smeared them on affected parts, or those preparations were given as an enema, and even scarification of affected parts was reported . Respondents reported that they were able to wear clothes, walk, and bathe, but had a feeding problem, had trouble in doing agricultural work, and had difficulty in using a toilet and difficulty in brushing their teeth . I am able to use a toilet, wear clothes, take a bath and take care of my body . I am able to use a toilet, wear clothes, take a bath and take care of my body . However, many said that they were not assisted by family member in feeding, using toilet, and wearing clothes . Although i can do my personal hygiene, i have trouble in going to the toilet . Sometimes due to swelling, i can't even do my daily chores but as i have no one in the family to assist me, i have to do them anyway with pain . Although i can do my personal hygiene, i have trouble in going to the toilet . Sometimes due to swelling, i can't even do my daily chores but as i have no one in the family to assist me, i have to do them anyway with pain . Some respondents said that they abstained from certain types of foods such as tea, sour items, onion, brinjal, kuvindo (vegetable), salt, fish, and yoghurt . This study was conducted in a small geographic area with diverse ethnic population in each district . The identified terminologies can be used to develop information, education, and communication (iec) materials that are locally appropriate . The majority of the respondents did not believe that the parasite in the body due to mosquito bites is the real cause of the disease . Quite the opposite, they attributed the disease to their past work and physical activity . Study conducted in south india reported that only 9% of people having lf and 20% of those without the disease knew that filariasis is caused through mosquito bites; the rest attributed it to many other causes . Similarly around 42% of people during the pre - mda period had accurate knowledge of the association of lf with mosquito bites . In general, people did not accept the mosquito theory of transmission, but they believed in other physical, spiritual, and hereditary causes . The current study showed that only a few knew about the preventive measures against the disease . About 83% of the affected and 87% of the unaffected individuals either were uncertain or felt that filariasis is not preventable . Krentel et al . Reported that the knowledge of the cause of the disease and its prevention is important for compliance with mass drug administration . Hence awareness activities during mass drug administration campaigns need to be emphasized for disseminating messages related to the cause and prevention of lymphoedema . Lymphoedema is debilitating in humans; it has several manifestations, including lymphoedema and hydrocele . The study participants had swelling of the legs and the hands and some even presented with knobs, lesions, and skin folds in the body . A study conducted in ghana found that the most common illnesses in the study communities were joint pains, lymphoedema, stomach pains, hernia: hydrocele, measles, malaria: fever, headache, dizziness, eye problems, diarrhoea, adl attacks, and waist pains . The greater the degree of lymphoedema, the lesser the quality of life they have . The morbidity management is one of the objectives of the national lf elimination program in nepal; there are no activities run at community level to provide treatment, care, and support for people with lymphoedema . We found that the majority of the respondents visited traditional faith healers prior to their seeking modern medicine . Ayurveda, homeopathy, unani, and amchi are the major alternative medicines that have long been practiced in nepal . Also, the ayurvedic treatment was also practised for lymphoedema management (25%) followed by home remedies and acupuncture in sri lanka . They are the first point of contact of care in rural areas of the country . There are public health programs on reproductive health and child morbidity to train the traditional faith healers to refer patients coming to them to the health centers . Therefore, trainings should be provided for alternative medicine providers so that they can provide counseling to manage lymphoedema and refer the cases to health centers . Self - care for the disease reduces health care utilization and potentially yields monetary benefits to a health plan . In conformity with an earlier study in india, washing affected limbs was common among the respondents, though it cannot be linked to lymphoedema care, as washing hands and feet is common in the hindu rituals . Respondents were not wearing larger size footwear and none of them reported massaging the affected limbs . A regular footwear might not prevent the individuals from external injuries; therefore, a larger size footwear should be recommended . Massaging affected limbs relieves pain and an elevation exercise prevents accumulation of fluids in affected limbs . Both should be recommended . Standard foot care practices can be incorporated into routine counseling services in health centers in endemic communities . Engaging in self - care as part of daily disease management is crucial in villages in nepal on account of villagers' poor access to health centers . Lymphoedema cases might have difficulty in traveling to health centers for their treatment because in most of the cases, the health centers are more than thirty minutes' walk from their place of residence . Thus, home - based management with a greater emphasis placed on foot care aimed at lymphoedema patients needs to be promoted . It is found that hydrocoele was the commonest manifestation among the lf cases in this study . In line with a study from ghana, most of the hydrocoele patients mentioned that the main reasons for refusing hydrocelectomy were the high cost of surgery and lack of money and to some extent fear of death and impotence and/or sterility that might result from the operation . Just as the previous studies reported, food taboos like refraining from taking certain foods and drinks, tea, sour items, onion, brinjal, kuvindo (vegetable), salt, fish, and yoghurt were reported . Abstinence reduces the supply of nutrients and minerals in the body, possibly leading to poor health . Psychological problems reported by the respondents in this study are in strong agreement with those in previous studies . A review literature reported fear, anxiety, frustration, and distress in people with lymphoedema . In our study population, illiteracy, lack of treatment, and poor or no counseling might be the causes of the reported cases having fear of transmitting disease to their children . While it was found that the respondents had some psychological conditions, the importance of social participation by those who have this condition needs to be emphasized more by peripheral health workers in nepal . Previous studies reported that the role of social participation is important in rehabilitation and reduction of psychological trauma and problems . This study is perhaps the first of its kind to report health - seeking behaviors and self - care practices of people living with filarial lymphoedema in nepal . Subjects with mild and moderate forms of filarial lymphoedema might not have been studied well in this study, for they did not participate for reasons, such as in part the hidden nature of the disease and in part our inability to indentify them . The health - seeking behavior and self - care practices reported in this study might not represent all of the practices across the diverse topography and people in nepal . Lymphoedema in the limbs and hydrocele were found to be major health problems in the studied communities . The majority of the respondents did not believe that the parasite in the body due to mosquito bites is the real cause of the disease . The traditional health care providers were the first contact of care for the majority of respondents . Active programmes for surgical management lymphoedema and standard foot care practices should be emphasized in lymphatic filariasis elimination programme by involving government bodies, ngos, and other community based organizations.
With technological advances in the field of implant dentistry novel treatment modalities and more efficient options became available . The custom 3d printed root analogue implant (rai) as defined by anssari moin et al . [1, 2] and figliuzzi et al . Is a futuristic treatment option for immediate implantation and immediate loading cases for a soon to be removed tooth . Advantages of the rai technique when compared to conventional screw shaped multipiece implants may encompass more cost efficiency, one - piece implant, and minimal traumatic surgical intervention [16]. An essential factor for realization of all implant - based prosthetic reconstructions is successful osseointegration of the implant . In particular, primary stability plays a fundamental role in one - stage implant surgery with or without immediate loading . Conventional screw type implants achieve primary stability through mechanical fixation by implant threads in bone . Numerous studies on the factors influencing primary stability (implant shape specifications, surface modifications, bone quality, and surgical technique) and the effect on the process of osseointegration have been performed [811]. However, primary stability for the rai technique is based on the (targeted) press - fit phenomenon for achieving successful osseointegration [13, 6]. Since the custom rai is based on cone beam computed tomography (cbct), computer aided design (cad), and 3d printing technology an unlimited array of designs for this custom implant approach is available . Every rai design option aimed at increasing initial mechanical stability for the root part of the rai will have a different biomechanical effect on the surrounding bone and influence on the relative microdisplacement at bone - to - implant interface consequently leading to diverse osseointegration results, bone resorption, or failures . Finite element analysis (fea) has become an effective method in investigating bone stress / strain around implants and relative microdisplacement between bone - to - implant interfaces . However, as with all fea studies the analysis is confined to a limited amount of factors and designs and cannot be generalised, specifically not for individual cases . Thus, the aim of this study is to analyse, with the means of fea, the influence of 5 custom rai designs on stress distribution of peri - implant bone and to evaluate the impact on microdisplacement for a specific patient case . A patient (male, 64 years of age) presenting a profoundly decayed upper right canine was selected and informed consent was obtained from the patient . Based on the method previously described by anssari moin et al . [1, 2] a 3d surface model of rai was constructed . In brief the procedure was as follows: the patient was scanned with the 3d accuitomo 170 cbct system (accuitomo 170, 90 kvp, 5 ma, 30.8 s, 4 4 cm field of view [fov], voxel 0.08 mm, morita inc ., kyoto, japan) using the recommended scan protocol . Amira software (v4.1, visage imaging, carlsbad, ca) was used for image analysis . A region of interest limited to the tooth and its surrounding was initially selected and a threshold segmentation algorithm based on histogram analysis of grey values was used to separate the tooth (root and crown) from surrounding bone and periodontium . Further semiautomated segmentation based on slice - by - slice analysis was implemented to enhance the segmentation by removing any residual artifacts (figure 1). The segmented dataset was converted to 3d surface model using the marching cube algorithm and saved in the standardized triangulation language (stl) file format . Based on the stl model five different (targeted) press - fit design rai fe models have been constructed using 3d cad software (inventor, autodesk gmbh, munich, germany). For the five rai models a standard identical abutment, based on morphological expectation of the original tooth crown and measurements on neighboring teeth, was designed at 2 mm distance coronal from the expected bone level after implantation . Subsequently, the following (targeted) press - fit design modifications were constructed: (1) nonmodified standard, (2) targeted press - fit prism, (3) targeted press - fit fins, (4) targeted press - fit plug, and (5) targeted press - fit bulbs, referred to as standard, prism, fins, five bone models surrounding 3 mm congruent to the respective rai models were built using femap software (v. 11.0.1, siemens plm software, plano, tx, usa). Finally, preprocessor software (femap v. 11.0.1, siemens plm software, plano, tx, usa) was applied to mesh the models with quadratic tetrahedral solid elements (figure 3). Mesh refinement based on convergence analysis resulted in a mesh size of 0.5 mm . The following assumptions were made for the rai fe models: composition of a titanium alloy ti6al4v, young's modulus e = 110 gpa, and poisson's ratio = 0.35 with the material being homogeneous, isotropic, and linearly elastic [13, 14]. The bone models were constructed using a homogenous isotropic linearly elastic material of 1 mm inner cortical layer (young's modulus e = 12.6 gpa, poisson's ratio = 0.3, and shear modulus g = 5.7 gpa) and a 2 mm outer trabecular layer (young's modulus e = 1.1 gpa, poisson's ratio = 0.3, and shear modulus g = 0.07 gpa) as proposed in the reviewed literature [13, 14]. Bone - to - implant interfaces were assumed to be frictional surfaces to represent a nonosseointegrated contact situation . A coulomb frictional method (coefficient of friction = 0.3) was adopted to define linear contact behavior [14, 15]. Two loads were applied to simulate anterior bite force: an oblique buccoapical force with a magnitude of 300 n set on 135 to the long axis of the implant and a vertical force in apical direction to the long axis of the implant with a magnitude of 150 n, as shown in figure 3 [16, 17]. The nodes in the outer surrounding layer of trabecular bone were constrained in all directions (zero nodal displacement). Numerical solving (nastran v. 8.0, siemens plm software, plano, tx) and postprocessor analysis (femap v. 11.0.1, siemens plm software, plano, tx, usa) was performed on the meshed bone - implant models to evaluate stress distributions on cortical and trabecular bone and deformed contact separation (micromotion) at the peri - implant region . Based on previous research the following measurements were recorded: the von mises equivalent stress (vm) at the bone peri - implant interface as a quantity of stress level for the load transfer mechanism [12, 1821], the tensile / maximum (t) and compressive / minimum (c) principal stresses as a criterion to evaluate the bone overloading [19, 20], and finally deformed contact separation (micromotion in m) as an indicator for initial implant stability [22, 23]. Figure 4 displays the average measured stress values (in mpa) of the principal and von mises stresses at the supporting tissues for all groups . Notably, on average the stress levels caused by oblique loading were higher when compared to vertical loading . The standard design rai exhibited the highest von mises stress and highest minimum principal stress values (highest compressive stress) under both loading conditions (vm = 252 and c = 50). The lowest von mises stress levels were numerically observed for the plug design under the different loading conditions (figure 4(a), vm = 82; figure 4(b), vm = 168), indicating a reduction of 67.4% and 33.3%, respectively, when compared to the standard design . Furthermore, the highest measured tensile stress in cancellous bone was 4 mpa for the standard design (data not shown). Comparing behavior of von mises stress distribution caused by vertical (figure 5(a)) and oblique loading (figure 5(b)), it can be observed that the cortical peri - implant bone exhibited greater stress concentration than trabecular bone . In tension stress, concentrations can be noted at the loaded side for the standard and prism under the oblique loading component (figure 6). However, under the same conditions the plug, fins, and bulb designs showed tensile stress intensities on the lingual side and in the buccal area of the protrusive extensions of the design (figure 6). The apical peri - implant area indicated high von mises stress concentrations in all designs (figure 7) and tensile stress peaks under both loading conditions for the standard, plug, and fin designs . Comparison of the minimum principal stress illustrated in all models the highest compressive stress concentrations on the lingual side (figure 8). Table 2 shows the microdisplacement of the various rai designs from the peri - implant bone with respect to the loading conditions . The highest magnitude of micromotion level was measured in the prism design, 32.10 m and 32.51 m under vertical and oblique loading, respectively . Remarkably, the lowest levels of contact separation were measured in the fins model followed by the bulbs design under vertical and oblique forces: 5.45 m, 6.25 m and 6.35 m, 6.42 m, respectively . Microdisplacement patterns were located at neck area in direction of the forces and in contra lateral direction in the apical area in all designs (images not shown). In this study five different designs of rai were analyzed for stress - based biomechanical behavior for a specific patient by means of finite element simulations . In the primary phase of endosseous healing multiple biomechanical mechanical factors the von mises stress was used as an indicator for the load transfer mechanism, principal stresses as indicator to bone overloading and micromotion as indicator for initial stability . Numerical results from the current study suggest that adding targeted press - fit design characteristics to the standard rai design will decrease the amount of maximum von mises stress in the surrounding peri - implant bone, subsequently leading to more favorable load behavior for this patient . Previous studies have assumed maximum bone strength as biological limit to bone failure and activation of bone resorption [15, 19, 21]. Correspondingly, it has been proposed that overloading of cortical bone occurs when the maximum compressive principal stress exceeds 190 mpa and maximum tensile principal stress exceeds 130 mpa [15, 19, 21]. Likewise, trabecular bone overloading will occur when the compressive and tensile principal stresses exceed 5 mpa and 5 mpa, respectively [15, 19, 21]. According to the result of this study it has been found that solitary prism design exceeded the maximum compressive stress criterion for cortical bone . The standard, fins, plug, and bulbs designs exceeded the tensile stress threshold in cortical bone . The threshold for trabecular bone overloading in tension was not reached . However, when observing the compressive stresses under oblique loading in trabecular bone, it can be noted that in the regions of the implant neck all implant designs exceeded the biological limit, inducing a risk to bone loss (figure 8). The fins and bulbs designs showed the lowest levels of micromotion, indicating the most favorable primary stability . Nonetheless, it must be noted that the influence of micromotion on osseointegration is of scientific debate as some studies have suggested a more positive effect on the tissue differentiation and bone formation around implants under controlled micromotion up to 50 m . Additionally, in our study it has been found that the higher oblique loading component causes more stress concentrations on cortical and trabecular bone when compared to vertical loading . Therefore, oblique loading in the primary stage after implantation will have a more negative effect on bone healing and should be minimized . In this current study was modeled and assumed as a homogeneous, isotropic, linearly elastic material . However, it is known that the biomechanical behavior of this living tissue is heterogeneous, anisotropic, and nonlinear [14, 19, 20]. Moreover, a 100% osseous contact between implant and bone was assumed . Contact relationship between implant and bone was defined as linear contact behavior by using a coulomb frictional model . Although contact behavior should be defined in a nonlinear method, several studies are in agreement about adopting a linear frictional model since non - linear contact analysis is highly complex [14, 25]. In clinical situations the actual bone - to - implant contact directly after insertion of the rai will be dependent on many factors, that is, accuracy of the rai technique on multiple levels, (periapical) bone defects, and surgical handling . The quantity of in situ osseous contact after implantation of the rai will have profound effect on primary stability and stress behavior . Furthermore, the herein applied loads were static one directional loads of amplitude of 150 n (vertical) and 300 n (oblique) whereas in clinical situations considerably variable loads can be observed depending on the location of the rai in the oral cavity and patient characteristics . Despite the fact that simulation methods and fe modeling were beyond the scope of this investigation, the current limitations can be considered as acceptable in a numerical sense and are in agreement with multiple studies [13, 14, 16, 17, 19, 25]. Especially with the rise of custom 3d printed implants questions concerning biomechanical behavior in each specific patient surface . Ideally for future implementation of custom 3d designed and printed implants easy accessible individual patients specific fea should be performed to get a better understanding of the biomechanical behavior of different implant designs for a specific case . Based on the results of this study and within the limitations of the applied methodology, it has been found that adding targeted press - fit geometry to the rai standard design, preferably fins or bulbs, will have a positive effect on stress distribution and lower concentration of bone stress and will provide a better primary stability for this patient case.
Children participating in this study were taking part in a larger project investigating family management of diabetes, which required them to complete surveys programmed on pda computers several times each day . Although later phases of the larger project involved interventions, including blood glucose awareness training, data for this study was collected during an earlier phase of the project that did not involve any intervention . Families were recruited for the study through pediatric endocrine clinics at the university of virginia and joslin diabetes center in boston, as well as through advertisements and regional parent support groups . Inclusion criteria for children were age 611 years, a diagnosis of type 1 diabetes for at least 1 year, willingness to perform glucose measurements 35 times daily, and ability to read, complete questionnaires, and use the pda . Inclusion criteria for parents included ability to complete the study protocol, role as main diabetes caregiver for the child, and the absence of any self - reported significant psychiatric disorder, including depression and substance abuse . Eligible families were invited to orientation meetings during which the study was explained and institutional review board a total of 77 families entered the study, and 66 parent - child pairs completed the protocol . Reasons for withdrawal for nine families included relocation, family stressors, and illness in the child . Two other families withdrew because the child did not want to continue completing the pda surveys . For two families, pda data were lost in the mail, and they chose not to repeat the procedure . Thus, the final sample of children with complete data was 61 (university of virginia n = 31, joslin n = 30). There were no significant demographic or clinical differences between families who completed and did not complete the study . The final sample included 31 girls and 30 boys, whose mean sd age was 8.83 1.6 years and diabetes duration was 4.7 2.6 years, with 25 children aged 68 and 36 aged 911 years . Almost all families were caucasian (95%), and most parents had at least some college education (mean years of school: 15.8 2.6). Children completed the state - trait anxiety scale and the children's depression inventory, measures that are widely used in research, with well - documented reliability and validity (13,14). Parents also completed a questionnaire about the child's diabetes history, including the past frequency of mild, moderate, and severe hypoglycemic episodes . Severe hypoglycemia was defined for parents as episodes during which their child was incapable of self - treatment or asking for treatment due to mental confusion, stupor, unconsciousness, or seizure . Moderate episodes were defined as those in which hypoglycemia significantly disrupted routine or ongoing behavior (e.g., the child could not continue with current activities). Mild episodes were defined as those associated with warning symptoms that quickly resolved after treatment and did not significantly disrupt function . Parents reported the frequency of severe and moderate episodes over the past year and reported the frequency of mild episodes over the past month . Families were provided with a visor pda programmed with cognitive tests and linked to a freestyle tracker bg meter to collect and store glucose readings (abbot diabetes care, abbott labs, alameda, ca). Families were asked to complete 35 pda trials each day, for a total of 70 trials over the next 46 weeks . Children were unable to complete trials unless the parent was present and entered a password to start the program . After children completed the cognitive tests, the computer instructed parents and children to measure glucose . All data were automatically stored and time stamped, providing a validity check to insure that children completed cognitive tests before glucose measurement . The pda presented two cognitive tests, a mental math task and choice reaction time . These tests were chosen based on previous studies (3,6,7) showing that these tasks were sensitive to cognitive - motor disruptions in performance caused by blood glucose extremes in adults . Both tests were adapted for use by children . The mental math task consisted of 10 math problems, 5 additions and 5 subtractions, presented in random order . Children entered their solution by tapping numbers displayed on a number pad on the screen . For children ages children aged 911 years were given math problems containing one double - digit number <20 and one single - digit number . In the choice reaction - time task, the symbols for the four card suits (hearts, diamonds, spades, and clubs) were shown in color in the four corners of the screen . One card suit would appear in the center of the screen, and children tapped the matching card suit in one of the corners, with a total of 10 to match presented in random order . The computer tracked two performance measures, time to complete the task (in seconds) and number of problems correct . After completing each task, children rated their difficulty completing the task (e.g., how hard it was) on a visual analogue scale, where 0 = not at all and 6 = very hard . After orientation and informed consent / assent, parents and children were given visor computers and freestyle meters and instructed on their use and were also given, questionnaires to complete and a stamped envelope for returning data . After return of pda data, a blood sample kit was mailed to parents, who obtained blood from their children and returned the sample to the university of virginia clinical laboratories for a1c measurement . Two dependent measures were computed for each cognitive test: 1) time (in seconds) to complete the task (math time and reaction time) and 2) number of correct responses (math correct and reaction time correct). For math time and reaction time, higher numbers indicate more seconds to complete the task and therefore poorer performance . For math correct and reaction time correct, higher numbers indicate more accuracy and better performance . The data were analyzed 1) across subjects at different blood glucose ranges to examine the impact of glycemic status on cognitive function and 2) within subject, using z scores, to determine whether individual subjects showed differences in the degree to which cognitive function declined at glucose extremes . For analyses across glucose levels, means for performance measures were computed for six clinically relevant ranges: <3.0 mmol / l (<54 mg / dl), 3.03.8 mmol / l (5469 mg / dl), 3.99.9 mmol / l (70179 mg / dl), 1016.6 mmol / l (180299 mg / dl), 16.722.1 mmol / l (300399 mg / dl), and> 22.2 mmol / l (> 400 mg / dl). Mmol / l were specifically chosen for investigation because, in the state of virginia, parents are called when children's glucose readings at school are higher than 16.7 mmol / l and sent home from school when readings are higher than 22.2 mmol / l (15). Iiss were also computed for each child, using the child's mean performance during euglycemia (4.39.9 mmol / l) as their individual baseline or normal performance . The difference, in z scores, between mean baseline performance and mean performance during hypo- and hyperglycemia was then computed . Iiss were computed for blood glucose levels <3.0 and> 22.2 mmol / l . Thus, these impairment scores represented the mean number of sds between performance during euglycemia and the most extreme blood glucose levels . The calculation of similar measures for individual impairment in adults with diabetes has been previously described in detail (6,7). Children completed the state - trait anxiety scale and the children's depression inventory, measures that are widely used in research, with well - documented reliability and validity (13,14). Parents also completed a questionnaire about the child's diabetes history, including the past frequency of mild, moderate, and severe hypoglycemic episodes . Severe hypoglycemia was defined for parents as episodes during which their child was incapable of self - treatment or asking for treatment due to mental confusion, stupor, unconsciousness, or seizure . Moderate episodes were defined as those in which hypoglycemia significantly disrupted routine or ongoing behavior (e.g., the child could not continue with current activities). Mild episodes were defined as those associated with warning symptoms that quickly resolved after treatment and did not significantly disrupt function . Parents reported the frequency of severe and moderate episodes over the past year and reported the frequency of mild episodes over the past month . Families were provided with a visor pda programmed with cognitive tests and linked to a freestyle tracker bg meter to collect and store glucose readings (abbot diabetes care, abbott labs, alameda, ca). Families were asked to complete 35 pda trials each day, for a total of 70 trials over the next 46 weeks . Children were unable to complete trials unless the parent was present and entered a password to start the program . After children completed the cognitive tests, the computer instructed parents and children to measure glucose . All data were automatically stored and time stamped, providing a validity check to insure that children completed cognitive tests before glucose measurement . The pda presented two cognitive tests, a mental math task and choice reaction time . These tests were chosen based on previous studies (3,6,7) showing that these tasks were sensitive to cognitive - motor disruptions in performance caused by blood glucose extremes in adults . Both tests were adapted for use by children . The mental math task consisted of 10 math problems, 5 additions and 5 subtractions, presented in random order . Children entered their solution by tapping numbers displayed on a number pad on the screen . For children ages 68 years, math problems and solutions contained only single - digit numbers . Children aged 911 years were given math problems containing one double - digit number <20 and one single - digit number . In the choice reaction - time task, the symbols for the four card suits (hearts, diamonds, spades, and clubs) were shown in color in the four corners of the screen . One card suit would appear in the center of the screen, and children tapped the matching card suit in one of the corners, with a total of 10 to match presented in random order . The computer tracked two performance measures, time to complete the task (in seconds) and number of problems correct . After completing each task, children rated their difficulty completing the task (e.g., how hard it was) on a visual analogue scale, where 0 = not at all and 6 = very hard . After orientation and informed consent / assent, parents and children were given visor computers and freestyle meters and instructed on their use and were also given, questionnaires to complete and a stamped envelope for returning data . After return of pda data, a blood sample kit was mailed to parents, who obtained blood from their children and returned the sample to the university of virginia clinical laboratories for a1c measurement . Two dependent measures were computed for each cognitive test: 1) time (in seconds) to complete the task (math time and reaction time) and 2) number of correct responses (math correct and reaction time correct). For math time and reaction time, higher numbers indicate more seconds to complete the task and therefore poorer performance . For math correct and reaction time correct, higher numbers indicate more accuracy and better performance . The data were analyzed 1) across subjects at different blood glucose ranges to examine the impact of glycemic status on cognitive function and 2) within subject, using z scores, to determine whether individual subjects showed differences in the degree to which cognitive function declined at glucose extremes . For analyses across glucose levels, means for performance measures were computed for six clinically relevant ranges: <3.0 mmol / l (<54 mg / dl), 3.03.8 mmol / l (5469 mg / dl), 3.99.9 mmol / l (70179 mg / dl), 1016.6 mmol / l (180299 mg / dl), 16.722.1 mmol / l (300399 mg / dl), and> 22.2 mmol / l (> 400 mg / dl). The ranges 16.722.1 mmol / l and higher than 22.2 mmol / l were specifically chosen for investigation because, in the state of virginia, parents are called when children's glucose readings at school are higher than 16.7 mmol / l and sent home from school when readings are higher than 22.2 mmol / l (15). Iiss were also computed for each child, using the child's mean performance during euglycemia (4.39.9 the difference, in z scores, between mean baseline performance and mean performance during hypo- and hyperglycemia was then computed . Iiss were computed for blood glucose levels <3.0 and> 22.2 mmol / l . Thus, these impairment scores represented the mean number of sds between performance during euglycemia and the most extreme blood glucose levels . The calculation of similar measures for individual impairment in adults with diabetes has been previously described in detail (6,7). 1 shows the means and sds for each of the four performance measures across blood glucose ranges . There were significant main effects for math time (f = 5.0, p <0.001) and a strong trend for reaction time (f = 2.2, p = 0.053). Contrasts showed that compared with performance at euglycemia, math time was significantly longer when blood glucose was <3.0 mmol / l (p = 0.017) and> 22.2 mmol / l (p = 0.0001). Reaction time was significantly longer at glucose levels <3.0 mmol / l (p = 0.01), with a trend toward significance when blood glucose was> 22.2 mmol / l (p = 0.08). For both math time and reaction time, seconds to task completion did not differ for glucose levels <3.0 and> 22.2 mmol / l, indicating that performance was equally poor in the lowest and highest blood glucose ranges for both tasks . Compared with performance during euglycemia, math time was an average of 12.6 and 16.8 s longer when blood glucose levels were <3.0 and> 22.2 mmol / l, respectively . Reaction time was an average of 2.6 and 1.5 s slower when glucose levels were <3.0 and> 22.2 mmol / l, respectively . In contrast to the results for time to perform tasks, there were no significant differences in the number of correct responses across ranges . Exploratory analyses were also conducted to identify practice effects over time on the two tasks . Math time and reaction time over the first 35 trials were compared with the second 35 trials at each of the above three blood glucose ranges: euglycemia (3.99.9 as expected, during euglycemia math time was significantly shorter for the second of 35 trials (56.9 vs. 66.8 s; f = 19.6, p = 0.001), but there was no improvement for reaction time (p = 0.13). When blood glucose was <3.0 or> 22.2 mmol / l, there were no significant differences in math time or reaction time between the first and second half of the study, indicating no practice effects over time . Mean difficulty ratings tended to be low across all blood glucose ranges; however, significant differences were still found . For the math task, average ratings were 0.44, 0.77, and 0.58 for euglycemia, hypoglycemia, and hyperglycemia, respectively, with a significant main effect across blood glucose levels (f = 4.3, p <0.0001). Mean difficulty ratings for the reaction time task were 0.15, 0.40, and 0.13, respectively, which also differed across blood glucose levels (f = 3.9, p <0.001). However, contrasts showed that for both tasks, difficulty ratings were significantly higher only when glucose was <3.0 this indicates that children perceived greater difficulty performing tasks during hypoglycemia but not when glucose levels were> 22.2 mmol / l, even though time to complete tasks increased significantly and equivalently at both blood glucose extremes . To examine individual differences in the impact of hypo- and hyperglycemia on performance, iiss were computed as described above for math time at blood glucose levels <3.0 and> 22.2 mmol / l . These scores were only computed for those children who had blood glucose readings <3.0 mmol / l (n = 34) or> 22.2 positive z scores indicated poorer performance compared with euglycemia, and negative scores indicated better performance . Mean iis for math time when blood glucose was <3.0 and> 22.2 mmol / l were 0.57 1.6 and 0.33 1.1, respectively . A total of 21% of children had iiss higher than 1.0, indicating that performance deteriorated on average> 1 sd when blood glucose was <3.0 impairment scores were not related to diabetes duration, blood glucse variability (as determined by several measures including the interquartile range, low and high blood glucose risk indexes), or depression and anxiety measures . The child's a1c correlated withimpairment scores for reaction time when blood glucose was> 22.2 mmol / l (r = 0.40, p = 0.02), indicating more impairment with poorer diabetes control . Number of severe hypoglycemic episodes over the past year correlated with impairment scores for both math time (r = 0.39, p = 0.04) and reaction time (r = 0.40, p = 0.02) when blood glucose was> 22.2 neither a1c nor frequency of severe hypoglycemia correlated with performance impairment exhibited when blood glucose was <3.0 1 shows the means and sds for each of the four performance measures across blood glucose ranges . There were significant main effects for math time (f = 5.0, p <0.001) and a strong trend for reaction time (f = 2.2, p = 0.053). Contrasts showed that compared with performance at euglycemia, math time was significantly longer when blood glucose was <3.0 mmol / l (p = 0.017) and> 22.2 mmol / l (p = 0.0001). Reaction time was significantly longer at glucose levels <3.0 mmol / l (p = 0.01), with a trend toward significance when blood glucose was> 22.2 mmol / l (p = 0.08). For both math time and reaction time, seconds to task completion did not differ for glucose levels <3.0 and> 22.2 mmol / l, indicating that performance was equally poor in the lowest and highest blood glucose ranges for both tasks . Compared with performance during euglycemia, math time was an average of 12.6 and 16.8 s longer when blood glucose levels were <3.0 and> 22.2 mmol / l, respectively . Reaction time was an average of 2.6 and 1.5 s slower when glucose levels were <3.0 and> 22.2 mmol / l, respectively . In contrast to the results for time to perform tasks, there were no significant differences in the number of correct responses across ranges . Exploratory analyses were also conducted to identify practice effects over time on the two tasks . Math time and reaction time over the first 35 trials were compared with the second 35 trials at each of the above three blood glucose ranges: euglycemia (3.99.9 as expected, during euglycemia math time was significantly shorter for the second of 35 trials (56.9 vs. 66.8 s; f = 19.6, p = 0.001), but there was no improvement for reaction time (p = 0.13). When blood glucose was <3.0 or> 22.2 mmol / l, there were no significant differences in math time or reaction time between the first and second half of the study, indicating no practice effects over time . Mean difficulty ratings tended to be low across all blood glucose ranges; however, significant differences were still found . For the math task, average ratings were 0.44, 0.77, and 0.58 for euglycemia, hypoglycemia, and hyperglycemia, respectively, with a significant main effect across blood glucose levels (f = 4.3, p <0.0001). Mean difficulty ratings for the reaction time task were 0.15, 0.40, and 0.13, respectively, which also differed across blood glucose levels (f = 3.9, p <0.001). However, contrasts showed that for both tasks, difficulty ratings were significantly higher only when glucose was <3.0 this indicates that children perceived greater difficulty performing tasks during hypoglycemia but not when glucose levels were> 22.2 mmol / l, even though time to complete tasks increased significantly and equivalently at both blood glucose extremes . To examine individual differences in the impact of hypo- and hyperglycemia on performance, iiss were computed as described above for math time at blood glucose levels <3.0 and> 22.2 mmol / l . These scores were only computed for those children who had blood glucose readings <3.0 mmol / l (n = 34) or> 22.2 positive z scores indicated poorer performance compared with euglycemia, and negative scores indicated better performance . Mean iis for math time when blood glucose was <3.0 and> 22.2 mmol / l were 0.57 1.6 and 0.33 1.1, respectively . A total of 21% of children had iiss higher than 1.0, indicating that performance deteriorated on average> 1 sd when blood glucose was <3.0 impairment scores were not related to diabetes duration, blood glucse variability (as determined by several measures including the interquartile range, low and high blood glucose risk indexes), or depression and anxiety measures . The child's a1c correlated withimpairment scores for reaction time when blood glucose was> 22.2 mmol / l (r = 0.40, p = 0.02), indicating more impairment with poorer diabetes control . Number of severe hypoglycemic episodes over the past year correlated with impairment scores for both math time (r = 0.39, p = 0.04) and reaction time (r = 0.40, p = 0.02) when blood glucose was> 22.2 neither a1c nor frequency of severe hypoglycemia correlated with performance impairment exhibited when blood glucose was <3.0 based on these findings, naturally occurring episodes of acute hypo- and hyperglycemia during daily routine can be associated with cognitive - motor disruptions in school - aged children with diabetes . To our knowledge, this is the first study comparing the negative impact of hypo- and hyperglycemia on cognitive function in this pediatric population . However, a significant decline in performance was not seen until hyperglycemia became quite profound . In addition, blood glucose extremes affected only the time to complete tasks and not the number of correct responses . This finding replicates results from adult studies (2,3) and adds to the data suggesting that the initial effect of blood glucose extremes is a decrease in mental efficiency and speed and not a decrease in accuracy . Thus, people with diabetes of all ages may compensate behaviorally for blood glucose related cognitive disruptions by first slowing down their performance and consequently sacrificing efficiency to preserve accuracy . A similar type of behavioral compensation plays a key role in models of aging and cognitive functioning (16). The extent to which people with diabetes are subjectively aware of these effects remains unclear and is an important area for ongoing research . In this study, children were aware that they were having more difficulty completing tasks during hypoglycemia but not during hyperglycemia, even though performance was equally affected . However, children's difficulty ratings were also extremely low (average <1.0 on a six - point scale) across all blood glucose ranges, which may indicate that they were reluctant to acknowledge problems in their performance . In a previous article (17), we have reported that young children show very poor ability to recognize mild to moderate hypoglycemia, failing to detect on average over 40% of blood glucose readings it is somewhat surprising that children in this study showed some awareness of increased difficulty performing the tasks when blood glucose levels were low . Although this study found statistically significant differences in performance at blood glucose extremes, it is also important to consider whether the observed level of deterioration is clinically significant . One approach to this question is to examine the degree of disruption by whatever objective standards are available . During euglycemia, it took children an average of just over 1 min to complete 10 relatively simple mental math problems . During hypo- and hyperglycemia, respectively, this task took an average of 12 and 16 seconds longer to complete, representing an 20% decrease in speed . It is not difficult to imagine that a 20% decrease in mental efficiency could be clinically meaningful, especially with more complex, demanding, or time - consuming tasks . Another approach to this question is to evaluate the number of individual children who showed effects that might be considered clinically significant . In this study, iiss indicated that performance declined> 1 sd during hypo- and hyperglycemia for> 20% of children . The finding that children varied greatly in the extent to which they were affected by glucose extremes replicates findings from studies of adults with diabetes (3,6). The mechanisms underlying these individual differences in vulnerability remain difficult to identify . In this study, exploratory correlations to examine the role of clinical variables indicated that higher a1c levels and frequency of severe hypoglycemia may be related to more impairment when blood glucose levels are very high . This finding is in contrast to the predictions some clinicians would make based on anecdotal evidence that type 1 diabetic individuals who are in better glycemic control experience more symptomatology and disruption with hyperglycemia . Neither a1c or history of severe hypoglycemia were related to the degree of impairment during hypoglycemia . Obviously, more research with much larger numbers of children is needed to gather more conclusive information regarding risk factors for acute blood glucose related cognitive disruption . More research is also needed to identify the neurobiological mechanismsunderlying the impact of acute hyperglycemia on cognitive function . While the effect of neuroglycopenia secondary to hypoglycemia is well defined however, there are several possibilities including microvascular dysfunction in the blood - brain barrier and alterations in the synthesis, availability, or reuptake of neurotransmitters, such as serotonin (18). Recent studies have identified a significant reduction in the plasma free fraction of l - tryptophan in children with type 1 diabetes, as well as differences in auditory cortical responses between children with and without diabetes, which may indicate brain differences in serotonergic neurotransmission (19,20). Other recent studies show that changes in extracellular brain glucose have a direct effect on orexin neurons in the lateral hypothalamus, which play a critical role in the regulation of wakefulness and arousal (21). Another purpose of this study was to determine whether pda procedures could provide an alternative field method for studying cognitive function in children at different blood glucose levels . Two questions need to be addressed for this purpose: feasibility and efficacy . In terms of feasibility, it appears that in general children can successfully perform repeated trials of brief pda - administered cognitive tests over a period of several weeks . Of 77 families who entered the study, 78% completed the protocol . In terms of efficacy, the mental math task appears to be sensitive enough to detect differences in children's cognitive performance associated with glycemic extremes . This replicates findings in previous studies of adults with diabetes (5,6) and indicates that even relatively simple tasks requiring working memory and problem solving can be disrupted by hyperglycemia . The reaction time task showed less sensitivity to the disruptive effects of blood glucose extremes and also no practice effects during euglycemia, which may indicate that it was not complex or difficult enough . Failure to find an effect on performance accuracy may also have occurred because of a ceiling effect for these relatively easy tasks, which were designed to avoid producing psychological burden and frustration for these young children . Future studies need to incorporate more complex and demanding cognitive tasks, while balancing the need to not overburden pediatric populations . The current study has several important methodological limitations that should be considered when interpreting these findings . First, we tested a relatively small number of children over a relatively short period of time, which yielded a limited number of extreme blood glucose readings to analyze . Only 56 and 67% of the children had blood glucose values in the lowest and highest ranges, respectively, for data analysis . Future studies are needed to test a larger number of children over a longer time period, or for repeated short periods over longer time, to capture more measures of performance during extreme blood glucose fluctuations . In addition, future studies would benefit greatly by using continuous glucose - monitoring devices in order to obtain a more comprehensive picture of glucose dynamics preceding cognitive testing . This approach would allow, for example, the opportunity to assess the impact of blood glucose variabililty and of antecedent episodes of hypo- and/or hyperglycemia on cognitive function . Finally, this study is limited by testing a fairly homogenous sample of almost all caucasian children with well - educated parents . Even with these limitations, the finding that routinely occurring episodes of acute hypo- and hyperglycemia can disrupt cognitive motor function in children, and that the impact of significant hyperglycemia equals that of significant hypoglycemia, has important implications . Nonetheless, these findings should be considered preliminary and interpreted with great caution . For example, these findings should not be interpreted as evidence that children's cognitive performance cannot be affected by blood glucose levels <22.2 mmol / l . Nor are these findings evidence that all children will experience significant impairments at 22.2 mmol / l . This study found large individual differences in degree of impairment at different blood glucose levels, and there are likely numerous, unidentified variables that influence the impact of an episode of acute hyperglycemia on cognitive function . What these findings do strongly indicate is that more research into the effects of acute hyperglycemia on cognitive function in children is warranted.
While there is a large body of evidence in favor of a clustering of representations of body effectors in the posterior parietal cortex, recent advances have revealed a hierarchical organization beyond a flat intentional map composed of functionally distinct subdivisions operating in parallel at same level . In particular, the parietal reach region (prr) and dorsal area 5 (area 5d) have been found to play distinct roles in visually - guided reach . Based on three lines of neurophysiological studies on nonhuman primates that utilize sophisticated behavioral tasks, including reference frame, effector choice, and sequential reach, this essay proposes that the prr and area 5d are involved in translation of general motor intentions into detailed motor programs at different stages . The posterior parietal cortex (ppc) has historically been considered a typical association cortex, important for spatial attention and multisensory integration in the generation of a unitary map whose output is relayed to the frontal lobe to guide behavior (critchley, 1953; ungerleider and mishkin, 1982; colby and goldberg, 1999). Since the 1970s, tremendous progress in neurophysiology, neuroanatomical tracing, functional imaging, and experimental intervention has yielded evidence of a variety of distinct functional subareas in the ppc, as opposed to a homogeneous area that constructs a single unified perceptual representation (rizzolatti et al ., 1997; mountcastle, 1998; andersen and buneo, 2002). Furthermore, the ppc has been shown to be involved in movement planning in a number of different contexts (andersen and cui, 2009; rizzolatti and kalaska, 2013). The ppc seems to be composed of a mosaic of intentional maps, each of which is thought to be specialized for a different kind of movement for a particular body part (andersen and buneo, 2002; scherberger et al ., 2013). Recent advances have further suggested that the individual effector - specific regions are heterogeneous (heed et al ., 2011; leone et al ., 2014), with a hierarchical organization among different subdivisions preferring the same effector (cui and andersen, 2011; verhagen et al ., 2013), indicating that some subareas in ppc are involved in sensorimotor integration at multiple levels, instead of working in parallel within a flat intentional map . This essay will focus on two reach - related areas in brodmann s area 5 (fig . 1a), the parietal reach region (prr), and dorsal area 5 (area 5d), and discuss the functional relationship between them based on recent neurophysiological studies using a variety of sensorimotor contingencies . B, three paradigms to distinguish general motor intention from detailed motor programs . In the reference frame task, general motor intention is represented in extrinsic space, while physical movement is prepared in intrinsic space . In the effector - choice task, multiple potential action plans can be formed prior to specification of the concrete motor parameters controlling the end - effector . In the sequential reach task, an action sequence encompassing all motor components at a cognitive level is formed before it is unfolded into a series of detailed programs of element movements . The prr was originally defined as an area medial and posterior to the lateral intraparietal area (lip), including more than one cytotectonic area, whose cells are more active for reaching arm movements than for saccadic eye movements (snyder et al ., 1997; quian quiroga et al ., 2006; cui and andersen, 2007). Many subsequent neurophysiological studies of the prr have targeted the medial bank of the ips (mip) (baldauf et al ., 2008; cui and andersen, 2007, 2011; pesaran et al ., 2006, 2008; reversibly inactivating the prr in monkeys causes miss - reach, similar to the optic ataxia reported in human patients (hwang et al . In addition, pre - movement activity is not simply related to the cue, because the prr has been found to encode the desired movement goal during an anti - reach task (gail and andersen, 2006). Moreover, the reaching plan embedded in the prr includes not only target locations or end - points, but also high - level kinematics such as movement trajectories (torres et al ., 2013), supporting the view that movement trajectories are geometrically formed, independently of physical motor commands (torres and zipser, 2002; torres and andersen, 2006). Area 5d, which is located caudal to the primary somatosensory cortex (si) and medial to the ips, is involved in representing spatial information for limbs and control of reaching arm movements (lacquaniti et al . 1995; kalaska et al . 1978; pearson and powell 1985), and is reciprocally interconnected with primary and premotor cortex (strick and kim, 1978). It was previously thought of as a higher somatosensory area that codes posture and joint positions (sakata et al . However, studies on behaving monkeys demonstrate that cells in area 5 became much more active during arm movements (mountcastle et al ., 1975), with strong directional tuning (kalaska et al ., 1983). During delayed reach, many area 5 cells exhibit sustained activity far before the earliest increase in electromyographic activity (kalaska and crammond, 1995). Compared with primary and premotor cortex, area 5 seems to be less directly involved in control of musculoskeletal dynamics: it appears to be insensitive to external force load, and to encode movement kinematics instead of dynamics (kalaska et al ., also, many area 5 neurons show strong firing patterns that continuously covary with hand trajectory (archambault et al ., 2011; hauschild et al ., 2012), a manually / brain controlled cursor (mulliken et al ., 2008), or even the seen position of a realistic fake arm (graziano et al ., 2000 although both the prr and area 5d have been found to be intimately involved in planning of reach, several recent studies of reference frame, decision making, and sequential movement suggest that they might play distinct roles in planning and control of reaching arm movement at different (abstract vs concrete) levels of complexity (fig . 1b), indicating a hierarchical circuitry that is involved in translation from cognitive intention into detailed motor programs to implement a key step of the complex sensorimotor transformations from receptors to muscle activation patterns . The first findings in favor of a hierarchical prrarea 5d circuitry emerged from experiments characterizing sensorimotor coordinates with a variety of combinations of target, eye, and hand positions (andersen at al ., 1997). Unlike an oculomotor plan in eyecentered coordinates that is always registered to its sensory target in a retinotopic map, goal - directed reach initially planned in visual space (flanagan and rao, 1995; wolpert et al ., 1995) requires a transformation from extrapersonal to joint / muscle coordinates . Recent studies demonstrate that different coordinate systems are employed in the prr and area 5d (batista et al ., 1999; bremner and andersen, 2012), suggesting that these areas are involved in reach planning in extrinsic and intrinsic spaces, respectively . Neurophysiological recordings during a delayed - reach task revealed a gradient distribution in the medial wall of the ips, with deep (superficial) neurons tending to be more modulated in the cue (movement) epoch (johnson et al ., 1996), suggesting that they are involved in different stages of the sensorimotor transformation (battaglia - mayer et al ., 2003). Reaching goals have been found to be represented in retinal coordinates (batista et al ., 1999; pesaran et al . 2006) in the prr, indicating a cognitive plan at a more conceptual level than at the subsequent stages of motor planning . That is, the prr only conveys abstract information (i.e., the spatial goal) about upcoming movements, without specifying physical movement parameters (i.e., joint angles). Nonetheless, accurate motor control must take into account intrinsic variables, such as posture, hand position, the dynamics and structure of muscles, etc . Therefore, abstract motor intentions ultimately must be converted into detailed physical motor plans, with movement parameters specified in muscle / joint coordinates to activate musculoskeletal system (kalaska et al ., 1997). The first step in converting target location in gaze - centered coordinates into limb motor commands for reaching is to incorporate proprioceptive and efference copy information about starting hand position and intrinsic arm posture . It seems likely that area 5d plays a key role in this stage by providing target information with respect to initial hand position to specify action parameters in musculoskeletal coordinates (buneo et al ., 2002). From the prr to area 5d, neuronal representations of the reaching goal progressively shift from eye - centered to hand - centered along the ventraldorsal axis of the medial bank of the ips (buneo and andersen, 2006), so that pre - movement activity in area 5d predominantly encodes the reach vector (bremner and andersen, 2012). However, this hand - centered coding is not static, but gradually emerges during the reach planning period (bremner and andersen, 2014), supporting the view that area 5d integrates the intrinsic biomechanics of the musculoskeletal system by dynamically incorporating information about intrinsic arm posture . Another approach to elucidating parietal sensorimotor circuitry is to identify distinct functional areas for decision making and motor planning . Decision making initially was assumed to be a neural process separate from action planning (miller et al ., 1960; keele, 1968). Nevertheless, attempts to distinguish decision making from motor planning with target selection tasks have proven frustrating, because virtually all motor - related areas seem to convey potential movements to candidate targets, and multiple potential plans exist concurrently before the final action is chosen (riehle and requin, 1989; platt and glimcher, 1997; basso and wurtz, 1998; cisek and kalaska, 2005). Consequentially, it is commonly believed that target selection and movement preparation involve overlapping brain circuits, and are performed in an integrated manner, as opposed to a serial model in which decision making occurs before action planning (shadlen and newsome, 2001; wang, 2008; cisek and kalaska, 2010). From the evolutionary point of view, forming multiple potential action plans prior to choosing among them might not only benefit reward prediction, but might also reduce reaction time (andersen and cui, 2009; cisek and kalaska, 2010). Nevertheless, the idea of integrated circuitry for decision making and motor planning has only been tested for spatial target selection (cisek, 2007), which involves spatial attention, and in turn engages numerous cortical areas (desimone and duncan, 1995). It is unclear if plan selection and action preparation are embedded in segregated areas for other kinds of decision making, such as nonspatial effector choice . To examine anatomical overlap for decision making and motor planning in nonspatial action selection, an effector - choice task was designed in which monkeys autonomously chose between a saccade and a hand reach to the same visual stimulus (cui and andersen, 2007). Monkeys were required to play a mixed - strategy game against a computer (barraclough et al ., 2004) to compensate for a potential bias due to movement costs . The effector - choice trials were pseudo - randomly interleaved with effector - instructed trials in which the monkeys were explicitly cued to make either a saccade or a reach to the target in the middle of a trial to discourage premature decisions . The effector - choice task allows potential motor plans to be formed without immediate specification of concrete motor parameters controlling a particular end - effector, providing an ideal paradigm to determine whether action selection and motor preparation are encoded in overlapping or in distinct areas in nonspatial decision making . If a brain region is involved in effector decision formation, then it should encode potential motor plans prior to effector selection . Conversely, if an area only carries selected motor plans, then it should only reflect the decision outcome after the effector is unambiguously specified . Interestingly, recent studies demonstrate remarkable qualitative differences between lip / prr and area 5d (cui and andersen, 2007, 2011). Whereas the lip and ppr encode potential motor plans, area 5d encodes only selected reach plans after the arm is chosen as the effector, suggesting that it is downstream to the prr in a serial visuomotor cortical circuit (cui and andersen, 2011). While the prr and area 5d presumably work in concert with premotor and primary motor areas, respectively (wise et al ., 1997), the functional relationship between the parietal and frontal areas in effector choice remains unclear . In contrast to the prefrontal cortex (tanji, 2001), the ppc has historically received little attention in studies of sequential planning . Nonetheless, neurological studies have suggested that the ppc is crucial for serial behaviour (buxbaum, 1998; zadikoff and lang, 2005). Patients with damage to the left parietal lobe appear normal in performing elementary movements, but are impaired in generating complex action sequences, indicating a profound role of the ppc in integrating multiple spatial goals into a motor sequence . To reveal the functional role of ppc in sequential arm movements, single - neuron activity from the prr (baldauf et al ., 2008) and area 5d (li and cui, 2013) has been recorded from monkeys performing a double - reach task . When the monkeys prepared to reach two simultaneously presented targets with sequential arm movements, most prr neurons carried information about both the immediate and the subsequent goals (baldauf et al ., 2008). Situated at the early stage of the visuomotor transformation, the prr presumably is involved in the first step to generate a coherent sequence, integrating information about all component movements into a high - level movement plan at an abstract level . Nevertheless, a conceptual sequential plan encompassing multiple goals in parallel ultimately must be decomposed into serial motor commands to drive the musculoskeletal system . In contrast to the prr, area 5d has been found to only encode the immediate upcoming reach, and not the subsequent movement (li and cui, 2013). Area 5d activity is tightly coupled only to the next upcoming movement, suggesting it might play a key role in integrating the visual goals from the prr with physical limb information to form a state estimation (mulliken et al ., 2008; moreover, the component reach encoded in area 5d, as opposed to the sequential information being conveyed in the prr and other cortical sensorimotor areas, suggests that unfolding of the movement sequence begins in the parietal - frontal cortex, including the ppc, prefrontal cortex, premotor cortex, primary motor cortex, and supplemental motor cortex, instead of being exclusively conducted by downstream subcortical and spinal circuits . Again, decomposition of the motor sequence appears to engage a larger sensorimotor network via mutual communication between parietal and frontal cortices (pesaran et al ., 2008). A series of recent experiments have been conducted on monkeys performing a variety of sensorimotor tasks to elucidate distinct roles of the prr and area 5d at different stages of the sensorimotor transformation . First, the reference frame task allowed us to isolate general motor intentions in extrinsic space and physical movement preparation in intrinsic space . Second, the effector - choice task allowed potential action plans to be formed without immediate specification of the concrete motor parameters controlling the end - effector . Third, the sequential reach task enabled us to isolate the cognitive action sequence from the detailed motor program of element movements . Three lines of evidence obtained with these tasks suggest that the prr and area 5d form a hierarchical sensorimotor circuitry that translates abstract intentions into detailed motor plans . Despite the markedly different functional roles between prr and area 5d, we should keep in mind that they are not sequentially involved in converting extrinsic stimuli into intrinsic motor plans in a hardwired fashion . First, although a serial neural process was observed in the sensorimotor tasks mentioned above, effector specificity and information flow might be different in behavioral contexts other than visually guided reach (e.g., swaminathan et al ., 2013; woloszyn and shadlen, 2013). Secondly, behavioral and computational studies have suggested that the sensorimotor transformation emerges through an intimate interplay between sensory inflow and motor outflow through paired forward and inverse internal models (wolpert and kawato, 1998; shadmehr and wise, 2005): the forward model translates motor commands into anticipated sensory outcomes, whereas the inverse model converts desired sensory consequences into motor commands (franklin and wolpert, 2011; shadmehr and mussa - ivaldi, 2012). Although the prevalent view posits that sensorimotor control largely relies on forward prediction of sensory consequences (wolpert et al ., 2011), direct neurophysiological evidence that persuasively links neural firing to predicted sensory consequences is still lacking, because the internal prediction of sensory consequences is inherently different from actual sensorimotor variables, and seems not to be behaviorally measurable . Since most previous studies have used reactive movements to stationary goals predefined by sensory cues, it is difficult to determine whether the neural activity observed reflects sensory stimuli or predicts impending movements . Although corollary discharges have been found in diverse species (crapse and sommer, 2008) for gating sensory inputs (e.g., lee and malpeli, 1998) and updating perception (e.g., duhamel et al ., 1992; sommer and wurtz, 2002; synofzik et al ., 2008), as well as for distinguishing sensory signals between active and passive motion (e.g., roy and cullen, 2004), it remains unclear how they are integrated with sensory inflow in recipient structures to form internal models for directing movement . Further elucidation of the ppc sensorimotor circuitry calls for novel behavioral tasks that are highly dependent on predictive spatiotemporal transformations . The author did not consent to publish the author response letter the decision was a result of the reviewing editor ranulfo romo, universidad nacional autonoma de mexico and the peer reviewers coming together and discussing their recommendations until a consensus was reached . A fact - based synthesis statement explaining their decision and outlining what is needed to prepare a revision is listed below . The following reviewers agreed to reveal their identity: christos constandtinidis and w. pieter medendorp the reviewers found your review paper of potential interest for publication in eneuro, provided you address their comments . In general, the reviewers think that you must lay down the scope of your review in the introduction section: input out - put organization of ppc and 5d . Also, there are many issues regarding what you mean by sensory - motor hierarchy, sensory - to - motor transformation, and integrated circuits for decision making, planning, and the link with frontal cortex . In the absence of a clear lay - out of these terms . It would be useful to place the prr-5d circuit in a broader context, at least in the introduction . It would be worth considering the inputs to the prr area from other parietal areas and outputs of area 5d to the premotor cortex . Line 77: please, discuss the effector specificity could change over time, and that the network is not hardwired . Line 88: be more specific about the involvements of ppc and 5d in the different stages of the sensory - motor transforms . Here and again, what is it a hierarchy and should refer also to caminiti and colleagues . But, there are so many examples of hierarchy and sensory - to - motor transformation and their roles in planning, and decision making, etc . Lines 123 - 125: it is unclear what is meant by integrated circuitry for decision making and motor planning for spatial target selection . The source being cited is a review (and a 20-year old one, as such). Line 145: what is the implication of this finding and the link with frontal cortices? Line 170: clarify the term parietal - frontal cortex, by spelling out the relevant areas . Line 171: expand communication via synchronization and de - synchronization between ppc and frontal cortices . Line 174: it is unnecessary to start the discussion with in summary . Line 195: could you please explain or consider that sensory consequences are not behaviorally measurable? The reviewers found your review paper of potential interest for publication in eneuro, provided you address their comments . In general, the reviewers think that you must lay down the scope of your review in the introduction section: input out - put organization of ppc and 5d . Also, there are many issues regarding what you mean by sensory - motor hierarchy, sensory - to - motor transformation, and integrated circuits for decision making, planning, and the link with frontal cortex . In the absence of a clear lay - out of these terms . It would be useful to place the prr-5d circuit in a broader context, at least in the introduction . It would be worth considering the inputs to the prr area from other parietal areas and outputs of area 5d to the premotor cortex . Line 77: please, discuss the effector specificity could change over time, and that the network is not hardwired . Line 88: be more specific about the involvements of ppc and 5d in the different stages of the sensory - motor transforms . Here and again, what is it a hierarchy and should refer also to caminiti and colleagues . But, there are so many examples of hierarchy and sensory - to - motor transformation and their roles in planning, and decision making, etc . Lines 123 - 125: it is unclear what is meant by integrated circuitry for decision making and motor planning for spatial target selection . The source being cited is a review (and a 20-year old one, as such). Line 145: what is the implication of this finding and the link with frontal cortices? Line 170: clarify the term parietal - frontal cortex, by spelling out the relevant areas . Line 171: expand communication via synchronization and de - synchronization between ppc and frontal cortices . Line 174: it is unnecessary to start the discussion with in summary . Line 195: could you please explain or consider that sensory consequences are not behaviorally measurable?
Nsclc is the most common type of lung cancer and the most common malignant neoplasm worldwide (1). During past decades, various studies have attempted to identify molecular biomarkers to predict the prognosis of nsclc (2, 3). Numerous promising biomarkers have been evaluated but none of these have been effective for clinical use (2, 3). The main cell population in anti - cancer immune response is the population of cytotoxic t lymphocytes (ctls) (4). The ctls population is represented by cd8 + lymphocytes, cd4 + lymphocytes, natural killer cells (nk), natural killer t cells (nkt) and lymphocytes b (5, 6). Cancer cells are killed by induction of apoptosis by cytolytic reaction or membrane - receptor induction of programmed death . The successful cytotoxic attack needs an effective antigen presentation by tumor cells and antigen presenting cells (apc). Anti - cancer defense is ineffective in clinically detectable cancers and the greater is the size of a tumor mass, the less effective anti - cancer response is (8). Lung cancer cells hide against cytotoxic attack by low antigen presentation and low co - stimulatory molecule expression . The lung cancer antigens are unstable and badly defined as a result of multiple genetic and epigenetic alterations during oncogenesis (9). Hepatitis b (10) and inflammatory bowel disease (11) are examples, leading to hepatocellular and colorectal cancer . Neutrophils, as a key component in inflammation, may play a crucial role in inflammation driven tumorigenesis (12). Neutrophils support angiogenesis via secretion of proangiogenic factors or by proteolytic activation of proangiogenic factors . Neutrophils are implicated in tumor growth through the proteolytic release of egf - epidermal growth factor, tgf1-transforming growth factor 1, and pdgf platelet derived growth factors from the extracellular matrix (ecm) (13). Immature neutrophils or g - mdsc (granulocytic myeloid derived suppressor cells) are implicated in the establishment of an immunosuppressive tumor microenvironment . Neutrophils kill tumor cells through direct or antibody dependent cell cytotoxicity (adcc) (13). They accumulate in large numbers in premetastatic organs and have a positive effect on tumor cell seeding (14 - 17). Also, it has been shown that neutrophils limit metastatic seeding by killing tumor cells (14, 16). Neutrophils do not affect the growth of the metastatic nodules (14, 16). There is a polarization of neutrophils in tumor promoting and antitumor phenotype which is mediated via cytokines in the tumor microenvironment (i.e. Tgf1 and ifns). Neutrophil abundance correlates with a better prognosis in some studies and a worse prognosis in others (18). Platelets play a significant role in cancer growth, progression and metastasis (19, 20). Significant attention has been given to the association between malignancies and coagulation (19, 20). A hypercoagulability is one of the signs of a more aggressive disease and thromboembolism is one of the major causes of mortality in cancer (21). A prognostic significance between the platelet count and lung cancer has been identified but not fully elucidated (22 - 27). Platelets release some growth factors such as platelet - derived growth factor, platelet factor 4, and thrombospondin which promote hematogenous tumor spread, tumor cell adhesion, invasion and angiogenesis and play an important role in tumor progression (22, 25 - 27). Platelets contain many active molecules and, as they adhere to sites of tumor activated or injured endothelium; many of these molecules are released into the local microenvironment leading to platelet - mediated effects on vascular tone and neo - angiogenesis (22 - 27). Platelets play important roles in the tumor microenvironment that may be thought of as a wound that never heals (28). Objective of this study is to compare neutrophil / lymphocyte ratio (nlr) and platelet / lymphocyte ratio (plr) in patients with nsclc (non- small- cell lung cancer) with and without metastases at the time of diagnosis to find out if there is the importance of these cell ratios in the assessment of severity nsclc . This is the retrospective analysis of nrl and prl in patients with nsclc at the time of the diagnosis of disease before any anti tumor treatment (chemotherapy, radiotherapy, surgery). X 10 / l), calculated nlr and plr in every patient and compared obtained values in patients with metastases and patients without metastases . In 57 patients there were 15 males (26%) mean aged 68, 64 with metastases, 28 males (49%) mean aged 64,63 years without metastases, 8 females (14%) mean aged 61,63 with metastases and 6 females (11%) mean aged 63,33 years without metastases . There were 23 patients with nsclc (40%) with metastases and 34 patients (60%) without metastases . The values of nlr in 34 patients without metastases were: 5,15; 8,57; 1,81; 1,45; 1,69; 3,77; 1,35; 1,83; 1,77; 2,11; 1,65; 2,26; 2,78; 4,25; 3,52; 1,92; 1,37; 1,77; 3,52; 1,22; 3,92; 3,28; 1,47; 3,74; 3,74; 1,82; 3,68; 2,39; 2,39; 2,67; 1,88; 1,70; 4,14; 2,84 . The values of nlr in 23 patients with metastases were: 1,51; 13,3; 0,58; 2,25; 1,71; 3,94; 2,47; 2,97; 2,58; 2,35; 2,35; 2,01; 4,46; 4,47; 3,23; 0,78; 2,79; 2,39; 3,67; 2,23; 2,25; 6,54; 1,56; (figure 2) nsclc with and without metastases in males and females nrl in patients with nsclc nlr in patients without metastases = 2.32 (1.75 to 3.69), in patients with metastases= 2.39 (2,01- 3.67). The values of plr in 34 patients without metastases were: 221,85; 261,0; 115,34; 69,8; 130,0; 166,9; 78,93; 50,29; 105,98; 137,99; 70,77; 106,9; 178,78; 168,4; 123,7; 72,5; 150,54; 83,02; 156,46; 76,12; 184,62; 156,46; 93,85; 225,78; 225,78; 68,09; 281,58; 105,56; 105,56; 116,94; 68,46; 92,71; 328,79; 107,69 . The values of plr in 23 patients with metastases were: 82,49; 402,56; 172,07; 167,60; 104,96; 220,53; 220,81; 201,15; 168; 171,65; 171,85; 133,09; 225,78; 307,84; 131,80; 204,90; 143,30; 128; 142,67; 110,90; 100,30; 124,60; 49,83 . Plr in patients without metastases = 116.14 (81.99 to 170.99), in patients with metastases=167.60 (124, 60- 204.90). Since there was no regularity in the distribution of obtained values of nlr and plr we made the mann - whitney u test . Mean values are not presented using the x and sd, but with a median and interquartile percentiles . The nlr in patients with nsclc without metastases amounted to 2.32 (1.75 to 3.69) and was not significantly different from the nlr in patients with metastases 2.39 (2,01- 3.67) (p=0.614; p = ns). The plr in patients with nsclc without metastases amounted to 116.14 (81.99 to 170.99) and was not significantly different from the plr in patients with metastases 167.60 (124, 60- 204.90) (p = 0.068; p = ns). Various studies have attempted to identify molecular biomarkers to predict the prognosis of nsclc (2, 3). Lymphocytes, macrophages and granulocytes are involved in the anti - cancer battle (1 - 8). The main cell population in anti - cancer immune response is the population of cytotoxic t lymphocytes (ctls) (4). Neutrophil abundance correlates with a better prognosis in some studies but a worse prognosis in others (18). Platelets release some tumor growth factors which play a significant role in cancer growth, progression and metastasizing (19, 20, 22, 25 - 28). Elevated pretreatment nlr, plr and mean platelet volume (mpv) in peripheral blood were identified as independent prognostic factors associated with poor survival with various cancers including nsclc (29). In 81 patients with lung cancer nlr and plr values were significantly higher compared to the healthy subjects (nlr: 4.42 vs 2.45 p=0.001, plr: 245.1 vs 148.2 p=0.002). No significant relationship was determined between these markers and histopathology or tnm stages (29). Pretreatment high nlr and plr were associated with significantly shorter disease - free and survival rates in study worked on 94 patients with nsclc; there was not impact on the response to chemoradiotherapy (30). In 149 patients with nsclc nonlocal failure rates were 11% for patients with plr less than 250 and 58% for plr . Patients with high pretreatment plr had shorter survival after stereotactic radiatiotherapy (31). Some studies indicated that the combination of nlr and plr could be a better prognostic factor . In study on 366 patients in iii and iv stage of nsclc, patients could be divided into three prognostic groups prior to treatment: poor: nlr> 2.68; moderate: nlr 2.68 and plr> 119.50; and good: nlr 2.68 and plr 119.50 (32). A high pretreatment plr (33) and nlr (34) might be a predictive factor of poor prognosis in nsclc a shorter survival after treatment . Some authors (35) failed to find the prognostic significance of nlr in nsclc and some (36) did not find correlation between plr and prognosis of nscl . We compared nlr and plr in patients with nsclc without and with metastases at the time of the diagnosis of diseases . Nrl in patients without metastases was 2.32 and plr 116.14; in patients with metastases nlr was 2.39 and plr 167.60 . Although there was not statistical significance these results show that nlr and plr could be useful in preliminary assessment of nsclc before any treatment . They can be useful predictors for worse prognosis but we still do not know reference values . If our sample were grater we might be given statistical significant results . Immune cells and their ratio influence prognosis and that could be clinically applied in nsclc . More investigations will improve the understanding of the lung cancer and may develop novel therapeutic opportunities.
Model organisms such as fruit flies, zebrafish, and mice have provided great insights into gene function in humans because they are easy to grow and genetically manipulate in the laboratory setting . By evaluating different mutations in these organisms, one can identify candidate genes that lead to disease in humans and develop models to better understand human disease pathogenesis . The mouse is an ideal model organism for human disease . Not only they are physiologically similar to humans, but a large genetic reservoir of potential models of human disease has been accumulated through the generation of radiation- or chemically induced mutant loci . Multiple technological advances have dramatically advanced our skills to create mouse models of human diseases . High - resolution genetic and physical linkage maps of the mouse genome have greatly facilitated the identification and cloning of mouse disease genes . Furthermore, transgenic approaches allowed us to ectopically express or make germline mutations in virtually any gene in the mouse genome by using homologous recombination in embryonic stem (es) cells [2, 3]. Inbred, congenic and transgenic strains are widely used in current research labs as very valuable tools to investigate human diseases pathogenesis and develop new effective therapeutical strategies . Pluripotency is the ability of a cell to give rise to progeny representing all types of cells in an organism . Murine embryonic stem cells derived from inner cell mass (icm) of the embryo exhibit two remarkable features in culture . First, under certain conditions, they can be propagated indefinitely as a stable self - renewing population where every cell undergoes symmetrical division . This immortalized phenotype allows es cells to be cultured over extended periods of time . Upon differentiation, this feature is lost and progeny undergoes cellular aging (hayflick limit) as has been previously documented for all other nontransformed primary cells . A second feature is that, during culture, es cells retain their pluripotency and can differentiate into the same range of cell types as those seen in the embryo from icm . The value of es cells is partly due to their amenability to extensive gene manipulation . Homologous recombination between genomic and the exogenous dna is a very inefficient and rare process, but it takes place in es cells with relatively higher efficiency than it does in other cell types . Gene targeting by homologous recombination in es cells has improved our ability to study many biological processes . Since es cells contribute to all tissues upon injection into a recipient blastocyst, including the germline [6, 7] modification in an es cell genome can be transmitted, by the breeding of es cell / wild - type chimaeras, to generate mice containing the desired mutations in all cells . In this way mice with a variety of modifications such as null and point mutations, chromosomal rearrangements and large deletions have been generated . In addition, it is possible to target reporter genes under the control of specific promoters to study gene expression patterns in different cell types . Furthermore, the ability of es cells to differentiate in vitro to many different mature somatic cell types, in combination with purification of the cell of interest by methods such as directed differentiation and lineage selection, opens up the opportunity to use these mature cell types for various basic and therapeutical applications . This makes it harder to investigate gene function and pathogenesis in those strains . With the advent of ips technology this issue has been overcome . In 2006, takahashi and yamanaka initially reported the direct reprogramming of murine embryonic fibroblasts (mefs) to pluripotent stem cells by introducing four transcription factors . Those factors, namely, oct4, sox2, klf4, and cmyc, that are important for self - renewal of embryonic stem cells (escs) have been shown to reprogram both mouse and human somatic cells into esc - like pluripotent cells (figure 1). Since then, a large number of laboratories have derived induced pluripotent stem cells from somatic cells, and many important advances have been made [915]. Most importantly these ips cells have shown properties very similar to the ones of es cells such as pluripotency markers expression, teratoma formation, chimeras contribution, and germline transmission . Moreover, the critical advantages of ips cells over es cells now seem to be obvious . First of all ips cells are being generated from the autologous recipient thus obviating the graft - versus - host problem in transplantation settings ., one can now generate es - like ips cells from human skin fibroblasts or hair - follicle cells without the need to resort to the human es cell lines and potentially (in the future) apply them to the therapeutic and/or basic science approaches . For the human disease animal modeling ips cell technology opened the way to even wider spectrum of available mouse model strains . In 2009, zhao et al . Reported the generation of all - ips - derived viable, fertile live - born progeny by tetraploid complementation which further proved them to be useful for the development of transgenic mice strains with desired gene defects homologous to those seen in human pathology at present, with this valuable tool in hand one can take literally any human disease mouse strain somatic cells (e.g., tail tip fibroblasts) and induce pluripotent stem cells from them . These disease - specific ips cells can be further used to explore given disease mechanisms both in vitro and in vivo . For example, human chronic lymphocytic leukemia (cll) (cd5 + b - cell malignancy) mouse model new zealand black mouse exhibits a defect in the mir-15a/161 gene on chromosome 14 which results in decreased levels of these micrornas, which is also seen in more than 50% of cll patients . Unfortunately, this mouse strain is refractory to true es cells derivation which makes it difficult to study the role of this microrna gene defect in b - cell development both in vitro and in vivo . In our lab now they can be used as subjects for gene targeting (correcting mir-15a/161 mutation and deletion) followed by in vitro differentiation towards b - lineage . This would help find out what role this particular gene defect plays in b - cell lymphogenesis and how its correction might alleviate malignant clonal expansion . Furthermore, nzb ips cells with corrected mir-15a defect could be differentiated into hematopoietic stem cells (hscs) followed by their adoptive transfer into appropriate recipients in order to observe the effect of gene correction on cll development in vivo . Another way to utilize ips cells to study human diseases in animal models is xenograft transplantation assay . In this case ips cells would be generated from patient's somatic cells (figure 3), differentiated into desired type of cells (e.g., hsc), and transplanted into immunodeficient murine recipients . In a recent report, yao et al . Have demonstrated a generation of human ips cells with zinc - finger nuclease, mediated disruption of ccr5 locus which is known to be a coreceptor for hiv entry . These patient - specific ips cells can now be differentiated into hsc and transplanted into animal recipients to study the role of ccr5 in hiv infection development in vivo . In another work, have used human ips - derived neural stem cells (nscs) in a mouse intracranial human glioma xenograft model . In this case, ips - derived nscs have been used as cellular vehicles for targeted anticancer gene therapy since they will home to the brain . As a proof of principle, hanna and colleagues have taken advantage of autologous ips cells derived from humanized mouse model of sickle cells anemia to correct human sickle hemoglobin allele by gene - specific targeting followed by their differentiation into hematopoietic stem cells and transplantation into irradiated recipients . This work has underlined the benefits of ips technology for the combined gene and cell therapy approach to study human disease in animal models . It is needless to say that currently various labs worldwide use patient - specific ips cells for animal modeling both in vitro and in vivo . Such pathological conditions as huntington disease, amyotrophic lateral sclerosis, spinal muscular dystrophy, gaucher disease type iii, down syndrome, type 1 diabetes, parkinson's disease, -thalassemia, and hepatic failure have been investigated using ips cells generation [2029]. Micrornas (mirs) are small noncoding rnas which are known to be critical for the expression control of more than a third of all protein coding genes by means of binding to the 3 untranslated region (utr) of target mrnas via an imperfect match to repress their translation and/or stability . They have been implicated in the regulation of many biological processes, including the stem cells self - renewal and pluripotency [3234]. Mirnas are generated from precursor transcripts primary mirnas (pri - mirnas)that are first processed in the nucleus into an intermediate pre - mirnas by the complex of enzymes containing drosha and dgcr8 proteins [3537]. The pre - mirnas are then transported by the exportin 5-rangtp shuttle into the cytoplasm, in which they are further processed by dicer, into mature mirnas . In es cells a set of micrornas (including mir-302 and mir-1792 clusters) closely interfere with the key pluripotency factors such as oct4, sox2, and nanog [39, 40] thereby preventing them from differentiation and controlling their proper self - renewal potential . Xu et al . Have demonstrated mir-145 to control the expression of oct4, sox2, and klf4 and repress self - renewal of human es cells . On the other hand, c - myc has been reported to repress mirnas such as mir-21, let-7a, and mir-29a during reprogramming . Tissue - specific mirnas often play important roles in normal tissues and organ formation [44, 45]. More importantly for the current review, micrornas proved to be effective tools for the ips generation . In particular, inhibition of mir-21, let-7a, or mir-29a has been shown to enhance the reprogramming efficiency . Alternatively, overexpression of the mir302/367 cluster has been shown to rapidly and efficiently reprogram both mouse and human somatic cells to ips state without any exogenous transcription factors delivery through oct4 gene expression activation and the suppression of hdac2 . Microrna gene expression profiling in human es cells revealed specific mir - signatures of elevated expression of mir-302 cluster, mir-200 family members as well as mir-520 cluster . This might imply the possibility of them to be used as tools to increase the efficiency of ips generation without any exogenous interventions into the genomic dna of the host cells and serve as additional ips quality control markers . Conversely, as the regulators of gene expression micrornas could be used to drive patient - specific ips cells down the specific cells lineage in vitro in order to produce the required cell type to be studied . Another promise that micrornas are holding is the development of microrna - based gene targeting for the temporal gene - of - interest silencing . For instance, aberrant expression of pax5 (also known as bsap), a critical regulator of b - cell development, is known to correlate with aggressive subsets of b - cell non - hodgkin lymphoma . It has been previously shown that overexpression of mir-15a/16 reduces endogenous c - myb levels and compromises pax5 function . Now one can produce ips cells from pax5-affected non - hodgkin lymphoma patient and apply in vitro b - cell differentiation protocol along with mir-15a/161 delivery to evaluate lymphomagenesis in the mouse xenograft model . Potentially, the similar approach could be employed for the discovery of leukemia (or more commonly cancer) stem cells . Finally, mirs can be used as biomarkers of human disease progression in mouse model settings . The importance of disease mouse models and their impact on medical research is hard to overestimate . Therefore the value of animal modeling is very critical for our understanding of human disease and development of new effective approaches to therapy . Induced pluripotent stem cells hold a great promise for both basic and applied science and open the road for many more opportunities for human disease research . Coupled with the use of fine - tune regulators of gene expression, micrornas, and mouse modeling they have a promising potential for subsequent discoveries and new therapies development in the complex field of human pathology.
Children with type 1 diabetes of at least 1 year duration, attending diabetes clinics at children s hospital new orleans, were invited to participate . Data regarding duration of diabetes, height, weight, date of birth, race, and sex were collected at the time of visit, and additional data regarding prior hba1c and mbg levels were extracted from patient medical records . The study was approved by the institutional review board at louisiana state university health sciences center and the children s hospital new orleans . At the time of clinic visit, the patient s glucose meter was inspected for proper operation . If no problems were found, the mbg from the 30 days prior to the visit was calculated and recorded . A blood sample at the time of the clinic visit was obtained for hba1c and glucose (blood glucose at time of clinic visit [cbg], measured by accu - chek inform, model 2001201, roche diagnostics, indianapolis, in). Hgi at the time of the clinic visit was calculated as the difference between a patient s observed hba1c minus the hba1c level predicted from the patient s observed mbg based on the population regression of hba1c on mbg (predicted hba1c = 0.021 mbg + 4.3) (16). We have previously shown the intra- and interindividual consistency over time of hba1c, mbg, and hgi collected from clinic data (16). During one clinic visit, sif levels were noninvasively measured from the volar surface of the left forearm from each subject using a scout ds instrument (veralight, inc . ). The device sequentially excited the skin surface using different light - emitting diodes (leds) that had peak excitation wavelengths of 375, 405, and 420 nm . Skin reflectance was measured for each excitation led, and a white light led was used to measure skin reflectance over the emission region . Spectral data collected by the instrument were transmitted directly to veralight, inc . For further analysis to adjust for the impact of skin pigmentation, hemoglobin content, light scattering, and other dermal characteristics . Data were adjusted mathematically using two different kx / km parameter sets on the measured reflectance at the excitation and emission wavelengths . For the first kx / km set (designated as set u), kx and km were 1.0 and 0.0, respectively . For the second kx / km set (set c), kx and km were 0.5 and 0, respectively, which is useful for populations with a wide range of skin melanin (17) (further technical detail in supplementary appendix). Sif data are reported in arbitrary relative fluorescence units as a function of excitation wavelength and which kx / km adjustment (u or c) used . Data were analyzed using hba1c, mbg, hgi, and cbg at the time of the visit when sif was measured . In addition, the means of mbg, hba1c, and hgi from all available prior clinic visits for the patient were also evaluated . As results were similar whether using data for that clinic visit or the mean of data from all available clinic visits, we report here the mean data . The influence of biological variation in hba1c on sif was evaluated in multivariate regression models using either hgi or by substituting hba1c adjusted for mbg in the model . For evaluation purposes, cbg was substituted in the models for mbg for single clinic visits . Models were further adjusted for chronological age, duration of diabetes, sex, race, and bmi z - score (z - bmi) using the glm procedure in sas software (sas institute, cary, nc). Children with type 1 diabetes of at least 1 year duration, attending diabetes clinics at children s hospital new orleans, were invited to participate . Data regarding duration of diabetes, height, weight, date of birth, race, and sex were collected at the time of visit, and additional data regarding prior hba1c and mbg levels were extracted from patient medical records . The study was approved by the institutional review board at louisiana state university health sciences center and the children s hospital new orleans . At the time of clinic visit, the patient s glucose meter was inspected for proper operation . If no problems were found, the mbg from the 30 days prior to the visit was calculated and recorded . A blood sample at the time of the clinic visit was obtained for hba1c and glucose (blood glucose at time of clinic visit [cbg], measured by accu - chek inform, model 2001201, roche diagnostics, indianapolis, in). Hgi at the time of the clinic visit was calculated as the difference between a patient s observed hba1c minus the hba1c level predicted from the patient s observed mbg based on the population regression of hba1c on mbg (predicted hba1c = 0.021 mbg + 4.3) (16). We have previously shown the intra- and interindividual consistency over time of hba1c, mbg, and hgi collected from clinic data (16). During one clinic visit, sif levels were noninvasively measured from the volar surface of the left forearm from each subject using a scout ds instrument (veralight, inc . ). The device sequentially excited the skin surface using different light - emitting diodes (leds) that had peak excitation wavelengths of 375, 405, and 420 nm . Skin reflectance was measured for each excitation led, and a white light led was used to measure skin reflectance over the emission region . Spectral data collected by the instrument were transmitted directly to veralight, inc . For further analysis to adjust for the impact of skin pigmentation, hemoglobin content, light scattering, and other dermal characteristics . Data were adjusted mathematically using two different kx / km parameter sets on the measured reflectance at the excitation and emission wavelengths . For the first kx / km set (designated as set u), kx and km were 1.0 and 0.0, respectively . For the second kx / km set (set c), kx and km were 0.5 and 0, respectively, which is useful for populations with a wide range of skin melanin (17) (further technical detail in supplementary appendix). Sif data are reported in arbitrary relative fluorescence units as a function of excitation wavelength and which kx / km adjustment (u or c) used . Data were analyzed using hba1c, mbg, hgi, and cbg at the time of the visit when sif was measured . In addition, the means of mbg, hba1c, and hgi from all available prior clinic visits for the patient were also evaluated . As results were similar whether using data for that clinic visit or the mean of data from all available clinic visits, we report here the mean data . The influence of biological variation in hba1c on sif was evaluated in multivariate regression models using either hgi or by substituting hba1c adjusted for mbg in the model . For evaluation purposes, models were further adjusted for chronological age, duration of diabetes, sex, race, and bmi z - score (z - bmi) using the glm procedure in sas software (sas institute, cary, nc). Characteristics of the patient population studied at the time of sif measurement in clinic are presented in table 1 . In the patient population, mean hba1c (mhba1c) was correlated with mean mbg (mmbg) (r = 0.5; p <0.001) but not the visit cbg (r = 0.07; p = 0.51). Mhba1c was correlated with mean hgi (mhgi) (r = 0.8; p <0.001). The sif data for the three excitation wavelengths were highly intercorrelated within the same adjustment set whether u or however, correlations between u and c data were considerably lower (supplementary table 3). Patient characteristics at time of clinic visit (n = 110) results from the single visit data were similar to the results using mean values from multiple visits . Simple correlation analyses were performed between u and c adjustments of sif with mmbg, mhba1c, mhgi, age, and duration of diabetes (table 2). Mmbg, cbg, and z - bmi were not correlated with any of the sif data . Pearson correlation between u and c set sif adjustments with mmbg, mhba1c, mhgi, age, and duration of diabetes the top number in each cell is the correlation coefficient (r), and the bottom number is the significance level (p value); n = 110 . Multivariate regression analysis showed that mhba1c adjusted for mmbg, or mhgi substituted in the model for mhba1c, was consistently associated with all sif excitation wavelengths, both u and c, after also controlling for presence of age, race, sex, z - bmi, and duration of diabetes (table 3). Mmbg and z - bmi were not statistically significant in relationship with any of the sif measures . Multivariate regression analysis between u- and c-adjusted sif levels at each wavelength as dependent variables with patient age, duration of diabetes (dod), sex, race, z - bmi, hba1c or hgi, and mbg statistically significant relationships, p = 0.05 or less, for the covariates in the model are indicated by x. the relationship between 405-nm excited sif using set age, duration of diabetes, and sex were statistically significant covariates for the c sif data at all three wavelengths . Sif levels increased with age and were higher in girls than boys at any given age (fig . Race was a significant covariate for all sif excitation wavelengths with the u adjustment, but only for 375 nm in the c-adjusted sif data, blacks being higher than whites . To our knowledge this is the first in - depth study of the relationship of skin ages (sages) with hba1c, mbg, hgi, and other characteristics in a biracial population of children with type 1 diabetes . We used a novel technology to estimate sages by measuring sif using a scout ds device at three different excitation wavelengths and two mathematical adjustment sets . Prior reports using this device have focused on its ability to identify adults with abnormalities of glucose tolerance (13,14) and the relationship of sif with coronary artery calcifications in type 1 diabetes (12). Spectral information read by the scout device from the skin is a combination of reflected light and fluorescence excited in the tissue by leds . Because of variability in skin pigmentation, skin thickness, hemoglobin, and other factors, the excitation and emission data returning to the device must be adjusted to minimize the distortion of the skin fluorescence by the aforementioned factors . U and c adjustments of the sif data (table 3). With the u adjustment, all data from the three excitation sif wavelengths evaluated were consistently higher in black than in white patients, consistent with differences in skin pigmentations . Such a difference was only found at the 375-nm sif excitation wavelength with the c adjustment . Sages and auto - fluorescence have been shown to increase with aging and duration of diabetes in dermal biopsy samples from patients with diabetes (15,18,19). However, the u adjustment from our sif data was insensitive to differences related to duration of diabetes and less sensitive to age - related change . Our finding of a significant increase in c-adjusted sif at all excitation wavelengths with patient age and duration of diabetes in children is in accordance with prior biopsy data, albeit from adults . C-adjusted sif levels between girls and boys that was independent of age, mbg, or hba1c level . There was no sex difference discernable using the u adjustment, which agrees with reported findings in adults using a similar method (20), with the exception of smokers (21). Potentially the detected sex difference in sif might represent a biochemical difference in accumulation of fluorescent ages between the sexes during childhood and adolescence . Alternatively this difference may represent non - age differences and be an artifact of the adjustment method or other factors . It is conventionally understood that formation and accumulation of glycated proteins such as hba1c (4) and ages (2) is a concentration - dependent function of glucose concentration . The sif data demonstrated a strong relationship with hba1c or hgi whether u or previous investigators have studied skin autofluorescence during oral glucose tolerance testing and noted no changes related to acute fluctuations in blood glucose (23). Initial nonenzymatic attachment of glucose to a protein such as hemoglobin occurs quickly and reversibly, but the formation of the stable amadori product takes much longer, and progression to ages even more time . Thus a randomly obtained glucose level, while correlated with the labile schiff base precursor to hba1c, is negligibly associated with stable hba1c levels (24). The lack of association of sif with mbg is more surprising . Both mbg and hba1c levels have been associated with the development of complications in type 1 diabetes (6). Hba1c is strongly correlated with mbg and widely used to estimate mbg (25). However in simple pearson correlation analysis, hba1c and hgi were correlated with the sif levels whereas mbg was not . In the multivariate models, both hba1c and hgi were significantly related to sif at that visit, but the visit mbg as a covariate was not . Considering the possibility that the 30-day average mbg from the clinic visit might have been insufficiently long to be involved with detectable age formation, we substituted the average of all available prior mbg measurements from patients into the model (along with hgi or hba1c). These findings suggest that mbg - independent, between - patient differences in hba1c are predictive of age burden in the skin and potentially influence the development of diabetes complications . Thus factors besides mbg that can influence nonenzymatic glycation (24) may be more influential in formation of skin ages and development of diabetes complications than just mbg exposure alone.
Therapeutic hypothermia has been shown to provide neuroprotection against ischemic injury after cardiac arrest in in vitro and in vivo models . In the previous issue of critical care, meybohm and colleagues demonstrate that cardiac arrest triggers the release of cerebral inflammatory cytokines in pigs' cerebral cortex . . The combination of hypothermia with sevoflurane post - conditioning does not confer additional anti - inflammatory effects compared with hypothermia alone . Cardiac arrest remains the leading cause of death in the us and europe, with an out - of - hospital cardiac arrest survival - to - discharge rate of less than 10% . In - hospital cardiac arrest presents a dismal prognosis . According to a large in - hospital registry, the survival - to - discharge rate is 18%, whereas that of a developing country is 6.9% . Without prompt care when immediate care is available and victims are successfully resuscitated, the majority of these initial survivors subsequently suffer crippling neurologic injury or die in the few days following the cardiac arrest event . Thus, improving survival and brain function after initial resuscitation from cardiac arrest remains a critical challenge . Therapeutic hypothermia, introduced more than six decades ago, remains an important neuroprotective factor in cardiac arrest . Laboratory studies have demonstrated that cooling after resuscitation from cardiac arrest improves both survival as well as subsequent neurologic and cardiac function and has few side effects . These findings have been reproduced using a variety of cooling techniques in different species, including rats, dogs, and pigs . However, physician use of hypothermia induction in patients resuscitated from cardiac arrest is low . In 2003, abella and colleagues reported that 87% of us physicians did not use therapeutic hypothermia following cardiac arrest . Various reasons for non - use were cited: 49% felt that there were not enough data, 32% mentioned lack of incorporation of hypothermia into advanced cardiovascular life support protocols, and 28% felt that cooling methods were technically too difficult or too slow . In 2002, a european group demonstrated an improvement in survival - to - discharge rate with favorable neurologic status in cooled patients, compared with normothermic patients surviving after cardiac arrest (53% versus 35%, respectively), and with no significant adverse events from cooling; thereafter, induced hypothermia was considered the best practice for patients following cardiac arrest . In 2005, the american heart association recommended the consideration of therapeutic hypothermia for unconscious adult patients with return of spontaneous circulation following out - of - hospital cardiac arrest due to ventricular fibrillation . In 2008, binks and colleagues reported that 85.6% of intensive care units in the uk were using hypothermia as part of post - cardiac arrest management . Clinical observation demonstrated that tumor necrosis factor - alpha (tnf) and interleukin-6 (il-6) protein were increased in cerebrospinal fluid following cardiac arrest . Animal studies showed that inflammatory markers were unregulated in rats' hippocampus tissue and pigs' serum and myocardial tissue after cardiac arrest [8 - 10]. Meybohm and colleagues go further to demonstrate anti - inflammatory and anti - apoptosis effects of therapeutic hypothermia via the reduction of the upregulation expression of il-1, il-6, il-10, tnf and intercellular adhesion molecule-1, bcl-2, and bax mrna and il-1 protein in cerebral cortex after cardiac arrest in a pig model . Small reductions in core temperature lead to vaso - constriction and shivering, effectively hindering hypothermia . Thus, prevention of vasoconstriction and shivering has become a major goal during induction of therapeutic hypothermia . Sevoflurane pre - conditioning and early post - conditioning reduced both cerebral infarct size and neurological defect score, reduced impairment of hippocampus long - term potentiation resulting from myocardial ischemia, and increased nuclear factor inhibitory kappabalpha content in thp-1 cells [11 - 13]. Sevoflurane pre - conditioning preserves myocardial function in patients undergoing coronary artery bypass graft surgery under cardiologic arrest . An in vivo study showed that combination hypothermia with sevoflurane attenuates the inflammatory response during endotoxemia . However, meybohm and colleagues could not provide evidence to support the view that sevoflurane post - conditioning confers additional anti - inflammatory effects in pigs' cerebral cortex after cardio - pulmonary resuscitation . In summary, meybohm and colleagues provide useful evidence to support the clinical use of therapeutic hypothermia for cardiac arrest, but they did not study the anti - inflammatory effects of sevoflurane in this model . It is even possible that in the setting of clinical practice, anesthetics may not provide significant neuroprotection beyond that which is already being produced by therapeutic hypothermia . Thus, at this time, it is difficult to recommend anesthetics for the purpose of neuroprotection in cardiac arrest.
A 72-year - old patient who had undergone bilateral uneventful cataract surgery 6 years ago, presented with visual loss in her left eye . Patient had a medical history of multiple intravitreal and periocular steroid injections for the treatment of recalcitrant uveitis that was diagnosed 21 years ago . Her visual acuity decreased to hand motion in left eye, and slit - lamp biomicroscopy revealed in - the - bag iol with an intact posterior capsule, and severe flare in the anterior chamber . An unclear image of macular edema was hardly detected on flourescein angiography (fa) that revealed flue images of posterior - segment obscured by media opacity . After obtaining a signed informed consent, we scheduled to perform the intravitreal ozurdex application under the sterile conditions of the operation room . A drop of 0.5% topical proparacaine hydrochloride with a drop of 5% povidone iodine was installed before the patient underwent an uncomplicated intravitreal dexamethasone implant application . After significant regression of inflammatory cells secondary to uveitis, mild macular edema and an epiretinal membrane were precisely demonstrated with both optical coherence tomography and fa at 2 weeks after ozurdex application . On the postoperative 5 week, best corrected visual acuity (bcva) was improved to 0.4 logmar, and slit - lamp examination revealed the implant mislocated just behind the iol in an intact capsular bag, although there was no reaction in the anterior chamber, and no corneal edema was observed [fig it was thought that such implant anteriorly migrated toward into the posterior chamber through weak zonules in the present case as she had a medical history of uneventful phacoemulsification surgery with the implantation of posterior chamber iol . Slit - lamp biomicroscopy revealed the dexamethasone implant mislocated just behind the intraocular lens in an intact capsular bag, and localized in the lower center of the visual axis (white arrow) any repositioning procedure was not considered since no sign of corneal edema or iop rise was present, as well as patient did not have any complaints although dexamethasone implant mislocated in the lower center of the visual axis . Close follow - up was scheduled for the case in order to find out any signs of anterior segment pathology . In the affected eye, bcva and iop were found as 0.5 logmar and 17 mmhg with a quiescent anterior chamber and a clear cornea at the 4 month of postoperative follow - up, besides dexamethasone implant completely degraded . Steroid - induced ocular complications such as cataract formation and iop elevation have been more commonly reported according to the accelerating ozurdex application in the treatment of macular edema . Anterior migration of dexamethasone implant has also been published as a rare complication in the literature . Migration of ozurdex into the anterior chamber was firstly described by pardo - lpez et al . In a patient with iris fixated iol . After the patient had been referred with blurred vision in his left eye at the postoperative 3 week, authors noticed anterior migration of the implant with a diffuse corneal edema . Patient had to be undergone corneal transplantation since corneal edema did not resolve even though surgical removal of the implant from the anterior chamber was performed . Jonas and schmidbauer reported dislocation of a steroid implant into the anterior chamber in an aphakic vitrectomized eye . Vela et al . Also reported the migration of ozurdex into the anterior chamber in a patient who previously underwent cataract surgery with iris - claw iol implantation . The anterior migration of the ozurdex implant can cause complications such as secondary corneal decompensation . In these cases, bansal et al . Published the migration of the implant into the anterior chamber in three noninfectious posterior uveitic eyes with the history of postlensectomy - vitrectomy aphakia . Authors reported that the implant was relocated in the vitreous cavity in two of the cases . Nonsurgical management of a dislocated dexamethasone implant into the anterior chamber with supine positioning after pharmacologic pupillary dilation was described by kishore and schaal . Published the scleral fixation of dexamethasone intravitreal implant with 10 - 0 nonabsorbable polypropylene suture in an angle - supported iol implanted case in order to prevent the risk of anterior segment complications associated with the migration of such implant into the anterior chamber . It was recommended that dexamethasone implant which migrated into the anterior chamber ought to be removed as soon as possible in cases with incipient corneal edema . On the other hand, no corneal edema was reported in patients with late migration of such intravitreally injected implant . Mentioned that only the patients in whom dexamethasone implant anteriorly migrated into the anterior chamber within 3 weeks after the intravitreal application developed corneal edema in their series . Any sign of corneal edema also did not occur in our case who was faced with the anterior migration of such implant 5 weeks after the uncomplicated intravitreal application . Anterior migration of a dexamethasone implant in eyes without perfect zonular or the posterior capsular integrity after cataract surgery, and with a history of the prior vitrectomy has been well - defined . Reported five patients with an anteriorly migrated dexamethasone implant despite within the same lens and capsular status, although they had previous uncomplicated dexamethasone implant injections without any anterior chamber migration . Anterior migration of a dexamethasone posterior implant into the anterior chamber through weak zonules was also described in cases with intact posterior capsule . Implant was surgically removed from the anterior chamber immediately by daudin and brzin, however turaka et al . Reported that, without any surgical intervention, implant was spontaneously relocated back into the vitreous cavity with significant resolution of the corneal edema . Herein, we report the first case with anterior migration of a dexamethasone intravitreal implant that mislocated just behind the iol in an intact capsular bag . In the present case, the migrated implant was well tolerated as there was no sign of the corneal complication, iop rise, and anterior chamber reaction . As this is a report of unique case with short follow - up, it is very difficult to comment that mislocation of a dexamethasone intravitreal implant on an intact posterior capsule is an innocent complication . It is mandatory to observe more cases with long follow - up in order to estimate possible side effects of ozurdex implant attached on the posterior capsule.
A 53-year - old woman presented to the emergency department with a 2-week history of abdominal distention and constipation . She had a previous history of having undergone a myomectomy 15 years ago and has been under thyroid hormone replacement therapy for 10 years . Seven years ago, she had visited our hospital due to the same symptoms . At the time, abdominopelvic computed tomography revealed markedly dilated entire colon with feces and tapered narrowing in the distal portion (fig . Colonoscopic mucosal biopsy showed mild lymphoplasmacytic infiltrates in the mucosa without muscularis mucosa component . At this visit, laboratory findings were within normal limits except for a slightly decreased tsh level (0.48 iu / ml; normal range, 0.55 to 4.2). Serologic study of anti - hu antibody, anti - ri antibody, anti - yo antibody, anti neuromyelitis optica antibody, anti antinuclear antibody, anti thyroid - stimulating hormone receptor antibody, anti - thyroglobulin antibody, and anti - thyroid microsomal antibody were all negative . On abdominopelvic computed tomography, the entire colon was severely dilated and there was no significant difference between the previous and current images . The differential diagnoses included colonic obstruction due to stricture from inflammatory bowel disease or ischemic colitis and a mild form of congenital megacolon . Proximal advancement of the scope was not possible due to much fecal material and the patient s pain . After 3 days of conservative management with slow bowel preparation with polyethylene glycol solution, the distended abdomen was decompressed and the symptoms subsided . Colon transit time study showed a slow transit constipation pattern . Given concern for the possibility of intractable benign luminal stenosis due to adhesive structure or ischemic enteritis there was no evidence of adhesion or mechanical obstruction except for focal physiologic attachment between the visceral and parietal peritoneum . The patient s postoperative course was uncomplicated and the patient s symptoms resolved after the surgery . The wall thickness appeared even without regional differences between the dilated and the narrowed portions . The average number of eosinophils was less than 4 per high power field (hpf). The muscularis mucosa was thickened up to 660 m in the dilated area in contrast to the thin muscularis mucosa in the narrow area (fig . Intermuscular auerbach s myenteric plexuses showed moderate eosinophilic infiltrates up to 22 per hpf (fig . 3b) and hypogangliosis with atrophic reduction of the volume of nerve bundle, which became more evident with synaptophysin immunostain (fig . The atrophic myenteric plexus was accompanied by increased fibrosis and admixed with some cd3 and cd4 dual positive lymphocytes (fig . Immunohistochemical study showed preserved positivity of c - kit and stronger but scantier glial fibrillary acidic protein (gfap) reaction in the myenteric plexus when we compared the immunostain results with a comparable descending colon of a 60 year old female patient who received hartmann s operation due to cancer (fig . There was no significant difference in the severity of the eosinophilic infiltrates in the myenteric plexus between the grossly dilated and narrow portions . However, there was regional difference in the eosinophilic infiltrates between the inner portion and the outer portion of the myenteric plexus . There was no eosinophilic infiltrates in the submucosa and the inner muscle proper in contrast to the increased number of eosinophils, lymphocytes, and histiocytes in the outer muscle layer, subserosa, and serosa . The wall thickness appeared even without regional differences between the dilated and the narrowed portions . The average number of eosinophils was less than 4 per high power field (hpf). The muscularis mucosa was thickened up to 660 m in the dilated area in contrast to the thin muscularis mucosa in the narrow area (fig . Intermuscular auerbach s myenteric plexuses showed moderate eosinophilic infiltrates up to 22 per hpf (fig . 3b) and hypogangliosis with atrophic reduction of the volume of nerve bundle, which became more evident with synaptophysin immunostain (fig . The atrophic myenteric plexus was accompanied by increased fibrosis and admixed with some cd3 and cd4 dual positive lymphocytes (fig . Immunohistochemical study showed preserved positivity of c - kit and stronger but scantier glial fibrillary acidic protein (gfap) reaction in the myenteric plexus when we compared the immunostain results with a comparable descending colon of a 60 year old female patient who received hartmann s operation due to cancer (fig . There was no significant difference in the severity of the eosinophilic infiltrates in the myenteric plexus between the grossly dilated and narrow portions . However, there was regional difference in the eosinophilic infiltrates between the inner portion and the outer portion of the myenteric plexus . There was no eosinophilic infiltrates in the submucosa and the inner muscle proper in contrast to the increased number of eosinophils, lymphocytes, and histiocytes in the outer muscle layer, subserosa, and serosa . Digestive motility, an important pathogenic factor of cipo, is a highly coordinated process and depends on smooth muscle contractility and the related pacemaker activity evoked by the interstitial cells of cajal (iccs). Both of these mechanisms are finely tuned by the intrinsic enteric nervous system (ens) and the extrinsic sympathetic and parasympathetic nerves, independently from the central and peripheral nervous systems . Based on histology, cipo can be classified into three major entities depending on the predominant involvement of enteric neurons, iccs, or smooth muscle cells: neuropathies, mesenchymopathies, and myopathies . Neuropathy can be classified into two major forms: (1) inflammatory neuropathies in which a significant inflammatory response is identified within nerve tissue and (2) degenerative neuropathies characterized by evidence of neurodegenerative aspects in the absence of an identifiable inflammatory response . Inflammatory neuropathy is also subdivided into lymphocytic and eosinophilic depending on the prominent infiltrating cells . Distinctive findings of eosinophilic neuropathy has mainly been reported in children showing no degenerative pathologic findings such as neuronal loss and no lymphocytic infiltrates . We report a case of eosinophilic myenteric ganglionitis which developed in a middle - aged female patient with the pathologic features of hypogangliosis and cd4-positive lymphocytic infiltrates . However, interleukin 5 (il-5), a well - known eosinophilic chemotactic factor, has been mentioned in eosinophilic myenteric ganglionitis . Although we did not evaluate il-5, the increased number of cd3 and cd4 dual positive cells may suggest the increased production of il-5 from the infiltrating cd4-positive helper t cells for eosinophilic chemotaxis . A few scattered cd8-positive lymphocytes may be involved in the cytotoxic reaction of the nerve plexus resulting in hypogangliosis . Atrophic plexus and cell loss in neuroinflammatory disease were mentioned as the result of the activation of a caspase dependent mechanism; however, immunoreactivity of bcl-2 in our case showed no definite reduced positivity . Enteric glial cells are the main constituent of the ens and the majority of the enteric glial cells are located in the myenteric plexus . Recently, the role of enteric glial cells as a neuroprotector and as an immune - modulator in inflammatory processes has been investigated and the myenteric gfap - expressing glial subpopulation was reported to be susceptible to systemic inflammation . In our case, the stronger but scantier expression of gfap seems to be related with the overproduction of gfap by activated enteric glial cells for neuroprotection as a damaged myenteric plexus . The role of the myenteric plexus as an immune - modulator and barrier was evident in the findings of scarce inflammatory reaction in the inner muscle layer in contrast to mild to moderate inflammatory reaction in the outer muscle layer and the subserosa . This regional difference suggests the barrier function of the myenteric plexus to diffusible and systemic immune reactants supplied by the mesenteric bloods through the subserosal and outer muscular layer vessels . There being no difference in the eosinophilic infiltrates in the myenteric plexus between the dilated and narrow areas was also meaningful . Similarly, no difference in ens abnormality between the affected and the nonaffected areas in crohn disease has been described . This finding suggests the eosinophilic reaction as a systemic reaction, and the possible discrepancy between the pathologic findings and functional impairment . The pathologic findings such as eosinophilic infiltrates or neuronal loss can be the underlying causes of cipo; however, the clinical functional impairment is more complex . Physiologic abnormality such as disturbed activity of neurotransmitters can be another factor to take into consideration . The increased number of mast cells in the submucosa may be the finding of the crosstalk between mast cells and eosinophils . The thickened muscularis mucosa in the dilated colon can be a compensatory response to prolonged high luminal pressure . The therapeutic effect of surgery is limited since cipo often involves the entire gastrointestinal tract with a progressive nature . As surgical approach can aggravate the underlying condition, it should be reserved for only carefully selected patients . If a confirmed diagnosis of ganglionitis is made through a full - thickness biopsy of the intestinal wall, the patient might benefit from an anti - inflammatory or immunosuppressive therapy before the surgical procedure . However, in the absence of a transmural tissue diagnosis, patients with enteric ganglionitis cannot be subject to these nonoperative treatment options . In conclusion, we think this colonic lesion might have begun as a systemic immune reaction showing eosinophilic myenteric ganglionitis and progressed into a chronic inflammatory lesion showing atrophic myenteric plexus, reduced ganglion cells, thickened muscularis mucosa, and subserosal fibrosis . The diagnosis of cipo is mainly clinical, and the histopathology of cipo has frequently been reported as a frustrating experience by pathologists due to limited experience . Further combined clinical and histopathological studies are needed in order to enrich the understanding and management cipo.
Neurologists are central to the provision of quality, patient - centered care for individuals with ms . The substantial majority of ms patients depend on neurologists for high quality treatment and education about their condition [1, 2]; for example, ms patients receiving care from neurologists are more likely to receive and use disease - modifying therapies (dmt), participate in rehabilitation clinics, and receive care from rehabilitation specialists (e.g., occupational and physical therapists) and urologists compared with those ms patients receiving care from primary care providers . Neurologists are also critical to quality ms care because of their detailed knowledge of treatment options for ms patients . Ms treatment options have become increasingly complicated as the available dmts increase and their benefits and safety profiles remain unclear [1, 4]. Among neurologists, little information is available regarding difference in treatment patterns or patient perceptions of care among individuals with ms receiving care from ms subspecialists versus from other neurologists . Although ms patients receiving care from neurologists and from ms subspecialists have been reported to experience similar use of healthcare services, those receiving care from ms subspecialists reported greater knowledge about and current use of a particular dmts (i.e., interferon beta-1b), expressed more confidence in their physicians, and indicated greater participation in nondrug research . Although neurologists are critical to providing ms care, shortages among the neurologist workforce (including both ms subspecialists and other neurologists) may restrict access to neurologists among individuals with ms . Anecdotal evidence from patient groups suggests that ms patients experience difficulty in gaining access to appropriate care, especially from ms subspecialists; new patients may experience substantial waits and difficulties in finding available neurologists . Understanding the factors that influence the number of ms patients seen by neurologists can aid in identifying challenges that ms subspecialists and other neurologists encounter in seeing ms patients and can suggest potential solutions for resolving these challenges . This study examines results from a recently completed study assessing factors associated with the number of ms patients seen by ms subspecialists and other neurologists . We developed the ms physician workforce neurologist survey initially by reviewing previously developed physician career surveys to identify items relevant to assessing neurologists' care for ms patients . We selected and adapted existing questions and crafted new items to create a draft survey, which included questions on respondents' demographic characteristics, medical training, attitudes toward providing ms patient care, ms subspecialist status, practice characteristics, and numbers of ms patients seen . To ensure comprehensiveness and usability of the survey, we sought input from an advisory panel of experts identified for this study and the american academy of neurology (aan) ms section executive committee . The advisory panel, including neurologists, reviewed and provided comments on the survey, which was then revised based on their feedback . Members of the aan ms section executive committee then reviewed the survey to ensure that it was feasible for aan members to complete . This final version of survey was approved by the rti international institutional review board prior to distribution . The sampling frame consisted of neurologists practicing in the united states who are members of the aan . The aan contacted a random sample of 1,700 neurologists who resided in the united states and were members of the aan, excluding retired members; those in medical school, residency, or fellowship; and aan committee members who participated in survey review . Seven respondents were later removed from the sample because they were not in practice or did not see adult patients . The aan administered the survey via email as well as postal mail or fax to the sample of neurologists in january 2012 . The aan sent neurologists an email describing the study and including a hyperlink to the online version of the survey . The paper version also included a cover letter signed by the chair of the aan ms section . Aan staff tracked responses to determine whether invited neurologists submitted surveys and, for nonresponders, distributed two email reminders to participate . Of the 1,693 neurologists invited to participate, 662 submitted responses (response rate of 39.1%). We used identification numbers to merge (deidentified) neurologists' survey responses with demographic information and practice characteristics from the 2008 aan member census . From the census, practice arrangement were categorized as solo practice, neurology group, multispecialty group, university based group, other, and unknown . The other category included staff - model hmo; government hospital or clinic; and other public or private hospital or clinic setting . The dependent variable for this analysis was self - reported total number of ms patients seen in an average week . Independent variables included demographic characteristics, practice characteristics, and attitudes toward ms patient care . Self - reported demographic characteristics included age group (categorized in quartiles), sex, race (white, asian, and other race including black / african american, american indian / alaska native, and native hawaiian / other pacific islander groups combined because of small numbers), ethnicity (hispanic / latino or not), and year began medical practice (categorized in quartiles). Urban / rural designation of practice area was coded into three categories: within a major city, suburban or moderate - sized city, and rural area or small city . First, based on fellowship training those who had completed a ms fellowship and reported seeing, on average, at least one ms patient per week were classified as ms subspecialists . As neurologists may also subspecialize in ms patient care without completing a ms fellowship, respondents who reported seeing, on average, more than 12 ms patients per week (the median number of ms patient seen per week among neurologists who did not complete a ms fellowship but considered themselves to be ms subspecialists) were also classified as ms subspecialists . Ten of these attitudes reflected factors that could limit the number of ms patients seen by respondents, while seven corresponded to positive aspects of providing ms care (table 2). Attitudes were coded dichotomously (i.e., zero or one), based on whether a respondent indicated that each attitude was applicable to his or her practice . This dependent variable (number of ms patients seen) was examined separately for ms subspecialists versus other neurologists (i.e., general neurologists and subspecialists in areas other than ms, referred to as non - ms subspecialists). Since this dependent variable was not normally distributed (shapiro - wilk w tests for normality, p <0.001), we computed nonparametric bivariate analyses to examine factors associated with ms patient seen using the wilcoxon - mann - whitney tests for independent variables with two categories and the kruskal - wallis tests for independent variables with three or more categories . We also conducted bivariate analyses to compare characteristics and attitudes of other neurologists (non - ms subspecialists and general neurologists) who did not see any ms patients with those who saw ms patients, using tests or, because of small cell sizes, fisher's exact tests to examine relationships between discrete variables (e.g., sex) and the proportion seeing any ms patients . We performed multivariate regression analyses to examine the association of neurologist characteristics and attitudes with numbers of ms patients seen while controlling for other potentially associated factors . Since residuals for the dependent variables were not normally distributed, we used negative binomial regression models with robust standard errors to identify predictors of number of ms patients seen . As factors associated with numbers of ms patients seen may differ between ms subspecialists and other neurologists, regression analyses were performed separately for these two populations . Results from the negative binomial regressions are presented as incidence rate ratios (irrs), that is, the rate of seeing individuals with ms associated with a predictor variable relative to the reference case for that variable . Irrs less than 1.0 indicate decreased rates of seeing ms patients compared with the reference case, while irrs greater than 1.0 indicate increased rates . Regression models included all independent variables (i.e., survey respondents' characteristics) that were significantly (p <0.05) or marginally significantly (0.05 <p <0.10) associated with the number of ms patients seen in bivariate analyses (tables 1 and 2). All variables included in regression analyses were examined for multicollinearity (vif> 5) prior to inclusion in the final model . Because of multicollinearity with age, the variable year began medical practice was removed from the regression model predicting ms patients seen per week among general neurologists and non - ms subspecialists (as age was not significantly associated with number of ms patients seen among ms subspecialists, age was not included in that regression model). Missing data generally comprised less than 3% of responses; individuals with missing data for descriptive, bivariate, or regression analyses were excluded from those analyses . After excluding neurologists who did not provide information about their subspecialty status, the study population consisted of 573 neurologists . Of these neurologists, 87 (15.2%) were categorized as ms subspecialists and 486 (84.8%) were other neurologists, including subspecialists in other areas of neurology (i.e., not ms) and general neurologists (table 1). Among the 87 ms subspecialists, 26 (approximately 30%) were classified as ms subspecialists based on having completed an ms fellowship, while the remaining 61 were classified in this group based on seeing, on average, more than 12 ms patients per week . After excluding neurologists who did not respond to the questions for other specified dependent variables, ms subspecialists saw approximately 25 ms patients per week on average (median = 20). Other neurologists saw approximately three ms patients per week on average (median = 3). Ms subspecialists and other neurologists indicated substantial differences in attitudes regarding ms patient care (table 2). Ms subspecialists saw, on average, more ms patients per week if they indicated the following attitudes: enjoy interacting with ms patients (marginally significant), and personal connection to individuals with ms who are not your patients (marginally significant) (table 2). Ms subspecialists who began medical practice between 1972 and 1985 were also marginally more likely to see more ms patients per week than ms subspecialists who began practice later . Practice arrangement, physician age, sex, race, ethnicity, urban / rural practice area, and the remaining attitudes toward providing ms patient care were not significantly associated with number of ms patients seen (table 2). Among survey respondents in the other neurologist group, general neurologists saw significantly more ms patients per week than did non - ms subspecialists (4.4 versus 3.2, resp ., physicians in a neurology or multispecialty groups also saw more ms patients that did those in solo practice . Younger neurologists, female neurologists, those practicing for fewer years, and those based within a major city saw fewer ms patients . General neurologist and non - ms subspecialist respondents who indicated the following attitudes saw significantly fewer ms patients per week: lack of sufficient knowledge to feel comfortable caring for this patient population; lack of sufficient knowledge regarding newer disease - modifying drugs; and seldom encounter ms patients other neurologist respondents saw more ms patients if they agreed that ms patient care involved ability to improve patient outcomes and quality of life, dynamic area with evolving treatment options, (marginally significant). As with ms subspecialists, general neurologist and non - ms subspecialist respondents based within a major city saw fewer ms patients . Race, ethnicity, and the remaining attitudes toward providing ms patient care were not significantly associated with number of ms patients seen . We used negative binomial regressions (as presented in section 2) to examine the association of neurologist characteristics and attitudes toward ms care with the number of ms patients seen per week while controlling for other potentially associated factors . Analyses were performed separately for ms subspecialists (table 3) and general neurologists / non - ms subspecialists (table 4). All survey items that had significant or marginally significant associations with number of ms patients seen in bivariate analyses (table 2) were included as independent variables in these regression models . Among ms subspecialists, the only factor significantly associated with number of ms patients seen per week in regression analysis was the attitude enjoy interacting with ms patients; ms subspecialists who indicated this attitude had a rate of ms patients seen per week that was 1.6 times greater than did those not indicating this attitude . Ms subspecialists who began medical practice between 1986 and 1993 saw fewer ms patients than did those who had been practicing longer, although this difference was only marginally significant (p = 0.060). In regressions analyses of the other neurologists group, the rate of ms patients seen per week among neurologists in a university - based group was approximately 60% (0.59 times) the rate of ms patient seen among female other neurologists was approximately 80% the rate among males . The rates of ms patients seen were significantly lower for non - ms subspecialists / general neurologists who indicated lack of sufficient knowledge to feel comfortable caring for this patient population and seldom encounter ms patients . The rates of ms patients seen were significantly greater for other neurologists who agreed with the following attitudes toward ms patient care: ability to improve patient outcomes and quality of life, dynamic area with evolving treatment options, and enjoy interacting with ms patients . Among other neurologists practicing in rural areas or small cities, the rate of ms patients seen per week was 1.30 times greater than among those practicing within major cities; practicing in suburban areas or moderate - sized cities was marginally associated with seeing more ms patients . Figure 1 graphically presents results from table 4, illustrating the incidence rate ratios (irr) and 95% confidence intervals for the association of non - ms subspecialist / general neurologist characteristics and attitudes with the number of ms patients seen per week . The solid horizontal line indicates irr of 1.0, corresponding to no significant difference from the reference group for that characteristics or attitude . Those in university - based practices had a significantly decreased rate of seeing ms patients, while those in small city / rural practices had increased rates . Examining physician characteristics, female neurologist had significantly decreased rates of seeing ms patients . Finally, for attitudes towards ms patient care, lack of sufficient knowledge for ms care and seldom encounter ms patients were associated with decreased rates of ms patients seen while ability to improve outcomes / quality of life, dynamic area with evolving treatment options, and enjoy interacting with ms patients were associated with increased rates . To our knowledge, this is the first published study examining factors influencing the numbers of ms patients seen among a broad sample of us neurologists . Given the importance of neurologist care for ms [2, 3, 7], barriers to access neurologists could have substantial impacts on symptom control, disease progression, and quality of life among individuals with ms . Several studies have described barriers that individuals with ms may experience in receiving needed care . For example, ms patients in rural areas have been reported to have more limited access to ms subspecialists and related care services and had longer travel times than did patients in urban areas to receive specialized ms care [3, 8, 9]. Racial and ethnic disparities are also barriers to ms patient care: african americans are less likely to have been treated by an ms subspecialist; latinos are less likely to have received rehabilitation care compared to caucasians . These barriers to care may have direct consequences on patient outcomes; for example, individuals with ms residing in rural areas had significantly greater reductions in their physical components of health - related quality of life . We found that ms subspecialists responding to this study's survey saw approximately 25 ms patients per week, while other neurologists saw approximately 3.4 patients per week . It is not surprising that general neurologists and neurologists who subspecialize in clinical areas other than ms see, on average, fewer ms patients than do ms subspecialists . However, as the population of individuals with ms in the us continues to increase and only limited numbers of ms subspecialists (particularly outside of large urban / suburban locations) are available to serve ms patients, more ms patients may seek care from these other neurologists . The results presented in this study, providing information on how much care these other neurologists are currently providing for ms patients, may be useful for estimating the future capacity of the neurologist workforce for care for individuals with ms . Among ms subspecialists, we identified only one factor that was significantly associated with number of ms patients seen per week in multivariate regressions (table 3). Those who indicated that they enjoyed interacting with ms patients saw more individuals with ms . This may relate to burnout among ms subspecialists; that is, subspecialists who do not enjoy interacting with ms patients likely experience less enjoyment from providing medical care and instead may focus on other activities (e.g., teaching or administration) and see fewer individuals with ms . More factors were found to be significantly associated with numbers of ms patients seen among other neurologists (i.e., general neurologists and non - ms subspecialists) (table 4). Neurologists in university - based practices had significantly lower rates of seeing ms patients compared with the reference group, neurologists in solo practice . This may reflect the nonpatient care activities that are required of university neurologists (including teaching, research, and administration) and the likelihood that many of the other neurologists at universities specialized in care for patients with conditions other than ms . Several attitudes toward providing ms patient care were also associated with numbers of ms patients seen among the other neurologists . Respondents who indicated that they lack sufficient ms knowledge or seldom encounter ms patients saw fewer ms patients . These attitudes may be linked; that is, physicians who feel they lack sufficient knowledge may be less likely to have ms patients referred to them . Among physicians who feel they lack sufficient knowledge regarding ms care, in contrast, those who indicated the ability to improve ms patient outcomes and quality of life, considered ms a dynamic area with evolving treatment options, or indicated that they enjoy interacting with ms patients were more likely to see more ms patients . For example, given the recent (and expected future) development of new ms disease modifying therapies [1, 4], this is clearly a dynamic area with the ability to improve patient outcomes . Knowledge of these newer treatment options as well as familiarity with the benefits of multidisciplinary approaches to ms care will likely increase physician interest and enjoyment in providing care to ms patients . Educational programs to increase neurologist familiarity with ms care may strengthen these positive attitudes and increase access to care for ms patients . Improving general neurologists' knowledge of new treatment options may also enhance ms patient care, as general neurologists may be less likely to discuss newer treatment options than are ms subspecialists . Surprisingly, other neurologists who practice in small cities or rural areas were marginally significantly more likely to see more ms patients than were those practicing in major cities . This appears to contradict previous research findings that ms patients in rural areas have decreased access to care and are more likely to choose other providers (e.g., primary care providers) to direct their care [3, 8]. Ms patients in rural areas likely have few neurologists available; they may therefore be more likely to see neurologists who practice in their local area rather than going to farther away ms subspecialists . Thus, non - ms subspecialist neurologists outside of large urban areas may be more likely to see ms patients as there are no ms centers or ms subspecialists easily available to provide this care . Although the population of neurologists invited to participate in the survey was randomly selected from the relevant members of the aan, only 39% responded to the survey; this group may not be representative of u.s . All information provided by survey participants was by self - report; we did not attend to validate any responses . In addition, as with all surveys, we limited the number of items asked to minimize respondent burden . There are likely additional factors that influence the number of ms patients seen by both ms subspecialists and other neurologists that were not captured in our survey . Finally, as our survey data are cross - sectional (i.e., collected at one time point), we are unable to assess causality in the analysis results . For example, we can only determine that an attitude of enjoy interacting of ms patients is associated with seeing more ms patients; we cannot determine whether this attitude preceded initiation of providing ms patient care or whether the attitude was generated based on providing ms patient care (or both). Future studies could involve longitudinal data collection from a cohort of neurology residents, to examine changes in attitudes and subspecialization decisions over time to assess causal relationships in this physician population . Despite these limitations, this study provides important information on factors affecting numbers of ms patients seen among the neurologist workforce and identifies potential actions to increase access to care for this population . Prior studies have suggested current or impending shortages among neurologists . Among individuals with ms, who may require frequent interactions with neurologists, barriers to access care may affect symptom control, quality of life, and potentially even survival . Our results indicate that several factors are associated with decreased provision of ms patient care among other neurologists these factors include demographic and practice characteristics (female neurologists, those in university - based practices, and those practice in major cities were less likely to provide ms patient care) and attitudes towards ms care . The significant associations of attitudes towards ms care with number of ms patients seen by neurologists suggest that increasing neurologist knowledge about treatments for individuals with ms may decrease barriers to seeing more patients . To ensure optimal health outcomes among individuals with ms, it is critical both to gain better insights regarding factors influencing provision of ms patient care by neurologists and to develop policies that ensure appropriate access to high - quality ms care.
All bacterial strains recovered from blood were identified from available microbiology department records of all 13 hospitals in san francisco county from january 1, 1996, to march 31, 1999 . For three hospitals (4,5,9), data from 1996 had been purged and were no longer available . For each isolate, information was also obtained on the ward, age, and gender of the patient . Only the first positive blood culture of a given species was included for a single patient throughout the study period, regardless of susceptibility pattern . Cultures positive for s. epidermidis were considered representative of clinical bacteremia if at least two isolates with identical susceptibility patterns were obtained from a minimum of two separate sets of blood cultures . All other cultures positive for s. epidermidis were excluded, as were other common skin contaminants (e.g., propionibacterium acnes, peptostreptococcus, corynebacterium). All but one hospital used automated systems (vitek [biomerieux vitek, hazelwood, mo] or microscan [baxter laboratories, west sacramento, ca]) for the susceptibility testing of gram - negative bacteria . All hospitals used kirby - bauer disk - diffusion techniques for the evaluation of susceptibility profiles for streptococcus pneumoniae and other streptococcal species according to national committee for clinical laboratory standards (nccls) guidelines . One hospital (12) performed all susceptibility testing using kirby - bauer disk - diffusion techniques . All microbiology laboratories used mic breakpoints established by the nccls . The annual number of blood culture sets processed by each microbiology laboratory was also obtained . Data from five hospitals were obtained as text files or microsoft excel (redmond, wa) files and subsequently imported into a microsoft access (redmond, wa) database . Data from four hospitals were obtained in printed form, scanned as image files, converted into text files using textbridge pro 98 (scansoft inc . Data from the remaining four hospitals were obtained from stored index cards, entered manually into the database, and verified for accurate entry . We also obtained bed size and census data for all 13 hospitals in san francisco county . Total hospital admissions were tabulated from quarterly administrative records from 1996 through 1998 . For each hospital, the number of isolates for each species was tabulated for each year and for the entire study period . Only species for which the total number of countywide isolates exceeded 100 during the study period was calculated as the yearly number of organisms with intermediate or full resistance divided by the total number of organisms isolated in san francisco county that year . Because of laboratory variability in susceptibility testing, not all isolates are included in descriptions of proportional resistance . Means were calculated from the annual countywide percentages in each of the 4 years studied . Percent annual resistance was determined for any antibiotic tested in 50 isolates and analyzed for increasing or decreasing annual trend from 1996 through 1999 . Strains with full or intermediate resistance to an antibiotic were counted as resistant in all statistical analyses . Organisms demonstrating increasing or decreasing annual resistance to a given antibiotic (p<0.05) were further described by calculating mean proportional resistance over the study period according to categories of hospital, ward, patient age (in 10-year intervals), and patient gender . Spearman rank tests were used to determine any correlation between hospital indices (beds, annual admissions, average length of stay) and proportional antibiotic resistance . P values remained unchanged with respect to alpha level (0.05) after removal of the three hospitals missing data from 1996 . The number of isolates for each species was tabulated for each year and for the entire study period . Only species for which the total number of countywide isolates exceeded 100 during the study period was calculated as the yearly number of organisms with intermediate or full resistance divided by the total number of organisms isolated in san francisco county that year . Because of laboratory variability in susceptibility testing, not all isolates are included in descriptions of proportional resistance . Means were calculated from the annual countywide percentages in each of the 4 years studied . Percent annual resistance was determined for any antibiotic tested in 50 isolates and analyzed for increasing or decreasing annual trend from 1996 through 1999 . Strains with full or intermediate resistance to an antibiotic were counted as resistant in all statistical analyses . Organisms demonstrating increasing or decreasing annual resistance to a given antibiotic (p<0.05) were further described by calculating mean proportional resistance over the study period according to categories of hospital, ward, patient age (in 10-year intervals), and patient gender . Spearman rank tests were used to determine any correlation between hospital indices (beds, annual admissions, average length of stay) and proportional antibiotic resistance . P values remained unchanged with respect to alpha level (0.05) after removal of the three hospitals missing data from 1996 . A total of 11,573 bacterial strains were recovered from blood cultures by the 13 hospitals . After excluding duplicate cultures, we had 8,072 remaining clinical isolates . Information on hospital size, census, and blood culture volume is provided in table 1 . Despite being distinct and nonadjoining, 74,600 sets of blood cultures were processed each year in san francisco county; 9.9% of these were positive for bacterial species . Staphylococcus aureus (1,858), escherichia coli (1,634), and s. pneumoniae (725) were the most common organisms . The numbers of s. aureus, enterococcus faecalis, bacteroides fragilis, e. coli, and serratia marcescens increased annually during the 4-year period . Fourteen species had> 100 isolates and were considered for further analysis (figure 1). All bacterial species that were isolated from blood in> 100 persons, january 1996 through march 1999, all hospitals in san francisco county, california . The proportion of mrsa and e. faecium resistant to vancomycin (vre faecium) increased annually (figure 2). Countywide, the proportion of mrsa isolates rose from 18.1% (1996) to 26.1% (1999) (p<0.001). In total, mrsa constituted 22.4% of all s. aureus isolates, including 19.6% of emergency department isolates and 15.9% of isolates in the outpatient setting (table 2). Methicillin resistance in s. aureus isolates was <15% in patients <20 years of age and> 20% in all other age groups, with the exception of 30- to 39-year - olds (17.6%) (table 3). Increase in percentage of staphylococcus aureus isolates resistant to methicillin and increase in percent of enterococcus faecium resistant to vancomycin on a yearly basis from 1996 through the first quarter of 1999 . Proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic . Proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic . Percentages for small numbers of total isolates should be cautiously interpreted . During the study period, 124 vre isolates and 157 vancomycin - sensitive enterococcus isolates were unspeciated . Among the speciated e. faecium isolates, the percentage resistant to vancomycin rose from 0% to 66.7% in the 4-year period (p<0.001). Vre faecium was most frequently isolated from inpatient adult wards, but six isolates were cultured from emergency department and outpatient settings . Over 66% of e. faecium isolates from skilled nursing facilities were resistant to vancomycin (table 2). Vre isolates exceeded 40% in all age groups with the exception of 10-year - olds (22.2%) (table 3). Of note, vre faecium isolates showed increasing annual resistance to doxycycline (from 30% to 68%; p = 0.02). Even after the skilled nursing facility (hospital 13) was excluded, the percentage of mrsa and vre faecium isolates varied substantially among individual hospitals (mrsa 12.5% to 37.5%, vre faecium 12.5% to 80.0%). There was no correlation between proportional resistance and number of icu or total hospital beds, number of annual admissions, or average length of icu or total hospital stay . However, for both organisms, there was increasing proportional resistance among adult wards in the following order: outpatient wards, emergency department, medical and surgical floors, medical and surgical icus, and skilled nursing facility wards . Notably, a substantial number of vre and mrsa isolates were cultured within the first 24 to 48 hours of hospital admission (figure 3). Plot of the number of methicillin - resistant staphylococcus aureus (mrsa) and vancomycin - resistant enterococcus (vre) isolates by hospital day of admission . An early peak is noted, corresponding to patients entering the hospital with mrsa or vre bacteremia . Proportional resistance increased from 10.8% (21 isolates) in 1996 to 14.6% (27 isolates) in 1998, but this increase was not statistically significant . Proportional resistance was highest at the extremes of age (16.4% in patients> 70 years of age and 19.3% in patients <10 years of age (table 3). Countywide, e. coli (1,634 isolates), klebsiella pneumoniae (428 isolates), and pseudomonas aeruginosa (260 isolates) were the most frequently isolated gram - negative bacilli . This was true in all inpatient and outpatient wards with the exception of skilled nursing facilities, where proteus mirabilis was the most common gram - negative isolate after e. coli . Proportional resistance by ward for selective gram - negative organisms is shown in table 4 . Icu = intensive care unit among e. coli isolates, resistance to trimethoprim - sulfamethoxazole averaged 28% and resistance to ciprofloxacin averaged 3% . Increasing annual resistance to ticarcillin - clavulanate was seen in both e. coli (6% to 16%, p = 0.03) and k. pneumoniae (0% to 18%, p = 0.007) isolates . P. aeruginosa isolates showed increasing annual countywide resistance to ciprofloxacin (7% to 21%, p = 0.005), ceftazidime (6% to 16%, p = 0.02), and imipenem (2% to 18%, p = 0.004) (figure 4). Resistance to each of these three antibiotics exceeded 10% in adult icu and adult medical and surgical wards . Resistance to gentamicin (15%) and piperacillin - tazobactam (12%) also increased but was not statistically significant . Yearly percent resistance to ciprofloxacin, ceftazidime, imipenem, and piperacillin in pseudomonas aeruginosa isolates from blood . Increasing proportional resistance occurred in three of the four antibiotics commonly used to treat this organism . Annual number of isolates tested to each antibiotic is given at the top of each graph . There were 182 e. cloacae and 116 s. marcescens isolates from january 1996 through march 1999 . S. marcescens isolates also showed increasing annual proportional resistance to gentamicin (0% to 14%, p = 0.02) and piperacillin (4% to 29%, p = 0.01). Resistance to ceftazidime, which can be predictive of inducible and extended - spectrum - beta - lactamases, was found in the following overall mean proportions in the study period: e. coli (1%), p. mirabilis (1%), k. pneumoniae (1%), s. marcescens (8%), p. aeruginosa (13%), and e. cloacae (39%). Only p. aeruginosa isolates demonstrated an increasing linear annual trend (p = 0.02). The proportion of mrsa and e. faecium resistant to vancomycin (vre faecium) increased annually (figure 2). Countywide, the proportion of mrsa isolates rose from 18.1% (1996) to 26.1% (1999) (p<0.001). In total, mrsa constituted 22.4% of all s. aureus isolates, including 19.6% of emergency department isolates and 15.9% of isolates in the outpatient setting (table 2). Methicillin resistance in s. aureus isolates was <15% in patients <20 years of age and> 20% in all other age groups, with the exception of 30- to 39-year - olds (17.6%) (table 3). Increase in percentage of staphylococcus aureus isolates resistant to methicillin and increase in percent of enterococcus faecium resistant to vancomycin on a yearly basis from 1996 through the first quarter of 1999 . Proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic . Proportional resistance refers to the proportion of isolates of that species that is resistant to the indicated antibiotic . Percentages for small numbers of total isolates should be cautiously interpreted . During the study period, 124 vre isolates and 157 vancomycin - sensitive enterococcus isolates were unspeciated . Among the speciated e. faecium isolates, the percentage resistant to vancomycin rose from 0% to 66.7% in the 4-year period (p<0.001). Vre faecium was most frequently isolated from inpatient adult wards, but six isolates were cultured from emergency department and outpatient settings . Over 66% of e. faecium isolates from skilled nursing facilities were resistant to vancomycin (table 2). Vre isolates exceeded 40% in all age groups with the exception of 10-year - olds (22.2%) (table 3). Of note, vre faecium isolates showed increasing annual resistance to doxycycline (from 30% to 68%; p = 0.02). Even after the skilled nursing facility (hospital 13) was excluded, the percentage of mrsa and vre faecium isolates varied substantially among individual hospitals (mrsa 12.5% to 37.5%, vre faecium 12.5% to 80.0%). There was no correlation between proportional resistance and number of icu or total hospital beds, number of annual admissions, or average length of icu or total hospital stay . However, for both organisms, there was increasing proportional resistance among adult wards in the following order: outpatient wards, emergency department, medical and surgical floors, medical and surgical icus, and skilled nursing facility wards . Notably, a substantial number of vre and mrsa isolates were cultured within the first 24 to 48 hours of hospital admission (figure 3). Plot of the number of methicillin - resistant staphylococcus aureus (mrsa) and vancomycin - resistant enterococcus (vre) isolates by hospital day of admission . An early peak is noted, corresponding to patients entering the hospital with mrsa or vre bacteremia . Proportional resistance increased from 10.8% (21 isolates) in 1996 to 14.6% (27 isolates) in 1998, but this increase was not statistically significant . Proportional resistance was highest at the extremes of age (16.4% in patients> 70 years of age and 19.3% in patients <10 years of age (table 3). Countywide, e. coli (1,634 isolates), klebsiella pneumoniae (428 isolates), and pseudomonas aeruginosa (260 isolates) were the most frequently isolated gram - negative bacilli . This was true in all inpatient and outpatient wards with the exception of skilled nursing facilities, where proteus mirabilis was the most common gram - negative isolate after e. coli . Proportional resistance by ward for selective gram - negative organisms is shown in table 4 . Icu = intensive care unit among e. coli isolates, resistance to trimethoprim - sulfamethoxazole averaged 28% and resistance to ciprofloxacin averaged 3% . Increasing annual resistance to ticarcillin - clavulanate was seen in both e. coli (6% to 16%, p = 0.03) and k. pneumoniae (0% to 18%, p = 0.007) isolates . Aeruginosa isolates showed increasing annual countywide resistance to ciprofloxacin (7% to 21%, p = 0.005), ceftazidime (6% to 16%, p = 0.02), and imipenem (2% to 18%, p = 0.004) (figure 4). Resistance to each of these three antibiotics exceeded 10% in adult icu and adult medical and surgical wards . No isolates resistant to ciprofloxacin were cultured from pediatric wards (table 4). Resistance to gentamicin (15%) and piperacillin - tazobactam (12%) also increased but was not statistically significant . Yearly percent resistance to ciprofloxacin, ceftazidime, imipenem, and piperacillin in pseudomonas aeruginosa annual number of isolates tested to each antibiotic is given at the top of each graph . There were 182 e. cloacae and 116 s. marcescens isolates from january 1996 through march 1999 . S. marcescens isolates also showed increasing annual proportional resistance to gentamicin (0% to 14%, p = 0.02) and piperacillin (4% to 29%, p = 0.01). Resistance to ceftazidime, which can be predictive of inducible and extended - spectrum - beta - lactamases, was found in the following overall mean proportions in the study period: e. coli (1%), p. mirabilis (1%), k. pneumoniae (1%), s. marcescens (8%), p. aeruginosa (13%), and e. cloacae (39%). Only p. aeruginosa isolates demonstrated an increasing linear annual trend (p = 0.02). San francisco county has a population of approximately 735,000 and covers 46.7 square miles (26). It comprises multiple racial and ethnic groups (black 10.9%, hispanic 13.9%, asian 29.1%, and native american 0.5%) and is served by 13 hospitals . We have shown that county surveillance of bacterial resistance is a useful addition to local hospital surveillance, particularly as antibiotic resistant bacteria increasingly spread from hospital to hospital and into the community at large . Across the county, annual proportions of mrsa and vre isolates significantly increased over the 4-year period . These data allow us to distinguish countywide outbreaks and trends from single - hospital changes in resistance patterns, and enable infection control efforts to expand or narrow to the appropriate scale . With awareness programs, county surveillance can broaden physicians knowledge of their hospital s effects on the community, as well as the effects of neighboring hospitals on resistance patterns in their particular hospital . Additionally, county surveillance that includes subcategorization of isolates by ward is invaluable in identifying patients at high risk and locations for transmission of resistant bacteria . Not surprisingly, we report our highest proportion of mrsa and vre isolates from icu and nursing home units . Large interhospital differences can lead to further study of ward practices that foster or abate transmission . Awareness can prompt hospital infection control personnel to ensure well - described preventive measures such as swabbing and isolation precautions for vre and mrsa in icu settings (2730) and nasopharyngeal swabbing and eradication of mrsa in hemodialysis wards (1,31). Whether or not these represent true community - acquired strains or strains from patients recently released from hospital settings, they suggest that highly resistant bacterial outpatient infections and infectivity are increasing, a result consistent with recent studies (19,21,22). We also evaluated whether the wide variability in the proportions of resistant bacteria among san francisco hospitals was linked to hospital indices . In contrast to previous nationwide sampling studies, none of this variability was correlated with the number of hospital icu beds (3), total beds (1,2,31), annual admissions (32), or annual mean length of stay . This may be due to our small number of hospitals, leading to limited power to detect such correlations . Alternatively, local community and hospital factors (e.g., increasing care of moderately ill patients at home, increasing home intravenous antibiotics, active transfer of patients between hospitals, and community - acquired resistant organisms) may now be diminishing the effect of hospital size, census and length of stay on proportional resistance . The 28% trimethoprim - sulfamethoxazole resistance in e. coli raises questions about the optimal empiric treatment of urinary tract infections in patients at high risk for bacteremia or urosepsis . Likewise, distinguishing resistance in the outpatient versus inpatient setting can guide empiric therapy in the appropriate setting . Second, in studying a larger populace, we can obtain sufficient numbers to study uncommon organisms . Similarly, countywide surveillance provides a means to identify and confirm novel resistant pathogens . In our study, one mrsa isolate with intermediate resistance to vancomycin and three vancomycin - resistant s. epidermidis isolates were noted in microbiology laboratory reports . As with the organisms recently reported in the united states (3537), these were reported from nontertiary hospitals . Nevertheless, our reports are unconfirmed and likely represent laboratory error . However, if surveillance could be expedited to real - time use, such reports could be investigated and confirmed rather than dismissed . At its worst, third, countywide surveillance engenders further hypotheses and research regarding interhospital and community transmission of resistant organisms . For example, our finding that 20% of emergency department s. aureus isolates are methicillin - resistant provides a flag to further study which county areas have the highest percentages of resistant s. aureus and which risk factors are involved (e.g., recent hospital admission, associated hemodialysis centers or nursing homes, or intravenous drug use). The finding that p. mirabilis bacteremia is more common in nursing home wards raises questions about preventing urinary tract infections in that setting . The relative lack of fluoroquinolone resistance in pediatric gram - negative isolates is likely due to the avoidance of fluoroquinolones in children because of potential detrimental cartilage effects . The study of fluoroquinolone - resistant organisms during the transition years from pediatric to adult medicine may provide insight into the quantity and duration of antibiotic needed to produce selection pressure, as well as the speed and durability of emerging resistance . Chart review would have been an invaluable addition in distinguishing between community - acquired and recent hospital or nursing home acquisition of resistant organisms . Second, we did not collect or confirm isolates; thus, although our results reflect microbiologic data actually presented to ordering physicians, they are subject to laboratory differences in speciation and susceptibility determination . Notably, not all organisms were fully speciated or tested against the antibiotics of interest . Third, we do not provide information on antibiotic use, which is known to be a major determinant of bacterial antibiotic resistance . Fourth, countywide trends can be driven by trends seen in the largest hospitals, particularly since smaller hospitals often lack sufficient numbers of isolates to make statistical analyses meaningful . This was notable for our data on proportional increases in vre, which was largely driven by three hospitals . On the contrary, mrsa trends were not limited to a few hospitals, nor were they limited to the largest hospitals in the county . This would expedite surveillance and allow real - time collection and identification of unusually resistant organisms, as well as provide sentinel data regarding countywide outbreaks . In addition, linking of patient information to microbiologic data would have expedited acquisition of sex, gender, and ward information . Furthermore, despite nccls guidelines, a fair amount of variability exists in laboratory practices and susceptibility panels . Further standardization of these practices would help ensure the reliability of merging data among hospitals . Without a doubt, the greatest utility of countywide surveillance lies in its ability to ask screening questions that prompt a more thorough investigation of specific hospitals, wards, or age groups at particular risk for acquiring or transmitting highly resistant organisms . We have shown how several such questions were raised by our surveillance of bacteremias in san francisco county and described its many advantages . We have further defined our limitations and difficulties in performing such surveillance, in the hope that this will be helpful to further similar surveillance efforts.
The difficulty with evaluating an intervention once it has become part of established practice is that, like servicing a car in motion, the method is inconvenient and the results unreliable . Moreover, the intervention is likely to have acquired both adherents and detractors, thereby ensuring maximal uncertainty while impairing individual equipoise . We are left with performing retrospective observational before - and - after studies, relying on large numbers to minimise confounding . Outcomes were obtained from the australia and new zealand intensive care society (anzics) database . From a pool of 172 australia and new zealand hospitals, the presence or absence of a medical emergency team (met) could be determined in 131, of which 84 (64%) had established an met . Of the 84 hospitals with an met, 24 provided adequate data to the anzics database to determine the number and rate of intensive care unit (icu) admissions following an in - hospital cardiac arrest, and the proportion of icu readmissions, one year before and one year after implementing the met . Comparisons were also possible with some of the hospitals that had participated in the medical emergency response improvement team (merit) study, the only prospective multicentre cluster - randomised study available . The authors found a reduction in the number and rate of post - cardiac arrest icu admissions for both the 47 met hospitals and 16 non - met hospitals during the two - year period . Icu readmission rates were unchanged, and there was no reduction in hospital mortality for either group . These findings are consistent with the merit study, which found that adverse outcomes improved in both the met and non - met hospitals . The authors suggest that the introduction of mets has been driven not by evidence of efficacy but by evidence of suboptimal care of acutely ill patients in hospital and an assumption that pre - emptive intensive care would either save lives or permit a dignified death . Should we wait 25 years before thrombolysis becomes the established best practice in guidelines and textbooks? Or should we follow nike's approach and' just do it'? This was what happened with mets, now referred to generically as rapid response teams (rrts). At first sight, the concept would seem to be an eminently sensible response to the problem of suboptimal care of acutely ill hospitalised patients: you take critical care expertise to the patient before, rather than after, multiple organ failure or cardiac arrest occurs . Why do we need evidence that neglect or inexpertise should be replaced by timely competent care? The answer to this question is as complex as the system to which the intervention has been applied . For some intensivists, the met was an unnecessary intrusion into the service they were already providing . For others others were concerned that it would just shift the burden of illness even more onto a service that could not cope . Ward staff could be positive, or suspicious;' deskilling' was a common phrase, although it was probably removing responsibility for patient care rather than skills that may never have existed in the first place . The problem is that the health care systems that funded mets did not want to spend additional money on finding out whether they were effective, and those of us involved from the beginning were unable at that time to persuade research funders otherwise . First, the main problem surrounding the entire literature on rrts is that the publications never specify the content of the intervention . An rrt is not an intervention: it is a vehicle for an intervention such as sepsis bundles or early antibiotics or a do - not - resuscitate (dnr) order or (perhaps even more importantly) education . As in the (similarly negative) pac - man study, the use to which the tool is put is largely unknown . Given the diffusion of best practice, we can be reasonably confident that non - met hospitals will also have doctors and nurses who want to provide good care and avoid burdensome futile care . Second, should we use process or outcome measures for evaluation? Processes of care are important not only because they may alter outcomes (the destination), but because they can change the way in which that outcome is achieved (the pathway). End - of - life care is the obvious example . Last week, our outreach nurses called one of us to an acute ward because a junior doctor was unwilling to implement a dnr order for a terminally ill 91-year - old man . We obtained full consensus on treatment limitation, provided comfort care, and contacted the patient's son, who then spent the few remaining hours by his father's bedside before death supervened . The alternative scenario would almost certainly still have resulted in a ward death, but without dignity and with less emotional resolution for the son . Third, it may be unwise to assume that the context in which the intervention is applied is similar across all hospitals . A well - staffed hospital with excellent senior staff relationships and teamworking might find little benefit from an rrt, whereas in another the converse conditions might prevent an rrt from having any effect on outcomes . Differences in patients' severity of illness could confound the results: further refinements of ward - based measures of severity of illness might help in this respect . Nurse - led, doctor - supported outreach care has transformed the way in which we in the uk provide support for acutely ill patients in ordinary wards in terms of relieving some of the workload on intensive care doctors, supporting timely delivery of care, improving pain relief and end - of - life care, enhancing communication, and teaching ward nurses and doctors . These qualitative aspects may not be reflected in immediate changes in mortality or icu readmissions, but they may still be very important to patients . We are aware of john galbraith's statement that' faced with the choice between changing one's mind and proving that there is no need to do so, almost everyone gets busy on the proof' . But perhaps the real problem is that we have not yet properly defined the content of the intervention, the context in which it is applied, or the research question . Anzics = australia and new zealand intensive care society; dnr = do not resuscitate; icu = intensive care unit; merit = medical emergency response improvement team; met = medical emergency team; rrt = rapid response team . Jfb declares an academic bias towards favouring outreach care as an effective method of improving the quality of acute care.
Bronchial asthma is a chronic disorder characterized by airway inflammation, remodeling of the airways, and their hyperresponsiveness to environmental stimuli . It is accompanied by thickening of the bronchial walls, epithelial damage, subepithelial fibrosis, and the increased deposition of extracellular matrix proteins . The bulk of these changes results from the phenotypic transitions of bronchial epithelial cells, smooth muscle cells, and fibroblasts [1, 2]. In general, phenotypic shifts, such as epithelial - mesenchymal transition (emt) and fibroblast - myofibroblast transition (fmt), in particular, erroneous function of myofibroblasts, representing the contractile subset of fibroblasts which have undergone fmt, was found to be crucial for the development of asthma [6, 7]. Fmt, which is also involved in wound contraction and participates in the development of fibrotic changes in several tissues [810], can be induced by a range of proinflammatory cytokines, in particular by factors belonging to the transforming growth factor (tgf-) family [11, 12]. On the other hand, an increase in the local concentration of tgf- was observed in bronchoalveolar lavage fluid sampled from asthmatic patients, and prolonged exposure to tgf- effectively induced fmt in hbfs in vitro models . It is generally accepted that tgf- regulates transcription of profibrotic genes, including cell adhesion molecules and extracellular matrix proteins [15, 16]. The canonical tgf- signaling pathway depends on the phosphorylation of r - smad proteins (smad2 and 3) by activated tgf- receptor, their interaction with smad4, and accumulation in the nuclei [17, 18]. Tgf- signaling activity is modulated by other signaling systems, such as the wnt - dependent pathway [19, 20], which has also been implicated in the pathophysiology of lung cancers, asthma, and lung fibrotic disease development [2125]. Tgf- was shown to induce fmt in human lung fibroblast populations; however, it attenuated the allergic inflammation and asthmatic symptoms in an ovalbumin - induced mouse model of asthma . Activation of wnt pathway results in the inhibition of gsk-3 (glycogen synthase kinase 3 beta) and subsequent cytoplasmic accumulation of -catenin . It forms complexes with transcription factors of the lef and tcf family activating the expression of wnt - responsive genes . A range of interactions between wnt and tgf- effectors have been identified, which conceivably account for synergic effects of tgf- and wnt signaling observed in vitro during fmt in mouse embryonic fibroblast populations . For instance, mapk can phosphorylate both r - smads and gsk-3 [25, 26], while -catenin was found to interact with i - smads . These interactions may also underline cellular context - dependent role of gsk-3 activity in asthma development . Lithium is a nonspecific gsk-3 inhibitor widely used in the therapy of bipolar affective disorder and other psychiatric diseases . Interestingly, lithium treatment of schizoaffective disorders coincided with the remission of asthmatic symptoms, whereas the recurrence of symptomatic asthma was observed after discontinuation of lithium therapy . Moreover, treatment with lithium led to a reduction in bronchial hyperresponsiveness in asthmatic patients . We have previously shown that hbfs derived from patients with asthma displayed more pronounced fmt in response to tgf- in comparison with nonasthmatic counterparts . This indicates susceptibility towards a tgf--induced phenotypic switch, an event crucial for the asthmatic process [14, 34]. In the current study, we aimed to elucidate the role of lithium compounds in tgf-1-induced fmt in hbf populations and the mechanisms responsible for this process . We demonstrate inhibition of gsk-3 activity by lithium attenuated tgf-1-induced fmt in hbf populations derived from asthmatic donors . This effect correlated with deficient nuclear localization of -catenin and p - smad2 in tgf-1/licl - stimulated hbfs . Bronchial biopsies were obtained from 11 patients with bronchial asthma (as) and 11 non - asthmatic subjects (na) during fiberoptic bronchoscopy performed in the department of medicine (jagiellonian university medical college). Primary hbf were isolated from bronchial biopsies as described previously [14, 34]. The first group consisted of patients with moderate to severe bronchial asthma (as group) with a mean age of 43.1 9.5 years (females 64%), the average duration of the disease was 11.3 9.4 years, and decrease in the first second forced expiratory volume (fev1: 78.4 22.4% of predicted). The second group comprised 11 subjects in whom diagnostic bronchoscopy ruled out any serious airway pathology, including asthma, chronic inflammatory lung disease, or cancer (nonasthmatics; na group), with mean age of 58.7 7.8 years (females 36%) and normal lung function tests (fev1: 97.3 12.8% of predicted). The study was approved by the jagiellonian university's ethics committee (kbet/362/b/2003), and informed consent was obtained from all study participants . Hbfs were cultured in dmem with 10% fetal bovine serum (fbs) at 37c in a humidified atmosphere with 5% co2 and used between 5 and 15 passages . For experiments, cells were plated at a density of 5000 cells / cm and cultured in serum - free dmem supplemented with 0.1% bovine serum albumin (bsa; sigma - aldrich, st . Louis, mo, usa). When indicated, human recombinant tgf-1 (bd biosciences, franklin lakes, nj, usa), lithium chloride (licl; sigma - aldrich) or their cocktail, and gsk-3 inhibitor xii, tws119 (calbiochem, la jolla, ca, usa) were administered 24 h after cell seeding . Lithium chloride was administered at the concentration of 10 mm, which remained in a range of serum concentrations observed in patients subjected to the chronic therapy of bipolar affective disorder [35, 36]. Myofibroblasts were identified by immunodetection of -sma as described previously . In brief, cells growing on glass coverslips were fixed in 3.7% paraformaldehyde, permeabilised in 0.1% triton x-100, blocked with 1% bsa, and incubated with a mouse monoclonal antibody against human -sma (clone 1a4, sigma - aldrich) and alexa fluor 488 goat anti - mouse igg (clone a11001, sigma - aldrich). Visualization of specimens mounted in polyvinyl alcohol (mowiol; sigma - aldrich) was performed with a leica dm ire2 microscope equipped with 40x, na-1.25 hcx plan apo objective, leica dc350fx camera, and leica fw4000 software . A similar protocol was used for immunodetection of other cellular antigens including tgf- receptor ii (with a mouse monoclonal antibody against tgf--rii, clone mm0056 - 4f14, abcam, cambridge, uk), -catenin (with a mouse monoclonal antibody against -catenin, sigma - aldrich) or p - smad (with a rabbit polyclonal antibody against smad2, phospho s467, abcam), and compatible secondary antibodies conjugated with alexa fluor 488 or alexa fluor 546 (all from invitrogen, carlsbad, ca, usa). When indicated, nuclei were stained with hoechst 33342 (sigma - aldrich). For the analyses of tgf- receptor ii expression on the surface of hbf, confluent cell monolayers were nonenzymatically detached using the cell dissociation solution (sigma - aldrich) according to the manufacturer's protocol, centrifuged, fixed and permeabilized with cytofix / cytoperm kit (bd biosciences), and stained with primary mouse monoclonal anti - tgf- receptor ii antibody (abcam). After washing with perm - wash buffer (bd biosciences), cell suspensions were stained with secondary fitc - conjugated goat anti - mouse igg (sigma - aldrich) and analysed by flow cytometry (coulter epics xl, beckman coulter, fullerton, ca). Results were expressed as a ratio of mean fluorescent intensity (mfi) of tgf--rii - specific staining to mfi of the corresponding goat - igg2a isotype control sample (expression index). Hbfs cultured in 6 cm petri dishes for 7 days (until confluency) in control conditions or stimulated with tgf-1 (5 ng / ml), licl (10 mm) and their mixture (tgf-1/licl), or tws119 (5 m) were washed, harvested using a cell scraper, centrifuged, and dissolved in lysis buffer (50 mm tris - hcl ph 7.4, 150 mm nacl, 2% tritonx-100, 0.02% nan3 with the addition of a proteinase inhibitor). Alternatively, total protein was isolated using m - per buffer (pierce, rockford, il, usa) with protease and phosphatase inhibitors (sigma - aldrich), and the nuclear fraction was isolated using neb - b buffer (29 mm hepes ph 7.9; 0.4 m nacl; 1 mm edta; 1 mm egta). In both cases, cellular proteins were separated on 15% sds - polyacrylamide gels and transferred to nitrocellulose membranes . After blocking in pbs - t (0.1% tween 80 in pbs containing 5% skimmed milk), membranes were incubated with mouse monoclonal antibodies (all from sigma - aldrich), anti--sma (1: 1000), anti--catenin (1: 2000), anti--tubulin (1: 1000), anti - gapdh (1: 10000), and rabbit polyclonal antibodies anti - human retinoblastoma - associated protein 46 (rbap46, 1: 2000). After washing, membranes were incubated for 1 hour with horseradish peroxidase - conjugated anti - mouse igg (1: 3000, invitrogen) or anti - rabbit igg (1: 3000, invitrogen) antibodies, treated with the chemiluminescent reagent super signal west pico substrate (pierce, rockford, il) and exposed to kodak x - omat film (sigma - aldrich). We have previously demonstrated that hbfs derived from patients with bronchial asthma display inherent features in vitro that facilitate fmt in response to prolonged incubation with tgf-1 [14, 34]. Here, we aimed to investigate the interference of lithium with tgf-1-induced fmt in hbf populations derived from asthmatic patients (as, n = 8) and non - asthmatic subjects (na, n = 8). Therefore, we first quantified -sma - positive cells in hbf populations which had undergone long - term tgf-1 stimulation . We have found a considerably higher fraction of -sma expressing hbfs in as as compared to na populations (figure 1(a)) which indicates their propensity towards a tgf-1-induced phenotypic switch . This effect was independent of the total density of tgf- receptor as both immunofluorescence microscopy and flow cytometry analyses did not reveal any significant differences in expression levels between as and na patients (figure 1(b)). Furthermore, cytoplasmic levels of p - smad2 protein, an effector of tgf- signalling, and its nuclear localisation were increased in tgf-1-treated hbf populations, both in na and as groups . However, we observed a slightly more pronounced nuclear accumulation of p - smad2 in tgf-1-stimulated asthmatic hbf populations (figure 1(c)). These results suggest that mechanisms downstream of the tgf- receptor which modulate nuclear transport of smad protein(s) determine the differences in tgf-1 reactivity between hbf derived from asthmatic and non - asthmatic patients . It has been found that gsk-3-dependent pathways interact with tgf- signaling in a variety of cellular systems [37, 38]. Similarly, lithium which at millimolar concentrations displays gsk-3 inhibiting activity, has been shown to interfere with the asthmatic process [32, 33]. Application of the experimental system based on quantification of fmt in hbf populations derived from as and na patients enabled us to elucidate the effect of cellular context on fmt - related cell reactivity to lithium (figure 2). We have not observed any significant differences in the viability and percentage of myofibroblasts between untreated (control) and treated by licl alone in hbf cultures, regardless of the source of cells (data not shown). In contrast to tgf-1-treated hbf from the na group, which reacted to the administration of lithium with the induction of fmt, as hbfs treated with tgf-1/licl displayed a decrease in the fraction of myofibroblasts compared to the cells stimulated with tgf-1 alone (43 to 85% of -sma positive cells in the presence of tgf-1 compared to 1542% in tgf-1/licl mixture; figure 2(b)). These data indicate that increased susceptibility of as hbfs to tgf-1-induced fmt is associated with a differential response to licl as compared to na and suggest the presence of a possible disturbance in crosstalk between signalling pathways regulated by tgf-1 and gsk-3. To check whether the attenuation of tgf-1-induced fmt in as hbf populations treated with lithium specifically results from its inhibitory effect on gsk-3 activity, we further treated the cells with tws-119, a specific gsk-3 inhibitor . Its effect on the number of myofibroblasts in tgf-1-treated na and as hbf populations was found to be similar to that exerted by lithium (figure 3) indicating the involvement of gsk-3 activity in the observed phenomena . Moreover, in na hbf populations treated with tgf-1/licl or tgf-1/tws-119, the percentage of -sma - positive cells corresponded to -sma protein levels (compare figure 2 to figure 4(a) and figure 3 to figure 4(b)). On the other hand, different results were obtained for as hbf populations . Despite lower numbers of -sma - positive cells, the same amounts of -sma this effect was not observed in tgf-1/tws-119-treated as hbf populations (figure 4(b)). This can be explained in terms of gsk-3-unspecific induction of supermaturation and hypertrophy of a subset of as hbf subjected to tgf-1/licl treatment . Such a sparse subpopulation characterised by a high degree of spreading and intense -sma - specific staining was found in as hbf populations treated with the tgf-1/licl mixture (data not shown). Moreover, it should be noticed that the effect of gsk-3 inhibition on fmt is much more striking when comparing the percentage of myofibroblasts to the -sma protein levels . The reason of it could be that not all expressed -sma is incorporated into stress fibers which are the marker of myofibroblasts . Altogether, these data indicate that gsk-3 participates in the attenuating effect of lithium on tgf-1-induced fmt in as hbf populations; however, lithium may exert a gsk-3-unspecific effect on the maturation of myofibroblasts . Inhibition of gsk-3 activity results, among others, in the hypophosphorylation of -catenin and its nuclear translocation leading to the activation of gene expression . Therefore, we further focused on the effect of lithium and tws119 on -catenin levels in na and as hbf populations stimulated by tgf-1 . Interestingly, as hbfs are characterized by a notably higher amount of total -catenin (figure 5) and -catenin cytoplasmic fraction (figure 6). Administration of tgf-1/licl and tgf-1/tws119 resulted in the additional increase of the -catenin level in na populations, in contrast to as cells (figures 5(a) and 5(b)). On the other hand, inhibition of gsk-3 regardless of tgf-1 treatment resulted in the nuclear accumulation of -catenin in na hbf, whereas this effect was not observed in as hbf populations (figures 6 and 7(b)). Thus, the impaired intracellular trafficking of -catenin may account for differences in cell reactivity to tgf-1 stimulation and fmt between na and as bronchial fibroblasts . To further clarify the mechanism of the observed phenomenon, we elucidated the effect of the licl and tgf-1/licl mixture on nuclear accumulation of p - smad2, an event crucial for the activation of tgf- signalling activity (figure 7). Treatment with the tgf-1 and tgf-1/licl cocktail induced p - smad2 nuclear accumulation in na hbf populations but not in as hbf populations . Tgf-1-induced nuclear translocation of p - smad2 was significantly attenuated by lithium in as hbf populations (figure 7(a)). Interestingly, this effect correlated with the pattern of nuclear accumulation of -catenin (figure 7(b)). These data suggest a role for the interference of the -catenin cytoplasmatic fraction with the function of p - smad2 during fmt in as hbf populations . Bronchial wall remodeling, a fundamental factor in the development of asthma, results from erroneous patterns of differentiation of bronchial cells, in particular fibroblasts [1, 2]. These changes are regulated by elevated production of proinflammatory cytokines, in particular tgf-, in regions of epithelial damage as reflected in bronchoalveolar lavage fluid from asthmatics [13, 42]. Fmt, a crucial event during bronchial wall remodeling and development of fibrotic changes observed in asthma, is supposed to depend on a concerted action of inflammatory mechanisms and inherent features of airway wall fibroblasts facilitating their phenotypic transitions in response to il-4 and tgf- [14, 34]. Our previous and current observations indicate that increased local concentrations of tgf-, resulting from inflammation, and differences in the expression levels of tgf- receptors are not necessarily implicated in bronchial wall remodeling during the asthma . In contrast, more pronounced differences in functional status of hbfs residing in healthy and asthmatic bronchi, for example, resulting from genetic patients' background or selection of clones epigenetically predisposed to fmt, can account for the differences in attitude towards fmt observed between hbfs derived from asthmatic and non - asthmatic patients . We show that inhibition of gsk-3 activity significantly attenuates tgf-1-induced fmt in hbf derived from asthmatic patients, whilst an opposite effect was observed in hbf isolated from non - asthmatic subjects . Thus, specific molecular mechanisms involving the interactions between smad proteins and effectors of gsk-3-dependent signaling determine fmt - related responses of hbf isolated from asthmatic patients . The observed augmentation of tgf-1-induced fmt by lithium in na hbf populations, along with a lack of differences in tgf- receptor expression levels between na and as hbfs, indicates the involvement of synergistic tgf-/gsk-3 signaling interplay in phenotypic shifts during differentiation of bronchial fibroblasts . Such an induction of fmt - related processes by synergic activation of tgf-/gsk-3 signaling was previously seen in mouse mesenchymal c3h10t1/2 cells, mouse embryonic fibroblasts, and pulmonary fibroblasts from individuals with chronic obstructive pulmonary disease . The inhibition of gsk-3 is a key event during the activation of canonical wnt pathway and leads to hypophosphorylation of -catenin, its cytoplasmatic accumulation, subsequent nuclear translocation of -catenin complexes with transcription factors, and induction of wnt - responsive gene expression . Our data, demonstrating a correlation between -sma expression, -catenin levels, and nuclear translocation of -catenin in the na hbf populations treated with tgf-1/licl and tgf-1/tws119 cocktails, reveals the involvement of the -catenin - dependent pathway in phenotypic transitions of bronchial fibroblasts . These data are in line with the in vitro observations of similar interrelations in human lung and skin fibroblast, and lung smooth muscle cell populations and with the in vivo data on reduced bleomycin - induced pulmonary fibrosis upon -catenin silencing in the murine model . Notably, our study was focused on -sma expression as a marker of fmt; however, wnt signaling controls the expression of a number of genes in lung cells, including fibronectin and collagen . Importantly, we have recently shown that the inhibition of gsk-3 activity by tws119 attenuates the secretion of ctgf by hbf derived from asthmatic patients . On the other hand, attenuation of tgf-1-induced fmt by lithium observed in hbf populations derived from asthmatic patients may indicate that inherent features facilitating fmt are accompanied by disturbed coherence of tgf-1/gsk-3 signaling pathways . Actually, an induction of fmt by tgf-1 coincided with an increased abundance of -catenin in as but not na hbf populations . In contrast, high levels of -catenin accompanied a relative decrease of -sma - positive cell numbers in tgf-1/licl - treated as hbf populations . Thus, the function of -catenin during fmt may depend on the activity of gsk-3 modulated by lithium or tws119 . The specific inhibition of gsk-3 by tws119 failed to induce the nuclear translocation of -catenin in as hbf populations . Therefore, the attenuating effect of lithium on fmt in as hbf populations may result from disturbed intracellular -catenin trafficking and its antagonistic effect on the function of downstream effectors of tgf-1 [29, 5153]. The phosphorylation of smad proteins and their subsequent accumulation in the nuclei are key events during the activation of tgf- signaling [17, 18]. Because we observed deficient nuclear accumulation of p - smad2 in as hbf populations treated with a tgf-1/licl cocktail, it is conceivable that cytoplasmically sequestrated -catenin may interact with smad proteins inhibiting their nuclear accumulation and tgf--specific signal transduction . Elevated levels of cytoplasmatic -catenin were previously demonstrated to inhibit p - smad2 nuclear accumulation via the inhibition of ismad phosphorylation and degradation . Therefore, further study is necessary to address the role of ismad proteins, in particular the possible involvement of their inhibitors, such as peptidyl - prolyl isomerases, in the observed differences between the lithium - evoked reactions of as and na hbfs . Our finding of an inhibitory effect of lithium on tgf-1-induced fmt in as hbf populations provides a potential explanation for the attenuating effect of lithium on the ongoing asthmatic process, as well as coincidental remission of asthmatic symptoms and their recurrence upon discontinuation of lithium therapy in psychiatric diseases, as revealed by epidemiological studies [3133]. However, it should be emphasized that numerous adverse effects of lithium have been reported . Nonetheless, other drugs targeting the wnt/-catenin pathway were reported to be potentially effective in treating cancer and tissue remodeling after injury . Significant progress has recently been made in identifying such molecules and inventing new strategies of inhibition of wnt signaling in therapeutic applications . Therefore, further research focused on the cell context - specific action of lithium or other wnt pathway activators during the asthmatic process, and the mechanisms of interference of these compounds with phenotypic transitions of bronchial fibroblasts would help to delineate borders of future clinical application in acute asthma treatment.
This study was approved by the institutional review board of loma linda university and was conducted in the center for implant dentistry, loma linda university school of dentistry . To be included in this study, the patients must: (i) be at least 18 years of age with good oral hygiene, (ii) possess one or more missing teeth in the maxillary or mandibular posterior region (excluding third molars), (iii) have adequate bone thickness to accommodate a 4.5 mm diameter implant, (iv) have the presence of opposing dentition . Those patients with: (i) implant insertion torque value of <35 ncm, (ii) a history of alcohol or drug dependency, or any medical, physical, or psychological factor that might affect the surgical or prosthodontic treatment and required follow - up examinations, (iii) history of bruxism, (iv) history of smoking, and/or (v) head and neck radiation treatment were excluded . A coin toss was utilized to randomize the abutment (ps or nps) placed in the patient . If patients were receiving more than one implant, the randomization was performed in such a way that the difference in the number of abutments of each group in the patient was not more than one . For example, if a patient was receiving 5 implants, 2 abutments would belong to one group and 3 abutments would belong to another group . Following the administration of local anesthetic (2% lidocaine with 1: 100,000 epinephrine [dentsply, york, pa, usa]), a full - thickness flap was reflected, and alveoloplasty was performed to level the alveolar crest prior to implant placement . The implants used in this study were 4.5 mm in diameter, threaded with sla surface, and an internal conical connection (superline, dentiumusa, cypress, ca, usa) (fig.1). The implants were placed 0.5 mm subcrestally with a minimum insertion torque of 35 ncm (fig.2). Resonance frequency analysis (rfa) multiple unit abutments (either ps or nps) were randomly selected and placed at time of surgery ., gangnam - gu, seoul, korea) with a horizontal mismatch of 0.6 mm was used as the control group (fig.1), while the nps multiple unit abutment (dentium co., ltd .) The abutments were torqued to 25 ncm (fig.3) and plastic healing covers (comfort cap, dentium co., ltd .) Placed . Flaps were approximated to allow for non - submerged healing using an absorbable polyglactin sutures (5 - 0 vicryl plus antibacterial suture [ethicon; johnson & johnson, somerville, nj, usa]). The distance between the iaj and the rl was 0.4 mm for the ps multiple unit abutment (a) and 0.1 mm for the nps multiple unit abutment (b and c). Antibiotics (amoxicillin 500 mg [ranbaxy laboratories ltd ., new delhi, india]) and analgesics (ibuprofen 800 mg [basf corporation, shreveport, la, usa]) were prescribed post - operatively . The patients were instructed to rinse with a 0.12% chlorhexidine gluconate solution (peridex, zila pharmaceuticals, inc ., phoenix, az, usa) twice daily and refrain from functioning over the surgical site for the initial 3 weeks . A soft diet was recommended throughout the remaining healing period (3 months). At 2 months, a definitive abutment level impression was made (aquasil monophase; dentsply, milford, de, usa). At 3 months, definitive screw - retained all ceramic crown (dentium co., ltd .) Was connected to the multiple unit abutment with a torque of 10 ncm (manufacturer's recommendation) (fig.4). Evaluations were made at the time of implant surgery (0) and at 3, 6, and 12 months following implant placement . The following parameters were evaluated at each follow - up appointment when applicable: implant success (smith & zarb 1989), marginal bone level (mbl) and marginal bone level change (mblc), rfa (sennerby & meredith 2008; zix et al . The implant success rates were evaluated according to the criteria proposed by smith and zarb (1989) where applicable . The mbls were measured on the mesial and distal aspects of each implant using sequential standardized periapical radiographs and the long - cone paralleling technique (strid 1985). A customized occlusal jig was made using a polyvinyl siloxane bite registration material (exabite; gc america inc, alsip, il, usa) to standardize the angulation and position of the film . The junction between the micro - roughened surface and the machined surface was used as the reference line (rl) (fig.5). The distance between the rl and the most coronal bone implant contact was measured . The value zero was designated when the mbl was at the same level or coronal to the rl and negative when the bone implant contact was apical to the rl . The average value of the mesial and distal measurements was used to represent the mbl for each implant . The mbls were measured at 0, 3, 6 and 12 months after implant placement (figs6 and 7). The mbls and mblcs were calculated and compared within group and between groups at designated time intervals . The intraexaminer reliability of the measurements was determined by using double assessments of mbl taken 2 months apart by one examiner and expressed as the intraclass correlation coefficient (icc). Reference line (rl) used to determine marginal bone level for the ps group (a) and the nps group (b). Radiographs taken at the day of implant placement (0) (a), 3 months (b), 6 months (c), and 12 months (d) for the ps group . Radiographs taken at the day of implant placement (0) (a), 3 months (b), 6 months (c), and 12 months (d) for the nps group . The rfa instrument (osstell isq, gothenburg, sweden) was used to evaluate implant stability immediately after implant placement (sennerby & meredith 2008; zix et al . 2008). Presence or absence of plaque was assessed at 6 sites (mesiolabial, labial, distolabial, mesiolingual, lingual, and distolingual) around the abutment or the definitive restoration (mombelli et al . Surgical complications were recorded and included but not limited to soft tissue problems, infection, or modifications of manufacturer's recommendations for implant placement . Prosthetic complications were documented, but were not limited to screw loosening, and/or repair of definitive restoration . The friedman test with post hoc pairwise comparisons was used to compare the mbls and mblcs within group, while the mann whitney u - test was used to assess the mbls and mblcs between groups . Pearson chi - square test was performed to evaluate the intragroup and intergroup differences in mpi . This study was approved by the institutional review board of loma linda university and was conducted in the center for implant dentistry, loma linda university school of dentistry . To be included in this study, the patients must: (i) be at least 18 years of age with good oral hygiene, (ii) possess one or more missing teeth in the maxillary or mandibular posterior region (excluding third molars), (iii) have adequate bone thickness to accommodate a 4.5 mm diameter implant, (iv) have the presence of opposing dentition . Those patients with: (i) implant insertion torque value of <35 ncm, (ii) a history of alcohol or drug dependency, or any medical, physical, or psychological factor that might affect the surgical or prosthodontic treatment and required follow - up examinations, (iii) history of bruxism, (iv) history of smoking, and/or (v) head and neck radiation treatment were excluded . A coin toss was utilized to randomize the abutment (ps or nps) placed in the patient . If patients were receiving more than one implant, the randomization was performed in such a way that the difference in the number of abutments of each group in the patient was not more than one . For example, if a patient was receiving 5 implants, 2 abutments would belong to one group and 3 abutments would belong to another group . Following the administration of local anesthetic (2% lidocaine with 1: 100,000 epinephrine [dentsply, york, pa, usa]), a full - thickness flap was reflected, and alveoloplasty was performed to level the alveolar crest prior to implant placement . The implants used in this study were 4.5 mm in diameter, threaded with sla surface, and an internal conical connection (superline, dentiumusa, cypress, ca, usa) (fig.1). The implants were placed 0.5 mm subcrestally with a minimum insertion torque of 35 ncm (fig.2). Resonance frequency analysis (rfa) multiple unit abutments (either ps or nps) were randomly selected and placed at time of surgery ., gangnam - gu, seoul, korea) with a horizontal mismatch of 0.6 mm was used as the control group (fig.1), while the nps multiple unit abutment (dentium co., ltd .) The abutments were torqued to 25 ncm (fig.3) and plastic healing covers (comfort cap, dentium co., ltd .) Placed . Flaps were approximated to allow for non - submerged healing using an absorbable polyglactin sutures (5 - 0 vicryl plus antibacterial suture [ethicon; johnson & johnson, somerville, nj, usa]). The distance between the iaj and the rl was 0.4 mm for the ps multiple unit abutment (a) and 0.1 mm for the nps multiple unit abutment (b and c). Antibiotics (amoxicillin 500 mg [ranbaxy laboratories ltd ., new delhi, india]) and analgesics (ibuprofen 800 mg [basf corporation, shreveport, la, usa]) were prescribed post - operatively . The patients were instructed to rinse with a 0.12% chlorhexidine gluconate solution (peridex, zila pharmaceuticals, inc ., phoenix, az, usa) twice daily and refrain from functioning over the surgical site for the initial 3 weeks . A soft diet was recommended throughout the remaining healing period (3 months). At 2 months, a definitive abutment level impression was made (aquasil monophase; dentsply, milford, de, usa). At 3 months, definitive screw - retained all ceramic crown (dentium co., ltd .) Was connected to the multiple unit abutment with a torque of 10 ncm (manufacturer's recommendation) (fig.4). Evaluations were made at the time of implant surgery (0) and at 3, 6, and 12 months following implant placement . The following parameters were evaluated at each follow - up appointment when applicable: implant success (smith & zarb 1989), marginal bone level (mbl) and marginal bone level change (mblc), rfa (sennerby & meredith 2008; zix et al . The implant success rates were evaluated according to the criteria proposed by smith and zarb (1989) where applicable . The mbls were measured on the mesial and distal aspects of each implant using sequential standardized periapical radiographs and the long - cone paralleling technique (strid 1985). A customized occlusal jig was made using a polyvinyl siloxane bite registration material (exabite; gc america inc, alsip, il, usa) to standardize the angulation and position of the film . The junction between the micro - roughened surface and the machined surface was used as the reference line (rl) (fig.5). The distance between the rl and the most coronal bone implant contact was measured . The value zero was designated when the mbl was at the same level or coronal to the rl and negative when the bone implant contact was apical to the rl . The average value of the mesial and distal measurements was used to represent the mbl for each implant . The mbls were measured at 0, 3, 6 and 12 months after implant placement (figs6 and 7). The mbls and mblcs were calculated and compared within group and between groups at designated time intervals . The intraexaminer reliability of the measurements was determined by using double assessments of mbl taken 2 months apart by one examiner and expressed as the intraclass correlation coefficient (icc). Reference line (rl) used to determine marginal bone level for the ps group (a) and the nps group (b). Radiographs taken at the day of implant placement (0) (a), 3 months (b), 6 months (c), and 12 months (d) for the ps group . Radiographs taken at the day of implant placement (0) (a), 3 months (b), 6 months (c), and 12 months (d) for the nps group . The rfa instrument (osstell isq, gothenburg, sweden) was used to evaluate implant stability immediately after implant placement (sennerby & meredith 2008; zix et al . 2008). Presence or absence of plaque was assessed at 6 sites (mesiolabial, labial, distolabial, mesiolingual, lingual, and distolingual) around the abutment or the definitive restoration (mombelli et al . Surgical complications were recorded and included but not limited to soft tissue problems, infection, or modifications of manufacturer's recommendations for implant placement . Prosthetic complications were documented, but were not limited to screw loosening, and/or repair of definitive restoration . The implant success rates were evaluated according to the criteria proposed by smith and zarb (1989) where applicable . The mbls were measured on the mesial and distal aspects of each implant using sequential standardized periapical radiographs and the long - cone paralleling technique (strid 1985). A customized occlusal jig was made using a polyvinyl siloxane bite registration material (exabite; gc america inc, alsip, il, usa) to standardize the angulation and position of the film . The junction between the micro - roughened surface and the machined surface was used as the reference line (rl) (fig.5). The distance between the rl and the most coronal bone implant contact was measured . The value zero was designated when the mbl was at the same level or coronal to the rl and negative when the bone implant contact was apical to the rl . The average value of the mesial and distal measurements was used to represent the mbl for each implant . The mbls were measured at 0, 3, 6 and 12 months after implant placement (figs6 and 7). The mbls and mblcs were calculated and compared within group and between groups at designated time intervals . The intraexaminer reliability of the measurements was determined by using double assessments of mbl taken 2 months apart by one examiner and expressed as the intraclass correlation coefficient (icc). Reference line (rl) used to determine marginal bone level for the ps group (a) and the nps group (b). Radiographs taken at the day of implant placement (0) (a), 3 months (b), 6 months (c), and 12 months (d) for the ps group . Radiographs taken at the day of implant placement (0) (a), 3 months (b), 6 months (c), and 12 months (d) for the nps group . The rfa instrument (osstell isq, gothenburg, sweden) was used to evaluate implant stability immediately after implant placement (sennerby & meredith 2008; zix et al . Presence or absence of plaque was assessed at 6 sites (mesiolabial, labial, distolabial, mesiolingual, lingual, and distolingual) around the abutment or the definitive restoration (mombelli et al . Surgical complications were recorded and included but not limited to soft tissue problems, infection, or modifications of manufacturer's recommendations for implant placement . Prosthetic complications were documented, but were not limited to screw loosening, and/or repair of definitive restoration . The friedman test with post hoc pairwise comparisons was used to compare the mbls and mblcs within group, while the mann whitney u - test was used to assess the mbls and mblcs between groups . Pearson chi - square test was performed to evaluate the intragroup and intergroup differences in mpi . A total of 30 implants (15 with ps abutments and 15 with nps abutments) randomly assigned to nine male and 10 female patients between ages of 23 and 76 (mean age 55.4 years) were included in this study (table 1). All implants possessed a diameter of 4.5 mm, with varied length (8, 10 and 12 mm). For the ps group, 5 implants were placed in the posterior maxilla and 10 implants in the posterior mandible, while for the nps group, 3 implants were placed in the posterior maxilla and 12 implants in the posterior mandible . After one year, all implants (30/30) were stable and none had lost osseointegration, which corresponded to an overall implant success rate of 100% . Patient distribution, locations, and implant dimensions the icc for marginal bone level measurements was 0.99, indicating that the measurements were reliable and reproducible . At baseline, the mbls were at or coronal to the rl for all mesial and distal sites for the ps (30/30) and nps (30/30) group, while at 12-months, the mbls of only 20/30 for the ps group and 15/30 for the nps group were still found at or coronal to the rl . For statistical analysis, one implant per patient was randomly selected accounting for eight independent implants in the ps group and 11 independent implants in the nps group (tables 25). The overall mbls at different time intervals and corresponding mblcs for the two groups are listed in tables 24 . For the ps group, changes in mbls were not statistically significant between all time periods (p> 0.05; table 2). For the nps group, significant differences were noted between all time points (p <0.05) except between 0 and 3 months (p = 0.066), and 6 and 12 months (p = 0.483) (table 3). When comparing mblc between ps and nps groups, statistically significant differences were noted at 012 months (p = 0.041) and 312 months (p = 0.026) (table 4). Comparison of the overall marginal bone level (mbl) and marginal bone level change (mblc) at different time intervals for the ps group using friedman test with post hoc pairwise comparisons at = 0.05 [] denotes mean sd of marginal bone level changes between the time intervals . Comparison of the overall marginal bone level (mbl) and marginal bone level change (mblc) at different time intervals for the nps group using friedman test with post hoc pairwise comparisons at = 0.05 statistically significant difference . [] denotes mean sd of marginal bone level changes between the time intervals . Comparison of marginal bone level changes (mblcs) at different time intervals between the ps and the nps groups (03, 06, 012, 36, 312, 612 months) using mann distribution and comparison of mpi scores at different time intervals using pearson chi - square test at = 0.05 p, comparison between groups; p, comparison within group . The mean isq value at the time of implant placement was 70 (range = 5782). The mpi scores of 0 and 1 were consistently observed throughout the study (table 5). No statistically significant difference was found within the group or between the two groups at the three time intervals (p> 0.05; table 5). Insertion torque of <35 ncm was observed with 4 implants during placement, and they were not included in the study . Prosthetic screw loosening was observed on 2 implants in two patients at 6-month follow - up and on 7 implants in four patients at 12-month follow - up . Each incidence of screw loosening was associated with a different implant for a total of 9 implants . No recurrence of prosthetic screw loosening on the same implant was noted in this study . Higher incidence of screw loosening was noted in the molar area (78% [7/9]) than the premolar area (22% [2/9]). All loose prosthetic screws were replaced and torqued to 10 ncm (manufacturer's recommendation). In this study, all implants remained osseointegration at 1 year, corresponding to a 100% (30/30) implant success rate . These findings are comparable to studies with various implant systems placed at healed sites with either ps (norton 2001; nentwig 2004; mangano et al . 2011) [95.6100%] or nps (naert et al . 2000; polizzi et al . The success rate of implants with sla surface used in this study is also comparable to that reported for implants with similar surface (98.8100%) (bornstein et al . 2011). In this study, although the difference in mblc at 12 months between the ps group (0.04 mm) and the nps group (0.19 mm) was statistically significant (p = 0.041; table 4), it was not clinically significant . It is interesting to note that the mblc reported in studies using implants with ps connection (ranged from 0.11 to 1.1 mm) (mangano et al . 2011a, b; norton 2006; donovan et al . 2010; canullo et al . 2012) was much less than those reported in studies using implants with the nps connection (ranged from 0.7 to 1.5 mm) (polizzi et al . Studies investigating the ps implant abutment interface have demonstrated that the greater the horizontal mismatch, the less marginal bone level changes were observed (baffone et al . . However, in this study, the similar mblcs observed in the two groups may be in part attributed to the conical connection being used for both the ps and the nps groups . Microbial leakage between implants and abutments has been identified as a causative factor for chronic inflammatory infiltration of the peri - implant tissues and subsequent bone loss (quirynen et al . Although microgaps have been noted at the implant prosthetic platform (jansen et al . 2008), implants with an internal conical connections may provide a more superior seal (jansen et al . 2000; norton 2000; hansson 2003), allowing less bacterial leakage (tesmer et al . 2009; assenza et al . The greatest amount of mblc observed in this study was during the first 6 months for both ps and nps groups (tables2 and 3). This is in accordance with studies that have shown most of the mblcs tend to occur within 36 months following one - stage implant procedures (cochran et al . 2010), and it had been suggested to be related to the establishment of proper physiological biological dimension (hartman & cochran 2004). In fact, during 612 months, the ps groups in this study showed bone gain (table 2). This can be attributed to one implant, which originally presented with distinct bone loss at 3 months, and resulted in some bone filled at 12 months . Few authors have related the peri - implant bone gain to the stimulating capacity of loaded implants in bone remodeling (brunski 1999) and to the implant surface (urdaneta et al . The rfa has been shown to be effective in evaluating implant stability (bischof et al . Study has shown that the rfa can reliably determine the implant stability with an isq 47 (nedir et al . 2004). As for predicting future osseointegration, it has been noted that implants with an isq 49 at placement, and loaded after 3 months, showed osseointegration after 1 year of function (nedir et al . Others have observed similar finding for successfully osseointegrated implants which had an isq of 4182 at placement using one - stage technique (guler et al . 2011). In this study, the isq of 5782 recorded during surgery was within the range of aforementioned studies, and all the 30 implants maintained osseointegration after 1 year, suggesting primary stability had been achieved at the time of implant placement . The relationship between oral hygiene and implant failure has been controversial (berglundh et al . 1993); however, it is generally agreed upon that plaque accumulation can cause an inflammatory response resulting in peri - implant bone changes (lindquist et al . The mpi scores observed throughout the course of this study were either 0 or 1 without significant differences noted between groups, implying that the patients were able to maintain a good level of oral hygiene . Therefore, the negative effect of plaque on the marginal bone levels for this study can be considered negligible . In this study, this may be partially attributed to the small prosthetic screw with a limitation of 10 ncm maximum torque used to connect the definitive crown to the prefabricated multiple unit abutments . With a similar prosthetic design, levine et al . (1999) also found high incidences of prosthetic screw loosening (22.2%) for single - tooth replacement . As all of the definitive crowns in this study were screw - retained, the screw loosening complications were easily resolved . Platform switching and conical implant - abutment connections have both been contributory to the maintenance of the peri - implant bone . Within the limits of this 1-year prospective clinical study, mean marginal bone level change at 12 months was similar for the ps (0.04 0.08 mm) and nps (0.19 0.16 mm) groups . Evidence from this study suggests that peri - implant marginal bone level change may not be related to the platform switch feature as much as the seal at the implant abutment interface . Nevertheless, due to the small sample size, the results should be interpreted with cautions, and long - term study with larger sample size is warranted . Additional supporting information may be found in the online version of this article: consort 2010 checklist of information to include when reporting a randomized trial.
.different factors can influence curing degree such as filler particle size, filler loading, polymerization initiator concentration, monomer type, amount of monomer, silane coupling agent, the shade and translucency of the material, intensity and distance of the incident light, wavelength of the light, irradiationtimes, design and size of the light guide and increment thickness . An inadequate curing degree affects the chemical and physical properties of the resin composite, such as water absorption, discoloration, wear resistance, strength, elution of the possible irritant, toxicity, hardness, marginal breakdown, bond between the tooth, adhesive and the restoration . In order to minimize these undesired effects, a composite resin should be cured to a high degree and to an appropriate depth as well . Because light intensity decreases as the light travels through composite resin, for proper polymerization a typical 2 mm thickness composite restoration requires a power density of at least 400mw / cm with 20 seconds irradiation time for halogen based light curing units . Therefore, the most commonly recommended thickness of resin composite placed with incremental layer technique is 2 mm in clinical practice [3, 6]. As expected, 40-second exposures led to significantly higher depths of cure than 20-second exposures for all curing units . However, ceballos showed that exposure time had no influence on the microhardness values for 0.5 to 2.5 mm depths . At higher depths, irradiation for 40 seconds produced greater microhardness values, but a further increase in the exposure time from 40 to 60 seconds did not result in significant microhardness improvement . However the total energy (intensity of curing unit exposure time) is important in the depth of cure, it has some disadvantages such as temperature rise and pulpal effects because of the heat . It has been shown for some products that a threefold difference in intensity only had a 15% difference in the depth of cure . So some manufacturers introduced core build - up composites and claimed that they can be used in high thickness with the bulk - curing technique because they have a high depth of cure . Some examples are 9 mm in 20 seconds for photocore and 4.4 mm in 10 seconds for quixfil . Polydorou evaluates the curing depth of two translucent composites; namely, quixfil and amament compared to a hybrid composite (tetric ceram). The curing depth of quixfil was 4.5 mm with a halogen based curing unite and 5.5 mm for an led curing unite . One of the indirect methods is microhardness which is the most popular method . For a specimen constructed from composite, in different thicknesses the bottom to top hardness ratios ranging from 0.800.90 have been used as criteria for the adequate degree of conversion at a specific sample thickness [9, 10]. The aim of his study was to evaluate the possibility of adequate curing - depth in bulk - curing of two translucent core build - up composites other than incremental technique, which are said to have a high depth of cure . The depth of cure was measured on the basis of vickers hardness of the top and bottom surface . The materials used in this study, with their respective compositions according to the manufacturers are given in table 1 . Two core build - up composites (photocore, kurary medical, okayama, japan) and (quixfil, denstsply detrey gubh, kontaz, germany) and a micro hybrid composite (z250, 3 m espe dental product, st . Paul mn, usa) were selected . As a halogen light source for all procedures a coltolux 75 light curing unit (coltene / whaledent, mahawash, nj, usa) with 800820 mw / cm output and 420510 nm wavelength was used . Before each curing, the power density was checked with a halogen - based radiometer (demetron 100, sds / kerr, usa). Eight polytetrafluoroethylene hollow cylindrical molds, 8 mm inner diameter and 10 mm outer diameter with different heights of 1,2,3,4,5,6,7 and 8 mm were provided . To prepare each specimen, the mold was placed on a clear glass slide, the resin composite was placed in the mold and then covered with a mylar matrix . Finally, a 1-millimeter - thick glass - slide was placed on the top of it immediately and was held by finger pressure to exude excess materials . Only the top side of the specimen was irradiated with visible light polymerization unit for 60 seconds . The head of the visible light cured unit was in touch with the glass - slide during exposure . In this way, we prepared 144 specimens of three selected resin composites for each composite type (n=48) and there were 8 subgroups according to their mold height (n=6 for each subgroup). The samples were removed from the mold and the bottom surfaces were marked to distinguish them from top surfaces . Samples were stored at room temperature in light - proof containers for 24 hours . Then the bottom and top vickers hardness were determined using a vickers hardness tester (beahler ltd, usa) with 100 gr load application for 15 seconds . For each sample, then for each thickness, the mean value and corresponding standard deviation of the vhn were measured . Besides, a bottom to top vh percentage was determined and a value of 80% was used to indicate acceptable curing . We conducted statistical analysis of data using two way analysis of variance (anova) to evaluate the hardness of the composite in different thicknesses . Tukey hsd was used as a post hoc test for multiple comparisons between the groups . The mean value and corresponding standard deviation of vhn as a function of depth is summarized in table 2 . Kolmogorov - smirnov test determined that data distribution in the top and bottom surfaces of the samples were normal . Then a two way anova studied the effect of the composite type and thickness on vhn . It showed that there was a significant interaction between composites and thicknesses (p<0.001) (fig . A tukey test demonstrated that the vhn of the top surface (thickness= 0) of the materials decreased in the following order: photocore> z250> quixfil photocore> z250> quixfil moreover, for 1 and 2 mm thicknesses, the bottom vhn of quixfil was significantly lower than the two other composites . In 3-mm thickness only the vhn for photocore was significantly higher than the two other composites, but in other thicknesses (4 and 5 mm) there were significant differences between all three composites in the following order: photocore> quixfil> z250 photocore> quixfil> z250 the satisfactory depth of cure for z250 was up to 3 mm and up to 5 mm for the two other composites (fig . None of them had an adequate curing depth in 6, 7 and 8 mm thicknesses; therefore, statistical analysis was not performed for these depths . The degree of polymerization plays an important role in physical and mechanical properties of composite materials . Infrared spectroscopy and laser ramon are direct methods and microhardness, scratching and visual inspection are some of the indirect methods . Direct methods are complex, expensive and time consuming; however, microhardness testing appears to be the most popular method because the other indirect methods tend to overestimate the curing depth . Surface microhardness (vickers or knoop) has been shown to be an indicator of the degree of conversion and correlates well with the infrared spectroscopy .the bottom to top hardness ratios ranging from 0.800.90 have been used as criteria for the adequate degree of conversion at a specific sample thickness [9, 10]. It means that the bottom to top surface microhardness ratio of 80% or more is adequate curing . In microhardness tests (vickers, koop), it should be in the range of 1grf to 1kgf and the most common is 100500 grf . The indenter with higher load penetrates deeper into the composite, reaches the harder layer and therefore measures a greater hardness . Because the optimum cure and therefore hardness is often reached slightly below the surface layer where the light transmission is high, no oxygen is present and a significant heat build - up occurs .in our study, the load was 100 grf and the dwell time was 15 seconds . The result of yoldaz study showed that a dwell time of 15 seconds could be accepted as an actual time of load application limit for the dental composite . Curing light irradiance, exposure time and composite are variables significantly affecting hardness and curing depth, although ceballos showed an exposure time higher than 40 seconds is not effective .in the present study although manufacturers recommended different exposure times for adequate curing depth, quixfil 20 seconds, photocore 10 seconds and z250 20 seconds, in our study the exposure time was 60 seconds in order to have maximum curing and the same experimental conditions . Yazici determined that the bottom knoop hardness number (knh) of a composite cured with led curing units is greater than halogen - based curing units . The distance between the light guide tip and the composite was 1 mm, in line with krishna study which said the distance can decrease the curing depth . In the present study, the hardness decreased with thickness and the acceptable depth of cure for z250 was up to 3 mm, but for photocore and quixfil it was up to 5 mm . Several researches showed that the depth of cure decreased with the increase in thickness, which is congruent with our study [8, 10]. Polydoraou showed a 4.4 mm depth of cure for quixfil using khn . In another research conducted by quixfil manufacturer, quixfil had a 4.4 mm satisfactory depth of cure and z250 had an up to 2.6 mm depth of cure . Ceballos showed a curing depth of 3 mm for z250 using vhn . In literature review, we did not find any research regarding the depth of cure for photocore . Owing to similarity in experimental conditions, the high depth of curing in quixfil and photocore can be because of the difference in organic matrix (monomer type, monomer concentration and photoinitiator concentration), greater filler size and translucency than z250 . As polydorou and ceballos demonstrated the effect of these factors in their study [2, 8]. Light scattering in composite with a smaller particle size can cause a lower depth of cure [3, 10], especially those similar in size to the wavelength of emitted light [10, 5]. So quixfil and photocore with a higher filler size (110 and 6, respectively) have a higher depth of cure than z250 with 0.01 3.5 filler particle size . The relationship between monomer conversion and inorganic filler loading is inversely proportional, as light transmission decreases with the increased filler loading . But boucschlicher showed b / t vhn ratios is independent of filler loading and size . He used fabricated composites with the same shade, resin matrix and photoinitiator, but a different filler loading and size . In our study, quixfil and photocore with the approximately same filler loading with z250 have a greater curing depth than z250 . Photocore contains silanated glass powder and silanated barium glass powder which are not found in the two other composites . Glass and it translucency can cause a high depth of curing and hardhness for the composite [2, 16] photocore had a higher hardness in all thicknesses than the two other composites; maybe, the glass fillers and translucency are the reason . It is suggested to evaluate the curing depth of these composites with led curing lights probably leading to a better curing depth . In addition, experiments are necessary to investigate the shrinkage behavior of these materials using bulk curing until their clinical advantages can be confirmed . Curing depth and microhardness were inversely related with thickness . Besides, the curing depth is a property which is material specific . So for being on the safe side, it is recommended to apply composite in the layering technique with 2 mm thickness in each layer.
Formulations were prepared using different polymers kollidone sr, cellulose acetate, acrycoat s 100, methocel k4 m, methocel k15 m, methocel k100 m . Drug and polymer in proportion 1:2, (drug: polymer) were dissolved in organic solvent (ethanol and acetone). This clear solution was poured slowly as a thin stream in oil phase; about 100 ml of liquid paraffin solution with continuous stirring at a speed of 500 rpm using mechanical stirrer at room temperature until complete evaporation of solvent took place . The floating microspheres were collected by decantation, while the non floating microspheres were discarded along with any polymer precipitates . Several preformulation trials were undertaken for various proportions of drug and polymer by variation of the ethyl acetate - acetone ratio and dichloromethane - ethanol ratio . Kollidone sr, acrycoat s 100 and cellulose acetate were selected as matrixing agent considering its widespread applicability and excellent gelling activity in sustain release formulations and also having the ph - independent and reproducible drug release profile . It was found that kollidone sr microspheres show desirable high drug content, yield, floatation and adequate release characteristics and hence was suitable for development of a controlled release system . In the present study, in vitro release studies of the floating microspheres were carried out in 0.1 n hydrochloric acid at 37c for a maximum period of 12 hours . At different time intervals, samples were withdrawn and cumulative% drug release was calculated . The percentage drug release of all the formulations is presented in figure 1 . Out of 9 formulations tried, the formulation k1 was found to be satisfactory; since it showed prolonged and complete release with 94.75% at end of 12 h.% drug release of all nine formulations multi unit gastroretentive drug delivery system has additional advantage of absence of dose dumping as in single unit drug delivery . The present investigation described the influence of the drug: polymer ratio on hydrochlorothiazide release . The release and drug entrapment efficiency of the microspheres were affected by the different polymers . It was found that the kollidone sr had a dominant role in the drug release from microspheres rather than acrycoat s 100 and cellulose acetate . And it can be given in hard gelatin capsule form . Therefore, it may be concluded that drug loaded floating microspheres in combination with kollidone sr are a suitable drug delivery system for hydrochlorothiazide.
Tumor necrosis factor (tnf) is a pleiotropic cytokine that plays an important role in mediating various immune functions including inflammation [1, 2], the regulation of apoptosis and necrosis, and induction of cytotoxicity . Tnf is capable of eliciting a variety of different immune responses by signalling via two types of membrane - bound receptors, type i (cd120a, tnfrsf1a) and type ii (cd120b, tnfrsf1b) receptors, with respective molecular weights of 55 and 75 kda [5, 6]. Type i tnf receptors (tnfri) are more widespread and expressed on all cell types in contrast to type ii tnf receptors (tnfrii) expressed mainly on cells of the immune system [6, 7]. Tnfri are activated via both soluble and membrane - bound (mtnf) forms of tumor necrosis factor alpha (tnf), whereas tnfrii are mainly activated by mtnf . Most biological effects of tnf (such as cytotoxicity and proliferation) are realized via tnfri activation . The intracellular tnfri domains, in contrast to the intracellular domains of tnfrii, contain a death domain (dd) associated with tnf - mediated cytotoxicity . The main function of tnfrii is proliferation induction in addition to apoptosis induction via a dd - independent mechanism . There also exist two soluble tnf receptor forms generated by proteolysis of membrane - bound receptors [12, 13] or alternative splicing that play an important role in tnf-mediated biological activity . Soluble tnf receptors (stnfr) do not allow binding to membrane - bound receptors thereby inhibiting tnf biological activity . The tnfri gene is located on chromosome 12p13 consisting of 10 exons [17, 18] and contains a housekeeping promoter with multiple transcription start sites, a high gc content, and missing consensus tata and caat box motifs . The tnfrii gene is located on chromosome 1p36 and also contains 10 exons [17, 20], a tnfrii promoter also high in gc content, but containing several consensus tata box motifs . What impact cytokines have on the nature of the developing immune response depends both on the percentage of cells expressing membrane - bound receptors and on receptor expression levels on respective cells . Single nucleotide polymorphisms (snps) occurring in promoter regions upstream of genes may potentially affect the process of transcription [2325]. Snps have important influence on mrna stability and translational efficiency and may influence susceptibility to many common diseases [2528]. The aim of this study was to establish associations between polymorphisms in the tnf receptor genes and membrane - bound type i and type ii tnf receptor expression levels on various mononuclear cell populations and to determine the levels of stnfrs in the serum of healthy individuals . Whole blood samples were obtained from the blood procurement station number 1 of the novosibirsk blood center and sampling was carried out from a population (n = 150 healthy individuals) between the ages of 19 and 55 years from the city of novosibirsk (south - western siberia). Also, 466 patients with rheumatoid arthritis (ra) were included in the study, of whom 86.5% were women and 13.5% were males, aged 18 to 70 years . Research was performed in accordance with the code of ethics of the world medical association (declaration of helsinki) and was approved by the local ethics committee of the fsbi tnf serum levels and the level of soluble types i and ii tnf receptors were determined . Soluble receptor levels were determined using enzyme - linked immunosorbent assay (elisa) kits . Specifically, the human stnf ri elisa kit and the human stnf rii elisa kit (raybiotech, norcross, ga, usa) were used according to the manufacturer's instructions . Tnf levels were determined using the -tnf - eia - best (jsc vector - best, novosibirsk, russia). Pbmcs were isolated from the blood of healthy individuals using a standard ficoll - urografin density gradient method (= 1.077 g / cm). Pbmcs were cultured at a concentration of 2 10/ml in 96-well flat - bottom plates (tpp, trasadingen, switzerland) in the absence or presence of lipopolysaccharide (lps) from escherichia coli serotype 055:b5 (sigma - aldrich, st . Louis, mo, usa) at a final concentration of 200 ng / ml . Cells were cultured in rpmi-1640 medium containing 10% fetal calf serum, 2 mm l - glutamine, 10 mm hepes buffer, 0.5 mm 2-mercaptoethanol, 80 g / ml gentamicin, and 100 g / ml benzylpenicillin for 24 h at 5% co2 and 37c . The number of cells expressing membrane - bound types i and ii tnf receptors was determined by flow cytometry as described previously . The antibodies labeled with phycoerythrin (pe) were used: anti - human tnf ri (r&d systems, minneapolis, mn, usa, cat number fab225p, clone 16803.1, mouse igg1) and anti - human tnf rii (r&d systems, cat number fab226p, clone 22235, mouse igg2a). The following antibodies from ebioscience (san diego, ca, usa) were used for immunophenotyping pbmc subpopulations: allophycocyanin (apc-) labeled anti - cd3 (cat number 17 - 0037, clone okt3, mouse igg2a), fluorescein isothiocyanate (fitc-) labeled anti - cd14 (cat number 11 - 0149, clone 61d3, mouse igg1), and phycoerythrin - cyanine 7 (pe - cy7) anti - cd19 (cat number 25 - 0199, clone hib19, mouse igg1). To obtain the calibration curve and convert the fluorescence intensity of cells expressing corresponding markers to absolute receptors numbers, bd quantibrite calibration particles (bd biosciences, san jose, ca, usa) were used . Flow cytometric analysis was performed using a bd facsaria flow cytometer (bd biosciences). We gated the populations for analysis on the basis of indices of forward (fsc - a) and side (ssc - a) scattering that were situated in the lymphocytic and monocytic regions . Subsequently, we selected subpopulations (cd3 t lymphocytes, cd19 b lymphocytes, cd14 monocytes) on the basis of the presence of markers of these subpopulations . Further, we established an interval gate on the control histogram, which was obtained with samples incubated in the absence of anti - human tnfri and tnfrii antibodies, and determined percent of positive events and mean fluorescence of cells expressing membrane - bound receptors for each of these subpopulations on pe / count histograms . Genomic dna was isolated from pbmcs harvested from healthy individuals using phenol - chloroform extraction methods . Snps selected for analysis for their association with receptor expression levels were selected from the ncbi dbsnp (http://www.ncbi.nlm.nih.gov/snp). Snp selection criteria were location within the promoter regions of the types i and ii tnf receptor genes and high minor allele frequency (maf) and by existence of associations with pathology . Additionally, snps were tested for the presence of transcription factor binding sites using software alibaba2.1 (http://www.gene-regulation.com/pub/programs/alibaba2/index.html). Genotyping polymorphisms at tnfri 609g / t (rs4149570), tnfri 1207c / g (rs4149569), tnfrii 1709a / t (rs652625), and tnfrii 3609c / t (rs590368) were conducted by polymerase chain reaction (pcr) in combination with rflp (restriction fragment length polymorphism) analysis . Sequences of primers specific for snps tnfri 609g / t, tnfri 1207c / g, and tnfrii 1709a / t were described previously [32, 33]. Sequences of primers specific for tnfrii 3609c / t were designed with the aid of the ncbi / primer - blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast). Primers specific for tnfri and tnfrii gene sequences were synthesized by biosan (novosibirsk, russia) (table 1). Pcr was carried out using a ptc-200 dna thermocycler (mj research inc ., watertown, ma, usa). The 20 l reaction volume contained 1 - 2 units taq dna polymerase (sibenzyme, novosibirsk, russia), 0.5 m of each primer, 0.25 mm of each desoxynucleoside - triphosphate, and 50200 ng of genomic dna . Reaction buffer was added to the dna polymerase containing 60 mm tris - hcl (ph 8.5, 25c), 1.5 mm mgcl2, 25 mm kcl, 10 mm 2-mercaptoethanol, and 0.1% triton x-100 . Pcr conditions were as follows: initial denaturation at 95c for 3 min followed by 30 cycles for tnfri 609g / t and tnfrii 1709a / t or 35 cycles for tnfri 1207c / g and tnfrii 3609c / t at 94c for 20 s; 61c for 15 s (tnfri 609g / t) or 58c for 15 s (tnfri 1207c / g) or 64c for 15 s (tnfrii 1709a / t) or 63c for 15 s (tnfrii 3609c / t); 72c for 20 s, and a final extension at 72c for 2 min . Amplification products were exposed to respective restriction enzymes (510 activity units) in a volume 2.55 l (sibenzyme). Restriction digestion of amplification products was carried out overnight at a temperature of 65c for tnfri 609g / t, 55c for tnfri 1207c / g, and 37c for tnfrii 1709a / t and tnfrii 3609c / t . Restriction products were analyzed by capillary electrophoresis using the qiaxcel system (qiagen, hilden, germany) or 2% agarose gel electrophoresis at a voltage of 140150 v for 2025 min . Qx dna markers (qiagen, valencia, ca) and the puc19 plasmid digested with msp i (sibenzyme) were used as molecular weight markers . The relationship of the respective genotypes with tnf receptor expression levels was tested using the kruskall - wallis anova h test, mann - whitney u test, and the median test . The tnf and soluble tnf types i and ii receptor levels in the serum of 150 healthy individuals were determined . These experiments demonstrated that serum levels of soluble tnf receptor type ii (2449.9 [1915.13768.9] pg / ml) were significantly higher than those of soluble tnf receptor type i (707.9 [497.8939.9] pg / ml) (p <0.001). This analysis also demonstrated that serum levels of stnfri in healthy individuals positively correlated with serum tnf levels (0.669 [01.9] pg / ml) (r = 0.32, p <0.05). The levels of tnf negatively correlated with the absolute number of tnfri expressed on cd3 t cells and cd19 b cells (r = 0.39 r = 0.22, resp ., we observed differences in the expression levels of membrane - bound tnf receptors on certain subpopulations of mononuclear cells, which may be indicative of different effector profiles of different immunocompetent cells in response to tnf. These potentially different responses are affected by the percentage of tnfr positive cells in the context of the absolute number of tnf receptors (table 2). Difference in receptor level expression may be both due to expression differences by different mononuclear cell populations or due to tnf receptor gene polymorphisms . Tnf receptor allele and genotype frequencies at the 609g / t and 1207g / c tnfri positions and the 1709a / t and 3609c / t tnfrii positions were studied in healthy inhabitants of novosibirsk (table 3). The genotype and allele frequencies of all four polymorphisms were consistent with hwe criteria (p> 0.05). We did not observe associations between snps present in the promotor region of tnf receptor genes and serum levels of tnf and stnfrii . When analyzing data regarding serum concentrations of soluble tnf receptors and respective genotypes, we observed that individuals homozygous at the t allele at position 609g / t tnfri (rs4149570) presented with lower levels of soluble tnf receptor type i compared to individuals presenting with the g allele (mann - whitney u test, tt versus gg, p = 0.006; kruskall - wallis h test, p = 0.032) (figure 1). The comparison of genotype frequencies at position 609g / t was also statistically significant with regard to differences in the percentage of cd19 cells expressing membrane - bound tnfri (median test, = 5.992, p = 0.05). The association between the expression level of membrane - bound tnf receptor type i and genotype was established for snp 1207g / c tnfri (rs4149569). The homozygous cc genotype was statistically more frequent in the group with lower densities of cd14 monocytes expressing surface tnfri (mann - whitney u test, cc versus gc, p = 0.012; kruskall - wallis h test, p = 0.025; median test, = 7.325, p = 0.025) (figure 2). We also demonstrated that frequencies in the genotypes of snp 1207g / c were associated with different stimulation index values (median test, = 6.283, p = 0.043). The stimulation index was calculated as a simple ratio of absolute number of tnfri receptors on cd14 cells in cultures with and without lps stimulation . When analyzing tnfrii genotype frequencies at snp 1709a / t (rs652625) we observed a statistically significant difference in the percentage of cd3 and cd19 cells expressing tnfrii in healthy individuals (median test, = 5.049, p = 0.024 and individuals with cc genotype at position 3609c / t (rs590368) of tnfrii had a lower percentage of intact cd14 cells expressing tnfrii compared to individuals with the ct genotype (mann - whitney u test, cc versus ct, p = 0.015; kruskall - wallis h test, p = 0.041) (figure 3). The frequencies of alleles and genotypes of tnfri promoter at positions 609 and 1207 and tnfrii at positions 3609 and 1709 had no statistically significant differences in ra patients and healthy individuals . However, the analysis revealed a combination of genotypes tnfri-609gt + tnfrii-3609cc . The frequency of this combination in patients was 10% and was significantly lower than that in the group of population controls 22% (= 11.6, p = 0.0006). The odds ratio for this combination of genotypes was or = 0,42 (ci95 = 0.250.71), and a relative risk of rheumatoid arthritis for carriers of this genotype was 10% lower . We have examined the association of combined genotypes with level of expression of tnf receptors in healthy donors . Individuals with the combination of gt+cc are characterized by an increase of membrane - bound tnfri on intact subpopulations cd19 b cells and cd3 t lymphocytes (figure 4) and reduced the percentage of cd3 t lymphocytes and cd14 monocytes expressing tnfrii (figure 5). Serum levels of tnf for combinations of genotypes had a trend to decrease in the series gg+ct - gt+ct - gt+cc . Analyses of signaling mechanisms associated with tnf are necessary to evaluate not only the cytokine itself and its soluble receptors but also membrane - bound receptors that confer different biological effects . It has been demonstrated that healthy individuals manifest quantitative differences in not only the percentage of cells expressing these receptors but also the quantity of receptors expressed . It can be inferred that different cell subpopulations would have different response to tnf depending on receptor expression densities . It is probable that cells expressing a greater receptor density or if a cell population expresses a greater percentage of these receptors it would enhance the effects conferred by tnf (on these cells). For this reason, the percentage of cells expressing tnf receptors does not always correlate with the absolute number of receptors . For example, comparison of tnfri expression by t and b lymphocytes and monocytes identified that cd19 b lymphocytes expressed the lowest number of total tnfri but as a population expressed the greatest density of receptors . By contrast, a greater percentage of cd3 t lymphocytes expressed tnfrii at the lowest density of any cell type examined . Previous work has demonstrated that cells cultured in the presence of lps for 24 h resulted in a significant enhancement in tnrii expression compared to tnfri expression in cd14 monocytes . Data presented in this report support these observations; that is, a higher percentage of monocytes cultured in the presence of lps expressed tnfrii (at a higher density) compared to tnfri expression (and density per cell) what testifies to a different involvement of tnf receptors in response to lps action . These data confirmed that lps significantly affected tnfrii expression on cd14 monocytes from healthy individuals . In addition, comparison of freshly isolated (unstimulated) cd14 monocytes to mock - stimulated cd14 monocytes cultured for 24 h revealed differences both in the percentage of positive cells and in the expression level of membrane - bound tnf receptors likely associated with microenvironment changes . For example, spinas et al . Established a correlation between tnf and stnfri levels but not with stnfrii and koga et al . Established a correlation between tnf and stnfrii but did not establish a correlation between tnf and levels of stnfri . Data presented in this report demonstrated that serum tnf levels positively correlated with stnfri levels in the serum of healthy individuals . We also demonstrated that serum stnfri (weakly) negatively correlated with that of the density of membrane - bound tnfri expressed on cell surfaces, suggesting an association with proteolytically derived membrane - bound receptors . Tnf levels also negatively correlated with the levels of membrane - bound tnfri on cells, supporting previous reports demonstrating that tnf decreased in the amount of mrna encoding for tnfri . Differences in the levels of receptor expression can also be affected by tnf receptor gene polymorphisms . A considerable number of snps located within the promoter region of tnf - tnfr superfamily gens can affect regulation by significantly impacting levels of gene expression [36, 37]. The presence of certain alleles within promoter regions of cytokine receptor genes can influence gene transcription rates and mrna stability resulting in increased or decreased levels of the synthesized protein . The snps analyzed during the course of this study were located within the tnf receptor gene types i and ii promoter regions and are therefore likely to affect tnfrs expression levels . Several studies have examined the association of polymorphisms at the tnfri 609g / t (rs4149570) locus with various pathologies . For example, the t allele was significantly associated with systemic lupus erythematous, poor survival outcomes in non - small - cell lung cancers, and t cell non - hodgkin's lymphoma; however, this polymorphism was protective against oral carcinoma, which decreased the risk of colon cancer and invasive pulmonary aspergillosis . Kim et al . Found out that the tnfri 609g / t polymorphism was strongly associated with primary hepatocellular carcinoma and that the t allele repressed tnfri expression . The present study demonstrated that individuals homozygous for the t allele of snp 609g / t located within the tnfri gene promoter presented with lower serum levels of soluble type i tnf receptors . It has been demonstrated that soluble receptors inhibit the biologic effects of tnf; therefore, when soluble receptors are present at lower concentrations there is less competition for membrane - bound receptors . A tendency has also been demonstrated to the lowering of the absolute numbers of membrane - bound tnfri on intact cd19 b cells in individuals with the tt genotype (mann - whitney u test, tt versus gt, p = 0.099) (figure 6). Considering that soluble tnf type i receptors are formed as by - products of proteolytic cleavage from membrane - bound tnf receptors [12, 13], it can be concluded that the smaller amounts of soluble tnfri associated with the tt genotype are directly associated with diminished expression levels of membrane tnfri levels . Reduced expression of tnf receptors appears to be associated with the g allele that encodes for the binding site of the interferon consensus sequence - binding protein (icsbp, also known as irf8 or interferon regulatory factor 8), a transcription factor that is involved in tnfri - mediated activation of nf-b signaling pathway . Miyagawa et al . Demonstrated that, for snp tnfri 1207g / c (rs4149569), the c allele frequencies in patients with systemic lupus erythematous were significantly lower than the frequencies in control groups . The present study demonstrated that cc genotype carriers at position 1207g / c of the tnfri gene presented with a reduced density of tnfri on cd14 monocytes . It has been demonstrated using the online alibaba2.1 (http://www.gene-regulation.com/pub/programs/alibaba2/index.html) program that this snp (in the context of the c allele) was associated with lack of transcription factor binding sites and that the g allele was associated with transcription factor binding sites for c / ebpalpha (also known as ccaat / enhancer - binding protein alpha), ap-2alpha (also known as tfap2a), and sp1 . It is quite probable that the differences in expression of tnfri on cells of individuals with different genotypes are associated with one of these transcription factors . A number of studies have established an association between snp tnfrii 1709a / t (rs652625) with pathology [45, 46]. Determined that the a allele in snp 1709a / t of the tnfrii gene increased the risk of severe infusion reactions to infliximab in crohn's disease patients . We examined the frequency of allelic variants of tnfri and tnfrii genes in patients with rheumatoid arthritis and demonstrated that ra patients (compared to controls) were significantly less likely to present with tnfri 609gt + tnfrii 3609cc combination of genotypes . Individual's predisposition to developing of disease may be determined by the individual characteristics of the expression regulation of tnf- and its receptors in the cells of the immune system . The present study identified statistically significant frequency differences in the percentage of cd3 and cd19 cells expressing tnfrii in individuals carrying aa genotype in snp tnfrii 1709a / t (rs652625). Individuals homozygous for the c allele in snp 3609c / t (rs590368) of the tnfrii gene had low percentage of cd14 cells expressing tnfrii . Using alibaba2.1 we demonstrated a difference at the binding site defined by the 1709a / t of tnfrii allele . Specifically, transcription factors did not bind to the sequence encoded by the t allele and the sequence encoded by the a allele resulting in cft binding (also known as transcription factor nf - i). The biologic effects of tnf result from interactions with two types of membrane - bound receptors: tnfri and tnfrii . It is known that simultaneous expression of tnfri and tnfrii results in the degradation of traf2 resulting in increased tnfri - mediated cytotoxicity [10, 48]. It is quite probable that cell populations expressing higher levels of tnfrii would be associated with higher rates of apoptosis . Thus, we have established that snps 609g / t and 1207g / c of tnfri gene promoter and 1709a / t and 3609c / t of tnfrii gene promoter are associated with expression level of tnf receptors what specifies that these polymorphisms are functional . Association of snps 1207g / c, 1709a / t, and 3609c / t of tnfr genes promoters with expression levels of membrane - bound tnf receptors types i and ii in the absence of association with level of soluble tnf receptors is established what testifies to existence of different mechanisms of regulation of soluble and membrane - bound receptors expression . Association of snps 1207c / t and 3609c / t with expression of tnfrs on cd14 population in the absence of association with expression on cd3 and cd19 subpopulations testifies to a functional role of these snps for separate subpopulations of mononuclear cells . A possible mechanism for determining the expression of the receptor is a cell - specific transcriptional regulation of a set of factors (enhancers and repressors) [49, 50]. Combination tnfri 609gt (rs4149569) and tnfrii 3609cc rarely is detected in ra patients and is associated with increased levels of tnfri and reduced level of tnfrii on the immune cells . Perhaps different levels of tnf receptors types i and ii on the cells determine the relationship of genetic variants with rheumatoid arthritis . This study identified differences in the percentage of cells expressing tnf receptors and in the absolute number of membrane - bound receptors expressed by pbmcs . Also we have established that the percentage of cells expressing tnfrs is not always associated with the absolute number of receptors . Furthermore, we determined that differences in expression levels of tnf receptors types i and ii could be associated with tnfri and tnfrii gene polymorphisms . Associations of snps located within the promoter regions of tnf type i and type ii receptor genes were established in the context of expression levels of membrane - bound receptors present on subpopulations of mononuclear cells and with the serum levels of soluble type i tnf receptors . These observations suggested that tnf receptor gene alleles represent one of the factors that affects variability in the expression of membrane - bound receptors that may explain differences in the effects mediated by tnf on different cell populations / subpopulations.
Intrusive thoughts are characteristic of a number of neuropsychiatric disorders, such as obsessive - compulsive disorder (ocd), posttraumatic stress disorder (ptsd), depression, schizophrenia, body dysmorphic disorder, eating disorders, and drug addiction . Such thoughts are involuntary images, ideas, or thoughts that can be difficult to manage or eliminate . For example, the intrusive desire in drug addicts to use drugs cannot be easily controlled and thereby causes behaviors that can lead to relapsing to drug use . In depression, unwelcome rumination on negative or sad events can precipitate a state of depression, and stress - associated cues can initiate difficult - to - manage thoughts that lead to a state of anxiety in ptsd . Paranoid delusions can be a particularly troubling phenomenon, disrupting thought processing in patients with schizophrenia . Finally, intrusive thinking has been best characterized in ocd, where it is a primary initiator of repetitive behavior that has a significant negative impact on a patient's life . Thus, intrusive thoughts act as triggers that initiate relapsing to un desired behaviors or internal states of mind that we characterize as one or another type of neuropsychiatric disorder . By considering that intrusive thoughts are a shared endophenotype of many neuropsychiatric disorders, it is clear that discovering the neurobiological underpinnings of how a thought becomes intrusive and can initiate maladaptive behaviors could have farreaching therapeutic impact on a number of disorders . While not likely to cure any of the disorders, a treatment rendering intrusive thoughts more controllable and less likely to trigger unwanted behaviors would broadly support current pharmacological and psychosocial therapies in many neuropsychiatric disorders . In this review, we explore questions pertaining to the neurobiology of intrusive thoughts, and how we might use this neurobiology to facilitate treatment of a number of neuropsychiatric diseases that are characterized in part by intrusive thinking . Is intrusive thinking adaptive in some situations where highly motivating stimulus demands a restricted focus on a single behavioral response? Why do people with neuropsychiatric disorders have difficulty controlling intrusive thoughts that trigger undesirable behaviors? How can the neurobiology of intrusive thinking be used to identify molecular targets for treating neuropsychiatric disorders characterized in part by intrusive thoughts? Because of recent neurobiological advances in animal models of addiction, this review will utilize drug addiction as an exemplar neuropsychiatric disorder for understanding the biological basis and pharmacological treatment of intrusive thoughts . Before delving deeply into the neurobiology of maladaptive intrusive thoughts, we first consider that thoughts becoming transiently intrusive can contribute to the execution of a normal, adaptive behavior . This viewpoint has proven very useful in conceptualizing drug addiction, where it is an axiom in the field that drugs usurp normal reward brain circuits . For example, this perspective has contributed to the rise of our current understanding of the critical role dopamine transmission plays in both normal reward learning and developing drug addiction accordingly, it may prove equally worthwhile exploring whether the circuitry underpinning the intrusive thoughts that trigger neuropsychiatric symptoms may have evolved to serve a biological purpose . When might the biology of intrusive thinking be used to generate an adaptive behavioral response? Consider that you encounter mortal danger, and the adaptive response is to run as far and as fast as possible from the source of danger . It could be argued that the thought of escape becomes intrusive since this thought will be prepotent in guiding your behavior until you escape . Thus, in the process of escaping, you will be unaware of other competing stimuli that might normally capture your attention and guide your behavior . Such competing stimuli might range from relatively minor stimuli, such as rain getting your clothes wet, to major stimuli, such as stepping on a nail while running . In this example, to support the likelihood of successful escape, the high motivational value of mortal danger cancels conscious perception of other less motivating, but also biologically relevant, stimuli . Importantly, for the intrusive nature of the thought of escape to be adaptive, the thought will be terminated, or at least adaptively modified once the goal of escape is achieved, and you then attend to the competing stimuli of wet clothes and a wound in your foot . In contrast, this pattern of thinking becomes pathological when the intrusive thought is not discontinued . For example, if you have an anxiety disorder, the life - threatening experience may trigger a pathological intrusion that you would have difficulty suppressing in order to focus on important competing stimuli and thoughts . Through this example of a prepotent thought generating a highly focused, adaptive behavior, we recognize that adaptive intrusive thinking consists of three components that we can dissect to determine neurobiological underpinnings . It is possible that the pathology of intrusive thinking leading to maladaptive behavior could arise from impairment in any of the three components . For example, the motivational value of the thought could be excessive, the motivational value of competing thoughts may be reduced, or the ability to adaptively devalue or suppress the intrusive thought may be weakened . Although the third component is most often evoked in clinical settings as a definition of intrusive thinking, next we will overview literature suggesting that all three components of an intrusive thought are harbored at least in part within corticostriatal glutamatergic projections from the prefrontal cortex (pfc) and allocortical regions (eg, amygdala and hippocampus) to the ventral striatum . Maladaptive intrusive thoughts initiating neuropsychiatric behavioral disorders have been characterized as an impairment in top - down control that is identified in neuroimaging studies by functional and morphological changes within corticostriatal projections . Clinical neuroimaging studies employing cognitive probes that might evoke intrusive thinking consistently indicate activation of pfc and amygdala . Imaging studies using functional magnetic resonance imaging (fmri) or positron emission tomography (pet) in addictive disorders, including drug addiction, gambling, and overeating, reveal that presenting cues associated with the addictive behavior increases activity in the anterior cingulate, amygdala, and ventral striatum (nucleus accumbens). Similarly, evoking sad thoughts in major depression or intrusive urges in ocd are associated with activation of anterior cingulate, orbital frontal cortex, amygdala, and ventral striatum . In the case of depression and ocd, there is also consistently reduced activity and morphometric volume in dorsolateral pfc, which has been particularly influential in characterizing these disorders as a loss of top - down control . Finally, evoking stress in subjects diagnosed with ptsd also reveals marked activation of anterior cingulate and amygdala, with less activation in ventral pfc, perhaps indicative of impaired extinction . Next, we will explore general theories of how the striatum is topographically organized to compute motivationally relevant information arriving from cortex and to translate this information into adaptive behavioral responses . The striatum harbors procedural memories, such as riding a bike, as well as habitual and stimulus - response behaviors, which makes this brain structure a likely site for the pathological intrusion of thoughts that lead to difficulty in controlling habitual behavior in disorders, such as ocd and addiction . Accordingly, there is a research focus on the striatum as a potential site of pathological impairment . Animal models of perseverative disorders, in particular models of addiction, have been used to examine in great detail the synaptic changes in the striatum produced by addictive drugs and to use this knowledge to uncover potential molecular pharmacotherapeutic targets for treating drug use and relapse . While these studies have not yet been overwhelmingly successful in bringing forth effective treatments for addiction, they have resulted in a deep understanding of how the striatum is organized to generate behaviors and how drugs of abuse and other experiences, such as stress, produce striatal cellular adaptations that are beginning to explain how motivationally relevant thoughts can become intrusive and difficult to control . The striatum can be divided into four functional regions that receive topographically organized inputs from the frontal cortex (figure 1). Divisions are not absolute, and all the compartments share functions and work in a coordinated manner to guide an organized, adaptive behavior . Given this caveat, work with experimental animals and, to a lesser extent, human imaging studies have assigned specific functions to each compartment . In the dorsal striatum, the dorsolateral striatum most strongly regulates habitual behavior and harbors classic procedural memories, while the dorsomedial striatum is critical for action - outcome learning of goal - directed behavior . In the ventral striatum, the shell subcompartment of the nucleus accumbens (nashell) is generally considered to play the largest role in predicting reward and reward learning, while the core subcompartment (nacore) is important for evaluating reward associations . The different functions of the striatal quadrants (figure 1) can perhaps be best illustrated through the preclinical addiction literature . When an animal is learning an action - outcome relationship for obtaining an addictive drug for example, learning to lever press for an intravenous infusion of cocaine the drug - induced release of dopamine into the nashell, and to a lesser extent nacore, is detected as motivationally relevant, thereby reinforcing lever pressing for the drug . In many experiments, a pavlovian cue, such as light and tone, is paired with the infusion of drug, and the animal learns to further associate lever pressing and drug infusion with the light / tone - conditioned cue . This pavlovian association strongly involves amygdala projections to the na - shell and na - core . Similarly, the animal makes an association between the drug and the environment in which the drug is delivered, and in the case of our example, the contextual association would be with the operant chamber . Importantly, once these associations (action - outcome, pavlovian, contextual) are learned, the motor pattern generator whereby the drug cue or context initiates the behavior (lever pressing) is in the dorsomedial striatum . With continued training, the stimulus - response relationship (cue triggering a lever press) becomes habitual and this relationship is ultimately stored as a procedural memory in the dorsolateral striatum . Importantly, reward learning in the nashell is relatively friable and easy to modify by changing environmental contingencies . However, as training continues, the stimulus - response associations with the reward gradually transfer to the dorsolateral striatum, where they become relatively stable procedural memories . While it is important to understand how drug associations are made and solidified as stimulus - response habits in the striatum, in the clinical setting, these habits are a heady formed by the time a person seeks treatment for a substance use disorder . Accordingly, it is a therapeutically more relevant question to ask: how does the striatum suppress or modify habits that no longer serve an adaptive purpose? Although a behavioral response that is repetitively experienced and is repeatedly associated with the desired outcome (eg, a drug infusion) becomes stored as a habit in the dorsolateral striatum, the behavior remains initiated by prefrontal and allocortical (amygdala and hippocampal) glutamatergic inputs to the nucleus accumbens, in particular the nacore (figure 1). This ongoing transfer of environmental information from the ventral to dorsal striatal quadrants reflects the hierarchical organization of the four divisions, with the nacore acting as a portal whereby motivationally relevant stimuli enter basal ganglia circuitry, and the dorsal striatum coordinating the appropriate behavioral response . For example, in an animal model of addiction where a rodent learns to press a lever for cocaine over many days, the response is well learned and can be initiated by presenting a cue that has been previously associated with cocaine delivery . To initiate a cue - induced lever press, the cue presentation is processed in the pfc and amygdala, communicated to the nacore, and then communicated to the dorsal striatum to access previously stored stimulus - response procedures coding the lever press response . Communication between the ventral and dorsal striatum is largely via well - characterized, topographically organized thalamo - cortico - striatal circuitry that processes information arriving to the accumbens and then engaging dorsal striatal motor pattern generators to elicit the desired behavior . Given the hierarchical organization of the striatum in processing stimuli that can elicit or modify a learned stimulus response, the nacore is positioned as the site where new information processed in pfc and allocortical brain regions arrives into the striatum to disrupt or suppress an intrusive thought . To illustrate the role of the nacore in how a stimulus response or habitual behavior can be disrupted by stimuli that require a behavior to change in order to remain adaptive, we again turn to the addiction literature . If the lever press for a drug is associated with an electric foot shock, the addicted animal will sustain substantially more foot shock than an animal trained to press for food . Indeed, how difficult it is for the electric shock to disrupt a drug - seeking behavior can be viewed as an estimate of the extent to which the stimulus (light / tone / context) is intrusive and is commandeering the animal to maintain a maladaptive behavior (pressing for drug) in spite of a competing motivationally relevant stimulus (shock). This indicates that chronic drug use has produced changes in how the striatum processes competing stimuli, thereby making the drug - associated stimulus - response behaviors more intrusive and difficult to disrupt . Thus, the capacity of the addicted animal to keep responding in the presence of foot shock is similar to the person in mortal danger described above who is having an intrusive thought of escape and does not notice a foot injury sustained by stepping on a nail . In contrast, the food - responding animal pressing for food is not experiencing intrusive motivation to seek reward and responds appropriately to the foot shock pairing by inhibiting lever pressing . Through the knowledge outlined above of how corticostriatal projections translate environmental stimuli and thoughts into behavior, we have identified the nacore as a portal for how motivationally relevant information is processed to guide behavior and to modify stimulus - response habits . Accordingly, the nacore has become a site in the brain for focusing reductionist technologies to more mechanistically understand how enduring molecular and cellular changes are permitting maladaptive thoughts to become intrusive and trigger symptoms of neuropsychiatric disorders . A recent animal study nicely illustrates that pfc synapses in the nacore are important in how competing, motivationally relevant information can disrupt an intrusive behavioral response, such as lever pressing, for an addictive drug . In this study, maladaptive intrusive thoughts related to addiction were modeled in rats trained to use cocaine, and a subpopulation of the rats was identified that sustained relatively large foot shocks and continued to seek drug delivery triggered by a conditioned cue . By optically stimulating pfc inputs to the nucleus accumbens, these investigators effectively increased the capacity of foot shock to disrupt cocaine seeking . In other words, the stimulated rats demonstrated improved top - down control and were able to attend to the foot shock and update their lever pressing behavior in an adaptive manner (in the case of this particular study, stop pressing for cocaine). The recent optogenetic study above was built upon nearly 20 years of previous animal research showing that enduring changes are produced in cortico - accumbens synapses by repeated administration of addictive drugs . The list of changes identified is prodigious and in some instances contradictory depending on the drug treatment protocol and the class of addictive drug the animal is using . For example, a very well - studied enduring synaptic effect of cocaine use is an increase in accumbens neuron dendritic spine density and size, but use of morphine or heroin produces the opposite effect on spine density and size . Since by definition addiction disorders with all drug classes share an endophenotype of the motivation to take drugs becoming intrusive, the long list of drug - induced synaptic adaptations can be simplified by considering only molecular adaptations that are shared between classes of drugs and that endure during drug withdrawal . This rationale is similar to how dopamine release in the accumbens was identified as an obligatory mediator of reward learning . Also, with these exclusion criteria, drug- and stress - induced adaptations at glutamatergic synapses have emerged as strong candidates for how a cue initiates a perseverative drug - seeking response . Importantly, in many experiments, it has been shown that these adaptations are not produced in animals trained to self - administer a biological reward such as sucrose, indicating that they are potentially pathological markers of substance use disorders . Figure 2 illustrates the primary adaptations elicited by presenting a drug - conditioned pavlovian cue to an animal withdrawn from training to self - administer an addictive drug, including rats and mice trained to use cocaine, heroin, nicotine, alcohol, or methamphetamine . When presentation of the drug - associated cue is processed in the pfc and allocortical regions, their glutamatergic projections to the nacore are activated to communicate the presence of a stimulus that will ultimately trigger a behavioral response (eg, lever pressing for drug). Since this is the same circuit for processing cues that achieve biological rewards, one characteristic shared between addictive drugs, but not food training, is that in the nacore, the drug cues elicit glutamate spillover from the synaptic cleft . Spillover occurs due to two separate enduring changes at glutamatergic synapses produced by repeated drug exposure . All drugs examined to date reduce the capacity of release - regulating presynaptic group ii metabotropic glutamate receptors (mglur2/3) to negatively regulate synaptic glutamate release . This is accomplished by a drug - induced, enduring, downregulation of mglur2/3 protein or by upregulating ags3 (activator of g - protein signaling 3), a g - protein - binding protein that inhibits mglur2/3 intracellular signaling by sequestering and thereby functionally inactivating gi. Simultaneously, chronic use of drugs or acute stress downregulates astroglial glutamate transporters (eg, glutamate type 1 transporter [glt-1]) in nacore . Because the glial glutamate transporters are densely distributed adjacent to the synaptic cleft, synaptic glutamate more effectively escapes uptake and enters the extrasynaptic space when glt-1 is downregulated . Regardless of the combination of enduring changes produced by addictive drugs or stress, once in the extrasynaptic space, glutamate stimulation of group i metabotropic glutamate receptor 5 (mglur5) appears to be critical in regulating cue - induced drug seeking, since mglur5 antagonists administered systemically or directly into the nacore or nashell inhibit cued reinstatement of seeking for all drugs tested to date . In addition, for at least some drugs (eg, heroin and nicotine), extrasynaptic stimulation of glun2b - containing n - methyl - d - aspartate (nmda) glutamate receptors contributes to cue - induced reinstatement of drug seeking . Activation of mglur5 or glun2b stimulates the catabolism and sculpting of the extracellular matrix (ecm) by activating gelatin - specific matrix metalloproteinase (mmp2, mmp9) activity in nacore . The mechanism for how these glutamate receptors stimulate mmps may involve synthesis of nitric oxide and n - nitrosylation of mmps (unpublished observations). Regardless, ecm digestion creates peptide ligands that bind to receptors on medium spiny neurons in the nacore, causing glutamatergic synapses to undergo transient synaptic potentiation (t - sp). How the ecm signals t - sp is under investigation, but signaling through integrin receptors is a likely mechanism . Three characteristics of cue - induced t - sp in the nacore make it a likely candidate mechanism for how a motivating environmental stimulus translates into an intrusive thought and maladaptive behavior . First, the transient nature of the potentiation means that it is eventually disrupted, and in animal models of cue induced drug seeking, the termination of synaptic potentiation parallels the animal no longer lever pressing for the cue . Second, when the drug goal is achieved (ie, the cue correctly predicts drug delivery), t - sp is terminated . This is akin to attending to your wounded foot after successful escape in the example above . Third, the extent of t - sp is correlated with the intensity of drug seeking (lever pressing) and does not occur in cue - induced sucrose seeking considering these three characteristics, we hypothesize that the transient potentiation of the majority of glutamatergic synapses in the nacore by the drug cue reduces the capacity of medium spiny neurons to process motivationally relevant stimuli arriving after the cue that would normally adaptively change the animal's behavior . In other words, because the pfc - nacore synapses normally coding a competing stimulus (as a shock would compete for the drug - conditioned cue) are already potentiated, these synapses will not properly code and transmit the information to dorsal striatal motor pattern circuits and will therefore not disrupt the ongoing behavior (lever pressing). Although the drug - associated cue is properly processed to initiate adaptive lever pressing, lever pressing becomes perseverative because competing stimuli cannot effectively modulate nacore neurons and disrupt the ongoing behavior . Thus, the already processed cue - induced lever pressing persists, and the drug cue becomes intrusive causing the animal to persistently relapse to drug seeking . Figure 2 provides a number of sites for pharmacological intervention to clinically evaluate in treating neuropsychiatric diseases characterized in part by intrusive thoughts . Indeed, every potential molecular target in figure 2 (glt-1, mglur2/3, mglur5, mmp, and integrins) has been examined pharmacologically in animal models and shown to reduce cue - induced drug seeking, and mmp inhibitors simultaneously prevent the induction of t - sp (none of the other targets have yet been examined in this regard). While many of these targets have generated compounds that have entered clinical trials for various disorders (eg, mglur2/3 agonist for schizophrenia, mglur5 antagonist for fragile x, and mmp inhibitors in cancer), only glt-1 has been studied in neuropsychiatric disorders characterized by intrusive thinking . Specifically, n - acetylcysteine (nac) restores glt-1 activity in the nacore in animal models of addiction and has been examined in clinical trials for treating drug addiction, gambling, trichotillomania, depression, ptsd (back se, unpublished data, 2016), and ocd . While nac reduced drug use in most of the studies, even when nac did not successfully reduce drug use or relapse, there was a positive reduction in the desire to use the drug (ie, intrusive thoughts related to drug use). Similarly, in a trial for comorbid ptsd and substance use disorder, nac reduced drug craving and ptsd, and was particularly effective at reducing the ptsd symptom domain of intrusive thoughts (back se, unpublished data, 2016). Thus, while a few trials with nac did not achieve the primary outcome of reducing or ameliorating the psychiatric disorder(s) being examined, when a measure of intrusive thinking is evaluated, such as drug craving in addiction, this endophenotype is consistently reduced by nac . The fact that nac does not appear to be curative in treating neuropsychiatric disorders, but rather ameliorates the domain of intrusive thinking, speaks to the design of future clinical trials evaluating nac or other compounds that restore glt-1 (see below). Thus, by reducing intrusive thoughts, patients will have greater opportunity to cognitively regulate the emergence of neuropsychiatric symptoms . For this reason, it seems logical to use nac in combination with other compounds with proven effectiveness in ameliorating a given disorder . For example, varenicline is the lead us food and drug administration - approved drug for treating cigarette addiction, and increases amygdala - accumbens resting state functional connectivity (rsfc), while nac increases pfc - accumbens connectivity, supporting a view that combining varenicline and nac may be especially beneficial in treating addiction to cigarettes . Similarly, antidepressants normalize amygdala hyperactivity in treating depression, pointing to the possibility that antidepressant treatment of elevated amygdala activity combined with pfc - accumbens normalization by nac might be beneficial . Although nac has a long history of clinical use as a mucolytic agent and in restoring glutathione after an acetaminophen overdose, and was therefore a convenient prototype for pilot clinical trials, it is not necessarily the ideal agent for treating intrusive thinking . Nac has a relatively short half - life, necessitating it be taken twice a day and thereby decreasing compliance in neuropsychiatric diseases such as addiction . Oral nac has relatively poor bioavailability to the brain, resulting in typical daily doses of 2 to 4 grams . Finally, nac has off - target effects, for example, as an antioxidant by promoting glutathione synthesis or as an activator of cystine- glutamate exchange . While no clinical trials to date indicate that these actions or the desired affect on glt-1 produce significant side effects compared with placebo, it is preferable to have a more specifically targeted action also, two other chemical scaffolds have been identified that produce compounds capable of activating glt-1 that can also be explored . -lactam antibiotics promote glt-1 in animal models of addiction or excitotoxic disease, such as amyotropic lateral sclerosis, and in animal models have been shown effective at reducing drug seeking . The other class of compounds restoring glt-1 are xanthine derivatives; notably, propentofylline reduced cue - induced cocaine seeking in an animal model of relapse . The capacity of thoughts to become intrusive has a biological substrate within the nucleus accumbens that is engaged by synaptic glutamate spillover and the widespread induction of t - sp at glutamatergic synapses . It is hypothesized that through this mechanism the accumbens becomes less responsive to stimuli that would normally compete with an intrusive thought and produce adaptive changes in thinking and behavior . Based on animal models of addiction, a key molecule that can be targeted to ameliorate cued drug seeking is the astroglial glutamate transporter glt-1 . Restoring glutamate transport inhibits reinstated drug seeking, and one compound capable of restoring glutamate transport, nac, has proven successful in many pilot double - blind clinical trials for treating neuropsychiatric disorders where intrusive thinking is an endophenotype that can trigger symptoms of the disorder . Thus, by targeting intrusive thinking, it may be possible to facilitate treatment for a number of neuropsychiatric disorders where intrusive thinking is a significant pathogenic characteristic, including drug addiction, stress disorders, eating disorders, gambling, ocd, and depression.
In western countries, breast cancer is a major concern and its incidence and mortality rates have been recently influenced by new therapeutic strategies . Our knowledge on cancer precursors, risk biomarkers, and cancer genetics has considerably increased, and prevention strategies are being successfully explored . Unfortunately, over the last decade, breast cancer prevention has been mainly focused on endocrine therapies using selective estrogen receptors modulators (serms) and aromatase inhibitors (ais). Available preventive strategies for nonhormonal breast malignancies, more frequently expressed in brca mutation carriers and, in general, in high - risk population, are needed . For these reasons, a great number of novel chemopreventative agents are currently under investigation in order to evaluate their efficacy in this particular cohort of patients . In accordance with their recognized role in the regulation of cell growth, differentiation, apoptosis, and their recognized inhibitory effect on cell growth in er positive and negative breast cancer cells, retinoids (either natural or synthetic compounds structurally related to vitamin a) have long been studied for their chemotherapeutic effect and for their chemopreventive potential in breast cancer setting . Only recently, retinoids have also been applied in this unaffected high - risk population and they have demonstrated to be able to suppress tumor promotion and modify some properties of fully transformed malignant cells by activating and/or repressing specific genes . Retinoids initiate ligand - induced dimerization of retinoid acid receptors (rar,, and) and retinoid x receptors (rxr,, and). Subsequently, receptors bind to retinoid response elements on dna, and they initiate transactivation of retinoids response target genes . Retinoid receptors are expressed in both normal and malignant breast epithelial cells and are critical for normal development . The mechanism by which retinoids inhibit breast cell growth has not been completely elucidated yet . Given the role played by rar- in the carcinogenesis of different tumors, its regulation by retinoids has also been advocated as a putative mechanism of action of these agents . However, they have been shown to affect multiple signal transduction pathways, including igf, tgf, and ap-1-dependent pathways [48], as showed in figure 1 . Several preclinical models suggest that retinoids inhibit mammary carcinogenesis in carcinogen - treated rats and in transgenic mice [911]. Recently, in order to reduce retinoids' side effects, rxr - selective retinoids, commonly known as rexinoids, have been studied as cancer preventive agents . In particular, preclinical studies have demonstrated that these compounds maintain retinoids' chemopreventive effect, but have greatly reduced toxicity . 9cra, a retinoid binding both rar and rxr, has significantly delayed the er - negative tumor development in sv40 tag mice and mnu - treated rats, although it induced significant cutaneous toxicity . In contrast, a rxr - selective retinoid, lgd1069, or bexarotene (targretin), has suppressed both er - positive and er - negative tumor development with minimal toxicity [14, 15]. One of the most promising retinoids to be used in chemoprevention trials is the synthetic amide of retinoic acid fenretinide, n-4-hydroxyphenyl retinamide (4-hpr) (figure 2). Its biologic activity was assayed by sporn et al ., who also showed the preferential accumulation of this drug in the breast, instead of in the liver . The inhibition of chemically induced mammary carcinoma in rats by fenretinide was first described in 1979 . Since then, promising in vitro data and a favorable toxicity profile compared with that of other retinoids have led to the extensive study of fenretinide in chemoprevention trials targeting different organs . Fenretinide has been found to have significant chemopreventive action in a large variety of in vitro and in vivo systems . Both fenretinide and its major metabolite, 4-metoxyphenyl retinamide (mpr), selectively accumulate in the human breast . It should be noticed that some fenretinide - based toxicities could be due also to its hydrolysis so that it returns to retinoic acid in vivo . High - dose fenretinide is cytotoxic for a variety of different tumor cells in preclinical studies [3537], although its accurate mechanism of action is not yet completely understood . However, it has been proposed that it might exert its inhibitory effects by means of both receptor - dependent and -independent mechanism (table 1, [1618]). Although rar influence on fenretinide action is highly debated, recent evidence would support the hypothesis according to which a mechanism does not require such relationship . In particular, we would stress the importance of the studies carried out by giandomenico et al ., explaining the role of the stable expression of the dominant negative rar; by anding et al ., showing that the use of a rar panantagonist influences an unhydrolyzable analogue of 4-hpr by inducing apoptosis with an independent rar signaling pathway; by delia et al ., assuming resistance to differential responsiveness is present in different cell lines thus indicating that fenretinide may act through different receptor types . Fenretinide characteristic feature is the ability to inhibit cell growth through the induction of apoptosis rather than differentiation, an effect that is strikingly different from that of the all - trans retinoic acid . Moreover, 4-hpr - mediated apoptosis seems to be tissue - specific, so that multiple mechanisms might operate within specific tissues . For example, in ovarian carcinoma cell lines, retinoids may induce apoptosis through the depolarization of the mitochondrial membrane and activation of caspase pathway [23, 24], while in the breast and in others cell lines apoptosis seems to be related with a direct molecular interaction with tubulin . Moreover, reactive oxygen species (ros), such as hydrogen peroxide and superoxide, seem to be critical in mediating apoptosis in different cancer cell types [2628]. The ability to increase ros levels, in particular nitric oxide (no) by no synthases (nos) over the elevation of sphingolipid ceramide levels, has been suggested as an explanation of the apoptotic effect of fenretinide . Recently, fenretinide has been shown to be able to induce no - mediated apoptosis in breast cancer (brca-1)-mutated breast cancer cells . Additional mechanisms are under investigations, such as the ability to inhibit cell growth by reducing the expression of growth - stimulating factors or by inducing the expression of growth - inhibitory factors . A recent proposed surrogate biomarker of fenretinide efficacy is circulating insulin - like growth factor 1 (igf1). The igf system plays a pivotal role in cell proliferation of both epithelial and mesenchymal tissues by stimulating mitosis, protecting cells from apoptosis, and maintaining transformed phenotype . Large prospective studies have shown that high circulating levels of igf1 and lower levels of its major binding protein (igfbp-3) are associated with a higher risk of developing subsequent premenopausal breast cancer and prostate, lung, and colorectal cancer [4447]. This indicates that circulating igf1 is a key regulator of cell and tumor proliferation for the vast majority of human epithelial cancer . Fenretinide has been shown to inhibit igf1-stimulated growth of breast cancer cell lines (bccls) and to downregulate the igf system in both er - positive and er - negative bccl . The expression of her2 has been recently observed to reduce fenretinide ability to induce apoptosis in breast cancer cells . Moreover, researchers found that her2 uses active human protein kinase (akt) to induce cyclooxygenase (cox-2) expression and that inhibition of akt or cox-2 increased 4-hpr induces apoptosis mediated by no production . Thus, a combination of 4-hpr with cox-2 inhibitors might be a new strategy to further investigate breast cancer chemoprevention . The 5-year administration of the milan study (see below) provided a vast corpus of information on the long - term safety and tolerability of this retinoid . As a major side effect, it induced a dose - related linear decrease of plasma retinol, associated with diminished retinal adaptation to darkness . In order to minimize this side effect, a 3-day treatment interruption at the end of each month was introduced to increase plasma retinol concentrations, allowing the partial recovery of retinal storage . However, an accurate and complete evaluation of toxicity was hampered by the lack of a placebo control group . Dermatological, gastrointestinal, visual, and ophthalmologic events were relatively frequent, but were mostly mild . In a recent analysis of the phase iii trial, the most common adverse events were diminished dark adaptation (cumulative incidence, 19%) and dermatologic disorders (18.6%), such as skin and mucosal dryness, pruritus, and urticaria . Less common events were gastrointestinal symptoms (13%) and alterations of the ocular surface (10.9%). Women in the control group complained of diminished dark adaptation, dermatologic disorders, gastrointestinal symptoms, and alterations of the ocular surface in 2.9%, 2.9%, 5.4%, and 3.2% of the cases, respectively . Interestingly, most side effects decreased with time and were significantly more frequent in postmenopausal women . Importantly, in contrast to other retinoids, prolonged administration of fenretinide is not associated with significant alterations of bone mineral density of the forearm . However, a trend towards an increase in bone resorption markers suggests the need for further assessment at different skeletal sites . After the completion of a phase i dose - ranging study, the 200 mg daily dose was chosen as the safest dose for prevention, as one case of a pathological electroretinogram after a 24-week administration was observed with the 300 mg / day dose . Higher doses, up to 400 mg, have been used in women with metastatic cancer in combination with tamoxifen, with no evident toxicity on liver and lipid profile, but with an increased incidence of nyctalopia [53, 54]. Peak levels of 4-hpr occur at approximately 6 h in adults with terminal half - life of approx . Since both 4-hpr and 4-mpr are selectively accumulated in the breast, evaluation of fenretinide as a chemopreventive agent in breast cancer has been particularly attractive . The most important clinical trials with fenretinide are mentioned in table 2 . As in the therapeutic setting, where drugs combinations are superior to monochemotherapy, the concept of combining agents with different mechanisms of action in the attempt to increase efficacy while minimizing side effects is a rational approach in chemoprevention . In preclinical models, combined administration of fenretinide and tamoxifen has proven additive and synergistic in both growth inhibition of mcf-7 cells and prevention of mnu - induced mammary carcinomas . Moreover, the activity of 9-cis - retinoic acid against mnu - induced mammary tumors in sprague - dawley rats is enhanced by the combination with tamoxifen or raloxifene . The safety and the tolerability of the combination of fenretinide and tamoxifen have been investigated in clinical trials in metastatic breast cancer patients and in women at increased risk . The concept of combining agents with different mechanisms of action in the attempt to increase efficacy on complementary molecular targets, while minimizing side effects is increasingly being pursued in breast cancer chemoprevention . A clinical randomized, double - blind, placebo - controlled phase iib trial with a 2 2 factorial design to test this interaction (fenretinide and low - dose tamoxifen) was conducted at the european institute of oncology . In spite of the favorable effects on plasma igf - i levels and mammographic density, this combination did not reduce breast neoplastic events compared to placebo, whereas both single agents, particularly fenretinide, showed numerical reduction in annual odds of breast neoplasms . Fenretinide (in combination with hrt) was also studied by our group in 226 postmenopausal healthy women, randomized in a two - by - two factorial design to either oral cee 0.625 mg / day or transdermal e2, 50 microg / day and to fenretinide 100 mg / twice a day or placebo for 12 months . Oral cee showed more favorable changes than transdermal e2 on circulating breast cancer risk biomarkers, while fenretinide exerted little modulation on most biomarkers . The most important study where 4-hpr was administrated as a single agent is a multicentric phase iii randomized trial, coordinated by the istituto nazionale dei tumori in milan, started in 1987 . Stage i (t1 - 2 n0) breast cancer patients, aged 3370 years, who had been operated on for breast cancer within the previous 10 years and had received no systemic adjuvant therapy were eligible . Women were randomly assigned to receive either no treatment or fenretinide given orally at a dose of 200 mg / day for 5 years . A placebo - control arm was not included in the study design because of the large capsule size and the objective nature of the main outcome measure . A 3-day drug stoppage at the end of each month was recommended in order to allow retinol recovery and to minimize dark adaptation impairment . The main outcome measure was the occurrence of contralateral breast cancer as first malignant event . The secondary endpoint was the incidence of ipsilateral breast cancer reappearance, defined as local recurrence in the same quadrant or the occurrence of a second breast malignancy in different quadrants from the primary tumor . Fenretinide showed no effect on contralateral breast cancer occurrence and a nonsignificant 17% reduction in ipsilateral breast tumor reappearance . However, a different effect was noted when the analysis was stratified by menopausal status, with a beneficial trend in premenopausal women on both contralateral and ipsilateral breast cancer (38%) and a reversed trend on contralateral breast cancer in postmenopausal women, as highlighted in figure 3 . Importantly, the protective effect persisted for up to 15 years (i.e., 10 years after retinoid cessation). Most notably, the younger the women were, the greater the benefit of fenretinide . Such benefit was associated with a remarkable 50% risk reduction in women aged 40 years or younger, whereas the benefit disappeared after 55 years of age . Interestingly, the incidence of ovarian cancer during the 5-year intervention period was significantly lower in the treatment arm . This effect has been confirmed in an important update . This phase iii trial suggested a possible role of fenretinide as a preventive agent acting at different levels of breast carcinogenesis . Admittedly, the results obtained during the phase iii trial on our subgroups had not been foreseen when the study was planned . While there are plausible biological explanations for this selective effect, our findings are hypothesis generating and do not have immediate practical clinical implications, although they do provide the rationale for testing the drug's efficacy in premenopausal women . Moreover, this protective effect was suggested in women with a high probability of carrying a brca-1 mutation . Indeed, fenretinide was highly effective in inhibiting the growth of brca-1 mutated breast cancer cell lines . When considering the protective activity of fenretinide on second breast cancer in young women and a similar trend on ovarian cancer, at least during intervention, it appears that women with germline brca-1 and brca-2 mutations may be ideal candidates for further investigation of this drug . All the collected data as well as fenretinide characteristics make this drug an excellent candidate for chemoprevention of the highlighted subgroup, that is, young healthy women with a high susceptibility to early - onset breast and ovarian cancer, such as brca1/2 mutation carriers or women with a significant familial risk . Since the drug activities are probably not strictly influenced by hormonal responsiveness, it may affect also hormone nonresponsive cancers several drugs used in prevention settings are usually the same as those used for treatment (adjusting dosage and/or route of administration). This is possible because their mechanism of actions is also active on early - phase carcinogenesis and not only on the inhibition of the tumor growth . This is confirmed by the reduction of second breast cancer found in the reported studies of veronesi et al . [40, 41]. These data make 4-hpr a surrogate marker of primary prevention and a favorable effect of the drug would provide a strong rationale for a primary prevention trial in unaffected women at high risk for breast cancer . Moreover, because its action does not seem to be influenced by the hormonal status, fenretinide might be active in hormone nonresponsive cancer prevention (as occurs in brca-1 mutation carriers). Although we obviously need to verify this hypothesis, we think it is an intriguing scenario that could be important in order to identify new pathways related to the efficacy of this drug . Our division of cancer prevention and genetics at european institute of oncology to milan has already activated a new phase iii prevention trial addressed to this particular cohort of women . A total of 764 healthy women at increased breast cancer risk will be randomized to 4-hpr 200 mg / day versus placebo for 5 years . The subjects will be stratified by participating center and breast cancer risk (brca1 mutation versus brca2 versus high risk subjects). The accrual estimated time is five years . The design of the study is explained in figure 4 . The aim of the proposed trial is to assess the efficacy of fenretinide, in reducing the incidence of breast cancer in healthy young premenopausal women at increased familial / genetic risk for breast cancer; the primary endpoint is to assess the incidence of histologically diagnosed invasive breast cancer and ductal intraepithelial neoplasia . Secondary endpoints are the incidence of other noninvasive breast disorders (i.e., intraepithelial lobular neoplasia and atypical hyperplasia), ovarian cancer and other cancers . Moreover, we propose an interdisciplinary research study to further investigate the mechanisms of action of fenretinide in preventing breast cancer . Early intermediate biomarkers of efficacy after 12, 36, and 60 months of treatment, genetic interactions with breast cancer risk modifiers will be explored with the primary goal to identify molecular biomarkers of response prediction . In particular, we will evaluate the percentage change in circulating biomarkers of the igf system, androgens, retinol binding protein (rbp-4), insulin, blood glucose, and vegf after 12, 36, and 60 months of treatment . In a subgroup of participants, fine needle aspirate breast biopsy or cells obtained from breast ductal lavage will be drawn at baseline and after a 12-month treatment and the percentage change in rar expression correlated with apoptosis (caspase-3) and proliferation (ki-67). Genotyping of single - nucleotide polymorphisms (snps) linked to breast cancer risk (mthfr, comt, gh, igfbp-3, ar, and tgf- genes), degree of methylation of rassaf1 and rar, and circulating progranulin will be assessed . The results will be correlated with mammographic instrumental measurements, plasma and tissue biomarkers after 1-year treatment . Should the results of this trial confirm that fenretinide is effective in reducing breast and ovarian cancer incidence in this very high risk population, that this effect lasts for many years after treatment, and that the tolerability profile is good, we will have a further preventive chance and a new risk reduction strategy.
Carpal tunnel syndrome (cts) is the most common entrapment neuropathy1,2,3, caused by compression and traction of the median nerve at the level of the carpal tunnel, which is a cylindrical, inelastic cavity, delimitated by the carpal bones and transverse carpal ligament4, 5.the compression results in impaired nerve conduction, paresthesia, and pain, which worsen at night and often wake the patient6, 7 . The treatment for cts can be broadly divided into surgical and non - surgical approaches . According to the findings of several important randomized controlled trials, carpal tunnel - release surgery is effective in 7075% of patients, but is relatively invasive and can be accompanied by complications10 . Furthermore, recurrence after surgery, although uncommon, can be difficult to treat11 . Moreover, surgical decompression leaves 8% of patients in a worse condition than they were previously12, and persistent pain in the scar or proximal palm 5 years after numerous non - surgical, less - invasive treatment options are available, including oral medication, splinting, exercise, corticosteroid injections, and mobilization interventions14,15,16,17 . Only a few complications result from non - surgical approaches in those with mild - to - moderate cts . Thus, non - surgical treatment might be indicated for patients with cts when surgery is not desired or for any other reason not immediately indicated18 . To the best of our knowledge, surgical decompression or carpal tunnel release . However, there is currently limited evidence indicating any benefit from splinting, exercise, and mobilization15, 16 . This study aimed at prospectively assessing the effectiveness of nocturnal splinting by using clinical scores and ncs . Of the 66 consecutive outpatients to our neurological clinic, 41 patients who met the inclusion criteria were finally enrolled (fig . All individuals provided informed consent to participate in this study, and the local ethics committee of the hospital approved this study . The inclusion criteria of the report of the quality standards subcommittee of the american academy of neurology were implemented19, whereby all the patients must meet at least one of the first 2 items and one of the remaining 5 items below to be included in the study:19, 20 (1) numbness in the median nerve territory or the whole hand; (2) pain or hypesthesiain the hand; (3) awakened from sleep by numbness or pain mid - night or early in the morning; (4) numbness relieved by shaking the hand and aggravated by flexing the wrist, or numbness becoming more severe in winter than in summer; (5) weakness of the hand; (6) atrophy of the thenar muscle; and (7) positive phalen or tinel sign . In this study, patients with the following conditions were excluded: (1) clinical or / and electrophysiological findings suggesting ulnar nerve lesion, cervical radiculopathy, polyneuropathy, systematic diseases (e.g., diabetes mellitus, hypothyroidism, and rheumatoid arthritis), and stroke20; (2) cts caused by wrist trauma or deformity20; (3) prior treatment for cts through any approach, either surgical or non - surgical; and (4) severe weakness or atrophy of the hand, and no response of compound muscle action potentials (cmap) elicited from the muscle of the abductor pollicis brevis (mapb) on ncs . The follow - up was completed in 20 patients with splinting (fig . 1). This study had a prospective design recommended by american association of neuromuscular and electrodiagnostic medicine (aanem)21 . All the subjects enrolled in this study underwent electrophysiological detection after oral informed consent was obtained . Electrophysiological detections were completed on keypoint 4 (detec, denmark) by one examiner . While ncs was performed, surface electrodes were used for stimulating or recording . While recording cmap, the median and ulnar nerves were both stimulated at the wrist . While recording the sensory nerve action potentials (snap), the median and ulnar nerves were also stimulated at the wrist, and ring electrodes were placed at digits 2 and 5, respectively . In addition, for comparing the difference in distal sensory latency (dsl), the median and ulnar nerves were stimulated at the wrist, recording the snap at digit 4 . The following parameters were analyzed: the distal motor latency (dml) from the wrist to mapb or abductor digiti minimi; the sensory conduction velocity (scv) from the wrist to digits 2, 5, and 4; and the dsl . Needle electromyographic detection was performed to observe abnormal spontaneous activities only when the amplitude of the cmap of mapb reduced . Considering stevens (aanem) and our previous study20, 22, the following electrodiagnostic criteria for cts were used in this study: (1) scv (wristdigit 2) <40.0 m / s or scv (wristdigit 4) <43.5 m / s; dml (wrist to mapb) 3.7 ms (distance 5.56.5 cm); or dsl 0.4 ms; and (2) normal sensory or motor conduction in the ipsilateral ulnar nerve . Symptom severity scale (sss) and functional status scale (fss) were used to evaluate the patients at each hospital visit23 . Sss consists of 11 questions with multiple - choice responses, which includes 6 critical domains for the evaluation of cts: pain, paresthesia, numbness, weakness, nocturnal symptoms, and over - all functional status . Fss consists of 8 functional activities, including writing, buttoning of clothes, household chores, and bathing and dressing . Each question was scored 1 point (mildest / no difficulty with the activity) to 5 points (most severe / unable to perform the activity). The overall score was calculated as the mean of the scores for all the individual items . Items that were left unanswered and if not applicable, they were not included in the calculation of the overall score23 . All the subjects were instructed to make stereoplasm splints of approximately 912 cm 57 cm size by themselves and bring them to us for assessment . Subsequently, they were asked to wear each splint on the dorsal and palmar surface of the hand, centered at the distal wrist crease, immobilizing the wrist in the neutral posture at bedtime, keeping the fingers relatively free.the wrists were fixed to ensure that they were neither too tight nor too loose . Meanwhile, the subjects were instructed to avoid flexing their wrists during any daytime activities such as washing clothes, riding a bicycle, working on a computer, and carrying heavy grocery bags . The follow - up was completed in 20 patients (31 wrists) with splinting . All statistical analyses were performed using statistical package for social sciences (spss) for version 17.0 (spss china, shanghai). One - sample t - test was used to compare the values with known population means, and paired - sample t - test was used to compare paired measurement data . In bivariate correlations, data of normality was analyzed by person correlations, else by spearman correlations . The shapiro - wilk test was used to test the normality of the measurement data . Values of p<0.05 were considered statistically significant; * represents p<0.05, * * p<0.01 and * * * p<0.001 . Of the 41 patients who met the inclusion criteria, 90% were female, and the mean age of all patients was approximately 50 years old . The clinical data are presented in table 1table 1.clinical data of the subjectsall 41 patients20 patients with splintingage (years)2072 (50.2 12.0)2672 (52.5 12.6)illness duration (years)1.1 1.71.5 2.3male / female (wrists)4 (5)/37 (59)1 (1)/19 (30)bilateral hands affected (cases)2411right hand affected (cases)114left hand affected (cases)6 (4 left - handed)5 (4 left - handed)numbness (or pain) at night (cases)216tinel sign (wrists)1811phalen sign (wrists)2014 . In 20 patients (31 wrists) with cts treated by splinting, sss (1.77 0.38, 1.55 0.38; t=5.956, p=0.000) and fss (1.53 0.31, 1.40 0.27; t=5.452, p=0.000) decreased . In addition, dml (4.53 1.25, 4.14 0.76; t=2.431, p=0.021) shortened and dsl (1.24 0.61, 0.97 0.60; t=2.978, p=0.006) decreased significantly after splinting (table 2table 2.comparison of clinical scores and ncs findings of splinting in 20 patients (31 wrists)parametersbefore splintingafter splintingclinical scoressss1.77 0.381.55 0.38fss1.53 0.311.40 0.27parameters of ncsdml (ms)4.53 1.25 4.14 0.76scv of wrist digit 2 (m / s)42.02 8.29 42.12 7.58scv of wrist digit 4 (m / s)38.20 6.72 38.51 6.42dsl (ms)1.24 0.610.97 0.60ncs: nerve conduction studies; sss: symptom severity scale; fss: functional status scale; dml: distal motor latency; scv: sensory conduction velocity; dsl: sensory latency difference between median and ulnar nervep<0.05, * p<0.01, * * * p<0.001). There was no improvement in the clinical scores in 9 patients (14 wrists, 45.8%) after splinting . There were significant correlations between sss and dml (r=0.420, p=0.019), scv of wrist digit 2 (r=0.425, p=0.017), and scv of wrist digit 4 (r= 0.519, p=0.003). No correlations were noted between sss and dsl (p>0.05), and between fss and all the parameters of ncs (p>0.05) (table 3table 3.correlation of clinical scores with ncs findings in 20 patients treated by splinting dml scv of wristdigit2 scv of wristdigit4dslsss0.4200.4250.519 * 0.189fss0.1920.1750.3190.124values represent correlation coefficientsp<0.05). Ncs: nerve conduction studies; sss: symptom severity scale; fss: functional status scale; dml: distal motor latency; scv: sensory conduction velocity; dsl: sensory latency difference between median and ulnar nerve p<0.05, * * p<0.01, * * * p<0.001 values represent correlation coefficients in this study, 90% of patients with cts were female, and the mean age was approximately 50 years, which is consistent with the epidemiological data3 . Studies have shown that females with the highest body mass index (bmi) are more likely to develop cts24, 25 . A bmi 30 kg / m almost doubled the risk of cts, and when bmi was assessed as a continuous variable, the hazard ratio increased approximately linearly with increasing bmi24 . Splinting is the most common method among the non - surgical treatments available for cts7 . In this study, a self - administered questionnaire was used to assess the severity of symptoms and functional status in cts, as questionnaires are reproducible, internally consistent, valid, and responsive to clinical change23 . The questionnaire for clinical evaluation was subjective, but it can be semi - quantitative . We recommend that the questionnaire should be used at least in the clinical research on cts, so that the outcome of each researcher is relatively comparable . In this study, both sss and fss were 3 points, which suggested that the condition in most patients was not severe . In a previous study, 89% of patients with severe cts experienced recurrence of the symptoms within 1 year after conservative treatment, whereas only 60% of patients with mild cts experienced recurrence26 . Therefore, the conservative approach is more successful in patients with mild nerve lesion than with severe cts . Conservative management with splinting should be initiated in patients with cts with only mild symptoms12 . Splinting is an acceptable method for patients in the early phases of cts, as it is simple and inexpensive and can be used at home . In this study, sss and fss decreased significantly, dml shortened, and dsl decreased . Moreover, 54.2% of patients with cts were effective after neutral wrist nocturnal splinting . Neutral wrist splinting can reduce the pressure on the median nerve and increase blood flow, especially when the wrist is held in a flexion position at night27 . In a review assessing the effectiveness of conservative therapy for cts, it was reported that splinting is an effective therapy especially when used for the whole day28 . No improvement was noted in the clinical scores of almost half the patients in this study . This could be because the splints made by the patients may not have immobilized the wrist . Additionally, patients low compliance for the complex course of splinting may have influenced the therapeutic effect, although we emphasized that the patients should follow our guidelines strictly . Splinting reduces latency, suggesting that the intervention may alter the underlying pathophysiological course of cts4 . Moreover, there was no correlation between sss and dsl, and between fss and the parameters of ncs . Thus, there was a weak correlation between the clinical scores and ncs, which is consistent with the findings of a previous study29 . Therefore, the use of both clinical scores and ncs may allow us to evaluate the therapeutic effect of splinting on cts through different aspects . Overall, conservative approaches such as splinting have a negligible incidence of serious complications and should be used more widely12 . Providing patients the facts about their conditions and nevertheless, patients with cts who do not show satisfactory improvement with non - surgical treatment should be offered surgery30 . The therapeutic effect of combination splinting and other conservative treatments on cts were not compared, and the number of patients enrolled in this study was small, which were the main limitations in this study . Moreover, patients made the splints by themselves, which may cause a variation in the therapeutic effect among individuals, although the splints that they made were checked . In conclusion, neutral wrist nocturnal splinting is effective in at least the short term in patients with cts . There is a weak correlation between the clinical scores and ncs, which suggests that both approaches should be used to assess the therapeutic effect of treatment on cts.
The oral environment is particularly favorable to metal biodegradation due to its chemical, thermal, microbiological and enzymatic conditions . When in contact with saliva and acid / basic foods at different temperatures, the metal components of orthodontic appliances may undergo corrosion . In addition, the orthodontic treatment, especially with fixed appliances, causes specific alterations in the oral cavity, including decrease of ph, increase of dental biofilm accumulation and elevation of the salivary microbial levels, which are favorable conditions to the occurrence of corrosion . Corrosion of orthodontic appliances may have serious clinical implications that range from loss of dimension of the appliance's components, which results in application of lower force to the teeth, up to possible failure of the appliance due to tension corrosion . In the corrosive process, the released metallic ions may get in contact with cells and tissues in the contiguous environment or pervade throughout the body . Absorption of toxic products released during corrosion of metallic orthodontic components by body tissues is undesirable . In dentistry, corrosion may manifest as biological, functional and esthetic effects, the biological being the most significant . Corrosion in orthodontic appliances has been extensively investigated by analysis of ion release both in vitro and in vivo . Considering that fixed orthodontic appliances may act as retention niches for microorganisms and are susceptible to corrosion, the development of in situ studies evaluating corrosion from a clinical standpoint is of paramount importance . This study evaluated in situ the corrosion of the metallic components of haas expanders by stereomicroscopy, scanning electron microscopy (sem) and energy dispersive x - ray spectroscopy (eds). Thirty - four patients of both sexes (14 male and 20 female) aged 6 to 12 years (mean age = 9 years and 7 months) from the orthodontic clinic of the ribeiro preto dental school, university of so paulo, brazil, who needed maxillary expansion with a haas expander for correction of posterior crossbite were enrolled in this study . The children were randomly assigned to two groups of 17 individuals each (groups i and ii). Patients who were using antimicrobial mouthwashes, presented any systemic disease and/or had used antibiotics within the previous 3 months were excluded from the trial . This research project was approved by the local ethics in research committee (process number 2006.1.391.58.9) and written informed consent was obtained from the parents or guardians . For construction of the haas expanders, the permanent first molars were banded and the metallic bands were silver welded (dental morelli, sorocaba, sp, brazil) to 0.9 mm stainless steel orthodontic wires (dentaurum, ispringen, germany). The active component was an 11 mm expanding screw (dental morelli, sorocaba, sp, brazil). The bands were cemented with zinc phosphate cement (ls cement; vigodent s / a indstria e comrcio, rio de janeiro, rj, brazil) and the orthodontic wires were fixed to the primary canines or molars with light - cured composite resin (tph; 3m / espe, st . After correction of the posterior crossbite, which lasted 20 days on average, the orthodontic appliance was maintained for additional 3 months in the mouth as a retention period . During the time of use of the appliances, the patients of group i were instructed to brush their teeth with a fluoride dentifrice 3 times a day and not to use any antimicrobial mouthrinse solution . For patients of group ii, in addition to toothbrushing with a fluoride dentifrice, 1-minute mouthrinses with 10 ml of a 0.12% chlorhexidine gluconate solution (periogard; colgate - palmolive company, new york, ny, usa) were prescribed twice a week (tuesdays and fridays). On tuesdays, mouthrinsing was performed at the dental school under the researcher's supervision, while on fridays mouthrinsing was performed at the child's home under parental supervision . After removal from the mouth, the fixed appliances of both groups were cut with a sterile pair of pliers (orthopli 021; dentaurum, pforzheim, germany) on either the right or the left side, according to a table of random numbers, in order to obtain a fragment containing a sample of the wire / silver brazing / band region . The fragments were placed in a 300-mm desiccator (vidrolabor, paulnia, sp, brazil) containing silica gel as an indicator of humidity for further analysis of the corrosive process . After desiccation, the specimens were examined with an optical stereomicroscope (leica l2; wetzlar, germany) coupled to a digital camera (sony cyber shot 3.3 mega pixels dsc-575, tokyo, japan) at x40 magnification to obtain images of the soldering point areas (silver brazing / band, silver brazing / wire and wire / silver brazing / band), as suggested by wichelhaus, et al . The images were analyzed with respect to the presence of areas with color change due to oxidation, suggestive of corrosion . This type of qualitative analysis has been described in several studies and allowed delimiting the areas to be examined by sem and eds . Data referring to the presence (+) or absence (-) of color change suggestive of in situ corrosion in both groups were compared by fisher's test using the sas (statistical analysis system, institute inc ., the analysis was undertaken with a scanning electron microscope (evo 50 ep; carl zeiss smt ag, germany) operating at 20 kv coupled to an energy dispersive x - ray spectrometer (ixrf systems inc ., houston, tx, usa), which allows analyzing the predetermined area at x60 to 194 magnification with four - quadrant electron - backscatter detectors (qbsd) using a pre - determined depth of penetration of the electron beam . This analysis was performed to identify the peaks of the following chemical elements: nickel (ni), iron (fe), chromium (cr), oxygen (o), carbon (c) and phosphorus (p) in the region delimited in the fragments of the appliances of groups i and ii . Graphs of the peaks of these chemical elements were obtained using the edx/2004 software . The analysis of the images obtained under optical stereomicroscopy revealed areas of color change suggestive of corrosion in the soldering point areas (wire / silver brazing / band region) in all specimens of both groups (figure 1a and b), with no statistically significant difference (p=1). A: stereomicroscopic analysis of a representative group i specimen showing color change in the band / silver brazing / wire joint . 20 magnification . B: close - up view of a showing color change suggestive of corrosion at the band/ silver brazing / wire joint . 40 magnification . C: scanning electron microscopy (sem) micrograph showing the band / silver brazing / wire joint to be analyzed by energy dispersive x - ray spectroscopy (eds). D: areas of ag solder (a), material - to - material interface (b) and orthodontic wire (c) analyzed by eds with four - quadrant electron backscatter detectors qbsd detectors the regions suggestive of corrosion were submitted to sem examination (figure 1c) with eds analysis (figure 1d). Graphs representative of the qualitative readings of the specimens of groups i and ii are presented in figures 2 and 3 . Quantitative energy dispersive x - ray spectroscopy (eds) reading representative of areas of color change suggestive of corrosion in the soldered region in contact with the band and the wire in a group i specimen (control) quantitative energy dispersive x - ray spectroscopy (eds) reading representative of areas of color change suggestive of corrosion in the soldered region in contact with the band and the wire in a group ii specimen (periogard mouthrinse) in both groups, the soldered areas in contact with the band and the wire presented fe, cr, o, c and p peaks . Figure 4 illustrates the reading obtained in a non - assayed region of an orthodontic wire of a haas expander . No area of corrosion was visible under optical stereomicroscopy, demonstrating how the peaks of chemical elements behave in this situation . Several studies have currently evaluate the resistance to corrosion of orthodontic wires and brackets in an attempt to find materials with greater compatibility with the oral environment . However, most studies are in vitro experiments that are performed with artificial saliva and methodologies that simulate the oral conditions, reaching results that cannot be extrapolated to the clinical situation . The present investigation was designed as a randomized clinical trial because it is the " golden standard " of studies that evaluate the clinical efficacy of materials and treatment techniques . Soldering is still the prevailing joining technique in the dental laboratory, although problems arise from the susceptibility to corrosion and the low strength of soldered joints . In the flame soldering technique, the wire elements are heated directly, so that the temperature of the wire mostly rises to levels exceeding the fusing temperature of the solder . Under these conditions, intergranular corrosion occurs due to the change in the composition of grain contours deriving from phase precipitations, concentration of impurities or elementary cr depletion close to the areas of grain contours . The results of the present study showed the occurrence of failure in the flame - soldered joints between the stainless steel wires occurred during the construction of the haas expanders . (2002) who evaluated in vitro different soldering techniques produced by various dental technicians and observed incomplete filling of the soldering gap, porosities resulting from the production process, local structural changes due to overheating and deficient alloying at the solder margin . The eds analysis qualifies chemically the composition of a certain material, being a complementary method to evaluate the presence of corrosion . Under ideal conditions, fe and cr peaks in the soldered joint graphs with high fe peaks and medium cr peaks and presence of o suggest the occurrence of corrosion due to formation of fe2o3 or hydrated ferric oxide (fe2o3 x h2o), indicating rust . The presence of p with high fe peaks and medium cr peaks suggests the occurrence of a process corrosive by formation of iron phosphate (fepo4). In the present study, peaks of these elements were observed in the soldered joints in all specimens of both groups . The deterioration of orthodontic appliances has been a matter of concern in orthodontic research due to the potential adverse biological reactions associated mainly to ni release . In the present in situ study, the eds analysis did not reveal ni peaks in the soldered joints of any of the haas expanders worn for approximately 4 months . Although no increase in the ni levels were observed, which would indicate a corrosive process, the presence of corrosion was detected on examination of the images under optical microscopy . This situation is further aggravated if one considers two other conditions that may contribute to potentialize corrosion: the mouth is an aggressive oral environment to the various types of sensitized alloys and crevice corrosion is intensified where gaps and/or cracks develop . In addition to intergranular and crevice corrosion, the stainless steel becomes susceptible to the corrosive process, since the alloy has its structure altered and the passivation film cannot be reestablished . This permits the occurrence of different types of corrosion: fatigue corrosion, which occurs frequently in orthodontic wires submitted to long intraoral periods under load; microbiological corrosion, which causes color change in the presence of microorganisms due to action of sulfur or reduction of sulfates to hydrogen sulfide and formation of insoluble dark sulfites, in addition to pitting (corrosive attack that occurs in points or small areas) and deposits that may occur in the presence of microorganisms; and galvanic corrosion, which is the deterioration of an alloy by transference of electrons due to difference of potential between two materials in the presence of a common electrolyte . The c peaks observed in the eds analysis were greater in group i, possibly due to the greater accumulation of bacterial biofilm . The p peaks were very expressive in all specimens, possibly originating from the saliva and/or the zinc phosphate cement used for cementation of the orthodontic bands . The presence of ag peaks in the graphs of soldered joints distant from material - to - material interface only suggests the presence of the metal used as a soldering material . Although the methodology of the present study does not allow determining accurately which factors caused corrosion, alterations occurred in the wire / silver brazing / band joints, since a dark line suggestive of corrosion was observed on the alloy by optical microscopy and sem, though without significant difference between the groups . Under the conditions evaluated in this randomized clinical study, it may be concluded that there were color change and peaks of chemical elements suggestive of corrosion in all specimens of both groups in the soldering point areas between the wire, silver brazing and bands of the haas expanders, with no difference between the groups . These results suggest that the use of chlorhexidine mouthrinses did not influence the occurrence of corrosion in situ.
Kartagener's syndrome (ks) is a subset of a larger group of ciliary motility disorders called primary ciliary dyskinesias (pcds). It is a genetic condition with an autosomal recessive inheritance, comprising a triad of situs inversus, bronchiectasis and sinusitis . Although siewart first described this condition in 1904, it was kartagener who recognized the etiological correlation between the elements of the triad and reported four cases in 1933 . The estimated prevalence of pcd is about 1 in 30,000, though it may range from 1 in 12,500 to 1 in 50,000 . In ks, the ultrastructural genetic defect leads to impaired ciliary motility which causes recurrent chest, ear / nose / throat (ent), and sinus infections, and infertility . A high index of suspicion is needed to make an early diagnosis so that timely treatment options may be offered for infertility in these young patients, wherever feasible . Also, although unproven, it seems likely that early diagnosis is important for the preservation of pulmonary function, quality of life, and life expectancy in this disease . This was a 34-year - old non - smoker male, born to non- consanguineous parents . He presented to the outpatient with chief complaints of recurrent episodes of common cold, sneezing, and cough with expectoration for past 10 years, exertional shortness of breath for last 5 years, and not having children despite being married for last 14 years . The patient also revealed that he frequently developed cough, cold, rhinorrhea, nasal blockade, and ear discharge during childhood . He received anti - tubercular treatment for these complaints 5 years back with no relief . Chest x - ray at that time was done, but was not available . On examination, physical examination revealed grade 2 digital clubbing and apex beat on the right side in fifth intercostal space . On auscultation, bilateral wheeze and right basal crackles were audible, with heart sounds being best heard on the right side of the chest . Chest x - ray postero - anterior (pa) view [figure 1a] revealed cardiac apex and aortic arch on the right side, suggesting dextrocardia along with left - sided bochdalek's hernia . An ultrasound of the abdomen revealed a normal liver and gall bladder on the left side and a normal spleen on the right side . Contrast - enhanced computed tomography (cect) chest [figure 1b] revealed dextrocardia, nodular opacities in right lower lobe suggestive of bronchiectasis, and left - sided bochdalek's hernia containing omentum and large bowel loops . A semen analysis revealed azoospermia, while the hormone profile was suggestive of hyperprolactinemia with normal luteinizing hormone and follicle stimulating hormone levels . (a) chest x - ray pa view showing dextrocardia and left - sided bochdalek's hernia; (b) cect - chest showing right - sided bronchiectasis, dextrocardia, and left - sided bochdalek's herina this was a 55-year - old non - smoker female . She presented to us with chief complaints of recurrent cold, cough with copious expectoration, anosmia, headache, and progressively increasing shortness of breath for last 30 years . She received anti - tubercular treatment for these complaints 12 years back for 6 months, with no relief . Her past history was significant as she had frequent visits to the pediatrician for recurrent chest infections . The patient had a blood pressure of 110/72 mmhg, pulse rate 92/minute regular, and oxygen saturation 76% on room air . General examination revealed bilateral pedal edema, raised jugular venous pressure, facial puffiness, and grade 3 clubbing . On auscultation, x - ray paranasal sinuses revealed mucosal thickening with hazy sinuses [figure 2a]. Chest x - ray [figure 2b] and high - resolution computed tomography (hrct) thorax revealed bronchiectasis and situs inversus . (a) x - ray pns showing opacified maxillary sinuses; (b) chest x - ray pa view showing dextrocardia and left - sided bronchiectasis a 40-year - old male patient, non - smoker, and born to non - consanguineous parents presented with recurrent productive cough, rhinorrhea, and headache since last 20 years with episodic fever and worsening of symptoms . He had been previously treated with antibiotics, antihistamines, bronchodilators, inhaled and oral corticosteroids, and even anti - tuberculous drugs, but the response was only partial and temporary ., he was febrile with nasal discharge, wheezy chest, and bilateral coarse crackles . Chest x - ray [figure 3a] showed cystic bronchiectactic changes in the lower and mid zones with dextrocardia . Ultrasound of the abdomen showed spleen on the right side of the abdomen, while liver on the left was suggestive of complete situs inversus . (a) cystic bronchiectactic changes in the lower and mid zones with dextrocardia; (b) axial ct image abdomen showing situs inversus with the liver and ivc on the left and the spleen and aorta on the right; (c) axial ct image paranasal sinuses showing mucosal thickening and opacified sinus cavities axial ct - chest showed dextrocardia with the inferior vena cava (ivc) and morphologic right ventricle on the left and the left ventricle on the right . Axial ct - abdomen showed situs inversus with the liver and ivc on the left and the spleen and aorta on the right figure 3b . Hematological and biochemical parameters were within normal limits except semen analysis which showed oligospermia with immotile live sperms . This was a 34-year - old non - smoker male, born to non- consanguineous parents . He presented to the outpatient with chief complaints of recurrent episodes of common cold, sneezing, and cough with expectoration for past 10 years, exertional shortness of breath for last 5 years, and not having children despite being married for last 14 years . The patient also revealed that he frequently developed cough, cold, rhinorrhea, nasal blockade, and ear discharge during childhood . He received anti - tubercular treatment for these complaints 5 years back with no relief . Chest x - ray at that time was done, but was not available . On examination, physical examination revealed grade 2 digital clubbing and apex beat on the right side in fifth intercostal space . On auscultation, bilateral wheeze and right basal crackles were audible, with heart sounds being best heard on the right side of the chest . Chest x - ray postero - anterior (pa) view [figure 1a] revealed cardiac apex and aortic arch on the right side, suggesting dextrocardia along with left - sided bochdalek's hernia . An ultrasound of the abdomen revealed a normal liver and gall bladder on the left side and a normal spleen on the right side . Contrast - enhanced computed tomography (cect) chest [figure 1b] revealed dextrocardia, nodular opacities in right lower lobe suggestive of bronchiectasis, and left - sided bochdalek's hernia containing omentum and large bowel loops . A semen analysis revealed azoospermia, while the hormone profile was suggestive of hyperprolactinemia with normal luteinizing hormone and follicle stimulating hormone levels . (a) chest x - ray pa view showing dextrocardia and left - sided bochdalek's hernia; (b) cect - chest showing right - sided bronchiectasis, dextrocardia, and left - sided bochdalek's herina she presented to us with chief complaints of recurrent cold, cough with copious expectoration, anosmia, headache, and progressively increasing shortness of breath for last 30 years . She received anti - tubercular treatment for these complaints 12 years back for 6 months, with no relief . Her past history was significant as she had frequent visits to the pediatrician for recurrent chest infections . The patient had a blood pressure of 110/72 mmhg, pulse rate 92/minute regular, and oxygen saturation 76% on room air . General examination revealed bilateral pedal edema, raised jugular venous pressure, facial puffiness, and grade 3 clubbing . On auscultation, diffuse ronchi and crackles (more on the left side) were heard . X - ray paranasal sinuses revealed mucosal thickening with hazy sinuses [figure 2a]. Chest x - ray [figure 2b] and high - resolution computed tomography (hrct) thorax revealed bronchiectasis and situs inversus . (a) x - ray pns showing opacified maxillary sinuses; (b) chest x - ray pa view showing dextrocardia and left - sided bronchiectasis a 40-year - old male patient, non - smoker, and born to non - consanguineous parents presented with recurrent productive cough, rhinorrhea, and headache since last 20 years with episodic fever and worsening of symptoms . He had been previously treated with antibiotics, antihistamines, bronchodilators, inhaled and oral corticosteroids, and even anti - tuberculous drugs, but the response was only partial and temporary . He also had similar complaints, off and on, during childhood . On examination, he was febrile with nasal discharge, wheezy chest, and bilateral coarse crackles . Chest x - ray [figure 3a] showed cystic bronchiectactic changes in the lower and mid zones with dextrocardia . Ultrasound of the abdomen showed spleen on the right side of the abdomen, while liver on the left was suggestive of complete situs inversus . (a) cystic bronchiectactic changes in the lower and mid zones with dextrocardia; (b) axial ct image abdomen showing situs inversus with the liver and ivc on the left and the spleen and aorta on the right; (c) axial ct image paranasal sinuses showing mucosal thickening and opacified sinus cavities axial ct - chest showed dextrocardia with the inferior vena cava (ivc) and morphologic right ventricle on the left and the left ventricle on the right . Axial ct - abdomen showed situs inversus with the liver and ivc on the left and the spleen and aorta on the right figure 3b . Hematological and biochemical parameters were within normal limits except semen analysis which showed oligospermia with immotile live sperms . Nearly 50% of pcd patients have situs inversus . Such cases of pcd with situs inversus are known as kartagener's syndrome . Pcd is a phenotypically and genetically heterogeneous condition wherein the primary defect is in the ultrastructure or function of cilia . Such defects are identified in approximately 90% of pcd patients and involve the outer dynein arms, inner dynein arms, or both . Pathophysiologically, the underlying defect which leads to accumulation of secretions and consequent recurrent sinusitis, bronchiectasis, infertility, and situs inversus is the defective ciliary motility / immotility . The severity of symptoms and the age at which the condition is diagnosed is quite variable, even though the symptoms are present from birth . Diagnostic criteria for this condition include clinical picture suggestive of recurrent chest infections, bronchitis, and rhinitis since childhood, along with one or more of the following: (1) situs inversus in the patient / sibling; (2) alive but immotile spermatozoa; (3) reduced or absent transbronchial mucociliary clearance; and (4) cilia showing characteristic ultrastructural defect on electron microscopy . Apart from fulfilling the criteria mentioned above, two types of tests are done for diagnosis of pcd screening tests (exhaled nasal nitric oxide measurement which is usually low in pcd, and saccharin test to assess mucociliary function of nasal epithelium) and diagnostic tests (ciliary beat pattern and frequency analysis using video recording, and electron microscopic confirmation of the ultrastructural ciliary defect). The samples for these tests for examining motility and ultrastructure of cilia may be obtained by biopsy of nasal mucosa and laparoscopic biopsies of tubal mucosa in females, as was done by halbert et al . In our cases, however, we could not perform these tests and the diagnosis was essentially clinico - radiological, with variation in view of azoospermia and oligospermia . These situations have been infrequently reported previously,[1618] and it could be possible that they are a variant associated with ks . Most infertile patients with ks have a normal spermatozoid count, but with a structural defect and a complete lack of motility . The hyperprolactinemia present in our first case could possibly be coincidental, as a search of literature has reported it to be a cause of infertility but has not revealed an association between this condition and ks . The issue of fertility was not addressed in the initial published reports of patients with ks until arge reported three male patients with this syndrome having immotile spermatozoa and sterility . Infertility in male ks patients is due to diminished sperm motility, while in females it is due to defective ovum transport because of dyskinetic motion of oviductal cilia, suggesting that the ciliated endosalpinx is essential for human reproduction . The development of assisted reproductive techniques has allowed rational treatment for these patients, and to date, there have been reported pregnancies using subzonal insemination (suzi) and intracytoplasmic sperm injection (icsi). The case report of kordus et al . Shows that even in severe cases of asthenozoospermia, icsi can overcome the inability of the spermatozoa to reach the ovum and produce healthy offspring . However, both suzi and icsi require expertise not available in all units and there are concerns over costs and outcomes . Until more is known with regard to the genetic control of pcd, it is suggested that treatment should be individualized depending on sperm motility . In cases where there is no sperm motility however, if sperm motility is present, a trial of in vitro fertilization (ivf) should be considered . One concern regarding the fertility treatment of men with pcd is the possibility that the resultant child has the risk of being affected by the same condition . It is therefore necessary to counsel couples regarding the possibility of genetic risks and to follow - up children fathered by men affected by pcd . To conclude, ks patients are frequently troubled by repeated infection episodes for which they have to seek medical attention and this is largely the reason for their morbidity . But infertility is also one important aspect that needs to be adequately addressed in their evaluation so that they may be offered a suitable option that could help them have children.
Regular assessment of hematological profile during pregnancy is an essential practice in antenatal care clinics . Physiologically, activation of renin - angiotensin - aldosterone system during pregnancy increases extracellular fluid and consequently plasma volume . The hematocrit (hct), plateletcrit (pct), counts of red blood corpuscles (rbc), white blood corpuscles (wbc) and platelets (plt) are expected to change according to the degree of plasma volume expansion and the amount of blood formed elements being added or removed from the circulation . Release of young rbc and activation of platelets affect the readings of some hematological indices like mean corpuscular volume (mcv), red cell distribution width (rdw), mean platelet volume (mpv) and platelet distribution width (pdw). However, the exact pattern of trimester change of these hematological indices, and others, remained ill - defined . This fact encouraged researchers in the field to investigate for trimester specific, reference range for hematological profile during normal pregnancy . Studies assessing detailed hematological profile during normal pregnancy are scarce before invention of automated hematology analyzer . Over the last decade, trimester specific reference ranges of hematological indices were reported in several western, eastern and african populations . We could not find a report exploring hematological profile among sudanese women with normal pregnancy, a part from a study exploring physiological variations of blood formed elements counts in 50 healthy pregnant sudanese women . The present study aimed to evaluate trimester pattern of change and reference ranges of fifteen important hematological parameters among sudanese women with normal pregnancy . We believed that the findings of this study would help in precise interpretation of laboratory results, correct diagnosis and appropriate management of blood disorders among pregnant sudanese women . A longitudinal study was conducted at the antennal care clinics of saad abu - alela hospital (khartoum, sudan) during the period of january - december 2015 . Women with singleton pregnancy were enrolled since early gestation and followed till the third trimester . After signing an informed consent, questionnaires were used to gather the medical and obstetrics history (age, parity, gravidity, gestational age). Women with thyroid disease, hypertension, renal disease, diabetes, liver disease and on medication(s) were excluded from the study . During each visit the weight and height measured during the first visit were used to calculate the body mass index (bmi), which was expressed as weight (kg)/height (m). During each visit, 2 ml of blood was taken from every women in an ethylene diamine tetra acetic acid, analyzed immediately for a complete hemogram using an automated hematology analyzer and following the manufacturers instructions as previously described . A total sample size of 140 women was calculated using a formula for longitudinal study and the difference in the mean of the proposed variables (mainly hemoglobin, wbc and platelets) that would provide 80% power to detect a 5% difference at = 0.05, and assumed that 10% of women will be lost during follow - up or will not respond . Statistical analysis was performed using statistical package for the social sciences (spss) for windows, version 16.0 (spss inc ., proportions of the studied groups were expressed in percentages (%). Means (m) and standard deviations (sd) were used to describe the studied variables . 5 - 95 centiles were used to identify reference ranges of hematological profile among sudanese women with normal pregnancy . The repeated measure anova and lsd post hoc analyses were used to evaluate the differences in the means of the hematological indices between different trimesters . Ethical clearance was obtained from the department of obstetrics and genecology, faculty of medicine, university of khartoum . Statistical analysis was performed using statistical package for the social sciences (spss) for windows, version 16.0 (spss inc ., m) and standard deviations (sd) were used to describe the studied variables . 5 - 95 centiles were used to identify reference ranges of hematological profile among sudanese women with normal pregnancy . The repeated measure anova and lsd post hoc analyses were used to evaluate the differences in the means of the hematological indices between different trimesters . Ethical clearance was obtained from the department of obstetrics and genecology, faculty of medicine, university of khartoum . Out of 175 pregnant women who were enrolled initially, 143 (81.7%) completed the follow - up till the third trimester . The rest (18.3%) were lost during follow - up due to address change . The mean age of the pregnant women enrolled in the study (n = 143) was 27.945.45 years (range 18 - 42 years). The other obstetric and socio - demographic characteristics of the studied pregnant women are summarized in table 1 . Table 2 shows the m (sd) [5 - 95 centiles] of rbc indices in the studied pregnant women . Rbcs counts were comparable in the first and second trimester (4.300.36 vs 4.353.03 10/mm, p=0.836), but decreased significantly during the third trimester (4.080.4410/mm, p <0.001). In comparison, hematocrit was lower in the second trimester (34.432.51%) compared with the first (35.383.52%, p<0.003) as well as the third trimesters (35.173.18%, p<0.008). Mcv increased steadily throughout pregnancy while mch and hchc were higher during the last two thirds of pregnancy compared with the first trimester . Rdw was lower during the third trimester (13.821.72%) compared with the first (13.991.76%) and the second (14.332.31%) trimesters . Table 3 shows the m (sd) [5 - 95 centiles] of the total and differential wbc counts in the studied pregnant women . The total wbcs counts were lower during first trimester (7.691.9610/mm) compared with the last two thirds of pregnancy (8.451.970/mm, p<0.001; 8.362.11 10/mm, p = 0.002). Lymphocyte count was least during the third trimester; however, the count of other types of leukocytes remained comparable throughout pregnancy . Table 4 shows the m (sd) [5 - 95 centiles] of platelets indices in the studied pregnant women . The platelets count and pct decreased steadily while mpv remained with no significant change as pregnancy progress . Pdw decreased significantly in the second compared with the first trimester (15.570.37 vs 15.680.36%, p=0.002), but remained with no significant change thereafter . It is evident from the current results that maximum decrease in rbcs count was in the third trimester . In comparison, lowest hb and hct as well as highest mcv, mch, mchc and rdw readings were achieved in the second trimester . Gradual decrement in rbcs count with progression of pregnancy was reported in a sudanese study and several previous reports but not others . Similar to the present results, lowest means of hb and hct were noted in the second trimester in at two separate jamaican and chinese studies . Assessment of 274 pregnant women attending lagos university teaching hospital in nigeria revealed comparable trimester peak of hct to present results; however, hb concentration showed progressive decline from the first to the third trimester . According to the same study, maximum dipping of mhc and mcv were in the second and third trimesters respectively . In another nigerian study, rbcs count and hb concentration decreased steadily throughout pregnancy, hct underwent marked drop in the second trimester and remained unchanged thereafter . The maximum mcv was in the second trimester while mch continued to rise up to the end of pregnancy . As shown in table 5, the m (sd) of rbcs derived hematological parameters in the present study are comparable with international reports with only few exceptions . The average hb concentration and hct reported by akingbola et al ., among nigerian pregnant women were markedly lower compared with our results as well as international records . Likewise, the drop in hb concentration and hct in the first trimester [11.24 (10.41) g / dl and 29.36 (4.22) respectively] compared to the third trimester [9.81 (1.32) g / dl and 29.36 (4.22)% respectively] were more than expected . Such significant drop in hb concentration and hct between first and third trimesters is difficult to explain on physiological basis and suggest an etiology other than the normal hematological response to pregnancy . Physiologically, increased vascular capacity secondary to systemic vasodilatation stimulates renin - angiotensin - aldosterone system, which explains blood volume expansion during pregnancy . Based on evans blue dye dilution methods, at least 10 - 15% increment in plasma volume occurs at the start of the second trimester . Maternal erythropoiesis is also enhanced during pregnancy; however, the increase rbc count (18 - 25%) is usually less compared with the ultimate expansion of plasma volume (50%). The resulted dilutional anemia causes significant drop in rbcs count, hb and hct during the second trimester, which remains unchanged thereafter in the third trimester . Absence of further drop over the third trimester is probably due to high levels of atrial natriuretic peptide and consequently contraction in maternal plasma volume . Enhanced maternal erythropoiesis during pregnancy results in release of more young erythrocytes to the circulation, which are usually larger in size compared with the mature rbcs . A longitudinal study among women with normal pregnancy demonstrated no variations in rdw between 16 and 34 weeks but significant increase afterward, up to the onset of labor . The unpredicted increase in the rdw over the 4 - 6 weeks before the onset of labor points to enhanced bone marrow activity; however, stimulus for this remained unexplained . Noteworthy, the physiological changes in rbcs count, hb, hct, mcv and rdw during pregnancy are further modified by nutritional, medical and obstetric complications, which explains trimester variations of hematological profile described in previous studies . The current results showed significant rise in the total wbc count during the last two thirds of pregnancy compared with first trimester . Lymphocytes counts followed reverse pattern of change, being comparable during the first two trimesters but dropped significantly over the last one . In contrast, the counts of gran and mid were comparable throughout the three trimesters . The relatively increased total wbc count demonstrated in the current results is supported by several previous reports . According to local and international studies, total wbc count is hardly less than 7000/mm during pregnancy (table 4). The exact cause of relative leukocytosis during gestation is yet to be explored by further researches; however, physical and emotional stress associated with pregnancy and depressed neutrophil apoptosis could be possible causes ., demonstrated slight elevation of lymphocytes count at the start pregnancy but gradual decrease over the following trimesters . In a longitudinal study, pitkin et al . Attributed the rise in total wbc count to synonymous increase in the number of neutrophils . Regarding other types of leukocytes, the study showed increased monocyte and decreased lymphocytes, eosinophils, and basophils as pregnancy advanced . Another report confirmed monocytosis during pregnancy, but denied significant rise in eosinophil and basophil counts, which disagreed with pitkin et al . Implications . The present data showed steady decrease of platelets count and pct, increase of pdw and unchanged mpv as pregnancy progress . Thrombocytopenia during pregnancy was attributed to plasma volume expansion, enhanced platelet activation and clearance . Theoretically, hemodilution and platelet activation associated with pregnancy can induce platelets swelling, pseudopodia formation and hence increase mpv and pdw . According to one study, pdw displayed substantial increase from the first to the third trimester of pregnancy; however, mpv failed to show the same trend . Noteworthy, comparable trimester changes of mpv and pdw were reproduced by the present study and others . The preferential rise pdw, but not mpv, as pregnancy advances suggests that variations in platelets size during gestation are principally due to platelet activation rather than hemodilution . Noteworthy, this study enrolled pregnant women with regular antenatal follow - up . Studied women received proper medical advice, regular checkup investigations and hematinic . The hematological reference range derived from the present study would be helpful in evaluating pregnant women with healthcare, socioeconomic and racial background comparable to our participants . However, a potential limitation remained because our results could not uncover pattern of trimester change in hematological profile of pregnant women with inadequate antenatal care . Another limitation of the study is the lack of controls for hematological profile readings (preconception or 6-weeks postpartum). The present study is the first to establish detailed, trimester specific, reference range for hematological profile among sudanese women with normal pregnancy . The reference range of rbc, wbc and platelets indices are mostly parallel to international records . The trimester pattern of change of hematological profile among sudanese is comparable with previous reports in this regard . The plasma volume expansion, physiological stress associated with pregnancy and preferential increased hematopoiesis of some blood formed elements, but not others, seemed to be the main grounds for hematological profile changes during pregnancy.
Success in endodontic treatment is best defined by the contemporary endodontic triad of diagnosis, anatomy, and debridement . In the learning curve of endodontics, the incisor tooth forms the basics due to its simple morphology of one root - one canal . The common developmental anomalies in a maxillary lateral incisor (mli) includes peg - shaped crown, radicular groove, talon cusp, and dens invaginatus (di). From an endodontic perspective considering the occurrence of additional canals, the literature shows that, mli with a maximum of four canals and with dens formation, three root canals have been reported . Teeth with di are often recognized clinically due to their unusual crown form or extremely marked foramen caecum and many a time may show no clinical signs of the malformation . However, due to the limitation of conventional radiographs in assessing root canal configuration, complex root canal morphology due to dental anomalies such as multiple root canals, two roots and dens formation poses a considerable diagnostic challenge to achieve the required success . Nevertheless, advanced radiographic techniques such as the cone beam computed tomogram (cbct) helps in a three - dimensional evaluation of a particular tooth to overcome the limitations of conventional radiography . The purpose of the present case report is to discuss the endodontic procedure adopted to negotiate the complex tooth morphology of a peg - shaped mli with di and five root canals (frc). The present case is about a 17-year - old male patient who was referred to the department of endodontics with a primary complaint of heaviness and pain in relation to the upper front tooth . Clinical evaluation revealed no significant extraoral findings . On intraoral examination, both the mlis appeared peg - shaped . However, the right lateral incisor was associated with a soft tissue swelling in the labial vestibule [figure 1a], tender on percussion and gave a negative response to electric pulp testing . Intra oral periapical radiograph of the maxillary right lateral incisor revealed ill - defined periapical radiolucency associated with an unusual canal morphology and di [figure 1b]. Based on the clinical and radiological findings (a) peg - shaped lateral incisor with intra - oral swelling in the labial vestibule . (b) radiograph revealing periapical radiolucency associated with right lateral incisor and presence of dens invaginatus . (d) radiograph with instruments in five canals the need for an endodontic therapy was explained to the patient and was advised to take a course of analgesics and antibiotics (amoxicillin 500 mg, metronidazole 400 mg, ibuprofen 400 mg thrice daily for 5 days). The patient was administered local anesthesia of 2% lidocaine with 1:100,000 epinephrine . Under rubber dam isolation, an access opening was made and an orifice (c1) was visualized after copious amount of purulent discharge was allowed to drain through the c1 . The endodontic explorer was placed in the c1 and verified with an apex locator (sybron endo mini, sybron endo, glendora, usa) and radiograph . However, as the drainage of purulent discharge persisted, a calcium hydroxide intra - canal medicament (calcicur, voco, cuxhaven, germany) was placed and access cavity was temporarily sealed . (cavit, 3 m espe ag, seefold, germany). After 3 days, the calcium hydroxide was removed from c1 and subtle changes were noted at some points over the sub - pulpal floor, which suggested the possibility of additional canals away from the c1 that was located on the labial half . In order to locate the possibility of a palatal canal, the access cavity was redefined in the distopalatal (dp) region and the second canal (c2) was identified and negotiated successfully . On further probing in the distolabial (dl) region, two additional orifices (c3 and c4) however, the orifices of c3 and c4 were interconnected . With four instruments placed in the respective canals, the radiograph showed the presence of another unidentified canal . The fifth canal orifice (c5) was located at the mesiopalatal (mp) aspect, summing - up to five canals all with separate apical exit [figure 1c and d]. Each time an orifice was identified, it was checked with an apex locator to rule out perforation . Coronal flaring alone was performed with protaper rotary ni ti instruments (dentsply, maillefer) and subsequent cleaning and shaping was completed by hand instrumentation (dentsply, maillefer) due to the constricted coronal pulp space . A cbct scanning was done to study the tooth morphology, which confirmed the presence of a single root with five canals with separate apical exit and a type iii di [figure 2a and b]. (c) radiograph taken after root canal filling and placement of temporary restoration in the access cavity . (d) follow - up radiograph after 12 months showing signs of healing one week later the tooth was obturated in the third visit, with gutta - percha and sealer (apexit, ivoclar vivadent) by cold lateral compaction technique [figure 2c]. The patient was periodically monitored, with satisfactory healing and the follow - up period of 12 months was uneventful [figure 2d]. Dens invaginatus is a developmental malformation resulting from invagination of the crown or root before calcification has occurred . With a reported incidence from 0.04% to 10%, di can be found affecting either the primary or the permanent dentition, and commonly involving the mlis . Cases of bilateral occurrence and rare cases are reported in molars, premolars, and maxillary central incisors . An indian study reported a prevalence of 0.5% of surveyed teeth; however, according to a chinese study, di was found in 517 permanent teeth from 67 chinese individuals, which account for 5% of di . The literature shows that mli with a maximum of four canals has been reported, but as far as the author's knowledge, this is the first case with frc, especially in a peg - shaped lateral incisor complicated by the presence of di . According to the literature, di in a mli is considered as unusual . Type 2 involves the root, but does not reach the apex, and in type 3, the invagination extends past the periapical region with and without manifesting an additional apical foramen . The pathways of infection in a di is found to be due to hypomineralized internal enamel and the thin canals or fissures in the dentin connecting the invagination to the pulp cavity through which microorganisms and irritating agents from the oral cavity gain access to the pulpal space leading to pulp alterations . The challenge posed in this case is the restricted access coupled with complex and unpredictable morphology of the root canal system . Krape and fidler emphasized the need and suggested incisal edge as the predominant location of straight - line access in anterior teeth . Various treatment techniques have been reported in the literature, including preventive sealing or filling of the invagination, root canal treatment with or without endodontic apical surgery, intentional replantation and extraction . Removal of di using hand files and gates glidden drills and with the aid of an operating microscope and subsequent root canal treatment has also been described . The presence of multiple root canals in a peg - shaped mli complicated by di poses substantial difficulty in diagnosis and treatment as well as achieving endodontic success for better prognosis . The present case has been evaluated and negotiated with the aid of conventional radiographs and by applying law of symmetry, cemento enamel junction and orifice location . The complicated tooth morphology with multiple root canals warrant use of cbct to define and confirm the canal morphology . In line with a molar tooth, the multiple canals in this case is named according to the surface in which it is located namely c1 mesiolabial, c2 dp, c3 and c4 dl1 and dl2, and c5 mp, respectively [figure 1c]. Case selection is the key in successful outcome of a treatment . Treating a tooth with a unique morphology definitely tests the clinical knowledge, skill of the operator and one should be ready for any kind of pulp space variant that could exist . Although, cbct would have been beneficial to precisely define and confirm the canal location in a complicated morphology, the present case illustrates the successful negotiation with conventional radiographs.
Systemic and topical corticosteroids are popularly used treatment methods in cutaneous sarcoidosis but are a poor long - term management strategy given the range of side effects . Treatment with biologic agents has recently been proposed as another treatment option for cutaneous sarcoidosis [25]. In the past decade, a number of case series has shown that infliximab is an effective and well - tolerated management strategy for this condition [326]. The authors report three cases of cutaneous sarcoidosis that were refractory to standard therapy but responded to infliximab treatment . A 43-year - old african american man with a history of pulmonary sarcoidosis was seen in the authors clinic in december 2007 because of a 4-month history of lesions on the patient s cheeks and legs . The patient was treated with increasing doses of prednisone, hydroxychloroquine, and intralesional corticosteroids without improvement . As a result of a combination of lack of efficacy and intolerable side effects of these medications, the patient was switched to infliximab 5 mg / kg intravenously on weeks 0, 2, and 6, and then every 8 weeks in november 2008 . After three infusions, the patient showed significant improvement of skin lesions, and prednisone was tapered back to 2.5 mg every other day . However, after 6 months of infusions, the patient began to experience some flaring of cutaneous lesions . Methotrexate 7.5 mg weekly was added in may 2009 in addition to infliximab in an effort to improve further the patient s cutaneous lesions . In addition, infliximab was increased to 5 mg / kg every 7 weeks, and shortly thereafter was increased again to 7.5 mg / kg every 7 weeks in november 2009 for flares near the time of infusion . The patient s cutaneous sarcoidosis was stable on this dose of methotrexate and infliximab for 9 months . In june 2011, the dosage again had to be increased to 10 mg / kg every 5 weeks due to flaring of lesions before infusions, which was able to control the disease better (table 1).table 1patient demographic datacase 1case 2case 3demographics43-year - old african american man53-year - old african american woman48-year - old african american womanother organ involvementpulmonarypulmonary and eyepulmonary and eyecutaneous features at diagnosisnodular lesions on face, eyelids, and earlobesmultiple erythematous to violaceous papules on eyelids, right cheek, tip of nose, and corner of mouthmultiple erythematous annular plaques around eyes, nose, perioral area, neck, arm, back and kneeshistopathologyfrom right cheek: granulomatous inflammatory infiltrate (lymphocytes, histiocytes, and giant cells)from right cheek: non - necrotizing granulomas (epithelioid cells and multinucleated giant cells)n / aprevious treatment before initiating infliximabprednisonehydroxychloroquineintralesional corticosteroidsprednisonehydroxychloroquinemethotrexateminocyclinetopical tacrolimustopical imiquimodmycophenolate mofetilprednisonehydroxychloroquinemethotrexatepulse methyl prednisonethalidomidetopical corticosteroidstherapeutic side effects or complicationsa significant amount of weight gainhypertensionnew onset diabetesa spontaneous hairline left 5th metatarsal fracturehypertensiondiabetesright hip avascular necrosiscorticosteroids - induced gastrointestinal upset, fatiguehydroxychloroquine - induced diarrhea and abdominal painmethotrexate - induced leucopenia and abnormal liver function teststhalidomide - induced peripheral neuropathyduration of disease before infliximab therapy2 years18 years8 yearstreatments used at the time of infliximab initiationhydroxychloroquine (200 mg twice a day)intralesional corticosteroidsmycophenolate mofetil (4 g / day)prednisone (15 mg / day)prednisone (40 mg / day)thalidomide (100 mg / day)infliximab dose, duration5 mg / kg on weeks 0, 2, and 6, then every 8 weeks 6 months later: add methotrexate 7.5 mg weekly after tapering prednisone 6 months later increased infliximab to 5 mg / kg and 7.5 mg / kg every 7 weeks 9 months later increased infliximab to 10 mg / kg every 5 weeks7.5 mg / kg on weeks 0, 2, and 6, then every 8 weeks7.5 mg / kg on weeks 0, 2, and 6, then every 8 weeks4 years later tapered to 5 mg / kg every 16 weeks3 years later taken off infliximabtime to achieve clinical response3 years5 months4 yearstreatments at the time of last follow - upinfliximab (10 mg / kg every 5 weeks)methotrexate (7.5 mg weekly)prednisone (2.5 mg / day every other day)infliximab (7.5 mg / kg every 8 weeks)discontinued infliximabfollow - upstill gradually increase new lesionsimprovement after mycophenolate mofetil and prednisone discontinuationno new lesions patient demographic data a 53-year - old african american woman with a history of pulmonary sarcoidosis for 15 years was referred to the authors clinic on april 2002 for management of 5 years of cutaneous sarcoidosis lesions . The patient s cutaneous lesions were primarily involving the nasal ala and bilateral cheeks in a distribution consistent with lupus pernio . The patient was treated with oral prednisone, hydroxychloroquine, methotrexate, minocycline, topical tacrolimus, and topical imiquimod at various points and only showed moderate improvement with systemic steroids . The patient was started on mycophenolate mofetil 500 mg twice a day in december 2003, increasing to as high as 4 g / day . However, the patient continued to experience refractory lesions at this dose (fig . 1), with multiple erythematous to violaceous papules and plaques on the patient s right cheek, tip of the nose, and corner of the mouth . Infliximab 7.5 mg / kg was started in may 2004 at weeks 0, 2, and 6, and then every 8 weeks, and the dosage of mycophenolate mofetil was decreased to 1 g twice a day and prednisone 10 mg / day . Five months after initiating infliximab, the patient s skin lesions showed moderate flattening without any adverse side effects . Prednisone was tapered over the course of 6 weeks and ended in november 2011 . The patient s lesions have since maintained improvement with infliximab 7.5 mg / kg every 8 weeks (fig . 2; table 1).fig . 2case 2 after missing two doses of infliximab and experienced a flare of the cutaneous disease the clinical manifestations of case 2 before initiating infliximab case 2 after missing two doses of infliximab and experienced a flare of the cutaneous disease a 48-year - old african american woman was first seen in the authors clinic in 2001 for management of cutaneous sarcoidosis lesions . The patient was diagnosed with cutaneous sarcoidosis by biopsy of the preauricular area in 1996 . The patient was originally treated with hydroxychloroquine and prednisone, but these medications were discontinued due to side effects . The patient was subsequently started on methotrexate 15 mg / week and minocycline 100 mg twice a day . Methotrexate was increased to 22.5 mg / week, prednisone was increased to 40 mg / day, and hydroxychloroquine 250 mg / day was initiated . Because of side effects from prednisone and methotrexate, these medications were discontinued . The patient was then lost to follow - up for approximately 2 years . In may 2004, the patient s lesions flared, and thalidomide 100 mg / day was initiated with prednisone 20 mg / day and topical corticosteroids . As a result of peripheral neuropathy, thalidomide was discontinued after 2 months of treatment . Infliximab 7.5 mg / kg at weeks 0, 2, and 6, and every 8 weeks thereafter was started in august 2004 . Figure 3 shows the lesions on the patient s upper extremities were significantly flattened compared with initial presentation.fig . 3the clinical manifestation of case 3, 2 years after initiating infliximab the clinical manifestation of case 3, 2 years after initiating infliximab the patient s lesions slowly improved with no new lesions manifesting; fig . 4 shows the lesions on the patient s upper extremities are dramatically flattened compared with initial presentation . Infliximab was tapered in october 2008 to 5 mg / kg every 12 weeks and was discontinued in november 2011 . 4the clinical manifestations of case 3, 6 years after initiating infliximab the clinical manifestations of case 3, 6 years after initiating infliximab a 43-year - old african american man with a history of pulmonary sarcoidosis was seen in the authors clinic in december 2007 because of a 4-month history of lesions on the patient s cheeks and legs . The patient was treated with increasing doses of prednisone, hydroxychloroquine, and intralesional corticosteroids without improvement . As a result of a combination of lack of efficacy and intolerable side effects of these medications, the patient was switched to infliximab 5 mg / kg intravenously on weeks 0, 2, and 6, and then every 8 weeks in november 2008 . After three infusions, the patient showed significant improvement of skin lesions, and prednisone was tapered back to 2.5 mg every other day . However, after 6 months of infusions, the patient began to experience some flaring of cutaneous lesions . Methotrexate 7.5 mg weekly was added in may 2009 in addition to infliximab in an effort to improve further the patient s cutaneous lesions . In addition, infliximab was increased to 5 mg / kg every 7 weeks, and shortly thereafter was increased again to 7.5 mg / kg every 7 weeks in november 2009 for flares near the time of infusion . The patient s cutaneous sarcoidosis was stable on this dose of methotrexate and infliximab for 9 months . In june 2011, the dosage again had to be increased to 10 mg / kg every 5 weeks due to flaring of lesions before infusions, which was able to control the disease better (table 1).table 1patient demographic datacase 1case 2case 3demographics43-year - old african american man53-year - old african american woman48-year - old african american womanother organ involvementpulmonarypulmonary and eyepulmonary and eyecutaneous features at diagnosisnodular lesions on face, eyelids, and earlobesmultiple erythematous to violaceous papules on eyelids, right cheek, tip of nose, and corner of mouthmultiple erythematous annular plaques around eyes, nose, perioral area, neck, arm, back and kneeshistopathologyfrom right cheek: granulomatous inflammatory infiltrate (lymphocytes, histiocytes, and giant cells)from right cheek: non - necrotizing granulomas (epithelioid cells and multinucleated giant cells)n / aprevious treatment before initiating infliximabprednisonehydroxychloroquineintralesional corticosteroidsprednisonehydroxychloroquinemethotrexateminocyclinetopical tacrolimustopical imiquimodmycophenolate mofetilprednisonehydroxychloroquinemethotrexatepulse methyl prednisonethalidomidetopical corticosteroidstherapeutic side effects or complicationsa significant amount of weight gainhypertensionnew onset diabetesa spontaneous hairline left 5th metatarsal fracturehypertensiondiabetesright hip avascular necrosiscorticosteroids - induced gastrointestinal upset, fatiguehydroxychloroquine - induced diarrhea and abdominal painmethotrexate - induced leucopenia and abnormal liver function teststhalidomide - induced peripheral neuropathyduration of disease before infliximab therapy2 years18 years8 yearstreatments used at the time of infliximab initiationhydroxychloroquine (200 mg twice a day)intralesional corticosteroidsmycophenolate mofetil (4 g / day)prednisone (15 mg / day)prednisone (40 mg / day)thalidomide (100 mg / day)infliximab dose, duration5 mg / kg on weeks 0, 2, and 6, then every 8 weeks 6 months later: add methotrexate 7.5 mg weekly after tapering prednisone 6 months later increased infliximab to 5 mg / kg and 7.5 mg / kg every 7 weeks 9 months later increased infliximab to 10 mg / kg every 5 weeks7.5 mg / kg on weeks 0, 2, and 6, then every 8 weeks7.5 mg / kg on weeks 0, 2, and 6, then every 8 weeks4 years later tapered to 5 mg / kg every 16 weeks3 years later taken off infliximabtime to achieve clinical response3 years5 months4 yearstreatments at the time of last follow - upinfliximab (10 mg / kg every 5 weeks)methotrexate (7.5 mg weekly)prednisone (2.5 mg / day every other day)infliximab (7.5 mg / kg every 8 weeks)discontinued infliximabfollow - upstill gradually increase new lesionsimprovement after mycophenolate mofetil and prednisone discontinuationno new lesions patient demographic data a 53-year - old african american woman with a history of pulmonary sarcoidosis for 15 years was referred to the authors clinic on april 2002 for management of 5 years of cutaneous sarcoidosis lesions . The patient s cutaneous lesions were primarily involving the nasal ala and bilateral cheeks in a distribution consistent with lupus pernio . The patient was treated with oral prednisone, hydroxychloroquine, methotrexate, minocycline, topical tacrolimus, and topical imiquimod at various points and only showed moderate improvement with systemic steroids . The patient was started on mycophenolate mofetil 500 mg twice a day in december 2003, increasing to as high as 4 g / day . However, the patient continued to experience refractory lesions at this dose (fig . 1), with multiple erythematous to violaceous papules and plaques on the patient s right cheek, tip of the nose, and corner of the mouth . Infliximab 7.5 mg / kg was started in may 2004 at weeks 0, 2, and 6, and then every 8 weeks, and the dosage of mycophenolate mofetil was decreased to 1 g twice a day and prednisone 10 mg / day . Five months after initiating infliximab, the patient s skin lesions showed moderate flattening without any adverse side effects . Prednisone was tapered over the course of 6 weeks and ended in november 2011 . The patient s lesions have since maintained improvement with infliximab 7.5 mg / kg every 8 weeks (fig . 2; table 1).fig . 2case 2 after missing two doses of infliximab and experienced a flare of the cutaneous disease the clinical manifestations of case 2 before initiating infliximab case 2 after missing two doses of infliximab and experienced a flare of the cutaneous disease a 48-year - old african american woman was first seen in the authors clinic in 2001 for management of cutaneous sarcoidosis lesions . The patient was diagnosed with cutaneous sarcoidosis by biopsy of the preauricular area in 1996 . The patient was originally treated with hydroxychloroquine and prednisone, but these medications were discontinued due to side effects . The patient was subsequently started on methotrexate 15 mg / week and minocycline 100 mg twice a day . Methotrexate was increased to 22.5 mg / week, prednisone was increased to 40 mg / day, and hydroxychloroquine 250 mg / day was initiated . The patient was then lost to follow - up for approximately 2 years . In may 2004, the patient s lesions flared, and thalidomide 100 mg / day was initiated with prednisone 20 mg / day and topical corticosteroids . As a result of peripheral neuropathy, thalidomide was discontinued after 2 months of treatment . Infliximab 7.5 mg / kg at weeks 0, 2, and 6, and every 8 weeks thereafter was started in august 2004 . Figure 3 shows the lesions on the patient s upper extremities were significantly flattened compared with initial presentation.fig . 3the clinical manifestation of case 3, 2 years after initiating infliximab the clinical manifestation of case 3, 2 years after initiating infliximab the patient s lesions slowly improved with no new lesions manifesting; fig . 4 shows the lesions on the patient s upper extremities are dramatically flattened compared with initial presentation . Infliximab was tapered in october 2008 to 5 mg / kg every 12 weeks and was discontinued in november 2011 . 4the clinical manifestations of case 3, 6 years after initiating infliximab the clinical manifestations of case 3, 6 years after initiating infliximab sarcoidosis is a multisystemic non - caseating granulomatous disease of unknown origin that is driven by t - helper type 1 immune responses . Cutaneous manifestations occur in 2535% of cases and may present at the onset of the disease process [2831]. Diagnosis is usually made by clinical suspicion in conjunction with biopsy and the exclusion of other conditions ., there are no definite guidelines for systemic therapy, but progressive, widespread, and disfiguring lesions should certainly be treated . Systemic and topical corticosteroids remain the mainstay of treatment for various manifestations of sarcoidosis, including cutaneous sarcoidosis . Unfortunately, the long - term side effects make corticosteroids a less than ideal long - term treatment option, and some patients remain refractory to this management strategy . Tumor necrotic factor (tnf)-alpha is an important proinflammatory cytokine involved in the pathogenesis of sarcoidosis . Macrophages of sarcoidosis patients have been implicated as major releasers of tnf - alpha in sarcoidosis [34, 35]. Higher serum tnf - alpha levels have been shown not only in patients with slow onset sarcoidosis compared with acute onset, but levels have been shown to fluctuate in correlation with disease activity [3638]. In addition, increased levels of tnf - alpha have been correlated with a greater risk of disease progression, relapse, and difficulty with treatment . As a result of evidence of tnf - alpha involvement in the pathogenesis of sarcoidosis [39, 40], alternative therapies for cutaneous sarcoidosis such as the tnf - alpha antagonists have been proposed [2, 3]. Reports on the efficacy of tnf antagonists used for cutaneous sarcoidosis are increasing, although at this time this treatment modality remains off label by the standards of the us food and drug administration . Although there have been no comparative trials between the biologic agents for sarcoidosis, infliximab is the most heavily reported medication in the literature [611], and overall has shown great promise in the treatment of sarcoidosis patients refractory to more conventional therapies [6, 7, 12, 26]. Favorable outcomes of infliximab therapy in patients who have renal sarcoidosis, vertebral sarcoidosis, joint sarcoidosis, optic neuropathy, pulmonary sarcoidosis [6, 17], retinal vasculitis, cutaneous sarcoidosis [35, 22], neurosarcoidosis, and cardiac sarcoidosis have also been reported . For sarcoidosis patients reported in the literature, the usual dose of infliximab has been 310 mg / kg per dose at 0, 2, and 6 weeks, followed by every 8 weeks for maintenance [25, 34]. If patients respond insufficiently to 5 mg / kg, reducing the treatment interval has been shown to lead to higher trough levels than increasing the dose . A randomized, double - blind, placebo controlled study on the efficacy of infliximab in 36 sarcoidosis patients with cutaneous and pulmonary involvement used a 6-week maintenance regimen . After 24 weeks of treatment, patients overall showed improvement in cutaneous findings compared with placebo, although this was not statistically significant (p = 0.09). In addition, a retrospective review of lupus pernio (13 courses of treatment with infliximab in nine patients) reported the success of a 6-week maintenance regimen, which was statistically more efficacious than corticosteroids used with and without other non - steroid immunosuppressive agents (p = 0.0015). All three patients in the current case study were able to control their cutaneous sarcoidosis, although doses had to be tailored to the severity and variably refractory nature of their diseases . However, it should be noted by the prescribing physician that increased doses of immunosuppression can of course lead to an increased tendency toward infection, and can also be associated with demyelination and worsening of multiple sclerosis lesions . Increasing doses of infliximab should not be given lightly . While tnf inhibitors have been shown to be of benefit in treating sarcoidosis, it is of some interest that a few cases have been reported wherein patients developed sarcoidosis while undergoing treatment with anti - tnf therapy . In the largest reported series of sarcoid - like granulomas developing during tnf inhibitor therapy, three of 10 patients treated with infliximab for ankylosing spondylitis developed pulmonary sarcoidosis 1451 months after the initiation of treatment . Other case reports of infliximab - treated patients include one patient with ankylosing spondylitis who developed pulmonary sarcoidosis after 5 years of therapy, and patients with psoriatic arthritis who developed pulmonary sarcoidosis and cutaneous sarcoidosis . The mechanisms of sarcoidosis development during anti - tnf - alpha therapy are unclear . In an article by saleh et al ., the following were offered as indications to initiate treatment with infliximab for refractory sarcoidosis: unsuccessful treatment with systemic corticosteroids;intolerant to systemic steroids side effects;unsuccessful treatment or intolerance to other systemic therapies;requirement for additional systemic therapy but intolerant to alternative agents . Unsuccessful treatment with systemic corticosteroids; intolerant to systemic steroids side effects; unsuccessful treatment or intolerance to other systemic therapies; requirement for additional systemic therapy but intolerant to alternative agents . In summary, the authors report three cases of patients with refractory sarcoidosis successfully treated with infliximab . Their clinical outcomes were good, sustained, and there was no associated morbidity from the medication . Definite indications, dosage, interval, and duration of treatment for cutaneous sarcoidosis are not firmly established, although altering the dose and schedule of infusions can be useful in bringing the disease under optimal control . Larger randomized controlled trials are warranted to validate the efficacy of infliximab in patients with cutaneous sarcoidosis . This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
Early passage dpscs (p1p2) isolated from deciduous teeth have been obtained from dr . Songtao shi (univ . Of southern california) and cultured in alpha - mem supplemented with 20% fetal bovine serum (v / v), 2 mm l - glutamine, 100 m l - ascorbate-2-phosphate, 50 u / ml penicillin and 50 g / ml streptomycin . Exponentially growing dpscs were treated with different concentrations of ethanol diluted from absolute ethanol (fw = 21.7 m). For acute exposure, cells were fed with media containing given concentrations of ethanol (0, 1, 5, 10, 20, 50 mm) for 24 or 48 h. total rna was isolated from dpscs treated with ethanol (0, 1, 5, 10, 20, 50 mm) for 24 or 48 h. rna was extracted using rneasy purification kit, following the manufacturer's instruction (qiagen, valencia, ca). Isolated rna was further purified by dnase treatment (ambion / life technologies, grand island, ny). Rna purity and concentration were determined by nanodrop, nd-1000 spectrophotometer (thermo scientific, indianapolis, in) and microfluidics - based platform 2100 bioanalyzer (agilent technologies, santa clara, ca). The sensitivity of the system was measured by% p using the 3 biased affymetrix hg - u133a 2.0 arrays .% p ranged from 41.1 to 45.1% demonstrating the ability to detect a large number of transcripts across a wide range of abundance . All 24 arrays were assessed for recommended standard quality control metrics by affymetrix including image quality, signal distribution and pair wise scatter plots and passed . Mas5.chp files were generated for each array by mas 5.0 (affymetrix, santa clara, ca) and combined to a final results.mas5.txt file . Degradation plot was prepared with each curve corresponding to a single chip and visualizing the chip - averaged dependency between probe intensity and probe position (fig . Raw data was initially analyzed for the quality of microarray analysis by log density estimates of the data across all arrays (fig . 2), quantile normalization and log transformation with robust multi - array average (rma) approach on affymetrix gene expression data using affy r package (fig . 3). We removed probes with expression lower than the overall sample median; 27,327 out of 54,676 probes were kept for further analysis (fig . 3). Given that the sensitivity of array platforms is generally considered lower than deep sequencing (with enough sequence depth), clearly a significant percentage of genes are either not expressed or beyond the detection limit in a typical array experiment . Filtering out this group of genes would potentially be beneficial to deg (differentially expressed gene) detection . Principal component analysis was performed to detect expression data separation by etoh treatment time (fig . Treatment time shows quite consistent grouping irrespective of doses, but doses in combination of treatment time show high degree of variations in gene expression . We constructed linear regression models to evaluate the effect of dose and time on gene expression (fig . We fit a separate model in terms of dose, time, and the dose by time interaction effect . In these models dose was treated as a continuous variable . In addition to the linear model, we performed a regression spline analysis with moderated f - test to detect the probes differentially expressed over time with increasing dose, with the assumption that expression changes smoothly along with the increasing of the dose rather than making discrete jumps from one dose to another . Limma q - values were generated to control the false discovery rate using qvalue r package (http://www.bioconductor.org/packages/release/bioc/html/qvalue.html). A set of significant probes with cut - off of p <0.05 for interaction effects was selected from each model for further functional analysis . Early passage dpscs (p1p2) isolated from deciduous teeth have been obtained from dr . Songtao shi (univ . Of southern california) and cultured in alpha - mem supplemented with 20% fetal bovine serum (v / v), 2 mm l - glutamine, 100 m l - ascorbate-2-phosphate, 50 u / ml penicillin and 50 g / ml streptomycin . Exponentially growing dpscs were treated with different concentrations of ethanol diluted from absolute ethanol (fw = 21.7 m). For acute exposure, cells were fed with media containing given concentrations of ethanol (0, 1, 5, 10, 20, 50 mm) for 24 or 48 h. total rna was isolated from dpscs treated with ethanol (0, 1, 5, 10, 20, 50 mm) for 24 or 48 h. rna was extracted using rneasy purification kit, following the manufacturer's instruction (qiagen, valencia, ca). Isolated rna was further purified by dnase treatment (ambion / life technologies, grand island, ny). Rna purity and concentration were determined by nanodrop, nd-1000 spectrophotometer (thermo scientific, indianapolis, in) and microfluidics - based platform 2100 bioanalyzer (agilent technologies, santa clara, ca). The sensitivity of the system was measured by% p using the 3 biased affymetrix hg - u133a 2.0 arrays .% p ranged from 41.1 to 45.1% demonstrating the ability to detect a large number of transcripts across a wide range of abundance . All 24 arrays were assessed for recommended standard quality control metrics by affymetrix including image quality, signal distribution and pair wise scatter plots and passed . Mas5.chp files were generated for each array by mas 5.0 (affymetrix, santa clara, ca) and combined to a final results.mas5.txt file . Degradation plot was prepared with each curve corresponding to a single chip and visualizing the chip - averaged dependency between probe intensity and probe position (fig . Raw data was initially analyzed for the quality of microarray analysis by log density estimates of the data across all arrays (fig . 2), quantile normalization and log transformation with robust multi - array average (rma) approach on affymetrix gene expression data using affy r package (fig . 3). We removed probes with expression lower than the overall sample median; 27,327 out of 54,676 probes were kept for further analysis (fig . 3). Given that the sensitivity of array platforms is generally considered lower than deep sequencing (with enough sequence depth), clearly a significant percentage of genes are either not expressed or beyond the detection limit in a typical array experiment . Filtering out this group of genes would potentially be beneficial to deg (differentially expressed gene) detection . Principal component analysis was performed to detect expression data separation by etoh treatment time (fig . Treatment time shows quite consistent grouping irrespective of doses, but doses in combination of treatment time show high degree of variations in gene expression . We constructed linear regression models to evaluate the effect of dose and time on gene expression (fig . We fit a separate model in terms of dose, time, and the dose by time interaction effect . In these models in addition to the linear model, we performed a regression spline analysis with moderated f - test to detect the probes differentially expressed over time with increasing dose, with the assumption that expression changes smoothly along with the increasing of the dose rather than making discrete jumps from one dose to another . Limma q - values were generated to control the false discovery rate using qvalue r package (http://www.bioconductor.org/packages/release/bioc/html/qvalue.html). A set of significant probes with cut - off of p <0.05 for interaction effects was selected from each model for further functional analysis.
The retina is susceptible to a variety of degenerative diseases, including age - related macular degeneration (amd), retinitis pigmentosa (rp) and other inherited photoreceptor degenerations, photoreceptor loss following retinal detachment, ganglion cell loss in glaucoma and optic neuropathies, as well as the loss of retinal neurons associated with nondegenerative conditions such as diabetic retinopathy (dr), macular edema and ischemia, vascular occlusions, trauma, and inflammatory diseases . Amd is a particularly prevalent cause of blindness among elderly persons, affecting more than 30 million people globally . That number is expected to double over the next decade in association with demographic shifts towards an older population, particularly in developed countries . Similar to the situation with many neurological diseases, little is available in the way of effective treatments for patients with amd or other blinding disorders of the retina . A large body of research has shown that the use of exogenous neurotrophic factors can reproducibly promote the survival of specific neurons in various parts of the central nervous system (cns), including the retina [2, 3]. Frequently investigated neuroprotective neurotrophic factors have included glial cell line - derived neurotrophic factor (gdnf), brain - derived neurotrophic factor (bdnf), and ciliary neurotrophic factor (cntf). Among these, gdnf has been associated with significant effects with respect to preventing cell death, including the protection of specific neuronal populations in the brain [5, 6], spinal cord, and retina [811]. Receptors for gdnf are known to be expressed within the mature retina [8, 11, 12]. Stem and progenitor cell transplantation has also shown considerable promise in animal models of neural degeneration . Subretinal transplantation of neural progenitor cells (npcs) has yielded intriguing evidence of cellular repopulation of damaged retinas, growth of neurites into the optic nerve head and retardation of ongoing retinal degeneration [1317]. Both unmodified, as well as genetically modified, cortical human npcs can survive for prolonged periods, migrate extensively, secrete growth factors, and rescue visual function following subretinal transplantation in the dystrophic royal college of surgeons rat, with sustained visual benefits following injection . More recently, subretinal transplantation of human forebrain progenitor cells has been extended to nonhuman primates, although this model used nondystrophic hosts and therefore did not lend itself to evaluation of neuroprotective efficacy . When used for transplantation therapy, npcs engineered to secrete gdnf contributed to reduced apoptotic death in vitro, enhanced survival in vivo, neuronal differentiation, and improved host cognitive function following traumatic brain injury as compared with nontransduced npcs [2124]. The visual system of the cat is quite sophisticated and one of the most extensively studied among higher mammals . There are many similarities to the human retina although that of the cat has a tapetum and is generally optimized for performance under scotopic conditions . Like humans, the cat retina has also been the subject of decades of anatomical and physiological studies and has been used as an animal model of binocular visual function as well as studies involving drug treatment and research on retina detachment [27, 28]. In addition, the feline eye is large relative to that of rodents thereby allowing the application of surgical techniques similar to those typically used clinically . Finally, there exist feline models of retinal degeneration caused by spontaneous mutations in genes known to be involved in retinitis pigmentosa in humans [29, 30]. These animals provide excellent models for exploring the therapeutic potential of stem cell - based neuroprotective strategies in an animal with highly developed visual capabilities . Previously, we showed that it is possible to derive npcs from the developing cat brain and that these cells are capable of integration into the retina of dystrophic feline recipients . To more fully exploit the potential of this model, it is useful to develop feline npcs capable of sustained growth factor delivery to the host retina . Here we use a bicistronic feline lentiviral vector to generate genetically modified feline neural progenitor cells that exhibit sustained overexpression of gdnf before and after differentiation . Cat neural progenitor cells (cnpcs) were originally isolated from 47 day cat fetuses as previously described . Briefly, forebrains were removed and finely minced with a surgical scalpel and the resulting tissue fragments digested for 20 minutes in 0.1% type i collagenase (invitrogen, carlsbad, ca). The supernatant containing dissociated cells was then passed through a 100 mm mesh strainer, centrifuged, and seeded in complete culture medium, designated here as standard medium (sm), consisting of advanced dmem / f12, 1% n2 neural supplement, 2 mm l - glutamine, 50 mg / ml penicillin - streptomycin, and epidermal and basic fibroblast growth factors (recombinant human egf and bfgf, invitrogen), both at final concentrations of 20 ng / ml . After initial isolation, all medium was changed to an ultraculture - based composition, identical to the above but in which dmem / f12 was replaced with ultraculture serum - free medium (lonza, basel, switzerland). Therefore, in the present study standard proliferation medium was ultraculture - based with growth factors and is designated (um), whereas differentiation medium was ultraculture - based as well, but did not contain added growth factors and did include 10% fetal bovine serum (um - fbs). Culture medium was changed every 2 days and proliferating cells passaged at regular intervals of 4 - 5 days . The lentiviral vector used in this study was an fiv - based bicistronic vector (genecopoeia, germantown, maryland) designated as lenti - gdnf - gfp, which carries a human gdnf gene driven by the cytomegalovirus (cmv) immediate - early promoter as well as an enhanced green fluorescent protein (gfp) reporter gene with an internal ribosome entry site (ires). Lenti - gdnf - gfp vectors were prepared by transient transfection of 293 t cells using a standard calcium phosphate precipitation protocol (clontech, mountain view, ca). Briefly, 293 t cells cultured in 10 cm tissue culture dishes (bd biosciences, san jose, ca) were transfected with 2 g of lentiviral transfer vector plasmid, along with 10 g of the mixed envelope and packaging plasmids . The viral supernatants were harvested 48 and 72 hours posttransfection and concentrated by centrifugation of virus - containing supernatant through a centricon plus-70 filter (millipore, billerica, ma) following the manufacturer's instruction . Titers of the concentrated lentivector were estimated by transducing cnpc cells with a serial dilution of the virus and flow cytometric identification of gfp - positive cells . Cat neural progenitor cells were transduced with lenti - gdnf - gfp vectors at a moi of 10 following the standard procedure . Briefly, cnpcs were seeded at a density that allowed them to grow to 90% confluency on the day of transduction . The cells were then transduced by 624 hours of exposure to virus - containing supernatant in the presence of 58 g / ml polybrene . Viral vector - containing medium was then replaced with fresh medium and cells were incubated at 37c in a co2 incubator . Cells were harvested using tryple express (invitrogen) and filtered through cell strainer caps (35 m mesh) to obtain a single cell suspension (approximately 10 cells per ml for analysis, 0.52 10 cells per ml for sorting). The stained cells were analyzed and sorted on a fluorescence - activated cell sorter facsaria (bd biosciences) using facsdiva software (bd biosciences). The fluorochromes were excited by the instrument's standard 488 nm and 633 nm lasers, and green fluorescence was detected using 490 lp and 510/20 filters . Prior to sorting, the nozzle, sheath, and sample lines were sterilized with 70% ethanol or 2% hydrogen peroxide for 15 minutes, followed by washes with sterile water . A 100 m ceramic nozzle (bd biosciences), sheath pressure of 2025 pounds per square inch (psi), and an acquisition rate of 1,0003,000 events per second were used as conditions previously optimized for neuronal cell sorting . The growth properties of transduced and nontransduced cnpcs were assessed by culturing both types of cells under proliferation conditions in ultraculture - based medium (um). Cells of identical passage number (p17) were seeded in four t25 culture flasks at a density of 0.25 million cells / flask . Cell numbers were graphed at each time point to compare the growth properties of transduced versus nontransduced cells . Transduced and nontransduced cnpcs of identical passage number were seeded in t25 culture flasks (0.25 million / flask). 4 hours), the original media were replaced with 3 ml of fresh media . Subsequently, 3 ml of conditioned media were collected and replaced with fresh media at 24 hour intervals and conditioned samples were saved at 80c for elisa analysis . Elisa was performed using a human gdnf duoset elisa kit and protocol (r&d systems, minneapolis, mn). Wells of microtiter plates were coated (overnight, room temperature) with 2 g / ml of gdnf capture antibody in 100 l of coating buffer (0.05 m na2co3, 0.05 m nahco3, ph 9.6) and then blocked with 0.1% bsa in pbs for 1 hour at room temperature . Samples (100 l) were loaded in triplicates and incubated for 2 hours at room temperature, followed by addition of 100 l antibody detection antibody (0.1 g / ml) for an additional 2 hours at room temperature . Hrp - conjugated streptavidin (1: 200) in blocking buffer was then added (20 minutes, room temperature) and the reaction visualized by the addition of 100 l of substrate solution for 20 minutes . The reaction was stopped with 50 l h2so4 and absorbance at 450 nm was measured with reduction at 540 nm using an elisa plate reader . Plates were washed five times with washing buffer (pbs, ph 7.4, containing 0.05% (v / v) tween 20) after each step . As a reference for quantification, a standard curve was established by a serial dilution of recombinant gdnf protein (31.25 pg / ml2.0 ng / ml). Total rna was extracted from each sample using the rneasy mini kit (qiagen, valencia, ca). Rna concentration was measured at a wavelength of 260 nm (a260) for each sample, and the purity of isolated total rna was determined by the a260/a280 ratio . Quantitative rt - pcr analyses were only performed on samples with a260/a280 ratios between 1.9 and 2.1 . Two micrograms of total rna in a 20 l reaction were used for reverse transcription using an omniscript cdna synthesis kit (qiagen, valencia, ca). A primer set for each gene (table 1) was designed using the cat genome browser (http://lgd.abcc.ncifcrf.gov/cgi-bin/gbrowse/cat/) and the primers synthesized commercially (invitrogen). Quantitative pcr was performed using an applied biosystems 7500 fast real - time pcr detection system (applied biosystems, foster, ca). Triplicate wells were used for each gene . A total volume of 20 l per well containing 10 l of 2x power sybr green pcr master mix (applied biosystems, foster, ca), 2 l of cdna and gene - specific primers were used . Cycling parameters for qpcr were as following: the initial denaturation was at 95c for 10 minutes, followed by 40 cycles of 15 s at 95c and 1 minute at 60c . To normalize template input, the relative expression of the gene of interest was then evaluated using 7500 fast system sequence detection software, version 1.4 . The value obtained for ct represents the number of pcr cycles at which an increase in fluorescence signal (and therefore cdna) can be detected above background and the increase is exponential for the particular gene . Error bars displayed the calculated maximum and minimum standard errors to the mean expression level of the triplicates . Transduced cnpcs were differentiated in um without added egf or bfgf and containing 10% fbs (um - fbs). Cells (0.2 million) in um were seeded in t25 culture flasks and allowed to attach, then culture medium was aspirated and replaced with either um - fbs for differentiation or fresh um for comparison . Conditioned media were collected and replaced with fresh media every 24 hours for 4 days and frozen for elisa analysis . At the end of day 4, cells were trypsinized, counted, and elisa analysis was performed on lysates as well as thawed media samples . For facs analysis, transduced cnpcs were cultured in either um - fbs or um for 10 days prior to processing . Transduced and nontransduced cnpcs were seeded in 4-well chamber slides (nalge nunc international, rochester, ny) and allowed to grow for 35 days . Cells were re - fed every 2 days and fixed with freshly prepared 4% paraformaldehyde (invitrogen) in 0.1 m phosphate - buffered saline (pbs) for 20 minutes at room temperature and washed with pbs . Cells were then incubated in antibody blocking buffer consisting of pbs containing 10% (v / v) normal goat serum (ngs) (biosource, camarillo, ca), 0.3% triton x-100, 0.1% nan3 (sigma - aldrich, saint louis, mo) for 1 hour at room temperature . Slides were incubated in primary antibodies (table 2) overnight at 4c . After washing the next morning, slides were incubated in fluorescent - conjugated secondary antibody (alexa fluor546 goat anti - mouse or goat anti - rabbit, 1: 800 in pbs, bd) for 1 hour at room temperature . After washing, dapi - containing vectashield hard set mounting medium (vector laboratories, burlingame, ca) was used to mount the slides for 20 minutes at room temperature . Negative controls for immunolabeling were performed in parallel using the same protocol but without primary antibody . Immunoreactive cells were visualized and imaged using a fluorescent microscope (eclipse e600, nikon, melville, ny). Recent development of feline immunodeficiency virus- (fiv-) based vectors could present a means for improved delivery of transgenes into cells of this species . Here, we employed an fiv - based bicistronic vector for delivery of glial cell line - derived neurotrophic factor (gdnf) to cat neural progenitor cells (cnpcs). Forty eight hours after lenti - gdnf - gfp viral vector transduction, approximately 50% of cnpcs expressed the gfp reporter gene based on direct observation via fluorescence microscopy . To enrich for transgene - expressing cells, cnpcs were trypsinized at 72 hours postviral vector incubation and sorted by facs based on gfp expression . The gfp - enriched population was subsequently cultured in ultraculture - based proliferation medium (um) for more than 60 days . High levels of gfp expression were sustained throughout this time period (figure 1). Gdnf is known to have a range of biological activities in the context of the nervous system and cultured neural cell populations . Because this activity might extend to neural progenitors, we examined the effect of gdnf transduction on cnpc behavior, specifically the ability to proliferate . Proliferation is an important consideration for large - scale expansion of modified donor cell populations for use in transplantation studies . Transduced cnpcs continued to proliferate in a logarithmic manner, similar to but slightly slower than the nontransduced cnpcs (figure 2). Conversely, the transduced cnpcs appeared to be somewhat more uniform, with less clumping and fewer floating cells, particularly when cells were cultured for more than 3 days in the same flask . Neuronal differentiation has been implicated in gene silencing; therefore facs analysis was performed to evaluate the effects of cell differentiation on gdnf transgene expression using the gfp reporter . Approximately 95% of transduced cnpcs expressed gfp, either when cultured in um (proliferation conditions) or 10% fbs - containing um (differentiation conditions). Among the cells expressing gfp there was no evidence of diminished gfp expression by the cells grown in the presence of fbs, thereby demonstrating maintained transgene expression was under differentiation conditions (figure 3). The levels of gdnf produced by transduced cnpcs, as present in conditioned culture medium and collected cell lysates, were analyzed by elisa and compared to nontransduced controls . High levels of secreted gdnf were present in the culture medium of transduced cnpcs, measured on days 28, 33, and 38 posttransduction (figure 4(a)). In addition, gdnf expression levels were considerably elevated in cell lysates extracted from transduced cultures on days 33 and 38 post - transduction (figure 4(b)). Hence, transduced cnpcs continued to produce elevated levels of gdnf over a sustained period of time . Having shown above that expression of the gfp reporter was sustained when transduced cnpcs were subjected to differentiation conditions, and that the transduced cells overexpress gdnf, we next verified that gdnf expression was sustained during cnpc differentiation (figure 5). Transduced cnpcs were cultured in um without added growth factors and containing 10% fbs to induce cell differentiation and media were collected for elisa . The level of gdnf produced under differentiation conditions was not diminished relative to proliferation conditions . Neural progenitor cells have shown great promise as a source of neural cell types in transplantation studies . We therefore investigated whether genetically modified cnpcs retained their neural progenitor phenotype in the presence of high levels of gdnf expression, as assessed by a gene expression profile (figure 6). Qpcr analysis showed that transduced cnpc cells exhibited approximately 14,000-fold gdnf upregulation at the mrna level compared to nontransduced controls . In transduced cells, expression levels of the progenitor cell markers nestin, vimentin, and sox2, as well as the neuronal marker 3-tubulin and the proliferation marker ki-67 remained similar to that seen in nontransduced cells . Transduced cells also exhibited increased transcript levels for stromal cell - derived factor-1 (sdf1, 4.2-fold), prominin (cd133, 2.9-fold), doublecortin (dcx, 2.4-fold), and hes1 (1.45-fold), as well as lower transcript levels for cxcr4, fabp7 and ncam . Immunocytochemical analysis demonstrated that cnpcs produced low levels of gdnf protein at baseline (figure 7(a)), but that expression of the protein was substantially elevated following transduction with lenti - gdnf - gfp (figure 7(b)). To investigate the effect of differentiation on gdnf protein overexpression, cnpcs were cultured in either serum - free um or um containing 10% fbs for 5 days . Following the induction of differentiation, the cells appeared larger in size and gdnf expression was sustained, although heterogeneity of expression levels across the population was evident (figure 7(c)). The expression of progenitor and lineage markers was also examined at the protein level, for both transduced and control cells, before and after induction of differentiation (figure 8). The neural progenitor cell marker nestin was only detected in cells grown in um and was not seen in um - fbs . Likewise, vimentin expression also decreased upon differentiation, although for this less - specific marker expression remained substantial . In contrast, -tubulin iii immunoreactivity was strikingly up - regulated in a subset of cells grown in um - fbs, suggesting the induction of neuronal lineage . The proliferation marker ki-67 was clearly downregulated in um - fbs cultured cnpcs, whereas the glial marker gfap was not detected under proliferation conditions, but was strongly up - regulated by a subset of cells cultured in um - fbs . Having confirmed the differentiating influence of the um - fbs conditions, the same immunocytochemical analysis was repeated on cnpcs of identical age that had been transduced using the lenti - gdnf - gfp vector . The results were equivalent, suggesting that the differentiation of cnpcs was not adversely influenced by transduction with gdnf (figure 8). Among mammals, the highly developed visual system of the domestic cat has been studied in particular detail, owing in part to greater similarities with the human visual system as compared to laboratory rodents . This body of work, combined with the availability of naturally occurring retinal dystrophic mutants, would serve to recommend the cat as a powerful model for retinal regeneration research . A major limiting factor to regenerative research in this species is the paucity of available donor cells of the type suitable for such work, including stem, progenitor, or precursor cells of allogeneic origin . Furthermore, the use of these cells in transplantation studies would benefit from the inclusion of a reporter gene and, in some cases, additional transgenes of potential therapeutic value . Here we demonstrate the feasibility of using feline lentiviral vectors to genetically modify cnpcs for sustained delivery of gdnf . These cells possess multiple desirable features for use in transplantation studies including ease of expansion in vitro, coexpression of a green fluorescence protein (gfp) reporter gene serving to both confirm gdnf expression as well as allowing easy tracking of donor cells after transplantation, and sustained transgene expression following differentiation . In addition, they are allogeneic with respect to the targeted host species and therefore likely to be well tolerated without for the need of exogenous immune suppression . The ability of a progenitor cell to sustain proliferation is important in order to avoid the necessity of repeated rederivation of the modified cell type . Importantly, the gdnf - gfp overexpressing cnpcs continued to exhibit log growth characteristics, indicating that neither the genetic modification process nor gdnf overexpression presents a major barrier to continued proliferation of these cells . Nevertheless, the growth of the gdnf - transduced cnpcs was less rapid than that of unmodified controls . This slower growth rate is also reflected in the lower number of cells that were ki-67 positive following introduction of the transgene construct . Since we have recently shown that exogenous gdnf tends to promote, rather than hinder, the growth of murine rpcs, it seems unlikely that a feedback signaling mechanism involving the overexpressed cytokine would explain the behavior seen here . Perhaps the particularly high levels of transgene expression maintained by the gdnf - gfp transduced cnpcs results in a metabolic load that slows growth relative to unmodified cells . Alternatively, genetic modification could introduce abnormalities to the host genome, for instance as a function of the sites of transgene integration . Another consideration in terms of clinical application of transduced cells is the regulation of transgene expression . Sustained overexpression might result in undesired effects such as decreased sensitivity to the gene product, as might result from down - regulation of the corresponding growth factor receptor or, alternatively, toxic responses to high levels of the cytokine, either within the eye or systemically . Titrating the dose of transplanted cells should set an upper limit on gdnf delivery, since the progenitor cells tend to cease proliferation in vivo, however, a more sophisticated approach would be the use of inducible promoters which allow for the dynamic regulation of transgene expression levels . Looking forward, the gdnf - gfp overexpressing cnpcs developed here are suitable for allogeneic transplantation to the vitreous cavity or subretinal space of cats with retinal disease . Of particular interest is the application of these cells to existing animals with photoreceptor dystrophy, such as the swedish abyssinian breed with the cep290 mutation, with the goal of ameliorating visual loss through the sustained intraocular delivery of a neurotrophic factor . In vivo experiments in this nonrodent species would more realistically model the prospective treatment of analogous human conditions and could yield valuable information pertaining to the mechanisms of graft - mediated effects on host visual function.
As the fourth most abundant cation in the body, magnesium fulfils an important role in multiple physiological processes . Over 300 enzymes require the presence of magnesium for their catalytic action, including many enzymes utilising or synthesising atp, or those that use other nucleotides to synthesise dna and rna . While clinical issues regarding magnesium disorders had received surprisingly little attention until the 1990s, a shift in focus led to some clinical investigations, especially in patients with chronic kidney disease (ckd). In 2012, several reviews on magnesium metabolism and disorders in magnesium balance were published in a special issue of the clinical kidney journal . Given the growing interest in the molecule since then the literature on the role of magnesium in ckd has continued to accumulate substantially . For instance, two cohort studies established hypomagnesaemia as a predictor of mortality (fig . 1) and kidney function decline in ckd patients as well as mortality in haemodialysis (hd) patients . Furthermore, magnesium was identified as an independent risk factor for non - recovery of renal function in a cohort of critically ill patients with acute kidney injury .fig . 1estimated survival probabilities in ckd patients with high (> 2.2 mg / dl/>0.90 mmol / l) serum magnesium concentrations (adapted and reprinted from with permission from elsevier) estimated survival probabilities in ckd patients with high (> 2.2 mg / dl/>0.90 mmol / l) serum magnesium concentrations (adapted and reprinted from with permission from elsevier) this review examines and reviews the clinical impact of magnesium on the health status of ckd patients, in particular taking into account the influence of magnesium on diseases such as metabolic syndrome, diabetes, hypertension, vascular calcification and cardiovascular events, fatigue and depression all of which are frequently present in ckd patients and/or contribute to ckd progress . Cardiovascular disease (cvd) is the leading cause of death in the ckd population . The molecular mechanisms leading to vascular calcification in ckd patients are still under investigation, but there is consensus that it is an active, multifactorial, cell - mediated and dynamic process [7, 8]. The progressive loss of kidney function is accompanied by elevated serum fibroblast growth factor 23 (fgf23) levels, a decrease in inorganic phosphate excretion and dysregulation of mineral and bone metabolism . These disturbances promote vascular calcification, whereby vascular smooth muscle cells (vsmcs) play a central role in the pathogenesis by undergoing an osteochondrogenic phenotype change in response to elevated phosphate levels . Several cell culture and animal studies suggest a protective role of magnesium through multiple molecular mechanisms [1012]. These results were extended by recent studies in which magnesium was shown to inhibit phosphate - induced calcification in vitro . Moreover, higher magnesium levels prevented calcification of bovine vsmcs, inhibited expression of osteogenic proteins, apoptosis and further progression of already established calcification . The first in vitro evidence in human aortic vsmcs for a protective role of magnesium on phosphate - induced calcification was based on the observation that living cells are necessary for magnesium ions to exert its protective effect . These studies suggested a potentially active intracellular role for magnesium ions in attenuating the vascular calcification process . Additionally, increasing magnesium concentrations improved cell viability and normalised the cellular release of proteins involved in vascular calcification . Inhibition of the wnt/-catenin signalling pathway was identified as one of the intracellular mechanisms by which the anti - calcifying effect of magnesium is achieved . In the context of these data, microcalcifications in human atherosclerotic lesions contain both calcium and magnesium (in the form of whitlockite and calcium phosphate / apatite); whereas ckd- accelerated calcification was associated with a predominant deposition of calcium phosphate / apatite . This suggests that in ckd, local magnesium homoeostasis is disturbed and potentially aggravates vascular calcifications . The clinical relation between serum magnesium and vascular changes including calcification was assessed in several recent studies . A prospective study in 47 hd patients revealed an association of magnesium serum concentration with the intima media thickness of carotid arteries . In addition, ckd patients with higher magnesium serum concentrations had a significantly lower pulse wave velocity (pwv). Similarly, a cohort study of 512 renal transplant recipients identified low magnesium levels as a predictor of pwv and thus of vascular stiffness, independent of clinically relevant covariates and especially in older patients . Furthermore, an observational cohort study, investigating 283 ckd patients, reported an association of high magnesium levels with less endothelial dysfunction . So far, one double - blind, placebo - controlled randomised trial examined the efficacy of oral magnesium oxide (440 mg three times per week for 6 months) on endothelial function in hd patients . While magnesium supplementation significantly decreased carotid intima media thickness, there were no significant effects on c - reactive protein or flow - mediated dilatation, i.e. A functional endothelial marker . The study was limited by the small sample size (less than 30 patients per treatment arm) and a significant baseline imbalance in intima media thickness between the groups . Another small study in 47 dialysis patients randomised to no therapy or 610 mg magnesium citrate orally every other day for 2 months also noted a reduction of carotid intima thus, more prospective interventional studies are warranted to assess the potential benefits of magnesium supplementation on vascular dysfunction and calcification . A meta - analysis of 19 prospective studies including 532,979 participants found a significant inverse association between magnesium intake and/or serum levels and the risk of cvd events in different patient populations . This relationship was further sustained by a comprehensive study in 7,216 spanish high - risk patients for cardiovascular disease . Again, an inverse association was noted between dietary magnesium intake and all - cause mortality . While the above studies did not focus on ckd patients, several smaller studies did . In 80 diabetic ckd stage 24 patients, low serum magnesium was identified as a significant risk factor for an elevated pulse pressure, an established marker for cardiovascular mortality . Analyses in 191 diabetics with ckd stage 13 have further underlined the relevance of this finding as lower magnesium levels are associated with increased mortality and accelerated progression of renal disease . A third observational cohort study in 283 ckd patients also identified magnesium as an independent predictor of future cardiovascular outcomes . In this study, kaplan meier curves showed significantly higher cardiovascular mortality rates in ckd patients whose serum magnesium levels were below 2.05 mg / dl (0.84 mmol / l). Finally, these results were confirmed in a recent registry - based cohort study of 142,555 hd patients that again identified low serum magnesium as a significant predictor of cardiovascular mortality . Thus, observational studies consistently identify low serum magnesium levels as a predictor or risk factor of vascular pathology, cardiovascular morbidity and all - cause mortality . However, recent large - scale randomised clinical trials [the fourth international study of infarct survival (isis 4) and magnesium in coronaries (magic)] in non - renal patients could not prove a benefit of intravenous magnesium after myocardial infarction, and magnesium therapy is presently only indicated in patients with life - threatening ventricular arrhythmias . Whether the situation is different in ckd patients with ischaemic heart disease remains to be tested . The association between magnesium and mineral metabolism was recently investigated in several animal studies . To elucidate the effect of magnesium on phosphate homoeostasis magnesium deficiency induced high serum fgf23 levels, possibly contributing to the observed decrease in renal phosphorus reabsorption . While direct effects of magnesium on fgf-23 are not well established, the administration of a calcium acetate / magnesium carbonate (camg) containing phosphate binder in dialysis patients lowered both serum phosphate and fgf-23; in another study magnesium oxide also lowered fgf-23 levels in dialysis patients; high magnesium concentrations can also activate the calcium - sensing receptor and thereby modulate pth secretion in similar manner to calcium, albeit less potently [30, 31]. Another study demonstrated increasing serum 1,25-dihydroxyvitamin d and decreasing pth as well as phosphate levels in response to magnesium loading in mouse models with genetic inactivation of pth or both, pth and the calcium - sensing receptor . Both studies indicate the importance of balanced magnesium intake in terms of regulated phosphorus levels . Since clinical studies have demonstrated the efficacy of magnesium - containing phosphate binders, and in line with the study by quinn and colleagues, some concern has arisen that high serum magnesium or magnesium loading might oversuppress pth secretion . In this context, an in vitro study with intact rat parathyroid glands is of importance, which showed that parathyroid glands were sensitive to an inhibitory effect of magnesium only when a moderately low calcium concentration was present . The general dialysis population, however, does not have low calcium concentrations; and this issue warrants more clinical studies . In addition to influencing pth secretion, magnesium affects the synthesis and metabolism of vitamin d . Many epidemiologic studies suggest that low vitamin d status may be associated with an increased risk of all - cause mortality [3537]. Importantly, the activities of three major enzymes determining 25-hydroxyvitamin d3 (25(oh)d3) level [3841] and vitamin d - binding protein are magnesium dependent . Indeed, a cohort study including 12 157 nhanes iii participants indicated that the inverse associations between serum 25(oh)d3 and risk of mortality could be modified by the intake level of magnesium . Although once again randomised controlled studies are missing, the importance of magnesium for the mineral metabolism seems likely . Effects of magnesium on bone in uremic patients have been reviewed recently . In summary, various authors have hypothesized that magnesium might contribute to osteomalacia and/or renal osteodystrophy in particular via suppressing pth . However, in vivo confirmation of this in dialysis patients is lacking and there is at present no conclusive evidence that magnesium administration in ckd is associated with adynamic bone disease . Of note, in the calmag trial, administration of a camg - containing phosphate binder to dialysis patients did not affect markers of bone turnover over 6 months . Epidemiological studies in non - ckd populations have linked magnesium deficiency to low bone mass and osteoporosis . Previous studies on the risk of hypertension and ischaemic heart disease have shown only a modest effect [4548], or inconsistent results [4954], regarding its correlation to dietary magnesium intake (and serum magnesium levels). Serum levels of magnesium only correlate weakly with its intake unless extreme conditions prevail (e.g. Excessive oral administration of magnesium salts). Thus, the use of urinary magnesium excretion as a more precise indicator of dietary magnesium uptake might provide a better insight into this association . In fact, results from a recent prospective population - based cohort study with 5,511 participants free of hypertension at baseline, demonstrated that urinary magnesium excretion was inversely associated with the risk of developing hypertension . The same study group investigated the association between magnesium uptake and ischaemic heart disease in 7,664 participants free from known cvd at baseline . The authors suggest that an increased dietary magnesium intake, in particular in those persons with a low urinary excretion of magnesium, could reduce the risk of ischaemic heart disease, lower blood pressure and prevent hypertension [56, 57]. A recently published cross - sectional study, involving 175 healthy subjects, supports the proposed pathophysiological role of magnesium in the development of hypertension: here a lower magnesium concentration was the only significant parameter in a multivariate analysis between pre - hypertensive and normotensive subjects . However, so far clinical trials have not detected significant antihypertensive effects of magnesium supplementation alone but rather suggest that magnesium might augment the response to antihypertensive drugs . Hypomagnesaemia occurs with an incidence of 1448% among patients with t2 dm compared with 315% among their counterparts without diabetes . A recent meta - analysis of thirteen prospective cohort studies involving 536,318 participants and 24,516 cases of diabetes provided further evidence that magnesium intake is inversely associated with the risk of t2 dm [relative risk (rr) 0.78 (95% ci 0.730.84)] in a dose response manner (fig . 2). Magnesium deficiency has also been linked to the development of the disease as well as its severity: the lower the magnesium level, the faster the deterioration of renal function in patients with type 2 diabetes mellitus (t2 dm).fig . 2meta - analysis of prospective cohort studies examining magnesium intake and the risk of developing type 2 diabetes . Copyright and all rights reserved . Material from this publication has been used with the permission of american diabetes association) meta - analysis of prospective cohort studies examining magnesium intake and the risk of developing type 2 diabetes . Copyright and all rights reserved . Material from this publication has been used with the permission of american diabetes association) correction of hypomagnesaemia via dietary magnesium supplementation improved glucose handling and insulin response in elderly and non - insulin - dependent diabetics and improved insulin sensitivity as well as metabolic control in t2 dm patients with decreased serum magnesium levels . The relationship between magnesium intake and metabolic parameters was further investigated in a cross - sectional study involving 210 elderly t2 dm patients, of which 89% exhibited a low magnesium intake and 37% had overt hypomagnesaemia . Metabolic syndrome and depression were associated with low intake, but not metabolic parameters, such as hba1c, low high - density cholesterol, triglycerides and blood pressure . In support of the well - known association between lower serum magnesium levels and impaired renal function, results from a cross - sectional study involving 51 t2 dm patients showed that ckd was accompanied by hypomagnesaemia including low intracellular magnesium content of red cells most likely as a result of low intake . In this study, magnesium deficiency was associated with poor blood glucose control and thus a potential increased risk of subsequent cvd events . In line with this, t2 dm is known to significantly increase the risk of ventricular arrhythmias, which represent a serious issue in ckd patients . Results from a recent cross - sectional health survey among 750 adults with high t2 dm prevalence showed that in diabetics the odds ratio of premature ventricular complexes was 0.24 (95% ci 0.060.98) if serum magnesium was above 0.70 thus, subnormal serum magnesium may be a contributor to arrhythmias among patients with t2 dm, and conceivably magnesium supplementation in adults with t2 dm may confer protection against ventricular arrhythmias . Despite the growing body of evidence on the relation between hypomagnesaemia and insulin resistance in t2 dm, transient receptor potential membrane melastatin (trpm)-6 is an ion channel which is crucial for magnesium homoeostasis and plays an essential role in epithelial magnesium transport as well as in the active magnesium reabsorption in the gut and kidney . Two rare single nucleotide polymorphisms in trpm6 (v1393i, k1584e) conferred susceptibility for t2 dm but only if magnesium intake was low (<250 mg / day). Insulin stimulates trpm6 activity via elevating the cell surface expression of trpm6 but this mechanism fails with the above genetic variants trpm6 . Thus, these studies identify a direct molecular link between diabetes and magnesium and thus potentially link magnesium homoeostasis to diabetic outcomes . Serum magnesium levels negatively correlated with hba1c, fasting plasma glucose and microalbuminuria . To better elucidate the relationship between magnesium deficiency and advanced t2 dm nephropathy, hypomagnesaemia was significantly associated with and independently predicted progression to esrd in patients with t2 dm nephropathy but not in those with non - diabetic ckd . In line with this observation, a later publication linked magnesium concentrations to the rate of kidney function decline . However, in this latter study, the effect of magnesium lost significance after adjustment for additional covariates, in particular diuretics . Thus, whether magnesium supplementation may delay the onset of t2 dm or its renal complications remains unknown at present . It is one of the most frequent dialysis - associated symptoms with prevalences reported from 60 and up to 97% . Fatigue is often accompanied by depression, and major depressive disorders are as well very common among ckd and esrd patients, affecting 20% of ckd patients compared to a prevalence of 210% in the general population . The fact that only a minority of affected ckd patients (~20%) receives an adequate diagnosis and treatment for depression, poses a major challenge for clinicians to develop strategies to better understand and manage depression in this population . Basic research reveals the involvement of magnesium ions in pathways which are connected to the known pathophysiology of depression . The n - methyl - d - aspartate (nmda)-ergic system received marked attention in the context of developing new compounds for mood disorders in recent years . The magnesium ion is a naturally occurring nmda - receptor antagonist, and reduced intracellular magnesium ions can be responsible for an increased nmda receptor sensitivity . Besides nmda targets involved in the antidepressant action of magnesium, there are brain - derived neurotrophic factors (bdnf) and glycogen synthase kinase-3 (gsk-3). Magnesium increases bdnf and both inhibit the activity of gsk-3, an enzyme involved in the mechanisms of action of antidepressants . Hence levels of serotonin and bdnf increase in the presence of magnesium, leading to an improvement of depressive symptoms . Animal data indicated that short - term administration of magnesium possesses potent antidepressant - like properties in the forced swim test in mice . An early study in non - renal patients with chronic fatigue indeed noted low intracellular magnesium levels, and magnesium supplementation led to some clinical improvement . Further evidence was gained in one randomised controlled trial comparing the efficacy of oral magnesium supplementation with the standard antidepressant imipramine in a 12-week treatment of newly diagnosed depression in twenty - three elderly with t2 dm and hypomagnesaemia . Here, oral magnesium supplementation with mgcl2 (equivalent to 450 mg of elemental magnesium) was as effective as 50 mg imipramine daily, the prototypic medication in depression . However, of note, no magnesium study has so far specifically targeted ckd - associated fatigue and depression . Given the paucity of the safety data of currently available antidepressants in ckd patients, magnesium supplementation provided that serum magnesium levels are controlled could become a safe therapeutic option in this population . The clinical role of magnesium in terms of benefits and harms of higher or lower serum magnesium levels continues to attract growing attention in research as evidenced by the increasing literature available on this topic . In ckd patients, vascular calcification, hypertension, diabetes, and diabetic nephropathy all of these factors are potentially affected by magnesium, and there is accumulating evidence for beneficial effects of magnesium supplementation and slightly elevated magnesium levels . However, it is important to stress that in most studies discussed above, causality cannot be inferred from the associations reported . For example, many of the associations between magnesium intake and outcome may represent true relationships but also could be confounded by magnesium intake reflecting different general dietary and/or life - style habits . As such, we need intervention studies to confirm or refute the hypotheses derived from these associations . In particular indications, thus, in hyperphosphataemia associated with advanced ckd, phosphate binders are often necessary to limit dietary phosphate absorption . Here, the combination of calcium acetate and magnesium carbonate has been shown to be as efficient and equally well tolerated as sevelamer hydrochloride in addition to reducing calcium intake compared to pure calcium - based phosphate binders . In an animal study, the ensuing mildly elevated magnesium levels resulted in beneficial effects in terms of calcification, pth levels and survival . Clinical data further demonstrated that camg does not negatively influence bone health and might even help to maintain it, causing neither an overstimulation nor a suppression of bone turnover (fig . 3). In view of the above discussion, therapy with camg in hyperphosphataemic ckd patients offers the exciting option to also evaluate whether the many pleiotropic actions of magnesium beneficially influence health issues beyond bone mineral disease.fig . 3impact of phosphate binders on serum levels of the bone turnover marker -ctx (beta - crosslaps). Time course of values at weeks 9 and 25 of the calcium acetate / magnesium carbonate group (n = 105) and of the sevelamer - hcl group (n = 99) is displayed in white and grey, respectively (adapted and reprinted from by permission of oxford university press) impact of phosphate binders on serum levels of the bone turnover marker -ctx (beta - crosslaps). Time course of values at weeks 9 and 25 of the calcium acetate / magnesium carbonate group (n = 105) and of the sevelamer - hcl group (n = 99) is displayed in white and grey, respectively (adapted and reprinted from by permission of oxford university press)
Cervical cancer is the third most common female cancer and the fourth leading cause of cancer death in women worldwide . It is the sixth most common female cancer and the seventh leading cause of cancer death in korea . At present, radical hysterectomy (rh) followed by tailored adjuvant therapy and primary chemoradiation therapy (crt) are the most frequent treatments employed for patients with bulky early - stage (stage ib2 and iia2) cervical cancer . Only a single randomized controlled trial reported to date has compared rh followed by tailored adjuvant therapy with primary radiation therapy (rt) in patients with early - stage cervical cancer . However, as the cited trial included only 40 patients with bulky early - stage cervical cancer in each treatment group, and was conducted before the era of crt, the results thereof cannot be generalized to all patients with bulky early - stage cervical cancer . To the best of our knowledge, only two small retrospective case - control studies have compared rh followed by tailored adjuvant treatment with primary crt in patients with bulky early - stage cervical cancer . However, recent larger series suggested that patients who underwent rh followed by tailored adjuvant therapy experienced better survival outcomes compared to primary crt . Moreover, it has been suggested by investigators in the usa that rh followed by tailored adjuvant therapy is potentially the most cost - effective treatment strategy for patients with bulky early - stage cervical cancer compared to other treatment strategies including crt, indicating that the role of rh in such patients should be re - evaluated . It is necessary to clarify whether rh followed by tailored adjuvant therapy, or primary crt, is the better treatment modality in such patients . We therefore compared survival outcomes and treatment - related morbidities in patients with bulky early - stage cervical cancer who underwent rh followed by tailored adjuvant therapy, and those who received primary crt . We retrospectively searched the records of two tertiary cancer centers located in seoul, korea (asan medical center and samsung medical center) and identified all consecutive patients with stage ib2 or iia2 cervical cancer who underwent rh followed by tailored adjuvant therapy, or primary chemoradiation therapy . Patients were included if they had: 1) histologically confirmed cervical cancer of stage ib2 or iia2 according to the international federation of obstetrics and gynecology (figo) staging system revised in 2009; 2) a tumor diameter> 4 cm on magnetic resonance imaging (mri); and 3) squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma . Patients with small cell neuroendocrine carcinomas, those with occult cervical cancer detected after simple hysterectomy, and those who received neoadjuvant chemotherapy, were excluded . Because current standard tailored adjuvant therapy after rh is rt or crt for intermediate risk groups and crt for high risk group, patients who did not receive rt or crt after rh in intermediate risk group and patients who did not receive crt after rh in high risk group were excluded . 1 shows the patient flow for this study . Of the 362 patients who were evaluated for the eligibility criteria of this study, 147 patients underwent rh and 68 patients received primary crt . Intermediate risk group after rh was defined according to gynecologic oncology group (gog) protocol 92 . High risk group after rh was defined as patients with parametrial involvement, lymph node metastasis, or positive resection margin . Recurrence - free survival (rfs) was defined as the time, in months, from the date of rh or crt, to the date of first documented recurrence, death or date of last contact . Overall survival (os) was defined as the time, in months, from the date of rh or crt to the date of death; or for living patients, the date of last contact regardless of whether or not this contact is on a subsequent protocol . All documented treatment - related toxicities were graded using the radiation therapy oncology group (rtog) criteria and the nci common toxicity criteria for adverse events (ctcae, version 3.0). Toxicities observed within 4 weeks of treatment were categorized as early complication, whereas those occurring later were considered to be late complication . All patients underwent piver - rutledge type 3 hysterectomy with pelvic and/or para - aortic lymphadenectomy . According to the pathologic risk factors, 48 (20.2%), 30 (33.8%), and 69 (46%) patients were low risk, intermediate risk, and high risk group, respectively . Of 30 patients in intermediate - risk group, 21 patients (13.1%) and 9 patients (26.2%) received adjuvant rt and crt, respectively (fig . 1). Of the 69 patients in high - risk group, received adjuvant crt (fig . The radiation dose ranged from 4,010 to 5,040 cgy in patients who received adjuvant rt or crt . Chemotherapy regimen in patients who received crt consisted of weekly cisplatin in 30 patients, 5-fluorouracil / cisplatin in 36 patients, or paclitaxel / cisplatin in 12 patients . Patients received external pelvic rt (radiation dose range, 4,140 to 5,040 gy), intracavitary brachytherapy (radiation dose range, 3,000 to 3,500 cgy), and parametrial booster dose (radiation dose range, 540 to 1,200 cgy). All patients received concurrent chemotherapy during external beam rt consisted of weekly cisplatin in 52 patients, 5-fluorouracil / cisplatin in 10 patients, or paclitaxel / cisplatin in 6 patients . Oncologic outcomes and treatment - related complications were compared between patients who underwent rh and those who received crt . Mean values in the two groups were compared using student's t - test or the mann - whitney u - test . Frequency distributions were compared using the chi - squared test or fisher's exact test . Rfs and os were estimated using the kaplan - meier method and group data were compared using cox's proportional hazards models . The data were initially compared using univariate analysis, and all variables significant in this exercise were included in multivariate analysis, again using cox's proportional hazards method . We retrospectively searched the records of two tertiary cancer centers located in seoul, korea (asan medical center and samsung medical center) and identified all consecutive patients with stage ib2 or iia2 cervical cancer who underwent rh followed by tailored adjuvant therapy, or primary chemoradiation therapy . Patients were included if they had: 1) histologically confirmed cervical cancer of stage ib2 or iia2 according to the international federation of obstetrics and gynecology (figo) staging system revised in 2009; 2) a tumor diameter> 4 cm on magnetic resonance imaging (mri); and 3) squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma . Patients with small cell neuroendocrine carcinomas, those with occult cervical cancer detected after simple hysterectomy, and those who received neoadjuvant chemotherapy, were excluded . Because current standard tailored adjuvant therapy after rh is rt or crt for intermediate risk groups and crt for high risk group, patients who did not receive rt or crt after rh in intermediate risk group and patients who did not receive crt after rh in high risk group were excluded . 1 shows the patient flow for this study . Of the 362 patients who were evaluated for the eligibility criteria of this study, 147 patients underwent rh and 68 patients received primary crt . Intermediate risk group after rh was defined according to gynecologic oncology group (gog) protocol 92 . High risk group after rh was defined as patients with parametrial involvement, lymph node metastasis, or positive resection margin . Recurrence - free survival (rfs) was defined as the time, in months, from the date of rh or crt, to the date of first documented recurrence, death or date of last contact . Overall survival (os) was defined as the time, in months, from the date of rh or crt to the date of death; or for living patients, the date of last contact regardless of whether or not this contact is on a subsequent protocol . All documented treatment - related toxicities were graded using the radiation therapy oncology group (rtog) criteria and the nci common toxicity criteria for adverse events (ctcae, version 3.0). Toxicities observed within 4 weeks of treatment were categorized as early complication, whereas those occurring later were considered to be late complication . All patients underwent piver - rutledge type 3 hysterectomy with pelvic and/or para - aortic lymphadenectomy . According to the pathologic risk factors, 48 (20.2%), 30 (33.8%), and 69 (46%) patients were low risk, intermediate risk, and high risk group, respectively . None of 48 patients in low risk group received adjuvant therapy . Of 30 patients in intermediate - risk group, 21 patients (13.1%) and 9 patients (26.2%) received adjuvant rt and crt, respectively (fig . 1). Of the 69 patients in high - risk group, received adjuvant crt (fig . The radiation dose ranged from 4,010 to 5,040 cgy in patients who received adjuvant rt or crt . Chemotherapy regimen in patients who received crt consisted of weekly cisplatin in 30 patients, 5-fluorouracil / cisplatin in 36 patients, or paclitaxel / cisplatin in 12 patients . Patients received external pelvic rt (radiation dose range, 4,140 to 5,040 gy), intracavitary brachytherapy (radiation dose range, 3,000 to 3,500 cgy), and parametrial booster dose (radiation dose range, 540 to 1,200 cgy). All patients received concurrent chemotherapy during external beam rt consisted of weekly cisplatin in 52 patients, 5-fluorouracil / cisplatin in 10 patients, or paclitaxel / cisplatin in 6 patients . Oncologic outcomes and treatment - related complications were compared between patients who underwent rh and those who received crt . Mean values in the two groups were compared using student's t - test or the mann - whitney u - test . Frequency distributions were compared using the chi - squared test or fisher's exact test . Rfs and os were estimated using the kaplan - meier method and group data were compared using cox's proportional hazards models . The data were initially compared using univariate analysis, and all variables significant in this exercise were included in multivariate analysis, again using cox's proportional hazards method . Mean patient age was significantly higher in the crt group (46.9 vs. 53.9 years, p<0.001), but there was no between - group difference in the body mass index (bmi), the presence of comorbid medical disease, eastern cooperative oncology group (ecog) performance status, figo stage, tumor histology, pretreatment serum scc ag concentration, mean tumor diameter, tumor diameter distribution as assessed using 2 cm intervals, parametrial invasion, or lymph node metastasis on mri . The mean and median follow - up times were 46 and 40 months (range, 3 to 130 months), respectively, for all patients; 47 and 40 months (range, 3 to 130 months), respectively, for patients in the rh group; and 42 and 40 months (range, 3 to 112 months), respectively, for patients in the crt group (p=0.253). Disease recurrence was observed in 27 rh (18.4%) and 20 crt (29.4%) patients (p=0.068); 23 (15.6%) and 17 (15.6%) patients, respectively, died of disease (p=0.101). The 5-year rfs rates were 77% in the rh group and 66% in the crt group (p=0.047) (fig . 2a); the 5-year os rates were 78% in the rh group, and 67% in the crt group (p=0.048) (fig . The pattern of recurrence was similar between rh group and crt group (p=0.409) (table 2). By univariate analysis, all of histologic type, and treatment group, were significantly associated with rfs; whereas none of age, bmi, the presence of comorbid medical disease, figo stage, pretreatment serum scc ag concentration, tumor size measured on mri, parametrial invasion, or lymph node metastasis as shown on mri was so associated (table 3). All of histologic type, lymph node metastasis as shown on mri, and treatment group, were significantly associated with os; whereas none of age, bmi, the presence of comorbid medical disease, figo stage, pretreatment serum scc ag concentration, tumor size measured on mri, or parametrial invasion as shown on mri showed a significant association (table 3). Multivariate analysis revealed that, after adjusting for histologic type, crt was associated with a significantly higher risk of recurrence (odds ratio [or], 2.26; 95% confidence interval [ci], 1.24 to 4.14; p=0.008) (table 3). Moreover, after adjusting for histologic type and lymph node metastasis as shown on mri, multivariate analysis showed that crt was associated with a significantly higher risk of death (or, 3.02; 95% ci, 1.53 to 5.98; p=0.001) (table 3). When dividing patients into three groups: those who underwent rh alone (n=48), those who underwent rh+(chemo) radiation therapy ((c)rt) (n=99), and those who received primary crt (n=68), rh alone group had significantly better rfs and os compared to rh+(c)rt group (p=0.012 for dfs, p=0.003 for os) and crt group (p=0.080 for dfs, p=0.020 for os) (fig . Group, 5 (10.4%) of 48 patients had recurrent disease and 3 (6.3%) of them died of disease . Therefore, 43 of 147 patients (29.3%) in rh group were cured by rh alone without rt . Grade 3 - 4, early complications were documented in 1 (2.1%), 24 (24.2%), and 21 (30.9%) patients of the rh alone group, rh+rt group, and crt group, respectively (p=0.001) (table 4). Grade 3 - 4, late complications were observed in 1 (2.1%), 1 (1%), and 6 (8.8%) patients of these groups, respectively (p=0.026) (table 4). Lymphedema of the lower extremities was documented in 6 (12.5%), 9 (9.1%), and 1 (1.5%) patients of these groups, respectively (p=0.058). Mean patient age was significantly higher in the crt group (46.9 vs. 53.9 years, p<0.001), but there was no between - group difference in the body mass index (bmi), the presence of comorbid medical disease, eastern cooperative oncology group (ecog) performance status, figo stage, tumor histology, pretreatment serum scc ag concentration, mean tumor diameter, tumor diameter distribution as assessed using 2 cm intervals, parametrial invasion, or lymph node metastasis on mri . The mean and median follow - up times were 46 and 40 months (range, 3 to 130 months), respectively, for all patients; 47 and 40 months (range, 3 to 130 months), respectively, for patients in the rh group; and 42 and 40 months (range, 3 to 112 months), respectively, for patients in the crt group (p=0.253). Disease recurrence was observed in 27 rh (18.4%) and 20 crt (29.4%) patients (p=0.068); 23 (15.6%) and 17 (15.6%) patients, respectively, died of disease (p=0.101). The 5-year rfs rates were 77% in the rh group and 66% in the crt group (p=0.047) (fig . 2a); the 5-year os rates were 78% in the rh group, and 67% in the crt group (p=0.048) (fig . The pattern of recurrence was similar between rh group and crt group (p=0.409) (table 2). By univariate analysis, all of histologic type, and treatment group, were significantly associated with rfs; whereas none of age, bmi, the presence of comorbid medical disease, figo stage, pretreatment serum scc ag concentration, tumor size measured on mri, parametrial invasion, or lymph node metastasis as shown on mri was so associated (table 3). All of histologic type, lymph node metastasis as shown on mri, and treatment group, were significantly associated with os; whereas none of age, bmi, the presence of comorbid medical disease, figo stage, pretreatment serum scc ag concentration, tumor size measured on mri, or parametrial invasion as shown on mri showed a significant association (table 3). Multivariate analysis revealed that, after adjusting for histologic type, crt was associated with a significantly higher risk of recurrence (odds ratio [or], 2.26; 95% confidence interval [ci], 1.24 to 4.14; p=0.008) (table 3). Moreover, after adjusting for histologic type and lymph node metastasis as shown on mri, multivariate analysis showed that crt was associated with a significantly higher risk of death (or, 3.02; 95% ci, 1.53 to 5.98; p=0.001) (table 3). When dividing patients into three groups: those who underwent rh alone (n=48), those who underwent rh+(chemo) radiation therapy ((c)rt) (n=99), and those who received primary crt (n=68), rh alone group had significantly better rfs and os compared to rh+(c)rt group (p=0.012 for dfs, p=0.003 for os) and crt group (p=0.080 for dfs, p=0.020 for os) (fig . Group, 5 (10.4%) of 48 patients had recurrent disease and 3 (6.3%) of them died of disease . Therefore, 43 of 147 patients (29.3%) in rh group were cured by rh alone without rt . Grade 3 - 4, early complications were documented in 1 (2.1%), 24 (24.2%), and 21 (30.9%) patients of the rh alone group, rh+rt group, and crt group, respectively (p=0.001) (table 4). Grade 3 - 4, late complications were observed in 1 (2.1%), 1 (1%), and 6 (8.8%) patients of these groups, respectively (p=0.026) (table 4). Lymphedema of the lower extremities was documented in 6 (12.5%), 9 (9.1%), and 1 (1.5%) patients of these groups, respectively (p=0.058). We found that 29.3% of patients with tumors> 4 cm in diameter were cured by rh alone without rt, consistent with the rates of 37 - 51% previously observed in patients with bulky early - stage cervical cancer . Many more patients than expected did well after surgery alone, and such patients experienced the best survival outcomes and the lowest morbidity rates, indicating that rh continues to play a significant role in patients with bulky early - stage cervical cancer . This is one of the best advantages of rh followed by tailored adjuvant therapy because it makes the patients avoid inadvertent radiation therapy . In our series, multivariate analysis showed that rh was associated with a significantly lower risk of recurrence (or, 2.26; 95% ci, 1.24 to 4.14; p=0.008) and death (or, 3.02; 95% ci, 1.53 to 5.98; p=0.001) compared to crt . Previous studies including a randomized controlled trial suggested that rh afforded survival outcomes similar to those seen after definitive rt or crt in patients with bulky early - stage cervical cancer [5 - 7]. However, the cited works included only a small numbers of patients with bulky early - stage cervical cancer, and did not evaluate important prognostic factors in survival analysis, such as lymph node metastasis status or parametrial invasion as seen on mri . Recently, a relatively large - scaled study suggested that rh would yield better survival outcomes compared to crt in bulky early - stage cervical cancer, and a multivariate analysis of surveillance, epidemiology, and end results (seer) data showed that rh was associated with a 49% improvement in survival compared to crt in bulky early - stage cervical cancer; an outcome consistent with our findings . This indicates that rh followed by tailored adjuvant therapy is a potentially more effective treatment modality than is crt for patients with bulky early - stage cervical cancer . In our series, the lymph node failure rate was higher in crt group than rh group although it was not statistically significant (14.7% vs. 8.2%). In addition, several studies suggested that the rate of residual disease on lymph node was 43.8% to 49% after crt followed by lymphadenectomy in patients with locally advanced cervical cancer [21 - 23]. We think that this may be one of the reasons for improved survival in rh group . In our study, the 5-year survival rates differed by about 10 percentage points (78% vs. 66%, p=0.002). Such a difference may be clinically significant and should be further evaluated in randomized controlled trials that include sufficient numbers of study subjects . We have calculated that recruitment of a total of 424 patients (212 patients per group), and the occurrence of 144 events, are required to show that a 10% difference in os by the fifth year (hazard ratio, 0.627) is statistically significant, with an alpha - value of 0.05 and a beta - value of 0.2 on two - sided tests . Assuming accrual of 85 patients / year, the study would require 10 years, 5 for accrual and 5 for follow - up . Such a trial will be launched soon by the korean gynecologic oncology group (kgog 1029). For successful surgical treatment for bulky early - stage cervical cancer in this trial, the radicality of surgery is of paramount importance and surgical procedures should be standardized . To achieve surgical radicality and standardize the surgical procedures, live surgery workshops on laparoscopic radical hysterectomy was held in each center by turns among kgog affiliated hospitals for the past ten years . Earlier studies have suggested that use of a combination of rh and rt was associated with the highest morbidity rates compared to rh alone group and crt group, including serious toxicity frequencies> 20% . Physicians were reluctant to perform rh in patients with bulky early - stage cervical cancer based on these results . However, recent other reports found that the rates of serious toxicity were lower after rh+rt; the rate of toxicity of grades 3 - 4 was only 7%, being 2% and 3% in terms of gastrointestinal and genitourinary complications, respectively . When we evaluated the occurrence of grade 3 - 4 toxicities that required treatment, we found that the rates of grade 3 - 4 early complications were not different between rh+rt group and crt group (24.2% vs. 30.9%, p=0.342). Rather, the rates of grade 3 - 4 late complications was lower in rh+rt group compared to crt group although the difference was not statistically significant (1% vs. 8.8%, p=0.019). Most complications were associated with radiation therapy per se . Because adjuvant rt in rh+rt group features only external pelvic rt, at doses of 4,140 - 5,040 cgy, whereas definitive rt in crt group consists of external pelvic rt followed by intracavitary brachytherapy and parametrial boosting; the radiation dose to the bowel, bladder, and vagina was much higher in the definitive rt group . Therefore, rt - related bladder, rectal, and vaginal complication rates should be greater in this group compared to the adjuvant rt group . Because our study is a retrospective one and included study subjects which is not big enough to confirm the complication rates between the two treatment groups, the complication rates associated with each treatment modality (rh+rt group vs. crt group) should be re - evaluated in a randomized controlled trial . Because we assessed patients over a long period of time, during which the selection criteria for initial treatment and adjuvant therapy varied, and this study was a retrospective one in addition, the treatment strategies for patients with bulky early - stage cervical cancer differed among the two centers, with one favoring rh followed by tailored adjuvant therapy, and the other permitting treatment to be at the discretion of the attending physician . Therefore, this may be a possible bias . However, we tested for associations between different clinicopathological variables that could be potential confounding factors in the two treatment groups . Apart from mean age, there was no significant between - group difference in any clinicopathologic factor or potential prognostic factor, including parametrial invasion status and lymph node metastasis . Further, we employed multivariate analysis after adjusting for all other factors that could significantly impact survival . Therefore, we think that the impact of selection bias on the outcomes was minimized . The strength of this study is that this is the largest one which compared the outcomes of patients with bulky early - stage cervical cancer between rh group and crt group and which was conducted in the era of crt . In conclusion, a significant proportion of patients with bulky early - stage cervical cancer were cured by rh alone . Rh followed by tailored adjuvant therapy resulted in a significantly better rfs and os, and significantly lower treatment - related morbidity rates than were afforded by primary crt in patients with bulky early - stage cervical cancer . A randomized controlled trial to compare these two treatment modalities
The mango is a commercial crop in many countries of the southeast asia, namely, india, pakistan, the philippines, indonesia, malaysia, thailand, burma, sri lanka, and bangladesh . Mango ranks third among the tropical fruits grown in the world with a total production of 23.87 million tons, to which bangladesh contributed by only 0.64 million tons . It is also gaining rapid popularity in the middle east, south east africa, south africa, florida, israel, and australia . Among the fruits, mango ranks first in terms of area and third in terms of production in bangladesh . Nutritionally, it contains substantial quantity of appreciable carotene, vitamin c, and dietary fibre as well as soluble sugars and different minerals which are used as good sources of nutrition, and readily available and easily assumable in human body, and therefore it is capable to prevent many deficiency diseases [7, 8]. Approximately 3050% fruits go wasted during postharvest handling, storage, and ripening . Among the fruits mango manifested high postharvest losses because of its high perishability and climacteric pattern of respiration . The marketability of this perishable fruit is closely linked with the development of suitable technology which reduces the losses at different stages of harvesting and storage condition . Losses in terms of quality and quantity of fruits occur at all stages in the postharvest system from harvesting to consumption . Reliable statistical data are inadequate especially in bangladesh to indicate the magnitude of postharvest losses of mango . Singh et al . Reported that the postharvest losses of mango fruit in india due to microbial decay ranged from 20 to 33% . Quality mangoes are produced in northwestern part of bangladesh, of which about 3538% of postharvest losses are caused due to inefficient handling during its transportation, storage, and marketing . Mango always decays after harvest, and postharvest losses can be considerably reduced by applying improved storage technology and prolonging the shelf life of fruits . Several researchers used bavistin df (bdf) for controlling spoilage of different fruits [10, 11]. The efficacy of bavistin against the fruit rot pathogen was reported by several workers [12, 13]. Although bavistin was observed to be the most effective treatment, there are numerous reports of the negative effects of using chemicals on farm income and the health of farm workers . Toxic contamination to the environment, particularly in developing countries, has also been reported . These treatments strongly impede in ethylene synthesis that resulted in low respiration and delay ripening . In addition, fungicidal treatments like bavistin df (bdf) are also excellent ethylene inhibitors . These treatments performed effectively in reduction of postharvest decay and extension of shelf life of mango . Apparently, these treatments deteriorate the qualities of fruits to some extent, but the reduction of losses and extension of postharvest life of mango will help to increase the market price in the off seasons which play a good role in the economic development . In this present investigation we tried to study the behavioral pattern of physicochemical properties of postharvest mango in the storage conditions . Especially the effects of bdf on ph, tritability, and sugar contents were studied in detail . It was also aimed to find out a desirable technology for extension of storage of mango . The two mango varieties, namely, langra and khirshapath, were selected as experimental materials . The mango varieties undertaken for investigation were collected from mango grower of kansart, shibganj upazila of chapainowabgonj district and charghat upazila of rajshahi district, and other material used as postharvest treatments, namely, bavistin df (bdf), were collected from royal scientific store at cooperative market, rajshahi city market . The experiment consisted of two factors and the required numbers of unblemished physically similar, more or less uniform size, shape and color fruits for the experiment were harvested manually from each plant of the varieties, langra and khirshapath . The skin of fruits was cleared with the help of a cloth just after harvesting . The 250, 500, and 750 ppm of bdf solution were prepared by dissolving 250, 500, and 750 mg of bdf (commercial product) in one litre of distilled water . It is noted that the active ingredient of bdf is carbendazim whose initial concentration is 500 g / kg, that is, 50% (product name the fruits of both varieties were dipped into the bdf solution for a period of 5 minutes . Care was taken to ensure enough quantity of bdf being absorbed by the fruits and stored at ambient condition on brown paper . The following reagents were used for determination of titrable acidity: (i)standard naoh solution (0.1 n), (ii)1% methyl red . Standard naoh solution (0.1 n), ten g of mango pulp was taken in a 100 ml beaker and then it was homogenized with distilled water in a blender . The blended materials were then filtered and transferred to a 100 ml volumetric flask, and the volume was made up to the mark with distilled water . Two to three drops of phenolphthalein indicator was added, and then the conical flask was shaken vigorously . It was then filtrated immediately with 0.1 n naoh solutions from a burette till a permanent pink color appeared . Percent titratable acidity was calculated by using the following formula: (1)%titratable acidity=(tnv1ev2w1000)100, where t is the titre, n is the normality of naoh, v1 is the volume made up, e is the equivalent weight of acid, v2 is the volume of extract, and w is the weight of sample . The following reagents were used for determination of total sugar: anthrone reagent: the reagent was prepared by dissolving 2 g of anthrone in one litre of concentrated h2so4,standard glucose solution: a standard solution of glucose was prepared by dissolving 10 mg of glucose in 100 ml of distilled water . Anthrone reagent: the reagent was prepared by dissolving 2 g of anthrone in one litre of concentrated h2so4, standard glucose solution: a standard solution of glucose was prepared by dissolving 10 mg of glucose in 100 ml of distilled water . Four g of mango pulp was cut into small pieces and immediately plunged into boiling ethyl alcohol and was allowed to boil for 5 to 10 minutes (5 to 10 ml of alcohol was used per gram of pulp). Then the extract was filtered through two layers of muslin cloths and the ground tissue was reextracted for three minutes in hot 80% alcohol, using 2 to 3 ml of alcohol per gram of tissue . The volume of the extract was evaporated to about 25% (1/4) of the volume over a steam bath and cooled . This reduced volume of the extract was transferred to a 100 ml volumetric flask and it was made up to the mark with distilled water . Aliquot of 1 ml of pulp extract was pipetted into test tubes and 4 ml of the anthrone reagent was added to each of this solution and mixed well . Glass marbles were placed on top of each test tube to prevent loss of water through evaporation . Then the tubes were placed in a boiling water bath for 10 minutes and then cooled . A reagent blank was prepared by taking 1 ml of water and 4 ml of anthrone reagent in a tube and treated similarly . The absorbance of blue green solution was measured at 680 nm in a colorimeter . A standard curve of glucose was prepared by taking 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of standard glucose solution in different test tubes containing 0.0, 10, 20, 40, 60, 80, and 100 g of glucose, respectively, and the volume was made up to 1 ml with distilled water . Then 4 ml of anthrone reagent was added to each test tube and mixed well . The absorbance was measured at 680 nm using the blank containing 1 ml of water and 4 ml of another reagent . The amount of total sugar present in the extract was calculated from the standard curve of glucose . Finally, the percentage of total sugar was determined by using the following formula: (2)%total sugar (g/100 g of mango) = (quantity of sugar obtainedweight of sample)100 t of acid, v2 = volume of extract, w = weight of sample . Reducing sugar content of mango reagent used: (i) dinitrosalicylic acid (dns) solution: simultaneously, 1.0 g of dns, 200 mg of crystalline phenol, and 50 mg of sodium sulphite were placed in a beaker and mixed with 100 ml of 1% naoh by stirring . When it was needed to store variation between varieties means in connection with titratable acidity was perceived to be highly significant at different days after storage (table 1). At various days of storage, the langra was noticed to be a higher producer of titratable acidity as compared to the khirshapat . The abating trend was hastil from the initial to 3rd day and, thereafter, it was slower . At initial day, higher (3.77%) was derived from the langra while lower (2.47%) was noticed from the khirshapat . At 12th day, higher (0.31%) was recorded from the langra while, the khirshapat gave lower amount (0.24%). The results of the present investigation might be possibly due to genetically dissimilarities between two varieties . Different doses of bdf solution imposed to this investigation in terms of titratable acidity showed significant variation among the means at various days of storage . At different days of storage, titratable acid content diminished hastily from initial to 3 days, and then it diminished steadily (figure 1). In all the storage period, higher titratable acidity (3.19, 1.20, 0.92, 0.74, and 0.42%) was derived from b3 treatment from initial to 12th days followed by 3.09, 0.68, 0.40, 0.22, and 0.11% from untreated mangoes . These phenomena happening might be possible due to b3 treatment delayed ripening that caused lower diminishing trend of titratable acidity while control treatment caused ripening fast resulting in high decreasing trend of titrable acid content . The combined effect of varieties and different doses of bdf solution in relation to titratable acidity of mango pulp demonstrated significant variation at different days after storage except initial days . At different days of storage, there appeared a decreasing trend of titratable acid content with the rising of storage period . At the 6th day, the highest (1.00%) quantity was recorded from the treatment combination of v1b3 and the lowest acid concentration (0.30%) was recorded from the treatment combination of v2b0 (table is not mentioned here). This occurrence might be probably due to the reduction of acid oxidation at v2b3 combination as well as genetic variation in between varieties . The analysis of variance between the varieties exhibited significant variation in terms of pulp ph of mango at different days after storage except at 6th day (figure 2). At various days of storage, a growing up trend of pulp ph with the increase of storage period was observed . In each storage period, higher pulp ph (6.96) was noted from the khirshapat at 12th day whereas lower (6.85) was noted from the langra . The growing up trend of pulp ph was also observed by shahjahan et al . . This phenomenon might be possible due to oxidation of acid during storage resulting in higher ph and also might have been genetical dissimilarities between varieties . Different doses of bdf solution subjected to this trial showed significant variation in pulp ph at different days after storage . The results indicated that the growing up trend of pulp ph was perceived from different treated and untreated mangoes at various days of storage (figure 2). Pulp ph was higher in control at all stages of storage followed by the fruits treated with b1, b2, and b3 treatments, respectively . The ph value of mango pulp was higher (7.05) in control which was statistically at par with b1 and b2 treatment whereas the fruits treated with b3 produced lower (6.57) value at 12th day . The results of the present investigation at b3 treatment interrupted the loss of acid oxidation resulting in lower ph value . The combined effect of varieties and different doses of bdf solution imposed to this study in pulp ph were noticed to be nonsignificant at different days after storage . There appeared a slightly rising trend of pulp ph from various treatment combinations at different days of storage . At 6th day, the highest (6.90) ph value was reported from the treatment combination of v2b0 which was statistically at par with v1b0 and the lowest (6.70) was reported from the treatment of combination of v1b3, respectively . Statistically highly significant variation was observed in tss content between two varieties at different days after storage . The results exhibited that tss content of mango pulp developed in a continuous stream with the expansion of storage period . The developing trend was hastily from initial to 6th day, thereafter; it increased slower . From initial to 6th day, the khirshapat enriched a better amount of tss than the langra, but, after 6th day, langra performed better than the khirshapat up to 12th day . At 9th day, higher (17.90%) tss quantity was noted from the khirshapat and lower (17.00%) was noted from the langra (table 2). Absar et al . Reported that tss was increased with maturity of mango fruit . Different doses of bdf solution implied to the postharvest mangoes in this study were noticed to be significant in terms of tss content at different days after storage . At different days of storage, the results showed that tss accumulation increased with the increase of storage duration . It also explored that tss content was hastily grown up from untreated mangoes from initial to 6th day and then it came down significantly (figure 3). The other treatment namely, b1, also increasingly produced tss from initial to 9th day, and thereafter it decreased sharply . Mango fruits treated with b2 also produced more or less similar enhancing trend from initial to 12th days . But the fruits treated with b3 treatment gave very slower motion in tss accumulation at various days after storage . The highest (21.25 and 21.30%) accumulation of tss content was perceived from b0 and b1 treatment at 6 and 9th days while the lowest (12.90 and 14.85%) was noted from b3 treatment (figure 3). The results of the present studies are strongly supported by the findings of dhemre and waskar also found the similar results . These happened possibly due to ripening condition resulting in maximizing tss accumulation in control and 750 ppm of bdf solution resisted in ethylene synthesis that caused delay ripening and ultimately in lower tss accumulation . It also revealed that tss accumulation is strongly related to ripening and it caused falling off owing to decaying . The combined effect of varieties and applied different doses of bdf solution in connection with tss content were perceived to be significant at different days after storage except initial and 3rd day . The highest accumulation (22.00, 21.80, and 21.60%) was obtained from the treatment combination of v1b0, v1b1, and v1b2 at 6, 9 and 12th days, while the lowest value (13.20, 14.50, and 17.50%) was notified from the treatment combination of v2b3 (table is not presented here), respectively . Highly significant variation was manifested between both varieties means in terms of total sugar content of mango pulp at different days after storage . This gathering trend was more or less hastil from initial to 9th days in both the varieties; thereafter; it expanded slightly slower . At all days of storage, the khirshapat produced more quantity of tsc than the langra . At initial day, the khirshapat had higher (6.09%) while; the langra provided lower (5.57%). At 12th day, it gave higher quantity (19.64%) and lower (19.07%) was noted in the langra . Reported that total sugar content was expanded gradually, when stored for 6 days at room temperature . The increase in tsc might be possible due to conversion of complex starch or carbohydrate into simple compound . Different doses of bdf solution subjected to the investigation in connection with total sugar content of mango pulp demonstrated significant variation at different days after storage except initial day . At different days, the results found that tsc increased hastily with expanding of storage period (figure 4). The increasing trend was very swift in untreated mango followed by other treatments, namely b1, b2 and b3 treatment, respectively . The highest quantity of tsc (21.06 and 21.36%) was obtained from control and b1 treated mangoes at 9 and 12th days, while the lowest (12.40% and 15.70%) was reported from b3 treatment . The results of the present investigation are in conformity with the reports of dhemre and waskar . The enhancing trend of total sugar at untreated mangoes might be perhaps due to breaking down of complex carbohydrate into simple compound but b3 treatment made delay ripening at storage period . The combined effect of varieties and implied different doses of bdf solution in this study in terms of total sugar content of mango pulp exhibited nonsignificant variation at different days after storage . The results indicated that total sugar content progressively accumulated with the advance of storage period (table is not mentioned here). At 9th day, the maximum (21.31%) quantity of tsc was formed from the treatment combination of v2b0 while the minimum (12.10%) was formed from the treatment combination of v1b3, respectively . Analysis of variance showed significant effect on reducing sugar content of mango pulp at different days of storage except at 6th day . The results showed an increasing trend of reducing sugar with the progress of storage period . It also annotated that the khirshapat was found better in enriching of reducing sugar than the langra at different days of storage . Higher (5.47%) quantity of this sugar was observed from the khirshapat while lower (5.20%) quantity was noticed from the langra at 12th day of storage (table 3). Khirshapat providing more reducing sugar might be possibly due to genetical variation in both varieties . Different doses of bdf subjected to this study were perceived to be significant in respect of reducing sugar content of mango pulp at different storage periods except initial day . The results explained that reducing sugar of mango pulp was increased progressively at different days of storage . It also stated that untreated mangoes were better in forming of reducing sugar as compared to other treatments . Control was found to be a more effective producer of reducing sugar up to 9th day and then it came down owing to starting decay . At 12th day, the maximum (6.27%) amount of reducing sugar was obtained from b1 treatment and the lowest (4.32%) was obtained at b3 treatment (table 3). The results of the present study are in inconformity with the findings of dhemre and waskar . Lower increasing trend of reducing sugar content treated with b3 treatment might be possibly due to delayed ripening that resulted in lesser conversion of carbohydrates into simple's molecules . The combined effect of varieties and different doses of bdf solution of mango pulp demonstrated nonsignificant variation in terms of reducing sugar content of mango pulp at different days after storage . The results exposited that reducing sugar content grew up progressively at three - day interval up to 9th day; thereafter, it came down from the treatment combination of v2b0 . At 9th day, the highest (6.44%) quantity was noticed from the treatment combination of v2b0 and the lowest (3.90%) was noticed from v1b3 (table 4). The variation between the varieties means demonstrated highly significant in respect of nonreducing sugar content at different days after storage . The results were noticed to be an enhanchable trend of nonreducing sugar content at different days of storage . At all the days, it revealed that the khirshapat was observed to be much better than the langra in receiving of nonreducing sugar content (table 3). At 12th day, higher (14.20%) amount of nonreducing sugar was recorded from the khirshapat and lower (13.88%) amount was recorded from the langra . Different doses of bdf solution imposed to this trial were noticed to be a significant variation in connection with nonreducing sugar content of mango pulp at different days after storage except at initial stage . The results stated that nonreducing sugar content of mango pulp was formed progressively at various days . It denoted that untreated fruits were noticed to be better in achieving more quantity of nonreducing sugar followed by the other treatments . This increasing trend was markedly up to 9th day, and thereafter it increased slowly owing to becoming hackneyed . Lower rising trend was perceived from the fruit treated with b3 treatment . At 12th day, the highest result (15.28%) was recorded from control and lowest value (11.39%) was recorded from b3 treatment (table 3). These events might be probably due to b3 treatment retarded ethylene synthesis of mango pulp resulting in delayed ripening and little amount of nonreducing sugar achieving . The combined effect of varieties and different doses of bdf solution exhibited nonsignificant in terms of nonreducing sugar content of mango pulp at different days after storage . The results showed a mild growing up trend of nonreducing sugar from different treatment combinations at various days (table 4). At 9th day, the highest (14.87%) quantity of nonreducing sugar was notified from the treatment combination of v2b0 while the lowest (8.42%) was notified from the treatment combination of v1b3, respectively . Different doses of bdf solution imposed to this investigation in terms of titratable acidity showed significant variation among the means at various days of storage . The combined effect of varieties and different doses of bdf solution imposed to this study in pulp ph were noticed to be nonsignificant at different days after storage . A slightly rising trend of pulp ph from various treatment combinations at different days of storage appeared . At different days of storage it also explored that tss content was hastily grown up from untreated mangoes from initial to 6th day, and then it came down significantly . The combined effect of varieties and different doses of bdf solution exhibited nonsignificant in terms of nonreducing sugar content of mango pulp at different days after storage . The results showed a mild growing up trend of nonreducing sugar from different treatment combinations at various days.
According to alzheimer s disease international, the worldwide costs of dementia ($604 billion in us dollars) amounted to more than 1% of the global gross domestic product in 2010 . Furthermore, dementia patients have increased healthcare utilization rates compared with patients with other major diseases . Previous research shows that informal costs make up a substantial part of the total annual costs of dementia . In the united states alone, the care provided by informal caregivers to people with dementia was valued at more than $202 billion in 2010 . Behavioral and psychological problems affect most individuals with dementia at some point during the progression of the disorder, adding to the cost and burden of caring for them . For example, as many as 83% of individuals with dementia suffer with depression; and as many as 77% suffer from anxiety . These problems may ultimately result inlong - term hospitalization, increased medication use, and decreased quality of life for caregivers and patients . Despite the fact that animal assisted therapy has been used for many years, some senior healthcare settings still do not accept animals, even though they acknowledge the positive benefits of animal assisted therapy and activities involving therapy pets . Many are concerned regarding the negative effects to human beings such as allergies, infections, biting, scratching, or even fear of the animals involved in therapy . The paro (short for personal robot in the japanese language) robotic pet has been in use in many countries since 2003 . The fda - approved device is designed to look like a baby harp seal, which is a non - familiar animal to most people . As a result covered in artificial fur, the robotic creature has a hard inner skeleton under which there are dual processors that control software for behavior generation and voice recognition . Paro, as a result, imitates animal behavior, but also responds to light, sound temperature, touch and posture and, over time, due to its artificial intelligence capability, develops its own character . Paro promotes the therapeutic results of psychological, physiological, and social effort from those who interact with it, lowering stress, improving depression, and reducing anxiety in many cases [5, 6].accurate data was needed regarding the effectiveness of robotic pet therapy . Therefore, the primary purpose of this study was to rigorously assess the effectiveness of paro robotic pet therapy in treating dementia - related symptoms such as anxiety and depression . The before and after outcome measures included: rating for anxiety in dementia (raid), cornell scale for depression in dementia (csdd), global deterioration scale (gds), pulse rate, pulse oximetry, galvanic skin response (gsr), and medication utilization . The interventional group received treatment with the paro robotic pet three times a week for 20 minutes, and the control group received the standard of care, which includes music, physical activity, and mental stimulation in 20-minute segments . Education was provided for the facility nurses and staff regarding the purpose of the study and the protocols for data collection . Subjects who met inclusion / exclusion criteria, or their significant family member, were approached and consented . Participation in the study was voluntary, and all residents had the right to refuse . Subjects routinely participated in group programming activities throughout the day . Subject groups were randomly assigned by the toss of a coin to receive either the paro robotic pet or standardized programming . Each participant was physician - diagnosed with mild to moderate dementia, utilizing standard diagnostic and statistical manual (dsm) or the national institute on aging alzheimer s organization criteria, and was 65 years of age or older . Patients with pre - existing psychiatric diagnoses (bipolar, schizophrenia, personality disorder, etc .) And those unable to participate in programming due to physical limitations were excluded from participating in the study . Consenting subject groups a sample of 60 was estimated based on an effect size (0.35) with a power of 0.80, and a 10% rate of attrition . The participants were equally divided into study and control groups and each group included 5 subgroups . Experimental group participants were exposed to treatment with the paro robotic pet once a day for three days a week . The pi and the trained facility nurses conducted the sessions with the paro robotic pets in the activity room of the assisted living memory care units . The 20-minute sessions involved seating 6 residents at a round table, placing the paro robotic pet in the center of the table, and encouraging the residents to interact with the robotic pet by demonstrating interaction . The demographic data and pre - tests of raid, csdd, and gds were administered by the pi and the trained facility nurses prior to the sessions with the paro robotic pet as well as after three months of exposure . The gsr, pulse oximeter, and pulse rate were recorded for each subject for every session throughout the three - month time period . The comparison group received what is considered the facilities standard of care which includesphysical activity, music, and mental stimulation . Facility nurses conducted these activities in small groups in the activity room on a daily basis . The demographic data, gds scores, and pre - tests of raid and csdd were administered and collected by the pi and the trained facility nurses and staff prior to the standardized program sessions as well as after three months of exposure . The gsr, pulse rate, and pulse oximetry readings were recorded for each subject before and after every session throughout the three - month time period . In summary, pulse oximetry, pulse rate, and gsr were collected before and after each 20-minute exposure to the robotic pet . The csdd and the raid were utilized for staff observations of selected behaviors prior to and after the study . Residents were assessed as to the severity of their dementia with the gds before and after the study . Staff observation and measurement of pulse oximetry, pulse rate, and gsr, along with the assessment of severity of dementia with the gds, occurred in the same manner with a control group of residents, who did not receive treatment with the paro robotic pet . The raid scale is a reliable and valid scale for measuring anxiety in dementia patients . In a previous study, inter - rater reliability and test / retest reliability were moderate, with an overall agreement of over 80% for individual items . Criterion validity and construct validity were established when the instrument was piloted on 51 inpatients and 32 day - hospital patients who had a dsm diagnosis of dementia . The sensitivity and specificity of the csdd has been reported as the 93% and 97%, respectively . The csdd was chosen for this study because its validity as a screening tool for depression dementia patients exceeds the geriatric depression scale in progressing dementia . Interrater reliability for the gds was found to be high, ranging from 0.87 to 0.97 in various studies . Concurrent validity of the gds was established by comparing scores of the gds to scores from the mini - mental state examination and showed high correlation between the two tools . Clinical / biological validity was also demonstrated to be satisfactory by comparing gds results with results from psychometric tests (r = 0.300.60), ct scan measures (r = 0.50 for sulcal enlargement and 0.60 for ventricular dilation), and cerebral blood flow (r = 0.700.80). Pulse rate and pulse oximetry have long been validated as indicators of stress and anxiety . As stress or anxiety decrease, gsr, or skin conductivity, can also be used as an indication of one s state of arousal . Gsr has been observed to continuously change over time and is correlated to the activity of the eccrine sweat glands . Located in the dermis, the eccrine sweat glands regulate body temperature by manufacturing and excreting sweat onto the skinssurface . Gsr can be measured through the collection of skin conductance and used as the quantitative indicator of anxiety . The development of bias potentials and polarization were minimized through the use of silver chloride cup electrodes . Retrospective and concurrent data was collected for utilization of pain, depression, sleep and behavior medications during the course of the study . A total of 61 patients (23% males, 77% females) with an average age of 83.4 years were randomized into the control and treatment groups . Table 1 summarizes the comparison in baseline characteristics and shows no difference between the two groups, thus relating homogeneity . Compared to the controlgroup, pulse oximetry and gsv were increased, while raid, csdd, pulse rate, pain medication, and behavior medication were significantly decreased in the treatment group . Table 2 displays the average changes in outcome measures in response to the therapy . No differences in gds staging or sleep medication and depression medication utilization were noted between the two groups . Table 3 contains the p values from multivariable regression representing the change in each outcome after the therapy . After adjusting for demographic variables, the group status showed a significant effect on all outcomes except gds, sleep medication and behavior medication . Table 4 represents a multivariable regression, using a mixed model, depicting gsv, pulse oximetry, and pulse rate, which were repeatedly measured throughout the study . After adjusting for demographics and time variable, the group status showed a significant effect on gsv, pulse oximetry, and pulse rate . The changes in gsv, pulse oximetry, and pulse rate over time were plotted for both groups in fig . 1 . The difference between the two groups was seen consistently throughout the study for pulse oximetry and pulse rate, while changes in gsv showed no difference between the two groups for several weeks . Using the data from the three - month study, researchers found that intervention with the paro robotic pet seal provided a viable alternative for controlling symptoms of anxiety and depression in elderly patients with dementia, often in lieu of pharmacological modalities . Oxygen saturation, pulse rate, gsv, raid, and csdd and medication use were all positively impacted in patients participating in the interventional group, indicating improvement in symptom control . This study significantly contributes to the body of knowledge regarding robotic biofeedback devices in the treatment of dementia . The study utilized a robust sample, measured a variety of outcomes, and used a randomized design . Previous studies lacked rigor with regard to sample size, design, and controlling for extraneous measures . Caution should be utilized in generalizing results beyond the sample in this study, however, due to the unique population of subjects . Implications for practice for providers working with this population include: 1) the average individual in the senior living environment consumes on average 16 to 28 medications per day at an average cost of 1200 to 1500 u.s . Dollars per month; and, 2) intervention with the paro robotic pet three times weekly for 20 minutes significantly reduced the need for these medications . As the literature suggests, the use of benzodiazepines in the elderly population result in falls, sedation, and physical dependence additionally, providers may use antipsychotics off - label to treat negative behaviors in individuals with dementia; these medications can cause or worsen heart arrhythmias in the older adult and worsen other chronic conditions such as renal impairment, gi distress, and liver impairment . Use of pain medications in the treatment group was significantly decreased as well, which may lead to further utilization of the paro . Other applications for the paro, in addition to reducing stress (as measured by gsv), may include improved oxygenation and improved cardiac status . The treatment group improved and maintained improvements in gsv, pulse oximetry, and pulse - rate over time (fig . 1). Whereas depression scores improved with the treatment groups, providers were reluctant to discontinue or reduce the amount of antidepressant medication . Further research is needed regarding provider awareness of evidence - based results of non - traditional methods of treating depression such as biofeedback therapy with robotic pets and provider willingness to discontinue or decrease medication use when depression scores improve with such therapy . Significant improvements in observed pain and decreased pain medication use were noted in the interventional group . Recent literature advises an observed overlap between pain and psychiatric disorders is common because some neurotransmitters, such as serotonin and norepinephrine (typically lower in individuals with dementia), are involved, albeit in different brain regions, in pain and sensory processing, as well as in modulating mood . Thus, it is likely that treatment with the paro, which decreases stress and anxiety, will also be effective in controlling or assisting in the relief of chronic pain . Future research could examine the impact of biofeedback therapy with robotic pets in acute care settings with various patient populations . Otherphysiological measures could be considered to measure the impact of interventions with the paro robotic pet seal, especially varying exposure of the treatment regimen.
Benign prostatic hyperplasia (bph) is a pathological term with no universally accepted epidemiologic definition . Lower urinary tract symptoms (luts) due to bph are a common problem, particularly in older men . The prevalence of luts / bph increases with age, affecting more than 70% of men older than 70 years . The socioeconomic burden of bph is tremendous, costing over $3 billion every year . As life expectancy increases, so does this burden . The prevalence of bph or luts has been studied in many nationwide surveys from various regions including europe, the united states, and korea . However, for the incidence of luts, not many reports have been published, and no study has reported the incidence of luts / bph previously in korea . The health insurance review & assessment (hira) service is a nationwide healthcare system in korea, including health insurance and medical aid, covering all citizens . Therefore, the incidence of luts / bph as well as treatment patterns can be determined from it without follow - up loss . Herein, we investigated the incidence of luts / bph and treatment patterns for patients who were diagnosed in a year with 3 years of follow - up using a nationwide korean database . Medication - free rate at 1 year after the surgery was also assessed . After the approval of institutional review board (h-1202 - 065 - 398), we extracted the data of patients who were diagnosed of bph in the year of 2008 and their follow - up visits for 3 years from hira database . The diagnosis of bph was defined as 2 or more records of reimbursement with the international classification of diseases, 10th revision (icd-10) diagnosis code of n40.0 used as a primary or secondary diagnosis . The first diagnosis was defined as an index diagnosis and patients with an index diagnosis of 2008 were traced for 3 years . Patients diagnosed with bph within the previous 12 months before the index diagnosis were excluded . Patients diagnosed with prostate cancer (icd-10, c61) within the 12 months after the index diagnosis or having claims suggesting a prior prostatic surgery, prostate cancers, inflammatory diseases of the prostate, neurological diseases or conditions that could affect luts were also excluded (table 1). Incidence was calculated as the number of new cases identified in 2008 divided by the number of at - risk individuals . Age, mean and median numbers of office visits and the tier of hospital where the index diagnosis was made were identified . To determine treatment patterns, patient status was determined every 3 months as medication, no medication, or surgery status . Medication status was defined as having a prescription record for 25% of the 3 months . Surgery was assigned as having a record of the following procedures: transurethral resection of the prostate (turp, r3975), photoselective vaporization of the prostate (pvp, r3976), holmium laser enucleation of prostate (holep, r3977), open prostatectomy (r3950), and thermal therapy (r3516). Alfuzosin, doxazosin, tamsulosin, terazosin, and silodosin were categorized as alpha - blockers . Oxybutynin, propiverine, fesoterodine, solifenacin, and tolterodine were categorized as anticholinergics . For those who underwent surgery once during the 3 years of follow - up, preoperative medication status was assessed 1 month prior to the surgery . For those who were taking medication 1 month prior to the surgery, the patient status such as medication, no medication and surgery were assessed as categorical variables . (ibm co., armonk, ny, usa) was used for all analyses, and a p - value <0.05 was considered statistically significant . After the approval of institutional review board (h-1202 - 065 - 398), we extracted the data of patients who were diagnosed of bph in the year of 2008 and their follow - up visits for 3 years from hira database . The diagnosis of bph was defined as 2 or more records of reimbursement with the international classification of diseases, 10th revision (icd-10) diagnosis code of n40.0 used as a primary or secondary diagnosis . The first diagnosis was defined as an index diagnosis and patients with an index diagnosis of 2008 were traced for 3 years . Patients diagnosed with bph within the previous 12 months before the index diagnosis were excluded . Patients diagnosed with prostate cancer (icd-10, c61) within the 12 months after the index diagnosis or having claims suggesting a prior prostatic surgery, prostate cancers, inflammatory diseases of the prostate, neurological diseases or conditions that could affect luts were also excluded (table 1). Incidence was calculated as the number of new cases identified in 2008 divided by the number of at - risk individuals . Age, mean and median numbers of office visits and the tier of hospital where the index diagnosis was made were identified . To determine treatment patterns, patient status was determined every 3 months as medication, no medication, or surgery status . Medication status was defined as having a prescription record for 25% of the 3 months . Surgery was assigned as having a record of the following procedures: transurethral resection of the prostate (turp, r3975), photoselective vaporization of the prostate (pvp, r3976), holmium laser enucleation of prostate (holep, r3977), open prostatectomy (r3950), and thermal therapy (r3516). Alfuzosin, doxazosin, tamsulosin, terazosin, and silodosin were categorized as alpha - blockers . Oxybutynin, propiverine, fesoterodine, solifenacin, and tolterodine were categorized as anticholinergics . For those who underwent surgery once during the 3 years of follow - up, preoperative medication status was assessed 1 month prior to the surgery . For those who were taking medication 1 month prior to the surgery the patient status such as medication, no medication and surgery were assessed as categorical variables . 21.0 (ibm co., armonk, ny, usa) was used for all analyses, and a p - value <0.05 was considered statistically significant . A total of 386,873 men were identified and included in the bph cohort of 2008 . The incidence of bph was 2,105 per 100,000 men, and increased with age (fig . Mean age was 59.7 years . For index patients who were diagnosed with bph in 2008, 27.1% of patients received medication for more than 9 months, 17.6% of patients took medication for more than a year . For the surgical treatment, 7,955 patients (2.1%) underwent surgery, including 252 patients who underwent surgery twice or more . The treatment rate increased with age until the age of 70 years (fig . Turp was the most commonly performed surgical procedure, followed by pvp and thermal therapy (fig . As our cohort comprised patients diagnosed with bph in 2008 and followed up until 2011, holep constituted only 2% of bph surgeries, as it has only been performed after 2010 . Although the symptom severity was not assessed, older patients were more likely to receive treatment, with an or of 1.56 for each additional decade (95% confidence interval, 1.551.58; p<0.001). To assess medication free - rate, patients taking medication at 1 month preoperatively were assessed and traced until 3 years after the surgery (fig . Were the most common type of medication used, followed by 5-alpha reductase inhibitors . Medication - free rate at 1 year after the surgery was 82% on average . To calculate medication - free rate, we excluded holep, because holep had only been used in this cohort after 2010 . Medication - free rate was highest for open prostatectomy (90.0%) and lowest for thermal therapy (78.1%). After 1 year, medication - free rate plateaued (average 82.9%), and at 36-month postsurgery, 83.9% of patients were not taking any medication . Among the patients who were taking medications at 36 months after the surgery, alpha - blockers were the most common type of medication used (alpha - blockers, a; 5-alpha reductase inhibitors, b; anticholinergics, c; a 5.2%, ab 3.9%, abc 0.7%, ac 1.1%), and anticholinergic - containing regimens were maintained in 4.0% of patients, including anticholinergic monotherapy in 2.1% of patients . For those who were taking anticholinergics preoperatively, medication - free rate after 1 year was 73.3%, which was lower than the average, and 11.8% of them had maintained anticholinergics - containing regimens . A total of 386,873 men were identified and included in the bph cohort of 2008 . The incidence of bph was 2,105 per 100,000 men, and increased with age (fig . Mean age was 59.7 years . For index patients who were diagnosed with bph in 2008, during the 3 years of follow - up, 27.1% of patients received medication for more than 9 months, 17.6% of patients took medication for more than a year . For the surgical treatment, 7,955 patients (2.1%) underwent surgery, including 252 patients who underwent surgery twice or more . The treatment rate increased with age until the age of 70 years (fig . Turp was the most commonly performed surgical procedure, followed by pvp and thermal therapy (fig . As our cohort comprised patients diagnosed with bph in 2008 and followed up until 2011, holep constituted only 2% of bph surgeries, as it has only been performed after 2010 . Although the symptom severity was not assessed, older patients were more likely to receive treatment, with an or of 1.56 for each additional decade (95% confidence interval, 1.551.58; p<0.001). To assess medication free - rate, patients taking medication at 1 month preoperatively were assessed and traced until 3 years after the surgery (fig . Were the most common type of medication used, followed by 5-alpha reductase inhibitors . Medication - free rate at 1 year after the surgery was 82% on average . To calculate medication - free rate, we excluded holep, because holep had only been used in this cohort after 2010 . Medication - free rate was highest for open prostatectomy (90.0%) and lowest for thermal therapy (78.1%). After 1 year, medication - free rate plateaued (average 82.9%), and at 36-month postsurgery, 83.9% of patients were not taking any medication . Among the patients who were taking medications at 36 months after the surgery, alpha - blockers were the most common type of medication used (alpha - blockers, a; 5-alpha reductase inhibitors, b; anticholinergics, c; a 5.2%, ab 3.9%, abc 0.7%, ac 1.1%), and anticholinergic - containing regimens were maintained in 4.0% of patients, including anticholinergic monotherapy in 2.1% of patients . For those who were taking anticholinergics preoperatively, medication - free rate after 1 year was 73.3%, which was lower than the average, and 11.8% of them had maintained anticholinergics - containing regimens . This study demonstrates the incidence of luts / bph, their treatment patterns, and medication - free rates after the surgery in a 3-year follow - up in a population - based bph cohort . The overall incidence of luts / bph was found to be 15 cases per 1,000 man - years in the triumph project from the netherlands from 1995 to 2000 . Our study showed similar or slightly higher incidence rates compared to those in these studies . Differences in time period, population size, and age distribution could have affected the result . A few reports have focused on the longitudinal changes of luts in population - based studies . In our study, less than one in 5 (17.6%) initially diagnosed patients continued medication for> 1 year, which could be partially explained by the dynamic nature of luts . Many previous studies have shown that patients with more profound or bothersome symptoms are more likely to visit clinics and seek medical care . Men who reported worse symptoms at baseline were four times more likely to be treated compared with those who reported less severe symptoms . In our study, about half of the surgeries were performed during the initial 6 months, which is in line with previous studies . Parsons et al . Reported that when community - dwelling older men having american urological association symptom index 8 or greater were followed up for 2 years, 4% underwent bph surgery and 13% started new prescription medication . In the proscar long - term efficacy and safety study, 5% of finasteride - treated patients and 10% of placebotreated patients with moderate - to - severe symptoms with an enlarged prostate underwent bph surgery during the 4-year follow - up period . Our study included 3-year followup data, and the reported surgery rate was lower compared to that in studies of symptomatic patients . In the urologic diseases in america project, 87,400 prostatectomies for bph were performed among 8 million visitors who visited urology clinics with a primary or secondary diagnosis of bph . As our study was longitudinal, it demonstrated the timing of surgeries during the 3 years of follow - up in a population - based cohort . Alpha - blockers were the most common single category of medication prescribed, and this was concordant with previous studies in korea . As han et al . Showed that older age, the presence of comorbidities, and preoperative anticholinergic usage could be associated with the continuation of medication after turp . Although detailed clinical information regarding the voiding status and the size of prostates could not be identified from this data, it is still meaningful considering the scarcity of nationwide data containing information on both prescriptions and surgical treatment . Detrusor underactivity or detrusor overactivity is contributable for this, and is considered to be one of important factors affecting the prognosis after transurethral prostatectomy in patients with bph . Detrusor underactivity was not changed by the surgery whereas detrusor overactivity was significantly decreased after the surgery . In our study, because medication free rate was calculated only for those who were taking medications preoperatively during one month before the surgery, the actual postoperative medication rate can be different from our result . The database did not include clinical variables; and the diagnosis of bph was based only on the physician's opinion . The incidence of luts may have been underestimated because only patients who had visited the clinic were included in the analysis . In addition, medical management can be underestimated because we assigned the patient's status as surgery if he underwent surgery during that period . Third, the incidence of surgery might be underestimated because we have excluded those diagnosed with acute urinary retention . Lastly, we could not analyze the clinical predictors for surgery and medication as this data lack clinical variables . The incidence of luts / bph increases with age until the 7th decade of life . About 1/5 of patients were taking medications for more than a year and 2.1% of patients were treated surgically . More than 80% of patients discontinued medications after the surgery, while patients having anticholinergics preoperatively are less likely to.
The purpose of bone scintigraphy is to portray areas of new bone formation within the skeleton . This is useful in imaging reaction of bone to tumor, fracture, and infection . Since approximately half of the administered radioisotope is excreted through renal filtration, abnormalities of the urinary system are also frequently noted during bone scintigraphy . In such cases, to reach an accurate diagnosis, the interpreting physician must first recognize which structures are involved in the uptake and the significance of the uptake . We describe a rare case in which extraosseous bone scan tracer accumulation was noted in a renal calculus . A 60-year - old man, who had difficulty in passing urine for the past 6 months and a complaint of lower back pain presented to our department . On ultrasound imaging, he was found to have an enlarged prostate . Transrectal ultrasound - guided biopsy revealed adenocarcinoma of prostate, gleason's score 3 + 3=6 . His psa was normal (1.3 ng / ml) and serum alkaline phosphatase was elevated 58 u / l (normal range 30 - 50 u / l). A 99 m technitium methylene diphosphonate (99 m tc - mdp) bone scan was performed . The scan [figures 1 and 2] showed mildly increased tracer uptake in lumbar vertebrae and focal accumulation of tracer in lower pole of the left kidney . Fusion imaging [figure 3], using single photon emission computed tomography along with x - ray computed tomography (spect - ct) of the lumbar spine was performed to characterize the vertebral tracer uptake . Lasix 40 mg was given 45 minutes prior to spect - ct to monitor the renal tracer accumulation . High tracer uptake in a lower pole of the left kidney was found localized in a calculus in the lower calyx of kidney . The ct attenuation factor was 1060 hounsfield unit, compatible with that of a renal calculus . (a) anterior and (b) posterior views, showing mildly increased tracer uptake in the lumbar vertebrae (black arrow) and intense, focal localization of the tracer in the lower pole of left kidney (red arrows). (a - b) lateral and (c - d) oblique views clearly demonstrate the focal tracer uptake outside the skeletal structures in left kidney (arrows). (a) coronal, (b) sagittal and (c) transaxial ct images; (d) coronal (e) sagittal and (f) transaxial spect images; (g) coronal, (h) sagittal and (i) transaxial spect - ct images . The ct attenuation factor was 1060 hounsfield unit, compatible with that of a renal calculus . We concluded this as extra - skeletal bone scintigraphy tracer uptake in a renal calculus . Bone scintigraphy is a valuable diagnostic tool in the evaluation of patients with a variety of osseous abnormalities . However, accumulation of bone scan tracer outside the skeleton can pose a difficulty in reporting for a nuclear medicine physician, especially if only planar imaging is performed and the tracer uptake is overlapping or is in close vicinity of the skeleton . Localization of bone scan tracer in a renal calculus has been reported in the past and its use in preoperative in vivo localization of the renal calculus has been explored . Retention of bone scan tracer on ureteric calculi and bladder calculus has been reported in the past. [35] in one of the studies, autoradiography of ureteric calculus demonstrated peripheral tracer distribution within the calculus . Accumulation of the radionuclide due to sluggish flow and its absorption onto the crystal surface, within the calculus, were suggested as possible mechanisms of tracer uptake . The other common causes of urinary system localization of the bone scan tracer are dilation of urinary - collecting system, bladder diverticulum, and presence of an ureterostomy bag. [69] conditions causing extraskeletal accumulation of bone scan tracer must be kept in mind while reporting a bone scan . Our case demonstrates a rare occurrence of extraosseous bone tracer accumulation in a renal calculus . It also highlights important role played by spect - ct in localizing the extraskeletal tracer uptake . Spect - ct can be used effectively when an extraskeletal uptake is encountered on planar bone imaging.
In most teleost fishes head kidney, pronephros plays an important role as main hematopoietic organ and blood cell reservoir (fange 1994; houston et al . 1996; fijan 2002a, b; romano et al . 2004; rombout et al . 2005; gangopadhyay and homechaudhuri 2011). According to wendelaar bonga (1997) and weyts et al . (1999), pronephros shows not only hematopoietic activity but also is a lymphoid and endocrine organ . However, head kidney is not the only site of hematopoiesis in fish . According to various authors (glomski et al . 1996; kobayashi et al . 2006, 2007, 2008; zapata et al . 2006 2007 and santos et al . 2011), other organs such as spleen, gut - associated lymphoid tissue (galt), mucosa - associated lymphoid tissue (malt), and intertubular tissue of trunk kidney (mesonephros) may also show hematopoietic activity . In some fish species, several hematopoietic organs are active, while in the others only one of them (liu et al . Morphology of hematopoietic tissue in fish is quite well known (e.g., fange 1994; 2002a, b), but little information is available on the effects of environmental factors on its structure and activity, and studies of hematopoietic tissue are seldom included in evaluation of physiological effects of toxic agents on fish . Hematopoietic activity of head kidney tissue involves proliferation of stem cells and early precursors of all cell lines, differentiation and maturation, as well as apoptosis, and the rate of all these processes is a key factor that determines efficiency of hematopoiesis . Stem cell renewal and precursor cell proliferation are counterbalanced by apoptosis in functionally inactive or terminally differentiated cells (mckenna and cotter 1997). Apoptosis plays an important role in regulating early progenitor and stem cells, and particularly for the development and function of lymphoid cells (domen 2000). The marker protein of apoptosis is caspase 3an enzyme participating in degradation of nuclear and cytoplasmic proteins during this process . This caspase is commonly defined as effector caspase (migliarini et al . 2005). Evaluation of both precursor cell proliferation and apoptosis rate is applied in hematological studies to evaluate the rate of cell turnover and hematopoietic activity (thiele et al . Have been proved to react with mammalian (mouse or rabbit) monoclonal antibodies, and these antibodies were successfully used for evaluation of proliferation and apoptosis of various cells in thalassoma pavo (monteiro et al . 2009), oreochromis niloticus (brunelli et al . 2011), and salmo salar (yousaf et al . Cadmium and copper are well known to induce hematotoxicity in fish, often resulting in anemia and immunosuppression (e.g., svobodova et al . 2004; seong - gil et al . 2004; ates et al . 2008; witeska et al . Sometimes the values of hematological parameters of intoxicated fish fluctuate, and their changes are not always directly related to metal concentrations and time of exposure or time post - exposure (ruparelia et al . These fluctuations may result from translocation of cadmium and copper within the organism, and their toxic action on various functions at different time . Cadmium and copper probably affect not only circulating blood cells, but also newly developing ones in hematopoietic tissue . Very scarce data concerning hematopoietic effects of heavy metals in fish (garofano and hirshfield 1982; ghosh et al . 2007; som et al . 2009) and mammals (lutton et al . 1984; mitsumori et al . 1998; van den heuvel et al . 1999; 2001; celik et al . 2005, 2009) indicate that they are cytotoxic to precursor cells, and various cell lineages show different sensitivity to metal toxicity . The aim of present study was to evaluate the effects of copper and cadmium under various exposure conditions (concentration and time) on hematopoietic potential of common carp head kidney . Common carp (cyprinus carpio l.) 6-month - old juveniles of body mass 21.5 8.3 g were harvested in october from the rearing ponds of the inland fisheries institute in abieniec and brought to the department of animal physiology, university of natural sciences and humanities in siedlce in plastic bags with water and supplied with pure oxygen . Before the experiment, the fish were acclimated for 4 weeks to the laboratory conditions in the flow - through tank, at 1718 c . Water was constantly aerated, and o2 concentration was 6.18.0 mg / dm of o2 (6687% saturation), while concentration of no2 0.020.06 mg / dm, and nh4 4.67.1 mg / dm . The fish were fed aller aqua classic 4 mm feed at the rate of 2% of stock mass per day . Prior to the experiment survival tests were performed, and 96hlc50 values were calculated using the probit method for both metals . Fish were exposed for 3 h to cd and cu concentrations equal to 100% of 96hlc50 (6.50 and 0.75 mg / dm for cd and cu, respectively)groups cd - s and cu - s or for 4 weeks to 10% of 96hlc50 (0.65 and 0.075 mg / dm, respectively)groups cd - l and cu - l . Control group (c) was kept in clean tap water (<0.31 g / dm of cd, 233 g / dm of cu). Experimental solutions were made using cdcl2 2 h2o and cuso4 5h2o, and 3/4 was renewed everyday without disturbing fish . The fish were kept in 100 dm aerated tanks (10 fish in each), and fed aller aqua classic 4 mm (1% of stock mass, once a day before water renewal). Five fish were sampled weekly for 4 weeks from each experimental group and killed for head kidney isolation . Water quality parameters were measured everyday (table 1) using portable do meter (hanna instruments hi 9143), ph meter (elwro prl tn 5123), and kits for nitrogen metabolites (visocolor ammonium 0,210 mg / dm and visocolor nitrite 0,052 mg / dm, machery nagel). Water hardness values were provided by the water supplier (www.pwik.siedlce.pl).table 1water quality parameters during the experimentparametergroupccu - s + cu - lcd - s + cd - ltemperature (c)17.0 0.517.1 0.517.2 0.6hardness (mg / dm caco3)179198ph6.87.0o2 (mg / dm)8.9 0.69.0 0.28.8 0.5no2 (mg / dm)0.03 0.010.06 0.020.04 0.02nh4 (mg / dm)3.9 0.65.2 0.86.1 1.6 water quality parameters during the experiment five fish were sampled weekly for 4 weeks from each experimental group and killed for head kidney isolation . The kidneys were sliced and smeared on degreased slides and dried at room temperature for 24 h. two preparations from each fish were stained giemsa and may - grunwald for cytological analysis . Hematopoietic precursor cells were identified (22 types of cells, according to fijan 2002a, b, and kondera 2011). Immunocytochemical staining was also performed to identify proliferating cells (showing pcna), and cells undergoing apoptosis (showing caspase 3) using monoclonal mouse antibodies anti - proliferating cell nuclear antigen clone pc 10 (dako cytomation) selectively binding to the pcna antigen, and anti - caspase 3 (active) antibody produced in rabbit, (sigma) binding to active caspase 3 . Dried preparations were hydrated with deionized water, and then peroxidase activity was blocked using 3% h2o2 (trace pur, merck). The antibody solution diluted 1:300 was applied and left on slides for 1 h at room temperature . Cells with antibody protein complexes were visualized using dako cytomation en vision + system - hrp for use with mouse primary antibodies, and dako cytomation en vision + system - hrp for use with rabbit primary antibodies, respectively, according to the producer s instruction . The pcna - positive and caspase 3-positive cells stained brownish and were easily distinguishable from other cells (stained light blue with hematoxylin). Negative control staining was also performed (the preparations were treated the same way except for incubation with antibodies) and resulted in no color reaction . The cells were counted in at least 6 fields, and percentage of proliferating and apoptotic cells was calculated per 300 cells in each preparation . All preparations were preserved with histokitt, glaswarenfabrik karl hecht gmbh germany, covered with cover glass, and viewed using nikon eclipse e600 microscope at 1,000 magnification . Eclipse e600 microscope connected with digital nikon coolpix camera and computer image analysis system coolview (precoptic, poland). The results were subjected to statistical evaluation of significance of differences in values of all parameters between the control and metal - exposed groups using mann the study obtained agreement of the iii local ethical committee at the warsaw university of life sciences (no 41/2008). Both metals induced significant changes in frequency of blast cells, as well as proliferating and apoptotic precursors . Short - term exposures to cd and cu resulted in an increase in the frequency of early blast cells in head kidney of carp in 1 week after the treatments (figs . 1, 2). The level of blasts in cd - s fish remained significantly elevated until the 3-week post - exposure, while in cu - s group decreased already in the 2 weeks . On the contrary, during long - term exposure, cd induced only transient increase in blast frequency (3 weeks), while the fish from cu - l group showed significantly elevated level of early blast cells beginning from the 2 weeks of exposure until the end of the experiment.fig . 1the effects of cd and cu on the frequency of early blast cells in head kidney hematopoietic tissue of common carp (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectivelyfig . 2early blast cells the effects of cd and cu on the frequency of early blast cells in head kidney hematopoietic tissue of common carp (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectively similarly, also the frequency of proliferating (pcna - positive) cells significantly increased in 1 week after short - term exposures (fig . 3, 4). In cd - s group, the level of cells undergoing mitosis remained elevated until the 2-week post - exposure, while in cu - s proliferation rate quickly decreased to the control level . During long - term cd exposure, a significant increase in precursor cell proliferation took place in 2 weeks, and their level remained elevated until the end of the experiment . The reaction of fish from cu - l group was different: a significant increase in frequency of proliferating cells took place at the end of the experiment (in 4 weeks of exposure).fig . 3the effects of cd and cu on the frequency of proliferating cells in common carp head kidney hematopoietic tissue (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectivelyfig . 4pcna - positive cells the effects of cd and cu on the frequency of proliferating cells in common carp head kidney hematopoietic tissue (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cu - l groups subjected to short- or long - term exposures, respectively both metals induced a significant increase in the frequency of apoptosis of hematopoietic precursor cells (fig . 5, 6). Both short - term and long - term exposures caused similar effects, but in case of cd exposures, the reaction was more pronounced.fig . 5the effects of cd and cu on the frequency of apoptotic cells in common carp head kidney hematopoietic tissue (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectivelyfig . 6caspase 3-positive cells the effects of cd and cu on the frequency of apoptotic cells in common carp head kidney hematopoietic tissue (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectively caspase 3-positive cells these changes resulted in a reduction of hematopoietic potential in all metal - exposed fish, measured as the ratio of frequencies of precursor cell proliferation and apoptosis (fig . 7). It is noteworthy that in case of short - term copper exposure, reduction in hematopoietic activity at the end of the experiment was deeper than in group subjected to short - term cadmium intoxication or to fish subjected to a long - term copper treatment . However, in all cases, hematopoietic activity was over than 1 showing that the frequency of proliferating cells was all the higher than percentage of precursors undergoing apoptosis.fig . 7the effects of cd and cu on cell turnover rate in common carp head kidney hematopoietic tissue (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectively the effects of cd and cu on cell turnover rate in common carp head kidney hematopoietic tissue (arithmetic mean s.e ., * different from the value before metal exposure (time 0), p 0.05, u mann whitney test). Cd - s, cu - s and cd - l, cu - l groups subjected to short- or long - term exposures, respectively the obtained results show that cadmium and copper disturbed hematopoiesis in carp but on the other hand indicate a considerable compensatory potential of carp hematopoietic system . The pattern of changes after short - term exposures (a rapid increase in cell proliferation rate and early blast frequency, accompanied by an increase in apoptotic rate) and during long - term treatments (gradual increase in the values of these parameters during the exposures) was different but the final effect reduction in cell turnover rate was very similar . The increase in apoptotic rate was higher when compared to acceleration of precursor cell proliferation . No anemia was observed in peripheral blood or a significant reduction in leukocyte count, and the most pronounced effect of metal exposures was significantly reduced frequency of peripheral phagocytes (neutrophils and monocytes), accompanied by reduction in their metabolic activity (kondera and witeska 2012, and unpublished data of the same study). These results indicate that hematopoietic potential of carp head kidney tissue was reduced due to the increase in apoptotic cell destruction since no necrosis or other visible cell damage was observed . Hematological effects of heavy metal intoxication of fish were extensively studied, but very little data concerning metal - induced alterations in hematopoietic system are available . According to garofano and hirshfield (1982), 2-h exposure of ictalurus nebulosus at 61.3 mg / dm of cd resulted in complete destruction and elimination of all hematopoietic precursor cells in head kidney hematopoietic tissue over 24-h post - exposure, and the only cells present after intoxication were mature erythrocytes . Also, saxena et al . Considerable hematopoietic disturbances were also observed in labeo rohita subjected to cu treatment: 50% 72hlc5025 mg / dm cu or 100% 72hlc5050 mg / dm cu (som et al . 2009). Sublethal exposure resulted in an increase of abundance of both erythrocyte and leukocyte precursors in head kidney hematopoietic tissue, while under lethal conditions, their number initially decreased, and then increased until the end of the experiment . The authors also observed an increase in blast cell abundance in cu - exposed fish . Studies of the effects of metals on hematopoietic tissue of other vertebrates also show their hematotoxic potential . Reduction of hematopoietic potential that resulted in anemia was observed in cadmium - treated rat (mitsumori et al . The results of in vitro study revealed that cadmium, lead, and silver adversely affected erythropoiesis in rat bone marrow (lutton et al . (1999, 2001) observed that cadmium was more toxic to human and murine hematopoietic progenitor cells than lead, erythroid cell being more sensitive than myeloid precursors . (2005, 2009) reported genotoxic and cytotoxic action of cadmium upon erythroid cells in rat bone marrow . Almost no data are available on the effects of heavy metals on proliferative and apoptotic activity of fish hematopoietic tissue . (2009) who observed an increase in labeo rohita blast cell apoptosis during both lethal and sublethal cu exposures, while their proliferation rate increased only under sublethal conditions . There are, however, some data showing the effects of cadmium and copper on proliferative and apoptotic activity in other fish tissues . According to brunelli et al . (2011), cadmium induced increase in proliferation and apoptosis rates in gill epithelium of thalassoma pavo which was followed by a decrease . (1999) reported a significant increase in proliferation and apoptosis rate of intestine epithelial cells of salmo salar fed cd - containing feed . Activation of both cell proliferation and apoptosis in the intestine of liza aurata from metal - polluted natural environment was also reported by ferrando et al . (2011), an increase in cell proliferation rate in cadmium - exposed s. salar and sparus aurata may be a protective mechanism reducing adverse effect of metal on fish tissues . According to hernandez et al . (2011), danio rerio larvae subjected to 19.5 mg / dm of cuso4 for 2 h showed an increase in frequency of apoptosis of various types of cells, head kidney cells being more sensitive than brain neurons and gill epithelial cells . Monteiro et al . (2009) reported an increase in proliferation rate of gill epithelial cells in cu - exposed oreochromis niloticus which was not accompanied by activation of apoptosis . Massive mitotic activity was detected using pcna immunostaining in olfactory epithelium of tilapia mariae following 4-day exposure to 20100 g / dm of cu (bettini et al . These observations confirm that cell proliferation is involved in tissue regeneration after metal - induced damage . Metal - induced activation of cell proliferation and apoptosis was reported also in mammals . According to habeebu et al . (1998), cadmium induced a dose- and time - dependent activation of murine hepatocyte proliferation and apoptosis . Tsangaris and tzortzatou - stathoupoulou (1998) observed that cadmium induced apoptosis of human immune cells, b lymphocytes being more sensitive than t cells and lymphoblasts . The data obtained in the present study indicate that cadmium and copper may affect hematological and immune status of fish organism by disturbing the process of hematopoiesis . Hematopoietic precursor cells are sensitive to intoxication and heavy metals enhance the rate of their apoptotic destruction . On the other hand, hematopoietic system of carp shows high homeostatic potential and tends to compensate cell loss by activation of mitotic divisions . The results showed that toxic effects of both metals were persistent and hematopoietic activity reduced after short - term exposures to cadmium and copper did not recover in 4-week post - treatment . They also showed that low sublethal concentrations of cadmium, and particularly of copper may significantly disturb hematopoietic processes in fish that do not show any other symptoms of intoxication . In conclusion, anemia and immunosuppression often observed in fish intoxicated with copper and cadmium may result from toxic effect of metals on hematopoietic system . Additionally, cellular parameters of hematopoietic tissue: frequency of blast cells and the rate of proliferation and apoptosis are sensitive indicators of sublethal intoxication.
Functional metabolic imaging of the brain using fluorine-18 fluorodeoxyglucose (fdg) positron emission tomography (pet) is one of the most useful tools in distinguishing the patient's symptoms from the various types of dementia by elucidating global and regional deficits in cerebral glucose metabolism . Magnetic resonance imaging (mri) often plays a role as well, as it is more widely available, and is used to evaluate structural abnormalities within the brain parenchyma . We present a case where hybrid imaging of pet / mri was used to diagnose a patient with semantic dementia (sd), which is one of the lesser known subtypes of frontotemporal lobe dementia (ftd). Sd is characterized by a loss of language and verbal skills with late stage behavioral changes . It is associated with predominantly temporal lobe atrophy (left greater than right) and is often referred to as temporal variant ftd . A 61-year - old, right - handed male presented with a sudden change in mental status preceded by a significant decline in functionality over the past 2 years . He was previously a highly functional person and worked as an executive for a fortune 500 company . His change in mental status was characterized by several behavioral changes; including cruelty toward family members and pets, promiscuity, adultery, drug abuse, wild spending habits, and lack of empathy . His family had also noticed a change in his speech patterns in terms of phonation and pronunciation of words . His initial diagnosis was bipolar disorder; however, he did not respond to mood stabilizing drugs . After losing his job, the mri [figure 1] demonstrated diffuse parenchymal atrophy, which was slightly more extensive in the temporal lobes . There was a lacunar infarct in the right thalamus that was suggestive of vascular dementia . 61-year - old male with change in mental status and decline in function diagnosed with semantic dementia . (a and b) the magnetic resonance imaging (mri) transaxial views of the t1-weighted and (c and d) t2-weighted images with proton density and fluid suppression sequences demonstrate diffuse parenchymal atrophy, slightly more substantial in the temporal lobes (arrows on a and c). These non - specific findings do raise the possibility of frontotemporal dementia (ftd). A thalamic infarct (arrows on b and d), along with subtle white matter abnormalities are also seen, which are suggestive of vascular dementia, though inconsistent with the negative magnetic resonance angiography findings . The images were co - registered with the mri using the institute of electrical and electronics engineers (ieee) mutual information algorithm . The resultant pet / mr images [figure 2] demonstrated a pattern of decreased fdg uptake that was predominantly confined to the temporal lobes and was particularly severe in the amygdalae and hippocampi . The decrease in activity was greater on the left side and extended superoposteriorly toward the parietal lobe and wernicke's area . The other atrophic areas and white matter changes seen on the mri had intact glucose metabolism . The lacunar infarct also had decreased fdg uptake, but that was considered to be an incidental finding . The decreased glucose metabolism affecting only the temporal lobes (worse on the left side) with the concordant areas of atrophy on the mri were highly specific for semantic dementia and allowed for a definitive diagnosis to be made . 61-year - old male with change in mental status and decline in function diagnosed with semantic dementia . The fluorine-18 fluorodeoxyglucose (f-18 fdg) positron emission tomography (pet) of the brain was obtained and coregistered with the t1-weighted sequences of the mri . Two separate triangulated cross - sections of the reconstructed images in (a and d) axial views, (b and e) coronal views, and (c and f) sagittal dimensions show significantly reduced uptake corresponding to atrophy in the temporal lobes (arrows on a), involving the amygdalae (arrows on b), hippocampi (arrows on e), and wernicke's area (arrow on c). The function is well preserved in the remaining areas of global atrophic change and white matter changes . There is also reduced activity in the location of the suspected right thalamic infract (arrow d). Although each type of dementia has its own constellation of symptoms, there is significant overlap and most patients present with features of several different psychiatric and neurologic disorders . The majority of patients present with some type of memory loss with a variety of non - specific behavioral changes . Anatomic imaging is often not useful, other than to exclude physical causes such as stroke or hydrocephalus . There may be subtle clues to a diagnosis, but nothing definitive . Functional imaging provided by fdg pet evaluation of brain glucose metabolism alzheimer's dementia (ad) is characterized by decreased glucose metabolism in the temporal and parietal lobes with sparing of the frontal lobes . Ftd, as the name suggests, demonstrates decreased activity in the frontal and temporal lobes with sparing of the parietal lobes . Dementia with lewy bodies (dlb) has pattern of decreased activity similar to ad, but also demonstrates decreased activity in the occipital lobe . However, many early stages of dementia may have atypical or variable appearances on fdg pet . For example, semantic dementia has involvement confined to the temporal lobe, but the deficits are often not as striking as in the other types of dementia on fdg pet . On mri, the aforementioned types of dementia may show global atrophy, with more extensive changes in the involved lobes (such as more frontal lobe atrophy in ftd). However, these structural changes often occur late in the disease progression and can be non - specific . Additionally, there is no typical structural abnormality in dlb . Often at times, the fdg pet will show metabolic deficits before atrophic changes become apparent on mri . In this case, we were able to combine metabolic deficits with the anatomic information provided by mri . This allowed us to corroborate the decreased activity confined to the temporal lobe with specific structures or areas within the brain that were involved . The amygdale, hippocampi, and wernicke's area all showed focal decreases in activity compared with the overall reduction of radiotracer activity in the temporal lobes . The fdg pet, on the other hand, showed that the decreased activity was greater on the left side, which is classic for semantic dementia, whereas the atrophy on the mri seemed relatively symmetric . Since the affected areas of the brain are involved in empathy (amygdala), meaningful word usage (wernicke's area), memory (hippocampus), and conceptual knowledge (amygdala and hippocampus interaction); the patient's symptoms could be easily explained . This case demonstrates an example of the value of combined pet - mri in clinical practice . Co - registration of images provides essential information that is needed to make a diagnosis in challenging cases . Thus, clinical symptoms can be related to lesions in specific parts of the brain.
Atherosclerosis and its vascular damage are the leading cause of mortality and morbidity in patients with diabetes . Recently, several studies have shown that cardiovascular diseases are the first cause of premature death among individuals with type 1 diabetes, demonstrating that although there have been substantial improvements in survival during the past 50 years, still challenges remain . Although diabetes - related vascular complications are uncommon in the pediatric age, early functional and structural abnormalities may be present just a few years after the onset of type 1 diabetes . Therefore, early diagnostic and therapeutic approaches are needed for youth with type 1 diabetes at risk for a premature vascular disease . A surrogate marker of cardiovascular disease is endothelial dysfunction, which is the inability of the artery to sufficiently dilate in response to an appropriate endothelial stimulus . The reactive hyperemia peripheral artery tonometry technique is used for a few years now as a noninvasive test to assess for early vascular changes in high - risk patient groups . In a previous study, we evaluated the prevalence of endothelial dysfunction measured as mean of reactive hyperemia index (rhi), in a cohort of adolescents with type 1 diabetes . Surprisingly a low rhi score was observed in 76.7% of patients, showing a dramatic prevalence of early endothelial dysfunction among type 1 diabetes individuals, even in the pediatric age . Endothelial cells have got numerous functions crucial for maintaining intravascular homeostasis: they play an important role in vascular tone regulation, hemostasis, and fibrinolysis and in the production of several substances [6, 7]. In patients with diabetes, hyperglycemia and related pathological biochemical processes trigger damage to the endothelial cells causing their dysfunction . The dysfunctional endothelium adopts prothrombotic, proinflammatory, and vasoconstrictive phenotype promoting the early development of atherosclerosis [3, 6, 7]. Alpha - lipoic acid is a potent mitochondrial antioxidant agent that acts by multiple mechanisms promoting anti - inflammatory and antithrombotic pathways and positively influencing the nitric oxide mediated vasodilatation . In most european countries alpha - lipoic acid is licensed and used as treatment in patients with neuropathic symptoms . Moreover, many studies suggest the potential role of alpha - lipoic acid in prevention and treatment of atherosclerosis and related cardiovascular disease [10, 11]. This hypothesis is based on similar results obtained either in animal models or in patients with diabetes mellitus, especially type 2 diabetes [1214]. In streptozotocin - induced rats, it may be assumed that alpha - lipoic acid treatment can protect against impaired vascular responsiveness . In type 2 diabetes patients, alpha - lipoic acid may influence angiogenesis through an effect on some circulating factors including vegf, bfgf, mcp-1, and il-10 . Therefore, the aim of our study was to investigate the effect of alpha - lipoic acid on endothelial dysfunction in youth with type 1 diabetes . This pilot study is a 6-month, double - blind, randomized controlled trial to test the efficacy of an antioxidant diet plus alpha - lipoic acid versus an antioxidant diet alone in improving endothelial dysfunction in adolescents with type 1 diabetes . This study was a 6-month, prospective, randomized, double - blind, controlled trial conducted in pediatric patients with type 1 diabetes, performed at the pediatric department of the university of milano . The trial was performed in accordance with the declaration of helsinki . Before any trial - related activities signed informed consent was obtained from all participants aged 16 years or older and from parents or guardians of participants aged younger than 16 years (assent was obtained from minors). Patients with type 1 diabetes were referred for study participation from the outpatient clinic of the university of milano between january 2013 and december 2013 . Seventy - one patients who had been previously evaluated for the presence of endothelial dysfunction were enrolled in the trial and completed the 6-month study period . Inclusion criteria were type 1 diabetes (diabetes onset was defined according to ada criteria), more than 1 year from diagnosis or confirmed c - peptide negative, age between 12 and 19 years, insulin requirement more than or equal to 0.5 u / kg / day, blood glucose checks more the 3 times per day, and insulin intensive therapy ongoing . Exclusion criteria were preexisting cardiovascular diseases or inflammatory systemic diseases, hypertension and prehypertension (bp 90th percentile for age, sex, and height), eating disorders, obesity (defined as bmi 95th percentile per age and sex in the 2000 cdc growth chart), celiac disease, inflammatory bowel disease or other significant gastrointestinal conditions, systemic glucocorticoid use (1-month cumulative use during last year) or ongoing treatment with glucocorticoid use, anemia, significant multiple food allergies, uncontrolled hypothyroidism or hyperthyroidism, significant mental illness, and pregnancy . Clinical history and physical examination, nutritional records, and biochemical sample were collected at baseline, after 3 months, and after 6 months; endothelial dysfunction was evaluated at baseline and after 6 months . Weight was measured unclothed to the nearest 0.1 kg using a calibrated balance scale . Blood pressure was measured using a mercury sphygmomanometer, according to the national high blood pressure education program working group . Moreover, all patients were in intensive insulin therapy, with either multiple daily injections or insulin pump therapy . Blood sampling was performed in the fasting state at 8 a.m. levels of lipids including triglycerides (tg), total cholesterol (tc), ldl - cholesterol, and hdl - cholesterol were evaluated in all subjects with standard laboratory methods . Blood was drawn for hba1c analysis using a fully automated high - performance liquid chromatography system (variant ii, bio - rad laboratories, munich, germany). At baseline and 3 and 6 months a nutritional visit was performed by the registered dietitian of the pediatric department of the luigi sacco hospital . Three - day dietary records with nutrition data were evaluated using dedicated software (metadieta, meteda, san benedetto del tronto, ap, italy). Data on dietary intake, reporting the types and amounts of all food and beverages consumed, with data on insulin doses and algorithms used at home, blood glucose results, and physical activity were collected . During the study period a dietary recall was collected every month; moreover a nutritional assessment was performed at 3 and 6 months to check the patients' compliance to the dietary plan assigned . Moreover, registered dietitian evaluated the body composition using tanita bc-418, il, usa . Peripheral endothelium - dependent vasodilator capacity was estimated by assessing the rhi by means of the endopat 2000 system (itamar medical ltd ., endothelium - mediated changes in vascular tone after occlusion of the brachial artery are reflecting a downstream hyperemic response, which is a measure for arterial endothelial function . The rh - pat score is calculated as the ratio of the average pulse wave amplitude during 1 min period starting 60 s after cuff deflation divided by the average pulse wave amplitude of a 210 s preocclusion baseline period . After the clinical assessment at baseline, each patient was randomly assigned to one of the three double - blind, study arms: 10.000 oxygen radical absorbance capacity units (orac) antioxidant diet plus alpha - lipoic acid (n = 25, group 1), 10.000 orac antioxidant diet plus placebo (n = 27, group 2), and controls (n = 19, group 3), with no changes in dietary habits . Supplementation with 400 mg of slow - release alpha - lipoic acid or placebo, both in a lyophilized formulation, was furnished by the ra of the pediatric department . Patients were advised to melt the lyophilized solution in a glass of water and to get it twice a day at least 1 hour before lunch and dinner, for 6 months . The compliance with the antioxidant diet was monitored by monthly dietary recalls and a full nutritional assessment was repeated, after 3 and 6 months . Both patients treated with antioxidant diet plus alpha - lipoic acid (n = 25, group 1) and patients treated with antioxidant diet plus placebo (n = 27, group 2) received the same diet plan, of 18002200 kcal per day, according to the age - group . The daily amount of carbohydrates was 60% of the total intake, with 15% of simple carbohydrates . The prescribed amount of fiber was 40 gr per day; fats were 26%, with a monosaturated percentage of 12, polysaturated percentage of 4, and saturated percentage of 8 . The ratio of the fatty acid was 8 gr for omega-6 and 2 gr for omega-3 . The daily amount of proteins was 14% of the total intake, with 54% of animal proteins and 46% of vegetable proteins . No particular nutrient source to elevate the dietary orac was administered, but a list of foods with high orac content was given to each subject with detailed instruction to reach the desired assumption of orac . A similar dietary plan was assigned to patients enrolled in the control group (n = 19, group 3), but no orac supplementation was suggested . Prior to data analysis, all metric variables were checked for normality by using shapiro - wilk test . We used unpaired t - test and manova (multiple analysis of variance) when appropriate . Results are presented as mean standard deviation (sd), or percentage, unless stated otherwise . All the 71 patients enrolled completed the 6-month study period and no one dropped out of study before completing it . Moreover no severe side effects have been reported by either alpha - lipoic acid group or placebo . Three patients of the alpha - lipoic acid group and 2 patients of the control group reported mild abdominal pain over the first 4 weeks of the study, with no need to withdraw from the study . Baseline and 6-month clinical characteristics are summarized in table 1 for each study group . At baseline, the mean age of the whole group was 16.3 3.4 years, the mean of diabetes duration was 8.1 5.2 years, the mean bmi was 21 3, and insulin requirement was 0.83 0.29 u / kg / day . All patients enrolled in the study reported in - range levels of triglycerides, total cholesterol, ldl - cholesterol, and hdl - cholesterol per age and sex over the study period . Moreover all patients showed normal blood pressure measurements per age, sex, and height . There was no statistical difference in all the demographic and clinical variables considered among the three study groups at baseline (table 1). After 3 months, a significant reduction in daily insulin requirements was observed only in group 1, both in the amount of the total daily dose (0.74 u / kg / day at 3 months versus 0.83 u / kg / day at baseline, p = 0.048) and in the percentage of bolus (22.0% at 3 months versus 26.3% at baseline, p = 0.047) (table 1). However these data were not confirmed at 6 months, and no differences were reported among the 3 groups after 6 months of diet in terms of daily insulin requirement . These results are supported by data collected during the nutritional assessment . Indeed, dietary recall to evaluate patients' compliance to the dietary plan showed a significant increase of orac intake at month 3 in both arms (treatment and placebo) but not in controls . After 6 months, orac consumption decreased in the treatment group but not in the placebo group, as reported in table 1 . Endothelial dysfunction was tested as rhi score in all groups at baseline and after 6 months . As described in a previous study, at baseline a low rhi score was reported in all patients enrolled, which resulted in a pathologic range (<1.67). At 6 months, rhi score significantly improved only in the group of patients treated with antioxidant diet plus alpha - lipoic acid (1.72 versus 1.4, p = 0.045), reaching a normal rhi value (table 1 and figure 1). The group of patients treated with antioxidant diet plus placebo reported a better rhi score at 6 months versus baseline as well, but it did not reach the significance . No differences in rhi score were showed in the control group at 6 months versus baseline . The analyses performed on the other clinical parameters collected showed no differences at baseline and after 3 and 6 months among the three groups: bmi, 24 h blood pressure, lipid profile, hba1c, dietary habits, and body composition resulted any different among the three groups and over the study period . To the best of our knowledge, this is the first study analyzing the effects of a dietary supplementation in improving the endothelial dysfunction in youth with type 1 diabetes . In this randomized, placebo controlled double - blind trial, we found a relationship between the consumption of an antioxidant diet plus alpha - lipoic acid and the improvement in endothelial dysfunction in youth with type 1 diabetes . In particular an antioxidant diet (10000 orac) plus alpha - lipoic acid (800 mg / day) seems to be effective in reducing insulin requirement and daily bolus rate after 3 months and in improving endothelial dysfunction after 6 months . The efficacy of alpha - lipoic acid in improving glucose disposal has been previously described in animal models [1215] and in type 2 diabetes adult patients [13, 14, 16]. It was 1970 when lipoic acid was shown to enhance glucose uptake into rat tissue [17, 18]. Gradually the effects of different formulas of alpha - lipoic acid were tested both in vitro and in animal models [16, 19] confirming the ability to increase glucose uptake and to enhance the glycogen synthesis [16, 19]. The molecule seems to work improving insulin sensitivity by modulating the signal transduction and by increasing the glucose uptake . In patients with type 2 diabetes, an acute intravenous administration of 1000 mg of lipoic acid moreover a 4-week oral treatment with alpha - lipoic acid successfully resulted in increasing insulin sensitivity in patients with type 2 diabetes . In our small study, a supplementation with 800 mg / day of alpha - lipoic acid for 3 months effectively resulted in reducing insulin requirement . The mechanisms of action are not completely clear, since, in our knowledge, studies having pediatric type 1 diabetes patients as target are lacking . However, as reported for type 2 diabetes, we hypothesize that alpha - lipoic acid can use nicotinamide adenine dinucleotide molecules (nadh) for the reduction to dihydrolipoic acid, resulting in an increased ratio of nicotinamide adenine dinucleotide (nad+) to nadh and thus stimulating the glycolysis pathway . In our study the alpha - lipoic beneficial effect on insulin requirement and thus on glucose uptake is reported at 3 months but it is not confirmed at 6 months . In our opinion this can be explained by a decreased compliance to the dietary supplementation, as confirmed by dietary recall and by data collected during the nutritional assessments . We also demonstrated for the very first time the power of alpha - lipoic acid in improving endothelial dysfunction in youths with type 1 diabetes . In our cohort of patients, the prevalence of endothelial dysfunction is largely represented, despite the pediatric age and the quite good metabolic control . Data from registries has recently shown that a large proportion of the type 1 diabetic population does not meet the age - associated hba1c targets across all countries, especially in the youth age . Moreover, data recently published by lind and colleagues showed that patients with type 1 diabetes and on - target hba1c still have a risk of death from any cause and from cardiovascular diseases more than twice the risks in the general population . These dramatic data ask for new insights into the pathogenesis of diabetes - related complications, as well as for new therapeutic approaches . Thus, during the last decades, many studies have been conducted to investigate the role of inflammation in diabetes onset and in diabetes - related complications . The opportunity for an early detection of endothelial dysfunction can lead to new insights into the very first steps of the cardiovascular complications . Indeed, endothelial cells play a wide spectrum of different functions, as regulating coagulation, leukocyte adhesion, and trafficking, modifying the tone of the vessel, and participating in the smooth - muscle growth . As a consequence, many different pathways can be affected by pathologic factors, leading to endothelial dysfunction . Several mechanisms have been suggested to explain endothelial dysfunction: hyperglycemia itself, increased oxidative stress and subsequent ages production and protein kinase c and polyol - pathway activation, lower vitamin c plasma concentration, and significantly higher circulating reactive protein c levels [27, 28]. Alpha - lipoic acid is reported to be effective in most of these reactions, getting the role of antioxidant, anti - inflammatory molecule . Indeed, although it is well defined as a therapy for preventing diabetic polyneuropathies and it is used in the management of diabetic peripheral neuropathy in both patients with type 1 and type 2 diabetes, alpha - lipoic acid has many biochemical functions . It acts as metal chelator, as scavenger of free radicals, and as reducer of the oxidized forms of other antioxidant agents such as vitamins c and e and it regulates several signal transductions . In particular alpha - lipoic acid was shown to increase the activity of enos, to downregulate the expression of the cell - adhesion molecules icam-1 and vcam-1 and of mmp-9 by the inhibition of age - induced nf - kb (nuclear factor kappa - light - chain - enhancer of activated b cells) adhesion factors [14, 22]. Supported by this evidence, we can speculate that a 6-month supplementation with alpha - lipoic acid 800 mg / day can positively impact endothelial dysfunction, decreasing oxidative stress and inflammation in type 1 diabetes, even in pediatrics . Despite this, these data must be carefully weighed against the lack of consensus regarding the most appropriate supplementation method for alpha - lipoic acid, dosage, and treatment duration . However, in a paper recently published a 3-month treatment with 600 mg of alpha - lipoic acid was demonstrated to provide significant beneficial changes in vegf, bfgf, mco-1, and il-10 serum levels in type 2 diabetic patients, confirming the efficacy of low - period treatment on endothelial outcomes . Moreover, several studies demonstrated the efficacy of 600 or more mg of alpha - lipoic acid administered for few weeks or months [30, 31] in the treatment of diabetic polyneuropathy . The strength of our study is the evidence for the first time of a positive association between alpha - lipoic administration and decreased endothelial dysfunction in pediatric patients with type 1 diabetes . Nevertheless the trial has some limitations, and we strongly advise a larger sample and a long - term follow - up to confirm these results . Moreover, the effects of alpha - lipoic acid on glycemic control need to be investigated as well, both as glycemic variability and hypo- and hyperglycemic events.
Posterior capsular opacification (pco) is the most common long - term complication following modern cataract surgery . It occurs in between <5% and 50% of uncomplicated senile cataracts during the first 2 years following cataract surgery [24]. Pco develops over a clear posterior capsule from a few months to a few years after uncomplicated cataract surgery . Regeneratory pco is caused by residual equatorial lens epithelial cells (lec) proliferation and migration from the equatorial region of the lens capsule to the posterior capsule surface . In regions of the anterior and posterior capsule junction, elschnig pearls can form, located behind the iris or filling the pupil space . The formation of elschnig pearls causes a decrease in visual acuity and sometimes double vision following the implantation of an intraocular lens (iol). Capsular fibrosis is less common and usually appears earlier than elschnig pearls; it is thought to be caused by lec metaplasia with myofibroblast development . Clinically, it is seen as a wrinkling on the posterior capsule, haziness and grey - white streaks and plaques on the surface of the posterior capsule . The incidence of pco is more common in young patients and those with uveitis, pseudoexfoliation syndrome or traumatic cataracts . Myopic eyes have been postulated to increase the risk of pco; this probably occurs because iol implantation was deferred in them, but a study of iol implantation in myopic eyes showed no association between the degree of myopia and the degree of pco . When compared with non - diabetic patients, diabetic patients had significantly more pco following cataract surgery, but the stage of diabetic retinopathy and the systemic status of the diabetes does not seem to correlate with the degree of pco . Patients with myotonic dystrophy required multiple capsulotomies following cataract surgery due to pco and progressive capsulorhexis contracture . Other factors that influence pco development are: the intraocular lens type (material, design, optic size and edge, haptic design), accurate hydrodissection and removal of cortical masses, anterior capsule polishing, in - the - bag fixation of the optic and the haptic iol part and anterior capsulorhexis localization and diameter . Numerous studies have examined the influence of physical properties of the iol and accurate surgical lens removal technique on the formation of the pco [1119]. Relatively few studies have investigated the influence of anterior capsulorhexis on the development of posterior capsule opacification [2022]. The present study evaluates the impact of anterior capsulorhexis diameter, localization and shape on pco development following cataract extraction with phacoemulsification . The incidence of pco is more common in young patients and those with uveitis, pseudoexfoliation syndrome or traumatic cataracts . Myopic eyes have been postulated to increase the risk of pco; this probably occurs because iol implantation was deferred in them, but a study of iol implantation in myopic eyes showed no association between the degree of myopia and the degree of pco . When compared with non - diabetic patients, diabetic patients had significantly more pco following cataract surgery, but the stage of diabetic retinopathy and the systemic status of the diabetes does not seem to correlate with the degree of pco . Patients with myotonic dystrophy required multiple capsulotomies following cataract surgery due to pco and progressive capsulorhexis contracture . Other factors that influence pco development are: the intraocular lens type (material, design, optic size and edge, haptic design), accurate hydrodissection and removal of cortical masses, anterior capsule polishing, in - the - bag fixation of the optic and the haptic iol part and anterior capsulorhexis localization and diameter . Numerous studies have examined the influence of physical properties of the iol and accurate surgical lens removal technique on the formation of the pco [1119]. Relatively few studies have investigated the influence of anterior capsulorhexis on the development of posterior capsule opacification [2022]. The present study evaluates the impact of anterior capsulorhexis diameter, localization and shape on pco development following cataract extraction with phacoemulsification . This retrospective analysis reviewed 297 patients above age 45, randomly chosen from a group of 3500 operated patients who had undergone phacoemulsification and iol implantation in the capsular bag at the department of ophthalmology, medical university of warsaw from september 2007 to september 2008 . Inclusion criteria were used to define the presence of a senile cataract in an otherwise normal eye . Exclusion criteria consisted of: previous eye surgery and ocular or systemic diseases (eg, diabetes mellitus, rheumatoid diseases and serious cardiovascular diseases). Visual acuity, intraocular pressure (iop), slit lamp examination, fundus examination, b - scan ultrasonography, keratometry, iol power calculation with a biometry measurement and systemic examinations were all evaluated prior to surgery in all patients . All patients used 0.1% diclofenac sodium solution eye drops (naclof; novartis) 3 times on the day before surgery and on the day of surgery . Phacoemulsification was performed by a single surgeon (e.l .- w .) Under topical anesthesia . In the first group of 97 patients, 53 patients (55%) were postoperatively assigned to the group whose capsulorhexis were classified as small (3.9 to 4.9 mm in diameter) and 44 patients (45%) were assigned to the group whose capsulorhexis were classified as large (5.0 to 5.9 mm in diameter). Another group of 99 patients was postoperatively assigned into 1 of 2 subgroups the first subgroup of 66 patients (66%) had capsulorhexis that were classified as centrally located and the second group consisted of 33 patients (34%) whose capsulorhexis were classified as paracentral . The capsulorhexis were defined as centrally located when the center of the capsulorhexis was in the center of the patient s own lens and later in the center of the implanted posterior chamber intraocular lens (pciol). The capsulorhexis were defined as paracentral when the center of the capsulorhexis was not in the center of the patient s own lens and the pciol . Capsulorhexis shift was at least 1.5 mm, which is half the radius of the optical part of implanted pciol . The capsulorhexis were small (3.9 to 4.9 mm in diameter) and regularly rimmed . A third group of 101 patients was also postoperatively assigned into 1 of 2 subgroups depending on the shape of their capsulorhexis rim . The first subgroup of 59 patients (58%) was classified as having a regularly rimmed capsulorhexis and the second subgroup of 42 patients (42%) was classified as having an irregularly rimmed capsulorhexis . The capsulorhexis were defined as regularly rimmed when the edge was smooth and the main perpendicular diameters were similar in length . The capsulorhexis were defined as irregularly rimmed when the edge was uneven at least on 1/3 of the capsulorhexis rim or the main perpendicular diameters were not similar in length . The capsulorhexis were small (3.9 to 4.9 mm in diameter) and centrally located . The edge of the capsulorhexis had to lie completely on the intraocular lens optic for 360 degrees, even if it was eccentric, in all groups . A 6.0-mm diameter optic, single - piece, hydrophobic acrylic intraocular lens (alcon, model sa60at) was implanted . Postoperatively, all patients used neomycin, polymyxin, and 0.1% dexamethasone solution eye drops (maxitrol; alcon pharmaceuticals) 4 times a day for 1 month . Postoperative follow - up visits were performed on days 1, 3, 14, 30, 90, and 180 . Patients were examined and refracted, and visual acuity was assessed using the snellen chart . Digital retroillumination imaging of the posterior capsule through dilated pupils was performed with our high - resolution digitized camera system . Standardized retroillumination images of the posterior capsule were taken 14 and 180 days after the operation . The images taken after 180 days were used for image analysis . At 6-month follow - up, posterior capsular opacification was classified as either none, mild, moderate, or severe, depending on the number of quadrants involved . Moderate pco occurred when it was present in 2 quadrants, and severe pco occurred when it was found in 3 or 4 quadrants . Special attention was given to central area as they are the most important to visual acuity . The postoperative relationship of the anterior capsulorhexis margin to the intraocular lens optic was classified as central or paracentral . The capsulorhexis rim was defined as either a regular anterior capsulorhexis or an irregular anterior capsulorhexis . Fisher s exact test was used to analyze the difference in pco between groups with a small and a large capsulorhexis, a central and a paracentral capsulorhexis, and regular and irregular capsulorhexis . Pco intensification was classed as either none, mild, moderate or severe . The fisher - freeman - halton test and the kruskal - wallis test were used to analyze the difference in data between the groups with different capsulorhexis diameter, localization and shape . A p value of less than 0.001 was considered as statistically significant . Before cataract extraction surgery all patients were adequately informed of the nature and possible consequences of the study and they provided signed consent for the use of medical records for research purposes . There were no surgical complications that would have led to patient exclusion . For 30 days after surgery, none of the patients reported infections, and within 180 days none of them had reported any serious fever episodes . The mean age was 66 years (range: 45 to 87 years) (figure 1); 188 of the patients were female and 109 were male . Ninety - seven patients were postoperatively recruited and assigned to each capsulorhexis size group 44 patients (45%) were found to have a large capsulorhexis and 53 patients (55%) were found to have a small capsulorhexis . In another group of 99 patients, it was the capsulorhexis localization that was assessed . In this group, 66 patients (66%) were found to have a central capsulorhexis and 33 patients (34%) were found to have a paracentral one . In the third group, which consisted of 101 patients, the first subgroup of 59 patients (58%) were found to have regularly shaped capsulorhexis rims and 42 patients (42%) were found to have irregularly shaped rims . At the 6-month follow - up, in the group in which the capsulorhexis diameter was observed, the edge of the large capsulorhexis lay at the edge of the intraocular lens optic in 19% (8 patients), while the rest of the patients had the edge of the anterior capsulorhexis on the intraocular lens optic for 360 degrees when the capsulorhexis was small as well as in large ones . The amount of pco at 6 months was significantly different between the groups with small and large capsulorhexis (figure 2) 86.79% of the patients with a small capsulorhexis had no or mild posterior capsule opacification, whereas 68.18% of the patients with a large capsulorhexis had moderate or severe pco . Patients with a small capsulorhexis had significantly less posterior capsular opacification than those with a large capsulorhexis (p<0.001) (table 1). The amount of pco in the groups with central (66 patients) and paracentral (33 patients) capsulorhexis localization was similar (figure 3) 89.4% of the patients with a central capsulorhexis had no or mild posterior capsule opacification, whereas 75.75% of the patients with a paracentral capsulorhexis had moderate or severe pco . Patients with a central capsulorhexis had significantly less posterior capsular opacification than those with a paracentral anterior capsulorhexis (p<0.001) (table 2). Figure 4 shows the amount of pco in the groups with regular and irregular anterior capsulorhexis rim shapes 86.44% of the patients with a regular rim of the anterior capsulorhexis had no or mild posterior capsule opacification and 13.55% had moderate or severe pco, while 69.04% of the patients with an irregular capsulorhexis rim had moderate or severe pco . Patients with a regular anterior capsulorhexis rim had significantly less posterior capsular opacification than those with an irregular anterior capsulorhexis rim (p<0.001) (table 3). In all examined parameters the posterior capsule opacification ratio was smallest when the capsulorhexis was small, centrally located and with a regularly shaped rim when compared to the large, paracentral and irregular ones . There was no statistically significant difference in the kruskal - wallis test results between the groups with small, central and regular shape of capsulorhexis (p=0.6997) (table 4). In the group of all operated patients, 38 (12%) of those experiencing severe postoperative posterior capsule opacification qualified for a neodymium: yttrium - aluminium - garnet (nd: yag) laser posterior capsulotomy . The effect of the size of the capsulorhexis on posterior capsular opacification was investigated by ravalico et al in a retrospective study of 107 patients who underwent extracapsular cataract extraction with capsulorhexis and capsular bag iol implantation (polyhema iols and pmma iols). They found that a capsulorhexis of a slightly smaller diameter than the iol optic appears to be better than a large - size capsulorhexis in reducing the incidence of pco . Due to different cataract extraction procedures and iol material, these results are not suitable for comparison with our research results . A procedure similar to that used in our investigation was carried out by hollic et al in a prospective study of 75 patients who underwent standardized phacoemulsification with capsulorhexis and in - the - bag placement of a 5.5-mm polymethylmethacrylate intraocular lens implant . The patients were randomly assigned to receive either a small capsulorhexis of 4.5 to 5 mm to lie completely on the intraocular lens optic, or a large capsulorhexis of 6 to 7 mm to lie completely off the lens optic . They suggested that large capsulorhexis were associated with a significant increase in the wrinkling of the posterior capsule and a worse posterior capsular opacification as compared to small capsulorhexis . At 1 year, the average percentage area of posterior capsular opacification was 32.7% for small capsulorhexis and 66.2% for large capsulorhexis . Patients with large capsulorhexis experienced significantly poorer visual acuities and a trend toward worse contrast sensitivities . Aykan at al found comparable results in their prospective study (496 eyes underwent standardized phacoemulsification with capsulorhexis and capsular bag foldable acrylic iol implantation). A small (4.5 to 5.0 mm) capsulorhexis and capsular bag implantation of 5.5 mm acrylic iol reduced pco incidence when compared to a 6.0 to 7.0 mm capsulorhexis . The effect of the position of the anterior capsulorhexis on posterior capsular opacification was investigated by wejde et al in a study of 119 patients who underwent cataract surgery with phacoemulsification performed by a single surgeon . The patients were randomized to implantation with either a silicone intraocular lens (iol) (si40nb, allergan) or an acrysof iol (ma60bm, alcon). Three years following the surgery, the rate of pco was analyzed using the evaluation of posterior capsule opacification computer software (epco). The results were related to the capsulorhexis position, which was assessed with a retroillumination photograph . If the capsulorhexis was located partially or completely off the optics of the iol as opposed to completely on the iol, significantly more pco was found, suggesting that the positioning of the capsulorhexis impacts the development of pco . A relatively small and central capsulorhexis that allows the complete coverage of the iol optics by the capsulorhexis edges seems to protect against pco in cataract surgery . This may be explained by lec mechanical blockade into the effect of the anterior lens capsule adhesion to the iol, which is especially strong when there is complete anterior capsule overlap on the iol optic in 360. however, hayashi reported that in a study of 100 patients, 81% showed complete anterior capsule overlap, and there was no significant difference in the pco value between the eyes with a complete anterior capsule overlap and those with partial or incomplete anterior capsule overlap . Furthermore, no significant correlation was found between the degree of the anterior capsule overlap and the extent of the pco . In our observations patients with a regularly shaped capsulorhexis rim had significantly less posterior capsular opacification than those with an irregular anterior capsulorhexis rim (p<0.001). We believe that when the capsulorhexis rim is regular, the disintegration of forces working on the lens capsule is identical, and wrinkling is much less likely to occur . In the event that the capsulorhexis rim is irregular, forces working on the lens capsule are different, which affects posterior capsular folds . Statistical analysis with the kruskal - wallis test shows that there was no statistically significant difference between the groups with small, central or regularly shaped capsulorhexis (p=0.6997). This result shows that not only the diameter, but also the localization and the shape of the capsulorhexis has an influence on the occurrence of posterior capsular opacification, and that the degree of influence of these seems to be at a comparable level of importance . This study indicates that not only a small capsulorhexis diameter, but also its central localization and its regular shape result in less posterior capsular opacification following the phacoemulsification procedure.
In our model, each particle consists of a core sphere with four tetrahedrally oriented arms (see fig . The central sphere and the arms are modeled as interpenetrating hard spheres with diameter, with the centre of each arm located a distance l from the centre of the core . On the surface of each arm the patch is modeled, via a standard kern - frenkel potential, as a square - well type attraction of depth: two particles are either bound or unbound, with no intermediate energy levels . The opening angle of the patch is given by cos = 0.95, and the maximum interaction range = 0.251. this choice ensures that each patch can form a bond with only a single other patch . Furthermore, in order to model arm flexibility, each arm can freely rotate around the core sphere within a maximum angle from its ideal (tetrahedral) position . Note that there are no hard - core interactions within a particle: spheres belonging to the same tetramer can overlap . Each mc simulation included translation moves, rotation moves rotating a full tetramer, rotation moves rotating only the core sphere, and rotation moves that rotate a single arm around the core . Additionally, we added volume moves for isobaric simulations, and particle insertion and deletion moves for grand - canonical successive umbrella sampling (sus) simulations . To detect ll phase separation, we used sus simulations, generating the relative free energy as a function of the density . To calculate the phase diagrams in fig . 4, and the crystallization lines in figs . 2 and 3 for the liquid phases, we used a high temperature fluid as a reference state, where we obtained the helmholtz free energy f by combining the chemical potential (evaluated from a sus simulation) and the pressure p at the same density (taken from an npt mc simulation), using: (1)ffn=p, where n is the number of particles . We then used thermodynamic integration by integrating the temperature along an isochore: (2)2f(,t2)=1f(,t1)+12du(,t), with u(, t) the average potential energy per particle, = 1/kbt and kb boltzmann s constant . Typically, at sufficiently low temperatures, the energy as a function of temperature takes the form (3)u=2+cexp(2), where 2 is the ground - state energy (where each particle has four bonds) and c is a (density - dependent) constant . This allows for straightforward extrapolation of the free energies to arbitrarily low temperatures after the regime in eq . This regime is accessible in our simulations for all cases where the fluid does not crystallize at low t (i.e. Cos 0.9). System sizes for the sus simulations were chosen such that near the ll transition the system contained n 150 particles . Other free - energy calculations were performed with similar or larger n. the investigated crystal structures were diamond and bcc (corresponding to ice ic and ice vii in water, respectively). Both are fully bonded structures and as such have the (same) lowest possible potential energy . Depending on cos and l, there will be a variety of other fully bonded crystal structures . However, the structures commensurate with an ideal tetrahedral geometry will always have the highest entropy and hence will be more stable at the densities where they are accessible . Thus, the only crystals which might compete with the ll phase separation are diamond and bcc . At high density, i.e. Beyond the bcc density, the stable crystalline phases will be controlled by a competition between packing, energy, and entropy, but as these phases will not change the stability of the ll phase transition, they are outside of the scope of this letter . Similarly, we do not expect these crystal phases to affect the phase diagrams in fig . 4, with the possible exception of the (dashed) high - density liquid - bcc lines in panels (c) and (d). To determine free energies for the crystal phases, we used an einstein crystal approach, for each t where a phase coexistence was calculated . Subsequently, using npt mc simulations, we employed thermodynamic integration along the density at constant temperature: (4)f(,t)=f(ref, t)+refd()p2, where ref is the density where the reference free energy was calculated . S4 in the si for examples). To determine whether spontaneous crystallization occured in our sus simulations, we used a bond - order parameter to find the largest crystalline cluster in the system . First, we determine the neighbors of each particle using a solid - angle based nearest - neighbor method . We then calculate for each particle the complex vector ql, the expansion of their environment in terms of spherical harmonics ylm, with l the order of the symmetry of the crystal, and l m l: (5)qlm(i)=1nb(i)j=1nbylm(r^ij). Here, nb is the number of neighbors of particle i, and r^ij is the normalized vector connecting particle i to particle j. for the purpose of finding crystalline clusters, two particles are considered bonded when their environments are sufficiently similar . To determine this, we calculate: (6)dl(i, j)=re(ql(i)ql(j)ql(i)ql(j)). To detect bcc clusters, we choose l = 6, consider two particles bonded if dl(i, j)> 0.6, and label any particle that has at least 5 bonds as a crystalline particle . For diamond, two particles are considered bonded if either dl(i, j) <0.87 or 0.3 <dl(i, j) <0.1, and only particles with 4 bonds are considered crystalline . Systems were considered to have crystallized spontaneously when more than 10% of the system was crystalline . Note that although the q6 based order parameter is sensitive to multiple crystal structures, visual inspection of the detected clusters consistently showed a bcc structure in all cases.
The public health system in india despite growing investments in every national 5-year plan (1.1% of gdp in 2012) and even after over 65 years of its functioning, has not yet delivered universal primary healthcare to the citizens of india . Around 70% of the indian population spend money for primary healthcare services from their own pockets . This article argues that it is necessary to urgently reform the content of public health system and make it more pluralistic . Medical pluralism in india is specially relevant because of the richness of india's medical heritage which offers a unique opportunity to integrate across 5 traditional systems of healthcare . A new national policy 2015 to replace the last policy formulated in 2002 is on the anvil . The 3 tiers operate through a large number of government, that is, taxpayer financed, primary secondary and tertiary healthcare institutions and a larger number of private (for - profit) institutions and a much smaller number of private (not for profit) organizations . At the base of the pyramid of the health system, are the primary healthcare institutions in the form of dispensaries and small - sized general hospitals . A substantial number of them are in the government - sector, but they have a larger presence in the private sector . Higher up the pyramid are the secondary institutions (like district and private hospitals) and at the top are the tertiary services provided by few well - equipped medical college hospitals and mostly by corporate super specialty establishments . Experts have identified a host of operational issues and gaps that plague the public health system . These relate to inadequate infrastructure, financing, human resources (hrs), drugs, hr policies, health information system, insurance and governance . The government is aware of the gaps in the functioning of the public health system as is evident from official reviews prepared by the planning commission . While the gaps do get addressed from time to time, through various schemes, the reform happens in the typical piece - meal fashion that characterizes government interventions . The officially declared goal of the public healthcare system is free and universal primary healthcare . However, even after 66 years around 70% of the population do not receive satisfactory or free primary healthcare and they are therefore forced to seek help from private providers and thus pay out of their own pocket . Public health experts in recent times have observed that safe drinking water, sanitation, nutrition, lifestyle, and the environment are key determinants of health and that the health system must address these basic needs . In practice however, the health system does not appear to have any influence, mechanism or programs, to address these key determinants of health because water, sanitation, nutrition, environment are domains managed by ministries other than the health ministry . It is almost wholly based on western bio - medicine . In fact 97% of the national health budget, since 1947 has been allocated to allopathy . Postindependence, the idea of integrating and mainstreaming seven other legally sanctioned health systems with allopathy has been mentioned in the introductory paragraphs, of all national 5 years plan and policy documents . In practice the eight systems of healthcare viz ., allopathy, ayurveda, siddha, sowa - rigpa, unani, yoga, naturopathy, and homeopathy function in silos . The seven ayush systems receive only 3% of the national health budget and the departments of ayush across all indian states operate with this meager funding . The ayush department despite their limited funding, operate a parallel national health service, unconnected to the mainstream 3 tier health system, with around 24,000 dispensaries and 3000 small general hospitals, across 30 states . The ayush public health services are planned and managed by the departments / directorates of ayush at the center and states . The planning and administrative machinery for ayush is distinct from the departments of health and family welfare that plan and administer the mainstream public health system, and thus ayush services are not aligned to national health priorities . The official ayush budget has sub - critical allocation for extramural research, education and for regulation of safety and quality . This is the reason why the ayush systems during the last 60 years have hardly generated any evidence - based clinical, pharmacological or pharmaceutical outputs and also the reason why the regulatory system is ineffective . The not for profit private sector in ayush is the public face of ayush . While there is no data on its growth and performance, judging from its visibility in the form of private dispensaries and secondary care hospitals, it is perceived to be a more effective provider of ayush health services to the community . The indian public availing ayush depends on this sector for quality health services . The limited evidence - based ayush research available in the public domain is generated by this sector through mostly, nongovernment funding . An overview of the indian public healthcare system thus clearly suggests that despite the fact that eight legally sanctioned health sciences operate within the health system, due to their skewed funding and poor integration, the public does not receive the advantage of synergy arising out of the richness of india's medical heritage . The public health system in india despite growing investments in every national 5-year plan (1.1% of gdp in 2012) and even after over 65 years of its functioning, has not yet delivered universal primary healthcare to the citizens of india . Around 70% of the indian population spend money for primary healthcare services from their own pockets . This article argues that it is necessary to urgently reform the content of public health system and make it more pluralistic . Medical pluralism in india is specially relevant because of the richness of india's medical heritage which offers a unique opportunity to integrate across 5 traditional systems of healthcare . A new national policy 2015 to replace the last policy formulated in 2002 is on the anvil . The 3 tiers operate through a large number of government, that is, taxpayer financed, primary secondary and tertiary healthcare institutions and a larger number of private (for - profit) institutions and a much smaller number of private (not for profit) organizations . At the base of the pyramid of the health system, are the primary healthcare institutions in the form of dispensaries and small - sized general hospitals . A substantial number of them are in the government - sector, but they have a larger presence in the private sector . Higher up the pyramid are the secondary institutions (like district and private hospitals) and at the top are the tertiary services provided by few well - equipped medical college hospitals and mostly by corporate super specialty establishments . Experts have identified a host of operational issues and gaps that plague the public health system . These relate to inadequate infrastructure, financing, human resources (hrs), drugs, hr policies, health information system, insurance and governance . The government is aware of the gaps in the functioning of the public health system as is evident from official reviews prepared by the planning commission . While the gaps do get addressed from time to time, through various schemes, the reform happens in the typical piece - meal fashion that characterizes government interventions . The officially declared goal of the public healthcare system is free and universal primary healthcare . However, even after 66 years around 70% of the population do not receive satisfactory or free primary healthcare and they are therefore forced to seek help from private providers and thus pay out of their own pocket . Public health experts in recent times have observed that safe drinking water, sanitation, nutrition, lifestyle, and the environment are key determinants of health and that the health system must address these basic needs . In practice however, the health system does not appear to have any influence, mechanism or programs, to address these key determinants of health because water, sanitation, nutrition, environment are domains managed by ministries other than the health ministry . It is almost wholly based on western bio - medicine . In fact 97% of the national health budget, since 1947 has been allocated to allopathy . Postindependence, the idea of integrating and mainstreaming seven other legally sanctioned health systems with allopathy has been mentioned in the introductory paragraphs, of all national 5 years plan and policy documents . In practice the eight systems of healthcare viz ., allopathy, ayurveda, siddha, sowa - rigpa, unani, yoga, naturopathy, and homeopathy function in silos . The seven ayush systems receive only 3% of the national health budget and the departments of ayush across all indian states operate with this meager funding . The ayush department despite their limited funding, operate a parallel national health service, unconnected to the mainstream 3 tier health system, with around 24,000 dispensaries and 3000 small general hospitals, across 30 states . The ayush public health services are planned and managed by the departments / directorates of ayush at the center and states . The planning and administrative machinery for ayush is distinct from the departments of health and family welfare that plan and administer the mainstream public health system, and thus ayush services are not aligned to national health priorities . The official ayush budget has sub - critical allocation for extramural research, education and for regulation of safety and quality . This is the reason why the ayush systems during the last 60 years have hardly generated any evidence - based clinical, pharmacological or pharmaceutical outputs and also the reason why the regulatory system is ineffective . The not for profit private sector in ayush is the public face of ayush . While there is no data on its growth and performance, judging from its visibility in the form of private dispensaries and secondary care hospitals, it is perceived to be a more effective provider of ayush health services to the community . The indian public availing ayush depends on this sector for quality health services . The limited evidence - based ayush research available in the public domain is generated by this sector through mostly, nongovernment funding . An overview of the indian public healthcare system thus clearly suggests that despite the fact that eight legally sanctioned health sciences operate within the health system, due to their skewed funding and poor integration, the public does not receive the advantage of synergy arising out of the richness of india's medical heritage . All over the world there is evidence of growing public demand for making available healthcare choices, based upon best knowledge and practices, drawn from different healthcare systems . In india also we see this trend reflected in the actual health seeking behavior of communities wherein people seek to combine or choose for different health conditions allopathy or ayurveda, siddha, sowa - rigpa, unani, homeopathy or yoga or a combination . For emergencies and surgery allopathy is the first choice, for common ailments it is ayurveda, sidha, yoga, unani, sowa - rigpa or homeopathy, for chronic conditions it may initially be allopathy and then a rebound to some other system, when there is insufficient relief . The public demand for pluralism in healthcare is probably based on a realistic assessment by laypersons of the inadequacy of any single system of healthcare to solve all their contemporary health needs . Governments and regulatory bodies also appear to have accepted the imperative for pluralistic approaches in healthcare with the caveat that all new, potentially useful healthcare interventions, must establish their safety, quality and efficacy . An objective manifestation of the global acceptance of medical pluralism is reflected in the creation of government - sponsored national research institutes for complementary and alternative medicine (cam) in the united states (like national center for cam) and in europe norway, (nafkam) sweden and in the introduction of introductory modules on integrative medicine (i m) in medical schools in countries like the us and uk . It is probably this public assessment that is responsible for the dramatic growth of the cam movement and the nascent evolution of different models of i m in both the public and private sector . From the globally observed health seeking behavior trends, it is apparent that the era of monoculture in healthcare is coming to an end . While the mainstream indian public health system relies largely on one single knowledge system viz ., modern medicine, for its services, at the frontiers of medical and life sciences the limitations of singular health knowledge systems are being recognized . The limitations of bio - medical sciences arise due to the reductionist theoretical framework of science . This framework imposes methodological limitations which permit only partial understanding of complex biological phenomena at the cellular level . Even today, underlying pathways for biological changes are hardly understood and therefore drug actions established after expensive clinical trials, have unpredictable side effects . Today, specially in the context of noncommunicable diseases, the world of medicine is no longer looking for blockbuster drugs aimed at single targets; it is looking for drugs that correct underlying physiological imbalances that manifest as syndromes . Even for infectious diseases, it is no longer looking for single molecules (that inevitably result in drug resistance) but rather for combinatorial drugs . This suggests that our understanding of life is still in its infancy, and intelligent ab initio design of therapy is difficult . It is tempting to suggest here that the approach of modern medicine which starts at molecular level and progresses toward building systems, now referred to as systems biology, and the traditional medicine's holistic understanding and approaches will intersect fruitfully if expertise and research are managed carefully leading to new and sustainable solutions and perhaps original contribution to the world of medicine and life sciences . In fact during the last decade on the knowledge plane, the tremendous potential of trans - disciplinary research in health sciences (integrating in an epistemologically informed manner ayurveda and modern biology) is already beginning to be demonstrated . The pioneering work of linking the ayurvedic phenotypes to genotypes has opened up huge possibilities for new understanding of human physiology, new design strategies for drug development, early detection of diseases and differential schemes for clinical management . Similarly, in the context of community health, it has demonstrated that the traditional advice for storing drinking water in copper vessels is probably the world's cheapest solution for microbial purification of drinking water . In the context of management of chronic diseases, a recent pilot clinical study from pune published in rheumatology (oxford) 2013 and another study sponsored by national institutes of health, usa have concluded that the systemic, holistic management of rheumatoid arthritis based on ayurveda, is as effective as the best biomedical treatment with specific drugs and has lesser side effects . The work of iits on classical herbo - mineral - metallic preparations of ayurveda called bhasmas, reveal that bhasmas prepared by these reputed institutions, in exactly the way prescribed in ayurvedic texts using rudimentary, home scale technologies, resulted in finished products that were of nanoparticle sizes . It was further observed that such microstructures as were produced through traditional technology could not be easily produced through conventional chemistry procedures in laboratories . These leads if pursued consistently and boldly have the potential to create new paradigms in modern science, technology, and medicine . Casual observers wonder how modernity can be advanced by combining modern western biology and biomedicine with traditional indian health sciences . The reason for doubt is because the mainstream schools of sociology have posited the modern and traditional as opposites . In fact historical analysis of european modernity as a case study, clearly reveals that the roots of modernity lie in tradition (just as the roots of the present lie in the past) and that in effect modernity is evolving tradition . Due to the recent history of colonialism, the colonized nations were led to believe that they needed to import modernity from their colonizers . But the colonial era is long over and in modern, independent nations it is essential for civil society and polity to realize that modernization of all societies must derive inspiration from their own traditional roots . While import and knowledge exchange, across different cultures, is desirable in a globalized world, neglect of one's own knowledge traditions, when they are of contemporary value is suicidal . India can be a world leader in this new emerging field of integrative healthcare because we have over the last century or so assimilated and achieved a reasonable degree of competence in biomedical and life sciences and we possess an incredibly rich medical heritage of our own . India has over the last 200 years successfully borrowed the modern western model of a3 tiered institutionalized structure from western nations . Casual observers wonder how modernity can be advanced by combining modern western biology and biomedicine with traditional indian health sciences . The reason for doubt is because the mainstream schools of sociology have posited the modern and traditional as opposites . In fact historical analysis of european modernity rooted in classical greek tradition, clearly reveals that the roots of modernity lie in tradition (just as the roots of the present lie in the past) and that in effect modernity is evolving tradition . Due to the recent history of colonialism, the colonized nations were led to believe that they needed to import modernity from their colonizers . But the colonial era is long over and in independent nations it is essential for civil society and polity to realize that modernization of all societies must derive inspiration from their own traditional roots . While import and knowledge exchange, across different cultures, is desirable in a globalized world, neglect of one's own knowledge traditions, when they are of contemporary value is suicidal . Today, the indian public health system is at crossroads . Despite massive investments over the last almost 65 years health seeking behavior studies reveal that the citizens of the world also recognize the limitation of a mono - cultural health system and are therefore exercising alternative choices . The moot question before the indian polity is, should the country further increase investment into a singular system of healthcare or should at this point of time india innovate and diversify its health system by evolving a new integrative healthcare system in the 21 century . It make sense, in the 21 century fora national government sensitive to social realities of public health seeking behavior which is already exercising pluralistic choices, to expand the scope of the wholly western medicine content of health care and refine it by deriving strategies, content and form, from our own traditional knowledge systems . India has had rich experience in managing healthcare for centuries in the longest surviving, and evolving health tradition in the world . Over 6500 species of medicinal plants, around 300 animal products, the traditional knowledge digital library, computerized by council of scientific and industrial research has already documented around 200,000 herbal formulations alongside their therapeutic indications . India possesses an estimated 100,000 medical manuscripts on medicine and surgery which includes sophisticated knowledge of pharmacology, pharmacy, diagnosis, therapies, prevention, and wellness ., it is necessary to shed unrealistic demands for immediate presentation of a large amount of clinical evidence about ayush systems . This is unrealistic because while limited evidence is certainly available, comprehensive clinical and pharmacological evidence is simply not available . The reason evidence is not available is that the state has for the last 200 years not invested in the creation of such evidence . The budget estimates for 20142015 of ayush department of government of india suggest that even in 2014 the extramural research budget of ayush is <rs . It can only be met when clinical research begins to get supported in a sustained way . Today, the integrative agenda needs to build on the national rural health mission (nrhm) 2005 policy framework . We need to select prioritized interventions, selected by ayush experts for introduction into the newly named national health assurance mission and into the 3 tiered public health system . Thus, the first step towards extending the social reach of healthcare in india is to urgently reform the existing 3 tiers of the public health system by infusing ayush content and hrs . This is a complex exercise as can be seen from the fact that although nrhm had the plan and strategy of co - location and co - posting, it has not worked because no homework was done to bring about the integration of health content derived from the different indian systems of medicine . The lesson to be learnt from 9 years of nrhm is that a new national integrative, public healthcare system not only needs logistical moves like co - location and co - posting but serious clinical exercises for identifying specific ayush interventions, orienting medical personnel in their use, developing protocols and cross referral guidelines and such operational details . The ayush interventions have to be selected for health services at primary, secondary and tertiary levels . In parallel, an integrative public health system will simultaneously require radical reform in medical education, medical research, regulations, and the legal framework for medical practice . In the 21 century, an integrative model for public health needs a 10-year budget, a detailed action plan, and strategy, in order to achieve this complex goal . A second radical step toward modernization of health care in india is to invest in and use its heritage to restore two more traditionally available tiers at the bottom of the pyramid to enrich the health system and demonstrate the efficacy of a uniquely indian, participatory public health system . These community - based and supported layers were existing until the beginning of the 20 century and are still functioning in eroded fashion . They have been overlooked and neglected in india since the country embraced the western model of public health . They will add millions of new health providers to the public health system at zero recurring cost . These tiers are to be managed, as was the case for centuries, by millions of households and traditional community - based health workers . The household was a repository of region - specific, self - help health practices based on the use of ecosystem - specific natural resources . Till recently, the indian households possessed knowledge of at least a 100 home herbal remedies, nondrug health practices and food and nutrition . The homes had competence to manage common ailments, preventive health practices, and healthy ethnic diets . The creation of this household tier to the public health system will require critical investment in a creatively designed, information and communications technology enabled health education strategy, for reaching millions of rural and urban households . The second additional tier to be introduced in a modern indian healthcare system is also a noninstitutional tier managed by community - based and community supported traditional health workers . The first step for restoration is to certify, accredit, and enrich the knowledge and skills of existing folk healers . Pilot experiments for certification and accreditation have already been demonstrated as recently as in 2013 . The community support base of the 1 million traditional health workers needs to be reinforced and care taken to avoid making them dependent on government support for their services to the community . The next step will be to motivate a new generation of folk healers to replace the older and ageing currently available generation . This is because while the government needs to invest in their revitalization, the action programs for developing these two tiers cannot be executed by the government . Thus, a very sensitive and long - term strategy is needed to revitalize and modernize india's public health system and make it both integrative and much more participatory . India has over the last 200 years successfully borrowed the modern western model of a3 tiered institutionalized structure from western nations . Casual observers wonder how modernity can be advanced by combining modern western biology and biomedicine with traditional indian health sciences . The reason for doubt is because the mainstream schools of sociology have posited the modern and traditional as opposites . In fact historical analysis of european modernity rooted in classical greek tradition, clearly reveals that the roots of modernity lie in tradition (just as the roots of the present lie in the past) and that in effect modernity is evolving tradition . Due to the recent history of colonialism, the colonized nations were led to believe that they needed to import modernity from their colonizers . But the colonial era is long over and in independent nations it is essential for civil society and polity to realize that modernization of all societies must derive inspiration from their own traditional roots . While import and knowledge exchange, across different cultures, is desirable in a globalized world, neglect of one's own knowledge traditions, when they are of contemporary value is suicidal . Today, the indian public health system is at crossroads . Despite massive investments over the last almost 65 years health seeking behavior studies reveal that the citizens of the world also recognize the limitation of a mono - cultural health system and are therefore exercising alternative choices . The moot question before the indian polity is, should the country further increase investment into a singular system of healthcare or should at this point of time india innovate and diversify its health system by evolving a new integrative healthcare system in the 21 century . It make sense, in the 21 century fora national government sensitive to social realities of public health seeking behavior which is already exercising pluralistic choices, to expand the scope of the wholly western medicine content of health care and refine it by deriving strategies, content and form, from our own traditional knowledge systems . India has had rich experience in managing healthcare for centuries in the longest surviving, and evolving health tradition in the world . Over 6500 species of medicinal plants, around 300 animal products, 70 metal and minerals the traditional knowledge digital library, computerized by council of scientific and industrial research has already documented around 200,000 herbal formulations alongside their therapeutic indications . India possesses an estimated 100,000 medical manuscripts on medicine and surgery which includes sophisticated knowledge of pharmacology, pharmacy, diagnosis, therapies, prevention, and wellness . It is necessary to shed unrealistic demands for immediate presentation of a large amount of clinical evidence about ayush systems . This is unrealistic because while limited evidence is certainly available, comprehensive clinical and pharmacological evidence is simply not available . The reason evidence is not available is that the state has for the last 200 years not invested in the creation of such evidence . The budget estimates for 20142015 of ayush department of government of india suggest that even in 2014 the extramural research budget of ayush is <rs . It can only be met when clinical research begins to get supported in a sustained way . Today, the integrative agenda needs to build on the national rural health mission (nrhm) 2005 policy framework . We need to select prioritized interventions, selected by ayush experts for introduction into the newly named national health assurance mission and into the 3 tiered public health system . Thus, the first step towards extending the social reach of healthcare in india is to urgently reform the existing 3 tiers of the public health system by infusing ayush content and hrs . This is a complex exercise as can be seen from the fact that although nrhm had the plan and strategy of co - location and co - posting, it has not worked because no homework was done to bring about the integration of health content derived from the different indian systems of medicine . The lesson to be learnt from 9 years of nrhm is that a new national integrative, public healthcare system not only needs logistical moves like co - location and co - posting but serious clinical exercises for identifying specific ayush interventions, orienting medical personnel in their use, developing protocols and cross referral guidelines and such operational details . The ayush interventions have to be selected for health services at primary, secondary and tertiary levels . In parallel, an integrative public health system will simultaneously require radical reform in medical education, medical research, regulations, and the legal framework for medical practice . In the 21 century, an integrative model for public health needs a 10-year budget, a detailed action plan, and strategy, in order to achieve this complex goal . A second radical step toward modernization of health care in india is to invest in and use its heritage to restore two more traditionally available tiers at the bottom of the pyramid to enrich the health system and demonstrate the efficacy of a uniquely indian, participatory public health system . These community - based and supported layers were existing until the beginning of the 20 century and are still functioning in eroded fashion . They have been overlooked and neglected in india since the country embraced the western model of public health . They will add millions of new health providers to the public health system at zero recurring cost . These tiers are to be managed, as was the case for centuries, by millions of households and traditional community - based health workers . Traditionally, the indian households were carriers and providers of healthcare to the family . The household was a repository of region - specific, self - help health practices based on the use of ecosystem - specific natural resources . Till recently, the indian households possessed knowledge of at least a 100 home herbal remedies, nondrug health practices and food and nutrition . The homes had competence to manage common ailments, preventive health practices, and healthy ethnic diets . The creation of this household tier to the public health system will require critical investment in a creatively designed, information and communications technology enabled health education strategy, for reaching millions of rural and urban households . The second additional tier to be introduced in a modern indian healthcare system is also a noninstitutional tier managed by community - based and community supported traditional health workers . The country still has an estimated 1 million community supported traditional health workers viz ., mid - wives, herbalists, bonesetters, and vishavaidyas . The first step for restoration is to certify, accredit, and enrich the knowledge and skills of existing folk healers . Pilot experiments for certification and accreditation have already been demonstrated as recently as in 2013 . The community support base of the 1 million traditional health workers needs to be reinforced and care taken to avoid making them dependent on government support for their services to the community . The next step will be to motivate a new generation of folk healers to replace the older and ageing currently available generation . This is because while the government needs to invest in their revitalization, the action programs for developing these two tiers cannot be executed by the government ., a very sensitive and long - term strategy is needed to revitalize and modernize india's public health system and make it both integrative and much more participatory.
In rural australia, undersupply and uneven geographic distribution of the specialist medical workforce has produced an apparent gap between service demand and the availability of radiologists.1 as a result, the responsibility for interpreting radiographic images often falls provisionally to the referring doctor, commonly a general practitioner . Radiographic image interpretation involves highlevel skills and complex decision making, and there is a risk that errors may compromise patient care.2, 3, 4 although some degree of error in radiographic image interpretation is unavoidable,5 these errors may be reduced by interprofessional collaboration2, 6 between the referrer and the radiographer . Effective communication is integral to interprofessional collaboration and has the potential to positively impact on patient care.7, 8, 9 radiographers' opinions about the presence or absence of pathology on a radiographic image could be valuable in a collaborative approach to diagnosis.3, 6, 10, 11, 12 however, it is often difficult for radiographers to navigate this interprofessional boundary because communication pathways for sharing radiographic interpretations are unclear.11 in part, this is because radiographic image interpretation was effectively removed from radiographers' scope of practice in 1925.13 at this time, it was formally decreed that radiographic interpretation was diagnostic work and therefore the responsibility of medically qualified practitioners.13 resultantly, few radiographers are now educationally prepared for image interpretation,14 although they may possess experiential knowledge in recognising abnormalities.15 radiographers have attempted to address the communication gap using radiographer abnormality detection schemes (rads). One welldocumented rads is the red dot system,16 which was designed as a means for radiographers to alert the referring doctor to an abnormality on a patient's radiographic image . More than 30 years after its introduction, the red dot system is not consistently used across australia.17 communication difficulties which contribute to adverse events in healthcare may arise from the hierarchy of power that exists between different health professionals.7, 8, 18 it seems that historical changes to radiography have set in place a restrictive, hierarchical professional structure in which radiographers now practice.19, 20 a consequence of this structure is that autonomy within radiographer practice is discouraged by paternalism and subordination from medical colleagues.21 this is a characteristic of medical dominance, where the medical profession holds significant power and authority over neighbouring occupations.22, 23 whenever issues related to interprofessional boundary delineation come into question, such as radiographic image interpretation and communication of results, historical hierarchical relationships may influence and shape the way the interactions take place . Awareness of the barriers and enablers of communication in rural radiographic practice will enable a move towards stronger collaborative partnerships between radiographers and their medical colleagues . A twophase doctoral study was undertaken to examine australian rural radiographers' and their experiences and perceptions of radiographic interpretation and subsequent disclosure . This article reports the research findings of the interpretive component of the study that answers the research question, how do rural radiographers construct ways to negotiate communication and disclosure of their radiographic opinion within their practice world? The study, which employed both descriptive and interpretive research methods had ethics approval from the human research ethics committee (tasmanian) network and was completed in 2012 . The inclusion criteria were possession of an australian institute of radiography statement of accreditation, or registration with the medical radiation technologists board of western australia (prior to national registration) and providing radiographic services to an area with a population of less than 100,000 . The first phase of the study employed a postal questionnaire to collect descriptive data for development of a demographic profile of the respondent rural radiographers and to examine the context in which this study was situated . Prior to dissemination, the questionnaire was piloted, and its content and construct validity were established . It was distributed by third parties to radiographers practising in rural new south wales, western australia and tasmania . The quantitative questionnaire data was analysed using spss (ms windows, version 15, chicago, il, usa) to produce descriptive statistics . The second phase of the study used semistructured interviews to elicit rich, indepth qualitative data . Qualitative research methods are well suited to the examination of professional communication24 and allowed an interpretive inquiry focused on understanding meaning, purposes and intentions in how individuals act and interact with others.25 the interview cues were developed initially from the key issues arising from the questionnaire data and later from the analysis of the interview data . The qualitative interview data and openended questionnaire responses were analysed thematically with crosscomparison assisted by nvivo 8 software (qsr international, melbourne, australia) to code, organise, explore and analyse the interrelated themes . The data collection, analysis and interpretation occurred as a continuous process allowing emerging themes to influence further data collection . Themes were derived that illustrate and describe the dynamics of interprofessional communications between rural radiographers and referring doctors . Twentythree of the respondents also volunteered for the indepth, semistructured interviews via the invitation included with the questionnaire . The interviewing ceased once the collective experience of subsequent informants confirmed earlier findings rather than leading to a new insight, indicating that theoretical saturation was reached.26 all interviewees were experienced rural radiographers with an age range of 3662 (mean 53.4) years and 1642 (mean 32.9) years' radiographic experience . At the time of the interview, all the radiographers were employed in either private practice or public hospitals in communities with a population of less than 50,000 . Patient advocacy emerged as an overarching theme and the key driver for radiographers to use various communication pathways with referrers . A number of factors shape professional communication and interaction, including the perceived seriousness of the patient's condition, delays in diagnosis, the degree of professional confidence and the relationship between the radiographer and the referrer . Communication pathways were either direct or indirect, depending upon the various barriers to communication encountered . Interceding on behalf of the patient to maximise healthcare outcomes was an overarching feature of rural radiographer's professional role.rr1: that's how i have looked at it, what's best for the patient rr 9: going all out and doing what you can to help the management of those patients rr1: that's how i have looked at it, what's best for the patient rr 9: going all out and doing what you can to help the management of those patients the radiographers clearly regard their role to be more than technicians responsible for image acquisition: patients are central to all of their actions and decision making . It is patient care that incentivises radiographers to ensure timely and appropriate treatment for the patient in the presence of radiographic abnormalities.rr 4: i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film, particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . Rr 9: i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film, particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . Rr 9: i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see well i think you have to although radiographers are strongly committed to patient welfare, patient advocacy is not easy . Direct communication pathways are the most transparent and effective means for achieving the best outcome for the patient . Recognising the risk of error in radiographic interpretation, the radiographers used direct communication to ensure that the doctors do not miss significant pathology.rr 1: because they miss so much if i wasn't sure i wouldn't say anything but if there is a definite crack there that i am pretty sure the doctor's going to miss, it might show in one view, i will say something to them it's been a few political decisions i've had to make over the years if i wasn't sure i wouldn't say anything but if there is a definite crack there that i am pretty sure the doctor's going to miss, it might show in one view, i will say something to them it's been a few political decisions i've had to make over the years collaborative practice provides an avenue for the referring doctor to actively seek the radiographer's opinion.rr 3: i do get asked for my opinion quite a few times rr 6: in spite of the fact that we have got teleradiology and pacs [picture archiving and communication system] in the first instance it is always your own view that is canvassed . Rr 9: certainly in rural areas not only do you feel that you should, you are actually asked if you can . Rr 3: i do get asked for my opinion quite a few times rr 6: in spite of the fact that we have got teleradiology and pacs [picture archiving and communication system] in the first instance it is always your own view that is canvassed . Rr 9: certainly in rural areas not only do you feel that you should, you are actually asked if you can . In other situations, radiographer's volunteer an unsolicited opinion to the doctor.rr 2: i am quite happy to ring them and say look your patient has got a pleural effusion rr4: i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture rr 2: i am quite happy to ring them and say look your patient has got a pleural effusion i am quite happy to do that or a fracture rr4: i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture these collegial, open and direct communications are considered as a collaborative approach to reaching an accurate diagnosis, even if the referrer was inexperienced, as one participant describes.rr 5: rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them don't necessarily have the experience rr 5: rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them don't necessarily have the experience deciding whether to directly and explicitly communicate any radiographic abnormality to the referrer is influenced by the perceived seriousness of the impending diagnosis and the urgency with which care is required.rr 2: a bad fracture or something needs to be treated straight away [whereas] someone with advanced cancer or whatever, even if i told them at four o'clock in the afternoon the doctor can't do anything, can he? Rr 1: this guy came in and he said my neck's really sore i looked at the first film, oh, you've got a fracture here so i went out to him, and it was really unstable, and i said i've got to ring the doctor but don't move. Within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . Rr 2: a bad fracture or something needs to be treated straight away [whereas] someone with advanced cancer or whatever, even if i told them at four o'clock in the afternoon the doctor can't do anything, can he? Rr 1: this guy came in and he said my neck's really sore i looked at the first film, oh, you've got a fracture here so i went out to him, and it was really unstable, and i said i've got to ring the doctor but don't move. Within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . In situations where a patient required urgent attention and interprofessional relationships allow, radiographers assume a proactive role and communicated directly with the referrer . However, sometimes individual traits became barriers to direct communication pathways . For example: rr 3: it depends a bit on the referrer and their rapport with the radiographer, some will ask our opinions, others rr 6: there are those [doctors] that set themselves apart and are just completely unwilling to accept any advice from anybody rr 3: it depends a bit on the referrer and their rapport with the radiographer, some will ask our opinions, others rr 6: there are those [doctors] that set themselves apart and are just completely unwilling to accept any advice from anybody other radiographers spoke about the way in which different referrers react to their disclosure of radiographic opinion . For example, one radiographer stated: rr 2: he just wouldn't have it . That's an old fracture, that's not new, it's an old fracture. You don't know what you are talking about, i'm the doctor. So you have no comeback at that because, yes, they are the doctor . That's an old fracture, that's not new, it's an old fracture. You don't know what you are talking about, i'm the doctor. So you have no comeback at that because, yes, they are the doctor . The radiographers revealed a lack of formal training in recognising and describing pathology on radiographic images.rr 6: well, i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . Rr 5: our interpretations skills came from discussion with colleagues; we had sessions where we would interpret films, so most of our training was discussion with colleagues, with seniors over pathologies rr 6: well, i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . Rr 5: our interpretations skills came from discussion with colleagues; we had sessions where we would interpret films, so most of our training was discussion with colleagues, with seniors over pathologies one consequence of limited knowledge in image interpretation is that radiographers are reticent to offer their radiographic opinion to referrers and this worked to impede communication.rr 7: i don't feel comfortable with chests so i rarely say much with a chest . Rr 8: if it's something that you are unsure of you don't offer rr 7: i don't feel comfortable with chests so i rarely say much with a chest . Rr 8: if it's something that you are unsure of you don't offer despite the lack of formal educational preparation for radiographic interpretation, the radiographers described intuitive recognition of radiographic abnormalities.rr 4: the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . Rr 4: the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . Radiographers' intuitive recognition of radiographic abnormalities could be useful for assisting referrers in reaching a diagnosis but in some instances a lack of a confident radiographic interpretation inhibits their use of direct communication pathways . In order to circumvent the barriers to communication the radiographers used indirect communication pathways to navigate complex interprofessional boundaries . When circumstances do not support direct communication, the radiographers choose either sidestepping direct communication with the referrer or to use a hint and hope approach in order to ensure timely patient care . One way of sidestepping direct communication with the referrer is for the radiographer to alert a radiologist of the need for an urgent report . In doing so, that particular patient's images move towards the front of the reporting queue and the radiologist's report is provided to the referrer more quickly.rr 2: i have looked at the films and one of them had a massive pleural effusion on the right side, no air at all i put a priority on it because, i mean the man couldn't breathe for a start but also you knew that something was happening in there that needed investigation . I have looked at the films and one of them had a massive pleural effusion on the right side, no air at all i put a priority on it because, i mean the man couldn't breathe for a start but also you knew that something was happening in there that needed investigation . Assigning a radiograph a higher reporting priority, does not necessarily result in faster diagnosis and significant delays can still occur.rr 2: the more that you've been in practice, the more that you know that it's not a perfect world even if you put a priority on it, some of them get missed and the doctor's left for the day all those things happen because it's not a perfect world . Rr 2: the more that you've been in practice, the more that you know that it's not a perfect world even if you put a priority on it, some of them get missed and the doctor's left for the day all those things happen because it's not a perfect world . To minimise the potential negative impact on patient care that these delays may create, some of the radiographers used the red dot system to subtly sidestep direct communication with the referrers, but still provide radiographic interpretation input.rr 1: i got to the stage where i used to put red dots on them, if there was something [abnormal]. Rr 6: if there is an abnormality in the films then we will red dot them . Rr 1: i got to the stage where i used to put red dots on them, if there was something [abnormal]. Rr 6: if there is an abnormality in the films then we will red dot them . Laudable when first developed, the red dot system is not universally accepted or even well understood by other health professionals . It is therefore flawed as a communication system, as suggested by this excerpt.rr 1: but other doctors weren't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . Rr 1: but other doctors weren't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . In situations where sidestepping would not result in timely medical intervention and the radiographer and referrer were less familiar with each other, radiographers sometimes take a more subordinate, but supportive role . They avoid direct language, and instead hint their opinion and hope the referring doctor will detect the abnormality.rr 8: sort of give the referrer an opportunity by heading them in a direction and quietly saying, now don't you think that looks a bit extraordinary or something like that . I would say that doesn't look right to me, or what do you think about that, does that bulge look normal i would let them make the decision . Rr 8: sort of give the referrer an opportunity by heading them in a direction and quietly saying, now don't you think that looks a bit extraordinary or something like that . I would say that doesn't look right to me, or what do you think about that, does that bulge look normal i would let them make the decision . The radiographers also allow the referring doctor to initially use their own radiographic interpretation skills, but intercede if the abnormality is overlooked.rr 4: so, you would take your films around and you would go, i think you'd better look at this, and then you would stand back and let them look at it . And, then if they went round and round whatever what was obviously wrong, i would either say if it was an obvious fracture or something, i would be quite happy to offer an opinion and say, what do you think of that?, and then lead them in to it that way . Rr 4: so, you would take your films around and you would go, i think you'd better look at this, and then you would stand back and let them look at it . And, then if they went round and round whatever what was obviously wrong, i would either say if it was an obvious fracture or something, i would be quite happy to offer an opinion and say, what do you think of that?, and then lead them in to it that way . The hint and hope strategy appears to be effective when communicating information about the patients needs, but also works to maintain the historical hierarchical professional boundaries between the radiographer and the referrer . Interceding on behalf of the patient to maximise healthcare outcomes was an overarching feature of rural radiographer's professional role.rr1: that's how i have looked at it, what's best for the patient rr 9: going all out and doing what you can to help the management of those patients rr1: that's how i have looked at it, what's best for the patient rr 9: going all out and doing what you can to help the management of those patients the radiographers clearly regard their role to be more than technicians responsible for image acquisition: patients are central to all of their actions and decision making . It is patient care that incentivises radiographers to ensure timely and appropriate treatment for the patient in the presence of radiographic abnormalities.rr 4: i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film, particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . Rr 9: i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see i feel that it is also inherent in the responsibility of whichever position i am in that should i be aware of something that is an abnormality on a film, particularly after hours if there is no radiologist to refer it to i would need to bring that to the attention of the referrer . Rr 9: i feel that actually as a professional i feel it is my duty really sometimes to mention things that i see well i think you have to although radiographers are strongly committed to patient welfare, patient advocacy is not easy . Direct communication pathways are the most transparent and effective means for achieving the best outcome for the patient . Recognising the risk of error in radiographic interpretation, the radiographers used direct communication to ensure that the doctors do not miss significant pathology.rr 1: because they miss so much if i wasn't sure i wouldn't say anything but if there is a definite crack there that i am pretty sure the doctor's going to miss, it might show in one view, i will say something to them it's been a few political decisions i've had to make over the years if i wasn't sure i wouldn't say anything but if there is a definite crack there that i am pretty sure the doctor's going to miss, it might show in one view, i will say something to them it's been a few political decisions i've had to make over the years collaborative practice provides an avenue for the referring doctor to actively seek the radiographer's opinion.rr 3: i do get asked for my opinion quite a few times rr 6: in spite of the fact that we have got teleradiology and pacs [picture archiving and communication system] in the first instance it is always your own view that is canvassed . Rr 9: certainly in rural areas not only do you feel that you should, you are actually asked if you can . Rr 3: i do get asked for my opinion quite a few times rr 6: in spite of the fact that we have got teleradiology and pacs [picture archiving and communication system] in the first instance it is always your own view that is canvassed . Rr 9: certainly in rural areas not only do you feel that you should, you are actually asked if you can . In other situations, radiographer's volunteer an unsolicited opinion to the doctor.rr 2: i am quite happy to ring them and say look your patient has got a pleural effusion i am quite happy to do that or a fracture rr4: i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture rr 2: i am quite happy to ring them and say look your patient has got a pleural effusion i am quite happy to do that or a fracture rr4: i will always preface it with this is the radiographer speaking the radiologist report will be out shortly but in my opinion they have a fracture these collegial, open and direct communications are considered as a collaborative approach to reaching an accurate diagnosis, even if the referrer was inexperienced, as one participant describes.rr 5: rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them don't necessarily have the experience rr 5: rather than offering a diagnosis i think it is largely to do with discussion and because a lot of the well a lot of the a&e doctors that come through are locums and a lot of them don't necessarily have the experience deciding whether to directly and explicitly communicate any radiographic abnormality to the referrer is influenced by the perceived seriousness of the impending diagnosis and the urgency with which care is required.rr 2: a bad fracture or something needs to be treated straight away [whereas] someone with advanced cancer or whatever, even if i told them at four o'clock in the afternoon the doctor can't do anything, can he? Rr 1: this guy came in and he said my neck's really sore i looked at the first film, oh, you've got a fracture here so i went out to him, and it was really unstable, and i said i've got to ring the doctor but don't move. Within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . Rr 2: a bad fracture or something needs to be treated straight away [whereas] someone with advanced cancer or whatever, even if i told them at four o'clock in the afternoon the doctor can't do anything, can he? Rr 1: this guy came in and he said my neck's really sore i looked at the first film, oh, you've got a fracture here so i went out to him, and it was really unstable, and i said i've got to ring the doctor but don't move. Within about 5 minutes there were about 12 people in the room trying to get him from sitting up to lying down because we had to put collars on he had a halo on two days later and was walking around again . In situations where a patient required urgent attention and interprofessional relationships allow, radiographers assume a proactive role and communicated directly with the referrer . Individual perceptions of role delineation within the health professions influenced radiographer and referrer interaction . For example: rr 3: it depends a bit on the referrer and their rapport with the radiographer, some will ask our opinions, others they look for other doctors around the hospital to discuss it with . Rr 6: there are those [doctors] that set themselves apart and are just completely unwilling to accept any advice from anybody rr 3: it depends a bit on the referrer and their rapport with the radiographer, some will ask our opinions, others they look for other rr 6: there are those [doctors] that set themselves apart and are just completely unwilling to accept any advice from anybody other radiographers spoke about the way in which different referrers react to their disclosure of radiographic opinion . For example, one radiographer stated: rr 2: he just wouldn't have it . That's an old fracture, that's not new, it's an old fracture. You don't know what you are talking about, i'm the doctor. So you have no comeback at that because, yes, they are the doctor . That's an old fracture, that's not new, it's an old fracture. You don't know what you are talking about, i'm the doctor. So you have no comeback at that because, yes, they are the doctor . The radiographers revealed a lack of formal training in recognising and describing pathology on radiographic images.rr 6: well, i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . Rr 5: our interpretations skills came from discussion with colleagues; we had sessions where we would interpret films, so most of our training was discussion with colleagues, with seniors over pathologies rr 6: well, i suppose you could say it is selftaught really but you just accumulate knowledge over a period of time . Rr 5: our interpretations skills came from discussion with colleagues; we had sessions where we would interpret films, so most of our training was discussion with colleagues, with seniors over pathologies one consequence of limited knowledge in image interpretation is that radiographers are reticent to offer their radiographic opinion to referrers and this worked to impede communication.rr 7: i don't feel comfortable with chests so i rarely say much with a chest . Rr 8: if it's something that you are unsure of you don't offer rr 7: i don't feel comfortable with chests so i rarely say much with a chest . Rr 8: if it's something that you are unsure of you don't offer despite the lack of formal educational preparation for radiographic interpretation, the radiographers described intuitive recognition of radiographic abnormalities.rr 4: the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . Rr 4: the more tricky ones are the abnormal chests and so on that you know are abnormal but my knowledge is limited as to why they are abnormal . Radiographers' intuitive recognition of radiographic abnormalities could be useful for assisting referrers in reaching a diagnosis but in some instances a lack of a confident radiographic interpretation inhibits their use of direct communication pathways . In order to circumvent the barriers to communication sidestepping direct communication with the referrer or to use a hint and hope approach in order to ensure timely patient care . One way of sidestepping direct communication with the referrer is for the radiographer to alert a radiologist of the need for an urgent report . In doing so, that particular patient's images move towards the front of the reporting queue and the radiologist's report is provided to the referrer more quickly.rr 2: i have looked at the films and one of them had a massive pleural effusion on the right side, no air at all i put a priority on it because, i mean the man couldn't breathe for a start but also you knew that something was happening in there that needed investigation . I have looked at the films and one of them had a massive pleural effusion on the right side, no air at all i put a priority on it because, i mean the man couldn't breathe for a start but also you knew that something was happening in there that needed investigation . Assigning a radiograph a higher reporting priority, does not necessarily result in faster diagnosis and significant delays can still occur.rr 2: the more that you've been in practice, the more that you know that it's not a perfect world even if you put a priority on it, some of them get missed and the doctor's left for the day all those things happen because it's not a perfect world . Rr 2: the more that you've been in practice, the more that you know that it's not a perfect world even if you put a priority on it, some of them get missed and the doctor's left for the day all those things happen because it's not a perfect world . To minimise the potential negative impact on patient care that these delays may create, some of the radiographers used the red dot system to subtly sidestep direct communication with the referrers, but still provide radiographic interpretation input.rr 1: i got to the stage where i used to put red dots on them, if there was something [abnormal]. Rr 6: if there is an abnormality in the films then we will red dot them . Rr 1: i got to the stage where i used to put red dots on them, if there was something [abnormal]. Rr 6: if there is an abnormality in the films then we will red dot them . Laudable when first developed, the red dot system is not universally accepted or even well understood by other health professionals . It is therefore flawed as a communication system, as suggested by this excerpt.rr 1: but other doctors weren't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . Rr 1: but other doctors weren't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . In situations where sidestepping would not result in timely medical intervention and the radiographer and referrer were less familiar with each other, radiographers sometimes take a more subordinate, but supportive role . They avoid direct language, and instead hint their opinion and hope the referring doctor will detect the abnormality.rr 8: sort of give the referrer an opportunity by heading them in a direction and quietly saying, now don't you think that looks a bit extraordinary or something like that . I would say that doesn't look right to me, or what do you think about that, does that bulge look normal i would let them make the decision . Rr 8: sort of give the referrer an opportunity by heading them in a direction and quietly saying, now don't you think that looks a bit extraordinary or something like that . I would say that doesn't look right to me, or what do you think about that, does that bulge look normal i would let them make the decision . The radiographers also allow the referring doctor to initially use their own radiographic interpretation skills, but intercede if the abnormality is overlooked.rr 4: so, you would take your films around and you would go, i think you'd better look at this, and then you would stand back and let them look at it . And, then if they went round and round whatever what was obviously wrong, i would either say if it was an obvious fracture or something, i would be quite happy to offer an opinion and say, what do you think of that?, and then lead them in to it that way . Rr 4: so, you would take your films around and you would go, i think you'd better look at this, and then you would stand back and let them look at it . And, then if they went round and round whatever what was obviously wrong, i would either say if it was an obvious fracture or something, i would be quite happy to offer an opinion and say, what do you think of that?, and then lead them in to it that way . The hint and hope strategy appears to be effective when communicating information about the patients needs, but also works to maintain the historical hierarchical professional boundaries between the radiographer and the referrer . One way of sidestepping direct communication with the referrer is for the radiographer to alert a radiologist of the need for an urgent report . In doing so, that particular patient's images move towards the front of the reporting queue and the radiologist's report is provided to the referrer more quickly.rr 2: i have looked at the films and one of them had a massive pleural effusion on the right side, no air at all i put a priority on it because, i mean the man couldn't breathe for a start but also you knew that something was happening in there that needed investigation . I have looked at the films and one of them had a massive pleural effusion on the right side, no air at all i put a priority on it because, i mean the man couldn't breathe for a start but also you knew that something was happening in there that needed investigation . Assigning a radiograph a higher reporting priority, does not necessarily result in faster diagnosis and significant delays can still occur.rr 2: the more that you've been in practice, the more that you know that it's not a perfect world even if you put a priority on it, some of them get missed and the doctor's left for the day all those things happen because it's not a perfect world . Rr 2: the more that you've been in practice, the more that you know that it's not a perfect world even if you put a priority on it, some of them get missed and the doctor's left for the day all those things happen because it's not a perfect world . To minimise the potential negative impact on patient care that these delays may create, some of the radiographers used the red dot system to subtly sidestep direct communication with the referrers, but still provide radiographic interpretation input.rr 1: i got to the stage where i used to put red dots on them, if there was something [abnormal]. Rr 6: if there is an abnormality in the films then we will red dot them . Rr 1: i got to the stage where i used to put red dots on them, if there was something [abnormal]. Rr 6: if there is an abnormality in the films then we will red dot them . Laudable when first developed, the red dot system is not universally accepted or even well understood by other health professionals . It is therefore flawed as a communication system, as suggested by this excerpt.rr 1: but other doctors weren't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . Rr 1: but other doctors weren't interested in it and they would send a fractured tibial plateau home and i had a red dot on the film this particular patient with the red dot and the tibial plateau fracture that was missed for 6 weeks his knee is stuffed . In situations where sidestepping would not result in timely medical intervention and the radiographer and referrer were less familiar with each other, radiographers sometimes take a more subordinate, but supportive role . They avoid direct language, and instead hint their opinion and hope the referring doctor will detect the abnormality.rr 8: sort of give the referrer an opportunity by heading them in a direction and quietly saying, now don't you think that looks a bit extraordinary or something like that . I would say that doesn't look right to me, or what do you think about that, does that bulge look normal i would let them make the decision . Rr 8: sort of give the referrer an opportunity by heading them in a direction and quietly saying, now don't you think that looks a bit extraordinary or something like that . I would say that doesn't look right to me, or what do you think about that, does that bulge look normal i would let them make the decision . The radiographers also allow the referring doctor to initially use their own radiographic interpretation skills, but intercede if the abnormality is overlooked.rr 4: so, you would take your films around and you would go, i think you'd better look at this, and then you would stand back and let them look at it . And, then if they went round and round whatever what was obviously wrong, i would either say if it was an obvious fracture or something, i would be quite happy to offer an opinion and say, what do you think of that?, and then lead them in to it that way . Rr 4: so, you would take your films around and you would go, i think you'd better look at this, and then you would stand back and let them look at it . And, then if they went round and round whatever what was obviously wrong, i would either say if it was an obvious fracture or something, i would be quite happy to offer an opinion and say, what do you think of that?, and then lead them in to it that way . The hint and hope strategy appears to be effective when communicating information about the patients needs, but also works to maintain the historical hierarchical professional boundaries between the radiographer and the referrer . Radiographers can play a valuable role in helping rural doctors to correctly interpret radiographic images . While this role may be familiar to most rural radiographers,10 the communication pathways for interprofessional collaboration are not well defined or standardised . This interpretive inquiry has revealed that the evolution of radiography under the medical dominance model21, 23 has influenced both radiographers preparation for interprofessional radiographic interpretation and impacted on their communication strategies . Radiographic image interpretation is intricately linked to diagnosis, and as a result, radiographers have not historically been formally educated and trained in identifying and describing pathology on radiographs . This leaves a gap in their knowledge, skills and abilities, which leads to a lack of confidence.14 furthermore, unequal power relationships may cause those positioned at the base of the hierarchical pyramid, such as radiographers, to remain silent.22 a flattened hierarchy enables individuals to voice their opinion,8 as is evidenced by radiographers use of direct communication pathways . This study indicates that in some cases, a perceived or evidenced hierarchy remains and, along with a lack of preparation for a role in radiographic interpretation, this has the ability to stifle interprofessional communication . Of concern is that as a consequence radiographers tend to enter into a radiographer nurse game,7, 27 which is an interprofessional communication strategy where doctors are overtly or covertly guided in their clinical decisions by nurses . In the same way that a nurse may make suggestions to guide the doctor's decision making, the radiographers, within the limitations of their knowledge base, use mechanisms designed to directly or indirectly communicate their radiographic opinion, influencing the subsequent radiographic diagnosis . As a patient advocate workaround strategies to communicate their opinion to the referring doctor and so engage in the radiographer in australia, the educational preparation of radiographers for the higher level competencies required in image interpretation and communication of their radiographer's opinion is currently ad hoc . This limits radiographers capacity to contribute to patient care and also limits the capacity of the health system to provide higher quality care through interprofessional teamwork . It is essential for safe care that communication is open, direct and transparent7, so that health professional, such as radiographers, do not feel the need to enter into game playing . Improving radiographers' image interpretation skills, and making the communication pathways between radiographers and referrers more explicit, will positively impact on patient care through the timely collegial sharing of knowledge.
The dental industry has a long history in the development of dental prostheses to recover a patient's tooth function.1 as a substitute for teeth, a dental prosthesis must show stable durability, aesthetic value, precise function, and convenient use, as well as biocompatibility in order to perform the desired function properly . In addition, these factors must be applied to a wide range of manufacturing methods used in the construction of dental prostheses.2 metal ceramic is a very common material used worldwide, and it has been successfully used as the gold standard for long - term clinical use; it provides excellent results in stability, aesthetic value, and marginal adaptation.345 in recovery using a dental prosthesis, marginal adaptation is an important factor.3 an inappropriate margin could cause a minute gap between the abutment tooth and prosthesis, which may lead to a periodontal lesion, plaque accumulation, secondary caries, microleakage, inflammation after endodontic treatment, or periodontal disease.5678 in addition, according to previous research, a defective margin may cause a failure of the long - term preservation of the prosthesis, resulting in an increase in the failure rate.9 conventional fabrication methods for a prosthesis is a series of processes that includes taking an impression of the patient's oral cavity, pouring stone, producing a wax pattern, and performing the investing, casting, and polishing . However, during this process, the risk of inaccuracy may increase due to the properties of the material used and the worker's ability . In addition, temporal labor and cost could increase as well.1345 therefore, to address these problems, an automated cad / cam system was introduced to the dental field.15 the cad / cam system is a type of subtractive manufacturing that cuts the materials to the desired shape and size . It enables a larger quantity of production than traditional methods, is easy to use, and saves the time . Because of these advantages, the cad / cam system is widely used.5 however, bornemann et al.10 showed that this system tends to reduce accuracy through the scanning process, software design, milling, and a number of other related processes . This results in too much consumption of raw material, and the waste of bur was increased . Accordingly, the additive manufacturing (am) method, which supplements labor - intensive conventional manufacturing methods and subtractive manufacturing methods with high raw material consumption, is being considered a technology - intensive alternative in the field . Multi - jet modeling, an additive manufacturing process used in the dental field, is the 3d printer, which is very advantageous in terms of manufacture speed and applicability with various materials compared with other 3d printings, as it has a number of jet nozzles.11 as a very professional 3d printer, a newly - launched additive manufacturing process " micro - sla " is characterized by high accuracy, and thanks to its minute ability in realization it is more appropriate for manufacturing the dental prosthesis than any other printers . Also, compared to the other 3d printings, it is cheaper and speedy printing (14 mm / hour on the basis of the vertical standard) is possible to shorten the time required . As a disruptive technology, am has the potential to revolutionize our lives, work, and international economy.12 only a few companies are applying am to dentistry, and, therefore, there are a limited number of studies done in this field . Identifying the limits and advantages of this manufacturing method is an important task in the prosthesis and dentistry fields . Thus, the purpose of this study was to verify whether the marginal and internal gap of a prosthesis made according to the am method is within the clinically allowable range by conducting a comparative evaluation of the conventional lost wax technique (clwt), the subtractive manufacturing system with wax blank milling (wbm), and am with multi jet modeling (mjm) and micro - stereolithography (micro - sla). The null hypothesis was that there is no difference in the marginal and internal gap among the 4 groups . An acrylic model (standard working model ag-3 zpvk 13, 14, 16, frasaco gmbh, tettnang, germany) with abutment teeth was used . Therefore, the maxillary right canine, first pre - molar, first molar were provided with a 360 1.0 mm chamfer preparation.13 the incisal and occlusal reductions were 1.5 - 2.0 mm . The maxillary right canine, first pre - molar, first molar were reproduced using duplication silicone (deguform, degudent gmbh, hanau, germany). In the reproduced area, the epoxy model (master model) was reproduced by pouring the epoxy (modralit 3k, dreve dentamid gmbh, unna, germany). For the reproduced epoxy model, 10 plaster molds for each tooth were produced using duplication silicone, and, as a result, a total of 30 molds were produced . After filling the plaster molds with type iv stone (dentona esthetic - base gold; dentona ag, dortmund, germany), a total of 30 study models were produced . The study models were scanned by a non - contact blue light scanner (identica; medit co., ltd ., based on the scanned files, a metal framework was designed by delcam power shape pro (delcam plc, birmingham, uk) according to the manufacturer's instruction, with the following parameters (thickness): 30 m for the cement film, 0.3 mm for the maxillary right canine, and 0.5 mm for the maxillary right first pre - molar and first molar . From this design, a standard template library (stl) file was created . For the clwt method, the lost wax technique was applied . After applying separating medium onto the study models and passing through the wax dipping process, each of the 10 abutment teeth, 30 in total, for the wbm method, the 30 wax patterns were produced, based on the stl files, using the cad / cam system (dwx-50, roland dg corporation, shizuoka, japan) and milling the wax blank (dmax co., ltd .,, the 30 resin patterns were produced using the mjm printer (projet - dp3000, 3d system, rock hill, sc, usa) and the stl files by jetting the light curing resin (build material visijet dp200, visijet, 3d system, rock hill, sc, usa) and wax (support material visijet s100, visijet, 3d system, rock hill, sc, usa) simultaneously through the inkjet print heads . Finally, for the micro - sla method, the 30 resin patterns were produced using a micro - sla printer (projet1200, 3d systems, rock hill, sc, usa) and the stl files, by projecting the desired metal framework via the beam projector onto the liquid uv curable plastic (visijet ftx green material, 3d systems, rock hill, sc, usa). The test piece was mounted on the uv curing station and photopolymerized for 10 minutes . The patterns were placed in the crucible former, covered with the metal ring, and invested in accordance with the proper water / powder ratio through phosphate - bonded investment (deguvest - impact - degussa - hls, hanau, germany). After passing through the burnout furnace (ring furnace, seki dental co., seoul, korea), the nickel - chromium (ni - cr) alloy (verabond 2v; aalbadent inc ., fairfield, ca, usa) was cast in the casting machine (seki dental co., seoul, korea), and each test piece was produced (fig ., skilled dental technician has used a silicone replica method . After mixing the light - body silicone (aquasil ultra xlv regular set, dentsply caulk, milford, de, usa), it was injected between the metal framework and model, and 50 n of finger pressure14151617 was applied . After hardening, the metal framework was separated carefully from the model, and heavy - body silicone (aquasil ultra rigid regular set, dentsply caulk, milford, de, usa) was injected into the circular tray so that the light - body and heavy - body silicone could combine through an even pressure, and embed the light - body silicone . In order to cut the equal part, the zig (modralit 3k, dreve dentamid gmbh, unna, germany) was made by duplicating each two epoxy models, cutting one in a bucco - lingual direction, and cutting the other in a mesio - distal direction . The silicone replica reproduced by using this method was cut in a bucco - lingual direction and mesio - distal direction, respectively, using a razor blade (fig . The thickness of the light - body silicone replica, which corresponds to the gap between model and the metal framework, was measured using a digital microscope (kh-7700; hirox, tokyo, japan) at 140 magnification . The margin gaps (mgs), which corresponded to the absolute marginal discrepancy and internal gap, rounded chamfer (rc), axial wall (aw), incisal area (ia), and occlusal area (oa) were measured . To confirm an accurate measurement, the measurement points (16 in total) 3). The total gap and the mean and standard deviation of the 16 points were determined, and this data met the hypothesis of a normal distribution (p>.01). The 16 points were divided into 4 regions as follows: mg, points 1, 8, 9, 16; rc, points 2, 7, 10, 15; aw, points 3, 6, 11, 14; and ia or oa, points 4, 5, 12, 13 . After each region's mean and standard deviation were determined, two - way analysis of variance (anova) was conducted to evaluate the difference in average values according to tooth type and fabrication method . As the reciprocal action between the tooth variable and fabrication method variable was significant (p<.05), this analysis allowed verification of the significance of the difference between groups as a full factorial model . The level of the type i - error for statistical significance was fixed at 0.05, and the statistical analysis was conducted by using ibm spss statistics 21.0 (ibm co., armonk, ny, usa). Results of the analysis of the marginal and internal gap according to tooth type and fabrication methods are listed as the mean and standard deviation (table 1). The mean value of mg, rc, aw, ia or oa according to the four fabrication methods and the three tooth types are shown in figures 4 and 5 . As a result of the two - way anova analysis of the means, there was a significant difference in mg according to tooth type (p<.001) and fabrication method (p<.037). In addition, there was a significant interaction (p<.001) between tooth type and fabrication method . Further, there were significant differences in rc, aw, and ia or oa according to tooth type (p<.001) and fabrication method (p<.001), with significant interaction (p<.001, table 2) between tooth type and fabrication method . The results of the post - hoc test using the tukey hsd method (table 2, fig . 5) indicate that mg was the lowest for the molar type fabricated using the clwt method, and was the highest for the canine type fabricated using the mjm method . In the case of rc, the canine type manufactured using the clwt method had the lowest value, while the molar type manufactured using the mjm method had the highest value . In aw, the premolar type fabricated using the wbm method showed had the lowest value, while the molar type fabricated using the clwt method had the highest value . Lastly in the case of ia or oa, the canine type fabricated using the clwt method had the lowest value, while the molar type fabricated using the mjm method had the highest value . To assess the results measured at individual points (16 points), results were classified into four regions: mg, rc, aw, and ia or oa . In mg, the canine and molar types showed the lowest value when fabricated using the clwt method, and the premolar type showed the lowest value using the micro - sla method . In the case of rc, the canine type showed the lowest value using the clwt method, while premolar and molar types showed the lowest value using the micro - sla method . In case of aw, the lowest value of the canine type was shown using the clwt method, while the lowest values for the premolar type and molar type were shown using the wbm method and the mjm method, respectively . In the case of ia or oa, the canine type showed the lowest value using the clwt method, while the premolar and molar type showed the lowest value using the micro - sla method (table 2, fig . 4, fig . 5). In addition, a whole am, mg, ia or oa, and rc showed a lower value (fig . This study evaluated the marginal and internal gaps according to four fabrication methods, in order to verify the applicability of am in dentistry . Clwt and micro - sla did not show a significant difference in the marginal gap, but, since these fabrication methods are significantly different from the other two methods, the null hypothesis was rejected . As shown in figures 4 and 5, the values increased in the following order: aw, mg, ia or oa, and rc . Because the abutment tooth was parallel to prosthetic appliance, aw showed a low value . However, because the occlusal surface has an irregular curve, ia or oa showed a relatively higher value compared to mg . Rc had the highest value because the shape of the margin forms a rounded chamber . According to the table 2, the marginal gap using either clwt or micro - sla was better than that using the other two methods . In other studies, clwt has been shown to achieve the most suitable marginal - adaptation value, and, therefore, clwt has been designated as the gold standard.5 however, considering that the micro - sla method shows no significant difference from the clwt method in the marginal gap, we infer that the micro - sla method has a better fitness value than the wbm and mjm methods . In the wbm method, it is difficult to reproduce the projection part, undercut part, and sharp edge accurately due to the positive error, as well as negative error resulting from the limits of the currently available bur diameters.216 in spite of the advantages of the mjm method in delicacy and precision, it combines wax and thermoset material in the fabrication process . Thus, the mjm method has several drawbacks such as weak solidity among the 3d printers and its deformation at high temperatures.12 as a modified method of digital light processing, the newly released micro - sla method projects a shaped light beam on the liquid photopolymer resin, hardens the resin as projected, builds the model layer by layer, and then, hardens the built shape by exposure to light again in the built - in uv curing station . The advantage of this method is that the manufacturing speed is even as the model forms and is comparatively fast . The micro - sla method shows high precision and surface roughness since the layers are applied at a thickness of 30 m . The sls method and sla method are currently the most widely used am method.17 according to the research relating to the stereolithography (sla) method, the mean (sd) of margin, axial wall, occlusal are 96.9 m (17.6 m), 84.7 m (16.8 m), 114.2 m (16.7 m) respectively.17 and as a result of the research, mjm method and micro - sla method showed better fitness value, except the occlusal part in the current study . According to the report of rtorp et al.,16 the mean (sd) value of the sls showed the best fitness value, with the measured value 133 m (89 m), 117 m (89 m), 166 m (135 m), and 84 m (60 m), at all measurement points in the conventional lost wax method, milled wax method, milled co - cr, and sls method . However, according to the earlier research, the mean (sd) value of the mg (absolute marginal discrepancy) part of premolar and molar made in the sls method showed 132.1 m (60.5 m) and 128.0 m (68.8 m) each . And it was found that the sls method has worse fitness than the mjm method and micro - sla method, showing the higher values than those of both methods used in the current study.15 also, kim et al.15 reported that the efforts to improve the sls method will be required since the current sls method is highly inferior to the conventional lost wax technique method and even has the gaps beyond the clinically allowable range . According to previous studies, no difference is observed in the marginal and internal gap between anterior, premolar, and molar teeth.1819 however, nakamura et al.20 showed that there are differences according to the tooth type . Our results on the canine, premolar, and molar teeth (table 2, fig . 4) indicate a difference in the marginal and internal gap according to tooth type . This is significant because each tooth type has a different morsal surface condition and appearance, although there is the uniformity in the tamper degree and chamfer margin . There are several ways to measure the fitness of a prosthesis, including a direct measurement after the cementation process of the prosthesis on the tooth model21 or observing the inside of the prosthesis using x - ray micro - computed tomography (micro ct).2223 in the present study, we used the cross - sectioning replica technique with silicone, which is considered the most suitable method to measure prosthesis prior to cementation.24 according to previous studies, there is much controversy as to the clinical validity of the size of the margin adaptation5 . For example, fransson et al.25 reported that the clinically allowable range is 100 m, and the value suggested by mclean and von fraunhofer26 and belser et al.27 is 120 m . Beuer et al.28 stated that the size of the marginal adaptation ranges from 100 to 150 m and boening et al.29 suggested the range is from 100 to 200 m based on the long - term preserved prosthesis . In this study, all of the mg results showed values within the clinically allowable range suggested by the preceding studies (table 2). In addition, the values for rc, aw, ia or oa were also within the clinically allowable range (table 1 and table 2). Therefore, all the four methods can be used clinically . A limitation to this study is that there could be an error in the resin pattern and wax pattern due to the characteristic contraction of the material itself . Thus, the development of suitable materials should be included in future studies, and, especially in the case of the am methods, further clinical studies should be conducted . All of the four fabrication methods have sufficient marginal adaptation, since the marginal and internal gaps were within the allowable clinical range . The results of the micro - sla method showed a statistically significant difference in outcome compared to the wbm and mjm methods and a significant difference from the gold standard clwt method in the internal gap, but no statistically significant difference in the marginal gap from the clwt method . Together, our results demonstrate that additive manufacturing can be used clinically as an alternative to the conventional lost wax - technique or subtractive manufacturing in the creation of dental prostheses.
Nasopharyngeal carcinoma (npc) is a nonlymphomatous squamous cell carcinoma that occurs in the epithelial lining of the nasopharynx . This neoplasm shows varying degrees of differentiation and is frequently seen in the pharyngeal recess (rosenmller's fossa), posteromedial to the medial crura of the eustachian tube opening in the nasopharynx . Npc is a distinct form of head and neck cancer that differs from other malignancies of the upper aerodigestive tract in terms of its etiology, epidemiology, pathology, clinical presentation, and response to treatment . Outside of endemic areas in southeast asia, npc is rare, occurring in less than 1/1,000,000 people . In north america, npc accounts for approximately 0.2% of all malignancies, with approximately 0.52 cases per 100,000 males and about one - third of that in females [46]. The incidence of npc reportedly remains high among chinese people who have emigrated to southeast asia or north america, but is lower among chinese people born in north america than in those born in southern china [7, 8]. This finding suggests that genetic as well as environmental factors play a role in the cause of the disease . The mainstay of npc treatment is radiotherapy, but treatment results for advanced npc is not satisfactory . The focus of this review is to provide an overview of npc, especially the recent insights regarding early detection of npc . It accounts for 2% of all head and neck squamous cell carcinomas, with an incidence of 0.5 to 2 per 100,000 in the united states . However, it is endemic in many geographical regions, including southern china, southeast asia, japan, and the middle east / north africa [10, 11]. Ho reported that npc is the third most common malignancy among men, with an incidence of between 50 per 100,000 in the guangdong province of southern china . Emigration from high- to low - incidence areas such as the united states and canada reduces the incidence of npc in first - generation chinese, but it still remains at seven - times the rate in caucasians . Npc presents as a complex disease caused by an interaction between chronic infection with oncogenic gamma herpesvirus epstein - barr virus (ebv) and environmental and genetic factors, involving a multistep carcinogenic process . Ebv exists worldwide, infecting over 95% of the global adult population . In hong kong, 80% of children are infected by 6 years of age, and almost 100% have seroconverted by 10 years of age . Although primary ebv infection is typically subclinical, the virus is associated with the later development of several malignancies, including npc . It is transmitted by saliva, and its primary infection occurs during childhood with replication of the virus in the oropharyngeal lining cells, followed by a latent infection of b lymphocytes (primary target of ebv). Elevated titers of ebv - associated antigens (especially of iga class), a latent ebv infection indentified in neoplastic cells of virtually all cases of npc, and the clonal ebv genome consistently detected in invasive carcinomas and high - grade dysplastic lesions suggest a critical role of ebv in the pathogenesis of npc in endemic areas . Nonviral exposure associated with the risk of npc involves the consumption of salt - preserved fish, a traditional staple food in several npc - endemic areas . In studies of chinese populations, the relative risk of npc associated with weekly consumption, compared with no or rare consumption, generally ranged from 1.4 to 3.2 per 100,000 whereas that for daily consumption ranged from 1.8 to 7.5 [1522]. Thus, this dietary staple pattern may explain part of the international distribution of npc incidence . The carcinogenic potential of salt - preserved fish is supported by experiments in rats, which develop malignant nasal and nasopharyngeal tumors after salted fish consumption [18, 24, 25]. The process of salt preservation is inefficient, allowing fish and other foods to become partially putrefied . As a result, these foods accumulate significant levels of nitrosamines, which are known carcinogens in animals [23, 26, 27]. Salt - preserved fish also contain bacterial mutagens, direct genotoxins, and ebv - reacting substances [2830], any or all of which could also contribute to the observed association . However, there have been no prospective studies of npc risk associations with salt - preserved fish consumption, or virtually any other environmental exposure, in endemic areas . Several associations have been described between the frequency of human leukocyte antigen (hla) class i genes in certain populations and the risk of developing npc . For example, increased risk of npc was observed in individuals with the hla - a2 allele, particularly hla - a0207 . Recent genome - wide association studies confirmed involvement of hla molecules in npc generation [32, 33]. Cellular gene alterations also contribute to development of npc, especially inactivation of tumor suppressor genes, splunc1, ubap1, brd7, nor1, ngx6, and ltf . In 1978, the histological classification guideline proposed by the world health organization (who) categorized npc into three groups: type 1 (keratinizing squamous cell carcinoma), type 2 (nonkeratinizing carcinoma), and type 3 (undifferentiated carcinoma). The 1991 who classification of nasopharyngeal carcinoma divided them into two groups: squamous cell carcinoma (keratinizing squamous cell carcinoma, type 1 of the former classification), and nonkeratinizing carcinoma (types 2 and 3 of the former classification combined into a single category). This classification is more applicable for epidemiological research and has also been shown to have a prognostic significance . Undifferentiated carcinomas have a higher local tumor control rate with treatment and a higher incidence of distant metastasis than do differentiated carcinomas [36, 37]. Published data indicate a higher proportion of keratinizing squamous cell carcinoma among all npc in nonendemic compared with endemic areas . Some studies reported that squamous cell carcinoma accounts for approximately 25% of all npc in north america, but only 1% in endemic areas; whereas undifferentiated carcinoma accounts for 95% of all cases in high - incidence areas, but 60% of cases in north america [9, 10, 38]. The control rate on conventional radiotherapy is 75 to 90% in t1 and t2 tumors, and 50 to 75% in t3 and t4 tumors . Because of the high incidence of occult cervical node metastasis, the control of cervical nodal regions is achieved in 90% of n0 and n1 cases, and about 70% of n2 and n3 cases . It is mandatory to keep the treatment schedule because interrupted or prolonged treatment reduces the benefits of radiotherapy . Recent studies have suggested that addition of chemotherapy to radiotherapy improves the treatment results in patients with nasopharyngeal carcinoma . Phase iii randomized intergroup study 0099 showed that patients treated with radiation alone had a significantly lower 3-year survival rate than those receiving radiation with cisplatin and 5-fluorouracil chemotherapy . A meta - analysis of chemotherapy for npc conducted by baujat et al . They reported a definite improvement of the 5-year survival rate due to the addition of chemotherapy (56% with radiotherapy alone versus 62% with chemoradiotherapy). In addition to these findings, other phase iii or meta - analysis studies also reported the superiority of concurrent chemoradiotherapy versus radiotherapy alone [4446]. The above - described reports suggest the benefits of the addition of chemotherapy, especially in advanced npc cases . However, there is still debate on the effectiveness of the addition of chemotherapy, and issues regarding the addition of adjuvant chemotherapy are even more controversial . Wei and sham divided symptoms presented by npc patients into four categories: (1) symptoms caused by the presence of a tumor mass in the nasopharynx (epistaxis, nasal obstruction, and discharge), (2) symptoms associated with dysfunction of the eustachian tube (hearing loss), (3) symptoms associated with the superior extension of the tumor (headache, diplopia, facial pain, and numbness), and (4) neck masses . Because symptoms related to npc in the early stage are usually nonspecific, most npc patients are diagnosed in the advanced stage . As treatment results for npc are not satisfactory in the advanced stage, early diagnosis and appropriate management are important to achieve favorable treatment results . The development of a good primary npc screening protocol may thus contribute to the early detection and improve the treatment outcome . The endemic form of npc is associated with ebv, although the exact role of ebv in the pathogenesis of npc remains unclear . Iga antibody titers to ebv viral capsid antigen (ebv - iga - vca) and ebv early antigen (ebv - ea) in immunofluorescent assays may be used for the serologic screening of npc [47, 48]. In recent years, enzyme - linked immunosorbent assays (elisa) employing purified recombinant ebv antigens these tests frequently precede the appearance of npc and serve as tumor markers of remission and relapse [50, 51]. They confirmed that elevation of the ebv antibody levels preceded the clinical onset of npc . They also reported that there is a window of about 3 years preceding the clinical onset, when the antibody level is elevated and maintained at high levels . However, none of these serologic screening tests appear satisfactory to date because of low - level sensitivity or specificity . Detection of the ebv gene in nasopharyngeal swabs from symptomatic patients has been shown to be highly predictive of symptomatic npc [54, 55]. Proteomic approaches have been applied for the analysis of malignant neoplasms . For practical usage in tumor screening recently, wei et al . Analyzed serum samples from patients with npc employing proteomic analysis . In their report, four protein peaks at 4,097, 4,180, 5,912, and 8,295 daltons (da) discriminated npc patients with a sensitivity of 94.5% and specificity of 92.9% . Furthermore, chang et al . Reported that the use of a three - marker panel (cystatin a, mnsod, and mmp2) could contribute to improved npc detection . Other potential markers for the diagnosis of npc include galectin-1, fibronectin, mac-2 binding protein, and plasminogen activator inhibitor 1 [57, 58]. There is a possibility that the incorporation of these tests in the routine screening of npc may enhance its early detection . The importance of clinical syndromes, history, and clinical examination for helping the early diagnosis of npc could not be ignored . Any adult presenting with unexplained unilateral serous otitis media should be carefully examined to rule out npc . Endoscopy plays a key role in detecting the early npc lesions, and endoscopic biopsy enables their definitive diagnosis . Reported an objective endoscopic score of abnormality of nasopharynx to predict the likelihood of npc . However, clinicians should keep in mind the fact that detection of npc is sometimes difficult with endoscopy . Endoscopic findings may be subtle in early npc lesions: only slight fullness in the rosenmller's fossa, or a small bulge or asymmetry in the roof . When npc is strongly suspected, considering early diagnosis of npc, appropriate imaging examinations and/or biopsy of the nasopharyngeal mucosa careful attention should be paid when mri is conducted for a patient with unilateral serous otitis media (stasis of secretions in unilateral middle ear) or cervical lymph node adenopathy . 60 to 96% of npc patients exhibited cervical lymph node adenopathy at the time of presentation [6163]. T1 tumors, confined to the nasopharynx, may be clinically occult, and also may be difficult to differentiate from the normal mucosa on a ct scan and mri . However, such small tumors are usually readily evident by their less intense enhancement by gadolinium than the normal nasopharyngeal mucosa . It has been suggested that mri is superior to 18-fluoro-2-deoxyglucose (fdg) positron emission tomography (pet) for the assessment of locoregional invasion and retropharyngeal nodal metastasis . Pet is not suitable for detecting small retropharngeal nodes or for distinguishing retropharyngeal nodes from adjacent primary tumors . To date, the modalities commonly used in the followup of patients with npc include clinical examinations and imaging studies . Inspection with a flexible fiberscope plays a primary role in followup examinations . However, mucosal reactions to radiotherapy make it difficult to find early recurrent lesions . Secretions and in addition, the detection of submucosal or deep - seated recurrent lesions is difficult with fiberscopic examinations . If recurrent npc lesions can be diagnosed properly and in a timely manner, these lesions may be treated by chemotherapy, reirradiation, such as further conventional external beam radiotherapy, brachytherapy, and stereotactic radiotherapy, or surgery . Regarding surgery, conventional nasopharyngectomy for recurrent npc lesions can still result in serious complications . However, early recurrent lesions (such as rt1 lesions) may be effectively treated with laser nasopharyngectomy . Diagnostic uncertainty may result in delayed treatment, which reduces the life expectancy of patients with recurrent npc lesions . Narrow - band imaging (nbi) is a novel technique that enhances the diagnostic sensitivity of endoscopes for characterizing tissues using narrow - bandwidth filters in a sequential red - green - blue illumination system . Superficial mucosal carcinoma lesions, which are rarely detected using conventional endoscopy, can be observed with nbi by viewing the nonangiogenetic, microvascular proliferation pattern [68, 69]. Recently, lin and wang applied this technique to the detection of early recurrent mucosal lesions of npc . They reported that early recurrent lesions of npc after radiotherapy were successfully detected by nbi coupled with conventional endoscopy . Regarding imaging studies after initial treatment, ct and mri are widely used for the detection of recurrent lesions . Generally, mri is superior to ct in the detection of soft tissue abnormalities . The baseline mri study is often conducted 2 to 3 months after termination of the initial treatment . After the baseline evaluation, close evaluation is recommended with further imaging followup every 3 to 6 months for the first 2 years posttreatment . However, any signal abnormalities in the nasopharynx on mri should be stable or reduced in this followup period . After 2-year followup without evidence of recurrence, the imaging interval is extended to be every 6 to 12 months . Recently, the effectiveness of fdg - pet in the detection of residual or recurrent npc lesions has been reported from several institutes . Fdg - pet is increasingly being used for detection of recurrent lesions in many types of tumor . Pet is reportedly useful to distinguish recurrent npc tumors from postirradiation changes, such as tissue necrosis, fibrosis, and edema [7073]. Liu et al . Reported that sensitivities of ct, mri, and pet for the detection of residual or recurrent npc lesions were 76, 78, and 95%, respectively . These findings suggest that pet can be a useful tool for the detection of recurrent npc lesions . However, there are also some limitations regarding the use of pet for the detection of early recurrent npc lesions furthermore, a recent cost - based analysis suggested that it is most cost - effective to perform pet if mri results are unclear . Npc detection in the early stage is often difficult because the symptoms are not specific . Ebv - related serologic tests are used as screening tools in high - risk populations, although the screening tests available in daily clinics are not satisfactory . Molecular biomarkers are under examination as a new tool for the detection of early npc lesions . Regarding imaging modalities, mri seems suitable for the detection of early lesions, and the routine use of pet for the initial diagnosis of npc does not seem to be justified . In addition to the new diagnostic modalities, improvement in the awareness of physicians and the general population regarding this carcinoma undoubtedly contributes to the earlier detection of the disease.
Resin - based composites are used worldwide in dentistry, mainly because of their aesthetic quality and good physical properties . Since resin composites were first developed, many efforts have been made to improve the clinical behaviour of this restorative material . Several studies have demonstrated that the degree of polymerization of light - cured resin composites depends on many parameters, such as, the specific formulation (i.e. Type and relative amount of monomer, filler and initiator / catalyst), the wavelength distribution, the intensity of the incident light and the irradiation time . Although both organic and inorganic phases might influence the material behaviour, the filler particles features and rate of curing are the most important factors related to an improvement in the mechanical properties of the resin composites . The intended areas of usage of resin composites have traditionally included a trade - off between composites polish ability and strength, based on filler size and loading . Recently, resin composites have been classified according to their filler particle size as hybrid (0,5 - 3 m), microhybrid (0,4 - 1 m) and microfilled (0,04 - 0,4 m). More recently, however, with the introduction of nanotechnology in dentistry, a new class of resin composite, the nanofilled composite resin, is available to clinicians, in an endeavour to provide a material presenting high initial polishing ability combined with superior polish and gloss retention . The degree of cure of visible light activated dental resins was recognized as important to the clinical success of these materials soon after these materials were introduced . A curing light intensity output depends on many factors (light guide, condition of the bulb, battery life) and the total energy determines the mechanical properties of the resin composites . Also, the distance of the light from the the resin composite is a crucial factor . In the last few years, curing light technology has advanced with the introduction of high intensity halogen lights, plasma arc lights and light emitting diode units (leds), with the aim of fast curing of resin composites and generating less heat . Led curing lights have recently become very popular since they have a number of advantages over conventional halogen units . Traditional modes use high initial irradiance and provide a higher degree of conversion (dc); on the other hand, a higher shrinkage stress may be induced during polymerization reaction . Gradual polymerization modes have been introduced in order to minimize polymerization shrinkage and consequent marginal gap formation . Physical properties of composite resin are also dependent on the dc of the resin matrix . A positive correlation has been demonstrated between increasing hardness and increasing dc; however, it was concluded that an absolute hardness number could not be used to predict dc when different resin composites are compared . A previously published study showed a significant correlation between the degree of conversion and hardness, modulus of elasticity and flexural strength of dental restorative resins . Direct methods that assess the degree of conversion, such as infrared spectroscopy and laser raman spectroscopy, have not been accepted for routine use as they are complex, expensive and time consuming . Incremental surface hardness has been shown to be an indicator of the degree of conversion and a good correlation between knoops hardness and infrared spectroscopy has also been reported . It has been used to predict the wear resistance of a material and its ability to abrade or be abraded by opposing tooth structures . To define depth of cure based on top and bottom hardness measurements, it is common to calculate the ratio of bottom / top hardness, and give an arbitrary minimum value for this ratio . In order to consider the bottom surface as adequately cured, the current in vitro study evaluated vickers hardness (vk) and depth of cure (hardness ratio) of three microhybrid, two nanohybrid and one nanofilled resin composites, polymerized with a led curing unit by three different polymerization modes . Six resin composites were selected for the present study and were chosen in accordance with their type of filler particles: three microhybrid (esthet.x hd, amaris, filtek silorane), two nanohybrid (grandio, ceram.x mono) and one nanofilled (filtek supreme xt). The materials evaluated and their manufacturers are shown in table 1 . During the whole experimentation, the resin composites were light cured with a led unit, celalux ii (voco, cuxhaven, germany). Three light polymerization modes were used for each material: standard 20 s: 1000 mw / cm for 20 seconds; standard 40 s: 1000 mw / cm for 40 seconds; soft - start 40 s: 0 to 1000 mw / cm for 5 seconds + 1000 mw / cm for 35 seconds . The hardness testing methodology used to assess samples of the respective materials were prepared by placing the material into a stainless steel mold (7 mm, h 2 mm), and were placed on a dark opaque paper background covered with a polyester matrix strip . The mold was filled with the resin composite and a second polyester matrix strip was placed on the top of the filled mold . A glass slide was pressed against the upper polyester film to extrude the excess resin composite and to form a flat surface . The distal end of the light guide was placed against the surface of the matrix strip and positioned concentrically with the mold; and, the material was then light - cured from the top . Materials used in the study and their manufacturers the cordless curing unit was maintained at full charge before use, and irradiance was checked with a radiometer (led radiometer, kerr, orange, ca, usa). After polymerization, the samples were stored for 48 hours in complete darkness at 37c and 100% humidity before the vickers hardness test (vk). The vickers hardness (vk) of the surface was determined with a microhardness tester (durometer zhu 0,2 zwick - roell, ulm, germany) using a vickers diamond indenter and a 200 g load applied for 15 seconds . Five vk readings were recorded for each sample surface (top and bottom); and the measurements were made in a sequential pattern, starting with the bottom surface of all specimens, and in 1 mm increments from the specimen centre and extending 2 mm in both x (east - west [e - w]) and y (north - south [n - s]) axes . Hardness measurements were not taken at more than 4 mm from the specimen centre to avoid any possible effect of the mold on polymerization . For a given specimen, the five hardness values for each surface were averaged and reported as a single value . The mean vickers hardness and hardness ratio of the specimens were calculated and tabulated using the formula: hardness ratio = vk of bottom surface / vk of top surface . The mean vickers hardness of top and bottom surfaces and hardness ratio associated with the standard 20 s polymerization mode is shown in table 2 . The mean vickers hardness of top and bottom surfaces and hardness ratio associated with the standard 40 s polymerization mode is shown in table 3 . The mean vk of top and bottom surfaces and hardness ratio associated with the soft - start 40 s polymerization mode is shown in table 4 . The influence of three curing modes (standard 20 s and 40 s and soft - start 40 s) on hardness ratio of six composite resins was compared . According to statistical analysis (t student test), it was observed that for all the materials there was no statistical difference (p> 0.5) in hardness values recorded on top surfaces . A statistical significant difference (p <0.01) was recorded on the bottom surfaces for all the materials tested; and this is due to the reduced energy reaching the lower layers, thus affecting the final hardness . Mean vickers hardness of top and bottom surfaces and hardness ratio recorded with the standard 20 s polymerization mode as seen in the study mean vickers hardness of top and bottom surfaces and hardness ratio recorded with the standard 40 s polymerization mode as seen in the study mean vickers hardness of top and bottom surfaces and hardness ratio recorded with the soft - start 40 s polymerization mode as seen in the study hardness ratio values for the different polymerization modes as seen in the study despite this drastic difference between the values recorded on the top and bottom surfaces for all the materials, hardness ratio was higher than the minimum value indicated in literature in order to consider the bottom surface as adequately cured (0.80). A statistically significant difference (p <0.01) was recorded comparing standard 20 s polymerization mode with both standard 40 s and soft - start 40 s polymerization modes for esthet.x hd, amaris and ceram.x mono . Comparing standard 40 s and soft - start 40 s polymerization mode, there was no statistical difference between hardness values recorded on top and bottom surfaces . Resin composites are widely used in restorative dentistry and specifically in posterior restorations, putting the material under constant masticatory stresses . One of the most important parameters deciding the resin composites resistance to stress is the depth of cure . The effectiveness of cure depends on the filler particle type, size, quantity and on the parameters (intensity, time and polymerization modes) of the light source . Effective cure of light - activated composites is also important to prevent cytotoxicity of inadequately polymerized material . The optimal degree of curing throughout the bulk of a visible light - activated dental resin composite is acknowledged to be important to the clinical success of a resin composite restoration . Unfortunately, the dentist has no means of monitoring the cure of the resin surfaces not directly exposed to the curing light . Stated that one brand of composite in flowable, hybrid and packable formulations did not achieve a 2 mm depth of cure with 20 s light exposure . De jong et al . Demonstrated that with high intensity light - curing units, exposure times of 10s/2 mm increment can be sufficient to obtain under in vitro conditions a high degree of conversion . These data suggest that a 2 mm buildup layering technique may not result in adequate curing of the bottom layer for such a wide range of materials and that manufacturers need to provide quantitative information about the degree of conversion at specific activation times and light intensities for their entire range of resin materials and shades so that the dentist can devise a placement technique that will ensure adequate cure of the bulk of a restoration . These findings where in disagreement with some studies that have shown that 2-mm increments were well polymerized . Concluded that the bottom - to - top surface microhardness ratios of a composite resin proved to be an accurate reflection of bottom - to - top degree of conversion; bottom - to - top microhardness; and, degree of conversion were independent of composite composition . The development of new technologies and polymerization modes for photo - activation of restorative composite resins has also caused a great interest among researchers . However, the real advantages of these techniques are not yet totally known . In the present study, 2-mm thick composite specimens were used as it ensured uniform and maximum polymerization . The degree to which light - activated composites polymerize is proportional to the amount of light to which they are exposed . Ideally, the degree of polymerization of the composite should be the same throughout its depth and the hardness ratio should be very close or equal to one . As light passes through the composite, the light intensity is greatly reduced due to light scattering, thus decreasing the effectiveness of cure at the bottom surface . It was suggested that the hardness ratio should be greater than 0.8% for light activated composites to be adequately polymerized . In the present study, the hardness ratio for all the tested materials was over 0.8% for standard 20 s and 40 s and soft - start 40 s polymerization . Denehy et al . Found that the top surface hardness of composites was less dependent on light intensity than the bottom surface . The top surface is actually receiving the maximum energy from the curing light . A statistically significant difference (p <0.01) was recorded comparing standard 20 s polymerization mode with both, standard 40 s and soft - start 40 s polymerization modes, at top and bottom surfaces, for esthet.x hd, amaris and ceram.x mono . These results depend on the total amount of energy reaching the composite layer and on chemical composition of the composites . At the the top surface, it has also been established that even relatively low intensity lights can cure the resin matrix to an extent almost equal to when high intensity lights are used . The general lack of significance between standard 40 s and soft - start 40 s curing modes in top vickers hardness found in this study corroborate the mentioned studies . At the top surface, sufficient light energy reaches the photoinitiator, thus starting the polymerization reaction . At the bottom surfaces, a significant difference in vk was observed for all the materials, but no statistical difference was observed between standard 40 s and soft - start 40 s polymerization modes . This may be due to the very fast increase (5 s) of light intensity in soft - start polymerization, while total light exposure was very similar between the two polymerization modes . About the properties of the composite resins, the results were generally dependent on the material evaluated, especially with regard to filler features . Suggested that no trend towards the size or shape of fillers affected hardness; and all materials generally presented different results in comparison with one another . Grandio, for instance, presented the highest values, probably because of its large particles and the highest filler content . Nanofilled filtek supreme xt showed significantly higher hardness values than esthet.x hd, filtek silorane and ceram.x mono, which were all conceived for posterior restorations . In this study, only one physical property was tested on a limited number of composite resins polymerized with one type of unit . More research involving the use of other materials and multiple combinations of polymerization modes is warranted . Curing time did not affect hardness ratio values for filtek silorane, grandio and filtek supreme xt . The effectiveness of cure at the top and bottom surface was not affected by soft - start polymerization mode.
Congenital epulis is a rare lesion of the newborn, presenting as mass in the oral cavity which can interfere with respiration and feeding . It should be distinguished from other lesions which can occur in newborns, both clinically and histopathologically . Here, we report a case of congenital epulis in a newborn female on the right alveolar ridge, along with an extensive review of literature and discuss the immunoprofiling . Early diagnosis of ce in a newborn is of paramount importance in the successful management of these rare cases . Congenital gingival granular cell tumor (cgct) of the newborn, also known as congenital granular cell lesion, congenital epulis, congenital myoblastoma (historically), or neumann's tumor, is a rare non - neoplastic lesion seen only in newborns (1). It presents in the mouth most commonly in the maxillary alveolar ridge as a smooth - surfaced sessile or pedunculated mass with normal to reddish colour mucosa . It varies in size from several millimeters to few centimeters in diameter and can interfere with respiration or feeding . In recent years, prenatal detection of such oral lesions has facilitated the narrowing down of differential diagnosis and proper treatment planning through multidisciplinary approach . Although, histopathologically this lesion shows similarity with granular cell tumour which occurs in adults, the two are separate entities with different histogenesis . A newborn female child was referred to our institute, immediately after delivery for examination of a mass protruding from her mouth . A round, soft pedunculated mass of 4 cm diameter, exhibiting a smooth erythematous surface was located on the right side of the maxillary alveolar ridge (fig 1). The mass prevented normal closure of the mouth and interfered with breastfeeding, but did not pose an immediate airway concern . Lesion attached to maxillary alveolar ridge protruding from the mouth on the second day after birth, the tumor was completely resected by surgical excision following anaesthesia, and subjected to histopathological examination . The newborn recovered with no complications, and breastfeeding was initiated on the subsequent day of operation . The gross specimen measured 3.5 cm 3.5 cm 2 cm and was pink in color with a smooth surface and firm consistency . This tissue was processed for routine histopathological examination and embedded in paraffin.4 m - thick sections were cut from these paraffin - embedded tissue blocks and stained with hematoxylin and eosin . Sections revealed lesional tissue comprising large sheets of polygonal or rounded cells with a centrally placed small dark basophilic nucleus with an abundant eosinophilic granular cytoplasm, abutting the overlying parakeratinized stratified squamous epithelium . The tissue was non - reactive to s-100 protein and cd68 (fig 3); but reactive to vimentin . H&e stained sections showing stratified squamous epithelium and underlying tissue with granular cytoplasm immunostaining showing negative staining for cd68 congenital epulis of the new born is a widely accepted term and few prefer it over congenital granular cell tumor, which is suggestive of a neoplasm (4). However, epulis is a non - specific term used to designate hyperplastic gingival tissue or gingival tumor masses . Since there are cases which are not exclusively related to the gingiva, (1,3) seems that the term congenital granular cell lesion would be a more appropriate term (5).since its first description in 1871 in germany as congenital epulis by neumann (6), over 200 cases of this rare lesion have been reported (2). Ce is usually seen at birth and has a site predilection for the maxillary alveolar process, lateral to the midline in the region of the primary canine and lateral incisor . Less frequently, it has been reported in the mandibular alveolus, tongue and one case with involvement of alveolar ridge as well as the tongue (6). Ce usually occurs as a solitary lesion, although in 10% of the cases, it occurs as multiple masses (4,7). It presents as a mass with a smooth normal colored surface, pedunculated, sometimes lobulated, and varying in size from a few millimeters to 9 cm (8). Ce is usually diagnosed at birth; although, if the lesion is large, it may be diagnosed in utero by 3d ultrasound and magnetic resonance imaging (mri) examinations . In utero diagnosis is important in choosing the delivery method, since large lesions may compromise a normal vaginal delivery and a cesarean operation may be necessary (8). Although there are studies that affirm successful prenatal diagnosis of ce, these studies actually obtained images of the tumor mass, but the diagnosis could not be conclusive (5). A list of differential diagnosis thus obtained is valuable in treatment planning and a multidisciplinary approach during delivery . The tumor is also postulated to originate from undifferentiated mesenchymal cells, fibroblasts, myofibroblasts, histiocytes, pericytes, schwann cells or odontogenic epithelial cells . Few immunohistochemical study findings support a mesenchymal origin (3, 5, 10).ultrastructural studies showed presence of many autophagosomes containing collagen precursors, suggesting the tumor cells represent early mesodermal cells that express pericytic and myofibroblastic features that undergo cytoplasmic autophagocytosis (11). There are usually no associated dental abnormalities or congenital malformations (2), except for occasional reports of a hypoplastic or absent tooth and the possibility of mild midface hypoplasia (2,4). Ce has been reported in infants with polydactyly, goiter, triple syndrome, maxillary hypoplasia, neurofibromatosis and polyhydraminos (2,12). Clinical differential diagnoses for congenital lesions of oral mucosa depend on site of involvement, size, velocity of growth, and possible accompanying lesions . This includes teratoma (epignathus)(13), hemangioma, fibroma, choristoma and hamartoma, melanotic neuroectodermal tumour of infancy, rhabdomyoma, rhabdomyoscarcoma, lymphangioma, osteogenic and chondrogenic sarcomas, and granular cell tumor (3,12,13). However, some congenital lesions occur predominantly on the alveolar ridge and others on tongue, thus narrowing the list of possible differentials in a particular site . Leiomyomatous hamartoma has the appearance of congenital epulis and is often seen on the median anterior alveolar ridge and the tip of the tongue (1). They are two different entities which may be differentiated on histological and epidemiological grounds (14). Granular cell tumor is more commonly seen, with few reported cases of malignant transformation, whereas ce is a rare lesion with an incidence of 0.0006% (9) and no evidence of malignant transformation . Ce occurs in neonates predominantly in females on the right side of the maxillary alveolar ridge, whereas, granular cell tumor occurs in adults on the tongue and a wide variety of visceral and cutaneous sites (orbit, lung, mastoid, tongue, infra and supraglottic regions), with no sex predilection . Granular cell tumour may show pseudoepitheliomatous hyperplasia often with squamous pearl formation in the epithelium and is less vascular with prominent nerve bundles . Ultrastructurally, in ce, membrane bound granules or phagolysosomes are present in the cytoplasm, many of which contain collagen precursors, but angulate bodies are absent unlike in granular cell tumor (11). Immunohistochemical study shows no reactivity of lesional cells to s-100 protien, ngfr / p75, and inhibin - alpha in ce but both ce and granular cell tumor have stained positive for macrophage markers like cd68 and ki - m1p . However, the statement is equivocal and few cases have demonstrated no reactivity to cd 68 (15). In line with these cases ce also shows positive immunohistochemical staining to hla - dr antigen, vimentin, nki / c3, and pgp9 and occasionally nse and cea(3,16,17,18). Although immunohistochemical profiling has not confirmed the cells of origin of this lesion, it has proved useful in confirming that ce is non - neoplastic and aids in differentiating it from granular cell tumor histologically (19). The treatment of choice is surgical excision, when the lesion is obstructing feeding or respiration . It can be excised either under general anesthesia within hours to days after birth or local anesthesia where intubation is not possible or in cases of small lesions (2). There is also the possibility of removal during the delivery, in cases where the lesion was detected during pregnancy (20). This approach provides the newborn a free airway and an unobstructed oral cavity immediately after birth eliminating additional procedures such as anesthesia and intubation . Surgical excision of ce using carbon dioxide laser and erbium, chromium: yetrium - scandium - galliumgarnet (er, cr: ysgg) laser have also been reported . There have been eight case reports that have documented spontaneous regression . In cases where there is no interference with feeding or respiration, regular monitoring of the lesion for regression has been advocated as an acceptable clinical approach . In our case, the lesion interfered with feeding and was thus excised at the earliest so to avoid any further dehydration in the newborn . Ce has not recurred even after incomplete excision, and has no tendency for malignant transformation.
The standard management for muscle - invasive transitional cell carcinoma (tcc) and non - muscle - invasive tcc of bladder with a high risk of progression is radical cystectomy with pelvic lymph node dissection . Orthotopic neobladder (onb) substitution is an accepted form of urinary diversion after radical cystectomy with a better quality of life as compared with cutaneous urinary diversion . There is a possibility of urethral recurrence after onb substitution, compromising its functional integrity . The reported recurrence rate of urethra after radical cystectomy has a wide variation and ranges from 0.5% to 18% . In initial studies, prostatic involvement was considered as a significant prognosticator for urethral recurrence, but later on it was shown that despite a positive endoscopic biopsy from the prostatic urethra, if the resected margin of the urethra on frozen section analysis (fsa) was negative, then the urethral recurrence is unlikely even after 10 years of follow - up . There is paucity of data on the robustness of intra - operative fsa, influencing the decision of urinary diversion . More often than not, the yield of fsa is lower than expected, and, in a majority of cases, it seems to be an unnecessary step . It is interesting to see that even a positive urethral margin on fsa does not predict the recurrence of tcc at the urethral stump . This raises an important issue of performing a step as a routine when it may not be necessary . We analyzed our data to assess the need for fsa in patients of radical cystectomy planned for onb . For this institutional ethics board approved study, data on radical cystectomy performed between january 2000 and june 2013 were retrieved from the hospital information system and internal data management source of our center . A total of 233 radical cystectomies were performed during the study period . Of these,, fsa was performed for presence or absence of malignancy at the urethra on the patient side . Based on the low yield of fsa, after 2010, this step was not performed as a routine and only those patients who had visible growth at the bladder neck had fsa . For statistical analyses, patients were grouped into three groups, i.e. Patients with fsa and positive margin, patients with fsa but negative margin and those in whom fsa was not performed . Pathological characteristics of these groups were compared for local staging and presence or absence of carcinoma in situ . A contrast - enhanced computed tomography (cect) imaging parameters like hydroureteronephrosis, tumor size at the time of presentation and site of the bladder tumor in relation to the bladder neck were assessed . Location of the tumor in relation to the bladder neck and trigone on cect was matched with the findings on the cut - open specimen . Patients were followed - up every 4 months with ultrasonography, serum creatinine, lower urinary tract symptoms, uroflowmetry, post - void residue and venous blood gas analysis . Survival curves for patients whose fsa was negative and patients who did not have fsa were compared with the log rank test . Binary logistic regression analysis was performed to analyze the effect of imaging parameters on the status of the urethral margin . All the statistical analyses were performed using spss 16 (chicago, il, usa). Survival curves for patients whose fsa was negative and patients who did not have fsa were compared with the log rank test . Binary logistic regression analysis was performed to analyze the effect of imaging parameters on the status of the urethral margin . All the statistical analyses were performed using spss 16 (chicago, il, usa). Survival curves for patients whose fsa was negative and patients who did not have fsa were compared with the log rank test . Binary logistic regression analysis was performed to analyze the effect of imaging parameters on the status of the urethral margin . All the statistical analyses were performed using spss 16 (chicago, il, usa). The mean age of 151 patients who were planned for onb was 56.3 10.7 years . Of those 109 patients, only three (2.7%) patients had a positive urethral margin . Two of them had ileal conduit diversion and one patient, after having a negative biopsy on the second fsa, had onb . Although none of the factors, i.e. Hydroureteronephrosis, tumor size and site, were found to be significant for predicting urethral margin status, all three patients with a positive urethral margin had tumor encroaching the bladder neck [figure 1]. All the patients with a positive urethral margin died of cancer at a mean follow up of 29.33 18.3 months, but none had urethral recurrence till the time of death . Patients who died of cancer had pelvic and/or distant metastasis . Pathological characteristics of the patients according to the urethral margin status outcome of the patients according to the frozen section analysis (fsa) of the urethral margin one of the patients with a positive urethral margin who had tumor involving the bladder neck (blue solid arrow) overall, 34/151 (22.5%) patients had growth at the bladder neck on cect . Of these, six tumors were actually found to be away from the bladder neck on the cut - open specimen . [figure 2, table 3] the false - positive report of bladder neck involvement on cect was 18%, (six of 34 cases). Therefore, only 28/138 (21.5%) patients had growth at the bladder neck [table 3]. Of these 28 patients, three (10.7%) had a positive urethral margin and all these three patients had tumor involving the bladder neck . Tumor involving the bladder neck on the contrast - enhanced computed tomography scan was found to be away from the bladder neck in the cut - opened specimen (white solid arrow) comparative results of bladder neck involvement found on cect and during naked eye examination in the surgical specimen regarding association of the urethral margin with the involvement of the prostate, it was found that of 141 male patients, 10 (7%) had their prostate involved but none had a positive urethral margin [table 4]. Similarly, two male patients who had a positive urethral margin had no prostatic involvement . Location of the tumor at the bladder neck did not correlate with the involvement of the prostate (p = 0.983) [table 4]. Relationship between location of the tumor and the involvement of the prostate gland (n=128) the 1- and 5-year overall survival of patients with negative fsa were 91.5% (95% ci, 8593.5) and 79% (95% ci, 7284%), and of those in whom fsa was not performed were 93% (95% ci, 8695%) and 82% (95% ci, 7489%). [figure 3] the mean follow - up times were 46.3 25.1 months and 36 9.3 months for the frozen section negative and not sent groups, respectively . None of the patients without fsa (42) had urethral recurrence at a mean follow - up of 36 9.3 months . Meir survival curve with log rank test comparing survival in patients with negative urethral margin and in patients in whom frozen was not sent only two patients had recurrence in the penile urethra: one patient after 6 years and the other patient 2 years following onb . In both the cases, the urethral biopsy was positive for malignancy but the neo bladder urethral junction was normal . The gold standard treatment for muscle - invasive or recurrent transitional carcinoma of the urinary bladder is radical cystectomy with urinary diversion . Among the various kinds of urinary diversions, onb substitution provides the most natural way to void, with a relatively better quality of life . The whole purpose of using an external sphincter as a continent mechanism for onb would be defeated if there is recurrence in the urethra . Therefore, it is a standard practice to send the urethral margin for fsa before performing onb to ensure that the urethral margin is negative . The reported rate of urethral recurrence after radical cystectomy has a wide variation, ranging from 0.5% to 18% . As recurrence at the urethral stump is known even after having a negative margin, it seems likely that the reason for recurrence is not the status of margin assessed by the available means . According to early reports, the most consistent risk factor in men for predicting urethral recurrence was the involvement of the prostate . Although urethral recurrence was not common in patients with prostatic urethral involvement, prostatic ductal and stromal involvement were associated with the urethral recurrence, ranging from 10% to 67% . Similarly, women with tumors located at the bladder neck or involving the anterior vaginal wall are at a higher risk for urethral recurrence . Later on, onb substitution was perfromed even in patients who had involvement of the prostatic urethra (confirmed on endoscopic biopsy), provided the fsa showed a negative urethral margin . After a minimum follow - up of 10 years, none of the patients had urethral recurrence . In another study of a similar kind, the positive and negative predictive values of the prostatic urethral biopsy prior to cystectomy were 12% and 99.9%, respectively . They concluded that presence of tumor in the prostatic urethra prior to cystectomy would not preclude orthotopic neobladder substitution if the urethral frozen section is negative . Following these studies, intraoperative frozen section biopsy of the urethral margin became the pre - requisite for an onb, disregarding the status of prostatic involvement . Interestingly, that the rate of urethral recurrence in patient undergoing onb has been found to be low . Although these studies are retrospective in nature, where bias of choosing a case with better tumor characteristics could be the reason, the inexplicable protective effect of onb against recurrence at the urethral margin is quite evident . Published their series and concluded that patients with onb have a lower risk of urethral recurrence than those who had cutaneous urinary diversion for tcc urinary bladder . This protective effect of onb for urethral recurrence was also demonstrated by stein et al . Although one would argue against not performing fsa, one needs to assess the overall impact of fsa in terms of decision making for or against the onb . Urethral recurrences have also been reported with the negative urethral margin, the incidence of which has been described to range from 0.9% to 2.8% . In one series, two of 13 patients with positive fsa had urethra recurrence as opposed to 13 recurrences in 222 patients who had negative fsa . Therefore, the urethral margin status failed to accurately predict recurrence at the urethral stump . In the present study too, we had a very low yield of frozen section biopsy, wherein we had only 2.7% of positive fsa . All three patients died of their disease, but none had urethral recurrence at the time of death . Among the survivors also, we had only two urethral recurrences at the mid penile urethra, and both the patients had negative fsa as well . Looking at the recurrence rate of less than 3% at the urethral stump and low positive predictive value of the frozen section, should we be performing fsa in all the patients? Moreover, an overall survival in patients where frozen was not sent was not different from those in whom frozen was sent and had a negative urethral margin . Interestingly, in our study, although the location and size of the tumor and hydroureteronephrosis did not predict the status of the urethral margin, three patients who did have a positive urethral margin had tumors located at the bladder neck . In our experience, carcinoma in situ (cis) changes are not found as frequently as reported in the literature; moreover, normal - looking mucosa on cystoscopy is unlikely to have cis changes . None of the patients who had tumor away from the bladder neck showed a positive margin, and about 77.5% of our patients had growth away from the bladder neck . The cut - section of the specimen immediately after the cystectomy is a useful way to determine the location of the growth at the neck, as six of 34 (18%) cases had a false - positive report on cect scan . Involvement of the prostate in the radical cystectomy specimen in the present study was just 7%, as against 17% in one of the largest series on radical cystectomy . None of the patients with prostatic involvement in the present series had a positive urethral margin, which contradicts the theory of synchronous involvement of the urethra . Visual impression of the specimen to determine the encroachment of tumor to the bladder neck, similar to the one we apply during partial nephrectomy, could be helpful in deciding against sending a routine frozen section . Limitations of the study are relatively less number of patients with positive urethral margin and relatively short follow - up of those in whom we did not send the frozen section . This study questions the ubiquity of performing a step that is unlikely to help in decision making . In the majority of cases, fsa seems to be an unnecessary step and mere visual impression of the tumor and urethra could be helpful in deciding about sending the urethral margin for fsa . Only patients who have growth located at the bladder neck on the cut - open specimen should have fsa.
Workers with occupational contact dermatitis (ocd) may have adverse outcomes . Not only may they continue to have disease and their overall quality of life may be impacted, but they may also have significant work disruption . A number of studies have provided disease outcome information and more recently there have been studies demonstrating an impact on quality of life . There are some studies reporting on work outcomes in populations of workers with ocd and one study examined barriers to return to work [110]. While overall work status has been reported, there is little information about the return to work process . The objective of the study was to describe the work outcomes and return to work process in workers with ocd over a six - month period following diagnosis . Potential participants were informed of the purpose, activities, risks, and benefits of the study and their signed consent to participate was obtained . Once the individual had been assessed and patch testing ordered, they were approached to participate in the study . Patients were invited to participate if (a) they had a possible diagnosis of contact dermatitis, (b) were employed or had been employed but stopped work because of their skin disease, (c) were undergoing patch testing, and, d) had hand involvement . This assessment collected information regarding the worker's clinical history and work status at the time of patch testing . The first follow - up assessment was carried out at three months by telephone and included a brief questionnaire regarding work status . The second follow - up assessment was carried out at six months, in person or by telephone, and included a detailed questionnaire about the workplace and return to work, workers' compensation and health care utilization over the previous six months . As the main purpose of the study was descriptive, the data were analyzed using standard statistical methods including means and frequencies . One hundred workers were enrolled in the study and completed the initial assessment . As the workers were enrolled prior to final diagnosis, 78 were determined to have ocd following their assessment and patch testing . Of the 78 workers with a diagnosis of ocd, 75 completed the second assessment and 60 workers completed the third assessment . The mean age was 40 with a range from 19 to 63 and 64% were male . The length of time with the rash prior to assessment at st michael's was 25 months . The arm was affected in 26% and the face, neck, or leg in 12% . Almost all described itching, pain, redness, and scaling, while 86% noted cracking and bleeding and 74% blisters . When asked about the relationship between symptoms and work, 91% noted their skin was worse at work and 87% noted improvement on vacation . A past history of any atopic condition was reported by 56% of workers including 46% with allergies, 19% with hayfever, 15% with asthma, and 14% with eczema . Family history of atopic disease was also common with 53% reporting any atopic condition including 19% with eczema . The mean total time at their current worksite was 79 months (range 2 to 238 months) and the mean time at their current job was 65 months (range 1 to 324 months). These included 91% with exposure to cleaning agents, 77% to metals, 72% to solvents, 46% to oils and greases, 37% to plastics, and 24% to other chemicals . Eighty - six percent reported wearing gloves at work and 21% reported wearing gloves while working at home . A diagnosis of irritant contact dermatitis was made in 83% and of allergic contact dermatitis in 51% . Additional diagnoses included 8% with atopic dermatitis and 5% with psoriasis . At the time of the initial assessment and patch testing 10% were not working . Of these 75% were not working because of their skin problem . Of those who were working . If they were working but had changed job, 67% had done so because of their skin . Forty - four percent reported talking to someone at their workplace about a workers' compensation claim and 41% reported that a workers'compensation claim had been submitted . At the three month follow - up interview we were able to contact 75 of the 78 workers . At this time 26% were not working, 95% of these because of their skin . Of those who were working, 78% were at the same job as when the problem started . If they were working but had changed job, all had changed jobs because of their skin . Information was obtained regarding advice received related to return to work (rtw) and the results are presented in table 1 . The minority of workers reported receiving recommendations regarding a change of job or job modification and, even if advice had been provided, it was not always implemented . Communication between the various parties involved in rtw was reported by a minority of workers . Workers reported that the physician wrote a letter or talked to the employer for 23% of the workers . Forty - four percent of the workers talked with their supervisor; if the worker belonged to a union, 31% talked with their union; 19% were involved in a meeting concerning their rtw involving their supervisor and others; 16% had some discussion about potential problems with rtw; and for 4% there was some discussion of their rtw with their coworkers . At the six month follow - up interview we were able to contact 60 of the 78 workers . To provide comparability of results across the 3 visits, the results of work status at the initial assessment, at three months and six months for the 60 workers that were assessed at all three visits are presented in table 2 . At six months 15% were receiving workers' compensation benefits, 3% employment insurance benefits, and for 10% the company had changed their job and no compensation claim had been filed . Of those who were working, . If they were working but had changed job, 92% had done so because of their skin . With respect to workers' compensation, 69% reported submitting a claim . Of those who submitted a workers'compensation claim, 70% were accepted, 6% denied, 15% pending, and 9% did not know the status of their claim . In addition to work status, information regarding work practices, communication amongst the parties involved in rtw and interactions with health care providers were obtained . Previous studies have demonstrated significant work disruption in workers with ocd [19]. Some workers had already experienced work disruption by the time of their initial assessment and significant further change in work status occurred in the six months following diagnosis . At the time of initial assessment,, the percentage not working had increased to 26% and at 6 months, the percentage not working had increased to 38% . At three months after diagnosis, the job modifications suggested including the use of gloves, changing the type of glove used and changes in skin care were suggested in the minority of cases and implemented in even fewer . When communication between the various parties was examined at three months, 44% had talked with their supervisor and this increased to 69% by six months . Similarly, at three months 31% of union members had talked with their union and this increased to 42% at 6 months . These gaps include lack of advice provided regarding needed job or workplace change, lack of implementation of modification and suboptimal between the workplace parties who are involved in the rtw process . Follow - up visits with their family physicians and, dermatologists, reported elsewhere, were 62% and 39%, respectively, even though workers are advised to see their physicians for follow - up . These findings suggest that there is significant room to improve the rtw (or stay at work) process . Further research, focused on the various aspects of rtw, including the identification of barriers and facilitators are needed to improve the rtw process and ultimately achieve better work outcomes for workers with ocd.
Acute heart failure (ahf) is one of the leading causes of unscheduled hospitalization in patients older than 65 years and is associated with frequent readmissions and substantial mortality.1 in recent years, several factors that may contribute to exacerbation of heart failure have been identified . Among these, acute coronary syndrome, arrhythmias and acute respiratory disease have been identified as being the most common precipitating factors.2, 3, 4, 5 as shown in the analysis of the optimizehf registry, which included patients admitted for ahf in 259 centres in the united states, precipitating factors of ahf influenced shortterm outcome.2 in particular, acute coronary syndrome and acute respiratory disease were associated with higher inhospital mortality . In contrast, little evidence is available about the effect of precipitating factors on readmission and longterm mortality . We recently showed that the effect on outcome of several prognostic factors was strongly influenced by age and was more pronounced in ahf patients younger than 75 years, compared with the older ones.6 the aim of the present study was to assess the effect of precipitating factors of ahf on readmission and longterm mortality in the overall population of patients admitted for ahf and in the subgroup of patients aged 75 years or younger . The biomarcoeur cohort included patients aged 18 years or older admitted to the emergency department of three tertiary centres (lariboisire university hospital in paris, france, fattouma bourguiba university hospital in monastir, tunisia, and cumhuriyet university hospital in sivas, turkey) between 1 january 2010 and 31 december 2013 . Ahf (including de novo ahf, acute decompensated heart failure or cardiogenic shock) was diagnosed by reviewing medical records after hospital discharge and was based on medical history, clinical examination, natriuretic peptides and additional tests obtained during hospitalization . Precipitating factors of ahf were assessed and classified in four main groups: acute coronary syndrome, atrial fibrillation with rapid ventricular conduction, acute pulmonary disease (including chronic obstructive pulmonary disease exacerbation, asthma and pulmonary infection) and other causes . The latter group includes undetermined precipitating factors and all precipitating factors not fitting in the previous three categories . The diagnosis of ahf in this cohort has been previously shown to be highly accurate.7 as detailed data about acute pulmonary disease were not included in the original database, the adjudication committee verified and confirmed the combination of ahf and acute pulmonary disease.7 followup visits were performed by phone contact at 1, 3, 6 and 12 months after hospital discharge . Hospital readmission during 90 days after hospital discharge and survival at 1 year after admission were analysed . The study was carried out in accordance with the declaration of helsinki and was approved by the institutional review board of each centre . The effects of the presence compared with absence of every precipitating factor on 1 year mortality and 90 days readmission rates were assessed using cox regression models without and with adjustment for potential confounding factors (age, sex and impaired renal function, defined as egfr <60 ml / min/1.73 m). Moreover, the effect of precipitating factors was assessed in the subgroups of patients older than 75 years and aged of 75 years or younger . Mortality and readmission were described with the kaplan meier curve, and differences between groups were assessed by the logrank test . Values are expressed as median (interquartile range) or as number (percentage), as appropriate . The biomarcoeur cohort included patients aged 18 years or older admitted to the emergency department of three tertiary centres (lariboisire university hospital in paris, france, fattouma bourguiba university hospital in monastir, tunisia, and cumhuriyet university hospital in sivas, turkey) between 1 january 2010 and 31 december 2013 . Ahf (including de novo ahf, acute decompensated heart failure or cardiogenic shock) was diagnosed by reviewing medical records after hospital discharge and was based on medical history, clinical examination, natriuretic peptides and additional tests obtained during hospitalization . Precipitating factors of ahf were assessed and classified in four main groups: acute coronary syndrome, atrial fibrillation with rapid ventricular conduction, acute pulmonary disease (including chronic obstructive pulmonary disease exacerbation, asthma and pulmonary infection) and other causes . The latter group includes undetermined precipitating factors and all precipitating factors not fitting in the previous three categories . The diagnosis of ahf in this cohort has been previously shown to be highly accurate.7 as detailed data about acute pulmonary disease were not included in the original database, the adjudication committee verified and confirmed the combination of ahf and acute pulmonary disease.7 followup visits were performed by phone contact at 1, 3, 6 and 12 months after hospital discharge . Hospital readmission during 90 days after hospital discharge and survival at 1 year after admission were analysed . The study was carried out in accordance with the declaration of helsinki and was approved by the institutional review board of each centre . The effects of the presence compared with absence of every precipitating factor on 1 year mortality and 90 days readmission rates were assessed using cox regression models without and with adjustment for potential confounding factors (age, sex and impaired renal function, defined as egfr <60 ml / min/1.73 m). Moreover, the effect of precipitating factors was assessed in the subgroups of patients older than 75 years and aged of 75 years or younger . Values are expressed as median (interquartile range) or as number (percentage), as appropriate . The median age was 75 (6484) years, 56% of patients were men and clinical signs of congestion were commonly reported: the respiratory rate was 26 per minute (2232 per minute), rales were audible during lung auscultation of 582 patients (82%), peripheral edema and jugular distention were reported in 431 (57%) and 310 (41%) patients, respectively . Concerning precipitating factors of ahf, acute coronary syndrome was present in 47 patients (6%), atrial fibrillation with rapid ventricular response in 127 patients (17%) and acute pulmonary disease in 149 patients (20%). In 459 patients (61%), ahf was precipitated by other causes . Of note, in 27 patients (4%), a combination of acute coronary syndrome and atrial fibrillation was identified as precipitating factor . Baseline characteristics at admission of patients with ahf acei, angiotensin converting enzyme inhibitor; ahf, acute heart failure; arb, angiotensin receptor blocker; bnp, brain natriuretic peptide; cabg, coronary artery bypass grafting; copd, chronic obstructive pulmonary disease; crp, creactive protein; dbp, diastolic blood pressure; egfr, estimated glomerular filtration rate; mra, mineralocorticoid receptor antagonist; pci, percutaneous coronary intervention; sbp, systolic blood pressure . As summarized in table 1, patients with ahf precipitated by acute pulmonary disease were older (78 years vs. 73 years), reported more often previous history of chronic obstructive pulmonary disease or asthma (42% vs. 12%), suffered from greater respiratory impairment (higher respiratory rate and lower peripheral oxygen saturation) and showed more often wheezing on lung auscultation (44% vs. 13%) than patients with ahf precipitated by nonpulmonary causes . As illustrated in figure 1, patients with ahf precipitated by acute pulmonary disease had higher levels of creactive protein [35 mg / l (10110 mg / l) vs. 14 mg / l (531 mg / l), p <0.001] but lower levels of bnp [899 pg / ml (4041445 pg / ml) vs. 1191 pg / ml (6432329 pg / ml), p <0.001] compared with the group of ahf precipitated by nonpulmonary causes . Levels of bnp and crp (creactive protein) at admission in the group of patients with acute heart failure precipitated by acute pulmonary disease (n = 149) compared with acute heart failure precipitated by nonpulmonary causes (n = 606). Median and 95% confidence interval are displayed . Precipitating factors significantly influenced the 90 days readmission rates, as shown in table 2 . In the overall population, acute pulmonary disease was associated with lower readmission rates (hazard ratio (hr) 0.61, 95% confidence interval (ci) 0.370.99, p = 0.049), whereas atrial fibrillation with rapid ventricular response, acute coronary syndrome and other precipitating factors showed a nonsignificant trend towards higher readmission rates . After adjustment for potential confounding factors, none of the precipitating factors significantly influenced the readmission rates . As shown in figure 2 (left panel), the subgroup of patients with ahf precipitated by acute pulmonary disease (n = 149) showed fewer readmissions (logrank p = 0.047) compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (n = 606). Precipitating factors and risk of 90 days readmission ahf, acute heart failure; ci, confidence interval . Risk of 90 days readmission in presence of predefined classes of precipitating factors of ahf compared with absence of the same factor . Readmissions of patients with acute heart failure precipitated by acute pulmonary disease compared with acute heart failure precipitated by nonpulmonary causes during 90 days after discharge in the overall population and in the subgroup of patients 75 years . In the subgroup of patients aged 75 years or younger, differences were even more pronounced: as shown in table 2, after adjustment for potential confounding factors, acute pulmonary disease was associated with markedly lower readmission rates (hr 0.20, 95% ci 0.060.63, p = 0.006), whereas atrial fibrillation (hr 2.23, 95% ci 1.293.85, p = 0.004) and acute coronary syndrome (hr 2.23, 95% ci 1.024.86, p = 0.044) were associated with higher readmission rates . As illustrated in figure 2 (right panel), younger patients with ahf precipitated by acute pulmonary disease showed fewer readmissions compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (logrank p = 0.003). As shown in table 3, acute pulmonary disease at admission was associated with higher mortality at 1 year in the overall population (hr 1.59, 95% ci 1.042.43, p = 0.034), whereas all three other groups of precipitating factors were not . After adjustment for potential confounding factors, acute pulmonary disease showed only a nonsignificant trend towards higher risk of death in the overall population (hr 1.46, 95% ci 0.942.25, p = 0.09). As illustrated in figure 3 (left panel), patients with ahf precipitated by acute pulmonary disease showed increased mortality during 1 year followup compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (logrank p = 0.032). Precipitating factors and risk of 1 year mortality ahf, acute heart failure; ci, confidence interval . Risk of 1 year mortality in presence of predefined classes of precipitating factors of ahf compared with absence of the same factor . Mortality of patients with acute heart failure precipitated by acute pulmonary disease compared with acute heart failure precipitated by nonpulmonary causes during 1 year after admission in the overall population and in the subgroup of patients 75 years . More interestingly, in the subgroup of patients aged 75 years or younger, acute pulmonary disease was associated with significantly higher risk of death (hr 2.52, 95% ci 1.175.41, p = 0.018). As illustrated in figure 3 (right panel), younger patients with ahf precipitated by acute pulmonary disease showed higher mortality compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (logrank p = 0.026). The median age was 75 (6484) years, 56% of patients were men and clinical signs of congestion were commonly reported: the respiratory rate was 26 per minute (2232 per minute), rales were audible during lung auscultation of 582 patients (82%), peripheral edema and jugular distention were reported in 431 (57%) and 310 (41%) patients, respectively . Concerning precipitating factors of ahf, acute coronary syndrome was present in 47 patients (6%), atrial fibrillation with rapid ventricular response in 127 patients (17%) and acute pulmonary disease in 149 patients (20%). In 459 patients (61%), ahf was precipitated by other causes . Of note, in 27 patients (4%), a combination of acute coronary syndrome and atrial fibrillation was identified as precipitating factor . Baseline characteristics at admission of patients with ahf acei, angiotensin converting enzyme inhibitor; ahf, acute heart failure; arb, angiotensin receptor blocker; bnp, brain natriuretic peptide; cabg, coronary artery bypass grafting; copd, chronic obstructive pulmonary disease; crp, creactive protein; dbp, diastolic blood pressure; egfr, estimated glomerular filtration rate; mra, mineralocorticoid receptor antagonist; pci, percutaneous coronary intervention; sbp, systolic blood pressure . As summarized in table 1, patients with ahf precipitated by acute pulmonary disease were older (78 years vs. 73 years), reported more often previous history of chronic obstructive pulmonary disease or asthma (42% vs. 12%), suffered from greater respiratory impairment (higher respiratory rate and lower peripheral oxygen saturation) and showed more often wheezing on lung auscultation (44% vs. 13%) than patients with ahf precipitated by nonpulmonary causes . As illustrated in figure 1, patients with ahf precipitated by acute pulmonary disease had higher levels of creactive protein [35 mg / l (10110 mg / l) vs. 14 mg / l (531 mg / l), p <0.001] but lower levels of bnp [899 pg / ml (4041445 pg / ml) vs. 1191 pg / ml (6432329 pg / ml), p <0.001] compared with the group of ahf precipitated by nonpulmonary causes . Levels of bnp and crp (creactive protein) at admission in the group of patients with acute heart failure precipitated by acute pulmonary disease (n = 149) compared with acute heart failure precipitated by nonpulmonary causes (n = 606). Median and 95% confidence interval are displayed . Precipitating factors significantly influenced the 90 days readmission rates, as shown in table 2 . In the overall population, acute pulmonary disease was associated with lower readmission rates (hazard ratio (hr) 0.61, 95% confidence interval (ci) 0.370.99, p = 0.049), whereas atrial fibrillation with rapid ventricular response, acute coronary syndrome and other precipitating factors showed a nonsignificant trend towards higher readmission rates . After adjustment for potential confounding factors, none of the precipitating factors significantly influenced the readmission rates . As shown in figure 2 (left panel), the subgroup of patients with ahf precipitated by acute pulmonary disease (n = 149) showed fewer readmissions (logrank p = 0.047) compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (n = 606). Precipitating factors and risk of 90 days readmission ahf, acute heart failure; ci, confidence interval . Risk of 90 days readmission in presence of predefined classes of precipitating factors of ahf compared with absence of the same factor . Readmissions of patients with acute heart failure precipitated by acute pulmonary disease compared with acute heart failure precipitated by nonpulmonary causes during 90 days after discharge in the overall population and in the subgroup of patients 75 years . In the subgroup of patients aged 75 years or younger, differences were even more pronounced: as shown in table 2, after adjustment for potential confounding factors, acute pulmonary disease was associated with markedly lower readmission rates (hr 0.20, 95% ci 0.060.63, p = 0.006), whereas atrial fibrillation (hr 2.23, 95% ci 1.293.85, p = 0.004) and acute coronary syndrome (hr 2.23, 95% ci 1.024.86, p = 0.044) were associated with higher readmission rates . As illustrated in figure 2 (right panel), younger patients with ahf precipitated by acute pulmonary disease showed fewer readmissions compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (logrank p = 0.003). As shown in table 3, acute pulmonary disease at admission was associated with higher mortality at 1 year in the overall population (hr 1.59, 95% ci 1.042.43, p = 0.034), whereas all three other groups of precipitating factors were not . After adjustment for potential confounding factors, acute pulmonary disease showed only a nonsignificant trend towards higher risk of death in the overall population (hr 1.46, 95% ci 0.942.25, p = 0.09). As illustrated in figure 3 (left panel), patients with ahf precipitated by acute pulmonary disease showed increased mortality during 1 year followup compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (logrank p = 0.032). Precipitating factors and risk of 1 year mortality ahf, acute heart failure; ci, confidence interval . Risk of 1 year mortality in presence of predefined classes of precipitating factors of ahf compared with absence of the same factor . Adjustment was performed for age, sex and impaired renal function . No risk calculation possible because of lack of events in this subgroup . Mortality of patients with acute heart failure precipitated by acute pulmonary disease compared with acute heart failure precipitated by nonpulmonary causes during 1 year after admission in the overall population and in the subgroup of patients 75 years . More interestingly, in the subgroup of patients aged 75 years or younger, acute pulmonary disease was associated with significantly higher risk of death (hr 2.52, 95% ci 1.175.41, p = 0.018). As illustrated in figure 3 (right panel), younger patients with ahf precipitated by acute pulmonary disease showed higher mortality compared with the subgroup of patients with ahf precipitated by nonpulmonary causes (logrank p = 0.026). The influence of precipitating factors on outcome, described in the present paper, extends the concept proposed by fonarow and coworkers after analysis of the optimizehf registry.2 fonarow and coworkers showed an association between acute coronary syndrome (acs), pulmonary infection and higher inhospital mortality, but no data exist on the influence of precipitating factors on postdischarge readmissions and longterm mortality . The present study demonstrated that precipitating factors of ahf may substantially influence outcome far beyond the duration of hospitalization, especially in patients aged 75 years or younger: readmission rates were low when acute pulmonary disease precipitated ahf and high when nonacute pulmonary disease, including atrial fibrillation or acute coronary syndrome, precipitated ahf . The influence of precipitating factors of ahf on postdischarge readmission rate, in patients aged 75 years or younger, is a novel observation . The existence of congestion is a frequent cause of hospital readmission in patients with chronic heart failure.8 in the present study, the subgroup with acute pulmonary disease, as precipitating cause of ahf, showed lower levels of bnp suggesting a lower level of congestion compared with patients with nonpulmonary precipitating factors . Furthermore, patients with acute pulmonary diseases, including infections, once treated, are usually discharged home with rare readmissions . The influence of acute pulmonary disease on postdischarge readmission rate was mostly seen in patients aged 75 years or younger, while no influence of precipitating factors on readmissions in older ahf patients . We recently showed that effect of several prognostic factors was influenced by age and was more pronounced in ahf patients younger than 75 years, compared with the older ones.6 results of the present study confirm the stronger relationship between prognostic factors and outcome in younger patients, although the reasons remain unknown: a more complex interaction between precipitating factors, multimorbidity and frailty of older patients with ahf could be a reasonable explanation . Surprisingly, the observed influence of acute pulmonary disease on longterm mortality was divergent from that on readmission: patients with ahf exacerbated by acute pulmonary disease showed higher mortality . Two recently published studies showed that pneumonia is associated with increased risk for cardiovascular events after hospital discharge.9, 10 a combination of endothelial dysfunction, plaque instability, activated coagulation,11 volume overload, inflammatory and ischemic myocardial injury12 and arrhythmias13 have been postulated to explain increased morbidity and mortality . It is still unclear whether the adverse prognosis of the subgroup of admitted for ahf precipitated by acute pulmonary disease is caused by the unfavourable association of ahf and pulmonary disease itself or is rather a marker of a more complex comorbidity profile in patients with such an association . Our study suggests that the peculiar pathophysiological background of ahf precipitated by acute pulmonary disease exposes those patients to increased risk of death despite fewer readmissions . As a consequence, those patients should benefit not only from aggressive initial treatment but also from a more intensive and interdisciplinary followup after hospital discharge . The study was conducted on a mediumsized cohort of patients with ahf from three tertiary centres . Therefore, the conclusions of this study should be confirmed in larger cohorts . However, this cohort was large enough to demonstrate significant differences between the different precipitating factors . Moreover, the group of other precipitating factors was quite large, as our database was not conceived for further differentiation among other causes of ahf . This group might include patients with ahf precipitated by other causes as hypertension, malcompliance and worsening renal function.3, 4, 5 despite outcome data were derived from telephonic followup and relevant censoring (287 patients, 38%) occurred, the median followup in the cohort was 320 days . In addition, the cause of readmission was not assessed during followup, and therefore, the risk of a potential bias derived from newonset precipitating factors, although small, cannot be excluded . In particular, patients with ahf precipitated by acute pulmonary disease showed fewer readmissions and higher 1 year mortality, especially in patients aged 75 years or younger.
The advancing age is associated with profound changes in body composition, including increased fat mass, decreased fat - free mass (particularly muscle), decreased total body water and decreased bone density (1). Bone itself presents an organ of particular interest in both medical and pharmacological sciences . In medical sciences because of its unique physiology, since it undergoes remodeling throughout the lifespan with faster formation in the youth and faster resorption by growing older (2). Bone is important in pharmacological sciences since different medications can either improve or worsen bone health, with different studies having analyzed the link between drug use and adverse effects in msh, some of which even resulted in potentially serious consequences such as drug - induced osteoporosis (3, 4, 5). Corticosteroids seem to be the leading cause of secondary osteoporosis (5, 6), with many other medication groups seeming to increase the risk, such as thyroxine overdose, gonadotropin - releasing hormone (gnrh) agonists, aromatase inhibitors, thiazolidines, ppi (proton pump inhibitors), loop diuretics, anticoagulant drugs, tricyclic antidepressants, anticonvulsant (7) which are still under scientific debates and investigations . While the regular controlled prescription presents a common standard in every developed country, in a developing, low income country such as kosova (8), it is generally believed that there are major problems regarding self - medication and usage of different medicines without the professional prescription . These might result in higher risk of unsafe usage of medicines, declining the efficacy and increasing the overall price of treatment . Unfortunately, there are very few published studies carried out in kosovo concerning drug use, side effects, drug - drug interactions (4, 9, 10, 11). This directly contributed to the main aims of this study, that are: having an overview regarding the msh status of mature adults (40 - 65 years old) in kosovo, finding out the impact of specific medications on adults msh . Having an overview regarding the msh status of mature adults (40 - 65 years old) in kosovo, finding out the impact of specific medications on adults msh . We hypothesized that our subjects should be physically active enough to fulfill the international recommendations, while some medication groups discussed for their possible (contradicting) effect on msh such as n05b, a02b (ppi, h2ra), h02ab, might have an impact on their msh, which could directly be affecting them . Especially since these medications are proved to be highly consumed within the studied group ages (40 - 65) (12, 13, 14), there is an increasing prevalence of poly - pharmacy by age (12), and the increasing risk of drug - drug interactions and adverse effects with the number of drugs used (12, 13). This is an observational, cross - sectional study, designed and implemented in accordance with the current version of the declaration of helsinki of ethical principles regarding human experimentation (14). This study was approved by the committee of ethical and professional issues of university clinical center of kosova (797 - 12/03/2015), while every participant was informed about procedures of this research and consciously signed a consent for participation and publication before the measurements . A total number of 162 subjects (53 or 32.7% males and 109 or 67.3% females) aged between 40 to 65 years old, residents of kosova, were recruited . Participants were randomly selected after announcements in the local media (radio and tv stations) and social networks (facebook and twitter), and after fulfilling the inclusion criteria . Each subject s weight and height was measured with clinical scale and stadiometer (respectively), with a precision of 100 g (weight) and 1 mm (height). Inclusion criteria were males and females aged 40 - 65, who have not been previously diagnosed with osteoporosis or osteopenia and (consequently) not being treated for that, patients with any conditions where x - ray radiation is counter - indicated and those with long term immobilizations . For analyzing medications effect on msh, patient that have been using the chosen medications for at least 6 months within the last 2 years were recruited . A random general healthcare status questionnaire provided by university clinical center of kosova (ucck), was used for inclusion criteria . Bmq1 (brief medication questionnaire 1) combined with additional questions for adherence of medicines use (15, 16) were used to evaluate medicine usage, timeline frame of their usage and the origin of prescription / suggestion, in order to analyze reference and reliability of the subjects to information regarding medicines use . Medications in this study are grouped according to atc code (anatomical therapeutic chemical classification system). International physical activity questionnaire (17), translated and updated in albanian from boshnjaku a et al (18), was used to assess the participants physical activity level, calculated as metabolic equivalent for task (met)/hour . Self - administered nutritional standard questionnaire (nsq) (19), modified by members of the department of health sciences, university of rome foro italico, was used to assess nutritional intakes in our study subjects . Bone density was measured on the distal radial bone of non - dominant arm by dxa scan (dual energy x - ray absorptiometry), with a host software version 3.9.4 . Isometric hand grip strength was measured with electronic dynamometer (saehan corporation, masan, korea), while in a sitting position when subjects squeezed for maximal isometric contraction in a duration of 45 seconds . All statistical analyzes were applied using the program graph pad prism 6 for statistical analysis and statistical significance set at p <0.05 . Differences between groups with continual data were performed using unpaired t test with welch s correction (to compare two groups), one - way anova (to compare three groups) and multi - way anova (to compare more than three groups), whereas the differences between categorical variables were made by using x test . Descriptive statistics for bio - anthropometric and physical activity (pa) level results are shown in table 1 . Male and female subjects enrolled in this study did not differ significantly in terms of age and weight (p>0.05), which was not the case in height and bmi (p<0.05). Participants characteristics surprisingly, there were no significant differences in terms of nutritional habits between genders, except for milk product consumption (males consuming on average 2.190.48 rations per day, comparing to females 1.910.7) (data not shown). Despite 41.5% of males and 22% of females reporting to be low alcohol consumers, since no differences in any parameter analyzed were detected, we decided to consider them as homogeneous groups . When analyzing variables amongst our study participants (table 2), significant differences (p<0.05) were found in bmd and t - score but not on z - score (p>0.05) between genders . Similar results were observed in hand grip (table 3), where males generated significantly higher force (p<0.05). 42% of total participants met the diagnostic criteria (20) of osteopenia (18.8% of males and 51.3% of females), out of which (total subjects) 6.8% are on the verge of getting osteoporosis (with t - score below -2) with menopausal woman being at the greatest risk (11.6%). No one met the diagnostic criteria of osteoporosis . When comparing the possible differences in study subjects that consumed n05b (anxiolytics) drugs comparing to those that do nt (table 3), no significant differences (p>0.05) in any variables were found in total, males and females except for hand grip in females (p<0.05). In contrary to that, when analyzing h02ab medication group (glucocorticoids) between consumers and non - consumers, significantly better results (p<0.05) were registered in all variables except hand grip strength (p>0.05) in total subjects, in females and male consumers (table 3). It was interesting to see that amongst the subjects consuming a02b drugs (drugs for peptic ulcer and gastro - oesophageal reflux disease gord) no significant differences in bmd, t - score and z - score were found (p>0.05) comparing to those that do nt, whereas significantly better results (p<0.05) were registered in hand grip isometric strength only (table 4). The same situation was encountered within males (consumers versus non - consumers) in bmd, t - score, z - score and hand grip, whereas significantly better results were recorded in all variables in females . When dividing the a02b drug consumers into two subgroups: ppi and h2 receptor antagonists, significantly higher (p<0.05) impact on bmd, t - score, z - score and hand grip isometric strength had ppi usage comparing to h2ra, but nevertheless this was nt the case when comparing each of these groups with the rest of this study subjects (non - consumers of a02b) (table 4). T - n05b+ total consumers t - h02ab+ total consumers, t - n05b- total non - consumers t - h02ab- total non - consumers, m - n05b+ male consumers m - h02ab+ male consumers, m - n05b- male non - consumers m - h02ab- male non - consumers, f - n05b+ female consumers f - h02ab+ female consumers, f - n05b- female non - consumers f - h02ab- female non - consumers, a02b medication group consumers, t - a02b+ total consumers- t - ppi+ total consumers, t - a02b- total non - consumers - t - h2ra+ total consumers, m - a02b+ male consumers - t - a02b+ total consumers, m - a02b+ male non - consumers, f - a02b+ female consumers . Our study shows that the prevalence of osteoporosis (po) and osteopenia (popen) in kosovo seems to be lower than in several developed countries, such as sweden po 6% in women and 2.5% men (aged> 50) (21), uk po 24% and popen 49% in women (7 decade, either hip, spine or both) (22), korea po 30.6% (45 - 64years old in lumbar spine) (23), as well as in other developing countries, including here the latin american countries - po vertebral 12 - 18% and 8 - 22% proximal femur (aged> 50) (24), as well as china the gender distribution of osteopenia was in line with the current world prevalence being much higher in females (51.3%) comparing to males (18.8%). In fact, a previous study of ours (18) raised concerns regarding the msh of young females (aged 17 - 30 years old) in kosova comparing to their counterparts in developing countries, but this was not the case in this study . Perhaps the higher levels of pa amongst our study participants (2.260.76 in total, 2.40.68 in females and 1.980.83 in males) comparing to young adult females from the other study (1.10.3) (18) might be playing a role in the general results . Within the contradicting studies regarding the effect of n05b drugs (including benzodiazepines: alprazolam, diazepam, bromazepam, lorazepam, midazolam) in msh of consumers comparing to non - consumers, the comparison within our study was in line with other similar studies results regarding the lack of association between benzodiazepines usage and reduce in bone mineral density (26, 27), as well as the association between benzodiazepines usage and the improve in hand grip strength (28). When comparing our study findings with other already published studies regarding the possible impact of the usage of h02ab drugs (methylprednisolone, dexamethasone) in their consumers msh comparing to non - consumers, similar significant results (p<0,05) were found with the majority of the up to date published data, allowing us to stay in line with the known facts of the existing correlation between glucocorticoids usage and osteoporosis (5, 6, 29). Interesting results were observed when analyzing the possible effects of a02b medication group (ppi inhibitors: pantoprazole, omeprazole, lansoprazole, esomeprazole and h2 antagonists: ranitidine) in subjects that have been consuming them, where no significant differences (p>0.05) were found in all bone variables and significant differences (p<0.05) were found only in hand grip isometric strength in total subjects and in males . Yet, significant differences (p<0.05) between female consumers and no consumers were found in all variables, supporting findings from van der hoorn study (30), and suggesting that the prescription of a02b medication group should be especially careful when prescribing in middle aged or elderly females . We believe that significant differences (p<0.05) found in hand grip isometric muscle strength in total and in gender based, could be linked to the possible association between ppi usage and hypomagnesemia which is related to muscle cramps, something that has been proofed previously by furlanetto & faulhaber and matsuyama et al . (31, 32). When investigating the effect of each of two subgroups of a02b (ppi and h2ra) on msh, significantly higher (p<0.05) impact had ppi usage comparing to h2ra, supporting previous findings from vestergaard (33). Finally, this study has a couple of limitations, such as the measurement site (only on distal radial bone). While recommendations suggest to perform measurements in hip and lumbar spine as well, due to the lack of accessibility to the device we had to use only distal forearm dxa . Another possible limitation could be the number of participants, but this was mainly due to the type of study, small population of kosova, the small percentage of this age group within this country (23.1%) (34), and the difficulties in recruitment process (the radiological exposure was a big concern amongst our study subjects). The po and popen amongst the mature adults in kosovo seems to be quite low, and together with the relatively high levels of pa (amongst the study participants), allows us to believe that this population stands a solid position against age related msh conditions such as osteoporosis and osteopenia . Nevertheless, future studies using other measurement sites (such as hip and vertebral region) should be done in order to prove and establish these results . One important recommendation resulting from this study would be that the appropriate benefit / risk assessment should be always made before prescribing and ordering medication, while the therapy ordination must be prescribed by qualified health professionals . One field of particular carefulness should be especially the patients that take medications discussed to be affecting msh for long periods of time, where precautions should always be considered . This includes recommendation of regular dexa scans, prescription of additional protective therapy, including here bisphosphonates, calcitonin or supplements as calcium - ca, vitamin c or d analogues (35, 36, 37), as well as prescription of specific physical activity for improving bone health such as multicomponent exercise with an emphasize on resistance exercise (38). It should also be invested in patients awareness raising and medical practitioners professional update, in order for them both to be informed about the positive and negative effects of medicines use and the steps that can be taken to minimize possible side effects.
This short communication is a consensus opinion - based review highlighting the discussions of an advisory board of renowned experts in the field of overactive bladder (oab). These professionals, who are all specialists in urogynecology or obstetrics and gynecology, were assembled to provide their perspectives on the importance of correct diagnosis and effective, appropriate treatment of patients with oab . The remit of the advisory board was to discuss factors affecting diagnosis and successful pharmacotherapy of oab with antimuscarinic agents . The participants who provided valuable input into the discussions and gave approval for this article are recognized in the acknowledgments . Oab comprises a group of symptoms (ie, urinary urgency usually accompanied by increased frequency of micturition and nocturia with or without urgency incontinence), in the absence of urinary tract infection or other obvious pathology.1 this condition is highly prevalent, affecting approximately 12% of adults in europe and canada.2 prevalence increases with advancing age and is similar for men and women, particularly in the population 60 years of age.2 oab can be a life - long, persistent (often lasting for at least 10 years), and progressive (from oab without incontinence to oab with incontinence) condition.3,4 furthermore, oab has a significant detrimental impact on patients quality of life, which is often greater than that resulting from other chronic diseases such as diabetes.5 it is therefore crucial that treatment strategies take into account that patients often require effective and well tolerated therapy for prolonged periods . Recent publications have focused on the fact that oab is undertreated, revealing that up to 76% of diagnosed patients remain untreated,6,7 but correct diagnosis is essential to ensure appropriate management of patients with urinary symptoms . Diagnosis may be hampered by patients failing to discuss their oab symptoms, perhaps through embarrassment or the misconception that this condition is a normal part of aging and cannot be treated.5 although physicians rely on patients complaints to trigger action, primary care physicians may not ask their patients about oab symptoms,8 possibly due to time constraints, or lack of knowledge or awareness of available effective treatments . To overcome these barriers, questions on incontinence and bladder habits should ideally be included in the primary care physicians list of standard screening questions for all adult patients.8 oab comprises a group of symptoms (ie, urinary urgency usually accompanied by increased frequency of micturition and nocturia with or without urgency incontinence), in the absence of urinary tract infection or other obvious pathology.1 this condition is highly prevalent, affecting approximately 12% of adults in europe and canada.2 prevalence increases with advancing age and is similar for men and women, particularly in the population 60 years of age.2 oab can be a life - long, persistent (often lasting for at least 10 years), and progressive (from oab without incontinence to oab with incontinence) condition.3,4 furthermore, oab has a significant detrimental impact on patients quality of life, which is often greater than that resulting from other chronic diseases such as diabetes.5 it is therefore crucial that treatment strategies take into account that patients often require effective and well tolerated therapy for prolonged periods . Recent publications have focused on the fact that oab is undertreated, revealing that up to 76% of diagnosed patients remain untreated,6,7 but correct diagnosis is essential to ensure appropriate management of patients with urinary symptoms . Diagnosis may be hampered by patients failing to discuss their oab symptoms, perhaps through embarrassment or the misconception that this condition is a normal part of aging and cannot be treated.5 although physicians rely on patients complaints to trigger action, primary care physicians may not ask their patients about oab symptoms,8 possibly due to time constraints, or lack of knowledge or awareness of available effective treatments . To overcome these barriers, questions on incontinence and bladder habits should ideally be included in the primary care physicians list of standard screening questions for all adult patients.8 as the pathophysiology of oab is not fully understood, the syndrome is a diagnosis of exclusion . Therefore, a holistic approach to patient assessment should be employed,5 including appraisal of a patient s genitourinary history as well as diagnostic tools and physical examination . When a patient presents with symptoms that are potentially indicative of oab, it is important to rule out other conditions that produce urological symptoms, such as bladder cancer, urinary tract infection, urethral inflammation, interstitial cystitis or simply urogenital atrophy, before initiating treatment . Initial assessments should focus on patients symptoms, but physical examination and diagnostic procedures should also be performed . Physical examination including pelvic examination (particularly in post - menopausal women) indeed, it has been estimated that 23%88% of patients with pelvic organ prolapse have varying combinations of urgency, urgency incontinence, frequency and/or nocturia.9 urinalysis should determine if urinary tract infection (uti) or urethral inflammation is present . Uti is the most common cause of bothersome urinary symptoms in women of all ages and treatable with antibiotics, however, symptoms of oab will persist even after successful treatment . In addition, routine measuring of post - void residual (pvr) volumes following micturition is often useful for determining urinary retention issues, especially in patients with bladder and prolapse symptoms, elderly patients with voiding symptoms and/or recurrent urinary tract infections, patients with neurogenic bladder and voiding dysfunction, and those patients with symptoms suggestive of decreased detrusor contractility or bladder outlet obstruction.5 the volume of residual urine can be measured either directly with a catheter inserted into the bladder (in - and - out catheterization), or indirectly with an ultrasound scan . In the majority of patients with symptoms indicative of oab, urodynamic assessment (beyond pvr testing) is not usually required . However, in some cases urodynamic assessment will provide a definitive diagnosis.5 cystometry and measurement of pvr will exclude detrusor hyperactivity with impaired contractility (dhic) during voiding, as 70% of women over the age of 70 years with symptoms of oab may have dhic.10 furthermore, in cases where treatment with antibiotics and antimuscarinics has failed to improve symptoms, cytology and cystoscopy would exclude bladder neoplasias and interstitial cystitis . Indeed, urodynamic studies are indicated for specific patient types: oab refractory to pharmacotherapy based on symptoms; women with suspected outlet obstruction; and oab associated with concurrent neurological disease . However, the role of urodynamic assessment in oab diagnosis is not clearly defined currently . Evidence has shown that urodynamic diagnosis of detrusor overactivity and symptomatic diagnosis of oab only co - existed in 54% of patients classified as having oab.11 bladder diaries or frequency / volume charts are very helpful but often underutilized tools in the diagnosis of oab that allow physicians to review patients accurate record of micturition frequency, number of incontinence episodes and urinary leakage, as well as fluid intake . Bladder diaries can be used to evaluate stress and urgency associated with incontinence (oab - related incontinence), thereby helping physicians to distinguish between stress incontinence and urgency incontinence . Stress incontinence should be managed with pelvic floor exercises for at least 36 months before referral for surgery . Recommended first - line therapy for oab includes behavioral interventions (lifestyle modifications, fluid management, bladder retraining, and pelvic floor exercises) with or without pharmacotherapy . Many patients with oab start on anticholinergic therapy with bladder training, then after 1 month are reviewed and advised on next steps . Determination of a patient s medical history is not only vital for assessing genitourinary history but also comorbidities, which may affect treatment options . Looking at full patient profiles rather than just their symptoms could aid treatment choice and, importantly, allow treatment to be individualized to each patient s needs . It is widely agreed that antimuscarinic agents have similar efficacy but different tolerability and safety profiles . One of the main differences is the varying potential of antimuscarinics to alter cognitive and cardiac functions.12,13 these potential effects are of particular concern for oab patients, who tend to be older and are often receiving concomitant medication for a variety of other conditions, including those relating to cognitive and cardiac function (figure 1).1416 therefore, the patient s concomitant medications and comorbidities should be carefully considered when selecting antimuscarinic therapy for the management of oab, which highlights the need for tailored therapy . Increasing brain barrier, which can leave patients more vulnerable to the central nervous system effects of drugs with anticholinergic properties.12 to date, darifenacin has the most clinical evidence (three randomized, double - blind, placebo - controlled studies in healthy participants, n = 302) for a lack of effect on cognitive function . In contrast, oxybutynin has been associated with a decline in cognitive function, especially memory loss, compared with placebo, darifenacin and tolterodine.12 memory loss is of particular concern in older patients who may be receiving concomitant medication . A recent case study reported that memory loss secondary to administration of oxybutynin in a 66-year - old female with oab led to non - adherence to medication regimens.17 specific data on cognitive function are limited for other antimuscarinic agents, although small clinical studies with tolterodine (n = 22 healthy participants), solifenacin (n = 12 healthy participants) and trospium (n = 12 patients with oab) have compared these agents favorably with both placebo and/or oxybutynin.12,18,19 impairment of cognitive function may also be increased as a result of anticholinergic effects of concomitant medications administered for a variety of comorbid conditions unrelated to oab . These medications add to the total anticholinergic burden on the patient and may cause non - degenerative mild cognitive impairment.20 commonly prescribed drugs that have anticholinergic effects include: cimetidine, prednisolone, digoxin, furosemide, isosorbide dinitrate, warfarin, and codeine . Therefore, awareness of concomitant comorbidities and associated medications is of particular importance and careful management of patients medication is required to avoid prescribing agents with potential drug drug interactions related to their mechanisms of action and/or side - effect profiles . For example, the administration of anticholinergics for oab therapy may potentially occur at the same time as cholinesterase inhibitors (eg, donepezil, rivastigmine), which are typically used to improve memory and cognition in alzheimer s disease . In addition, medications often used in persons with diabetes, such as thiazolidinediones for glucose control and gabapentin for neuropathic pain, can lead to nocturia; and the use of anticholinergic agents in the treatment of parkinson s disease can impair bladder emptying.21 in addition to the decline in cognitive function, advancing age is associated with increasing prevalence of cardiovascular (cv) disease.16 moreover, cv disease occurs more frequently in patients with oab than those without oab.22 in a recent us database analysis of> 6000 patients with oab, 39% had some type of cv comorbidity at the time of oab diagnosis compared with 21% of age - matched non - oab patients (p <0.0001).22 hypertension (21%), diabetes (8%), cv symptoms (6%), and ischemic heart disease (6%) were the most common cv conditions.22 antimuscarinics have the potential to increase cv risk through prolongation of the qt interval, which may lead to potentially fatal cardiac tachyarrhythmia or torsade de pointes,23 and increased heart rate.13,24 faster resting heart rate, even by single digit increases, is associated with an increased risk of cv events and death in patients with and without cv disease (figure 2).25 in an observational longitudinal study, an increase in heart rate of only 5 bpm was associated with a 16%17% increase in mortality (p = 0.03).25 added to this, analysis of a separate us database indicated that almost 40% of patients with oab had an elevated heart rate of 80 bpm before receiving antimuscarinic treatment.22 hence, any further increases in heart rate resulting from antimuscarinic treatment may add to the cv risk . Selected antimuscarinic agents, such as tolterodine, have been associated with increased heart rate (between 212 bpm) compared with placebo in clinical studies enrolling healthy volunteers.13,24 in contrast, darifenacin did not increase heart rate from baseline compared with placebo or tolterodine.13,24 although the magnitude of heart rate effects observed in these studies13,24 may not be of consequence in healthy volunteers, the same changes may have a greater impact in patients with an established higher risk for cv events and cv comorbidities, such as patients with oab . These data demonstrate that patient comorbidities should be considered carefully during the treatment of oab . However, as there are no guidelines for the treatment of patients with oab and comorbidities, the medication needs of each patient should be individually assessed and resulting treatment tailored appropriately . The expert panel noted that ideally more patients should be treated successfully in the primary care setting, which would leave only those patients requiring specialist help being referred as and when appropriate . However, the main findings from the advisory board centered on the need for more comprehensive assessment of patients, which should assist in the correct diagnosis of oab, and suitable pharmacotherapy for patients with oab and comorbidities . In particular, increased awareness of the differences between antimuscarinic agents should allow for appropriate treatment of patients with oab and comorbidities, who may be receiving concomitant medications . Antimuscarinics have differing potential to negatively impact on patients cv and cns function, which could result in a reduction of quality of life, hence patients may benefit from treatment tailored towards their individual needs.
Since the 1990s, the average length of postnatal hospital stay has declined, both in denmark and internationally . The most prominent reasons are a renewed focus on the fact that giving birth is not a disease and the general need for cost savings in the healthcare system [14]. In denmark, the average length of postnatal hospitalization has decreased from 92 hours in 2007 to 77 hours in 2012 . A danish questionnaire study (n = 1, 507 women) identified that 44.3% of the women who were discharged early (within 24 hours) from postnatal care experienced a lack of follow - up support; that is, they felt that they did not receive the support needed to care for the newborn; 37.5% did not receive support for postnatal self - care, and 46.1% did not receive adequate support around breastfeeding . These findings concur with results in international research [2, 6, 7]. Studies show that new parents experience concerns, uncertainty, doubts, and feelings of insecurity during the postnatal period and are in need of follow - up support after early discharge [2, 6, 810]. Underline that one of the mediating factors in becoming a mother is the nature of social support available, which includes partner, family, friends, and health professionals . A sense of security is a central element to support as it might influence a parent's journey towards becoming a successful parent . Persson et al . Have developed the concept parents' postnatal sense of security . They identified the following dimensions as important for both parents' postnatal sense of security: empowerment from staff, affinity within the family, and the health and wellbeing of the family . An empowering organisation was fundamental for strengthening this [9, 1215]. If the parents feel insecure it can have a negative effect on parental self - efficacy (pse). The definition of pse is as follows: beliefs or judgments a parent holds of their capabilities to organize and execute a set of tasks related to parenting a child . For parents to employ parenting behavior positively, they must have confidence in performing the specific behavior . Parents with high self - efficacy are likely to make a greater effort than parents with low self - efficacy . Bandura has clarified what it is that enables an individual to build self - efficacy beliefs . Important aspects are mastery learning, where you can gain positive experiences, when you are doing things yourselves, vicarious experiences, that is, seeing others perform, and verbal persuasion, where others assure you that you hold the ability to perform a certain task [17, 18]. They experience that they have too little time to support new parents and to give individualised and timely information [6, 19]. In 2011, the region of southern denmark issued a new policy regarding the postnatal period, in which early postnatal discharge (i.e., from four to six hours; max . 24 hours) was to become general practice following uncomplicated delivery for first - time and multiparous mothers . This shift in the postnatal care presents a challenge in terms of finding new ways to provide the sufficient support that meet the needs of the new parents with a postnatal follow - up that can enhance pse and a sense of postnatal security . One possibility is the use of telemedicine, which can provide an innovative solution [2022]. It seems that telemedicine has the potential to provide appropriate support to early discharged mothers and their families, because it offers the possibility for new parents to be guided by healthcare professionals in their transition into parenthood . Findings by lindberg show that both parents and healthcare professionals find that telemedicine has the potential to provide appropriate support because it presents new ways to communicate that can substitute for face - to - face contact and it can be a valuable and functional complement to usual practice [25, 26]. We wanted to explore this potential and therefore designed and developed a software application (app), which was tested in a pilot study prior to the intervention . The aim is to explore how nurses experience using an app in nursing practice and how it impacts their ability to offer support and information to postnatal mothers who are discharged early and their families, in a way that will enhance the families' sense of security and self - efficacy . This study applied a participatory design (pd). It combines the use of qualitative methods and intervention, based on collaboration with users . The pd approach involves defining problems and indicating solutions in designing sustainable it solutions for practice together with the users . An essential aspect of designing and developing a new technology is the intervention phase, where the actual technology is tried out in practice and concrete experiences with the use of the new technology are gained . Participatory design can be viewed as hermeneutics, where new understanding is developed through a circular collaboration between the researcher's understanding and an attempt to interpret a certain phenomenon in collaboration with the participants . Action research spans a wide landscape of differentiated, but primarily qualitative, research strategies for bringing about change through action, developing and improving practice . This study was an intervention study where an app was tested between hospital staff and new parents at home following early postnatal discharge . The content, format, and style of the app were designed on the basis of the parents' identified needs, in close cooperation with the nurses on the postnatal ward, and with the assistance of a team of computer programmers ., new families requested an individualised postnatal follow - up, timely information and guidance, and accessibility to, and new ways to communicate with, healthcare professionals . This reflected the professional concern that the nurses had as to how they can ensure a postnatal care, which will ensure a sense of security, wellbeing, and parental self - efficacy, when the new parents are being early discharged . The app was designed with the following functionalities that should accommodate the needs of the early discharged parents. (1) asynchronous communication, online chat, where the families could send text messages to the healthcare professionals as well as photos and videos and receive an answer within four hours . This method of communication may diminish the barrier in accessing healthcare professionals after hospital discharge. (2) a knowledgebase consisting of information material with a search function for easier access to information . The information material was evidence - based and written and compiled by the nurses on the ward . The information material consisted of written material about the postnatal period, for instance, information about breastfeeding, skin - to - skin contact, the mother's restitution after giving birth, and practical advice about baby care . The knowledgebase also contained instructions videos with guidance about breastfeeding, skin - to - skin contact, the wellbeing of the baby, baby clues, and how to bathe the baby. (3) messages sent out automatically every 12 hours from the time of birth . The messages relate to the age of the baby and should be relevant to the new parents providing them with information about breastfeeding, the baby's first bowel movement, and so on . The nurses had written down what they would normally inform and instruct the new parents about in the first postnatal days . It was rewritten into short messages that the new families would receive every 12th hour for the first 4 days after their baby was born . In the messages there are relevant links to the knowledgebase with more thorough information . Asynchronous communication, online chat, where the families could send text messages to the healthcare professionals as well as photos and videos and receive an answer within four hours . This method of communication may diminish the barrier in accessing healthcare professionals after hospital discharge . A knowledgebase consisting of information material with a search function for easier access to information . The information material was evidence - based and written and compiled by the nurses on the ward . The information material consisted of written material about the postnatal period, for instance, information about breastfeeding, skin - to - skin contact, the mother's restitution after giving birth, and practical advice about baby care . The knowledgebase also contained instructions videos with guidance about breastfeeding, skin - to - skin contact, the wellbeing of the baby, baby clues, and how to bathe the baby . The messages relate to the age of the baby and should be relevant to the new parents providing them with information about breastfeeding, the baby's first bowel movement, and so on . The nurses had written down what they would normally inform and instruct the new parents about in the first postnatal days . It was rewritten into short messages that the new families would receive every 12th hour for the first 4 days after their baby was born . In the messages there are relevant links to the knowledgebase with more thorough information . Your baby will often wake up and show signs of hunger, if not you [sic] have to wake him up, read more about that here: your baby will often wake up and show signs of hunger, if not you [sic] have to wake him up, read more about that here: the parents were given an ipad to take home on loan on which the app was installed . They were to return the ipad to the hospital after seven days in a prestamped package . Prior to the intervention, we tested the app in a pilot study, where the nurses were instructed in the use of the app and the accompanying website . The nurses registered the new parents on the website and used it to check for messages . The nurses were responsible for the online chat, which in practice meant that they had to check it every four hours and send replies to the families . These responsibilities were additional to the nurses' assigned duties involving caring for the patients admitted to the postnatal ward . No extra time was allocated in their shift for the additional work involved in answering messages . The study took place on a postnatal ward that handles approximately 1,000 births a year and included nurses employed on the ward . The management at the ward had initiated the project after the implementation of the new postnatal policy in the region of southern denmark . The nurses at the ward were all involved in the project and willing to participate in the intervention . During the course of the study, four nurses moved job and three were employed . The newly employed nurses were introduced to the intervention . At the onset of the intervention, their professional postnatal experience varied from less than one year to 30 years, with a mean of 10.2 years . At the end of the intervention their professional postnatal experience varied from under one year to 30 years, with a mean of 7.1 years . Participant observation was carried out on the postnatal ward from march to august 2013, on average one day a week, in all 20 days . The data were primarily collected during day shifts, though five times were also during evening shifts . The nurses were not followed through an entire shift, because the focus was how they experienced using the app in nursing practice and how it affected their ability to offer support and information to postnatal mothers who are discharged early . The informal conversations took place during the nurses' coffee or lunch breaks or in the nurses' office . Sometimes they spontaneously started talking about the app, and other times we would ask a question to initiate a talk . Occasionally we were also assisting them with practical advice or help concerning the ipads or the webpage, which automatically led to conversations about the app and how they experienced using it . Field notes were taken concurrently with a focus on place, participants, and activity . The following served as a guideline for the observations: what happens at the time of observation and what intentions and feelings occur in the situation . All the nurses on the ward who had taken part in the study were invited to a focus group interview . The other nurses could not attend on the given dates, due to either work or personal matters . The number of participants who could attend on the chosen dates determined the size of each group, which ended up being four and five . The focus group interviews were held in the employee staff - room on the postnatal ward . Before each focus group interview commenced, the moderator (the first author) introduced the purpose of the interview and clarified the guidelines and the focus: experiences using the app in nursing practice and how it affects their ability to offer support and information to postnatal mothers who are discharged early . The overall theme focused on the nurses' experiences, which formed the basis of the discussion [37, 38]. The development nurse on the ward participated as a comoderator, made notes during the interviews, and evaluated the atmosphere and interaction . The focus group interviews lasted 44 and 55 minutes, respectively, and were audio - recorded and transcribed verbatim . The participants received oral and written information about the study and were included after providing their informed consent, in compliance with the helsinki declaration . The first author asked the nurses if they would like to participate in a focus group interview, and they were given time to think it over . They were told that participation was voluntary and that the focus group interviews would be held during working hours . The committee decided that approval from an ethics committee was unnecessary according to the national legislation in denmark (s-20110171). The danish data protection agency registered and approved the study (2008 - 58 - 0035). The data analysis was inspired by malterud's systematic text condensation (stc) and organised according to the steps taken in the analysis, as shown in table 1 . Stc is a descriptive and explorative method used in the analysis of qualitative data, such as interview studies, observational studies, and in the analysis of written texts . He developed the descriptive phenomenological method in psychology [38, 41, 42]. Stc is a development of giorgi's principles, including four comparable steps of analysis . It is pragmatic in the sense that it is easy to both follow and share due to the elaborated steps of the analysis . Firstly, we captured an overall impression of the data and extracted a preliminary set of main themes . Secondly, the data was divided into meaningful topics, which were relevant to the study question . Next, the meaningful topics were condensed and coded . Finally, the findings were synthesized, involving a shift from condensation to descriptions and categories . The codes were developed based on the preliminary themes identified in the first step and the theoretical framework . In order to optimise validation, the categories that emerged from the data analysis were as follows: an app as a means of providing support, an app as a means of conveying timely and accessible information . An app as a means of providing support, an app as a means of conveying timely and accessible information . The categories are presented below and are illustrated by quotations from the two focus group interviews (fgi) and from conversations that took place during the participant observation (po). The nurses were hesitant at first when they had to chat online with the families, that is, using written instead of verbal communication . The following example occurred during a lunch break on the ward at the very beginning of the intervention . One of the nurses (nurse t) related that she had answered a message: it was all new to me and normally i would just talk on the phone, but i really had to think twice before sending the message . One of the other nurses (nurse k) supplemented this with: yes, it does take quite some time and the mother who wrote, well, how would i put it, the message wasn't well articulated . It wasn't that it was difficult, but it just felt so different to write to a family instead of just talking . Nurse k then said: it is probably also a matter of time we have to get used to it . (field note, march 2013, po) one of the nurses (nurse t) related that she had answered a message: it was all new to me and normally i would just talk on the phone, but i really had to think twice before sending the message . One of the other nurses (nurse k) supplemented this with: yes, it does take quite some time and the mother who wrote, well, how would i put it, the message wasn't well articulated . It wasn't that it was difficult, but it just felt so different to write to a family instead of just talking . Nurse k then said: it is probably also a matter of time we have to get used to it . (field note, march 2013, po) another concern was that when communicating in writing, one uses fewer of the senses . I answer their questions () i look at the photo of the umbilicus, for instance, or whatever it is . (nurse i, fgi) i answer their questions () i look at the photo of the umbilicus, for instance, or whatever it is . (nurse i, fgi) though, after a period of time using the app, the nurses no longer felt that it was such a big challenge or that it involved changes to their work . Maybe you have to have some ping - pong, to ask the right questions, like you would have asked, if you were in the room [i.e. Face to face]. (nurse d, fgi) maybe you have to have some ping - pong, to ask the right questions, like you would have asked, if you were in the room [i.e. Face to face]. (nurse d, fgi) however, they did state that a lot depended on the type of questions that they had to answer on the online chat . Messages that were accompanied by, for example, a photo of an umbilicus were considered easy to answer, whereas questions about breastfeeding were more difficult, since more information and dialogue were required in order to make a judgment and give the appropriate support . Well, they can get answers to something very specific, but it is also the intention that where it is very complicated, and there are a lot of problems, we need to see them . Well, they can get answers to something very specific, but it is also the intention that where it is very complicated, and there are a lot of problems, we need to see them . (nurse a, fgi) the nurses stressed that the written communication cannot stand alone, but they emphasized that there was always the option to invite the parents to come to the ward for more guidance face - to - face and that this occurred on occasion . The nurses had to check the chat for messages every 4 hours, which showed to be a constant challenge . Explanations given for forgetting to check the online chat were that the nurses were too busy and there were challenges to adjust to the new procedures; the nurses had to go to the office to check the chat, and they usually spend most of their time in the patients' rooms or the nursery room . We do delegate who is responsible for the chat during the shift, but then (nurse a, fgi) we do delegate who is responsible for the chat during the shift, but then oh no we have forgotten it . (nurse a, fgi) the app gave the parents the option to stay at home, while, for instance, having the baby's umbilicus assessed, because they could send a photo . The nurses found that the possibility to send photos was an advantage instead of the parents having to explain how the umbilicus looked like, over the phone . It provided the nurses with a more accurate impression of the umbilicus, and they experienced that it increased their possibility to provide the appropriate advice and support . The following example shows the differences between the distinctive forms of contact the nurses used . One of the nurses had assessed an umbilicus based on a photo sent using the online chat . She could see that the baby was red in the groin, so she also wrote a note on that to the family . To which another replied: but, if they had been here [on the ward], you could have seen the whole baby, not just the groin, and then you could also check the armpits, for instance . (field note, march 2013, po) one of the nurses had assessed an umbilicus based on a photo sent using the online chat . She could see that the baby was red in the groin, so she also wrote a note on that to the family . To which another replied: but, if they had been here [on the ward], you could have seen the whole baby, not just the groin, and then you could also check the armpits, for instance . (field note, march 2013, po) the nurses agreed that families often found it difficult to contact healthcare professionals, because they did not want to disturb, which they ascribed to cultural factors or general expectations in society . (nurse d, fgi) i also think it is just a cultural thing . Nowadays, (nurse d, fgi) the nurses also discussed that the new parents were reluctant to call the ward for help, even though the nurses told them that they should always call, if they had any doubt when they had been discharged . They thought that it was because the parents had experienced that the nurses were busy, and then they did not want to disturb . (nurse v, fgi) the nurses experienced that the app gave the families an opportunity to make contact with them after discharge, where they did not feel that they were intruding . (nurse v, fgi) and i think that's a help . They feel that it is ok that they make contact . (nurse v, fgi) the nurses emphasized that one of the advantages of the app was that the information material for the parents was in digital instead of paper form . (nurse i, fgi) paper, it is all over, a mess, whereas the ipad they know where that is . (nurse i, fgi) the nurses expressed that there was a lot of information material handed out at the hospital, and they questioned how much of it the families actually read . They considered it an advantage that it was now in digital form, as it seemed to appeal more to the families, because they could easily access it on the ipad and they could also search within the material in the same way as using google or other search engines . The nurses experienced that this was a suitable way for the new parents to be guided . For instance, the nurses at the ward showed the admitted parents how to bathe the baby, but this was at a fixed time during the day, and if the parents watched the video, they could watch it whenever they wanted . When they are admitted for such a short time, it becomes very hectic to tell and show them everything . This way they can do it, when they want to and also when they are at home . (nurse k, fgi) when they are admitted for such a short time, it becomes very hectic to tell and show them everything . This way they can do it, when they want to and also when they are at home . (nurse k, fgi) they also found that it was easy for them to refer to a video or a written instruction . Well she wrote me a question, and i answer her back, but i also wrote that i thought she should read the information, it was easy to do, because i knew that she could find it easily on the app . (nurse s, june 2013, po) well she wrote me a question, and i answer her back, but i also wrote that i thought she should read the information, it was easy to do, because i knew that she could find it easily on the app . (nurse s, june 2013, po) the nurses told that the parents reported that they felt secure with the app . They knew where to look for the information, and at the same time they knew that they could easily get in contact with the nurses at the ward . And then she [a mother] told me that she was so secure, because it was just like having a nurse standing outside the door . (nurse b, august 2013, po) and then she [a mother] told me that she was so secure, because it was just like having a nurse standing outside the door . (nurse b, august 2013, po) the nurses had to adjust to the new policy with the early discharge . Well they come from the delivery ward, and then they are here for such a short period of time . (nurse v, fgi) well they come from the delivery ward, and then they are here for such a short period of time . (nurse v, fgi) the nurses expressed that it was reassuring to know that when the families were discharged with the app they were drip - fed information in the form of automated messages . It relieved some the pressure they might feel when discharging mothers early, in terms of the duty to have informed thoroughly enough . I think that there is so much information that they need in such a short time . Then you are just talking and talking, while you think, how much do they remember, when they come home . (nurse a, fgi) i think that there is so much information that they need in such a short time . Then you are just talking and talking, while you think, how much do they remember, when they come home . (nurse a, fgi) the nurses often had a feeling that the families could not retain all the general information . The nurses considered that the automated messages seemed to meet this challenge by providing families with timely information . Knowing that, if there is something that i have forgotten, they get the pop - up messages, which means they get the information one more time, that's great . (nurse a, fgi) knowing that, if there is something that i have forgotten, they get the pop - up messages, which means they get the information one more time, that's great . (nurse a, fgi) the nurses regarded the automated messages that the families received as a tool to stimulate the families' curiosity and also their capacity to take control of their situation . The nurses believed that because of the interactive links in the automated messages, when the parents read the messages, they could easily read additional information material in the knowledgebase or they could address a question to the nurses on the postnatal ward . The nurses experienced that the parents took control of their situation and the messages made the parents feel well prepared for the postnatal period . The messages served either to reassure them or to allow them to react, if they required more information or support . In this study, we found that the nurses consider that the app gives them the possibility to offer support to the families discharged early, as it provided easier access to timely information and support, and it enhanced opportunities for families to initiate contact after discharge . The nurses state that the written asynchronous environment offers an easy way to offer families support . They feel that it connects the hospital setting with the home and goes some way towards reducing the gap, which families can experience as a barrier, in the fact that they are reluctant to contact the hospital staff for support after discharge [2, 6, 43]. Other studies have also found that when new families are discharged, it is essential that they are able to get professional support whenever they need it [25, 44, 45]. Persson et al . Have identified that accessibility to support from healthcare professionals is an essential part of experiencing a postnatal sense of security [9, 14, 15, 46] (table 2). The nurses regard the app as a lifeline for families because it increases access to professional support . This is in line with the conclusions from a study by bjoernes et al . In 2012 that explored the possibilities involved in online contact between nurses and men with prostate cancer (n = 34). The patients experienced a feeling of partnership in dialogue (via e - mail) that supported their ability to be active and it gave them a feeling of freedom and security . They saw the written asynchronous contact as providing a flexible and calm communication environment and as a way to substitute for the reduction in face - to - face contact at the hospital . Yet an important aspect is that the new parents are depending on the fact that the nurses do check the chat every 4 hours in order to have access to support, and the study showed that it was a constant challenge . Even though the nurses thought they just had to get used to the new routine, we discussed new ways of remembering the chat, because it was critical for the parents' sense of security that they could rely on it . The nurses in our study also found that when the face - to - face contact was reduced due to the early discharge the automated messages and the use of instructions videos were a suitable way for informing the new parents . This relates to bandura's viewpoints on interactive computer - assisted feedback as a convenient means to inform, enable, motivate, and offer support . It offers a way to reassure parents that their newborn is healthy and to help parents to feel in control of their new situation, which are factors that enhance a postnatal sense of security as well as pse [17, 18, 48] (table 2). Another aspect of the instruction videos is the potential of enhancing pse through vicarious experiences, where the parents can see others perform, for instance, breastfeeding positions and bathing the child (table 2). The results revealed that the nurses feel the app enhances patients' curiosity and, to some extent, it encourages parents to act more independently, because they can easily search for information themselves . The nurses experienced that the new parents are more likely to seek for information themselves, when it is digitalized than in a paper pamphlet . According to bandura, acting independently and thereby gaining one's own experience are a way of achieving mastery experiences, which strengthen pse (table 2). The nurses found that the automated messages serve to reassure parents, and this suggests that the messages could potentially have the effect of encouragement . According to bandura, verbal persuasion contributes to pse because the parents are convinced that they can cope successfully . Also personal messages with encouraging feedback from healthcare professionals could to some extent substitute for the verbal persuasion that the families would receive if they were admitted for a longer duration after childbirth . Bandura also states that because it is readily accessible and convenient, there are advantages in offering internet - delivered guidance . This is reflected in our study, where the nurses point out that the asynchronous communication is essential to their view of the app as a lifeline . It is easy to seek help; the families do not encounter a barrier in contacting the nurses for advice . This is because they do not feel that they are disturbing the nurses, as opposed to making a synchronous phone call . This is described in the literature as an issue in healthcare, because patients are often reluctant to contact healthcare professionals, even when they have something important to ask or discuss [2, 49]. It seems that the app has potential to be more efficient in ensuring access to healthcare than a phone . Other studies have tested videoconferencing in the postnatal period [23, 24, 26]; it was valued as a supplement to traditional practice . The midwives saw that communicating via videoconferencing was almost equivalent to having a face - to - face meeting . The same was found in other studies that involved videoconferencing; the healthcare professionals experienced that it is possible to create an intimate relationship and proximity in technology - mediated care and that it provides a tool for patients to develop a sense of security at home [50, 51]. A photo can say more than a 1000 words, where the nurses can actually see and observe instead of both families and nurses having to rely on written or oral descriptions over the phone . Other studies have pointed out further advantages for patients in staying at home instead of going to the hospital, in terms of time saved on travelling and waiting for a consultation . The use of online communication such as e - mail or text messaging involves a language - analogue mediation it is a dialogue, but not like a dialogue that two people have face - to - face or mediated by the phone . The nurses addressed that the online chat function changed their way of communicating with the families, which they experienced to change their support to the new parents . This can be explained by applying ihde's postphenomenological theory, where he underlines that the technological mediation of human practice shapes our experiences of the situations in which we are engaged . Technology is not a neutral tool; it provides a framework and invites us to employ certain use - patterns [52, 5456]. When communicating face - to - face or on the phone, they felt they could use more of their senses to assess the patient's expressions or voice and evaluate their emotional or mental state as when communicating online . In this situation, as compared to when conducting a written dialogue, they felt it would be more natural for them to extend the dialogue to issues other than the one initially addressed . However a report from the institute for healthcare informatics on the use of social media shows that patients also use social media for emotional support, which indicates that it is no longer only through face - to - face dialogue that people feel they can get emotional support . The report concludes that there have been essential changes in the way people communicate, and as a consequence the new technologies will change how healthcare operates on a global scale . This development is also underpinned by a review by plantin and daneback that showed the majority of today's parents search for not only information, but also social support on the internet . As a result of this development and because of the reduction in face - to - face contact, it has become more common for hospital staff to both communicate online [27, 59] and offer telephone support following early discharge . However the participatory design process with involving the participants in the design of the technologies was valuable . We could use the concrete experiences with the use of the app in the intervention in the further design process, where there had to be adjustments to the chat function . The new adjustments mean that the nurses do not have to check the computer for new messages, but they got an iphone, where they receive a notification, whenever there is a new message . There is a potential to assess the app in a randomized controlled trial for a more generalizable knowledge . She was newly employed and had not been a part of the intervention . Yet some of the nurses at the ward were familiar with her, which could contribute to a comfortable and safe atmosphere during the interview [35, 61]. The app gives the nurses the possibility to offer support and information to the parents being early discharged, as the app is experienced as a lifeline that connects the homes of the new parents with the hospital . The written asynchronous communication provides an easy way for the nurses to offer the new parents support, when they are being early discharged, because the parents find it easier to contact the nurses through the app than the phone . This provides access to the healthcare professionals, which is essential in order to ensure parents' postnatal sense of security . The automated messages are a suitable way for informing the new parents and it encourages them to act independently, which can enhance parental self - efficacy because the parents are inspired to take action thereby gaining mastery experiences . The nurses experience that the app offers an efficient way to provide information to the parents as compared to pamphlets, because the parents were more likely to seek information when it was digitalized . The nurses generally tend to focus their actions around providing information, and they do not consider that written communication lends itself to a more open and extended dialogue . This could be a question of needing more time to adapt to this new way of communicating . With more time, they could possibly use the asynchronous communication not only to convey information and for observation purposes, but also to offer emotional support.
Phakic intraocular lens (piol) provides internal compensation of the dysfunctional refractive condition of the phakic eye and reduces or eliminates the dependence on glasses or contact lens . An implantable lens consisting of a biocompatible collagen copolymer (visian implantable collamer lens [icl]; staar surgical, nidau, switzerland) was developed in 1993 as a posterior chamber piol and was called the implantable contact lens, as initially it was thought that it would come into contact with the anterior surface of the crystalline lens . Staar (monrovia, ca, usa) patented this material made of 60% poly - hydroxyethylmethacrylate hema, water (36%), benzophenone (3.8%), and 0.2% porcine collagen, and called it the collamer (collagen - copolymer).1, 2 icl is a posterior chamber phakic iol which is a soft, flexible gel - lens ushering an era of reversible refractive surgery . Icls are ciliary sulcus placed posterior chamber piols that can be implanted through a small (3.0 mm), self - sealing limbal / clear corneal incision . In contrast with refractive lens exchange, icl implantation does not impair natural accommodation or increase the risk of retinal detachment above the background rate for untreated patients with high myopia, and have a good safety profile . Icl is a boon in achieving spectacle independence in patients who are unsuitable for laser refractive procedures like those with high myopia (> 13diopter d), thin corneas, those with expected residual stromal bed thickness less than 300, and severe dry eye . With its increasing acceptance and establishment of safety profile, icl implantation has become an increasingly popular choice for the correction of moderate to high myopia.4, 5 the convexo - concave design of the icl creates a vault between it and the anterior lens surface . However, the previous v4b icl model is known to cause pupillary block, and so either a preoperative / intraoperative laser / surgical peripheral iridotomy / iridectomy (pi) is required . To overcome this additional step, the v4c model with a central hole (0.36 mm) was developed in 2011 . It has a central hole in addition to two additional holes outside the optic facilitating aqueous outflow and removal of ophthalmic viscosurgical device (ovd) during surgery . However, complications of icl implantation such as cataract formation (anterior subcapsular lens opacities - typical butterfly cataract), endothelial cell loss, pigment dispersion, intraocular pressure (iop) elevation, and secondary glaucoma have been reported, and these complications are expected to increase with time.4, 5 studies have shown good acceptance profile of both the icl models.4, 5, 7 in view of the increasing prevalence of this surgical procedure, we conducted this study to evaluate the visual outcome, complication rate and safety indices of both the v4b and v4c icl models for the correction of high myopia in a tertiary eye care center in south india over a follow - up period of 9 months . All patients undergoing icl implantation for the correction of high myopia (manifest spherical equivalent mse 6 d) were included in the study . The following were the inclusion criteria: (a) age between 21 and 45 years, (b) stable refraction within the past 1 year, (c) patients not suitable for corneal - based laser refractive procedures those with abnormal corneal topography and keratoconus, predicted thin residual stromal bed thickness of less than 300, high refractive errors of> 13 d, severe dry eye, (d) corneal diameter> 11 mm, (e) internal anterior chamber depth acd (measured from endothelium)> 2.9 mm . Bilateral implantation of the same icl model for the correction of bilateral moderate - high myopia was preferred in an individual patient . A detail preoperative assessment was carried out including uncorrected distant visual acuity (udva), corrected distant visual acuity (cdva), iop measurement with non - contact tonometry nct (nt-510 nidek technologies, japan) and a gonioscopy to ensure wide open angles . A detailed slit - lamp examination to rule out any ocular pathology was done . A detailed fundus examination to rule out any myopia - related or other fundus pathology was done, and prophylactic barrier laser, if required, was given . Automated and manual keratometry values were recorded using topcon kr-8800 and ultrasound pachymetry using tomey pachymeter sp2000 . Corneal topography was performed using optikon 2000 keratron scout topographer (optikon, italy) and axial length and anterior chamber depth (acd) by sonomed pacscan 300a (sonomed, inc . The white - to - white (wtw) diameter was measured using a digital biometric ruler - digital calipers . Endothelial cell count (ecc) was measured using tomey em-3000 specular microscope (tomey corporation, japan). The icl power was calculated using the staar surgical customer service department formula that uses the acd, mean keratometry or simulated keratometry values, central corneal pachymetry, horizontal wtw distance, and refraction 12 mm from the corneal vertex . The horizontal axis was marked with the patient sitting at the slit lamp prior to the surgery . Two dots were placed on the corneal - limbal area with a surgical marker indicating 0 and 180 meridians as reference for later toric icl (ticl) alignment . Pupillary dilatation was achieved with a combination eye - drop containing 1% tropicamide and 2.5% phenylephrine . Two 1 mm paracenteses were made using angled keratome or 15 side port knife at 12 and 6 o'clock positions . Hypromellose 2% (viscomet pf, unimed technologies) viscoelastic was injected into the anterior chamber taking care not to overfill the chamber . The paracenteses were used to position the footplates under the iris using the special manipulating instruments like vukich's manipulator . It was ensured that all haptics were posterior to the iris . In case of v4b icl, the pupil was constricted with carbachol (mio - chol, 0.01% preservative free, usa, marketed in india by appasamy associates), and a single pi at 1 the o'clock position was done with vitrector under viscoelastic cover . In case of v4c icl, this step was skipped . In case of ticl proper alignment was ensured . At the end of surgery, viscoelastic was cleared from the ac . A standard postoperative regime consisting of topical prednisolone acetate 1% (pred forte, allergan, usa) 4 times a day for 5 days tapering over 2 weeks and topical gatifloxacin 0.3% (zymaxid, allergan las, irvine, usa) 4 times a day for 2 weeks was started . Timolol maleate eyedrops 0.5% (timolet, sun pharmaceuticals, india) was also started 2 times per day for 3 days . Postoperatively, the patient was examined at 4 h to check for proper icl positioning and vaulting on slit lamp, and iop was checked . The patient was then followed on postoperative day 1, 1 week, 1 month, 3 months, 6 months, and 9 months . The main surgical outcomes were evaluated at 1, 3, 6, and 9 months follow - up . At each of these visits, udva, cdva, mse, iop, icl vault was measured by anterior segment optical coherence tomography (as - oct) rtvue (model - rt100 version 6.9, fremont, usa). Patients were asked about subjective symptoms of glare and haloes at the end of follow - up period of 9 months . The safety index was calculated by dividing the postoperative cdva (in decimal) at 9 months by the preoperative cdva (in decimal). The efficacy index was calculated by dividing the postoperative udva (in decimal) by the preoperative udva (in decimal). The data were analyzed using spss version 20 (statistical package for social sciences) with paired t - test for intragroup comparison and mann whitney u value test for intergroup comparisons . Pupillary dilatation was achieved with a combination eye - drop containing 1% tropicamide and 2.5% phenylephrine . Two 1 mm paracenteses were made using angled keratome or 15 side port knife at 12 and 6 o'clock positions . Hypromellose 2% (viscomet pf, unimed technologies) viscoelastic was injected into the anterior chamber taking care not to overfill the chamber . The paracenteses were used to position the footplates under the iris using the special manipulating instruments like vukich's manipulator . It was ensured that all haptics were posterior to the iris . In case of v4b icl, the pupil was constricted with carbachol (mio - chol, 0.01% preservative free, usa, marketed in india by appasamy associates), and a single pi at 1 the o'clock position was done with vitrector under viscoelastic cover . In case of v4c icl, this step was skipped . In case of ticl proper alignment was ensured . At the end of surgery a standard postoperative regime consisting of topical prednisolone acetate 1% (pred forte, allergan, usa) 4 times a day for 5 days tapering over 2 weeks and topical gatifloxacin 0.3% (zymaxid, allergan las, irvine, usa) 4 times a day for 2 weeks was started . Timolol maleate eyedrops 0.5% (timolet, sun pharmaceuticals, india) was also started 2 times per day for 3 days . Postoperatively, the patient was examined at 4 h to check for proper icl positioning and vaulting on slit lamp, and iop was checked . The patient was then followed on postoperative day 1, 1 week, 1 month, 3 months, 6 months, and 9 months . The main surgical outcomes were evaluated at 1, 3, 6, and 9 months follow - up . At each of these visits, udva, cdva, mse, iop, icl vaulting, and ecc were evaluated . Icl vault was measured by anterior segment optical coherence tomography (as - oct) rtvue (model - rt100 version 6.9, fremont, usa). Patients were asked about subjective symptoms of glare and haloes at the end of follow - up period of 9 months . The safety index was calculated by dividing the postoperative cdva (in decimal) at 9 months by the preoperative cdva (in decimal). The efficacy index was calculated by dividing the postoperative udva (in decimal) by the preoperative udva (in decimal). The data were analyzed using spss version 20 (statistical package for social sciences) with paired t - test for intragroup comparison and mann whitney u value test for intergroup comparisons . A total of 62 eyes of 32 patients with a mean sd age of 24.56 4.8 years underwent v4b icl implantation (21 non - toric, 41 toric icl - ticl) with intraoperative peripheral iridectomy (pi), and 10 eyes of 5 patients with a mean sd age of 26.13 3.8 years had implantation of v4c icl with central hole (p = 0.81) (4 non - toric, 6 ticl). The large difference in the number of eyes in the two groups is due to the later development of the v4c model . The mean preoperative manifest spherical equivalent (mse) was 9.98 2.8 d and 9.14 2.4 d in the v4b and v4c groups, respectively (p = 0.51), which reduced to postoperative values of 0.24 1.3 d and 0.2 1.18 d, respectively (p = 0.09). The mse reduced significantly in the two groups (p <0.001 in both the groups). The mean preoperative astigmatism was 1.7 1.5 diopter cylinder (dcyl) and 1.8 1.5 dcyl which respectively reduced to 0.7 0.7 dcyl and 0.8 0.4 dcyl at 9 months (p <0.001 in both the groups) (table 1). A gain of 1 line of cdva was seen in 10% and 11.76% eyes in v4b and v4c groups, respectively (p = 0.08), while no change in cdva was seen in 90% and 88.24% of eyes (p = 0.07). No eye had loss of lines of cdva post surgery . At the end of 9 months follow - up, mean ecc loss was 6.4% and 6.1% (fig . 1, fig . 2) (p = 0.08), mean vault was 573.13 241.13, and 612 251.14, respectively, in the v4b and v4c groups (p = 0.02) (fig . 3). Anterior subcapsular opacities were present in 6.9% and 3.14% of eyes with v4b and v4c groups, respectively (p <0.01). Four eyes from v4b (9.75%) and 1 eye from v4c (16.66%) groups had rotation of more than 30 and required re - alignment surgery which was done successfully (fig . 4). Two eyes (3.22%) with v4b icl implantation had high postoperative iop (> 35 mm hg) due to blocked pi and required nd: yag laser iridotomy which was done with successful control of iop . The safety indices were 1.11 and 1.14 and efficacy indices were 1.4 and 1.5 in the v4b and v4c groups, respectively, at the end of 9 months . At the end of 9 months, on questioning, the most common subjective symptom reported was glare and haloes in 23% and 25% in the two groups, respectively (p = 0.09). However, they were not annoying enough to cause visual disability . Due to the large difference in the sample size between the two groups, the results of the intergroup comparisons done by the mann whitney u value test with p values is limited and need to be considered accordingly . Phakic intraocular lens implantation is so far the only refractive treatment for high myopia that offers preservation of accommodation and potential reversibility . In our study, we found that both the types of icl with or without the central hole showed a satisfactory visual outcome which was maintained at the end of 9 months follow - up period . . Also found similar results with the two models with both providing good visual outcome and no difference in the objective scatter index and higher order aberrations . Icls have emerged as a successful and promising modality for the treatment of moderate to high myopia especially in candidates unsuitable for laser refractive procedures.10, 11 though being an intraocular procedure, it provides the advantage of reversibility and an acceptable safety profile . With the advent of toric icl, a significant amount of astigmatism can be corrected . The ticl have shown to be stable over a long term period with the haptics enforcing stable lens position in the ciliary sulcus . The ticl is fundamentally different from toric intraocular lenses as it is not subject to contraction of the capsular bag . The soft footplates of the icl conform to the normal undulating contours of the ciliary sulcus with a kind of lock - and - key situation where the footplates will drape over and into the tiny irregular features of the sulcus ., we had 4 from v4b (9.75%) and 1 eye from v4c (16.66%) groups requiring realignment surgery with successful outcomes . Found an incidence of 1.7% of rotation in excess of 10 with 98.3% showing excellent rotational stability without decrease in visual acuity . The iop was maintained below 21 mm of hg in both the groups over 9 months . Higueras - esteban et al . Found no significant changes between the v4b and v4c models with respect to iop stability . Studied the fluid dynamics of aqueous humor in v4c model and suggested that hole - icls improve the circulation of aqueous humor to the anterior surface of the crystalline lens . Sanders reported approximately 67% of eyes developing anterior subcapsular opacities at 7 year following icl implantation but only 12% had progressed to clinically significant cataract in the same period, especially in high myopes and older patients . Fernandes et al . Also found cataract as the major complication . In our study, none of the eyes had visually significant cataract at the end of 9 months follow - up period . In a study conducted by pothireddy et al . In india, the safety index was 0.75, and the efficacy index was 1.04 twelve months postoperatively . Icl was thus evaluated to be a safe and effective procedure in terms of visual outcome . Pineda - fernandez et al . Reported mse in 61.1% and 22% of eyes within 1.00 d and 0.50 d of emmetropia . In their study, the mean residual sphere was 0.25 d, and mean residual cylinder was 0.12 dcyl . Insignificant refractive change during follow - up after icl implantation with similar results was obtained by igarashi et al . Demonstrated rapid cell loss until 1 year postoperatively, after which the rate of loss was no longer statistically significant.20, 21 in our study, the vitrector pi had clean cuts with minimum pigment dispersion and a chance of being incomplete or getting blocked . Iop was stable throughout the 9 months of follow - up in both the groups . In the v4c group, this could be attributed not just to the centraflow technology, but also to the negligible pigment dispersion due to avoidance of pi . None of our cases developed secondary glaucoma following excessive vault or pigment dispersion during the follow - up . In our study, the icl vault was maintained at 9 months follow - up period.22, 23 kamiya et al . Found the vault of the new central hole piol to be essentially equivalent to the vault of the conventional piol, suggesting that the presence of the central hole did not significantly affect the vault or the refractive accuracy . Kamiya et al.25, 26 found the v4c icl with aquaport essentially equivalent in the optical quality variables to conventional icl implantation . They suggested that the presence of the central artificial hole does not significantly affect the optical quality and the intraocular scattering after surgery . Other studies27, 28, 29, 30 have also found good optical quality results with v4b and v4c icls . Studied piols and found that v4b icl implantation leads to decreased night vision performance with glare and haloes . Many studies33, 34, 35, 36, 37, 38 have shown good visual performance and quality of life after icl implantation . In our study, patients in both groups experienced glare and haloes, but they were visually insignificant and non - annoying . Icl thus offers a safe, effective, and reversible option for correction of high myopia . However, evaluation of the incidence of cataract formation and endothelial cell loss over a decade should be carried out . The small sample size and unequal number of patients in the two groups precludes definite conclusion . Also, including both eyes of one patient in the study, limits the conclusion . Icl with a central hole offers an added advantage of annulling a pi and providing a stable iop . However, a larger sample size undergoing v4c implantation with a longer follow - up is required for confirming the results.
The number of men and women worldwide who identify as lesbian, gay, or bisexual (lgb) has increased in recent years . According to a survey undertaken in 2011 by the williams institute at the ucla school of law, approximately 9 million people (3.5% of adults) in the united states identify as lgb and an additional 0.3% of adults identify as transgender (1). In 2003, a study on australia reported that among adults aged 1659, 1.6% and 0.9% of males identified as gay and bi - sexual, respectively, and 0.8% and 1.4% of females identified as lesbian and bisexual, respectively (2). In denmark in 1992, 2.7% of adult men and women reported having had same - sex sexual experiences (3). In france in 1992, 4.1% of men and 2.6% of women reported at least one same - sex sexual experience during their lifetime (4). In ireland in 2006, 2.7% of men and 1.2% of women identified as either homosexual or bisexual (5), while in a 2003 norwegian survey, 12% of respondents reported having had a same - sex sexual experience (6), and 6.1% of people surveyed in the united kingdom in 1992 had had a same - sex sexual experience (7). In summary, the number of men and women worldwide who identify as lesbian, gay, or bisexual (lgb) has increased in recent years, with up to 12% of respondents reporting having engaged in same - sex sexual activity (1 7). Locally, even though statistics for the lgbt population cannot be determined for certain korean cultures, of a sample of 1,748 adolescents in south korea, 12.7% had problems with their gender identity (8). A number of studies have shown higher rates of diseases such as hiv / aids among the lgb population (911), as well as a higher prevalence of asthma (12), obesity (13), and poor health behavior, indicating a relationship between this group s atypical sexual orientation and health - related lifestyle (1415). For this and other reasons, however, there is currently almost no research in south korea on the prevalence of lgb identity and sexual behavior, meaning that the korean government has no information regarding possible public health concerns among the lgb population . Therefore, the purpose of the present study was to determine whether lifestyle - related factors, including physical activity (pa), differ between lesbian, gay, bisexual, and heterosexual korean adolescents . The 8 korea youth risk behavior web - based survey (kyrbws - viii) is an annual epidemio - logical and cross - sectional study that assesses the public health of korean adolescents . It uses a complex sample design of 43 clustering techniques, 129 stratification techniques, and multistage sampling to cover all of south korea while limiting the sample size to a manageable size: 400 middle schools and 400 high schools . All of the details regarding the data collection procedure have been described by the ministry of education, science, and technology, the ministry of health and welfare, and the korea centers for disease control and prevention (16). This survey has well - established validity and reliability for this population (1718). In the kyrbws - viii, students are assigned unique identification numbers by their classroom instructors, and then access the survey web page using these numbers . On the web page, they are asked if they are willing to participate; students who choose to participate complete the questionnaire at their schools anonymously . Because the kyrbws - viii does not collect any private information (e.g., students names, social security numbers, school names, telephone numbers, or home addresses), ethical approval was not required . The overall response rate for participation in the kyrbws - viii was 96.4% (74,186 out of 76,980 students; 38,221 males and 35,965 females). However, only 11,829 of these 74,186 participants provided enough information about their romantic and sexual behavior to be categorized as gay, lesbian, bisexual, or heterosexual . Sexual identity was evaluated using the item select all of the following items that you have experienced . Kissing and fondling someone of the opposite sex, sexual intercourse with someone of the opposite sex, kissing and fondling someone of the same sex, sexual intercourse with someone of the same sex, respondents who responded yes to only or (i.e., respondents who reported sexual or romantic behavior with people of the same sex only) were categorized as homosexual (gay or lesbian), respondents who responded yes to both or and or (i.e., respondents who reported sexual or romantic behavior with people of both sexes) were categorized as bisexual, and respondents who responded yes to only or (i.e., respondents who reported sexual or romantic behavior with people of the opposite sex only) were categorized as heterosexual . The subjects demographic characteristics subjects who reported that they had never engaged in sexual or romantic behavior (responding yes to), or who reported only experiences of sexual violence (responding yes to or) were excluded, as these items provided no information on sexual identity . Using these criteria, males were divided into gay (n = 323), bisexual (n = 243), and heterosexual (n = 6,501) groups, and females were divided into lesbian (n = 208), bisexual (n = 113), and heterosexual (n = 4,441; table 1) groups . The data were then analyzed according to the groups sexual orientation for any significant relationships with certain key lifestyle behaviors: smoking cigarettes, drinking alcohol, eating breakfast, mental stress, and exercise (vigorous, moderate, muscular strength exercises and walking at least 10 minutes per week) through the kyrbws - viii . Possible responses to these items are given as follows . Frequency of vigorous pa, frequency of moderate pa, and frequency of muscular strength exercises per week were 1 = never, 2 = once, 3 = 5 = four times, and 6 = five times or more . Vigorous pa included activities such as digging, aerobics, heavy lifting, or fast cycling; moderate pa included activities such as cycling at a regular pace, carrying light loads, or playing doubles tennis; and muscular strength exercises included activities such as sit - ups, push - ups, and weight lifting or weight training . Frequency of smoking and frequency of drinking per month had possible responses of 1 = never, 2 = 2029 days, and 7 = every day for . For frequency of breakfast consumption and frequency of walking at least 10 minutes per week, response options included 1 = never, 2 = once, 3 = twice, 4 = three times, 5 = 4 times, 6 = 5 times, 7 = 6 times, and 8 = every day . Finally, self - reported mental stress was assessed using responses of 1 = very high, 2 = high, 3 = normal, one - way analysis of variance (anova) was used to identify differences in lifestyle factors, especially physical activity, among the three sexual identity groups (homosexual [gay or lesbian], bisexual, and heterosexual). The analyses were performed using spss version 18.0 (chicago, il, usa) and statistical significance was set at p <0.05 . All study results are given as the mean standard deviation or number (%). Sexual identity was evaluated using the item select all of the following items that you have experienced . Possible responses were: none of these, kissing and fondling someone of the opposite sex, sexual intercourse with someone of the opposite sex, kissing and fondling someone of the same sex, sexual intercourse with someone of the same sex, respondents who responded yes to only or (i.e., respondents who reported sexual or romantic behavior with people of the same sex only) were categorized as homosexual (gay or lesbian), respondents who responded yes to both or and or (i.e., respondents who reported sexual or romantic behavior with people of both sexes) were categorized as bisexual, and respondents who responded yes to only or (i.e., respondents who reported sexual or romantic behavior with people of the opposite sex only) were categorized as heterosexual . The subjects demographic characteristics subjects who reported that they had never engaged in sexual or romantic behavior (responding yes to), or who reported only experiences of sexual violence (responding yes to or) were excluded, as these items provided no information on sexual identity . Using these criteria, males were divided into gay (n = 323), bisexual (n = 243), and heterosexual (n = 6,501) groups, and females were divided into lesbian (n = 208), bisexual (n = 113), and heterosexual (n = 4,441; table 1) groups . The data were then analyzed according to the groups sexual orientation for any significant relationships with certain key lifestyle behaviors: smoking cigarettes, drinking alcohol, eating breakfast, mental stress, and exercise (vigorous, moderate, muscular strength exercises and walking at least 10 minutes per week) through the kyrbws - viii . Possible responses to these items are given as follows . Frequency of vigorous pa, frequency of moderate pa, and frequency of muscular strength exercises per week were 1 = never, 2 = once, 3 = twice, 4 = three times, 5 = four times, and 6 = five times or more . Vigorous pa included activities such as digging, aerobics, heavy lifting, or fast cycling; moderate pa included activities such as cycling at a regular pace, carrying light loads, or playing doubles tennis; and muscular strength exercises included activities such as sit - ups, push - ups, and weight lifting or weight training . Frequency of smoking and frequency of drinking per month had possible responses of 1 = never, 2 = 1019 days, 6 = 2029 days, and 7 = every day for . For frequency of breakfast consumption and frequency of walking at least 10 minutes per week, response options included 1 = never, 2 = twice, 4 = three times, 5 = 4 times, 6 = 5 times, 7 = 6 times, and 8 = every day . Finally, self - reported mental stress was assessed using responses of 1 = very high, 2 = high, 3 = normal, one - way analysis of variance (anova) was used to identify differences in lifestyle factors, especially physical activity, among the three sexual identity groups (homosexual [gay or lesbian], bisexual, and heterosexual). The analyses were performed using spss version 18.0 (chicago, il, usa) and statistical significance was set at p <0.05 . All study results are given as the mean standard deviation or number (%). The differences in lifestyle factors, especially physical activity, among the sexual identity groups are shown in table 2 . Using one - way anova, we found that males showed significant differences by sexual identity group for frequency of smoking (p = 0.029), alcohol consumption (p <0.001), muscular strength exercises (p = 0.020), and walking for at least 10 minutes (p <0.001). Post - hoc test indicated that gay males performed muscular strength exercises less frequently (p <0.05) and walked for at least 10 minutes less frequently (p <0.001) than heterosexual males . Bi - sexual males consumed alcohol more frequently (p <0.001) and walked for at least 10 minutes less frequently (p <0.001) than heterosexual males . We also found, again using one - way anova, significant differences among females according to sexual identity group for frequency of smoking (p <0.001), alcohol consumption (p <0.001), vigorous pa (p <0.001), moderate pa (p <0.001), and muscular strength exercises (p <0.001). There were also significant inter - group differences for self - reported mental stress (p <0.001). Post - hoc test showed that lesbian females reported more frequent vigorous pa (p <0.001), moderate pa (p <0.001), and muscular strength exercises (p bisexual females engaged more frequently in smoking (p <0.001), alcohol consumption (p <0.001), vigorous pa (p <0.001), moderate pa (p <0.001), and muscular strength exercises (p <0.001) than did heterosexual females, in addition to higher self - reported mental stress (p <0.001). This study examined whether there were differences in lifestyle factors by sexual orientation in korean adolescents . The most notable finding in the current study was that 887 adolescents (7.5% of the sample) had some same - sex romantic or sexual experience, such as kissing, fondling, or sex . This statistic indicates that the lgb adolescent population in korea is larger than that of many other countries that were surveyed (9). This comparably large lgb population may pose a public health risk in terms of sexually transmitted diseases . For this reason, we recommend that the korean government put into place school- and community - based programs for preventing social and health problems in the lgb community . The differences in lifestyle factors among the sexual identity groups prior studies have shown that gay and bisexual men have poorer general and mental health (9), higher rates of smoking (19) and higher rates of alcohol use (20) than do heterosexual men . Studies have also indicated that lesbians and bisexual women have poorer overall and mental health (13), higher rates of smoking (21 22), and higher rates of alcohol use (2324). The results of the present study support these previous findings, in that gays, lesbians, and bisexual adolescents of both genders had higher rates of smoking and alcohol consumption than heterosexual adolescents did . However, although gay and bisexual males reported lower rates of muscular strength exercises and walking than their heterosexual counterparts, which might be indicative of poorer physical health, lesbians and bisexual females reported higher rates of vigorous pa, moderate pa, and muscular strength exercises than did their heterosexual counterparts, which might be indicative of better physical health . Although the kyrbws - viii did not gather any information that might help us to better understand the reason for this difference, we hypothesize that this might be because lesbians may tend to assume male roles or lifestyles, leading lesbians and bisexual females to engage in more physical activities than heterosexual females . This study is limited in that the kyrbws - viii was a cross - sectional, retrospective cohort study; as a result, we cannot make any statements regarding causality . Furthermore, although sexuality in adolescence may remain consistent throughout the lifespan, it is well known that sexual orientation in childhood and adolescence is not likely to persist into adulthood . Therefore, findings from this study cannot be generalized to the adult population in korea . Moreover, since only participants who met the criteria for inclusion took part in the survey, findings from this study cannot be generalized to the entire youth population in south korea . However, as the first national study on the lifestyle behaviors of the adolescent lgb population in south korea, we believe that this study will both encourage additional inquiry in this area and provide valuable information to promote the public health of this population . An additional limitation could be the fact that, at such a young age and with so little sexual experience, the sexual experiences reported by participants may not reflect their true, respective sexual orientations . We concluded that gay, lesbian, and bisexual males and females fare poorly with regard to healthy lifestyle behaviors, as compared to their heterosexual peers ., we suggest that efforts be undertaken by schools and communities to modify and prevent unsuitable lifestyle behaviors among lgb adolescents, using social and health education programs that specifically target this group . 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.
Hamstring injuries are common in sports that involve sudden bursts of running, and can be frustrating for both athletes and physicians due to the unpredictability and wide variability of return to play in any given athlete . Of all muscle injuries, hamstring strains have one of the highest recurrence rates . Biomechanical analysis of hamstring function is complex due to its ability to influence movement at multiple joints, which in turn makes identification of clear risk factors and development of effective injury prevention scenarios a challenge . The purpose of this review is to describe hamstring injuries as they relate to an athletic population and to summarize the current literature with respect to risk stratification, treatment decisions, and prevention of recurrent injury . The hamstring is actually a group of four muscles, three of which originate in common at the ischial tuberosity and then diverge to attach distal to the knee . Semimembranosus and semitendinosus originate at the mid - portion of the ischial tuberosity and attach distally at the posteromedial tibia, while the long head of biceps femoris arises from both the lateral portion of the ischial tuberosity and the sacrotuberous ligament and attaches distally at the fibular head, in common with the short head of biceps femoris . The short head of biceps femoris arises from the lateral intermuscular septum and lateral femoral cortex at its distal third . The multiple attachments of the hamstring allow this muscle to impact function throughout the pelvis and lower extremities . In addition to flexion and extension of the knee, the hamstrings affect pelvic tilt and rotation, sacral rotation and extension and rotation of the hip [2, 3]. Much of the biomechanical analysis of hamstring function has been done using sprinters running on a treadmill . These analyses consistently show that the maximal lengthening of the hamstring occurs at the end of the swing phase of gait, just prior to foot contact, when the hip is flexed and the knee flexion moment is reducing . During sprinting, which involves more hip flexion, emg analysis confirms that the maximal hamstring contraction also correlates to this portion of the running phase, as the hamstrings apply a braking force to the quadriceps and hip flexors . The period of maximal eccentric contraction in the running cycle, when the muscle is both lengthening and contracting at the same time, seems to carry a higher risk for muscle injury . A low hamstring - to - quadriceps strength ratio will increase the extension moment through the knee, potentially stretching the eccentrically contracting hamstring beyond its elastic capabilities . Most epidemiological studies show a relatively higher rate of injury to the biceps femoris as compared to the medial hamstring muscles [2, 4, 8, 9]. This is felt to relate to the fact that the hip and knee flexion moments are less for biceps femoris than for its medial counterparts . Athletes who bend forward at the hips to accelerate while running tilt their pelvis forward and increase tension on the hamstrings . Over - lengthening of the long head of biceps femoris has been documented as the cause of acute hamstring strain in injuries captured on video analysis . Tightness of the iliopsoas is directly responsible for a relative anterior pelvic tilt that places the hamstring at a mechanical disadvantage by increasing the tension at the muscle when the end of the swing phase is reached . Different activities recruit the hamstring in different ways, and the implications for injury also vary . Water skiers are at a higher risk for proximal injury when being pulled out of the water . This occurs if they are forcefully pulled into hip flexion while maintaining their knees fully extended . Hockey players, whose feet are constantly on ice, are thought to develop hamstring problems that stem from core muscle weakness . Weakness of the abdominal muscles allows an anterior pelvic tilt, placing the hamstrings at a mechanical disadvantage that can lead to overuse injury . Marathon runners have been shown to develop decreased eccentric strength at the hamstrings during the course of a race . Most epidemiological research on athletes and hamstring strains has been performed on soccer or australian rules football players [8, 9, 1315]. Both sports demand frequent bursts of sprinting over a prolonged period of time, with a higher risk for muscle fatigue . The kicking aspect of these sports also predisposes to a muscle injury that can require more prolonged recovery times, since the forces driving the hip into flexion during the acceleration phase of the kick are relatively greater . Likewise, demand for the eccentric braking action of the hamstring muscles is greater during the kicking motion as well . Risk factors that have been associated with hamstring injury include competition (versus practice), decreased quadriceps flexibility (which keeps the knee relatively extended), older age (which has been shown to be associated with increased hip flexor tightness and increased body weight), over - striding during sprint acceleration, black race, low hamstring: quad ratio, muscle fatigue, hamstring tightness (though it is unclear whether this is a cause or consequence of injury), decreased tendon compliance, later stages of competition, and previous injury, which is consistently one of the highest predictors of subsequent injury risk . The incidence of recurrent injury runs between 12 and 14% in soccer players, and as high as 30% in australian rules football . Premature return - to - play is also cited as a risk [9, 13, 1618]. Sprinters have been shown to average 16 weeks for full return, while dancers in the same study averaged 50 weeks . The diagnosis of hamstring strain is usually readily apparent on clinical exam, with tenderness at the muscle belly, and occasionally subcutaneous ecchymosis or a palpable muscular defect noted . A diagnostic test for biceps femoris strains has been recently described whereby the patient is asked to remove the shoe of the affected leg while standing by levering the heel against the medial tibial border of the contralateral (stance) leg . The need for imaging of suspected hamstring injuries is dependent on the patient being evaluated . Patients with open physes and suspected proximal injury should have radiographs obtained to rule out apophyseal avulsion fractures of the ischial tuberosity . Surgical repair of fractures displaced more than 2 cm is debated, but advocated by some . Older patients with suspected common tendon rupture should either be sent on for surgical consultation or imaged with mri to rule out the injury . Delayed repair of proximal hamstring tendon ruptures that have been missed clinically often leads to suboptimal outcomes because of the interval shortening of the muscle - tendon unit . This shortening can also lead to secondary complications such as sciatic nerve injury or compartment pressure syndrome of the thigh [21, 22]. There have been several studies looking at the extent of injury noted on mri to predict recuperative time . Although it appears that the length of injury noted on coronal views does have predictive value, there has been no clear benefit established over clinical evaluation alone [2325]. Initially, the treatment of acute hamstring injuries is similar regardless of the mechanism of injury, after which, management tends to diverge based on the activity requirements of the individual . Otherwise, early treatment goals focus on minimizing intramuscular bleeding and controlling the inflammatory response . Ice massage and compression wraps are both helpful in this regard [16, 26]. Anti - inflammatory medication can be used acutely to try to moderate (but not eliminate) the effects of the inflammatory cascade . Nsaid use has been shown in animal models to be detrimental to tissue healing when used for long periods of time, but for shorter periods can help to minimize inflammatory - mediated damage to healthy tissue that is peripheral to the injury . Anti - inflammatories also reduce tissue soreness and probably allow earlier progression from total rest to early rehabilitation . It has been suggested through uncontrolled case studies that intramuscular steroid injections speed return to play in professional football players . Balanced against this is the knowledge that corticosteroid injections have the potential to inhibit collagen linkage and slow overall tissue healing . In most studies, prophylactic stretching has not been shown to influence the rate of hamstring injury, but stretching after an injury may be more helpful . One controlled study that looked at this found an increased benefit when sets of four hamstring stretches (begun 48 h after injury) were repeated four times per day in comparison to once per day . Hamstring flexibility tends to decrease after an acute injury, in part due to an increase in intramuscular scar tissue formation . Restoration of muscle length reduces eccentric loads that are imposed at peak joint torque once activity is resumed . Stretching the hamstrings with the pelvis maintained in an anterior tilt there are no controlled studies comparing different return - to - play strategies for hamstring injury, though some uncontrolled studies have been published that suggest the value of using objective recovery of strength and optimization of the hamstring: quadriceps ratio determined through isokinetic testing [32, 33]. Functional rehabilitation of hamstring injuries must be tailored to the individual needs of the patient . General goals include restoration of pre - injury muscle strength and flexibility, as well as assistance with pain relief . Supervised strength programs gentle concentric strengthening should be initiated first, followed by progressive open - chain eccentric strength exercises . Eccentric strength exercises are more effective than concentric exercise and can assist standard stretching in re - establishing muscle length after injury [34, 35]. The biarticular nature of the hamstring muscle, as well as its wide array of muscle attachments, requires attention beyond the hamstring itself . Abdominal and core muscle strengthening keeps the pelvis in an optimal position for hamstring function and increases the overall stability of the pelvic platform an important goal for athletes such as hockey and soccer players, who are in closed - chain positions for longer periods of time . Rehabilitation programs that emphasize core stability have been shown to outperform those that focus on isolated progressive hamstring strength training . Return - to - play decisions are usually supported by a measurable return of strength to 9095% of pre - injury levels, a demonstrated ability to perform progressive sport - specific challenges without pulling up, and readiness on the part of the athlete . Generally, athletes who are involved in ballistic power activities such as sprinting (with less ability to use other muscle groups to compensate around an injured hamstring) should be held from practice or competition for longer periods of time, until demonstrated strength of the injured leg is approaching that of the uninjured side . Preventative measures such as the nordic hamstring exercises are advocated for use with athletes wishing to reduce their overall risk of injury . Several early studies have shown an apparent treatment benefit, but these are still awaiting confirmation through larger trials [8, 34]. One interventional study done on australian rules football players that demonstrated a positive benefit incorporated anaerobic high - intensity running drills, frequent hamstring stretches, sport - specific training drills, and counseling on resistance training after an initial 2-year period of pre - interventional injury analysis . Surgical treatment should be reserved for widely displaced avulsion fractures of the ischial tuberosity or complete proximal hamstring tendon rupture . Repair of partial common tendon ruptures has also been shown to obtain good functional outcomes in selected patients who are failing conservative attempts at care . Hamstring mechanics are complex, making efforts at predicting injury patterns and developing injury treatment and prevention protocols difficult . Efforts at management should incorporate proven standards of acute muscle injury care with rehabilitation efforts that consider the total activity - specific demands on lower extremity and pelvic kinetics.
As a consequence of the availability of whole - genome expression methodologies, regulation of gene expression is at the core of current post - genomic studies . Once a set of genes is clustered on the basis of similar expression profiles, a logical next step is that of searching their upstream regions for potential binding sites for transcriptional regulators . The predicted binding sites in dna can then be mutated or used to fish out the dna - binding regulatory protein . Different methods exist for finding binding sites, with a recent rapid increase in different methods with small variations and improvements . However, as the computational biology community has long been aware, a common limitation of such methods is the high rate of false - positives that they generate as a result of the low degree of conservation of the dna sequences of binding sites . This work is a contribution towards a more detailed evaluation of the performance of these methods, with the aim of finding the best selection of thresholds to provide reliable predictions . On the basis of our evaluations, we suggest improved methods to search for novel binding sites that give a much lower rate of false positives . We use information gathered in regulondb, a database on regulation of transcription in escherichia coli compiled from the literature . The database contains data on regulons - sets of genes in transcription units whose expression is regulated by the same regulatory proteins - with different types of evidence and different levels of description . For instance, at the time of writing, the database contains information on 112 regulatory proteins, but binding sites in dna are only described for 60 of these . The data for 26 of the regulatory proteins includes information on at least three regulated genes, with at least one binding site per gene (table 1). As explained below, we distinguish between pattern discovery and pattern search and evaluate each separately . One is dyad - analysis, a program developed to find over - represented small words separated by a given distance . We also describe and evaluate an elaboration of this method that aims to search for probable binding sites using the dyads generated (dyad sweeping). The other method uses consensus, a program that generates optimized ungapped multiple alignments for sets of known or suspected regulatory sequences and builds matrices representing the frequency of each base at each position of the aligned sequences . Its companion program' patser' uses the matrices generated to scan for similar new sequences . The evaluations take into account the interest in minimizing the false - positive rate, as even a very small false - positive rate can overshadow true positives because of the small number of genes expected to be part of each regulon (see below). As most regulatory sites for dna - binding proteins are found 200 to 400 base - pairs (bp) upstream of the regulated genes, we built two sets of upstream regions . One contained 200 bp of the region upstream of the genes' start sites plus 50 bp downstream (200 + 50 set); the other contained 400 bp upstream plus 50 bp downstream of the start sites (400 + 50 set). Repressor sites are located near the promoter site, whereas activators tend to occupy a larger region upstream of the promoter . It is therefore potentially useful to evaluate the performance of the methods with these two different ranges of sequence . Additional information can also influence the decision of the experimentalist to select the length of upstream region to analyze . For instance, some proteins tend to have a single binding site per promoter, which has to be proximal to the promoter (for example lexa), whereas other proteins tend to have several binding sites per upstream region, with some of them farther upstream of the promoter (for example arac, lrp and metj). Another factor that influences the size of region to analyze is whether the precise site of transcription initiation (the + 1 position) is known . When the promoter is known, the search can be limited to 200 bp upstream from the + 1 position . If it is not known, then the reference point has to be the start codon and the 400 bp upstream of this are used - which assumes an average of 50 to 100 bp between the promoter and the beginning of the gene . We used the total set of upstream regions containing at least one reported binding site in regulondb as the basic data for evaluation . In each case, upstream regions of genes regulated by the same protein (regulons) were separated from the collection and constituted the' training sets' . For each set, the remaining upstream regions, known to be regulated by other proteins, are assumed to be the collection of' known negatives' . Though there is still a risk that the known negatives contain genes that also pertain to the regulon we are contrasting them with, the fact that they have been the subject of experimental work allows us to think that this risk is minute . Because of the small amount of data for each protein, we could not leave out a set of known positives to evaluate the rate of true positives, except in the case of the regulatory protein crp . For those families having at least five upstream regions we were able to apply a' leave one out' procedure as described below . We also have information, in some cases, on genes regulated by a given protein in the regulons analyzed, but with no reported binding site . The upstream regions of these genes were used to search for binding sites and provide further evaluation . A more detailed analysis was performed for lexa, comparing our predictions with a recent report in the literature . Depending on the information available, there are basically two computational approaches to predicting binding sites for transcription initiation factors in dna . In the best cases, there is information on experimentally determined examples of binding sites for a given regulatory protein . In such cases, the search programs can be trained using the sequences corresponding to the binding sites, and the information obtained (dyads, weight matrices) can then be used to find similar sequences, and thus other genes that might be under the control of the same regulatory protein . On the other hand, a common scenario at present is that a set of apparently co - regulated genes is identified from transcriptome experiments . In this case, a program would be trained with a collection of upstream regions from these genes with the goal of identifying probable shared regulatory sites . If the data come from transcriptome experiments, the collection of co - regulated genes might not be complete . Because of the noise inherent to such experiments, and/or to the limitations of clustering algorithms, a researcher might wish to try to find other genes likely to be under the control of the same protein . However, other genes regulated by the same protein might display a different pattern of expression as a result of complications such as regulation by more than one regulatory protein . On the basis of these considerations, the analyses we present contemplate the use of experimentally determined binding sites as training sets to study pattern search, and the use of upstream regions of co - regulated genes to study pattern discovery . More precisely, we use the set of binding sites in dna for each regulatory protein reported in regulondb to try to find additional genes in the genome with similar sites . We also use the data on known co - regulated genes to try to find the binding site within the genes' upstream regions . As training sets, we ran the dyad or matrix search programs on the sequences of known regulatory binding sites and on upstream regions of 200 + 50 and 400 + 50 bp from genes regulated by a given regulatory protein . Families corresponding to a given regulatory protein were evaluated only if there were at least three sequences in the corresponding training set (40 in the collection of binding sites; 26 in the 200 + 50 and the 400 + 50 datasets). Subsequently, the dyads and matrices were evaluated against the complete collections of 200 + 50 and 400 + 50 upstream regions . This gives a total of 3 2 = 6 evaluations for each regulon analyzed . The evaluations included regions 200 + 50 or 400 + 50 only if there was at least one reported binding site within that range; thus, the total set of 200 + 50 regions contained 172 sequences, and the 400 + 50 set contained 189 . We used the dyad - analysis program to find dyads within each training set . The options used were to find dyads of 3 bp long separated by distances of 0 to 16 bp, with any kind of dyad (direct repeat, inverted repeat, asymmetric), searching in both dna strands . Further analyses were limited to the training sets where the program found at least one dyad with a significance equal to or above 1.0 (see for a detailed description of significance). This left 19 families from the binding - sites training sets, 11 from the 200 + 50 regions, and 14 from the 400 + 50 regions (the program dyad - analysis did not find any dyad in about 75% of the rejected families, and found just one in most of the rest of them). The program consensus was run to obtain alignments and matrices 20 bp long - the most frequent size among binding sites for regulatory proteins . To assign match scores, we used an' alphabet' based on the frequency of each base at upstream regions of 200 + 50 and 400 + 50 of all genes in e. coli . The search was done in a single strand . Although we also ran the program to find symmetric patterns, we first present results of pattern discovery, then concentrate on the selection of the best thresholds, analyzing their performance on the basis of the evaluation criteria described above . As most regulatory sites for dna - binding proteins are found 200 to 400 base - pairs (bp) upstream of the regulated genes, we built two sets of upstream regions . One contained 200 bp of the region upstream of the genes' start sites plus 50 bp downstream (200 + 50 set); the other contained 400 bp upstream plus 50 bp downstream of the start sites (400 + 50 set). Repressor sites are located near the promoter site, whereas activators tend to occupy a larger region upstream of the promoter . It is therefore potentially useful to evaluate the performance of the methods with these two different ranges of sequence . Additional information can also influence the decision of the experimentalist to select the length of upstream region to analyze . For instance, some proteins tend to have a single binding site per promoter, which has to be proximal to the promoter (for example lexa), whereas other proteins tend to have several binding sites per upstream region, with some of them farther upstream of the promoter (for example arac, lrp and metj). Another factor that influences the size of region to analyze is whether the precise site of transcription initiation (the + 1 position) is known . When the promoter is known, the search can be limited to 200 bp upstream from the + 1 position . If it is not known, then the reference point has to be the start codon and the 400 bp upstream of this are used - which assumes an average of 50 to 100 bp between the promoter and the beginning of the gene . We used the total set of upstream regions containing at least one reported binding site in regulondb as the basic data for evaluation . In each case, upstream regions of genes regulated by the same protein (regulons) were separated from the collection and constituted the' training sets' . For each set, the remaining upstream regions, known to be regulated by other proteins, are assumed to be the collection of' known negatives' . Though there is still a risk that the known negatives contain genes that also pertain to the regulon we are contrasting them with, the fact that they have been the subject of experimental work allows us to think that this risk is minute . Because of the small amount of data for each protein, we could not leave out a set of known positives to evaluate the rate of true positives, except in the case of the regulatory protein crp . For those families having at least five upstream regions we were able to apply a' leave one out' procedure as described below . We also have information, in some cases, on genes regulated by a given protein in the regulons analyzed, but with no reported binding site . The upstream regions of these genes were used to search for binding sites and provide further evaluation . A more detailed analysis was performed for lexa, comparing our predictions with a recent report in the literature . Depending on the information available, there are basically two computational approaches to predicting binding sites for transcription initiation factors in dna . In the best cases, there is information on experimentally determined examples of binding sites for a given regulatory protein . In such cases, the search programs can be trained using the sequences corresponding to the binding sites, and the information obtained (dyads, weight matrices) can then be used to find similar sequences, and thus other genes that might be under the control of the same regulatory protein . Is that a set of apparently co - regulated genes is identified from transcriptome experiments . In this case, a program would be trained with a collection of upstream regions from these genes with the goal of identifying probable shared regulatory sites . If the data come from transcriptome experiments, the collection of co - regulated genes might not be complete . Because of the noise inherent to such experiments, and/or to the limitations of clustering algorithms, a researcher might wish to try to find other genes likely to be under the control of the same protein . However, other genes regulated by the same protein might display a different pattern of expression as a result of complications such as regulation by more than one regulatory protein . On the basis of these considerations, the analyses we present contemplate the use of experimentally determined binding sites as training sets to study pattern search, and the use of upstream regions of co - regulated genes to study pattern discovery . More precisely, we use the set of binding sites in dna for each regulatory protein reported in regulondb to try to find additional genes in the genome with similar sites . We also use the data on known co - regulated genes to try to find the binding site within the genes' upstream regions . As training sets, we ran the dyad or matrix search programs on the sequences of known regulatory binding sites and on upstream regions of 200 + 50 and 400 + 50 bp from genes regulated by a given regulatory protein . Families corresponding to a given regulatory protein were evaluated only if there were at least three sequences in the corresponding training set (40 in the collection of binding sites; 26 in the 200 + 50 and the 400 + 50 datasets). Subsequently, the dyads and matrices were evaluated against the complete collections of 200 + 50 and 400 + 50 upstream regions . This gives a total of 3 2 = 6 evaluations for each regulon analyzed . The evaluations included regions 200 + 50 or 400 + 50 only if there was at least one reported binding site within that range; thus, the total set of 200 + 50 regions contained 172 sequences, and the 400 + 50 set contained 189 . We used the dyad - analysis program to find dyads within each training set . The options used were to find dyads of 3 bp long separated by distances of 0 to 16 bp, with any kind of dyad (direct repeat, inverted repeat, asymmetric), searching in both dna strands . Further analyses were limited to the training sets where the program found at least one dyad with a significance equal to or above 1.0 (see for a detailed description of significance). This left 19 families from the binding - sites training sets, 11 from the 200 + 50 regions, and 14 from the 400 + 50 regions (the program dyad - analysis did not find any dyad in about 75% of the rejected families, and found just one in most of the rest of them). The program consensus was run to obtain alignments and matrices 20 bp long - the most frequent size among binding sites for regulatory proteins . To assign match scores, we used an' alphabet' based on the frequency of each base at upstream regions of 200 + 50 and 400 + 50 of all genes in e. coli . The search was done in a single strand . Although we also ran the program to find symmetric patterns, no clear improvement was observed . In the results section, we first present results of pattern discovery, then concentrate on the selection of the best thresholds, analyzing their performance on the basis of the evaluation criteria described above . Finally, we present some specific predictions . Pattern discovery starts with a collection of co - regulated genes for which no binding sites are yet known . To evaluate the methodology, we counted the number of times a sensor can locate a known binding site in a collection of 200 + 50 or 400 + 50 regions . Over - represented words would be expected to occur at the binding sites, and thus the first step was to determine if the resulting dyads match the binding sites . We found that there are significant dyads all along the sequences analyzed, with most of them matching at or near the known binding sites . Figure 1 shows, using the purr family, that most dyads were found at distances very close to or overlapping the true binding sites . We thus decided to search for stretches of contiguous matches, which we call' regions of overlapping matches' (roms), in the upstream sequences being analyzed by counting (sweeping), base by base, the number of matching dyads . As seen in figure 2, the roms with the highest number of matching dyads overlap the true known binding sites in the dna . This result motivated us to use the highest number of matches within a rom as the score . We call this method dyad sweeping . As the highest - scoring roms frequently overlap reported binding sites (figure 2, table 2), we decided to keep, for subsequent analyses, the dyads found within the highest - scoring roms of each upstream region, as long as the rom contained at least two dyads . In table 3 it can be seen that, except in a few of the regulons, the fraction of regions with known binding sites found is quite high . In other words, the set of dyads that result after keeping only those that contribute to the highest rom in each family is able to recover a large fraction of all the known binding sites in the family . It is important to keep in mind that a given dyad can match several positions - and therefore sites - in a single region or family . Thus, selecting only those dyads appearing in the highest peak does not restrict their ability to find more than one site per region . The number of dyads that describe the set of known binding sites in a given regulatory family is quite variable . For instance, if we use the known binding sites as training sets, the tyrr family involves 14 different dyads whereas arca has 65 . There is no clear correlation between the number of dyads per site and the total number of sites in the training set for any given family, or any other property of the regulatory site, such as its size . Consensus is a program designed to find and align shared stretches of sequence among a given set of sequences . The searching method based on the results of consensus is already available; the weight matrix generated can be used to search, with the companion program patser, for sites in other upstream regions . The search using patser was made using the first matrix (highest informational content) obtained in the final cycle of consensus . This cycle requires all regions to contribute at least one sequence to the matrix . Using patser, we searched for the highest - scoring sequence in each region in the training set . The lowest value among these results was set as the minimal score and a second search was performed with this threshold in order to find new sites above this limit within each upstream region in e. coli for further searches and analyses . The capacity for pattern discovery of the two methods can be estimated by calculating the fraction of binding sites found when the training sets were the 200 + 50 or 400 + 50 bp regions, as shown in table 4 . A site was considered found when the predicted pattern overlaps 20% of the binding site . We also show the results of using the sequences of the binding sites with 10 bp extensions on each side as training sets, so we could distinguish between pattern discovery and pattern abstraction or identification . In the case of dyad - analysis / sweeping we evaluated whether the filtered dyads overlap the set of true sites . In the case of consensus / patser we evaluated whether the set of sites selected by consensus / patser overlaps the set of known sites . Consensus / patser is able to abstract a pattern for each of the 25 families, whereas dyad - analysis / sweeping can only do it for 19 of the families . In 11 of these 19 families consensus / patser finds more sites, in two families dyad - analysis / sweeping finds more sites, and in the remaining six both methods perform equally well . The real pattern discovery situation is that of the 450/sites cases (see legend to table 5 for definition), where consensus generates matrices for 24 of the families and dyad - analysis finds significant dyads for 11 of them . Dyad sweeping finds on average more than 70% of the binding sites (when dyad - analysis obtains significant dyads) as compared to around 60% with patser . Note that using shorter regions to search for dna binding sites (200 + 50), improves the performance of both methods by about 5 - 7% . Once table 4 was generated, we estimated the fraction of upstream regions recovered (table 3). A region is considered found when at least one site in that region is found . Therefore, the results differ from those in table 4 because of the occurrence of multiple sites in some upstream regions . A clear case of this is the argr regulon, where each of the six regions has two binding sites . The methods detect from 17% to 58% of the sites, but find from 33 to 100% of the regions . Detection of new members of regulons requires the selection of an optimal threshold to accept a sequence as a predicted binding site, and the genes downstream of such sequences as new members of the regulon family . The selection of the best threshold requires the evaluation of the following parameters: sensitivity (rate of true positives), specificity (rate of true negatives), accuracy (overall rate of true results), and, very important in this case, the positive predictive value (rate of true positives among the total number of positives, true and false). We used a leave one out (loo) procedure to evaluate the true - positive and false - negative rates with families containing at least five reported genes with binding sites . The loo method consists of leaving one gene at a time out of the training set; then, with the matrix or dyads built with the remaining sites, a search is made for a probable binding site within the upstream region of the gene that was left out . We combined the results of the left - out regions to build the total set of known positives for evaluation of true positives and false negatives . The evaluation of true negatives and false positives was carried out using the whole set of known positives as training sets and all the remaining regions known to be regulated by any other protein, as known negatives . Instead of calculating an average of the scores, and defining the threshold on the basis of standard deviations, we scanned the scores scale form the minimum score obtained in the collection of positive, to the maximum one, calculating the evaluation parameters noted above at each point of the scale . There is no point in searching at lower scores as there is no effect on sensitivity at such values . In figure 3 we show the results of the analyses of the purr regulon using dyad sweeping . Here, the minimum number of matches evaluated was one . Note that, as the dataset of known negatives exceeds that of known positives, high accuracy coexists with a large number of true negatives . Nevertheless, at the threshold of 10 matches, despite a very low false - positive value (less than 10%), and a very high accuracy (approximately 95%) and sensitivity (90%), the positive predictive value (ppv) shows that the total true positives in the whole' predicted' set is about 60% . As most regulatory proteins regulate just a few genes in comparison with the whole set of genes in a given organism, such a difference means that false positives might dilute reliable predictions even at very low false - positive rates . The ppv alone would leave results with very little recovery of true binding sites . Therefore, calculating an optimal point for prediction requires the use of a balanced evaluation criterion . After examining several graphs, we noticed that the average between accuracy and ppv (which we call the overall performance or op) would be a good criterion . This makes sense, as op represents a trade - off between those two statistical measures . Other criteria, such as the product of accuracy and ppv, might be used instead, but op worked well for our purposes . In a few cases, the point of highest op leaves a very small sensitivity value (around 50% in purr, for instance). If the sensitivity value was less than 60%, we used the last point where the sensitivity was above 60% . In figure 4 we show the results of sensitivity and false - positive rate for all regulons at their best op value using dyad sweeping . The use of weight matrices derived from consensus (with patser) is not illustrated, as the selection of the best threshold is the same as in dyad sweeping . In figure 5 we show the results of sensitivity and false - positive rates of each regulon at the best overall performance point of each regulon analyzed using patser . In table 6 we give the fraction of sites found per family in regions of 400 + 50 bp when starting from different training sets using the threshold chosen as described above . Dyad - detection / sweeping still performs better at finding the sites within an upstream region, while consensus / patser trained with binding sites finds the sites at an average of almost 77% . An interesting finding here was that, when trained with all the upstream 400 + 50 sequences, consensus finds an alignment and matrix that clearly discriminates between the sequences used in the training set, or regulon, from any other upstream sequence in e. coli . However, in some families, the matrix matches at sites different from the experimentally determined dna binding site of the regulon under analysis (figure 6), and such sites do not correspond to any known site, motif or region annotated in regulondb in the upstream sequence . We also verified that they do not match conserved regions in between pairs of sites . It will be indeed interesting to find out if these sequences have any biological meaning . Once the optimal threshold was obtained, we proceeded to predict other members of each regulon using the complete collection of upstream regions (200 + 50 and 400 + 50) of the e. coli genome . In order to further evaluate the predictions obtained, we used the recent annotations of cellular functions assigned by monica riley and her group to known e. coli genes . About 30% of the genes in e. coli have no function assigned, and each gene or gene product can be assigned to more than a single cellular role . In table 7 we show the consistency between the functional annotations of genes experimentally demonstrated to belong to each regulon as compared with the functional annotations of the set of predicted genes . In the cases of predictions of high confidence (for example, argr, crp and purr - all with correspondences above 90%), a putative function can be reliably assigned to genes of unknown function . For instance, in the case of the purr family, the genes without functional annotations might be assigned to macromolecule (dna / rna) biosynthesis . This is an example of functional gene prediction based on analysis of its regulatory elements . Annotations like' active transporter' would require other kinds of evidence (see additional data files). Functional annotations might be quite helpful in cleaning up wrong predictions, or adjusting the proposed thresholds, although limited by the genomic coverage of the functional assignments . Regulondb contains information on a few genes belonging to some of the regulons studied but with no mapped binding site for the relevant regulatory protein . As further evaluation, we show the results of dyad sweeping and patser, trained with the known binding sites of each regulon, for all of these genes (tables 8,9). In the tables we indicate whether the gene would be included in the corresponding predictions, the highest scoring rom (dyad sweeping, table 8) or pattern match (patser, table 9) found in the 400 + 50 region of the gene, and the actual sequence suggested as part of the possible binding site . Some genes would be rejected as predictions, but the small amount of data makes it impossible to appropriately evaluate this problem . A researcher might choose to use a different, perhaps lower, threshold if the intention is to find every gene for a given regulon experimentally, and such a decision would depend on how many confirmatory experiments it is possible to perform (an example is shown in the next section). Lower thresholds can also be used if the intention is to confirm new members suggested by other data, like clustering of a gene or genes with known members of a regulon . The latter case is exemplified by the results with those regulon members lacking a mapped binding site . Most contain roms or patterns scoring above the minimal score obtained for a known member of the regulon (no search is performed below this lower limit), often just below our suggested threshold . Thus, if there is additional evidence that a gene belongs to a given regulon, the roms found can be proposed as the putative binding sites . A recent attempt has been made by fernandez de henestrosa et al . To locate all the members of the lexa regulon by a combined strategy that included prediction of probable binding sites and experimental confirmation . Experimental confirmation showed that only 10 of the 49 predicted new members responded to lexa . The authors also give a table of previously found members of the lexa regulon, which includes a few genes not annotated in regulondb . We could analyze only five of their experimentally confirmed genes and 31 of their wrong predictions (predictions they later found experimentally not to be regulated by lexa) because of the lack of updating of the e. coli k12 genome annotations . In table 10 we present our results for the genes noted as previously determined members of the lexa regulon in, plus the five new members found by this study . In table 11, we find 20 out of the 23 confirmed members of the lexa regulon, whereas we would reject 20 out of their 31 wrong predictions . With consensus, we detect 18 of the 23 confirmed members of the regulon, while rejecting 19 of their wrong predictions . Pattern discovery starts with a collection of co - regulated genes for which no binding sites are yet known . To evaluate the methodology, we counted the number of times a sensor can locate a known binding site in a collection of 200 + 50 or 400 + 50 regions . Over - represented words would be expected to occur at the binding sites, and thus the first step was to determine if the resulting dyads match the binding sites . We found that there are significant dyads all along the sequences analyzed, with most of them matching at or near the known binding sites . Figure 1 shows, using the purr family, that most dyads were found at distances very close to or overlapping the true binding sites . We thus decided to search for stretches of contiguous matches, which we call' regions of overlapping matches' (roms), in the upstream sequences being analyzed by counting (sweeping), base by base, the number of matching dyads . As seen in figure 2, the roms with the highest number of matching dyads overlap the true known binding sites in the dna . This result motivated us to use the highest number of matches within a rom as the score . We call this method dyad sweeping . As the highest - scoring roms frequently overlap reported binding sites (figure 2, table 2), we decided to keep, for subsequent analyses, the dyads found within the highest - scoring roms of each upstream region, as long as the rom contained at least two dyads . In table 3 it can be seen that, except in a few of the regulons, the fraction of regions with known binding sites found is quite high . In other words, the set of dyads that result after keeping only those that contribute to the highest rom in each family is able to recover a large fraction of all the known binding sites in the family . It is important to keep in mind that a given dyad can match several positions - and therefore sites - in a single region or family . Thus, selecting only those dyads appearing in the highest peak does not restrict their ability to find more than one site per region . The number of dyads that describe the set of known binding sites in a given regulatory family is quite variable . For instance, if we use the known binding sites as training sets, the tyrr family involves 14 different dyads whereas arca has 65 . There is no clear correlation between the number of dyads per site and the total number of sites in the training set for any given family, or any other property of the regulatory site, such as its size . Consensus is a program designed to find and align shared stretches of sequence among a given set of sequences . The searching method based on the results of consensus is already available; the weight matrix generated can be used to search, with the companion program patser, for sites in other upstream regions . The search using patser was made using the first matrix (highest informational content) obtained in the final cycle of consensus . This cycle requires all regions to contribute at least one sequence to the matrix . Using patser, we searched for the highest - scoring sequence in each region in the training set . The lowest value among these results was set as the minimal score and a second search was performed with this threshold in order to find new sites above this limit within each upstream region in e. coli for further searches and analyses . The capacity for pattern discovery of the two methods can be estimated by calculating the fraction of binding sites found when the training sets were the 200 + 50 or 400 + 50 bp regions, as shown in table 4 . A site was considered found when the predicted pattern overlaps 20% of the binding site . We also show the results of using the sequences of the binding sites with 10 bp extensions on each side as training sets, so we could distinguish between pattern discovery and pattern abstraction or identification . In the case of dyad - analysis / sweeping we evaluated whether the filtered dyads overlap the set of true sites . In the case of consensus / patser we evaluated whether the set of sites selected by consensus / patser overlaps the set of known sites . Consensus / patser is able to abstract a pattern for each of the 25 families, whereas dyad - analysis / sweeping can only do it for 19 of the families . In 11 of these 19 families consensus / patser finds more sites, in two families dyad - analysis / sweeping finds more sites, and in the remaining six both methods perform equally well . The real pattern discovery situation is that of the 450/sites cases (see legend to table 5 for definition), where consensus generates matrices for 24 of the families and dyad - analysis finds significant dyads for 11 of them . Dyad sweeping finds on average more than 70% of the binding sites (when dyad - analysis obtains significant dyads) as compared to around 60% with patser . Note that using shorter regions to search for dna binding sites (200 + 50), improves the performance of both methods by about 5 - 7% . Once table 4 was generated, we estimated the fraction of upstream regions recovered (table 3). A region is considered found when at least one site in that region is found . Therefore, the results differ from those in table 4 because of the occurrence of multiple sites in some upstream regions . A clear case of this is the argr regulon, where each of the six regions has two binding sites . The methods detect from 17% to 58% of the sites, but find from 33 to 100% of the regions . Detection of new members of regulons requires the selection of an optimal threshold to accept a sequence as a predicted binding site, and the genes downstream of such sequences as new members of the regulon family . The selection of the best threshold requires the evaluation of the following parameters: sensitivity (rate of true positives), specificity (rate of true negatives), accuracy (overall rate of true results), and, very important in this case, the positive predictive value (rate of true positives among the total number of positives, true and false). Definitions of these terms are given in the legend to table 5 . We used a leave one out (loo) procedure to evaluate the true - positive and false - negative rates with families containing at least five reported genes with binding sites . The loo method consists of leaving one gene at a time out of the training set; then, with the matrix or dyads built with the remaining sites, a search is made for a probable binding site within the upstream region of the gene that was left out . We combined the results of the left - out regions to build the total set of known positives for evaluation of true positives and false negatives . The evaluation of true negatives and false positives was carried out using the whole set of known positives as training sets and all the remaining regions known to be regulated by any other protein, as known negatives . Instead of calculating an average of the scores, and defining the threshold on the basis of standard deviations, we scanned the scores scale form the minimum score obtained in the collection of positive, to the maximum one, calculating the evaluation parameters noted above at each point of the scale . There is no point in searching at lower scores as there is no effect on sensitivity at such values . In figure 3 we show the results of the analyses of the purr regulon using dyad sweeping . Here, the minimum number of matches evaluated was one . Note that, as the dataset of known negatives exceeds that of known positives, high accuracy coexists with a large number of true negatives . Nevertheless, at the threshold of 10 matches, despite a very low false - positive value (less than 10%), and a very high accuracy (approximately 95%) and sensitivity (90%), the positive predictive value (ppv) shows that the total true positives in the whole' predicted' set is about 60% . As most regulatory proteins regulate just a few genes in comparison with the whole set of genes in a given organism, such a difference means that false positives might dilute reliable predictions even at very low false - positive rates . The ppv alone would leave results with very little recovery of true binding sites . Therefore, calculating an optimal point for prediction requires the use of a balanced evaluation criterion . After examining several graphs, we noticed that the average between accuracy and ppv (which we call the overall performance or op) would be a good criterion . This makes sense, as op represents a trade - off between those two statistical measures . Other criteria, such as the product of accuracy and ppv, might be used instead, but op worked well for our purposes . In a few cases, the point of highest op leaves a very small sensitivity value (around 50% in purr, for instance). If the sensitivity value was less than 60%, we used the last point where the sensitivity was above 60% . In figure 4 we show the results of sensitivity and false - positive rate for all regulons at their best op value using dyad sweeping . The use of weight matrices derived from consensus (with patser) is not illustrated, as the selection of the best threshold is the same as in dyad sweeping . In figure 5 we show the results of sensitivity and false - positive rates of each regulon at the best overall performance point of each regulon analyzed using patser . In table 6 we give the fraction of sites found per family in regions of 400 + 50 bp when starting from different training sets using the threshold chosen as described above . Dyad - detection / sweeping still performs better at finding the sites within an upstream region, while consensus / patser trained with binding sites finds the sites at an average of almost 77% . An interesting finding here was that, when trained with all the upstream 400 + 50 sequences, consensus finds an alignment and matrix that clearly discriminates between the sequences used in the training set, or regulon, from any other upstream sequence in e. coli . However, in some families, the matrix matches at sites different from the experimentally determined dna binding site of the regulon under analysis (figure 6), and such sites do not correspond to any known site, motif or region annotated in regulondb in the upstream sequence . We also verified that they do not match conserved regions in between pairs of sites . It will be indeed interesting to find out if these sequences have any biological meaning . Once the optimal threshold was obtained, we proceeded to predict other members of each regulon using the complete collection of upstream regions (200 + 50 and 400 + 50) of the e. coli genome . In order to further evaluate the predictions obtained, we used the recent annotations of cellular functions assigned by monica riley and her group to known e. coli genes . About 30% of the genes in e. coli have no function assigned, and each gene or gene product can be assigned to more than a single cellular role . In table 7 we show the consistency between the functional annotations of genes experimentally demonstrated to belong to each regulon as compared with the functional annotations of the set of predicted genes . In the cases of predictions of high confidence (for example, argr, crp and purr - all with correspondences above 90%) for instance, in the case of the purr family, the genes without functional annotations might be assigned to macromolecule (dna / rna) biosynthesis . This is an example of functional gene prediction based on analysis of its regulatory elements . Annotations like' active transporter' would require other kinds of evidence (see additional data files). Functional annotations might be quite helpful in cleaning up wrong predictions, or adjusting the proposed thresholds, although limited by the genomic coverage of the functional assignments . Regulondb contains information on a few genes belonging to some of the regulons studied but with no mapped binding site for the relevant regulatory protein . As further evaluation, we show the results of dyad sweeping and patser, trained with the known binding sites of each regulon, for all of these genes (tables 8,9). In the tables we indicate whether the gene would be included in the corresponding predictions, the highest scoring rom (dyad sweeping, table 8) or pattern match (patser, table 9) found in the 400 + 50 region of the gene, and the actual sequence suggested as part of the possible binding site . Some genes would be rejected as predictions, but the small amount of data makes it impossible to appropriately evaluate this problem . A researcher might choose to use a different, perhaps lower, threshold if the intention is to find every gene for a given regulon experimentally, and such a decision would depend on how many confirmatory experiments it is possible to perform (an example is shown in the next section). Lower thresholds can also be used if the intention is to confirm new members suggested by other data, like clustering of a gene or genes with known members of a regulon . The latter case is exemplified by the results with those regulon members lacking a mapped binding site . Most contain roms or patterns scoring above the minimal score obtained for a known member of the regulon (no search is performed below this lower limit), often just below our suggested threshold . Thus, if there is additional evidence that a gene belongs to a given regulon, the roms found can be proposed as the putative binding sites . A recent attempt has been made by fernandez de henestrosa et al . To locate all the members of the lexa regulon by a combined strategy that included prediction of probable binding sites and experimental confirmation . Experimental confirmation showed that only 10 of the 49 predicted new members responded to lexa . The authors also give a table of previously found members of the lexa regulon, which includes a few genes not annotated in regulondb . We could analyze only five of their experimentally confirmed genes and 31 of their wrong predictions (predictions they later found experimentally not to be regulated by lexa) because of the lack of updating of the e. coli k12 genome annotations . In table 10 we present our results for the genes noted as previously determined members of the lexa regulon in, plus the five new members found by this study . In table 11 we show our results with their wrong predictions . Using dyad sweeping, we find 20 out of the 23 confirmed members of the lexa regulon, whereas we would reject 20 out of their 31 wrong predictions . With consensus, we detect 18 of the 23 confirmed members of the regulon, while rejecting 19 of their wrong predictions . Stringent evaluations of pattern discovery and pattern searching methods should be carried out to establish the confidence of a given prediction . Here we take advantage of the availability of reasonable negative samples - all other known regulons described in regulondb, except the one under study - in order to use standard statistical measurements of performance such as specificity and ppv . The ppv allowed us to stress how important even low rates of false positives might become in a large population . The small proportion of genes expected to be regulated by a given regulatory protein makes it important to emphasize the need for a stringent threshold to admit new members of regulons, as the true positives might be diluted in a high number of false positives . Nevertheless, if additional independent evidence is available, thresholds can be relaxed to include as many predictions as the confirmation procedure (genetic evidence of the regulatory effect, for instance) would allow . For instance, if the two computational methods were combined, only one of the genes known to be regulated by lexa (see previous section) would be rejected by both methods (ybfe in table 10), while 16 of the wrong predictions are rejected by both methods (table 11). A very striking observation that deserves experimental analysis the program discovers patterns that discriminate, very specifically, the upstream regions used as training sets from the other regions . These results imply the existence of new sequence elements specific to each family, different from those reported in the literature . We have not yet found (data not shown) any additional property that could suggest their function; their distance from the start site of transcription to known binding sites is not conserved; in some cases the predicted motif occurs upstream of the known sites in some promoters and downstream in other promoters . We have, of course, verified these observations twice, and find no additional property to associate with such families . In the comparison of the two methods we have not found that one of them performs better in all the evaluations and scenarios considered (pattern search, pattern abstraction and pattern discovery). This implies that one could consider combining the different methods to make the best use of their respective strengths . For instance, if there is evidence of co - regulation only, we would suggest using dyad - analysis / sweeping first to find the binding sites . If dyad - analysis finds significant dyads, the dyad sweeping methodology can be used to extract possible binding sites . After that, the predicted sites can be used to train consensus and search for further co - regulated genes . In cases where the dna binding sites are known, consensus / patser, which are both very fast and simple to use, can give very reliable results in a short time . The combination of computationally more confident predictions, together with additional independent evidence - for example, functional classes or operon organization - is an intelligent strategy for making more robust predictions . These more robust upstream regulatory analyses can be used to assign function to unknown genes, as illustrated here with the argr, crp and purr regulons . One can envisage highly relevant genomic applications of these predictions, such as distinguishing orthologs within families of paralogous genes, based on their differential regulation, or identifying non - orthologous gene displacement on the basis of regulatory comparisons . The goal in computational biology is twofold: to provide, on the one hand, methods that generate useful and evaluated predictions, and, on the other hand, to use such methods as models of the biology under study . This latter virtue could generate new ways of understanding fundamental processes in gene regulation, along with, as suggested here, new properties of gene regulation at the genomic level . Each algorithm should be tested on well - defined problems in order to find their strengths . Thus it should be possible to choose which method, or combination of methods, is best suited for the problem at hand . Additional data files containing the functional annotations (using dyad analysis and consensus analysis, respectively) associated to the genes within each regulons, and of those genes downstream of predicted binding sites are available . Functional annotations using dyad analysis click here for additional data file functional annotations using consensus analysis click here for additional data file this research was supported by grants from conacyt no . 0028 and from dgapa to j.c.v . Position of dyads found by the dyad - analysis program in relation to the binding sites in dna for the whole purr family . The graph shows the distances between all the dyads found in relation to the known binding sites of the purr regulon . Contiguous regions of overlapping matching dyads (roms) frequently overlap with the known binding sites . This example shows results after finding significant dyads in the 200 + 50 regions of the purr regulon, and finding the roms within the same regions . The two roms with the highest peaks completely overlap with the two reported regulatory binding sites in this region (sites lie at positions -59 to -43 and at 29 to 45). It can be seen, for instance, that blue dyads occur only in the two true binding sites . Different thresholds, defined as number of overlapping matches, were evaluated for all regulons . This graph shows the case of the purr regulon when the dyads are obtained from the known binding sites and the evaluation is carried out on the 400 + 50 regions . The only dyads used in the search were those found at the roms with the highest value per region in the purr regulon . The statistical parameters (see table 5) are plotted as percentages instead of fractions . The arrow indicates the point of maximum overall performance (op) (see text). Performance of dyad - analysis / dyad sweeping at the best threshold defined for each family . Sensitivity and false - positive rate (expressed as percentages) at the highest overall performance for each regulon are shown, using the binding sites as training sets, and the 400 + 50 regions as evaluation sets . Sensitivity, and false - positive rate (expressed as percentages) at the highest overall performance for each regulon are shown, using the binding sites as training sets, and the 400 + 50 regions as evaluation sets . The positions found by consensus / patser . If consensus is run to find an alignment within the 400 + 50 regions, the resulting matrix finds sites within each region (indicated here by the sites labeled' matrix') that do not always match the binding sites for the relevant regulatory protein (arac in the case illustrated here), but are very specific to the gene family . The sequence found does not correspond to known binding sites for other regulatory proteins (for example crp) within the regions nor to the promoter . Summary of the datasets in regulondb regulondb contains information for the 86 regulons shown in this table . Of these, only 60 have at least three known binding sites for their corresponding regulatory protein . The second column indicates the total number of known sites, which are distributed in upstream regions (fourth column). The last column indicates the number of upstream regions for which there is experimental evidence suggesting regulation, but no direct proof of binding of the regulator to the upstream site is yet available . For instance, there are 12 known sites for argr located in only six regions (with two sites per region), plus one region for a different gene for which there is evidence of regulation by argr . Pattern discovery using roms (regions of overlapping matches) with maximal score to find binding sites in dna the total number of genes in the regulon with a known binding site (in the 400 + 50 upstream regions). The number of regions where a rom (region of overlapping matches) with the highest number of matches (max rom) touches a known binding site . Number of regions where either a rom or dyad touches a known binding site, but the max rom does not . The percentage of all upstream regions in which any rom touches a binding site . Number of regions with dyads, but no match between known binding sites and roms . Pattern discovery at the level of upstream regions for each family, we show the results with dyad - analysis / sweeping and with consensus / patser . The data shown are obtained using different training sets - the 200 + 50 and 400 + 50 regions (250 and 450) and a comparison with training sets of known binding sites (sites) as a reference standard . Results are given as the number of regions where at least one binding site was found divided by the total number of regions, and expressed as percentages . Note that only the dyads extracted from the max roms within each region are used here . In each column heading, the first word refers to the training set and the second refers to the regions where the patterns were searched . For instance, columns headed 450/sites show the results of pattern discovery when consensus or dyad - analysis has as input the 450 + 50 bp regions, and the sensor is evaluated with the files of known sites . We counted only those regions containing known binding sites within the range covered (that is, if a known binding site is present more than 200 bp upstream of the gene start site, the corresponding 200 + 50 region is not counted). Dashes mean that either there was no binding site within the region, or the programs failed to provide a matrix (consensus) or significant dyads (dyad - analysis). A region is considered found if at least one of its binding sites is matched . Pattern discovery at the level of binding sites for each family, we show the results of applying dyad - analysis / sweeping and consensus / patser to the problem of discovering binding sites . The results contain pattern discovery data similar to those in table 3, but this time counting the number of binding sites found per total number of sites . Definitions of parameters used in evaluating the predictions fn, false negative; fp, false positive; tn, true negatives; tp, true positives . Binding sites remaining at best threshold for each family, we show the results with dyad - analysis / sweeping and with consensus / patser accepting a match only if its score exceeds the defined best threshold . A comparison between the functional annotations of genes known to be regulated by a given protein and the functional annotations of the predicted set of genes . The percentage with related function is calculated against all the genes with functional annotations, while the percentage without functional annotations is calculated against the whole set of predicted genes . The number in parentheses excludes genes known to be part of the corresponding regulon . In cases with high correlation of functional annotations we can propose a related function for genes without functional annotations, as in the consensus / patser predictions of argr, crp and purr (all with correspondences above 90%). . Dyad - analysis / sweeping predictions in regions without binding sites reported in regulondb sequences and positions of binding sites predicted by dyad sweeping in genes with experimental evidence for co - regulation in regulondb, but with no binding site experimentally identified . Genes follow the alphabetic order of the regulatory proteins, with the name of the protein separating each group . The number in parentheses after the regulator is the value of the threshold - derived from requesting best overall performance . The site coordinates are' i' for initial base,' f' for final position relative to the start codon . The score is given as the maximum number of matching (' m') dyads within a rom . The number of families used was the same for any method, but we only show families where the methods provided significant results . Consensus / patser prediction in regions without binding sites reported in regulondb data and analysis as described in table 8 . Sc, the number of families used was the same for any method, but we only show families where the methods provided significant results . The table shows our binding - site predictions with dyad sweeping and with patser, using their corresponding best overall performance thresholds . Sc, score as obtained by patser; m, maximum number of matching dyads . Note that most genes clearly have roms with 10 or more matches and with scores of patser above 10 . Contrasting predictions: regions known to lack lexa sites after experiment, fernandez de henestrosa et al . Rejected this set of genes in which they had predicted lexa sites using other computational methods . We tested the capacity of dyad sweeping and patser to also reject these false positives . Sc, score as obtained by patser; m, maximum number of matching dyads . Note that for both methods, most of the genes here show much smaller scores than genes belonging to the lexa regulon (see table 10).
The success of root canal therapy is dependent on understanding the anatomy of root and root canal morphology.1,2 awareness and understanding of the presence of unusual external and internal root canal morphology largely contributes to the successful outcome of the root canal treatment . In literature, description of the mandibular first premolar is typically of a single - rooted tooth.3,4 two - rooted, three - rooted, and four - rooted varieties have also been reported, but are rare.5,6 the root frequently has developmental depressions or grooves on both the mesial and distal surfaces . According to slowey, the mandibular premolars may present the greater difficulty of all teeth for a successful endodontic treatment.7 a study at the university of washington in 1955 assessed the failure rate of non - surgical root canal therapy (nsrct) in all teeth . Numerous endodontic failures after a routine treatment and flare - ups during the course of nsrct are cited as evidence . It could be because of variations in root canal morphology and difficult access to additional canal systems.8 this case report presents a successful, non - surgical management of mandibular left second premolar with three roots and root canals and a first premolar with two roots using a cone beam computed tomography (cbct). A 31-year - old male patient was reported to the department of conservative dentistry and endodontics, mnr dental college and hospital with a chief complaint of pain and pus discharge in the lower left back tooth region since 3 months ., there was a gingival inflammation with recession in relation to tooth numbers 34, 35, 36 . Electric pulp testing and thermal testing of the teeth indicated non vitality of 34, 35, and 36 . A confirmatory diagnosis of acute apical abscess in relation to 34, 35, and 36 was established and endodontic therapy was planned . The mesiodistal width of the crown is lesser than mesiodistal diameter of the root and for further confirmation a cbct (gentex), was planned . Cbct with a 3d reconstruction confirmed a three rooted 34 and two rooted 35 (figure 1b - d). (a) pre - operative radiograph showing first and second premolar and first molar . The area was anaesthetized using 2% lignocaine with 1:80,000 adrenaline (lignox) and isolated with a rubber dam . An access cavity was prepared with a modification that it had a cut at the buccoproximal angle from the entrance of the buccal canals to the cavosurface angle, so that it is results in a t - shaped outline (figure 2a). A working length was determined with an apex locator (root - zx) (figure 2b). With crown down technique, cleaning and shaping were performed with twisted file and protaper rotary instruments (dentsply mailfilter). Abundant irrigation with 3% sodium hypochlorite solution is done . Biomechanical preparation is done up to f2 . Before obturation irrigation with 17%, edta and saline are done . Obturation is done with gutta - percha and resin sealer (ah plus, dentsply) by cold lateral condensation (figure 2c). The coronal access was restored with resin composite (3 m espe) (figure 2d). Successful and predictable endodontic treatment requires knowledge of biology, root canal anatomy, and careful radiographic evaluation in order to determine number of roots and root canals . Preoperative parallel radiographs, as well as mesial or distal angled radiographs, can help to determine number of roots . The diagnosis and management of extra roots or root canals in mandibular premolars pose an endodontic challenge . Failing to locate and obturating a root canal is the major cause of failure in endodontic therapy . Hoen and pink found 42% incidence of missed roots or canals in the teeth that needed retreatment.9 according to cleghorn, the incidence of three rooted mandibular first premolars is 0.2%.10 hence, it is import that all the canals be located and treated during the course of nonsurgical endodontic therapy . Rodig and hulsmann have reported a case of mandibular second premolar with three separate roots and root canals diagnosed using intraoral periapical radiographs.2 wong and al - fouzan have published cases of mandibular second premolars with four canals.11,12 tzanetakis et al . Have reported endodontic management of mandibular second premolar with four root canals diagnosed with the aid of operating microscope.13 it has been established that a root with a tapering canal and a single foramen is the exception rather than the rule . Serman and hasselgren (1992) reported a high incidence of multiple roots (18.1%) and root canals in mandibular premolar teeth in a series of radiographic surveys with mandibular first premolars involved in 15.7% of patients and mandibular second premolars in 7% of patients.14 studies by lu et al . Are similar to slowey suggestions that mandibular premolars are the most difficult to treat endodontically and also the apical configuration of these teeth was found to be complex.15 thus, a careful understanding and diagnosis of canal anatomy is of utmost importance for successful management of such cases . Accurate preoperative radiographs of good quality along with an occlusal view, into the access opening and down the chamber of a mandibular premolar tooth, rarely shows any chamber floor, even when a suspected bifurcation of the canal is seen on the radiograph . The surgical operating microscope sometimes aids in canal visualization of a canal system branching off the main canal . A fine, curved stainless steel file with a good tactile sense is the best guide to the detection of the accessory canals.16 a modification in the access cavity preparation is often needed for unhiding the additional orifices of the root canals or the orifices of the extra roots for a better instrumentation . When present the roots are usually mesiobuccal, distobuccal, and a lingual root . At least two radiographs, with the second radiograph angulated from 15 to 20 either mesial or distal from the horizontal long axis of the root, are required to reliably diagnose more than one root or root canal system in premolar teeth . A sudden narrowing of the main canal on a parallel radiograph was a good criterion to judge root canal multiplicity.17 however, martinez - lozano et al . Recommend up to 40 mesial angulation from horizontal as more reliable in identifying the extra canals.18 deviation of the x - ray angle from the vertical axis of 15 to 30 was effective only in the mandibular first premolar in helping to visualize canal anatomy of premolar teeth . Dyes, fiber - optic transillumination, magnifying loupes, and sodium hypochlorite bubbling in the extra canals help in locating additional canals . With the advent of advanced imaging techniques, an understanding of complex anatomies cbct is better of digital radiographic techniques in identifying multiple root canal systems in the mandibular incisor, mandibular first premolar, and maxillary first molar teeth.19 management of teeth with morphological variations presents a challenge, which requires proper instruments and the knowledge to use the instruments effectively . Advanced imaging techniques like cbct are valuable tools in diagnosing and managing cases, which deviate from the regular pattern . The present case report emphasizes the need to understand, interpret, and manage a three rooted mandibular first premolar with three roots which has been successfully managed using cbct.
The left atrium (la) plays a major role in left ventricle performance; hence, la mechanical function is a surrogate marker of left ventricular (lv) dysfunction . The components of la function are divided into: reservoir, conduit, and contractile phase . Reservoir corresponds to the difference between maximal and minimum la volumes occurring in the interval just before the opening mitral valve and just before aortic valve opening . The contribution of three phases of the la function changes according to the diastolic properties of left ventricle . In normal conditions, the contribution of la to lv filling is 40, 35, and 25%, respectively . In relation to the lv valve movements, la activity can be divided in four phases: a) isovolumetric relaxation period occurring between the aortic closure and the opening of the mitral valve; b) lv rapid filling, which begins when lv pressure falls below the atrial pressure and the mitral valve opens; c) diastasis, this corresponds to the equality between la and lv pressures; and d) atrial systole, which corresponds to la contraction and ends at the mitral valve closure . Even though conventional, noninvasive techniques (as echocardiography and tissue velocity imaging derived parameters) are widely used to evaluate la function; 2d - speckle tracking echocardiography (2d - ste) has emerged as a new, noninvasive method for the assessment of cardiac function . It is an imaging technique that provides accurate and angle - independent informations also regarding la deformation and motion . Recent reports suggested that 2d la longitudinal strain obtained with ste (2d - laste) is an effective method for quantification of la function . It is known that lv diastolic pressure increases with advancing age inducing some changes in la dimensions and function . To define the changes of atrial function dependent on the ageing fifteen consecutive persons (12 males (m) and three females (f)) aged from 70 to 82 years (mean age = 79 5) were chosen among those afferent to our department of internal medicine and geriatrics between may 2012 and april 2014 . These were actually free of any cardiovascular, respiratory, and/or metabolic derangements (group i). Nineteen healthy adult controls (11 m and eight f) ranging in age from 44 to 61 years (mean age = 55 6 years). None of the enrolled subjects had a history of ischemic heart disease or significant valvular abnormalities, peripheral vascular disease, cerebrovascular disease, systemic hypertension, or diabetes mellitus (group ii). In addition, none of both groups subjects received cardioactive drugs at the moment of the study . Both groups were in sinus rhythm with heart rate (hr) <100 beat / min subjects and gave their written informed consent for participation to the study . Echocardiographic examinations were performed by experienced sonographers using a philips ie33 machine (eindhoven, nl). All measurements were performed in m- and b - mode in accordance with the american society of echocardiography criteria . Ejection fraction% (ef%) was also defined according to the modified simpson's criteria . The peak of early (e) and late (a) waves of diastolic mitral inflow were measured, and the e / a wave ratio were calculated . Mitral annular plane systolic excursion (mapse) was measured using m - mode echocardiography in the mitral annular lateral approach by apical four - chamber view . Left atrial volume (lav) was determined by the biplane area - length method . Its value was indexed for body surface area in m and evaluated in ml / m . Values of peak early (e) and late (a) diastolic annular velocities were also obtained . Measurements were obtained during end expiration to eliminate respiratory variations and an average of three beats measured . The leading epidemiological, metabolic, and conventional echocardiographic characteristics of two groups are reported in table 1 . Clinical, echocardiography and metabolic aspects of the two enrolled groups lvedv = left ventricular end - diastolic volume, lvesv = left ventricular end - systolic volume, lvef = left ventricular ejection fraction, mapse = mitral annular plane systolic excursion, ns = not significant speckle tracking images of the left atrium were obtained both in aged patients (group i) and in healthy adult controls (group ii) by activating ste on the same echocardiographic machine . The left atrium endocardial surface was manually traced using a point - and - click approach in apical four - chamber view that automatically allowed a region of interest (roi). The cardiac cycle was demarcated by indicating qrs onset afterwards, roi was divided into six segments (two corresponding to the interatrial septum; two to the lateral wall; and two to the roof of the left atrium). If adequate images quality were not obtained, the records were rejected by the software and excluded from the analysis . By using la volume, the following la dynamic volumes were calculated: maximum la volume (lav max), minimal la volume (lav min), and the la volume before the atrial contraction (lav pre - a). Precisely, lav max was recorded just before mitral valve opening; lav min was recorded at mitral valve closure; and lav pre - a is lav at onset of atrial systole (p wave). These values were corrected for body surface area as lav index (lavi) and consequently, lavi maximum; lavi minimum; and lavi pre - a were obtained . The phases of left atrial function, corresponding to reservoir, conduit, and booster pump were calculated by the lavi volumes, as follows: left atrial emptying fraction (laef) total (corresponding to atrial reservoir function) laef passive (corresponding to atrial conduit function) laef booster (corresponding to atrial contractile booster pump) in addition, lavi passive emptying and passive fraction, and lavi active emptying and active fraction were calculated . In particular, lavi passive emptying was measured as the difference between lavi maximum - lavi pre - a . Passive fraction was obtained as the ratio between lavi passive emptying / lavi maximum 100% . Active fraction was calculated as lavi active emptying / lavi pre - a 100% . Finally, la global systolic strain (s); systolic strain rate (srs); early diastolic strain rate (sre); and late diastolic rate (sra) were calculated in both groups . Data referring two - dimensional (2d) echocardiographic finding in both groups were expressed as mean standard deviation (sd). Comparison of lavi (max, min, and pre - a) and the different phases of lavi in two groups were performed using the student's t - test for unpaired data . This was also employed to evaluate the differences between two groups referring lavi passive emptying, passive fraction, lavi active emptying, and active fraction . The mean values of reservoir, conduit and booster pump phases were also calculated in two groups . Likewise, the values of s, srs, sre, and sra obtained in two groups were compared . All statistical analyses were performed using the statistical package for social sciences (spss) software . Data were analyzed using analysis of variance to assess for age - related changes in echocardiographic variables applying a bonferroni's correction between two groups . Echocardiographic examinations were performed by experienced sonographers using a philips ie33 machine (eindhoven, nl). All measurements were performed in m- and b - mode in accordance with the american society of echocardiography criteria . Ejection fraction% (ef%) was also defined according to the modified simpson's criteria . The peak of early (e) and late (a) waves of diastolic mitral inflow were measured, and the e / a wave ratio were calculated . Mitral annular plane systolic excursion (mapse) was measured using m - mode echocardiography in the mitral annular lateral approach by apical four - chamber view . Left atrial volume (lav) was determined by the biplane area - length method . Its value was indexed for body surface area in m and evaluated in ml / m . Values of peak early (e) and late (a) diastolic annular velocities were also obtained . Measurements were obtained during end expiration to eliminate respiratory variations and an average of three beats measured . The leading epidemiological, metabolic, and conventional echocardiographic characteristics of two groups are reported in table 1 . Clinical, echocardiography and metabolic aspects of the two enrolled groups lvedv = left ventricular end - diastolic volume, lvesv = left ventricular end - systolic volume, lvef = left ventricular ejection fraction, mapse = mitral annular plane systolic excursion, ns = not significant speckle tracking images of the left atrium were obtained both in aged patients (group i) and in healthy adult controls (group ii) by activating ste on the same echocardiographic machine . The left atrium endocardial surface was manually traced using a point - and - click approach in apical four - chamber view that automatically allowed a region of interest (roi). The cardiac cycle was demarcated by indicating qrs onset afterwards, roi was divided into six segments (two corresponding to the interatrial septum; two to the lateral wall; and two to the roof of the left atrium). If adequate images quality were not obtained, the records were rejected by the software and excluded from the analysis . By using la volume, the following la dynamic volumes were calculated: maximum la volume (lav max), minimal la volume (lav min), and the la volume before the atrial contraction (lav pre - a). Precisely, lav max was recorded just before mitral valve opening; lav min was recorded at mitral valve closure; and lav pre - a is lav at onset of atrial systole (p wave). These values were corrected for body surface area as lav index (lavi) and consequently, lavi maximum; lavi minimum; and lavi pre - a were obtained . The phases of left atrial function, corresponding to reservoir, conduit, and booster pump were calculated by the lavi volumes, as follows: left atrial emptying fraction (laef) total (corresponding to atrial reservoir function) laef passive (corresponding to atrial conduit function) laef booster (corresponding to atrial contractile booster pump) in addition, lavi passive emptying and passive fraction, and lavi active emptying and active fraction were calculated . In particular, lavi passive emptying was measured as the difference between lavi maximum - lavi pre - a . Passive fraction was obtained as the ratio between lavi passive emptying / lavi maximum 100% . . Finally, la global systolic strain (s); systolic strain rate (srs); early diastolic strain rate (sre); and late diastolic rate (sra) were calculated in both groups . Data referring two - dimensional (2d) echocardiographic finding in both groups were expressed as mean standard deviation (sd). Comparison of lavi (max, min, and pre - a) and the different phases of lavi in two groups were performed using the student's t - test for unpaired data . This was also employed to evaluate the differences between two groups referring lavi passive emptying, passive fraction, lavi active emptying, and active fraction . The mean values of reservoir, conduit and booster pump phases were also calculated in two groups . Likewise, the values of s, srs, sre, and sra obtained in two groups were compared . All statistical analyses were performed using the statistical package for social sciences (spss) software . Data were analyzed using analysis of variance to assess for age - related changes in echocardiographic variables applying a bonferroni's correction between two groups . Left ventricular end - diastolic volume (lvedv; 107 14 ml) and left ventricular end - systolic volume (lvesv; 47 10 ml) measured in aged subjects were not significantly different from the mean values (lvedv = 96 17 ml and lvesv = 40 13 ml) recorded in healthy adults (not significant (ns)). Ef% resulted 53 3.5 in aged group and = 58 2.4 in controls . Differences between two values were ns too . On the other hand, left atrial diameter of aged (41.4 3.7 mm) was significantly greater (p <0.05) than that recorded in healthy controls (35 41 mm). Lavi also increased (p <0.05) in elderly subjects (31 3 ml / m) in comparison to healthy adults (23 2.4 ml / m). E wave recorded in aged group (74.2 1.4 cm / s) was lower (p <0.05) than that recorded in controls (89.4 2.3 cm / s). On the contrary, a wave significantly (p <0.05) increased in healthy aged (83.3 2.1 cm / s) compared with the mean value recorded in healthy controls (75 1.8 cm / s). In agreement with these results, e / a waves ratio (p <0.001) decreased in aged group (0.89 1.8) compared to control group (1.1 2.1). Finally, mapse recorded in aged patients (10.0 0.7) was not significantly different (ns) from the mean value obtained in adult controls (12.7 08) [table 1]. The e wave velocity decreased (p <0.001) from 11 2.6 cm / s in controls to 7.5 1.4 cm / s in aged subjects . On the contrary, the a wave velocity increased (p <0.05) from the normal adults (8.6 1.8 cm / s) to the healthy aged (9.8 1.4 cm / s). The e / e ratio also decreased (p <0.05) with advancing age (9.1 1.5 in adults and 11.7 1.2 in healthy aged) [table 1]. Lavi max was = 30.7 7.5 ml / m, in comparison to 24.7 3.5 ml / m found in adult controls (p <0.05). Lavi minimum recorded in old people was 17.5 6.1 ml / m with respect to the controls (13.9 5.2 ml / m). Lavi pre - a recorded in aged group was 26.2 3.8 ml / m . This value was significantly higher (p <0.001) then that recorded in healthy adults (17.2 6.7 ml / m). In addition; reservoir, conduit, and booster pump fractions of la were recorded in two groups . Reservoir phase resulted superimposable in two groups (42.9 1.5 in aged subjects vs 43.7 1.8 in controls). On the contrary, conduit fraction reduced (p <0.001) in oldest individuals (14.6 1.9) in comparison with adult persons (30.3 1.7). Booster pump significantly increased (p <0.001) in aged group (33.2 2.1) with respect to controls (19.1 1.7) [table 2]. Finally, lavi passive emptying was 4.5 0.4 ml / m in old subjects and 7.5 0.6 ml / m in healthy adults . Passive emptying fraction was 14.7 1.7 in aged, whereas its value was 30 1.9 in controls . Lavi active emptying calculated in the elderly persons was 9.2 0.7 ml / m and 6.3 0.2 ml / m in adults without significant differences (ns). Active emptying fraction was 33.2 1.9 in aged individuals, and 25.4 1.3 in healthy adults, with significant differences (p <0.001) [table 2]. Values of lavi (maximum, minimum and pre - a), reservoir, conduit and booster pump in two groups . Lavi passive and active emptying fraction recorded in healthy aged and adult controls with statistical significance lavi = left atrial volume index, srs = systolic strain rate, sre = early diastolic strain rate, sra = late diastolic strain rate, ns = not significant, lav max = maximum la volume, lav min = minimal la volume, lav pre - a = la volume before the atrial contraction left ventricular end - diastolic volume (lvedv; 107 14 ml) and left ventricular end - systolic volume (lvesv; 47 10 ml) measured in aged subjects were not significantly different from the mean values (lvedv = 96 17 ml and lvesv = 40 13 ml) recorded in healthy adults (not significant (ns)). Ef% resulted 53 3.5 in aged group and = 58 2.4 in controls . Differences between two values were ns too . On the other hand, left atrial diameter of aged (41.4 3.7 mm) was significantly greater (p <0.05) than that recorded in healthy controls (35 41 mm). Lavi also increased (p <0.05) in elderly subjects (31 3 ml / m) in comparison to healthy adults (23 2.4 ml / m). E wave recorded in aged group (74.2 1.4 cm / s) was lower (p <0.05) than that recorded in controls (89.4 2.3 cm / s). On the contrary, a wave significantly (p <0.05) increased in healthy aged (83.3 2.1 cm / s) compared with the mean value recorded in healthy controls (75 1.8 cm / s). In agreement with these results, e / a waves ratio (p <0.001) decreased in aged group (0.89 1.8) compared to control group (1.1 2.1). Finally, mapse recorded in aged patients (10.0 0.7) was not significantly different (ns) from the mean value obtained in adult controls (12.7 08) [table 1]. The e wave velocity decreased (p <0.001) from 11 2.6 cm / s in controls to 7.5 1.4 cm / s in aged subjects . On the contrary, the a wave velocity increased (p <0.05) from the normal adults (8.6 1.8 cm / s) to the healthy aged (9.8 1.4 cm / s). The e / e ratio also decreased (p <0.05) with advancing age (9.1 1.5 in adults and 11.7 1.2 in healthy aged) [table 1]. Lavi max was = 30.7 7.5 ml / m, in comparison to 24.7 3.5 ml / m found in adult controls (p <0.05). Lavi minimum recorded in old people was 17.5 6.1 ml / m with respect to the controls (13.9 5.2 ml / m). Lavi pre - a recorded in aged group was 26.2 3.8 ml / m . This value was significantly higher (p <0.001) then that recorded in healthy adults (17.2 6.7 ml / m). In addition; reservoir, conduit, and booster pump fractions of la were recorded in two groups . Reservoir phase resulted superimposable in two groups (42.9 1.5 in aged subjects vs 43.7 1.8 in controls). On the contrary, conduit fraction reduced (p <0.001) in oldest individuals (14.6 1.9) in comparison with adult persons (30.3 1.7). Booster pump significantly increased (p <0.001) in aged group (33.2 2.1) with respect to controls (19.1 1.7) [table 2]. Finally, lavi passive emptying was 4.5 0.4 ml / m in old subjects and 7.5 0.6 ml / m in healthy adults . Passive emptying fraction was 14.7 1.7 in aged, whereas its value was 30 1.9 in controls . Lavi active emptying calculated in the elderly persons was 9.2 0.7 ml / m and 6.3 0.2 ml / m in adults without significant differences (ns). Active emptying fraction was 33.2 1.9 in aged individuals, and 25.4 1.3 in healthy adults, with significant differences (p <0.001) [table 2]. Values of lavi (maximum, minimum and pre - a), reservoir, conduit and booster pump in two groups . Lavi passive and active emptying fraction recorded in healthy aged and adult controls with statistical significance lavi = left atrial volume index, srs = systolic strain rate, sre = early diastolic strain rate, sra = late diastolic strain rate, ns = not significant, lav max = maximum la volume, lav min = minimal la volume, lav pre - a = la volume before the atrial contraction the morphological changes happening in the cardiovascular system with advancing age are responsible for increased myocardial stiffness and lv hypertrophy further evolving in lv diastolic dysfunction typical of the elderly . For these changes, in our healthy aged individuals lv systolic function resulted to be within the normal limits with preserved lv volumes and ef% was> 50%, as previously described . Since atrial function is related to increased lv filling, e wave velocity of diastolic mitral flow decreased and a wave significantly increased in comparison to the controls, with the inversion of the e / a wave ratio . That happens for a strongest atrial contraction in response to the increased end - diastolic lv pressure dependent on increased lv filling pressure (lvfp). It is known that mapse, reflecting longitudinal myocardial shortening, is a simple and sensitive echocardiographic parameter for assessing global longitudinal lv wall function . Particularly, mapse <8 mm was associated with depressed left ventricular ejection fraction (lvef; <50%), whereas mean mapse> 10 mm was linked with preserved lvef (> 55%). In our aged persons, we have found a mapse value of 10.0 0.7 that is indicative of ef%> 50 . In accordance with the study by tighe et al ., and successively, munagala et al ., in our healthy aged individuals; we found that the e velocity decreases, the a velocity increases, and the e / e ratio is> 10 . It is known that the e / e ratio is able to estimate lvfps in patients with preserved systolic function . Patients with e / e> 15 can be classified as having elevated filling pressure . An e / e <8 suggests normal filling pressure . In the range of e / e of 8 - 15 other informations, including systolic function, chambers dimensions, and all doppler variables thus, the e / e value (11.7 1.2) found in our aged subjects has an uncertain significance; but, considering all other echocardiographic data, the obtained value can be considered indicative of normal lvfps . 2d - ste is a relatively new technology that tracks speckles in the myocardium frame - by - frame basis throughout the cardiac cycle, resulting in a noninvasive calculation of global and regional velocity, displacement, strain, and strain rate . In this study, we applied this echocardiographic technique on la walls of healthy aging hearts to define la volumes (max, min, and pre - a) indexed for body surface area (lavi anatomy). Lavi passive / active emptying and functions were also defined in order to evaluate the influence of lavi function on reservoir, conduit, and booster pump function . In our healthy aged individuals, reservoir is maintained; while conduit phase decreases and booster pump function increases [figure 1]. Specifically, srs (correlated with reservoir function) remained unchanged with respect to controls; sre (correlated with conduit phase) decreased, whereas sra (corresponding to the contractile function) increased . Changes in la function occurred in conjunction with age - related changes in lv diastolic function . The preservation of la function during ventricular systole (reservoir) is important to maintain the cardiac output . La acts as a conduit during the phase of early lv diastolic filling, evidencing a decline in la passive emptying fraction . This decline corresponds to the age - related inversion in e / a ratio described in our elderly individuals . Ageing also induces both prolonged lv relaxation and impairment of lv passive properties responsible for an augmentation of la contribution to transmitral flow (booster pump). Therefore, the advanced age is associated with depressed lavi passive emptying function inversely, lavi active emptying function increases with age in order to maintain systolic ventricular volume, in accordance with a previous report of rossi et al ., and likewise to the diabetic cardiomyopathy, as we already described . 2d - laste represents an easy, noninvasive tool to characterize the morphological and functional la changes to compensate healthy aged lv diastolic dysfunction . In other words, since la is directly exposed to lv diastolic dysfunction through the mitral valve, it is evident that the changes of la function reflects the duration and severity of increased la pressure following the increased age - related lv diastolic dysfunction . Must be also added that la dimensions and functions can be better evaluated with a multimodality imaging approach including cardiac magnetic resonance (cmr) and computed tomography (ct). Finally, the recently developed three - dimensional speckle tracking could more easily and simultaneously define the effective motion of speckles in all directions.
Breast cancer is the most prevalent malignancy in women and affects about 1 in 8 women around the world (1). Therefore, investigation of early biomarkers and molecular aspects is valuable for improvement of breast cancer therapy and outcome . Cancer - testis antigens (cta) are proteins with physiological expression restricted to adult testicular germ cells . They are down - regulated in somatic adult tissues but may be aberrantly re - expressed in various malignancies . The first cta was discovered by taking advantage of a newly developed dna - cloning method to identify targets of t - cell recognition . Cytotoxic t lymphocytes (ctl) recognizing autologous tumor cells were obtained from a patient bearing melanoma with an unusually favorable clinical course . Using the melanoma cell line mz2- mel and autologous ctl clones cytolytic to this line, mage-1, subsequently was renamed as mage - a1, and was identified as the target antigen . This was the first molecularly characterized tumor antigen eliciting autologous ctl responses in a cancer patient . Further analysis of the mage - a family revealed 12 closely related genes clustered at xq28 . A growing number of tumor - associated antigens (taa), with similar characteristics, identified by cellular or serological screening techniques, have been reported since . Although some of them may be expressed in placenta as well, they are collectively referred to as cta . Cta presently include 44 distinct gene families, some comprising multiple members, such as mage - a and gage1, as well as splice variants, such as xage1a and xage1b, for a total of 89 transcripts . Cta can be classified into those that are encoded on the x chromosome (x - cta) and those that are not (non - x ctas) (2). To date, almost 100 genes and gene families encoding ctas have been identified . Ctas mapping to chromosome x are referred to as x -ctas and are distinguished from non - x ctas located on other chromosomes (2 - 4). X - ctas expression in breast cancer tissues is associated with a poor outcome and is more prevalent in higher grade and advanced stage tumors (5 - 9). Due to testis blood barrier and the immune privileged status of germinal cells (10), expression of ctas in tissues other than testis can trigger an immune response . These antigens are also expected to become new candidates for cancer - specific immunotherapy, but little information is available on the comprehensive expression of ctas in a large number of samples of gastrointestinal and breast carcinomas (11). Expression of ctas of the mage family has been also reported in human breast carcinomas although only to a limited degree (12). Breast cancers, especially triple - negative cancers, show higher expression of ct genes, which is the prerequisite for any immunotherapeutic approach . Ct genes have also gained attention for immunoprevention in high - risk patients (13). The purpose of the present study is to assess immunohistochemical expression of cta mage-1 in tissue samples of invasive breast cancer and its correlation with known prognostic factors . A total of 113 patients with invasive breast cancer (112 ductal and one lobular) were included . All patients were surgically treated at omid hospital in mashhad university of medical sciences, iran between 2011 and 2013 . Data related to tumor size, grade, stage, estrogen receptor (er) and progesterone receptor (pr) status, human epithelial growth factor receptor 2 (her-2/neu), and axillary lymph node status are summarized in table 1 . Immunohistochemical staining of mage-1 was performed on the invasive breast cancer . For the detection of mage-1 protein, we used undiluted ncl - mage-1 monoclonal antibody (mab), staining is described in detail elsewhere (14). Briefly, tissue slides from paraffin embedded breast cancer tumor samples were places on silane (3 aminopropyltriethoxysilane, a 3648, sigma, st ., slides were heated in an 800-w microwave oven at maximum power for 30 minutes, held in 10 mmol / l edta buffer (ph 6.0) for 5 minutes and then rinsed with a tris buffer solution (pbc, ph 7.2). To suppress endogenous peroxidase activity, after additional rinsing with pbc, they were incubated for 20 minutes with a 1:10 dilution of normal rabbit serum (dakox0902, dako a / s) in a wet chamber at room temperature for 20 minutes to prevent non - specific binding of immunoglobulin . Slides were then treated with undiluted mabs at room temperature for 90 minutes . The envision (dako) system was used as a secondary detection tool and diaminobenzidine tetrahydrochloride served as a chromogen . Sections of normal human testis with intact spermiogenesis were used as positive controls for mage-1 mabs . This method takes into account percentages of positive cells (scored on a 0 - 3 scale) and the intensity of their staining (scored on a 0 - 3 scale). The percentage of positive cells is then multiplied by the intensity of staining, and the final score ranges from 0 (no staining) to 9 (diffuse and strong staining). The final results were further classified as 0 (no staining), 1 (score 1, 2, 3), 2 (score 4, 5, 6) and 3 (score 7, 8, 9). For statistical analysis, mage-1 scores of 0 and 1 were considered negative, whereas scores 2 and 3 were considered positive . The association between immunohistochemical data and different clinicopathological parameters were evaluated by chi2 and t - test . The computer program spss 16 software (spss for windows, 2007) was used . A total of 113 patients with invasive breast cancer (112 ductal and one lobular) were included . All patients were surgically treated at omid hospital in mashhad university of medical sciences, iran between 2011 and 2013 . Data related to tumor size, grade, stage, estrogen receptor (er) and progesterone receptor (pr) status, human epithelial growth factor receptor 2 (her-2/neu), and axillary lymph node status are summarized in table 1 . Immunohistochemical staining of mage-1 was performed on the invasive breast cancer . For the detection of mage-1 protein, we used undiluted ncl - mage-1 monoclonal antibody (mab), staining is described in detail elsewhere (14). Briefly, tissue slides from paraffin embedded breast cancer tumor samples were places on silane (3 aminopropyltriethoxysilane, a 3648, sigma, st . Louis mo, usa). After de paraffinization, slides were heated in an 800-w microwave oven at maximum power for 30 minutes, held in 10 mmol / l edta buffer (ph 6.0) for 5 minutes and then rinsed with a tris buffer solution (pbc, ph 7.2). To suppress endogenous peroxidase activity, after additional rinsing with pbc, they were incubated for 20 minutes with a 1:10 dilution of normal rabbit serum (dakox0902, dako a / s) in a wet chamber at room temperature for 20 minutes to prevent non - specific binding of immunoglobulin . The envision (dako) system was used as a secondary detection tool and diaminobenzidine tetrahydrochloride served as a chromogen . Sections of normal human testis with intact spermiogenesis were used as positive controls for mage-1 mabs . This method takes into account percentages of positive cells (scored on a 0 - 3 scale) and the intensity of their staining (scored on a 0 - 3 scale). The percentage of positive cells is then multiplied by the intensity of staining, and the final score ranges from 0 (no staining) to 9 (diffuse and strong staining). The final results were further classified as 0 (no staining), 1 (score 1, 2, 3), 2 (score 4, 5, 6) and 3 (score 7, 8, 9). For statistical analysis, mage-1 scores of 0 and 1 were considered negative, whereas scores 2 and 3 were considered positive . The association between immunohistochemical data and different clinicopathological parameters were evaluated by chi2 and t - test . A p value <0.05 was considered significant . For all statistical analyses, the computer program spss 16 software (spss for windows, 2007) was used . Mage-1 expression (score 2 +) was detected in 34/111 (30.1%) of patients (figure 1). A, strong nuclear and cytoplasmic staining of most of neoplastic cells (h & e, 100); b, strong nuclear and cytoplasmic staining of most of neoplastic cells (h & e, 100). Mage-1 expression (score 2 +) was detected in 36/111 (31.8%) of patients (figure 1). Expression of mage-1 was significantly associated with lymph node (p = 0.003) breast cancers, but no association was found between mage-1 cytoplasmic expression and tumor size, age, her-2 status, tumor stage, grade, and er/ pr status . Expression of mage-1 was significantly associated with tumor size (p = 0.018) and lymph node (p = 0.042) breast cancers . No association was found between mage-1 nuclear expression and age, her-2 status, tumor stage, grade, and er/ pr status . Mage-1 expression (score 2 +) was detected in 34/111 (30.1%) of patients (figure 1). A, strong nuclear and cytoplasmic staining of most of neoplastic cells (h & e, 100); b, strong nuclear and cytoplasmic staining of most of neoplastic cells (h & e, 100). Mage-1 expression (score 2 +) was detected in 36/111 (31.8%) of patients (figure 1). Table 3 presents associations between mage-1expression with clinicopathological variables . Expression of mage-1 was significantly associated with lymph node (p = 0.003) breast cancers, but no association was found between mage-1 cytoplasmic expression and tumor size, age, her-2 status, tumor stage, grade, and er/ pr status . Expression of mage-1 was significantly associated with tumor size (p = 0.018) and lymph node (p = 0.042) breast cancers . No association was found between mage-1 nuclear expression and age, her-2 status, tumor stage, grade, and er/ pr status . Breast cancer is among the leading causes of death in women worldwide (16). The search for human tumor antigens as potential immunotherapeutic targets represents an appealing therapeutic concept since decades ago . Recent advances in molecular characterization of human tumor - associated antigens have paved the way toward active specific immunotherapy of cancer (17). Ctas are of particular interest, because they are expressed in a very limited number of healthy tissues typically including hla class i negative spermatogonia, while they are expressed in a wide range of malignancies (18)., we analyzed expression of mage-1 antigen on archival paraffin - embedded samples of invasive breast cancer tissue of 113 patients and correlated their expression with other clinicopathological variables . To our knowledge, this is the first report specifically examining expression of mage-1, at the protein level, in breast cancer . Mage-1 cytoplasmic expression (score 2 +) was detectable in 30.1% and nuclear expression (score 2 +) was detectable in 31.8% of patients . Data on mage - a and ny - eso-1 expression in literature are highly variable . The frequency of multispecific mage - a and ny - eso-1 positivity in published studies ranges between 17 and 74% and 2% - 40%, respectively (2). Stefan found a 18% positive cta7 (mage - c1), defined as immunoreactivity in more than 50% of tumor cells, in 124 women with invasive breast cancer (16). In the study of recurrent ductal breast cancer, bandic found 74% of mage - a and 40% of ny - eso-1-positivity in samples (19). These discrepancies observed between studies may be due to different antibodies used for ct detection or difference in scoring system . Some authors define positive x - cta expression according to percentage of cells (19 - 21), while others combine the extent and intensity of cta expression using semi - quantitative scoring systems (22, 23). Studies exploring potential prognostic significance of cta expression in breast cancer have yielded contradictory findings . Some authors found that expression of cta is associated with poorly differentiated histological phenotypes (20, 22). Others found no association between their expression and various pathological parameters (19) or only an association between mage - a1 and ki-67 labeling index (23). According to the present study (table 3), a positive nuclear expression mage-1 status correlated significantly with lymph nodes status (p = 0.042), and positive cytoplasmic expression mage-1 status correlated significantly with lymph nodes (ln) status (p = 0.003). Positive nuclear expression mage-1 status correlated significantly with tumor size (p = 0.018). However, the expression of mage-1 was not associated with other typical adverse clinicopathological features, namely tumor stage, and pathological grade . In badovinac crnjevic study (2) mage - a10 expression was significantly associated with er - negative (p = 0.002), pr - negative (p = 0.002) and her-2-negative (p = 0.044) tumors . They showed that mage - a10 was frequently expressed in the triple negative (tn) subgroup of patients, where the majority (85.7%) of tumors expressed cta (19). Curigliano showed a significantly higher expression of mage - a (26%) in tn breast cancers compared with er - positive tumors (10%) (p = 0.07) (23). Although the exact biological function of ctas is still unknown, future studies will hopefully allow more insights into the activities of ctas in tumor cells on the molecular level . In conclusion, due to mage-1 expression (score 2 +) in about 30% of our patients, even more frequent than her2 positivity in breast cancer
Various techniques of fixation have been described and used successfully in treatment of patients with atlantoaxial instability4678101618). Although posterior wiring techniques have been used in stabilization of the atlantoaxial joint, these simple procedures have been associated with high fusion failure rates due to their limited stiffness in rotation and require rigid postoperative immobilization71314). For this reason, technically demanding rigid screw fixation techniques, such as c1 - 2 transarticular screw fixation and the c1 lateral mass screw combined with c2 pedicle screw fixation have been developed4581114). Both techniques are biomechanically superior to wiring techniques; however, they may carry a risk of iatrogenic injury to the vertebral artery (va)101621). The incidence of va injury during posterior screw fixation of the c1 - 2 complex will probably remain unknown and under - reported1113). Vascular injury presented immediately after the penetration, but delayed onset of vascular symptom after cervical fusion is extremely rare19). We present here an extremely rare case of delayed onset of vascular symptom caused by an embolism and vessel dissection after cervical fusion without definite the screw breach in cervical traumatic atlantoaxial instability patient . A 56-year - old woman visited our hospital complained of neck pain after traffic accident . There was no apparently definite abnormal result in neurologic examination with motor grade 5 in all extremities . The c - spine radiograms, computed tomography (ct) and magnetic resonance (mr) images revealed cervical type ii odontoid process fracture with traumatic spondylolisthesis type i (fig . 1a). Because the patient complained the neck pain with excessive movement at the junction between c1 and c2, the posterior cervical fusion with pedicle screws (c1 and c2) and lateral mass screws (c3) were performed after intraoperative odontoid process reduction under the guide of fluoroscopy (fig . All pedicle screws were inserted with a free - hand technique . After creating a pilot hole and confirming no arterial bleeding or cerebrospinal fluid leakage, an appropriate pedicle screws were inserted . After the surgery, all screws were appropriately inserted without breach, injury of vertebral arteries, and no bleeding sign observed in postoperative ct (fig . 1c, d, e and f). On the sixth postoperative day, the patient had a seizure event with right side weakness . The checked brain ct revealed no definite abnormalities in intracranial lesions, but mr imaging showed multi - focal acute infarction in bilateral occipital lobe, left thalamus, left corpus callosum splenium and bilateral cerebellar hemispheres (fig . Mr angiograms showed stenosis in left vertebral artery and basilar artery (white arrow in fig . 2d), and an angiography confirmed ischemic symptom by thromboembolism due to left va (v3 segment) dissection (black arrows in fig . The injury of vertebral artery (va) by cervical screws was not observed in three dimensional digital subtraction angiographys (fig . Six weeks after the parent artery occlusion, no evidence of acute infarct was observed in follow - up mr images . There are some reports concerning the risk of cerebral infarction subsequent to va injury or embolism caused by cervical fusion14161922). Complete occlusion or penetration of the va at the injured site is thought to be responsible for the infarction18). Posterior fusion of the cervical spine is usually performed using a pedicle screw, lateral mass screw, and a spinous process plate; however, they may carry a risk of direct or indirect iatrogenic injury to the va10161921). Vascular injury presented immediately after the penetration, but delayed onset of vascular symptom after cervical fusion is extremely rare19). To our knowledge, this is the first report of a delayed onset of vertebral artery dissection after posterior cervical fusion in cervical traumatic event . Iatrogenic vertebral artery injury (vai) most cases of va laceration in posterior cervical spine surgery have occurred during the c1 - 2 transarticular screw fixation procedure13). Insertion of transarticular screws into the c1 - 2 facet joint could injure the va, because of the proximity of the va in the bony groove and the blind passage of the screws through the structures142024). A radiological analysis showed that placement of a c1 - 2 transarticular screw was unsafe in about 20% of the patients, secondary to an aberrant course of the va or thin c2 pars / pedicle8). Five (8%) va lacerations out of 61 patients with atlantoaxial instability who had undergone this procedure were also reported by madawi et al.14). In a large retrospective survey, but, the delayed onset of vascular symptom after cervical fusion due to pseudoaneurysm or arteriovenous fistula formation could happen as we presented16). In fact, traumatic injury of the vertebral artery, although rare, can result in laceration, occlusion, dissection, pseudoaneurysm formation or more rarely arteriovenous fistula formation21215). Reported the majority of traumatic injuries of the vertebral artery had a delay in presentation of more than one day (61%)1). This presented case also visited the hospital after traffic accident and had no blunt penetration of screws during the operation, so delayed onset of vascular symptom could be happen by traumatic injury of the vertebral artery . Unfortunately, the authors did not checked preoperative angiography which could be conclusive evidence; however, no hematoma at the operation site in the postoperative ct images is also evidence of not traumatic cause of delayed va dissection . But, another possible hypothesis is present, such as the impending artery rupture followed by the intimal injury after traumatic cervical event . So, traumatic injury of the vertebral artery by preoperative traffic accident was not a conclusive cause, but a subjective cause . Nevertheless its ambiguous cause of delayed va dissection, this case informed the spine surgeon that the possible unusual hemorrhage event after cervical fusion in traumatic cervical patient . Unilateral va injury rarely leads to a lethal complication because the contralateral va feeds cerebral circulation . Neo et al.17) also reported that 15 patients with va injury had no neurologic sequelae except for one patient who complained of vertigo . Taneichi et al.23) reported that va occlusions were rarely symptomatic, based on a prospective observation of 11 va occlusions induced traumatically . Recently, endovascular management, such as coil embolization, stent - assist coil embolization, and the use of stent grafts or covered stents, has been introduced1359). It can be a good alternative in the treatment of va injury as this presented case5). Fortunately, this presented case illustrated a postoperative seizure with hemiparesis in 6 days after the operation, but improved to no neurological deficit with mildly decreased comprehension . When a cervical operation performed in the cervical trauma patient, even if no apparent va injury occurs before and during the operation, the surgeon must take caution not to risk cerebral infarction because of the delayed va dissection.
Do people maintain exercise? . Indeed, the majority of elderly subjects in germany or the us fall far short of the exercise doses recommended for consistently impacting bone mineral density [46]. A novel training technology, called whole - body electromyostimulation (wb - ems), may increase effects of moderate exercise on the musculoskeletal system and thus might be a time - saving and feasible option for subjects unable or unwilling to perform strenuous conventional exercise . Unlike local ems application, wb - ems technology simultaneously stimulates up to 1418 regions or 812 muscle groups with up to 2.800 cm electrode area . Although ems - technology primarily focuses on muscle by activating muscle contraction and directly stimulating muscle protein synthesis rate, there is some evidence that this mode of muscle stimulation also impacts bone . However, human trials determined the effect of ems - application on bone only in the case of disuse - induced bone loss in spinal cord injury (sci) patients . In these studies ems was applied either locally only on plantarflexors or as functional electrostimulation (fes) activating isolated thigh muscles to induce leg cycling movements on ergometry or knee extention movements on strength machines . One meta - analysis and two reviews [10, 11] analyzed and discussed the effect of ems and fes on bmd in sci and found positive results . Basic mechanisms of bone adaptions to disuse after sci and following ems training are reviewed by dudley - javoroski and shields . However, conclusions drawn from studies determining the effect of ems on bone in the specific situation of disuse in paralyzed patients are not valid for the normal population and, furthermore, the transferability of results of local ems studies on the wb - ems technology is rather questionable . In the present study we evaluated the impact of wb - ems on bmd in sedentary, lean, and osteopenic elderly females, a cohort at high risk for fractures . The main hypothesis of this trial was that wb - ems training significantly increases bmd at the lumbar spine compared with a control group . Our secondary hypothesis was that wb - ems training significantly impacts bmd at the proximal femur compared with the control group . The training and electrostimulation trial (test) project is a series of studies that determine the effect of wb - ems on parameters related to risk factors and diseases of elderly subjects . In the present study, test - iii is a randomized, controlled trial (rct) that evaluated the effect of whole - body electromyostimulation (wb - ems) on osteoporosis in lean, sedentary elderly females with osteopenia . To determine the isolated effect of wb - ems we implemented an active control group which performed identical movements as carried out during the wb - ems session . The study protocol was approved by the ethical committee of the friedrich - alexander university erlangen - nuremberg (fau), germany (ethik antrag 4184), and the german radiation safety agency (z 5 - 22462/2 - 2010 - 027). Test - iii was conducted from november 2010 through july 2012 at the institute of medical physics, fau, germany, and is fully registered under www.clinicaltrials.gov (nct01296776). All female subjects (9256 women) 70 years and older, living in the area of erlangen, germany, were contacted by personal letters . Though eligibility criteria for the trial (figure 1) already had been listed in the letter, 272 subjects of the 451 women who responded had to be excluded after phone interviews, because they (a) did not meet our criteria for leanness (body weight (kg)> (body height (cm) 100)), (b) had exercised more than 1 hour per week during the last 10 years, (c) reported contraindications (i.e., total endoprosthesis, abdomen / groin hernia, epilepsy, and cardiac arrhythmia) for wb - ems intervention, and (d) reported diseases or medication affecting our primary endpoints . 179 women were invited to our lab to determine body composition and bone status . After measuring body weight and height with calibrated devices and determining bmd at the lumbar spine and proximal femur, 103 women had to be excluded because they did not meet our inclusion criterions of leanness or osteopenia (bmd <1 sd t - score) (figure 1). The 76 remaining subjects were assigned into two study groups using computerized block randomization stratified for age (block size: n = 2) by an external statistician to ensure allocation concealment . Wb - ems group performed 54 weeks of consecutive wb - ems intervention, while in parallel the control group (cg) carried out an intermitted not strenuous gymnastics program . The underlying rationale for this procedure was to validate the isolated effect of wb - ems versus a motivated and blinded control group that performed gymnastics containing identical low intensity / low amplitude movements as the wb - ems group . Both interventions were performed at the institute of medical physics (imp), which could be easily reached by the subjects . All the sessions were supervised by certified trainers who recorded the attendance of the participants . In order to check for parameters that may impact our study endpoints, lifestyle parameters (i.e., dietary intake, physical activity, etc .) Were inquired by questionnaires at baseline and follow - up [16, 17]. After analyzing standardized dietary intake protocols over 4 days (prodi-4.5, wissenschaftlicher verlag, freiburg, germany), both groups were provided with a maximum of 1,200 mg / d calcium and 800 iu / d of cholecalciferol (rottapharm / madaus, cologne, germany). The wb - ems group performed supervised wb - ems training (18 - 19 min / session; bipolar current; frequency: 85 hz; pulse - breadth: 350 sec) 3 times in 2 weeks (each monday or tuesday and every second thursday or friday) for 54 weeks using the wb - ems technology of miha bodytec (augsburg, germany). Eight muscle groups were simultaneously activated by electrostimulation: upper legs, upper arms, bottom, abdomen, chest, lower back, upper back, and latissimus dorsi . Wb - ems sessions consisted of easy, not strenuous dynamic exercises performed in a standing position, with 6 s dynamic movements under ems intermitted by 4 s of static rest without current . 1014 exercises (e.g., dead lift with elbow - extension or arm - flexion; squat with trunk flexion; squat with lat pulleys or military press; squat with crunch and butterfly or reverse fly; squat with vertical chest press or vertical rowing) were structured in 1 - 2 sets of 8 repetitions . Total time under load (current) averaged 11 min / session during which 110 stimulation intervals 6 sec were completed . Motion amplitude as well as corresponding intensity generated by the movements was set low (i.e., squat: leg - flexion: <35) to prevent effects from the exercise per se . In a wb - ems session 3 subjects underwent supervised and video - guided exercise (at three wb - ems devices) at the same time . Current intensity was individually adapted for each region during the first sessions to a rate of perceived exertion (rpe) of 1416 of 20 (somewhat hard to hard). Intensities were saved on chip cards that allowed fast and reliable setting of the devices in the subsequent sessions . If necessary, current intensity was increased in the following sessions to maintain the predefined rate of perceived exertion . Subjects of the cg performed two 10-week blocks of easy, not strenuous gymnastics with 1 session (60 min)/week with 10 weeks of rest between the blocks . After 5 min of walking variations, subjects performed the identical low intensity / low amplitude dynamic exercises as during the ems sessions . After this, flexibility, general coordination, and balance exercises were carried out for 20 min . The aim of the cg protocol was to ensure a blinding by carrying out a multifaceted, attractive exercise and relaxation program . Primary and secondary endpoints were determined at baseline and after 54 weeks of intervention . Primary outcome measures bone mineral density at the lumbar spine (ls),bone mineral density at the proximal femur (total hip region of interest (thip - roi)). Bone mineral density at the lumbar spine (ls), bone mineral density at the proximal femur (total hip region of interest (thip - roi)). Secondary outcome measures total lean body mass (lbm),grip strength . Total lean body mass (lbm), all assessments and analysis were carried out in a blinded mode . Research assistants were not informed about the status of the participants (wb - ems or cg) and were not allowed to ask either . Body weight was measured to the nearest 0.1 kg on a digital scale (inbody 230, seoul, korea). Height was determined barefoot to the nearest 0.1 cm with a stadiometer (holtain, crymych dyfed, great britain). Body composition was assessed with dual energy x - ray absorptiometry (dxa, qdr 4500a, discovery - upgrade; hologic inc ., grip strength of the dominant arm was assessed with a jamar dynamometer (jamar, bolington, il). Bone mineral density (bmd) was also determined by dxa at the lumbar spine (l1l4, a.p .) And the proximal femur (total hip roi) at baseline and after 1 year using standard protocols specified by the manufacturer . Long - term reliability was 0.4% (coefficient of variation) as determined by 177 lumbar spine phantom scans conducted during the study period . In order to detect a relevant between - group difference of 2.0% (standard deviation (sd): 2.8%) 31 subjects / group were required (5% error probability, 80% statistical power). Analyses were performed on an intention - to - treat basis for all the participants who completed the baseline and at least one follow - up measurement (finisher analysis). The treatment effect was defined as between - group differences in changes from baseline to 12 months . In order to obtain normally distributed data all endpoints (bmd, lbm) analysis of covariance with baseline value, age, height, and fat and muscle mass as covariates were carried for statistical comparison of the two study groups . We used spss 19.0 (spss inc, chicago, il) for all the statistical procedures . N = 10) of 76 subjects were unable or unwilling to visit the one - year control assessment and were thus lost to follow - up (figure 1). Altogether 6 women suffered fractures, surgery, and/or serious diseases (cancer, chd). One subject of the cg lost interest, and five subjects listed personal reasons for their withdrawal, two of them related to the cg protocol, one related to the wb - ems protocol . One participant of the cg died during the interventional period and one woman of each group moved . Thus 28 subjects of the cg (74%) and 32 subjects of the wb - ems group (84%) were included in the analysis . Summing up, as total attendance in the wb - ems group, 61 out of 78 sessions (79 18%) were conducted . The corresponding attendance rate in the cg was slightly lower (74 18%; 14.9 of 20 sessions). Although the total number of sessions was much lower in the cg, the difference in total exercise volume was smaller with an average of 24 min / week in the wb - ems group and 18 min in the cg . No intergroup differences were recorded for anthropometric and clinical parameters at baseline (table 1). With respect to confounding parameters, no significant changes between baseline and follow - up were observed for lifestyle parameters (physical activity, e.g.) And/or medication as recorded by questionnaires or dietary intake as assessed by 4-day dietary protocol and analyzed using prodi-4.5/03 expert software (wissenschaftlicher verlag, freiburg, germany). Bmd at the ls increased in the wb - ems groups (0.6 2.5%) and decreased in the cg (0.6 2.4%). At the total hip roi bmd decreased in both groups (wb - ems: 0.9 1.9% versus cg: 1.0 2.3%). Similar negative changes were observed for the proximal femur subregions (femoral neck, trochanter). Differences between wb - ems and cg - group were borderline nonsignificant for ls - bmd (p = 0.051, es: d = 0.49) and not significant for bmd of the total hip roi (p = 0.771, es: d = 0.04). Total lbm as assessed by total body dxa scans increased in the wb - ems group (0.7 1.6%) and decreased in the cg (0.8 2.5%). Corresponding differences between wb - ems and cg - group were significant (p = 0.006, es: d = 0.71). The changes in grip strength were 10.5 12.6% in the wb - ems group and 2.2 8.19% in the cg (p = 0.000, es: d = 0.71). This is the first trial that determined the effect of (wb-) ems on bmd at lumbar spine or / and proximal femur in elderly females with osteopenia . We found a borderline significant effect (p = 0.051) for the lumbar spine bmd, but not for the femoral neck site . In view of the high effects of wb - ems on muscle mass and strength in previous studies [20, 21] and the close interaction of muscle and bone, we had expected stronger effects on bone . In the present study the effect of wb - ems on lbm was significant but rather moderate, showing a 1.5% net gain . However, maximum isometric leg and trunk extension strength significantly increased by more than 10%, as published elsewhere . All the other studies examining the effect of ems in bmd exclusively determined (functional) electromyostimulation (fems) under disuse conditions (e.g., sci) [2232]. In the recent meta - analysis by chang et al . The authors found a significant increase in bmd after 3, 6, and 12 months of fes leg cycling or fes knee extension exercises in sci patients . A longer period of exercise and a higher training frequency were associated with higher effectiveness . Reviewed 10 studies determining the effect of fes leg cycling on bmd, taking into account the time after injury . Two of two studies report effects of fes therapy in the first 2 months after injury; only one of three studies showed effects at an average of 36 years after injury and 4 out of five 913 years after injury . In line with the results of dolbow et al ., biering - srensen et al . Concluded in a systematic review including 19 ems studies that there may be some effects of electrical stimulation especially in the early phase . This review includes studies with ems of the lower limb (4 studies), leg cycling fes (7 studies), knee extention fes (5 studies), fes during treadmill gait (1 study), or a combination of leg cycle and knee extension fes . Improvement is seen in a longer period of training or with higher training frequency . Although the studies included in the meta - analysis and reviews differ widely with respect to subjects or measurements, the large majority of trials which ensured sufficient time for bone adaption (612 months) reported positive bmd changes at skeletal sites stimulated and loaded by ems or fes cycling or fes leg - extension exercises . However, it can be assumed that in paralyzed subjects the strain threshold is low due to inactivity and it remains unclear if the positive bmd changes were caused more by ems induced muscle contraction producing joint reaction forces or just by resulting external reaction forces leading to axial loading of bones during exercises like leg - extension or fems cycling . And again, the relevance of the results of these studies for older people without serve functional limitations is rather questionable . A comparison of the effect of wb - ems with gold - standard exercise protocols might be helpful for estimating the relevance of wb - ems programs for fighting osteoporosis . Reviewing the literature for exercise - induced bmd changes in subjects 60 years and older as assessed by dxa (review [33, 34]) shows that our wb - ems effects were lower compared with conventional exercise effects in particular when specific resistance exercise was applied . Comparing the results of the present wb - ems results with data of our recent 18-month sefip exercise trial that used identical measurements and included comparable subjects with respect to age (69 4 yrs) with higher bmi (26 4 kg / m) revealed more favorable data for the sefip cohort (net bmd difference eg versus cg at ls and thip 1.52.1%; both p = 0.001) that had carried out a combined resistance / aerobic / balance protocol . Little is known about the optimum ems protocol for impacting bone, and the low effect on bone mineral density in the present study might be due to a suboptimal setting of current parameters . Although the most favorable composition of ems parameters for triggering bone adaptation still has to be established, a recent study that directly compared different frequencies of ems in an animal disuse model determined the most favorable effect on bone parameters (e.g., volume fraction, connectivity, and trabecular number / thickness) especially at 20 and 50 hz . With respect to stimulation intensity dudley - javoroski and shields suggested supramaximal amperage (200 ma) to elicit very strong contractions . Because each pair of electrodes was regulated separately with different intensities and due to differences in electrode size, we were unable to control and describe the stimulation intensity (ma) in the present study . In ems studies in sci patients, where isolated muscles were stimulated, external forces were measured and put into relation with body weight to estimate intensity . Because of the simultaneous activation of agonists and antagonists and resulting co - contraction we applied ems for 3 20 min in 2 weeks using a bipolar current at 85 hz at a pulse width of 350 sec whereas 6 sec of stimulation was intermitted by 4 sec of rest . The highly significant effect of this protocol on muscle mass and strength and its acceptance [20, 21, 37] supported the application of this ems protocol . Considering that enthusiasm for conventional exercise is rather low in the cohort of elderly women, one key factor for the relative high compliance with ems training may have been the low total volume of the program (in total 30 min / week). Furthermore, wb - ems programs are applied under rather individualized conditions and it may be also the exclusiveness of the exercise program that resulted in a high compliance . (a) we evaluated the effect of ems in the cohort of lean elderly females with osteopenia and the assignability of our results to other cohorts is questionable . (b) although dxa is still the gold standard technology for assessing bmd, qct technique may be the more appropriate method for assessing bmd at lumbar spine in the cohort of subjects 70 years and older due to degenerative changes of the spine . (c) no x - ray examinations have been performed to detect vertebral body fractures . However, we presume that bmd increases in wb - ems group were not the result of training induced compression fractures . In the analyzed ls - scans further, during wb - ems the mechanical loading of vertebrae due to muscular tension is rather moderate compared to classic high impact training contents . (d) a semiactive control group that performed a comparable exercise volume and identical movements was implemented to ensure blinding and to determine the effect of wb - ems per se . For reasons of attractiveness and compliance the cg did not carry out only identical movements but also performed exercises for mobility, coordination, and relaxation; and the training schedule differed . However, the movements of the wb - ems program and all the contents of the cg sessions were not strenuous and designed not to impact our endpoint . Thus, in our opinion, the influences of the differences in exercises contents and schedule on the validity of the studies are rather low . (e) due to a lower number of subjects included (76 instead of 80) and a slightly higher drop - out rate than expected we failed to realize our estimated sample size of 31 subjects / group . (g) it is not possible to objectify intensity of muscle contraction during wb - ems and it is not clear if subjects realized the requested high intensity . In summary, we found a borderline nonsignificant effect of wb - ems on bone mineral density at the lumbar spine and no effect at the hip . However, taking into account the high impact of this technology on muscle mass and strength, wb - ems may be an option for musculoskeletal prevention / rehabilitation at least for (elderly) subjects unable or unwilling to exercise conventionally . Nevertheless, due to the higher impact of mixed exercise programs on bmd and their comprehensive effect on multiple risk factors and diseases of advanced age, classic exercise should be favored for elderly subjects.
Quantitative changes in longitudinal (t1) and transverse (t2) relaxation times in discrete brain regions have been described in a host of different pathologies 1 . However, reproducibility for quantitative mri measurements has proved a challenge and requires lengthy scan times to obtain parameters sequentially . Consequently, quantitative parameters are rarely acquired, and most evaluations of mri images are based on visual inspection without quantitative evaluation of underlying values of t1 and t2 . Recently, a new approach of estimating multiple parameters at once, called mr fingerprinting (mrf), has been proposed 2 . This method has the potential to be used for quantitative mri routinely in both clinical and preclinical environments, enabling new tools for research and diagnosis . Mr fingerprinting is based on the response of tissues to repeated acquisition sequences without attaining the steady state . Voxelwise responses are compared with a dictionary of simulated responses calculated from particular parameters (including, but not limited to, t1 and t2) and voxels are assigned the parameters from the best match to produce multiparametric maps . To date, mrf has been performed with fast gradient echo sequences based on steady state free precession (ssfp). For fully rewound sequences, the simulations for the fingerprint database must include an estimate of the voxelwise frequency inhomogeneity for the correct signal evolution 2 . To apply the technique of mrf in small animals, where less b0 homogeneity is usually achieved, a gradientspoiled approach has been used . Due to data redundancy between frames, this characteristic can be used to achieve aliasfree maps in short times . However, antialiasing the mri images before mrf pattern recognition is also possible 5, 6 . Here, we tested a reconstruction method with a simple antialiasing technique, sharing the edges of kspace between neighboring mrf frames before comparing signal evolution traces with the fingerprint database . Current mrf methods, both in the clinical and preclinical environment, have been demonstrated assuming a perfectly rectangular slice profile and homogeneous excitation . Indeed, it has been shown that different pulse shapes used for slice selection produce different t2 values 4 . In addition, inhomogeneous radiofrequency excitation profiles are a common problem in both preclinical and clinical highfield scanners . We have extended the mrf approach by including a parameter for b1 in the signal evolution dictionary . In addition, we have implemented a threedimensional (3d) acquisition and tested a new antialiasing strategy applied before pattern recognition . The 3d acquisition has the advantage of broader coverage (particularly important for whole brain imaging) and the considerable reduction of slice profile effects . As well as b1 estimation our scheme builds on ssfp mrf as recently reported 3 . Briefly, an inversion pulse is applied before a train of fast imaging with steady state precession readouts with variable flip angles and repetition time delays . These rapid changes prevent a steady state from being achieved, but rather lead to signal variations dependent on local magnetic properties and the applied b1 field . Phase encoding is applied before each readout and rewound afterward so that each train of readouts has the same phase encoding value and the whole sequence is repeated for each phase encoding step . In our implementation, we used a train of 1000 frames taking 10 s to acquire a single kline for all frames . To allow for return to equilibrium, we inserted a delay of 5 s before repeating the acquisition with a different level of phase encoding . To increase the sensitivity to different b1 values, we changed the final part of the sequence from the original mrf approach as shown in figure 1 . By using abrupt changes of the nominal excitation flip angle, our scheme introduces oscillations of signal the frequency of which is proportional to the obtained flip angle (fig . We used alternating blocks of 15 pulses of flip angle 90 followed by 15 pulses of flip angle 0 to exploit the oscillatory behavior of the signal to resolve the b1 field . C: the modified fa pattern demonstrated here, including abrupt changes in flip angle to increase the sensitivity to the b1 field . Simulation of the signal obtained with our novel mrf scheme (t1 = 160 ms t2 = 44 ms) at the end of the mrf train, in correspondence with abrupt changes in flipangle . Flip angle changes between blocks of 45 and 0 (a), between 90 and 0(b), between 135 and 0(c). Two different phantoms were used, one to investigate 2d mrf and another to assess the accuracy of the measurements across partitions of a 3d acquisition . We used a bruker biospec 47/40 system (bruker inc ., ettlingen, germany) equipped with 400 mt / m gradients and a 12 cm diameter quadrature birdcage coil used as a transceiver . Each signal acquisition was preceded by an adiabatic inversion pulse (15 ms hyperbolic secant). Mrf acquisitions were performed at the magnet isocenter on a single slice (3 mm slice thickness; field of view 7 cm; 64 64 matrix yielding 1.1 mm resolution with 50 khz receiver bandwidth). To assess 3d mrf acquisitions, we used a matrix of 64 64 64 for 0.5 mm isotropic resolution of a uniform gel with a t1 of 640 ms and a t2 of 74 ms . We measured the average value obtained in a region of interest through the slice direction (z). We compared the following methods: the original mrf method (with no b1 in the dictionary); the original mrf scheme including b1 in the dictionary; our new method with abrupt changes in flip angle; and classical methods (described below). A formalinfixed listerhooded adult rat brain was imaged using a 35mmdiameter linear birdcage coil for both signal transmission and reception . For 2d mrf, we tried three different excitation pulse shapes: sinc, hermite, and gauss each of 2 ms and bandwidth factors 6.21, 5.40, and 2.74, respectively . For 3d acquisitions, we used a 64 64 64 matrix with 0.5 mm isotropic resolution (a 2 ms sinc excitation pulse as above was used). As in the phantom acquisitions, each signal acquisition was preceded by an adiabatic inversion pulse (hyperbolic secant, length 15 ms, bandwidth factor 77.86). The receiver bandwidth was 50 khz in each case . The scan time for the fully sampled 3d acquisition was 17 h 4 min (10 s mrf train + 5 s recovery time) (64 64) phase encoding steps . To test our acceleration strategy, we retrospectively undersampled the 3d acquisition of the ex vivo rat brain . Our undersampling scheme consisted of a binary mask in the phase encode directions (kykz plane) as shown in figure 3 . Nonlinear sampling based on a gaussian distribution around the kspace center was used with a uniform angular distribution . The gaussian function had a standard deviation of 45% of the full ky and kz axes . This could be implemented readily on a scanner by acquiring a list of preplanned klines varying for each frame . A: the undersampling scheme used for our accelerated 3d acquisitions acquiring 9% of kspace . C: the values corresponding to the kspace location (32, 15, 15) for all frames when zerofilling . D: kspace data for the same location as (c) when using our viewsharing method, applying a nearestneighbor interpolation through time ., the correction was obtained using the inverse of the sampling probability density function used for generating the kspace masks . Finally, we implemented a viewsharing strategy where unacquired kspace points were borrowed from the nearest frame with an acquired point at that location . As a result of our sampling density, points closer to the center, containing most of the contrast information, were acquired with higher temporal resolution . On the other hand, the edges of kspace, changing less during the mrf signal evolution, were acquired at lower temporal resolution . To test our undersampling strategy we prepared multiparametric mrf maps using data from the fully sampled kspace . We compared maps obtained from 18%, 9%, and 5% of kspace to values found using the full dataset . Values were measured in the whole brain and muscle tissue, which were automatically masked using thresholding . The thresholding criterion was of a t1 between 0 and 1 s, which included all brain and muscle for our formalinfixed sample . To perform a timematched comparison of our view sharing technique, we compared the use of only 8% of kspace for all frames with fully acquiring only the first 80 frames of the mrf sequence . For this experiment, aimed at evaluating the efficiency of the acquisition only, we excluded b1 from the dictionary . Signal simulations were performed using extended phase graphs 8, including gradient dephasing as well as radiofrequency pulses and signal evolution . All code for simulation and pattern recognition was written in matlab (mathworks, natick, ma). The dictionary contained values of t1 ranging from 80 ms to 1000 ms in steps of 20 ms, and from 1 to 2.5 s in steps of 50 ms . Values of t2 ranged from 10 ms to 100 ms in steps of 2 ms and from 105 ms to 250 ms in steps of 5 ms . B1 was modelled by a flipangle factor, a linear factor equal to the obtained flip angle divided by the desired flip angle . The quantitative maps using each scheme were compared with standard estimation of t1, t2 and b1 using manufacturerprovided sequences . T1 and t2 were estimated using spinecho sequences with the same field of view (fov) and matrix as the mrf acquisitions . The mrf scan times were compared with scan times from the manufacturerprovided t1+t2 rare relaxometry sequence, with unchanged sequence parameters [multiecho spin echo; echo time (te): 11, 33, 55, 77, 99 ms; repetition time (tr) 200, 400, 800, 1500, 3000, 4500 ms; number of excitations (nex), 1] on the same geometry . For more accuracy, in our classical measurements we increased the number of sampled points and averages and used single echoes rather than multiecho to reduce the impact of system imperfections . However, for comparison of the time taken we used the acquisition time of the unmodified sequence . T1 was estimated with a spin echo sequence with variable tr (tr: 10,000, 3000, 1500, 800, 400, 200 ms; te: 11 ms; receiver bandwidth 48 khz, 3 nex). T2 was estimated with singleecho spinecho acquisitions with variable te (tr: 2500 ms; te: 12, 36, 48, 60, 84, 108, 216, 324, 500 ms; receiver bandwidth 60 khz, 1 nex). Two gradient echo images were acquired with a flip angle of 45 and 90 on with the same fov of the mrf acquisitions (tr / te 15,000/2.9 ms, excitation with sinc pulse of 2 ms duration, receiver bandwidth 78 khz, 1 nex). Due to slice profile imperfections, the flip angle seen by spins varies across the slice and includes some contribution from spins outside the slice . To account for these effects in the mrf dictionary the shinnarle roux algorithm 15 was used to create a slice profile of 128 partitions based on the pulse waveforms used by the scanner . The extended phase graph simulation was performed for the central flip angle of each of the 128 partitions producing an mrf train for each . The signal of each frame was summed over each partition to create a dictionary for each pulse waveform that takes account of the variability in flip angles experienced across the slice . Our scheme builds on ssfp mrf as recently reported 3 . Briefly, an inversion pulse is applied before a train of fast imaging with steady state precession readouts with variable flip angles and repetition time delays . These rapid changes prevent a steady state from being achieved, but rather lead to signal variations dependent on local magnetic properties and the applied b1 field . Phase encoding is applied before each readout and rewound afterward so that each train of readouts has the same phase encoding value and the whole sequence is repeated for each phase encoding step . In our implementation, we used a train of 1000 frames taking 10 s to acquire a single kline for all frames . To allow for return to equilibrium, we inserted a delay of 5 s before repeating the acquisition with a different level of phase encoding . To increase the sensitivity to different b1 values, we changed the final part of the sequence from the original mrf approach as shown in figure 1 . By using abrupt changes of the nominal excitation flip angle, our scheme introduces oscillations of signal the frequency of which is proportional to the obtained flip angle (fig . We used alternating blocks of 15 pulses of flip angle 90 followed by 15 pulses of flip angle 0 to exploit the oscillatory behavior of the signal to resolve the b1 field . C: the modified fa pattern demonstrated here, including abrupt changes in flip angle to increase the sensitivity to the b1 field . Simulation of the signal obtained with our novel mrf scheme (t1 = 160 ms t2 = 44 ms) at the end of the mrf train, in correspondence with abrupt changes in flipangle . Flip angle changes between blocks of 45 and 0 (a), between 90 and 0(b), between 135 and 0(c). Two different phantoms were used, one to investigate 2d mrf and another to assess the accuracy of the measurements across partitions of a 3d acquisition . Ettlingen, germany) equipped with 400 mt / m gradients and a 12 cm diameter quadrature birdcage coil used as a transceiver . Each signal acquisition was preceded by an adiabatic inversion pulse (15 ms hyperbolic secant). Mrf acquisitions were performed at the magnet isocenter on a single slice (3 mm slice thickness; field of view 7 cm; 64 64 matrix yielding 1.1 mm resolution with 50 khz receiver bandwidth). To assess 3d mrf acquisitions, we used a matrix of 64 64 64 for 0.5 mm isotropic resolution of a uniform gel with a t1 of 640 ms and a t2 of 74 ms . We measured the average value obtained in a region of interest through the slice direction (z). We compared the following methods: the original mrf method (with no b1 in the dictionary); the original mrf scheme including b1 in the dictionary; our new method with abrupt changes in flip angle; and classical methods (described below). A formalinfixed listerhooded adult rat brain was imaged using a 35mmdiameter linear birdcage coil for both signal transmission and reception . For 2d mrf, we tried three different excitation pulse shapes: sinc, hermite, and gauss each of 2 ms and bandwidth factors 6.21, 5.40, and 2.74, respectively . For 3d acquisitions, we used a 64 64 64 matrix with 0.5 mm isotropic resolution (a 2 ms sinc excitation pulse as above was used). As in the phantom acquisitions, each signal acquisition was preceded by an adiabatic inversion pulse (hyperbolic secant, length 15 ms, bandwidth factor 77.86). The scan time for the fully sampled 3d acquisition was 17 h 4 min (10 s mrf train + 5 s recovery time) (64 64) phase encoding steps . To test our acceleration strategy, we retrospectively undersampled the 3d acquisition of the ex vivo rat brain . Our undersampling scheme consisted of a binary mask in the phase encode directions (kykz plane) as shown in figure 3 . Nonlinear sampling based on a gaussian distribution around the kspace center was used with a uniform angular distribution . The gaussian function had a standard deviation of 45% of the full ky and kz axes . This could be implemented readily on a scanner by acquiring a list of preplanned klines varying for each frame . A: the undersampling scheme used for our accelerated 3d acquisitions acquiring 9% of kspace . C: the values corresponding to the kspace location (32, 15, 15) for all frames when zerofilling . D: kspace data for the same location as (c) when using our viewsharing method, applying a nearestneighbor interpolation through time ., the correction was obtained using the inverse of the sampling probability density function used for generating the kspace masks . Finally, we implemented a viewsharing strategy where unacquired kspace points were borrowed from the nearest frame with an acquired point at that location . As a result of our sampling density, points closer to the center, containing most of the contrast information, were acquired with higher temporal resolution . On the other hand, the edges of kspace, changing less during the mrf signal evolution, were acquired at lower temporal resolution . To test our undersampling strategy we prepared multiparametric mrf maps using data from the fully sampled kspace . We compared maps obtained from 18%, 9%, and 5% of kspace to values found using the full dataset . Values were measured in the whole brain and muscle tissue, which were automatically masked using thresholding . The thresholding criterion was of a t1 between 0 and 1 s, which included all brain and muscle for our formalinfixed sample . To perform a timematched comparison of our view sharing technique, we compared the use of only 8% of kspace for all frames with fully acquiring only the first 80 frames of the mrf sequence . For this experiment, aimed at evaluating the efficiency of the acquisition only, we excluded b1 from the dictionary . Signal simulations were performed using extended phase graphs 8, including gradient dephasing as well as radiofrequency pulses and signal evolution . All code for simulation and pattern recognition was written in matlab (mathworks, natick, ma). The dictionary contained values of t1 ranging from 80 ms to 1000 ms in steps of 20 ms, and from 1 to 2.5 s in steps of 50 ms . Values of t2 ranged from 10 ms to 100 ms in steps of 2 ms and from 105 ms to 250 ms in steps of 5 ms . B1 was modelled by a flipangle factor, a linear factor equal to the obtained flip angle divided by the desired flip angle . The quantitative maps using each scheme were compared with standard estimation of t1, t2 and b1 using manufacturerprovided sequences . T1 and t2 were estimated using spinecho sequences with the same field of view (fov) and matrix as the mrf acquisitions . The mrf scan times were compared with scan times from the manufacturerprovided t1+t2 rare relaxometry sequence, with unchanged sequence parameters [multiecho spin echo; echo time (te): 11, 33, 55, 77, 99 ms; repetition time (tr) 200, 400, 800, 1500, 3000, 4500 ms; number of excitations (nex), 1] on the same geometry . For more accuracy, in our classical measurements we increased the number of sampled points and averages and used single echoes rather than multiecho to reduce the impact of system imperfections . However, for comparison of the time taken we used the acquisition time of the unmodified sequence . T1 was estimated with a spin echo sequence with variable tr (tr: 10,000, 3000, 1500, 800, 400, 200 ms; te: 11 ms; receiver bandwidth 48 khz, 3 nex). T2 was estimated with singleecho spinecho acquisitions with variable te (tr: 2500 ms; te: 12, 36, 48, 60, 84, 108, 216, 324, 500 ms; receiver bandwidth 60 khz, 1 nex). Two gradient echo images were acquired with a flip angle of 45 and 90 on with the same fov of the mrf acquisitions (tr / te 15,000/2.9 ms, excitation with sinc pulse of 2 ms duration, receiver bandwidth 78 khz, 1 nex). Due to slice profile imperfections, the flip angle seen by spins varies across the slice and includes some contribution from spins outside the slice . To account for these effects in the mrf dictionary the shinnarle roux algorithm 15 was used to create a slice profile of 128 partitions based on the pulse waveforms used by the scanner . The extended phase graph simulation was performed for the central flip angle of each of the 128 partitions producing an mrf train for each . The signal of each frame was summed over each partition to create a dictionary for each pulse waveform that takes account of the variability in flip angles experienced across the slice . Reconstruction time for the parametric maps (including b1) from each slice was less than 1 min . Figure 4 compares estimates of t1, t2, and b1 from our acquisitions with the original mrf train of delays and flip angles, our modified scheme and the standard spin echo method . Good agreement was seen between techniques for t1 with less than 5% discrepancy between methods . We found that the previously reported mrf sequences did not successfully resolve t2 and b1 effects with substantial errors in the estimation of both . Our new method, as a result of abrupt changes in flip angle, was able to better discriminate between these parameters . However, this 2d mrf acquisition did not provide a complete separation of t2 and b1 due to slice profile effects, and b1 maps systematically underestimated (> 10%) the true values when using a 2d acquisition . Results comparing the original mrf scheme, our modified scheme with abrupt changes in flip angle (dfa) and the maps using classical methods (described in methods). The original scheme confounds t2 and b1 effects, while scheme 2 can, though here with 2d acquisitions a systematic underestimation of b1 is seen relative to the double angle method . The effect of slice profiles on mrf results is clear in figure 5 which shows mrf maps from the rat brain without slice profile correction for sinc, hermite, and gauss pulses with 3d mrf and spin echo methods for comparison . As in the phantom, mrfderived t1 measurements do not appear to be sensitive to the slice profile but the estimates for t2 and b1 vary considerably . Compared with 3d for sinc, hermite, and gauss shapes, t1 values differ by 3.8%, 3.8%, and 4.3%; t2 values differ by 6.5%, 17%, and 8.7%, and b1 differs by 8.4%, 6.7%, and 36% (rms errors). Comparison between 2d mrf with different slice profiles, our 3d mrf acquisition, and classical methods . When performing 2d imaging, only using a sinc excitation agreed with the 3d acquisition, despite a constant negative bias in b1 . 3d mrf slightly underestimated both t1 and t2 compared with spin echo measurements, but agreed well with doubleangle b1 measurements . The discrepancies in the b1 estimation are considerably reduced when slice profiles are taken into account (fig . The corresponding discrepancies for t1 values are 3.8%, 3.8%, and 4.6%; for t2 24%, 14.9%, and 7.7% and b1 4.0%, 2.6%, and 5.3% for sinc, hermite, and gauss shapes, respectively (rms compared with 3d). Comparison between mrf results for t2 and b1 with and without slice profile correction (labeled slr). Figure 7 shows profile plots of t1, t2, and b1 measured from a uniform phantom with the original mrf approach, mrf with correction for b1 with and without the abrupt flip angle changes in addition to the standard measurements . T1 measurement is accurate for mrf methods across the slice, where the spinecho measurement is inaccurate away from the center . Without modeling b1 in the dictionary b1, results are similar to figure 4 with confounded estimates unless abrupt flip angle changes are introduced into the scheme . Quantitative measurements on a uniform phantom plotted through the 3d slice direction . The original mrf scheme with no b1 estimate (in blue) presented a bias dependent on b1 . The original scheme including b1 estimation (dotted green) could not discriminate between t2 and b1 effects . Our new mrf acquisition including abrupt changes in flipangle (black, labelled dfa) had no significant bias, and compared well with the expected values (magenta). At the edges of the slice, some t2 underestimation can be observed . Figure 8 compares the different approaches to deal with undersampled data, and it can be seen that viewsharing significantly outperforms the other methods . A comparison of the viewsharing method on 8% kspace with a timematched acquisition fully acquiring just the first 80 mrf frames revealed that spatial undersampling has a high efficiency . A view sharing method using only 8% of kspace gave lower errors (t1 = 35 ms; t2 = 4 ms; rms compared with full) than a timematched comparison using only the first 80 elements of the mrf sequence (t1= 450 ms; t2= 50 ms; rms compared with full). Quantitative t1 maps from an axial slice of our multiparametric 3d mrf acquisition, comparing fully sampled data with maps obtained acquiring only 8% of kspace . Zero filling generates artifacts due to nonuniform sampling of kspace; density correction of the zerofilled data recovers the details in the image but results in a noisy map; sharing kspace points between neighboring time frames reduces the noise . Acquisitions using 18% of kspace (3 h long) and 9% of kspace (1.5 h long), generated quantitative maps (fig . All the undersampled mrf scans were faster than the manufacturerprovided t1+t2 rare relaxometry sequence on the same geometry (4.5 h). Mrf maps from only 5% of kspace are shown in supporting figure s1, which is available online . This corresponds to an acquisition of approximately 50 min for the whole brain . For this acquisition, some areas of the b1 and t2 maps presented errors superior to 10%, mostly in remote areas of the head where b1 is changing more rapidly and in areas with low snr (e.g., due to the inclusion of air in the ex vivo sample). Comparison of a full 3d dataset of a fixed rat brain with the maps reconstructed using only 18% of the data and 9% of the data . Values for mean error due to undersampling, as well as 5% and 95% percentiles are reported in table 1 . Undersampling the acquisitions did not significantly bias the t1, t2, and b1 estimates . Error of undersampled acquisitions with respect to full, calculated on a mask including the brain and musclea data are expressed as mean error [5% percentile; 95% percentile]. We have shown that b1 estimation can be used in an mrf framework and that this acquisition can be extended to 3d . This is particularly important for the application of mrf to smallbore preclinical scanners where small rf coils may have significant inhomogeneity . The inclusion of b1 effects will be of growing importance, as these are problematic for body imaging at both 3 t and 7 t 10, 11 and brain imaging at 7 t 12 . Images in mrf scans can be reconstructed from undersampled kspace, as the mrf reconstruction sees through aliasing 2 . However here we have successfully demonstrated a simple strategy to deal with undersampled kspace acquisitions . In our scheme, nonacquired kspace points in one frame are borrowed from neighboring frames where these had been acquired . This concept, similar to keyhole imaging 13, is based on the fact that the image contrast is mainly in the center of kspace, while the image details, which are unchanged between frames, are in the edges of kspace . Therefore, the signal evolution is estimated well when only the central part of kspace is updated in subsequent frames . When doing slice selective imaging, different locations in the slice are excited with a different flip angle leading to partial volume effects . In mrf, partial volume effects have a different behavior with respect to conventional imaging 14 . In conventional imaging, different subvoxel areas are averaged within a voxel, while mrf tends more toward the most represented subvoxel area, as it can be noted when using different pulses in figure 5, where b1 is not homogeneous throughout the voxel . We demonstrated that the slice profile can be directly included in the simulation, performing independent calculations for single subvoxel areas using slice profiles derived from the shinnarle roux algorithm 15 . . To check this, we used actual flip angle imaging 16 to measure the mean flip angle in a uniform phantom achieved when different pulse shapes were used . The measured flip angles were found to be factors of 1.07 for sinc, 1.06 for hermite, and 0.96 for gauss pulses . Introducing these factors for flip angles into the dictionary reduced the errors but further investigation of imperfect rf transmission was beyond the scope of this study . The adverse effects of slice distortion can be largely mitigated when selecting a large slab for 3d imaging, as in this case the slice profile problem becomes part of the b1 estimation . When estimating parameters through the slice, we saw unbiased results with our new method . Previous work in the context of radial kspace acquisition found that 3d scans offer more possibility of undersampling the kspace with respect to 2d slices 17 . Brain imaging methods to compare groups of subjects usually benefit from isotropic 3d acquisitions to meaningfully compare regions after image registration 18 . In addition, wholebrain coverage can be achieved in a single acquisition when using 3d mrf methods . By accelerating the acquisition more than 10fold, the undersampling technique described here can be used to significantly shorten scan times, and achieve acquisition durations suitable for in vivo imaging of rodents . Current techniques for voxelwise comparison of rodent brains include voxelbased morphometry 19 and tensorbased morphometry 20 . However, both approaches are more concerned with image geometry rather than the signal levels seen, as a consistent widespread approach for quantitative imaging between centers has not appeared to date in the literature . Fully quantitative, 3d acquisitions could be used in this context to obtain standardized multicenter data for analysis of different disease models and treatments . The scan times obtained here, of the order of 1.5 h, can be used in the preclinical environment, however, they would be prohibitive in human studies . The efficiency of our method is limited when compared with the gold standard of human quantitative imaging featuring acceleration with compressed sensing and parallel imaging . The introduction of array coils, as well as more sophisticated antialiasing strategies, could be used to further accelerate mrf acquisitions . Using iterative reconstruction such as compressed sensing is a promising strategy for antialiasing the images before pattern recognition 5, 6 . However, the large size of 4d datasets represents a challenge for iterative algorithms, and new strategies are needed to deal with the high computational demand . For instance, new compressed sensing algorithms based on fast, dedicated processing units of highperformance graphics cards (gpus) developed for cardiac mri hold promise for reconstruction of large mrf datasets 17 . In addition, fast algorithms such as split bregman could be used to accelerate compressed sensing of large datasets 21, perhaps including spatiotemporal total variation constraints 22 . In the current implementation, we waited for full relaxation (5 t1) between acquiring kspace lines . However, this is not necessary to perform mrf . In the future, optimized mrf acquisitions could be used to further shorten scan times . In addition, noncartesian methods have been demonstrated in the preclinical environment as well as for clinical scanners . Noncartesian acquisitions yield better efficiency and antialiasing of undersampled data with respect to cartesian in several preclinical applications, e.g., buonincontri et al 23 . Mr fingerprinting has already been demonstrated using arbitrary gradient waveforms, for instance derived from music to increase patient's comfort 24 . Although the use of spiral trajectories has shown great speed benefits for mrf in clinical scanners, we are unaware of implementations of spiral mrf in smallbore mri scanners . The use of novel 3d noncartesian strategies could further accelerate the acquisition bringing 3d, isotropic mrf methods into both preclinical and clinical applications . Direct reductions in scan time can also be achieved by reducing the number of frames 25 . Including the slice profile correction increased the computational burden of the dictionary creation proportionally to the number of partitions used . However, this correction had no impact on the pattern recognition algorithm, as the dictionary size remained unchanged . It took approximately a minute per slice to reconstruct the mrf data here, and this is a reasonable time frame . It is possible to include further parameters in the reconstruction though this is prohibitive as the reconstruction time will scale exponentially with the number of parameters . New methods to meaningfully compress the dictionary 26 and perform more sophisticated matching 27 are currently being studied . New reconstruction methods could permit the measurement of more parameters simultaneously, such as t2 * and diffusion, enhancing the sensitivity of novel mrf techniques . We have demonstrated a method for incorporating b1 estimation into mrf and extended the protocol to 3d imaging . Our methods greatly reduced problems seen with particular pulse shapes and b1 inhomogeneity, improving the accuracy of parameters estimated from mrf . We showed that view sharing between mrf frames produces accurate results acquiring less than 10% of the full dataset . Supporting figure s1 . Performance of 3d mrf acquiring only 5% of kspace per frame (50 min), compared with full acquisition (17 h). Some sparse areas of the t2 and b1 maps have errors between 10 and 20% compared with the full acquisition.
Hypertension is considered as the first leading risk factor for the global burden of disease . Epidemiological observations have led to the hypothesis that the risk of developing some chronic noncommunicable diseases (ncds) in adulthood is influenced not only by genetic and adult lifestyle factors but also by environmental factors acting in early life . Concept, several factors determine the blood pressure (bp) level in childhood, which is associated with the adult bp levelrough the tracking phenomenon . Therefore, prevention of ncds, namely, cardiovascular diseases in adulthood should begin from childhood . In addition, hypertension in childhood may induce target organ damages such as left ventricular hypertrophy, thickening of the carotid vessel wall, retinal vascular changes, subtle cognitive changes, and even premature development of atherosclerosis . The prevalence of hypertension is increasing in the pediatric population in line with the childhood obesity epidemic and lifestyle widespread changes . Alarming data exist on the considerable prevalence of overweight children and its metabolic consequences not only in the industrialized countries but also in developing countries . The strong relationship of even the early stages of hypertension with obesity and environmental factors such as air pollution, noise pollution, and passive smoking suggest that its prevalence will be escalating in the near future . There is a growing body of evidence, which shows that impaired maternal nutrition may negatively influence vascular health in later life . More specifically, the dietary calcium (ca) intake of pregnant women may be associated with the bp of their infants, and ca intake is inversely correlated to systolic bp in young children . Some recent experimental and observational studies in humans and animals have reported an association between maternal ca intake during pregnancy and bp in the offspring, but others did not confirm it . We conducted a systematic review of studies reporting the association of maternal ca intake either by food or supplements with bp in the offspring . We searched pubmed and scopus as the main international electronic data sources . The medical subject headings (mesh) including entry terms of pubmed and emtree of scopus were used for most comprehensive and efficient searches . We searched the databases using the following strategy: for scopus [title - abs - key (maternal or mother) and title - abs - key (calcium) and title - abs - key (blood pressure)], and for pubmed [others (mesh)) or maternal) and blood pressure(mesh) and dietary supplements(mesh)) or calcium]. Duplicates were removed; the relevant papers were selected in three phases . In the first and second phases, titles and abstracts of the papers were screened and irrelevant papers were excluded . In the last phase, the full text of recruited papers was explored intensely to select only relevant papers . For any additional pertinent studies, the reference list of all reviews and relevant papers was screened as well . In the next step, identification of main findings of the studies was conducted on a case - by - case basis and included consideration of any statistical analyses that might have been conducted and consistency of the general pattern across exposure groups . The required information that was extracted from all eligible papers were as follows: general characteristics of the study (first author's name, publication year, study year, study design)participantssupplement, age of the offspring, loss to follow - up, andoutcomes . General characteristics of the study (first author's name, publication year, study year, study design) age of the offspring, loss to follow - up, and we searched pubmed and scopus as the main international electronic data sources . The medical subject headings (mesh) including entry terms of pubmed and emtree of scopus were used for most comprehensive and efficient searches . We searched the databases using the following strategy: for scopus [title - abs - key (maternal or mother) and title - abs - key (calcium) and title - abs - key (blood pressure)], and for pubmed [others (mesh)) or maternal) and blood pressure(mesh) and dietary supplements(mesh)) or calcium]. Duplicates were removed; the relevant papers were selected in three phases . In the first and second phases, titles and abstracts of the papers were screened and irrelevant papers were excluded . In the last phase, the full text of recruited papers was explored intensely to select only relevant papers . For any additional pertinent studies, the reference list of all reviews and relevant papers was screened as well . In the next step, identification of main findings of the studies was conducted on a case - by - case basis and included consideration of any statistical analyses that might have been conducted and consistency of the general pattern across exposure groups . The required information that was extracted from all eligible papers were as follows: general characteristics of the study (first author's name, publication year, study year, study design)participantssupplement, age of the offspring, loss to follow - up, andoutcomes . General characteristics of the study (first author's name, publication year, study year, study design) age of the offspring, loss to follow - up, and based on our search strategy, we found 636 records . After removing duplicates, during the three refine steps on the papers titles, abstracts, and full texts and considering the inclusion criteria, flowchart of study selection summary of maternal calcium supplementation and offspring blood pressure this systematic review of the literature identified four randomized trials and three observational studies . Three studies included infants less than 1 year of age and seven included children between 1 year and 9 years of age . A number of maternal ca supplementation trials have been conducted on pregnant women in recent years, primarily to investigate the potential for reducing the risk of preeclampsia . All of the four trials have published follow - up data on the offspring, focusing on bp as an outcome . The study of belizan et al . Is a good quality randomized trial, which randomized 1,194 women during early pregnancy to either 2 g (four tablet of calcium carbonate of 500 mg) of oral ca supplementation or placebo . The follow - up only included the 614 participants from the private hospital (approximately 50% of the original sample). The proportion of children with high systolic bp was lower in the ca group (11.4%) than in the placebo group (19.3%) [relative risk (rr) 0.59; confidence interval (ci) 0.39 - 0.90]. The proportion of children with high diastolic bp was also lower in the ca group [10.2% vs 12.7% (0.80; 0; 49 - 1.30)]. A large reduction in the incidence of hypertension in children at 7 years of age was found in this study . There was a modest, statistically insignificant effect on systolic bp but a clinically and statistically significant effect on the incidence of high systolic bp at 7 years of age . This study also reported that the effect was stronger among overweight children; other studies did not observe this though the sample sizes were too small to exclude such a difference . For children under 12 months, randomized 4,589 women during early pregnancy to receive either 2 g of oral ca supplementation or placebo . Patients from only one out of five medical centers were included in the follow - up (559 out of 4,589 subjects). Loss of follow - up was 53% at 3 months and 90% at 2 years . The authors acknowledged this to be a problem, adding that a large proportion of the cohort had not reached 2 years of age by the end of the study . The trial provided evidence that maternal ca supplementation was associated with lower systolic bp in the offspring at 2 years . Systolic bp was 2.2 mmhg lower in the ca - supplemented infants than in the placebo group (p> 0.05). At 2 years of age, systolic bp was 4.8 mm hg lower in the ca supplemented group (p <0.05), whereas diastolic bp was lower by 3 mmhg . Designed their study to explore the association between infant bp and maternal dietary intake of ca, potassium, and magnesium . The authors measured the offspring bp in hospital when the babies were 2 - 4 days old and at home at 1 month, 6 months, and 12 months . There were no significant associations between the mothers pregnancy intake of these cations and the newborns bp . However, at 1 month of age maternal prenatal ca intake had a significant inverse association with systolic bp . At 6 months of age, maternal prenatal intakes of all three cations were significantly and inversely associated with diastolic bp . At 12 months of age, gillman et al . Used the data from a cohort study of pregnant women conducted in the united states . This study was designed to assess the effects of the mother's diet on the mother's and the offspring's health . It assessed maternal ca intake during the first and second trimesters using a validated semi - quantitative food frequency questionnaire (ffq), and measured offspring bp at birth and at 6 months . The authors reported figures for ca from food sources and from prenatal supplements, and then performed two independent analyses accordingly . After further adjustment for demographic, anthropometric, dietary, social, and economic variables, the decrease in 6-month systolic bp was 3.0 mmhg (95% ci, 4.9 to 1.1) for each 500 mg increment of maternal supplemental ca intake during pregnancy . Morley et al . Used the data from a population - based survey in tasmania designed to investigate sudden infant death syndrome . Mothers of all live - born twins during the study period were approached after birth for data collection including nutritional supplement consumption during pregnancy . Data on ca consumption from other sources (i.e., foods) were not available . Children were assessed at a mean age of 9 years and their bp was measured . Conducted a study with 389 children from a rural area of gambia whose mothers received ca supplement (1,500 mg ca / day from 20 weeks of gestation until delivery) or placebo in west africa . The study was part of a european union consortium investigation of the early life nutritional determinants of disease (framework 6: early nutrition programming project). At the time of this follow - up study, the surviving offspring were aged between 5 years and 10 years (mean age: 7.4 years, standard deviation (sd) 6 1.2]. This study found no evidence that maternal calcium supplementation (1,500 mg ca / day) of rural gambian women during the second half of pregnancy was associated with the offspring's bp at 5 - 10 years of age . Hiller et al . Followed up 414 participants with live - born infants who lived in south australia . Mothers and their children attended the women's and children's hospital for anthropomorphic examination . There was no indication that ca supplementation during pregnancy had an impact on either systolic or diastolic bp at follow - up among women with preeclampsia, pregnancy - induced hypertension (pih), or severe pih . However, some evidence showed that their children tended to have lower bp at follow - up . This interaction between maternal ca supplementation and the child's bp at follow - up was the strongest for women with more severe pih and preeclampsia . The evidence indicated by this body of research suggests a connection between dietary ca intake during pregnancy and the offspring's bp . A good quality trial found a large reduction in the incidence of hypertension in children at 7 years of age . For infants under 1 year of age, it is well - known that the determinant of bp varies with age, and it has been shown that the impact of factors affecting the fetal environment are seen particularly after adolescence . This problem is magnified because of the difficulties in measuring bp accurately in early ages . It is unclear whether this is a true biological differential effect or a statistical artefact resulting from the greater variability within individuals of diastolic pressure compared with systolic pressure . Among the seven studies reported, we found four randomized trials . The validity of the evidence from observational studies for assessing the effect of interventions is controversial . Apart from the methodological problems of the original articles, other limitations of this analysis should be pointed out . The sources and dose of dietary ca vary widely among the observational studies, and so do the methods used to assess the amount consumed . It is well - known that the determinant of bp varies with age, and it has been shown that the impact of factors affecting the fetal environment are seen particularly after adolescence . This problem is magnified because of the difficulties in measuring bp accurately at an early age . Six out of the seven studies included in the current review were conducted in developed countries, and on populations in which the maternal ca intake was adequate or even higher than the recommended levels during pregnancy . Given the evidence that the effect of ca might be apparent only when there is a deficit, the external validity of these results might be compromised . In hawkesworth's study from a region of habitually very low dietary calcium intake, there was no association between maternal calcium supplementation and offspring bp . As a whole, the main limitation in all the observational studies was loss to follow - up . For two of these studies, higher maternal calcium intake during pregnancy was associated with lower offspring systolic bp in all studies but the effect was statistically significant in only three of them . The argentinean follow - up reported an interaction between the intervention and childhood bmi; for individuals with a bmi above the mean (15.7), maternal calcium supplementation was associated with lower systolic bp . These studies did not observe an increase in effect estimates as the body mass index increased . Gillman's study suggested that the bp - lowering effect of ca might be limited to the three quartiles of the offspring's body mass index . Most of the protective effect of ca supplementation during pregnancy is concentrated in children with high body mass index; if this finding is confirmed, it might have important preventive implications because this subgroup is at higher risk of elevated bp and adult hypertension . High maternal ca intake with lower maternal bp could reduce fetal exposure to maternal hormones or substances related to elevated bp . The observed effect could reflect a long - term programming of elevated bp in utero by ca supplementation in the mother . This is consistent with the results obtained from observational studies that an early fetal exposure to calcium reduces bp during childhood . In general, our findings confirm the association of maternal ca intake during pregnancy and offspring bp . However, more research is needed to confirm these findings, given the small sample sizes and the methodological problems in many of the studies conducted so far . More studies on populations with low ca intake are also needed . If confirmed, these findings could have important public health implications . Ca supplementation during pregnancy is simple and inexpensive and may be a way to reduce the risk of elevated bp and its sequels in the next generation . The study was funded as a thesis of isfahan university of medical sciences, isfahan, iran . The study was funded as a thesis of isfahan university of medical sciences, isfahan, iran . All authors contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.
Drug rash with eosinophilia and systemic symptoms (dress) syndrome was first described by bocquet et al . In 1996 . However, the most frequent causes are anticonvulsants, sulfonamides, dapsone, allopurinol, minocycline, and gold salts [13]. In addition to hematologic, hepatic, cardiac, neurological, gastrointestinal, and endocrine abnormalities, pulmonary involvement is observed rarely . In this study, dress syndrome was presented with multiorgan involvement based on carbamazepine use in a 14-year - old female patient . Pulmonary involvement presented in the form of pleurisy and atelectasis, different from the literature, and as a long - term sequela, type 2 diabetes was observed in our case . A 14-year - old female patient presented at our clinic due to skin rash and fever lasting for 1 week . It was found in her history that carbamazepine treatment had been initiated owing to epilepsy 15 days before her symptoms began . There had been no transmitted chronic disease history such as viral, bacterial, or parasitic infection before the patient's reaction in question . On the physical examination, she had 38.5c core temperature, and there was a bilateral crepitant rale condition determined by listening on her respiratory system examination ., there were maculopapular rashes in the process of healing with desquamation that involved> 50% of the body (fig . There was microscopic hematuria in her total urinalysis . In the viral serological evaluation of the case, ebstein - barr virus (ebv), herpes virus type 1 - 2, hepatitis a - b - c virus, hiv, and toxoplasma were established as negative . Immunoglobulins, c3 and c4 levels, urinalysis were normal . In the punch biopsy obtained from the skin lesions, due to the drug intake history and the clinical, laboratory, and histopathological findings, carbamazepine treatment was discontinued in our patient . Desloratadine antihistaminic 5 mg / day treatment and systemic steroid 1 mg / kg / day (40 mg / day) were added to the therapy . Owing to the chest pain and the reduction in lung sounds in basals and since the sinuses were monitored as closed in the chest radiography, thorax ultrasonography was carried out, and bilateral pleural effusion 6 mm thick was determined . In the thorax ct performed on the patient, a local consolidated area in the middle lobe segment of the right lung, atelectatic changes in the lower lobes of both lungs, and thickness in the left fissure were seen . Since there was symptomatic hyperglycemia on the second day of steroid treatment, the steroid was discontinued . The patient was found hyperglycemic (fasting blood glucose: 120 - 180 mg / dl, postprandial blood glucose:160 - 250 mg / dl). The 33-year - old mother was diagnosed with type 2 diabetes at the age of 26 years . Physical examination revealed a weight of 52.8 kg (64 percentile, 0.38 sds), a height of 151 cm (8 percentile, 1.38 sds), body mass index of 23.56 (1.52 sds) and normal vital signs . Islet cells cytoplasmic autoantibodies (ica) were negative as tested by indirect immunofluorescence and her glutamic acid decarboxylase autoantibodies (gada), which was measured by radioimmunoassay, were also negative and fasting levels of c - peptide and insulin (electrochemiluminescent immunoassay; roche diagnostics, penzberg, germany) remained detectable throughout the observation period (c - peptide 2.1 - 6.23 ng / ml). With an hba1c value of 8.6%, she was diagnosed with diabetes mellitus . The combination of long - standing non - ketotic hyperglycemia, glycosuria, at a relatively high fasting and postprandial blood glucose, and negative pancreatic auto - antibodies in a child with a diabetic mother raised the possibility of mody . Direct dna sequencing of all exons and intron - exon bounders of the mody genes revealed no mutation in our patient . Insulin treatment was discontinued as the hyperglycemia disappeared, and metformin treatment was initiated with the type 2 diabetes diagnosis . The patient was discharged from the hospital after the lung, skin, liver, and renal findings regressed . A patch test was performed with carbamazepine 10% concentration 6 weeks later . As a result of the evaluation carried out 48, 72, and 96 h later, + 1 sensitivity was determined (fig . 2). Although the skin, liver, renal and lung findings resolved during the 1-year follow - up period, regulation of type 2 diabetes with an oral antidiabetic continued . A) maculopapular lesion in the recovery process with desquamation on the erythematous base . B) skin biopsy determined by lymphocytic infiltration in the papillary dermis epicutaneous patch test with carbamazepine dress syndrome, known as drug hypersensitivity syndrome, is a quite rare acute, idiosyncratic, and life - threatening drug reaction characterized by fever, skin rash, and single or multiple internal organ involvement [3, 4]. There is an average 3- to 9-week latent period (0.5 - 16 weeks) between drug use and the emergence of symptoms . These findings can persist or exacerbate although the responsible drug is discontinued [1, 3, 5]. Fever, skin rash, liver involvement, hypereosinophilia, and lymphadenopathy can be seen in almost all patients . There was pulmonary involvement presented by pulmonary atelectasia and pleurisy as well as maculopapular rash, fever, and hypereosinophilia, liver involvement manifested with transaminase elevation, and renal involvement was characterized by microscopic hematuria in the case . It has been suggested that there is a delayed hypersensitivity reaction related to t lymphocytes against toxic metabolites of drugs, and viral infections (ebv, human herpes virus, types 6 and 7) can also play a role in the etiology . It has been reported in the literature that there can be a genetic predisposition, and families of patients diagnosed with dress syndrome are also at risk in terms of this syndrome . It has been maintained in recent years that human herpes virus type 6 reactivation can also be used as a diagnostic marker [1, 3, 6, 7]. The positive result on the patch test with carbamazepine conducted 6 weeks later supports that there was a delayed hypersensitivity reaction . The main principle in the treatment of dress syndrome is to discontinue the drug or drugs thought to be suspicious immediately and provide supportive care . Systemic steroid is particularly recommended in internal organ involvement [2, 3, 7]. Hyperglycemia thought to be dependent on steroid treatment in the first place preceded although steroid treatment was discontinued and insulin treatment had to be commenced . Type 1 diabetes and pancreatitis have been reported in patients with dress syndrome in the literature [6, 9]. However, our patient's lipase level was normal, and no laboratory finding supporting type 1 diabetes was diagnosed in our patient . Although the hyperglycemia finding in the patient was not linked to dress syndrome in the first place, it was evaluated as dress syndrome related to type 2 diabetes by the endocrine department since it had a clinical table regulated by oral antibiotic in 1-year follow - up period . In the literature, various long - term sequelae concerning dress syndrome have been observed . In a study by chen et al ., a long - term sequelae rate was reported as 11.5% in analyses of 52 patients of whom 9 were lost and 43 recovered . The researchers reported that a total of 4 patients had autoimmunity disease, including 2 with grave's disease, 1 with type 1 diabetes, and 1 with autoimmune hemolytic anemia . They also observed that lifetime hemodialysis was necessary due to renal failure in 2 patients with organ involvement . In a study in which 34 patients were evaluated, however, the researchers reported that autoimmune disease developed in 2 patients, including 1 with lupus erythematosus and 1 with autoimmune thyroiditis . Reported 11 patients with type 1 diabetes related to dress syndrome and observed that half of the patients developed non - autoimmune fulminant type 1 diabetes . However, no case of type 2 diabetes associated with dress syndrome has been reported thus far . To the best of our knowledge, this case is the first in whom dress syndrome initially developed followed by type 2 diabetes . Dress syndrome is an acute, life - threatening, and rarely seen drug reaction . It should be considered a definitive diagnosis in patients with fever, diffuse skin rash, and internal organ involvement . The drugs used by patients should be questioned . It should be kept in mind that a variety of organ involvement as well as long - term sequelae can also be seen in patients with dress syndrome.
In february 2010, a 39-year - old man fractured both bones of the forearm after his arm was caught in a conveyor belt; he underwent a surgery at another hospital . An intramedullary forearm rod (acumed, hillsboro, or, usa) nineteen months after surgery, an attempt was made to remove the implant; it failed because the threaded portion of the nail remover was broken while being connected to the nail (fig . Two years after the failed surgery, the patient visited us with complaints of occurrence of left elbow pain, without having any new trauma . A partial defect of the triceps tendon with severe pain and tenderness around the scar of nail insertion was noted . Ultrasonography revealed partial rupture of the triceps tendon, and bursitis around the tender area . Based on these observations, the authors planned to remove the nail and repair the tendon . Surgery was performed under general anesthesia, with the patient in the supine position; a tourniquet applied to the affected area . The forearm was placed on the patient's chest, with the shoulder joint and elbow joint each flexed at 90. a longitudinal skin incision exposed the hole of the nail insertion and the interlocking screws . The inlet site of triceps brachii muscle was thinned not torn; it was not possible to grip since it was already covered by the bone . We removed the bone at the inlet site and created another bony window using an osteotome, to expose the interlocking screw holes . We inserted an impactor in the middle interlocking hole, after which we tapped the impactor towards the proximal side . The nail moved one inter - interlocking hole distance, and its end point passed through the triceps brachii tendon . We gripped the end point of the nail by a vise grip and used a hammer to remove it; however, the proximal part of the nail was damaged, and the vise grip and nail were separated (fig . Finally, we hit the distal most part of the interlocking hole, and moved the nail to a two inter - interlocking hole distance . Based on the authors' experience, we propose a surgical technique that can easily remove the nail, and minimize size of the bony window and damage to the triceps brachii . The distal incision is approximately the size of 2 inter - interlocking holes, and the proximal incision is about 1 cm, big enough to pass the nail (fig . 4). Only needs one inter - interlocking hole length of the bony window . It needs to be placed on the proximal 2 interlocking holes, and not on the distal hole (fig . An impactor is inserted on the distal hole, and tapped proximally by the mallet (fig . 6a). When the hole is moved to proximal part of the bony window (fig . 6b), a new interlocking screw hole appears in the distal part of the window (fig . The impactor is then laid on the new distal interlocking hole and tapped again (fig ., the triceps brachii tendon can be carefully monitored, without being impacted by the nail in any way . The nail is then exposed enough to be gripped firmly (figs . 3 and 6e). After the nail removal, the triceps brachii tendon is sutured and the bony window is covered, using the bone that was removed to make the window . The distal incision is approximately the size of 2 inter - interlocking holes, and the proximal incision is about 1 cm, big enough to pass the nail (fig . It needs to be placed on the proximal 2 interlocking holes, and not on the distal hole (fig . In the bony window, an impactor is inserted on the distal hole, and tapped proximally by the mallet (fig . 6a). When the hole is moved to proximal part of the bony window (fig . 6b), a new interlocking screw hole appears in the distal part of the window (fig . 6c). The impactor is then laid on the new distal interlocking hole and tapped again (fig ., the triceps brachii tendon can be carefully monitored, without being impacted by the nail in any way . The nail is then exposed enough to be gripped firmly (figs . 3 and 6e). After the nail removal, the triceps brachii tendon is sutured and the bony window is covered, using the bone that was removed to make the window . Intramedullary nailing can be used to treat fractures with minimal skin incision . Owing to its reliable biomechanical results, it is widely used in cases of long bone fracture . However, its direct placement into the bone can sometimes cause difficulty in handling . A hooked - shaped, modified guide pin was used when the nail's middle portion had broken . Levy et al.2) reported a method of removing the distal piece of the broken nail, using another nail less than 1 mm . Georgiadis et al.3) reported on the management of failed proximal piece of the nail removal in the leg . Despite reports of such related cases, there were no case reports in the existing literature about failure of retrieval of an ulnar intramedullary nail . When the extraction device is broken during removal of the ulna nail, only a bony window the size of 2 inter - interlocking holes at the most proximal part of the nail can be used to remove the nail with minimal damage of the triceps brachii tendon and soft tissue . In spite of the simplicity and usefulness of this method, care should be taken when making the bony window, because there is potential risk of a fracture around the olecranon, especially in osteoporotic old patients.
Auditory neuropathy / dyssynchrony (an / ad) is a hearing disorder characterized by an absent or atypical auditory brainstem response (abr), with preservation of the cochlear microphonics (cm) and/or otoacoustic emissions (oaes).1 2 in 1996, starr et al first described this rare entity, drawing on their observations in 10 patients.3 the authors suggested that these patients were probably similar to those previously reported cases with a paradoxical absence of abrs and only a slight impairment of pure tone thresholds but in whom cms or oaes had not been recorded.3 4 5 also in 1998, doyle et al reported eight patients with normal transient evoked oaes (teoaes) and distortion product oaes (dpoaes) combined with the absence or marked abnormalities of abrs.6 starr et al suggested that this type of hearing impairment is due to a disorder that impairs auditory nerve function and may have as one of its causes a neuropathy of the auditory nerve, occurring either in isolation or as part of a systemic neuropathic process.3 clinically, the diagnostic criteria of an / ad is defined as (1) sensorineural hearing loss, usually bilateral, of any degree; (2) normal outer hair cell function as evidenced by the presence of oaes and/or cm; (3) absent or atypical abr; (4) understanding of speech worse than would be predicted from the behavioral or pure tone audiometry; (5) absent acoustic reflexes to the ipsilateral and contralateral tones a 110-db hearing level.1 7 8 9 in this retrospective study, we investigated the audiological findings, history, and clinical manifestations of patients diagnosed with an / ad at our clinic . Fifteen patients with an / ad were included in this study, and their records were retrospectively investigated . All of the patients' medical and otologic histories were recorded . Otoscopic examinations were done by an otolaryngologist before testing to rule out any external or middle ear pathology that could affect audiometric measurements . Then pure tone audiometry (250 to 8,000 hz), tympanometry, and acoustic reflex measurement (500 to 4,000 hz) were done in a standard fashion (interacoustic ac 40 and az 26, denmark, assens). Dpoaes were measured at click levels of 65 (l1) and 55 db (l2) peak sound pressure for the f1 and f2 components (homoth medizinelektronik gmbh&co, kg, germany, hamburg). Dpoae - grams were recorded in one - quarter - octave steps over a frequency range of f2 from 0.5 to 6 khz . Dpoae values were plotted on a dpoaegram, which shows the emission level as a function of the f2 frequency . Electrodes were placed on the forehead as a ground electrode and on both mastoids as active electrodes . Alternate polarity clicks of 100-millisecond duration were presented monaurally with a repetition rate of 16.4/s ., we did not include genetic research; we investigated only according to the history of patients . Eleven male and four female patients diagnosed with an / ad (age range: 2 to 52 years and median age: 19.3 years) were included in this study . These patients had a history of exchange transfusion because of bilirubin levels over 20 mg / dl . Three patients had family history of hearing loss, two patients had consanguineous marriage, two patients had head trauma, one patient had mental motor retardation (psychomotor retardation), one patient had cerebrovascular disease, and there was no apparent causes in three patients . None of the patients with an / ad had middle or inner ear anomalies on computed tomography or magnetic resonance imaging . Table 1 summarizes the patients' demographic, clinical, and audiological features (see also fig . 1 for a left and right ear abr and dpoae recording of a case of an / ad). Left and right ear auditory brain stem response and distortion product otoacoustic emission recording of a case of auditory neuropathy / dyssynchrony . Abbreviations: dbspl, decibels sound pressure level; dp, distortion product; f1 and f2 used as formulation markers . Abbreviations: abr, auditory brain stem response; an / ad, auditory neuropathy / dyssynchrony; dpoae, distortion product otoacoustic emission . Eight of 15 patients' pure tone audiometric results showed profound hearing losses; however, seven of them had mild to moderate hearing loss . An / ad is characterized by a unique pattern of hearing loss and distorted abr with preservation of outer hair cell function.10 11 an / ad comprises a spectrum of pathology affecting the auditory pathways anywhere from the inner hair cells to the brainstem . Increased clinical suspicion supported by appropriate diagnostic tests is needed to establish an accurate diagnosis.12 13 the clinical findings for auditory neuropathy are associated with several diagnoses including hyperbilirubinemia, neurodegenerative diseases, charcot - marie - tooth syndrome, and other sensorimotor neuropathologies, mitochondrial disorders, and ischemic - hypoxic neuropathy resulting from asphyxia.14 also, experimental animal models for auditory neuropathy have been proposed using the carboplatin ototoxicity and ischemic - hypoxic neuropathy methodologies.12 15 16 in our series, three patients with an / ad had neonatal hyperbilirubinemia, three patients had family history of hearing loss, two patients had consanguineous marriage, two patients had head trauma, one patient had mental motor retardation, and one patient had cerebrovascular disease . Recently two mechanisms have been proposed for explaining the abnormalities of auditory function: (1) impaired synchrony among nerve fibers and/or (2) reduced neural input.17 18 19 it is generally thought that absent or severely distorted abr is highly likely to be related to impairment of neural synchrony in the auditory pathways . Similar to the previous reports, we could not obtain any response to the click stimulus . The prevalence of an / ad in patients with hearing loss ranged from 0.5 to 15% according to studies in literature.10 11 because there are so many possible causes for an / ad, it is difficult to estimate its exact prevalence.14 davis and hirsh suggested that 1 in every 200 hearing - impaired children had abr findings inconsistent with pure tone findings.20 permanent bilateral hearing loss is seen in 1.4 per 1,000 live births . Therefore the incidence of an / ad is likely to be 1.4 per 10,000 live births . According to rance et al, an / ad would be present in 2.3 per 1,000 infants with risk factors for hearing loss . Thus, if oae tests alone are used for hearing screening of infants with risk factors, 11% of infants with permanent hearing loss will be missed.14 21 these data are important for designing the newborn hearing screening protocols especially in developing countries . Patients with an / ad complain of hearing disability, especially in the presence of noise, and tend to have word - recognition scores that are disproportionately poorer than would be expected by audiometric thresholds . In our series, all of the patients were admitted to our department with the complaint of hearing and speech disability . Pure tone audiometric thresholds are variable in patients with an / ad, and the degree of hearing loss can range from mild to profound sensorineural type . Eight of our patients' pure tone audiometric results showed profound hearing losses, and seven had mild to moderate hearing loss . Because oaes and cm are dependent on the integrity of cochlear outer hair cells and are preneural events, they may be present and normal in an / ad; however, absent or grossly abnormal abrs are seen.14 22 similar findings were observed in our cases . The hallmark of an / ad is an abnormal abr reading together with a normal oae reading . However, there is a lack of actual diagnostic procedures for an / ad . Other tests may also be used as part of a more comprehensive evaluation of an individual's hearing and speech - perception abilities . Electrocochleography (ecog), which objectively assesses cochlear potentials, is the indicated clinical procedure to analyze cms . Although transtympanic ecog yields recordings with higher amplitudes and lower test retest variability, it has the disadvantage of being an invasive procedure . Extratympanic ecog, therefore, is clinically more useful in this context, supporting an audiological diagnosis and increasing knowledge about cochlear function in an / ad . It is necessary and helpful to diagnose the sites of lesion in patients with an / ad by analyzing the patterns of cm amplitudes . Therefore, recently ecog has been used for diagnosing an / ad.8 23 some patients with an / ad lost their oae over the period of time but there was no associated change in pure tone thresholds . It has also been reported that some patients with an / ad do not have oaes but rather evidence of hair cell function was evident from cm . Therefore these authors suggested that presence of cm with absent abr seems to be reliable criteria for diagnosing an / ad.24 25 there has been controversy regarding whether to provide hearing devices (hearing aids, personal radiofrequency [frequency modulation] systems, or cochlear implants) to children with an / ad and whether to offer aural - oral or visual - manual modes of habilitation.14 26 berlin et al suggested that conventional amplification has little beneficial effect on an / ad patients.27 on the contrary, cone - wesson et al concluded that nearly 50% of children demonstrate some benefit from the use of conventional hearing aids and that a trial of amplification is warranted.14 21 cochlear implantation may also be an option for hearing rehabilitation . Although the outcome of cochlear implantation in children with an / ad might vary, it is favorable in most cases . Cochlear implantation seems a justified hearing rehabilitation option for children with an / ad and limited benefits from conventional hearing aids.28 29 30 in our series, we obtained some development in hearing and speech abilities using a conventional hearing aid in one case . The current position statement of the joint committee on infant hearing (2007) calls for (1) physiological hearing screening of all infants before they are 1 month old, (2) confirmation of the hearing loss before 3 months of age, and (3) commencement of an interdisciplinary intervention program before the infant is 6 months old . Moreover, the scope of disorders targeted for identification has been expanded to include neural hearing loss (especially an / ad) in addition to sensorineural and permanent conductive hearing loss.31 although an / ad affects only a small portion of all persons with hearing loss, the infant, child, or adult with an / ad is often most disabled by the hearing disorder because of the lack of knowledge about its cause and, more importantly, its treatment . Continued research regarding the causes and pathologies underlying this disorder is needed.10 however, it is also necessary to develop methods to reduce false - negative screening results and to provide accurate diagnosis for the disorder.2 a combined oae and abr screening procedure may be considered to overcome the limitations of oae - only procedures especially in high - risk infants . Also, it should be kept in mind that adult an / d cases may be related with systemic neurological diseases.
Confident peptide identification through tandem mass spectrometry (ms) is one of the most important components in ms - based proteomics . For this reason, a great amount of effort has been invested to develop automated data analysis tools to identify peptides through tandem ms (ms) spectra . Among available because each search method uses a different algorithm and proceeds from a different view of what spectrum components contain the most critical information for identification, the search results for one spectrum from various search engines may differ significantly . However, it is also well - recognized that such difference may be turned into positive use: it would be useful to find complementary engines and combine the results in an effective way to enhance peptide identification. (1) combining search results from different methods, if feasible, definitely bears the possibility to improve the peptide identification confidence via reducing noise and utilizing complementary strengths . The difficulty in combining results from different search methods largely comes from the lack of a common statistical standard. (2) the importance of having a community standard has been stressed, and efforts in reaching such community standard have been invested . Using iterative expectation - maximization (em), keller et al . (3) proposed a statistical model to estimate the probability, determined through a global analysis of ms spectra from an experiment, for a given spectrum to have correct peptide identification . In principle, results from different search methods may go through the same analysis and thus compared . However, if after the statistical analysis two different methods report different confident identifications for the same spectrum, one ends up needing to invent an ad hoc rule to decide which identification should be kept . Furthermore, to use the em approach, one needs to assume or guess, without theoretical / statistical foundation, the forms of the score distributions for true positives and false positives . This, unfortunately, must weaken the validity of any statistical significance assignment obtained from such type of analyses . In our recent work,(4) it was shown possible to calibrate the statistics (e - value) of various search methods to reach a universal standard that is in agreement with the fundamental definition of e - value . For a given query spectrum and quality score cutoff s, e - value is defined as the expected number of hits, in a random database, with quality score being the same as or larger than the cutoff . A realistic e - value assignment thus provides the user with the number of false positives to anticipate when setting a quality score threshold . Most importantly, this peptide - centric statistical calibration allows one to combine search results even if the top hits from various methods disagree . Another possible approach to establish common statistical standard is through equating the false discovery rate (fdr) (5) of various methods considered . This approach, however, does not provide statistical significance for each peptide hit and thus is not directly applicable to peptide - centric combination of different search results . To be explicit, one may refer from fdr the e - value of a peptide hit with score identical to the first false positive, but any peptide hits with score better than the first false positive cannot have their e - value assigned . Furthermore, for peptides with scores falling in the range [sk+1, sk], where sk represents the kth best score of false positives, one cannot distinguish them statistically well except by using some ad hoc interpolations . There also exist other issues concerning misleading inference using fdr, but this is not the focus of the current paper and we refer the readers to a few relevant literatures . In this paper, in addition to providing a universal protocol to combine search results, we also carry out the performance assessment for all possible combinations, among seven database search methods, of two and three search methods using the receiver operating characteristic (roc) curves . The database search methods employed in our analysis are sequest(8) (v27 rev12), probid(9) (v1.0), inspect(10) (v20060505), mascot(11) (v2.1), x! Tandem(12) (v2007.07.01.2), omssa(13) (v2.0) and raid_dbs. (14) to better illustrate the main points of this paper, we have relegated to the supporting information a large number of roc curves that convey similar information of that exhibited in the plots of the main text . Since the centroid mode seems to be the dominant mode in ms database searches today, we present in the main text only results from centroid mode data and results from profile mode data are shown in the . Throughout the paper, we use dalton (da) as the unit for molecular weight . In the following, we will then describe briefly in the implementation, followed by our main results: best combinations within search methods tested . In this section, we will start with the definitions of p - value and e - value, which will be frequently used for the rest of this paper . We then describe the mathematical underpinnings of how to combine the p - values of different database search methods to result in a final e - value . We should note that the mathematical formulation employed here was first introduced by fisher,(15) and its extensions and applications to other research areas also exist . To the proteomics community let us define the p - value and e - value in the context of peptide identification in database searches . For a given spectrum and a score cutoff sc, one may ask what is the probability for a qualified (with molecular weight in the allowed range) random peptide to reach a score larger than or equal to sc . This probability p(sc), a function of sc, is called the p - value . For spectrum, if a database contains n qualified, unrelated random peptides, one will expect to have e(sc) = np(sc) number of random peptides to have quality score larger than or equal to sc . This expectation value e(sc) is by definition the e - value associated with score cutoff sc . If one further assumes that the occurrence of a high - scoring random hit is a rare event and thus can be modeled by a poisson process with expected number of occurrence e(sc), one may then define another p - value, which is called the database p - value, via the database p - value pdb(sc) represents the probability of seeing at least one hit in a given random database with quality score larger than or equal to sc . Note that, at the level of pdb, one may compare the statistics from different search methods using different sizes of random databases . Because of the differences in the choices of optimal search parameters, it is likely that different search methods, for the same query spectrum, may search over different number of qualified peptides, that is, having different effective database sizes . Therefore, combining the database p - values is the natural choice if one were to merge results from different search methods . Suppose that one wishes to combine the search results from l different search methods, each peptide candidate will have in principle l different p - values reported by the l search methods . The formula in eq 6 of the next subsection provides us the final combined p - value pcomb from the list . Once pcomb is obtained, we may invert the formula in eq 1 to get a combined e - value ecomb via having outlined how to obtain the final quantity of interest, ecomb, we now turn to the mathematical underpinnings of how to combine a list of, ideally independent, p - values reported by different database search methods . Consider an event labeled by a list of l independent quantities s1, s2,..., sl . Each quantity si may have an associated p - value pi depending on the statistics of the variable si . An important issue to address is how one should combine all the pi values to obtain an overall p - value . In the context of combining search results of different methods to assign statistical significance to a certain candidate peptide pl) uniformly distributed in the interval (0, 1)], what is the probability of finding their product to be smaller than a certain threshold . To put it in a more concrete framework, one may consider a unit hypercube whose interior points having coordinates (x1, x2,..., xl) with 0 xi 1 for all 1 i l. one then asks what is the volume bounded by the hypersurfaces xi 0 and (i=1lxi) with = i=1lpi . We may express this volume f() mathematically as an integral: where (x) is a step function with (x> 0) = 1 and (x <0) = 0 . Let f() f()/, we have with (x) being the dirac delta function that takes zero value everywhere except when x = 0 where its value approaches infinity . For the ease of computation, we make the following changes of variables: e and xi e. after this change, all the new variables t and ui are in the range (0,). Equation 4 now becomes (with = e understood) where the identity (e e) = e(t c) is used . Using the integral expression of the delta function, we may rewrite eq 4 as (with = e understood) where the last equality results from choosing the integration path to enclose the lower half of the complex k plane . We may now go back to f() by integrating f(). With = i=1lpi while combining the lp - values p1, p2,..., pl . As specific examples, when l = 2, we have f(p1p2) = p1p2[1 ln (p1p2)], and when l = 3, we have f(p1p2p3) = p1p2p3[1 ln(p1p2p3) + ln(p1p2p3)]. We will provide in the more examples to elucidate the consequence of the formula provided in eq 6.
Human lagochilascariosis is caused by lagochilascaris minor, an infection that has been reported in individuals of both sexes . Lagochilascariosis is considered an emerging helminthosis described in the american continents and its diagnosis is underestimated . The main clinical symptoms in humans are chronic lesions with abscess formation, usually affecting the neck and head tissues [2, 3]. Sometimes the parasite invades the pulmonary tissue and central nervous system, leading to death [4, 5]. Frequently, l. minor lesions contain multiple stages of the parasite (eggs, larvae, and adult worm), indicating autoinfection and favoring the development of chronic disease . The natural life cycle of l. minor and its infection mechanism remain unknown . An experimental heteroxenous life cycle for the parasite has been described in mice and domestic cats [5, 6] as well as in wild rodents and cats . The capacity of l. minor to migrate across different human tissues is also observed in animal models of the disease . In mice orally inoculated with l. minor infective eggs, hatched larvae can be observed migrating in the intestinal tract (612 h). After hatching, 3rd stage larvae (l3) migrate through intestinal mucosa into vessels and hepatic parenchyma (12 h) and disseminate to other tissues . Granulomatous lesions containing encysted l3 larvae have been found 30 days after infection in lungs, skeletal muscles, subcutaneous tissues, and lymph nodes . In cats that eat carcasses of infected mice, l3 migrate from the stomach (6 h) to the upper portions of the digestive tract, where they develop into 4th - stage larvae (l4), reaching maturity after 12 days of infection . Adult worms located in the esophagus, pharynx and trachea, and rhino - oropharynx and cervical lymph nodes, can expel eggs that will be found in feces of infected cats . We recently showed that balb / c mice are more resistant to l. minor infection than c57bl/6 mice, having less severe lesions in the lungs, lower numbers of nodules with encysted larvae, fewer adult worms, and higher serum levels of ifn- . The availability of isogenic strains of mice with different genetic backgrounds and the same h-2 haplotype has enabled the study of this host - parasite relationship, which is crucial to the establishment of susceptibility or resistance to infection . The manipulation of the host immune response system by l. minor antigens may be a key determinant of survival within the mammalian host . Therefore, the aims of this study were to evaluate infection in b10.a and a / j mice (both h-2), compare the survival rate and tissue lesions, and analyze the proliferative response and production of ifn- and il-10 in cultures of spleen cells from normal and immunized mice stimulated with the crude extract of the parasite (ce) or concanavalin a (cona). Six- to eight - week - old a / j and b10.a male mice were purchased from the university of so paulo animal facility . Eggs from the parasite were collected from feces of felis domesticus experimentally infected with a human isolate of l. minor . Feces from infected animals were subjected to hoffman's method and kept in culture in formalin solution (1%) at room temperature for 30 days . After the development of infective eggs containing third - stage (l3) larvae, cultures were submitted to faust's method for optimal recovery of eggs free from fecal debris . Egg suspensions were washed five times (20 minutes/4.000 rpm) with phosphate - buffered saline (pbs, ph 7.4) and transferred to a graduated centrifuge tube . The final concentration was adjusted to 2 10 eggs / ml and then used to infect the mice . In total, 60 a / j and 66 b10.a mice were orally inoculated with a suspension of 2 10 200 l. minor eggs per animal . In total, 20 a / j and 26 b10.a animals were followed for one year to determine survival rates . Forty animals were sacrificed (5 per day) at different time points (30, 45, 60, 90, 120, 150, 180, and 210 days postinfection) and submitted to necropsy for collection of organs for histopathology . Additionally, 40 a / j and 40 b10.a uninfected mice received saline orally and were used as follows: 20 animals served as controls for mortality (followed for one year), and 20 were necropsied and served as controls for histopathology (5 animals sacrificed at each time point: 30, 90, 150 and 210 days after infection). Sections of spleen, lung, lymph node, liver, muscle, and subcutaneous nodules derived from groups of 5 uninfected and 5 infected b10.a and a / j mice were collected 30 to 210 days after infection, fixed in 10% neutral buffered formalin, embedded in paraffin and subsequently stained with hematoxylin and eosin (h & e), masson's trichrome for detection of fibrosis and lunas' staining for characterization of eosinophils . The granulomas were classified as primary or exudative granuloma, secondary or exudative - productive granuloma and tertiary or productive - fibrotic granuloma . The secondary granuloma was characterized by a necrotic zone of variable extension, a cellular exudation zone, and presence of epithelioid cells . The tertiary granuloma was characterized by less necrotic cells and diminished cellular exudation with predominance of conjunctive tissue [14, 15]. L. minor - infected mice were euthanized 6090 days postinfection . Encysted l3 larvae were collected from nodules under sterile conditions upon puncturing to allow spontaneous release of the parasite . After 20 washes in pbs, live l3 larvae were resuspended in pbs and sonicated in an ika - tk8 disruptor . The crude extract (ce) of l3 larvae was then centrifuged at 50000 g for 1 h at 4c . The supernatant was collected and the protein content was estimated by the micro bca method (pierce, usa). Seven naive mice of each strain were immunized with 10 g of ce of l. minor in a volume of 0.1 ml . Mice were immunized subcutaneously four times at one - week intervals, and their spleens were collected one week after the final immunization for evaluation of lymphocyte proliferation and cytokine production . Spleens from a / j and b10.a mice were collected in rpmi medium supplemented with 2 mm l - glutamine, 1 mm sodium pyruvate, 100 u / ml penicillin, 100 g / ml streptomycin, 2 10 m 2-mercaptoethanol, 1% nonessential amino acids and 5% fetal calf serum (fcs) and were passed through nylon mesh to obtain spleen cells . After erythrocyte lyses, the cell suspension was washed twice in supplemented rpmi medium and plated in triplicate at 5 10 cells / well of 96-well round - bottomed plates in a total volume of 200 l medium . Spleen cells were stimulated with 5 g / ml of cona (sigma) or 5 g / ml of ce of l. minor . This concentration of cona and ce produced optimum responses in immunized animals when tested in within a range of 0.5 and 50 g / ml . Mitogen- and antigen - stimulated cultures were maintained for 5 days at 37c and 5% co2 in a humidified atmosphere . H - thymidine was added (0.5 ci per well) for the last 18 hours of culture . Cells were then harvested on glass fiber filters, and the incorporated radioactivity was measured in a liquid scintillation counter . Data are indicated as counts per minute (cpm). For analysis of secreted cytokines, 5 10 spleen cells / well (as above) were plated in 96-well u - bottom tissue culture plates (costar) and stimulated with cona or ce (as above). After 48 hours, cell - free culture supernatants were collected and stored at 80c until use . Il-10 and ifn were measured by a sandwich elisa using the opeia kit (bd bioscience, usa) according to the manufacturer's instructions . Cytokine analysis was performed based on a standard cytokine concentration curve with a detection limit of 15 pg / ml for both il-10 and ifn. Results are expressed in pg / ml . The survival curve was analyzed using the kaplan and meier method, and the differences between groups were tested using the log - rank test . B10.a mice began to die on day 13 postinfection, reaching only 26% survival on day 340, with a median survival time of 246.5 days . In contrast, a / j mice began to die later, on day 252 of infection, reaching 90% survival on day 340 (figure 1). A / j mice displayed significantly greater survival rates than b10.a throughout the entire period of infection (p = .0003). In the control group, only two b10.a mice and one a / j mouse died by day 340 postinfection . In the initial infection phase (0 to 30 days postinfection), a / j mice presented a primary granuloma in the lungs containing preserved larvae, perivascular and peribronchial moderate inflammatory infiltration of macrophages and discrete infiltration of neutrophils (figure 2(a)). In the intermediate infection phase (45 to 90 days postinfection), a secondary granuloma was observed with severe and diffuse inflammatory infiltration of foaming macrophages, multinuclear giant cells and a small number of eosinophils and fibroblasts in the lungs (figure 2(c)). In late infection phase (120 days postinfection), a tertiary granuloma was observed in the lungs, containing disrupted third stage larvae with moderate necrosis and destruction of the tissue, concentric fibrosis, and inflammatory infiltration foci composed of macrophages . In contrast, b10a mice in the initial phase of the infection (0 to 30 days postinfection), presented severe and diffuse inflammatory infiltration in the lungs, composed of macrophages, neutrophils and small number of eosinophils (figure 2(b)). In the intermediate phase (45 to 90 days postinfection), a secondary granuloma with severe and diffuse inflammatory infiltration was observed in the lungs with a predominance of macrophages and a small number of neutrophils (figure 2(d)). In late - phase infection (120 days postinfection) tertiary granuloma with concentric fibrosis intercalated by foaming macrophages and multinuclear giant cells was observed in the lungs . The moderate inflammatory infiltration of the lungs was diffuse and composed of macrophages and few neutrophils . In both mouse lineages, the liver developed large hepatocytes with bilobular nuclei and congestion with occasional perivascular inflammatory infiltrate . Spleen cells from a / j mice immunized with the crude extract of the parasite showed higher levels of proliferation in the presence of ce (p = .01) when compared to nonimmunized controls (figures 3(a) and 3(b)). A / j immunized mice also exhibited a greater proliferative response against ce antigen compared to b10.a immunized mice (p = .008). In addition, spleen cells of a / j and b10.a mice immunized with the crude extract (ce) of the parasite showed higher levels of proliferation in the presence of cona when compared to nonimmunized mice, although the difference was not statistically significant . Spleen cells from a / j immunized mice produced higher levels of il-10 induced by ce than did nonimmunized mice, but in the b10.a strain, the difference in il-10 production was not statistically significant (figure 4(a)). Additionally, a / j immunized mice produced more il-10 against ce antigen compared to b10.a immunized mice (p = .04). In a / j immunized mice, the il-10 production by spleen cells stimulated with cona was higher than that of nonimmunized animals, although the results were not statistically significant (figure 4(b)). In contrast, b10.a immunized mice produced lower amounts of il-10 when compared to nonimmunized animals, but these results were also not statistically significant . However, il-10 production induced by cona stimulation of spleen cells from a / j immunized mice was significantly higher than the amounts produced by b10.a mice (p = .02). Levels of ifn- induced by ce in spleen cells of a / j immunized mice were higher than nonimmunized mice, although not statistically different . Ifn- production induced by ce stimulation of spleen cells from b10.a immunized mice was similar to from nonimmunized animals . Moreover, the ifn- production induced by ce stimulation of spleen cells from a / j immunized mice was higher compared to b10.a mice (p = .049) (figure 5(a)). In addition, levels of ifn- produced by spleen cells of immunized a / j mice stimulated with cona were higher than those presented by nonimmunized mice (p = .014). In contrast, spleen cells of b10.a immunized mice produced lower levels of ifn- induced by cona than did nonimmunized control animals (p = .02). The ifn- production induced by cona stimulation of spleen cells from a / j immunized mice was also higher than that of b10.a mice (p = .005) (figure 5(b)). This paper provides evidence that the mhc complex (h-2 haplotype) of mice infected with l. minor does not influence survival during experimental infection . The survival rates of infected a / j mice were significantly higher than those of infected b10.a mice, although these isogenic mouse strains have different genetic backgrounds and the same h-2 haplotype (h-2). H-2 is an important factor, but we must consider that susceptibility to parasitic infection is polygenic . In fact, host genetic factors have a major influence on the susceptibility of mammals to infection by a variety of microorganism . The genetically determined differences between individuals in a population affect the efficiency of the immune response, and thus the host phenotype of susceptibility . In experimental paracoccidioidomycosis, b10.a mice behave as susceptible and a / j as resistant strain to fungal infection, that correlates with differences in the immune response, since resistant mice showed prevalent type 1 immunity whereas susceptible b10.a presented a progressive form of infection with impaired ifn- secretion [18, 19]. Acquired immunity against leishmania donovani has been shown to be under the control of genes within the h-2 locus: in a b10 genetic background, mice carrying the h-2 haplotype present a cure phenotype; whereas, the h-2 haplotype strains developed a noncure disease, in a manner dependent of the cytokines produced . The influence of the h-2 linked as wells as non - h-2 linked genes on the immune response was also shown by the infection of mice with trichinella spirallis, which affects the expulsion of the nematode from inbred and congenic mice [21, 22]. The lesions detected in infected b10.a mice were similar to those described by semerene et al . In c57bl/6 mice . Severe perivasculitis, vasculitis, and interstitial inflammation were visualized in the lungs, in the early phase of infection . We also observed organized granulomas around intact l3/l4 larvae with concentric fibrosis, macrophages, neutrophils, and small number of eosinophils in the lungs of these mice, in the intermediate phase of the infection (4590 days after infection). In contrast, the lungs of infected a / j mice had moderate perivasculitis, vasculitis, and interstitial inflammation, in the early phase of the infection, while during the intermediate phase of infection we detected less severe organized granulomas around intact or disrupted l3/l4 larvae with discrete fibrosis and presence of macrophages, plasma cells, few neutrophils and rare eosinophils, these results indicate that pulmonary lesions are of greater severity in the b10.a strain and correlate with mortality . The differences noted in mouse susceptibility to l. minor infection with distinct survival rates and tissue lesions may also be influenced by individual host immune response to the parasite . Different cell populations are involved in the immune response to the parasite: t and b cells, and mononuclear phagocytes . Th2 cells produce il-4, il-5, and il-13, which stimulate humoral responses and are known to be involved in resistance to extracellular helminths such as nipostrongylus brasiliensis . Antibodies to l. minor (igg, igm and iga) were detected in higher levels in a / j infected mice, when compared to b10.a infected mice, and it may reflect its greater resistance to infection . The recently described th17 cells that produce il-17 play a key role in inflammation and the activation of neutrophils and immunity to microorganisms, particularly at mucosal surfaces . Regulatory t cells (tr) of different subtypes produce il-10 and/or tgf-, and can regulate th1, th2 and th17 cells, b cells, and macrophages [2628]. Ifn- is the signature cytokine produced by th1 cells, but can be also derived from tc, nk cells, and b cells in small proportion . Il-10 is the major cytokine produced by tr cells, but can be also derived from b cells and monocytes [29, 30]. Considering that the participation of these different cell populations and the manipulation of the host immune response system by l. minor antigens may be a key determinant to their survival within a mammalian host, we immunized mice with the ce parasite antigen and analyzed the immune response . In this paper, we investigated the proliferative response and cytokine production of spleen cells from mice immunized with the ce of l. minor l3 larvae . Spleen lymphocytes from a / j mice immunized with ce and stimulated with cona (positive control) or ce of the parasite showed better proliferative response when compared to b10.a mice, particularly against the ce of the parasite . Ce is composed of intracellular and membrane components of the parasite that may stimulate specific spleen cells from a / j mice better than b10.a mice . Immunized a / j mice showed significantly increased production of il-10 against cona and ce . On the other hand, spleen cells of immunized spleen cells from immunized a / j mice produced more ifn- when stimulated with cona or ce than did spleen cells from b10.a immunized mice . Moreover, immunized b10.a mice produced less ifn- when stimulated with cona when compared to nonimmunized controls . In conclusion, the a / j mice, which are more resistant to infection, displayed a better proliferative response and greater production of il-10 and ifn- against ce antigen and cona when immunized with l. minor antigens, compared to the susceptible b10.a mice which presented a lower proliferative response and less il-10 and ifn-. This intense inflammatory response may be a consequence of an uncontrolled reaction caused by the small amount of il-10 produced in b10.a mice . In a / j mice, the concomitant production of il-10 and ifn may control the tissue lesions and the number of parasites, limiting collateral tissue damage caused by a robust antiparasitic immune response . In fact, when we compared experimental lagochilascariosis in c57bl/6 and balb / c mice, the latter were more resistant to infection and produced more ifn and il-10 . The high mortality observed in b10.a infected mice may be a consequence of the intense inflammatory reaction without control by regulatory cytokines . We are currently investigating the kinetics of cytokine production in a / j and b10.a mice infected with l. minor to better understand the immunological mechanisms involved in the resistance to the infection.
In an experimental study, fifty male weaning wistar rats (pasture institute, iran), 21 days of age on arrival were housed individually in wire bar - floor cages . The animals (weight: 35.59 0.39) were allowed 1 week of acclimatization in a standard environment at 22c, 50% humidity and 12-h light / dark cycles with free access to food and water . During the first week, all animals were fed a standard laboratory chow (pasture institute, iran) and afterwards, they were randomly assigned to five groups . The composition of standard diet (sd) was completely the same as the commercial ain-93 g . Diet treatments included high fat diets (hfd) with soy oil (hf - s), butter (hf - b), fish oil (hf - f) or olive oil (hf - o). High fat diets were prepared to provide equal vitamins and minerals per calorie, and contained equal percentages by weight of fiber . After an 8-week feeding period, blood samples were collected from the retro - orbital veins into polypropylene tubes containing sodium edta (1-mg / ml blood) and aprotinin (500-unit / ml blood, bayer) in the non - fasted and 24 h - fasted state for measurement of serum parameters and were then centrifuged for 15 min at 3000x g. plasma samples were stored at -80c . Animals were anesthesized with co2 to minimize the potential impact of anesthesia on hormone levels . The experimental hfds were almost isocaloric (table 1), and composed of a fat - free basal diet based on american institute of nutrition rodent diets -growth purified diet-(ain93-g),25 containing carbohydrate (corn starch, dextrinized corn starch, dyets company, usa), protein (98.5% casein hydrolisate and 1.5% l - cyctine, choline bitartrate, dyets companay, usa), fiber (cellulose, dyets company, usa), vitamin - mineral mix, and tertbutylhydroquinone (dyets company, usa) corn starch . Dietray fats were also added in order to provide a balanced diet and included fish oil and olive oil which were a generous gift from nooshdarooye darya institute, iran, soy oil (purchased from ladan institute, iran) and butter (obtained locally). The diets were prepared weekly and stored as vacuum packed (500 g) at 20c . The food was offered daily at the beginning of the dark phase, and the remains were weighed and removed after an 48 hours . All experiments were carried out in accordance with standards approved by the local ethics committee of the research institute for endocrine science of shahid behesti university of medical sciences . Plasma glucose was determined by an enzymatic (glucose oxidase) colorimetric method (pars azmoun co, tehran, iran). The assay sensitivity was 1 mg / dl, the intra- and interassay coefficients of variation were 1.2% and 1.8%, respectively . Plasma insulin was determined by an elisa method (mercodia ab, uppsala, sweden). G / l, the intra- and interassay coefficients of variations were 2.5% and 4.1%, respectively . Blood samples were immediately transferred to chilled tubes containing na2-edta (1 mg / ml) and aprotinin (500-unit / ml blood, bayer) centrifuged in 2000 rpm for 15 min at 4c . Hydrogen chloride was added to the samples at a final concentration of 0.1 n immediately after separation of the plasma . Unacylated form of ghrelin was measured using dag elisa kit according to the manufacturer's protocol (mitsubishi kagaku iatron, inc). The minimal detection limit of dag in this assay system was 12.5 fmol / ml . The assay used to detect dag has less than 0.1% of cross - reaction with acylated ghrelin . Insulin resistance was estimated by homeostasis model assessment (homa), according to the formula: insulin resistance index = fasting insulin (g / l) fasting glucose (mg / dl)/405.26 gas chromatography analyses were carried out on gas chromatography (younglin instrument, 6000 series, south korea) equipped with a split - injector and flame ionization detector . Methyl - esters of fatty acids (fame) were prepared using methanolic koh, according to the standard method (iso5509:2000). The fatty acid profile was determined by gas chromatographic separation of their methyl esters (iso 5508: 1990) on a capillary column (j&w scientific db-23, 30 m 0.25 mm 0.25 m). Chromatography software (unicam 4880 chromatography data system) was used for data collection and processing . Data are presented in table 2 . Fatty acid composition (%) of the diets analyzed by gas - liquid chromatography . Statistical analyses were performed using spss 16.0 software (chicago, il, usa). The kolmogorov smirnov test was applied to determine the normality of the distribution of the data to be used in the parametric test . One - way anova, paired t test and tukey test were used to compare the diet groups and p value<0.05 was considered statistically significant . Nonparametric tests (kruskal - wallis, wilcoxon signed - rank and mann - whitney u) were also employed for insulin level and homa - ir variables and p value<0.05 was considered significant . The spearman's rank correlation coefficient was also used to determine whether a significant relationship existed between insulin and dag concentrations . In an experimental study, fifty male weaning wistar rats (pasture institute, iran), 21 days of age on arrival were housed individually in wire bar - floor cages . The animals (weight: 35.59 0.39) were allowed 1 week of acclimatization in a standard environment at 22c, 50% humidity and 12-h light / dark cycles with free access to food and water . During the first week, all animals were fed a standard laboratory chow (pasture institute, iran) and afterwards, they were randomly assigned to five groups . The composition of standard diet (sd) was completely the same as the commercial ain-93 g . Diet treatments included high fat diets (hfd) with soy oil (hf - s), butter (hf - b), fish oil (hf - f) or olive oil (hf - o). High fat diets were prepared to provide equal vitamins and minerals per calorie, and contained equal percentages by weight of fiber . After an 8-week feeding period, blood samples were collected from the retro - orbital veins into polypropylene tubes containing sodium edta (1-mg / ml blood) and aprotinin (500-unit / ml blood, bayer) in the non - fasted and 24 h - fasted state for measurement of serum parameters and were then centrifuged for 15 min at 3000x g. plasma samples were stored at -80c . Animals were anesthesized with co2 to minimize the potential impact of anesthesia on hormone levels . The experimental hfds were almost isocaloric (table 1), and composed of a fat - free basal diet based on american institute of nutrition rodent diets -growth purified diet-(ain93-g),25 containing carbohydrate (corn starch, dextrinized corn starch, dyets company, usa), protein (98.5% casein hydrolisate and 1.5% l - cyctine, choline bitartrate, dyets companay, usa), fiber (cellulose, dyets company, usa), vitamin - mineral mix, and tertbutylhydroquinone (dyets company, usa) corn starch . Dietray fats were also added in order to provide a balanced diet and included fish oil and olive oil which were a generous gift from nooshdarooye darya institute, iran, soy oil (purchased from ladan institute, iran) and butter (obtained locally). The diets were prepared weekly and stored as vacuum packed (500 g) at 20c . The food was offered daily at the beginning of the dark phase, and the remains were weighed and removed after an 48 hours . All experiments were carried out in accordance with standards approved by the local ethics committee of the research institute for endocrine science of shahid behesti university of medical sciences . Plasma glucose was determined by an enzymatic (glucose oxidase) colorimetric method (pars azmoun co, tehran, iran). The assay sensitivity was 1 mg / dl, the intra- and interassay coefficients of variation were 1.2% and 1.8%, respectively . Plasma insulin was determined by an elisa method (mercodia ab, uppsala, sweden). G / l, the intra- and interassay coefficients of variations were 2.5% and 4.1%, respectively . Blood samples were immediately transferred to chilled tubes containing na2-edta (1 mg / ml) and aprotinin (500-unit / ml blood, bayer) centrifuged in 2000 rpm for 15 min at 4c . Hydrogen chloride was added to the samples at a final concentration of 0.1 n immediately after separation of the plasma . Unacylated form of ghrelin was measured using dag elisa kit according to the manufacturer's protocol (mitsubishi kagaku iatron, inc). The minimal detection limit of dag in this assay system was 12.5 fmol / ml . The assay used to detect dag has less than 0.1% of cross - reaction with acylated ghrelin . Insulin resistance was estimated by homeostasis model assessment (homa), according to the formula: insulin resistance index = fasting insulin (g / l) fasting glucose (mg / dl)/405.26 gas chromatography analyses were carried out on gas chromatography (younglin instrument, 6000 series, south korea) equipped with a split - injector and flame ionization detector . Methyl - esters of fatty acids (fame) were prepared using methanolic koh, according to the standard method (iso5509:2000). The fatty acid profile was determined by gas chromatographic separation of their methyl esters (iso 5508: 1990) on a capillary column (j&w scientific db-23, 30 m 0.25 mm 0.25 m). Chromatography software (unicam 4880 chromatography data system) was used for data collection and processing . Data are presented in table 2 . Fatty acid composition (%) of the diets analyzed by gas - liquid chromatography . Statistical analyses were performed using spss 16.0 software (chicago, il, usa). The kolmogorov smirnov test was applied to determine the normality of the distribution of the data to be used in the parametric test . One - way anova, paired t test and tukey test were used to compare the diet groups and p value<0.05 was considered statistically significant . Nonparametric tests (kruskal - wallis, wilcoxon signed - rank and mann - whitney u) were also employed for insulin level and homa - ir variables and p value<0.05 was considered significant . The spearman's rank correlation coefficient was also used to determine whether a significant relationship existed between insulin and dag concentrations . In an experimental study, fifty male weaning wistar rats (pasture institute, iran), 21 days of age on arrival were housed individually in wire bar - floor cages . The animals (weight: 35.59 0.39) were allowed 1 week of acclimatization in a standard environment at 22c, 50% humidity and 12-h light / dark cycles with free access to food and water . During the first week, all animals were fed a standard laboratory chow (pasture institute, iran) and afterwards, they were randomly assigned to five groups . The composition of standard diet (sd) was completely the same as the commercial ain-93 g . Diet treatments included high fat diets (hfd) with soy oil (hf - s), butter (hf - b), fish oil (hf - f) or olive oil (hf - o). High fat diets were prepared to provide equal vitamins and minerals per calorie, and contained equal percentages by weight of fiber . After an 8-week feeding period, blood samples were collected from the retro - orbital veins into polypropylene tubes containing sodium edta (1-mg / ml blood) and aprotinin (500-unit / ml blood, bayer) in the non - fasted and 24 h - fasted state for measurement of serum parameters and were then centrifuged for 15 min at 3000x g. plasma samples were stored at -80c . Animals were anesthesized with co2 to minimize the potential impact of anesthesia on hormone levels . The experimental hfds were almost isocaloric (table 1), and composed of a fat - free basal diet based on american institute of nutrition rodent diets -growth purified diet-(ain93-g),25 containing carbohydrate (corn starch, dextrinized corn starch, dyets company, usa), protein (98.5% casein hydrolisate and 1.5% l - cyctine, choline bitartrate, dyets companay, usa), fiber (cellulose, dyets company, usa), vitamin - mineral mix, and tertbutylhydroquinone (dyets company, usa) corn starch . Dietray fats were also added in order to provide a balanced diet and included fish oil and olive oil which were a generous gift from nooshdarooye darya institute, iran, soy oil (purchased from ladan institute, iran) and butter (obtained locally). The diets were prepared weekly and stored as vacuum packed (500 g) at 20c . The food was offered daily at the beginning of the dark phase, and the remains were weighed and removed after an 48 hours . All experiments were carried out in accordance with standards approved by the local ethics committee of the research institute for endocrine science of shahid behesti university of medical sciences . Plasma glucose was determined by an enzymatic (glucose oxidase) colorimetric method (pars azmoun co, tehran, iran). The assay sensitivity was 1 mg / dl, the intra- and interassay coefficients of variation were 1.2% and 1.8%, respectively . Plasma insulin was determined by an elisa method (mercodia ab, uppsala, sweden). G / l, the intra- and interassay coefficients of variations were 2.5% and 4.1%, respectively . Blood samples were immediately transferred to chilled tubes containing na2-edta (1 mg / ml) and aprotinin (500-unit / ml blood, bayer) centrifuged in 2000 rpm for 15 min at 4c . Hydrogen chloride was added to the samples at a final concentration of 0.1 n immediately after separation of the plasma . Unacylated form of ghrelin was measured using dag elisa kit according to the manufacturer's protocol (mitsubishi kagaku iatron, inc). The minimal detection limit of dag in this assay system was 12.5 fmol / ml . The assay used to detect dag has less than 0.1% of cross - reaction with acylated ghrelin . Insulin resistance was estimated by homeostasis model assessment (homa), according to the formula: insulin resistance index = fasting insulin (g / l) fasting glucose (mg / dl)/405.26 gas chromatography analyses were carried out on gas chromatography (younglin instrument, 6000 series, south korea) equipped with a split - injector and flame ionization detector . Methyl - esters of fatty acids (fame) were prepared using methanolic koh, according to the standard method (iso5509:2000). The fatty acid profile was determined by gas chromatographic separation of their methyl esters (iso 5508: 1990) on a capillary column (j&w scientific db-23, 30 m 0.25 mm 0.25 m). Chromatography software (unicam 4880 chromatography data system) was used for data collection and processing . Fatty acid composition (%) of the diets analyzed by gas - liquid chromatography . Statistical analyses were performed using spss 16.0 software (chicago, il, usa). The kolmogorov smirnov test was applied to determine the normality of the distribution of the data to be used in the parametric test . One - way anova, paired t test and tukey test were used to compare the diet groups and p value<0.05 was considered statistically significant . Nonparametric tests (kruskal - wallis, wilcoxon signed - rank and mann - whitney u) were also employed for insulin level and homa - ir variables and p value<0.05 was considered significant . The spearman's rank correlation coefficient was also used to determine whether a significant relationship existed between insulin and dag concentrations . Food, calorie intakes and body weights of the groups are shown in table 3 . High fat butter and hf - s groups had significantly higher food and calorie intake in comparison with sd, hf - f and hf - o groups (f=6.38, p=0.00). Initial body weights were not different among dietary groups but the weight gains in the hf - b and hf - s were 26.6 and 17.74 g higher, respectively, than the weight gain of sd group animals (p=0.01 and p=0.04, respectively). High fat butter had also 21.2 and 21.7 g higher weight gain compared with the hf - f and hf - o groups, respectively (p=0.00 and p=0.00, respectively). Weight gain of hf - f and hf - o groups were 12.3 and 12.8 g higher than the weight gain of hf - s group, respectively (p=0.04 and p=0.01, respectively). Final body weight in hf - s and hf - b groups was higher than that of ds, hf - f and hf - o groups (p=0.04). Body weight, food and calorie intake of rats consuming high fat diets (hfds) for 8 weeks1 the plasma dag concentration was significantly decreased in sd, hf - f, hf - o and hf - s diets in the ad libitum fed status, compared with fasting condition (p=0.00, figure 1a). Plasma dag levels at fed state in hf - b and hf - s diet were higher than those of sd, hf - f and hf - o diets; however, these findings were not significant . Plasma desacyl - ghrelin (1a), glucose (1b) and insulin (1c) levels and insulin resistance (homa - ir) (1d) in rats consuming high dietary fats (hfds) for 8 weeks (n=10 in each group). High fat fish, sd and hf - o diet rats showed 47.3, 42.7 and 38.6 g / ml higher dag concentrations than the hf - b diet rats at fasted state, respectively (p=0.00); meanwhile, plasma dag level in the hf - s diet rats was 16.4 g / ml lower than the hf - f diet rats (p=0.00). Ad libitum fed plasma insulin concentration in the hf - b diet was significantly higher than that of the sd, hf - f and hf - o diets (p=0.00) and the increase in the hf - s diet group was 0.35, 0.37 and 0.31 g / l higher than that of the sd, hf - o and hf - f diets, respectively (p=0.01, figure 1b). Furthermore, plasma insulin levels were decreased by fasting in all diet groups, although the decreases were not significant . Rats on the sd, hf - f and hf - o diets had significantly lower fasting insulin plasma levels than those on hf - b (p=0.04). Plasma glucose levels were significantly decreased by fasting in all of diet groups, compared with fed state (p=0.00, figure 1c). However, plasma glucose levels were not significantly different among dietary groups . After 24 h fasting, hf - b diet had significantly higher homa - ir in comparison to the hf - f, hf - o and sd diets (p=0.05, figure 1d). There was no significant correlation between changes in insulin and changes in dag levels in the feeding (spearman correlation=0.16, p=0.3) and 24 hour fasting status (spearman correlation=-0.34, p=0.06). Food, calorie intakes and body weights of the groups are shown in table 3 . High fat butter and hf - s groups had significantly higher food and calorie intake in comparison with sd, hf - f and hf - o groups (f=6.38, p=0.00). Initial body weights were not different among dietary groups but the weight gains in the hf - b and hf - s were 26.6 and 17.74 g higher, respectively, than the weight gain of sd group animals (p=0.01 and p=0.04, respectively). High fat butter had also 21.2 and 21.7 g higher weight gain compared with the hf - f and hf - o groups, respectively (p=0.00 and p=0.00, respectively). Weight gain of hf - f and hf - o groups were 12.3 and 12.8 g higher than the weight gain of hf - s group, respectively (p=0.04 and p=0.01, respectively). Final body weight in hf - s and hf - b groups was higher than that of ds, hf - f and hf - o groups (p=0.04). Body weight, food and calorie intake of rats consuming high fat diets (hfds) for 8 weeks1 the plasma dag concentration was significantly decreased in sd, hf - f, hf - o and hf - s diets in the ad libitum fed status, compared with fasting condition (p=0.00, figure 1a). Plasma dag levels at fed state in hf - b and hf - s diet were higher than those of sd, hf - f and hf - o diets; however, these findings were not significant . Plasma desacyl - ghrelin (1a), glucose (1b) and insulin (1c) levels and insulin resistance (homa - ir) (1d) in rats consuming high dietary fats (hfds) for 8 weeks (n=10 in each group). High fat fish, sd and hf - o diet rats showed 47.3, 42.7 and 38.6 g / ml higher dag concentrations than the hf - b diet rats at fasted state, respectively (p=0.00); meanwhile, plasma dag level in the hf - s diet rats was 16.4 g / ml lower than the hf - f diet rats (p=0.00). Ad libitum fed plasma insulin concentration in the hf - b diet was significantly higher than that of the sd, hf - f and hf - o diets (p=0.00) and the increase in the hf - s diet group was 0.35, 0.37 and 0.31 g / l higher than that of the sd, hf - o and hf - f diets, respectively (p=0.01, figure 1b). Furthermore, plasma insulin levels were decreased by fasting in all diet groups, although the decreases were not significant . Rats on the sd, hf - f and hf - o diets had significantly lower fasting insulin plasma levels than those on hf - b (p=0.04). Plasma glucose levels were significantly decreased by fasting in all of diet groups, compared with fed state (p=0.00, figure 1c). However, plasma glucose levels were not significantly different among dietary groups . After 24 h fasting, hf - b diet had significantly higher homa - ir in comparison to the hf - f, hf - o and sd diets (p=0.05, figure 1d). There was no significant correlation between changes in insulin and changes in dag levels in the feeding (spearman correlation=0.16, p=0.3) and 24 hour fasting status (spearman correlation=-0.34, p=0.06). Food, calorie intakes and body weights of the groups are shown in table 3 . High fat butter and hf - s groups had significantly higher food and calorie intake in comparison with sd, hf - f and hf - o groups (f=6.38, p=0.00). Initial body weights were not different among dietary groups but the weight gains in the hf - b and hf - s were 26.6 and 17.74 g higher, respectively, than the weight gain of sd group animals (p=0.01 and p=0.04, respectively). High fat butter had also 21.2 and 21.7 g higher weight gain compared with the hf - f and hf - o groups, respectively (p=0.00 and p=0.00, respectively). Weight gain of hf - f and hf - o groups were 12.3 and 12.8 g higher than the weight gain of hf - s group, respectively (p=0.04 and p=0.01, respectively). Final body weight in hf - s and hf - b groups was higher than that of ds, hf - f and hf - o groups (p=0.04). Body weight, food and calorie intake of rats consuming high fat diets (hfds) for 8 weeks1 the plasma dag concentration was significantly decreased in sd, hf - f, hf - o and hf - s diets in the ad libitum fed status, compared with fasting condition (p=0.00, figure 1a). Plasma dag levels at fed state in hf - b and hf - s diet were higher than those of sd, hf - f and hf - o diets; however, these findings were not significant . Plasma desacyl - ghrelin (1a), glucose (1b) and insulin (1c) levels and insulin resistance (homa - ir) (1d) in rats consuming high dietary fats (hfds) for 8 weeks (n=10 in each group). High fat fish, sd and hf - o diet rats showed 47.3, 42.7 and 38.6 g / ml higher dag concentrations than the hf - b diet rats at fasted state, respectively (p=0.00); meanwhile, plasma dag level in the hf - s diet rats was 16.4 g / ml lower than the hf - f diet rats (p=0.00). Ad libitum fed plasma insulin concentration in the hf - b diet was significantly higher than that of the sd, hf - f and hf - o diets (p=0.00) and the increase in the hf - s diet group was 0.35, 0.37 and 0.31 g / l higher than that of the sd, hf - o and hf - f diets, respectively (p=0.01, figure 1b). Furthermore, plasma insulin levels were decreased by fasting in all diet groups, although the decreases were not significant . Rats on the sd, hf - f and hf - o diets had significantly lower fasting insulin plasma levels than those on hf - b (p=0.04). Plasma glucose levels were significantly decreased by fasting in all of diet groups, compared with fed state (p=0.00, figure 1c). After 24 h fasting, hf - b diet had significantly higher homa - ir in comparison to the hf - f, hf - o and sd diets (p=0.05, figure 1d). There was no significant correlation between changes in insulin and changes in dag levels in the feeding (spearman correlation=0.16, p=0.3) and 24 hour fasting status (spearman correlation=-0.34, p=0.06). Our findings showed that insulin concentration at fed state was significantly lower in the hf - f and hf - o groups, in comparison to the hf - b and hf - s diets, although in fasting state, insulin level differences were similar . Insulin resistance (homa - ir) was also higher in the hf - b diet, in comparison to the sd, hf - f and hf - o groups . On the other hand, food, energy intake and the following weight gain in hf - f and hf - o diets were lower than those of the hf - b and hf - s groups . Although, the effect of different fat sources on appetite is controversial and limited, but previous studies have reported that each energy unit from fat had weaker satiety effects compared to carbohydrate and protein; on the other hand, high fat foods usually have higher energy density, so these foods can lead to more food consumption and weight gain and obesity.27 alfenas and mattes have been shown that saturated fatty acid (sfa) absorption is less effective than unsaturated fatty acids in rats and they assumed that polyunsaturated fatty acid (pufa) has more satiety effect than monosaturated fatty acid (mufa) and mufa more than sfa.28 therefore, higher insulin levels or insulin resistance in rats receiving hf - b (containing sfa) or hf - s (containing -6 pufa) groups, probably, is related to higher weight or appetite.2930 so, in this study the satiety effect of pufas was not found in -6 pufa . This finding probably was related to the scientific fact that high proportion of -6 pufa to -6 pufa in the diet shifts the physiological state in the tissues toward the pathogenesis of many diseases like obesity.3132 latown and co - workers also reported that food containing sfa, compared to pufa, was accompanied by higher food and energy intake33 and may be, because of this, most previous studies suggest that fish oil high in polyunsaturated -3 fatty acids normalizes the insulin action and prevents insulin resistance induced by a hfd in rats33436 or can lead to better glucose response and obesity parameters in mice.37 furthermore, animal and human studies have demonstrated that sfa increases insulin resistance3839 and animal studies also showed that -6 pufa in comparison to -3 pufa could decrease insulin sensitivity.635 however, other investigators have suggested that higher intakes of sfa and -6 pufa do not adversely affect insulin secretion4041 and homa - ir.34 in addition, some studies have demonstrated that hfd with mufa does not improve insulin secretion and sensitivity in rats.2342 it should be noticed that the majority of these studies have used adult rats (125300 g body weight), fed more total calories than controls,243 while the present study utilized weaning, prepubertal rats fed an ad libitum diet . Furthermore, duration of the study and composition of basal diet, especially fiber content, could affect insulin sensitivity and basal glucose metabolism.4445 these differences may explain the apparent contradictions among different researches . The present study has shown that the decrease in fasting plasma insulin and insulin resistance in the fish oil and mufa groups was associated with an increase in fasting plasma dag, suggesting that the increased dag levels observed in these rats may be due to decreased insulin secretion . Although, the mechanisms responsible for the differential effects of fatty acids on insulin action and glucose homeostasis have not been fully elucidated,46 ghrelin levels were found to be reciprocal to those of glucose and insulin1447 and previous data available suggested a negative association between systemic ghrelin and insulin levels, with ghrelin inhibiting insulin secretion both in vitro and in vivo and in most human or animal studies.48 it should be pointed out that ghrelin and dag are two separate peptides but they can modify the actions of each other on glucose handling.16 in this study, it is reasonable to postulate that the low fasting dag levels in hf - b fed rats partially relieve its inhibition on insulin production . The insulin level therefore, is increased to compensate the peripheral insulin resistance caused by high sfa consumption as previously reported.39 nevertheless, this study could not show any significant correlations between insulin and dag concentrations . On one hand, this may be related to different metabolic status in growing rats and on the other hand, previous studies have measured insulin concentration in the portal circulation following intravenous injection of dag, which may affect the findings.244950 gauna et al . Have hypothesized that assessment of insulin concentration in the portal vein might be more informative than that in the systemic circulation.24 as we said above, calorie intake, food intake and weight gain were higher in hf - b and hf - s diets in comparison to sd, hf - f and hf - o groups and on the other hand, hf - f and hf - o had higher dag and lower insulin and insulin resistance in comparison to the hf - s and hf - b diets . As, documented, ghrelin and insulin are two gut hormones playing an affective role in body weight regulation;6 dag could hence decrease food intake and gastric emptying in mice and rats.51 food intake modulator actions in the central nervous system have been suggested for insulin.5253 according to the findings of this study, food intake, calorie intake and weight gain were lower in groups with higher dag, which is consistent with recent report that dag induces a negative energy balance by decreasing food intake and delaying gastric emptying.51 on the other hand, in groups with higher insulin concentration and insulin resistance, food intake, calorie intake and weight gain also were higher . These findings are comparable with the buettner (and coworkers) study in which they have shown that groups with higher weight gain, had higher plasma glucose levels and less effective insulin - induced glucose disposal.2 rats fed with hfds containing mufa and -3 pufa had significantly lower weight gain, food and calorie intake and these changes were associated with increased fasting plasma dag concentrations, concomitant with lower insulin concentration and insulin resistance . Results of this study suggest that hfds containing fish oil and olive oil can increase the dag which may play a role in weight, appetite control and insulin resistance improvement in young rats . As carried out the design of study, coordinated, carried out all the experiments and also writing the manuscript . Mk, sz carried out the design, writing the manuscript and also supervised the project . Mv carried out the design and coordinated the study, participated in most of the experiments and prepared the manuscript . Ag, msd and mz participated in data collection and provided comments on the laboratory analysis of samples.
Antibodies and reagents anti - human gpvi monoclonal antibody (mab) 204 - 11 has been described previously (25). Anti - syk polyclonal ab (pab) (26) was kindly provided by j. b. bolen (dnax, ca). Anti - phospho - syk (tyr) pab and anti - myc mab were purchased from cell signaling technology (new england biolabs uk ltd ., anti - phosphotyrosine mab 4g10, anti - fcr -chain pab, and normal rabbit igg were purchased from upstate biotechnology (milton keynes, uk). Anti - pecam-1 mab ab468 was from autogen - bioclear (wiltshire, uk). Horseradish peroxidase - conjugated donkey anti - rabbit secondary ab and enhanced chemiluminescence reagents (ecl) were purchased from amersham biosciences . Fluorescein isothiocyanate - conjugated anti - mouse igg secondary antibody was purchased from sigma . The src kinase inhibitors used were, pp1, purchased from biosource europe (nivelles, belgium); pp2, purchased from calbiochem (nottingham, uk); and pd0173952, a gift from pfizer global research and development (ann arbor, mi). Fcr -chain knock - out mice were bred as heterozygotes as described (28). 10 mm pervanadate was freshly prepared on the day for use by mixing sodium orthovanadate and hydrogen peroxide in phosphate - buffered saline to final concentrations 10 mm, then left for 5 min at room temperature and kept on ice . Other reagents were from previously described sources (6, 8, 24). Constructs the human prc / gpvi, pef6/fcr -chain, pef6/clec-2, and mutant clec-2 (y7f) expression plasmids have been previously described (8). The human pcdna3/-g6b - b (kindly given by prof r. d. campbell, oxford, uk) has been described (9) and the human pcdna3/pecam-1 (kindly given by c. d. buckley, birmingham, uk) has also been described (29). The nfat luciferase reporter containing three copies of the distal nfat site from the interleukin-2 promoter has been described (30). Making myc - tagged fcr -chain the c - myc epitope tag (eqkliseedl) was fused at the amino terminus of fcr -chain by using pef6/fcr -chain as a template and the primers: forward (5-gaa caa aaa ctc atc tca gaa gag gat ctg ctg gga gag cct cag ctc-3) and reverse (5-cag atc ctc ttc tga gat gag ttt ttg ttc ggc cgc tgc ttg ttc aac-3), and subcloned into pef6 . Site - directed mutagenesis of g6b - b site - directed mutagenesis of g6b - b was performed by a quikchange site - directed mutagenesis kit (stratagene, cambridge, uk). The primers g6b - b - y211f - forward (5-ccg agc ctg ctc ttt gcg gat ctg gac-3) and g6b - b - y211f - reverse (5-gtc cag atc cgc aaa gag cag gct cgg-3) were used for tyrosine to phenylalanine mutation in g6b - b (y211f) using wild - type human pcdna / g6b - b as a template . The primers g6b - b - y237f - forward (5-gat gcc tcc acc atc ttt gca gtt gta gtt tg-3) and g6b - b - y237f - reverse (5-caa act aca act gca aag atg gtg gag gca tc-3) were used for tyrosine to phenylalanine mutation in g6b - b (y237f) using wild - type human pcdna / g6b - b as a template and they were also used for tyrosine to phenylalanine mutation in g6b - b (y211f / y237f) using mutant pcdna / g6b - b (y211f) as a template . Cell culture wild - type (wt), syk - deficient (31), shp1 and shp2 double - deficient (32) (kindly donated by l. meyaard, utrecht, the netherlands), ship - deficient (33) (kindly donated by d. k. newman, milwaukee, wi) dt40 chicken b cells were grown in rpmi supplemented with 10% fetal bovine serum, 1% chicken serum, 100 units / ml penicillin, 100 g / ml streptomycin, 50 m -mercaptoethanol, and 20 mm glutamine . Luciferase assay the nfat reporter assay was performed as described (8, 24). The indicated amount of dna of each construct and 15 g of nfat - luciferase reporter construct were transfected by electroporation at 350 v and 500 microfarads into 2 10 cells of wt, syk - deficient, and shp1/shp2-deficient dt40 cells . Twenty hours after transfection, live cells were counted by trypan blue exclusion, and samples divided for luciferase assay (2 10 cells / ml), flow cytometry (5 10 cells / sample), and western blotting (1 10 cells / sample). Collagen was used at 10 g / ml and rhodocytin was used at 50 nm . Luciferase activity was measured with a centro lb960 microplate luminometer (berthold technologies, germany). Cell surface expression of transfected cells was analyzed by flow cytometry using 1 g / ml, gpvi mab, pecam-1 mab, and myc mab to detect clec-2 and fcr -chain, t7-tag mab to detect g6b - b, or mouse igg followed by staining with 4 g / ml fluorescein isothiocyanate - conjugated anti - mouse igg secondary antibody, and assessed on a facscalibur (becton dickinson, san jose, ca). Human and mouse platelets washed preparations of human and mouse platelets were prepared as previously described (5, 8). Platelets were resuspended in a modified tyrodes - hepes buffer at concentrations of 4 10/ml (human) or 2 10/ml (mouse). Platelets were prewarmed to 37 c for 5 min and incubated with inhibitors or solvent controls for up to 10 min . Immunoprecipitation and western blotting transfected cells (1 10/ml) were incubated for 30 min in rpmi at 37 c before stimulating . After stimulation, transfected cells or platelets were lysed with ice - cold 2 lysis buffer (2% triton x-100, 2% dodecyl maltoside, 4 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 20 g / ml aprotinin, 20 g / ml leupeptin, 2 g / ml pepstatin, 10 mm sodium orthovanadate, ph 7.5) and insoluble material was removed by centrifugation . For immunoprecipitation, lysates were precleared with protein a (g)-sepharose beads for 30 min at 4 c and mixed with 2 g of the indicated antibodies and protein a - sepharose beads (protein g - sepharose). Whole cell lysates or immunoprecipitated lysates were added to 2 laemmli sample buffer . Samples were separated by sds - page on 10 or 412% bistris gels (invitrogen) and transferred to polyvinylidene difluoride membrane . Statistical analysis experiments were performed on at least three occasions and results are shown as mean s.e . With the exception of the representative western blots and flow cytometry histograms . Constitutive signaling of gpvi - fcr -chain in dt40 cells we set out to extend our previous characterization of the gpvi - fcr -chain signaling pathway in the dt40 b cell line (24) by testing whether activation of gpvi by collagen is dependent on its associated fcr -chain . Activation was monitored in the absence and presence of collagen and compared with mock - transfected cells that had been allowed to settle on fibronectin using a nfat - luciferase reporter assay . Adhesion to fibronectin alone did not induce nfat activation (not shown). As anticipated, collagen stimulated nfat activation in dt40 cells expressing both gpvi and an nh2-terminal myc - tagged version of fcr -chain, whereas it had no effect when either subunit was transfected on its own (fig . The myc - tagged version of fcr -chain supports a similar level of activation to that of the wild - type protein (not shown) and has the advantage that it can be used to monitor surface expression by flow cytometry, which is not altered by expression of gpvi (fig ., gpvi was not expressed on the surface of dt40 cells in the absence of the fcr -chain (not shown), as is the case for platelets (34). These results are consistent with a model in which cross - linking of gpvi by collagen leads to tyrosine phosphorylation of the fcr -chain and activation of the tyrosine kinase syk . These studies further revealed the unexpected observation that expression of the myc - tagged or wild - type fcr -chain caused a significant increase in luciferase activity that approached almost 10% of the response to collagen (fig . Furthermore, the constitutive signal from the fcr -chain was not altered by co - expression with gpvi (fig . Thus the increase in luciferase activity that was observed in the absence of a stimulatory agonist reflects signaling by the itam - containing protein . To investigate constitutive signaling in further detail, dt40 cells were transfected with varying concentrations of the fcr -chain plasmid and the level of nfat activation monitored . Increasing levels of transfection of fcr -chain led to a corresponding increase in both nfat activation (fig . 1b, panel i) and fcr -chain expression as shown by western blotting (fig . Furthermore, western blotting of gpvi - fcr -chain - transfected cells demonstrated an increase in tyrosine phosphorylation of a band of 72 kda that comigrates with syk, along with constitutive tyrosine phosphorylation of fcr -chain (fig . Confirmation that the 72-kda protein corresponds to syk was confirmed using a phosphospecific antibody (phospho - syk tyr) that binds to the activated tyrosine kinase (not shown). Tyrosine phosphorylation of syk and fcr -chain were inhibited in the presence of the src family kinase inhibitor pd0173952 (fig . These results demonstrate that the fcr -chain generates constitutive signals in dt40 cells that lead to tyrosine phosphorylation of syk and nfat activation . Stimulation of gpvi - fcr -chain - transfected cells with collagen leads to a further increase in tyrosine phosphorylation of syk (fig . 1c, panel ii), consistent with the activation of syk by the collagen receptor complex, as described in earlier studies . The above studies were extended to clec-2, which is a recently identified platelet glycoprotein receptor that mediates activation through a single yxxl motif in its cytosolic tail . Clec-2 is a receptor for the snake venom toxin, rhodocytin, and the lymphatic marker podoplanin . Both agonists induce activation of clec-2-transfected dt40 cells, with the response being dependent on the conserved tyrosine in the cytoplasmic yxxl motif (7, 8). In the present study, we have confirmed that rhodocytin stimulates nfat activation in clec-2-transfected dt40 cells, with the level of response increasing with the level of surface expression of clec-2 (fig . Moreover, as is the case with the fcr -chain, expression of clec-2 was sufficient to increase nfat activity in the absence of agonist stimulation, with the level of response increasing in parallel with that of clec-2 (fig . The level of constitutive activity approached between 10 and 20% the response to rhodocytin . Constitutive signaling by clec-2 is abolished following mutation of the tyrosine in the clec-2 yxxl sequence to a phenylalanine (y7f) (fig . 2b, panel i), as is the case for stimulation by rhodocytin (8). Flow cytometry confirmed a similar level of expression of wild - type and the y7f mutant of clec-2 in these studies (fig . These results demonstrate that constitutive and agonist - induced signaling by clec-2 is dependent on the yxxl motif . A, panel i, constitutive and collagen - induced signaling in dt40 cells transfected with plasmids (10 g) encoding gpvi and/or myc - fcr -chain (fcr) was analyzed using an nfat - luciferase reporter assay . Cell lysates were analyzed for luciferase activity following incubation with media or collagen (10 g / ml) for 6 h. data are expressed as -fold increase over the basal of mock - transfected cells . Panel ii, gpvi and fcr surface expression was measured by flow cytometry following transfection with 10 g of plasmid of gpvi / myc - fcr or myc - fcr only (corresponding to a, panel i) relative to a control igg . Data are representative of three experiments.b, constitutive signaling by the fcr alone . Panel ii, western blotting (wb) for fcr -chain in dt40 cells transfected with increasing amounts of fcr -chain plasmid . C, panel i, constitutive tyrosine phosphorylation of syk and fcr. Mock and gpvi - fcr-transfected dt40 cells were pre - treated with pd0173952 (20 m) for 5 min . Whole cell lysates were western blotted for tyrosine phosphorylation using mab 4g10 and then reprobed for syk and fcr. Tyrosine phosphorylated bands that comigrate with syk and fcr are shown . Panel ii, tyrosine phosphorylation of syk in gpvi - fcr-transfected dt40 cells lysates by collagen (30 g / ml) is partially inhibited by cotransfection with g6b - b . Cells were stimulated by collagen for 90 s. experimental conditions were as for panel i. data are representative of three experiments . A, panel i, constitutive and collagen - induced signaling in dt40 cells transfected with plasmids (10 g) encoding gpvi and/or myc - fcr -chain (fcr) was analyzed using an nfat - luciferase reporter assay . Cell lysates were analyzed for luciferase activity following incubation with media or collagen (10 g / ml) for 6 h. data are expressed as -fold increase over the basal of mock - transfected cells . Panel ii, gpvi and fcr surface expression was measured by flow cytometry following transfection with 10 g of plasmid of gpvi / myc - fcr or myc - fcr only (corresponding to a, panel i) relative to a control igg . Data are representative of three experiments.b, constitutive signaling by the fcr alone . Panel ii, western blotting (wb) for fcr -chain in dt40 cells transfected with increasing amounts of fcr -chain plasmid . C, panel i, constitutive tyrosine phosphorylation of syk and fcr. Mock and gpvi - fcr-transfected dt40 cells were pre - treated with pd0173952 (20 m) for 5 min . Whole cell lysates were western blotted for tyrosine phosphorylation using mab 4g10 and then reprobed for syk and fcr. Tyrosine phosphorylated bands that comigrate with syk and fcr are shown . Panel ii, tyrosine phosphorylation of syk in gpvi - fcr-transfected dt40 cells lysates by collagen (30 g / ml) is partially inhibited by cotransfection with g6b - b . Cells were stimulated by collagen for 90 s. experimental conditions were as for panel i. data are representative of three experiments . Constitutive signaling by gpvi - fcr -chain and clec-2 is mediated by src and syk tyrosine kinases we and others have previously reported that collagen stimulates phosphorylation of the fcr -chain itam in platelets through the src kinases, fyn and lyn, leading to recruitment and tyrosine phosphorylation of syk (3537). Similarly, phosphorylation of clec-2 and syk by rhodocytin in platelets is mediated through a src kinase pathway, although the identity of the src kinase is not known (5). Consistent with this, we observed constitutive and collagen - stimulated tyrosine phosphorylation of syk in gpvi - fcr -chain - transfected dt40 cells (fig ., we have not been able to detect constitutive phosphorylation of clec-2 in our studies, possibly because of a lower level of expression relative to that of fcr -chain, as shown using a myc - tagged version of both proteins (supplemental fig . S1), and because the c - type lectin receptor has a single yxxl motif . However, we have observed a small increase in constitutive phosphorylation of syk in dt40 cells expressing clec-2, which was further increased in the presence of rhodocytin (not shown). Experiments were designed to investigate whether nfat signaling by the gpvi - fcr -chain complex and clec-2 in transfected dt40 cells is mediated through src and syk kinases . As shown in fig . 3a, constitutive and agonist - induced nfat activation by both receptors is blocked in the presence of the src family kinase inhibitor pd0173952 . Furthermore, constitutive and agonist - induced signaling by the two receptors was also markedly inhibited in the absence of syk (fig . Thus, these results demonstrate that constitutive and agonist - induced signaling by gpvi - fcr -chain and clec-2 are both dependent on src family and syk tyrosine kinases . G6b - b inhibits constitutive and agonist - induced signaling by gpvi - fcr -chain and clec-2 through its two itims a series of experiments were undertaken to investigate the possible regulation of constitutive and agonist - induced signaling through the gpvi - fcr -chain complex and clec-2 by the platelet immunoglobulin receptor g6b - b, which contains two itims in its cytosolic tail (9, 10). Xpression of g6b - b significantly inhibited constitutive signaling by both receptors by more than 60% (fig . 4, panel i), as well as inhibiting the response to collagen and rhodocytin by 35 and 60%, respectively (fig . 4, . The greater degree of inhibition of the response to rhodocytin may reflect the lower level of expression of clec-2 relative to gpvi - fcr -chain (supplemental fig . S1). Expression of g6b - b had no effect on the level of expression of either clec-2 or gpvi (supplemental fig . Expression of g6b - b also partially inhibited collagen - induced tyrosine phosphorylation of syk (fig . 1c, panel ii), demonstrating a molecular basis for its inhibitory action . These results demonstrate that g6b - b inhibits constitutive and agonist - induced signaling induced by gpvi - fcr -chain and clec-2 . Figure 2.constitutive signaling by clec-2 in dt40 cells is dependent on its yxxl motif . A, panel i, constitutive and rhodocytin - induced signaling in dt40 cells transfected with varying amounts of the clec-2 plasmid was analyzed by nfat - luciferase reporter assay . Cells were stimulated with 50 nm rhodocytin for 6 h. values are mean s.e . Panel ii, clec-2 surface expression following transfection with varying amounts of clec-2 plasmid was assessed by flow cytometry using anti - myc tag mab relative to a control igg . B, panel i, constitutive and rhodocytin - induced signaling in cells transfected with wild - type (wt) and mutant (y7f) clec-2 . Panel ii, clec-2 surface expression (corresponding to panel i) was assessed by flow cytometry using anti - myc tag mab relative to a control igg . A, panel i, constitutive and rhodocytin - induced signaling in dt40 cells transfected with varying amounts of the clec-2 plasmid was analyzed by nfat - luciferase reporter assay . Cells were stimulated with 50 nm rhodocytin for 6 h. values are mean s.e . Panel ii, clec-2 surface expression following transfection with varying amounts of clec-2 plasmid was assessed by flow cytometry using anti - myc tag mab relative to a control igg . B, panel i, constitutive and rhodocytin - induced signaling in cells transfected with wild - type (wt) and mutant (y7f) clec-2 . Panel ii, clec-2 surface expression (corresponding to panel i) was assessed by flow cytometry using anti - myc tag mab relative to a control igg . G6b - b contains two itims in its cytosolic tail, which independently mediate binding to shp1 and shp2 following incubation with the protein - tyrosine phosphatase inhibitor, pervanadate (9). Mutation of the two conserved tyrosines at positions 211 and 237 to phenylalanine inhibits association with the two sh2 domain - containing protein - tyrosine phosphatases (9). To investigate whether either or both of the itims mediate the inhibitory effect of g6b - b, the y211f / y237f double mutant was expressed in dt40 cells together with the gpvi - fcr -chain complex and clec-2 . Flow cytometry was used to confirm similar levels of expression of the g6b - b mutant, gpvi and clec-2 in the transfected cell lines to that in cells transfected with wild - type g6b - b and the two yxxl - containing receptors (supplemental fig . Mutation of the two conserved tyrosines in the g6b - b itims abolished the inhibitory effect of g6b - b against constitutive and agonist - induced activation of nfat induced by gpvi - fcr -chain and clec-2 (fig . 4), whereas mutation of either of the two tyrosines alone had a partial or neglible effect (not shown). In line with this, we were able to detect weak tyrosine phosphorylation of g6b - b in the dt40-transfected cells (not shown). These results demonstrate that g6b - b inhibits constitutive and agonist - induced signaling through the conserved tyrosines at positions 211 and 237 . The ability of g6b - b to inhibit constitutive and agonist - induced responses mediated through gpvi - fcr -chain and clec-2 was further investigated in the combined absence of the two sh2 domain - containing tyrosine phosphatases, shp1 and shp2, or in the absence of the 5-inositol phosphatase, ship . Unexpectedly, the absence of the two protein - tyrosine phosphatases led to an increase in the level of constitutive and agonist - induced nfat activation through gpvi - fcr -chain and clec-2, suggesting that both receptors are under dynamic inhibitory feedback through the two tyrosine phosphatases (fig . Nevertheless, g6b - b was still able to inhibit constitutive and agonist - induced signaling through gpvi - fcr -chain and clec-2 in the absence of shp-1 and shp-2 (fig . 5a). Indeed, the inhibitory action against rhodocytin was enhanced in the combined absence of shp1 and shp2 (fig . 5a), although this may reflect the increased response to rhodocytin that is observed in the absence of the two protein - tyrosine phosphatases . G6b - b also inhibits constitutive and agonist - induced nfat activation by gpvi - fcr -chain and clec-2 in dt40 cells deficient in the sh2-domain - containing inositol 5-phosphatase (ship) (fig . Indeed the inhibitory action of g6b - b against clec-2 was also enhanced by the absence of the 5-inositol phosphatase (fig . 5b), as was the case in the absence of shp1 and shp2 . These results demonstrate that g6b - b inhibits gpvi and clec-2 signaling in dt40 cells by a shp1/shp2-independent and ship - independent mechanism . Figure 3.constitutive and agonist - induced signaling by gpvi - fcr -chain and clec-2 is dependent on src and syk tyrosine kinases . Constitutive and agonist - induced signaling in wild - type (wt) and syk - deficient dt40 cells transfected with gpvi - fcr-chain or clec-2 was analyzed using an nfat - luciferase reporter assay . Cell lysates were analyzed for luciferase activity following agonist stimulation for 6 h. a, wt dt40 cells were either mock - transfected or transfected with (panel i) gpvi - fcr -chain (10 g of each plasmid) or (panel ii) clec-2 (10 g) and stimulated with 10 g / ml collagen or 50 nm rhodocytin in the presence and absence of the src kinase inhibitor, pd0173952 (20 m). B, basal signaling in wt or syk - deficient dt40 cells transfected with mock or transfected with (panel i) gpvi - fcr -chain (10 g of each plasmid) or (panel ii) clec-2 (10 g) and incubated for 6 h to monitor the degree of constitutive signaling . Values are mean s.e . Constitutive and agonist - induced signaling by gpvi - fcr -chain and clec-2 is dependent on src and syk tyrosine kinases . Constitutive and agonist - induced signaling in wild - type (wt) and syk - deficient dt40 cells transfected with gpvi - fcr-chain or clec-2 was analyzed using an nfat - luciferase reporter assay . Cell lysates were analyzed for luciferase activity following agonist stimulation for 6 h. a, wt dt40 cells were either mock - transfected or transfected with (panel i) gpvi - fcr -chain (10 g of each plasmid) or (panel ii) clec-2 (10 g) and stimulated with 10 g / ml collagen or 50 nm rhodocytin in the presence and absence of the src kinase inhibitor, pd0173952 (20 m). B, basal signaling in wt or syk - deficient dt40 cells transfected with mock or transfected with (panel i) gpvi - fcr -chain (10 g of each plasmid) or (panel ii) clec-2 (10 g) and incubated for 6 h to monitor the degree of constitutive signaling . Figure 4.the novel platelet itim transmembrane protein g6b - b inhibits constitutive and agonist - induced signaling through gpvi and clec-2 . The effect of the wild - type (wt) and double itim - mutant transmembrane protein g6b - b (y211f / y237f) on (panel i) constitutive and (panel ii) agonist - induced signaling by gpvi - fcr -chain and clec-2 in transfected (10 g of each plasmid) dt40 cells, measured using an nfat - luciferase reporter assay . Cells were stimulated with 10 g / ml collagen or 50 nm rhodocytin as appropriate . The double itim mutant contained inactivating point mutations (y / f) at tyrosines 211 and 237 . The results are mean s.e . Of between 4 and 7 independent experiments . , p <0.05; and , p <0.01 compared with the gpvi - fcr -chain or clec-2-transfected samples . The novel platelet itim transmembrane protein g6b - b inhibits constitutive and agonist - induced signaling through gpvi and clec-2 . The effect of the wild - type (wt) and double itim - mutant transmembrane protein g6b - b (y211f / y237f) on (panel i) constitutive and (panel ii) agonist - induced signaling by gpvi - fcr -chain and clec-2 in transfected (10 g of each plasmid) dt40 cells, measured using an nfat - luciferase reporter assay . Cells were stimulated with 10 g / ml collagen or 50 nm rhodocytin as appropriate . The double itim mutant contained inactivating point mutations (y / f) at tyrosines 211 and 237 . The results are mean s.e . Of between 4 and 7 independent experiments . , p <0.05; and * , p <0.01 compared with the gpvi - fcr -chain or clec-2-transfected samples . Figure 5.the inhibitory effect of g6b - b is independent of shp1, shp2, and ship . Constitutive and agonist - induced signaling in shp1/shp2 double - deficient (a) and ship - deficient dt40 cells (b) transfected with gpvi - fcr -chain, clec-2, and g6b - b was analyzed using a nfat - luciferase reporter assay . Cells were stimulated with 10 g / ml collagen or 50 nm rhodocytin for 6 h. , p <0.05 relative to response in the absence of g6b - b . The inhibitory effect of g6b - b is independent of shp1, shp2, and ship . Constitutive and agonist - induced signaling in shp1/shp2 double - deficient (a) and ship - deficient dt40 cells (b) transfected with gpvi - fcr -chain, clec-2, and g6b - b was analyzed using a nfat - luciferase reporter assay . Cells were stimulated with 10 g / ml collagen or 50 nm rhodocytin for 6 h. *, p <0.05 relative to response in the absence of g6b - b . Pecam-1 does not inhibit constitutive signaling in dt40 cells a similar set of studies were undertaken to investigate whether the major platelet itim receptor, pecam-1, also inhibits constitutive and agonist - induced activation of nfat downstream of gpvi - fcr -chain and clec-2 . S3a, pecam-1 had no effect on constitutive or agonist - induced activation of either receptor in dt40 cells, possibly because of a low level of expression (supplemental fig . A, human washed platelets were incubated at 37 c with the syk kinase inhibitor, r406 (1 m), and src inhibitor pp2 (20 m) for 1 min . B, wild - type (wt) or fcr -chain deficient mouse (ko) platelets (pretreated with egta to block aggregation) were stimulated with 100 m pervanadate (pv) for 90 s in the presence or absence of src inhibitor pp2 (20 m). C, human washed platelets were incubated at 37 c with the src kinase inhibitors pp1 (10 m) or pd0173952 (10 m) for 5 min . Cell lysates were immunoprecipitated with anti - syk ab or an igg and probed for tyr(p). D, human washed platelets were stimulated with 30 nm rhodocytin for 5 min in the presence and absence of the src kinase inhibitor, pp2 (10 m). Cell lysates were immunoprecipitated (ip) with anti - clec-2 ab and probed for tyr(p). A, human washed platelets were incubated at 37 c with the syk kinase inhibitor, r406 (1 m), and src inhibitor pp2 (20 m) for 1 min . B, wild - type (wt) or fcr -chain deficient mouse (ko) platelets (pretreated with egta to block aggregation) were stimulated with 100 m pervanadate (pv) for 90 s in the presence or absence of src inhibitor pp2 (20 m). C, human washed platelets were incubated at 37 c with the src kinase inhibitors pp1 (10 m) or pd0173952 (10 m) for 5 min . Cell lysates were immunoprecipitated with anti - syk ab or an igg and probed for tyr(p). D, human washed platelets were stimulated with 30 nm rhodocytin for 5 min in the presence and absence of the src kinase inhibitor, pp2 (10 m). Cell lysates were immunoprecipitated (ip) with anti - clec-2 ab and probed for tyr(p). Src and syk kinase - dependent constitutive signaling in platelets experiments were designed to investigate whether there is evidence for constitutive signaling through src and syk kinases in platelets . In this context, it is important to emphasize that platelets express several classes of receptor that signal through src and syk tyrosine kinases, including the major platelet integrin, iib3, and the gpib - ix - v complex, the levels of which are over 1 order of magnitude greater than that of gpvi - fcr -chain and clec-2 . We first asked the question as to whether the tyrosine phosphorylation that is seen in the absence of agonist stimulation in platelets is regulated by src or syk tyrosine kinases . Western blotting for phosphotyrosine using the monoclonal antibody 4g10 reveals the presence of a broad band of proteins in non - stimulated platelets of between 50 and 60 kda (fig . This region includes src kinases, which are constitutively phosphorylated in resting platelets by the src kinase - regulatory protein, csk, at an inhibitory site that promotes intramolecular binding to the sh2 domain . In addition, several other proteins, which have been shown to be constitutively phosphorylated in nonstimulated platelets, migrate in this region, including dok-2 (38). Additional phosphorylation bands that lie outside of this region can also be seen in non - stimulated platelets in longer exposures, as illustrated in fig . Tyrosine phosphorylation of the majority of these bands is markedly inhibited in the presence of the src family kinase inhibitors, pp1 and pp2, and the syk family kinase inhibitor, r406 (39), with the exception of the broad band of proteins that runs at 5060 kda, which is only partially reduced (fig . The residual phosphorylation that is seen in this region is likely to reflect phosphorylation of src kinases on their inhibitory site by csk . Significantly, csk is not known to be regulated downstream of src and syk kinases . The reduction in phosphorylation in this region is presumably due to loss of phosphorylation of proteins such as dok-2, as discussed above . The similar pattern of reduction in tyrosine phosphorylation that is induced by r406 to that of pp2 cannot be due to direct inhibition of src kinases, as r406 does not inhibit collagen - stimulated tyrosine phosphorylation of the fcr -chain,5 which is mediated through src kinase activation . These results demonstrate that constitutive signaling by src and syk family kinases underlies tyrosine phosphorylation of the majority of proteins in non - stimulated platelets, thereby providing evidence of constitutive signaling by src and syk kinases . We used the protein - tyrosine phosphatase inhibitor, pervanadate, to further investigate the contribution of src and syk family kinases to constitutive tyrosine phosphorylation in human and mouse platelets, on the grounds that this would potentiate protein tyrosine phosphorylation by constitutively active tyrosine kinases . 6b), with similar observations made for human platelets (not shown). As previously reported (4042), pervanadate induces a marked increase in tyrosine phosphorylation of a large number of proteins in platelets (fig . Strikingly, protein tyrosine phosphorylation induced by pervanadate was dramatically inhibited in the presence of the src kinase inhibitor, pp2, confirming that src family kinases are the major family of kinases that contribute to constitutive tyrosine phosphorylation in platelets . A small number of tyrosine - phosphorylated proteins are seen in longer exposures of pervanadate - treated platelets in the presence of pp2, including a band of 72 kda (fig . 6b) that was identified as syk by immunoprecipitation of the kinase and western blotting for phosphotyrosine (fig . Confirm that src kinases are constitutively active in nonstimulated platelets and demonstrate that they underlie the major increase in tyrosine phosphorylation that is induced in the presence of protein - tyrosine phosphatase inhibition . Further experiments were designed to investigate the possible dependence of constitutive signaling through src and syk family kinases on the gpvi - fcr -chain complex . For these experiments, syk was immunoprecipitated from resting human platelets and western blotted for phosphotyrosine in the absence and presence of the src family kinase inhibitor, pd0173952 . 6c demonstrate association of constitutively tyrosine - phosphorylated syk and constitutively tyrosine - phosphorylated fcr -chain, which is dramatically reduced in the presence of the src kinase inhibitor pd0173952 . On the other hand, treatment of fcr -chain - deficient mouse platelets with pervanadate revealed that constitutive tyrosine phosphorylation in mouse platelets is minimally altered in platelets deficient in the itam - containing protein (fig . 6b), demonstrating that the gpvi - fcr -chain complex makes a relatively minor contribution to the overall level of tyrosine phosphorylation . Constitutive phosphorylation of clec-2 is observed in human platelets and is reduced in the presence of the src kinase inhibitor, pp2 (fig . The contribution of clec-2 to constitutive tyrosine phosphorylation in mouse platelets cannot be assessed, as mice lacking the c - type lectin receptor have not been made . The present study demonstrates that the gpvi - fcr -chain complex and clec-2 generate constitutive signals in transfected dt40 cells leading to nfat activation with the degree of signaling corresponding to the level of expression . Furthermore, the dependence on src and syk family kinases provides strong evidence that constitutive signaling by both receptors is mediated through the same pathway as used by agonists to mediate activation . In line with this, constitutive signaling by clec-2 is inhibited by mutation of the conserved tyrosine in the yxxl sequence in its cytosolic tail . Furthermore, the response to the gpvi - fcr -chain complex is mediated by fcr -chain, and is independent of gpvi, consistent with it being generated through the tandem yxxl sequence of the fcr -chain . Constitutive and agonist - induced signaling through gpvi - fcr -chain and clec-2 is reduced by co - expression of the itim - containing platelet protein, g6b - b (9). The inhibitory effect of g6b - b is dependent on the conserved tyrosines in its two itims, as shown by abolition of the inhibitory response upon mutation of the itim tyrosines to phenylalanine . However, unexpectedly, this inhibitory effect is retained in dt40 cells in the combined absence of the sh2 domain - containing tyrosine phosphatases, shp1 and shp2, which have been shown to bind to g6b - b (9, 10). The inhibitory effect of g6b - b is also retained in the absence of the sh2 domain - containing inositol phosphatase, ship, which has been shown to inhibit b cell receptor signaling through association with the fcriib itim (43, 44). An alternative pathway of inhibition could be through recruitment of the sh2 domain - containing inhibitor of src kinases, csk, to the two itims in g6b - b . Such a mechanism has been proposed to underlie the inhibition of fcri signaling by the itim - containing transmembrane protein, lair-1, in rat rbl mast cells (45). However, we were unable to demonstrate association of csk with g6b - b in transfected dt40 cells (not shown), possibly because of the low level of constitutive phosphorylation of the itim - containing protein . Access to csk - deficient dt40 cells is required to directly test the role of the src kinase regulatory protein in mediating the inhibitory effect of g6b - b . It is also possible that the inhibitory effect of g6b - b is mediated by association with several proteins, including a combination of shp1, shp2, ship, and csk, such that loss of any one is compensated by expression of the others . Such a mechanism would explain why g6b - b is still able to mediate inhibition in dt40 cells deficient in shp1 and shp2, which have both been shown to bind to its cytosolic itims . Interestingly, we observed a small reduction in tyrosine phosphorylation of syk by g6b - b in dt40 cells stimulated by collagen, suggesting that this underlies the inhibitory effect of the itim - containing protein . The observation that the fcr -chain and clec-2 generate constitutive signals is in line with reports that itam receptors generate weak, constitutive signals in other hematopoietic cells . For example, in mouse thymocytes and peripheral t cells, there is limited constitutive phosphorylation of tcr chains leading to association with the syk - related tyrosine kinase zap-70 (46, 47). This gives rise to constitutive signaling in resting mouse thymocyte and jurkat t cell lines, as shown by suppression of rag gene expression via extracellular signal - regulated kinase (erk) and abl kinases, and prevention of further recombination of t cell receptor genes (48). The b cell antigen receptor also signals independently of ligand engagement and this provides functionally relevant signals in immature b lymphocytes (49) and in diffuse large b - cell lymphoma (50). Significantly, this constitutive signaling is mediated through the b cell receptor itams (51, 52). Thus, constitutive signaling appears to be a common feature of yxxl - containing receptors . Evidence of constitutive signaling through src and syk tyrosine kinases in platelets has been demonstrated in the present study in association with a low level of tyrosine phosphorylation of the fcr -chain and clec-2 . Studies in mice deficient in the fcr -chain demonstrate that the gpvi receptor complex makes only a minimal contribution to this phosphorylation, most likely because of the association of src kinases with other surface receptors, including the iib3 complex that is expressed at over 20 times the level of gpvi . The demonstration of constitutive signaling via src and syk kinases in platelets emphasizes the need for inhibitory signals to oppose activation occurring in healthy vessels . We speculate that this function may be mediated, at least in part, by the itim receptor, g6b - b, which is markedly phosphorylated in resting platelets (10). The ligand for g6b - b is not known, but if this is expressed on platelets, it is possible that this may account for the high level of constitutive phosphorylation of g6b - b through a cis - interaction . A similar function to that of g6b - b may be mediated in platelets by the itim - containing receptor, pecam-1 . This is in line with studies that have reported inhibition of agonist - induced platelet activation by the immunoglobulin receptor (2123). However, unexpectedly, we were unable to observe inhibition of constitutive and agonist - induced signaling by pecam-1 in the dt40 cell line, possibly because of too low a level of expression . In summary, the present study reports that both fcr -chain and clec-2 generate weak, sustained constitutive signals that are inhibited by co - expression with g6b - b . We speculate that this could represent an important physiological role of g6b - b and other platelet itim proteins in helping to prevent platelet activation in vivo.
All animal procedures were performed in accordance with recommendations in the arvo statement for the use of animals in ophthalmic and vision research, in the guide for the care and use of laboratory animals by the national institutes of health, and under an approved protocol from the institutional animal care and use committees at the scripps research institute . Mixed - background tmod1 mice used in this study all contained a cardiac - restricted -myosin heavy chain (mhc) promoter - driven tmod1 transgene, as previously described . Genotyping was as described, and for brevity, mouse genotypes are referred to as tmod1 and tmod1 . Mixed - background mice carried an endogenous mutation in the bfsp2/cp49 gene leading to a loss of beaded intermediate filaments in the lens . We restored wild - type bfsp2/cp49 alleles to tmod1 mice by backcrossing with wild - type c57bl6 mice, as previously described . All mice used in this study were littermates that carried the mhc - tmod1 transgene and wild - type bsfp2/cp49 . Regarding rabbit polyclonal primary antibodies, anti - pan - fimbrin was a generous gift from paul matsudaira (national university of singapore), and anti - human tmod1 was prepared in our laboratory . With respect to mouse monoclonal primary antibodies, anti--actinin (nonsarcomeric, actn1) was from sigma - aldrich corp . Louis, mo, usa), anti - arp3 was from bd biosciences (612134; san jose, ca, usa), anti - ezrin from sigma - aldrich corp . Rat monoclonal primary antibody anti - n - cadherin was a generous gift from dietmar vestweber (max - planck - institute for molecular biomedicine). Secondary antibodies were alexa-488conjugated goat anti - rabbit (a11008; thermo fisher scientific, grand island, ny, usa), alexa-488conjugated goat anti - mouse (115 - 545 - 166, minimal cross - reaction; jackson immunoresearch, west grove, pa, usa), alexa-647conjugated goat anti - rat (112 - 605 - 167, minimal cross - reaction; jackson immunoresearch), and alexa-647conjugated goat anti - mouse igg (a21236; thermo fisher scientific). Rhodamine - phalloidin (r415, thermo fisher scientific) was used to stain f - actin, and hoechst 33258 (b2883; sigma - aldrich corp .) Three - month - old wild - type lenses were prepared for scanning electron microscopy (sem) as previously described . Briefly, lenses were removed from enucleated mouse eyes and immersed in 2.5% glutaraldehyde 2% formaldehyde in 0.1 m sodium cacodylate buffer at room temperature for 30 minutes to 1 hour . After critical point drying, lenses were split into quarters along the anterior posterior axis, yielding a freshly fractured surface that revealed a surface spanning a complete equatorial lens radius . Imaging was conducted with a phillips xl30 tmp scanning electron microscope (fei company, eindhoven, the netherlands). To observe interlocking protrusions and paddles along the short sides of fiber cells, freshly isolated lenses from 2-month - old tmod1 and tmod1 mice were fixed in 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer, ph 7.3, at room temperature for 48 to 72 hours . Each lens was properly oriented and fractured in an anterior posterior orientation with a sharp razor blade to expose the longitudinal features of the fiber cell short sides along the anterior, equatorial, and posterior regions of the lens . Lens halves were then postfixed in 1% aqueous oso4 for 1 hour at room temperature, dehydrated in graded ethanol, and dried in a samdri-795 critical point dryer (tousimis, inc ., lens halves were mounted on specimen stubs and coated with gold / palladium in a hummer 6.2 sputter coater (anatech, inc . Micrographs were taken with a jeol 820 scanning electron microscope at 10 kv (jeol, tokyo, japan). The lens nucleus was used as a reference to determine the location of images taken and to ensure that images were from comparable areas of different lenses (i.e., comparable regions were located based on measurements from the center outward). Freshly enucleated eyes were collected from 6-week - old mice . A small opening made at the corneal scleral junction facilitated fixative penetration . After fixation, samples were washed in pbs, cryoprotected in 30% sucrose, frozen in optimal cutting temperature (oct) medium (sakura finetek, torrance, ca, usa), and stored at 80c until sectioning . Frozen sections (12 m thick) were collected with a leica cm1950 cryostat (wetzlar, germany). Prolong gold antifade reagent (thermo fisher scientific) was used to mount the slides, and images were collected using a zeiss lsm780 confocal microscope (oberkochen, germany). Frozen sections with cross - sectionally oriented fiber cells near the lens equator were identified based on the thickness of the lens epithelium . Staining was repeated on three samples from different mice for each genotype, and representative data are shown . After fixation, lenses were cut into quarters using a sharp scalpel along the anterior posterior axis . Lens quarters were postfixed in 1% paraformaldehyde in pbs for 15 minutes at room temperature and then washed two times briefly with pbs . Lens quarters were then permeabilized and blocked using 3% normal goat serum, 3% bovine serum albumin, and 0.3% then, samples were washed three times, 5 minutes per wash, with pbs . Lens quarters were incubated in secondary antibodies diluted in blocking solution for 3 hours at room temperature, followed by washing (four times, 5 minutes per wash) with pbs . Lens quarters were stored in prolong gold antifade reagent at 4c until imaging . Images and z - stacks with 0.2-m steps were collected of single fiber cells or small bundles of fibers from lens quarters using a zeiss lsm780 confocal microscope . Fiber cell morphology and cell location in relation to the bulk lens quarter were used to approximate the maturity of imaged fiber cells . Staining was repeated on at least three lenses from three different mice for each genotype, and representative data are shown . Z - stack confocal images were analyzed using volocity 6.3 (perkinelmer, waltham, wa, usa). Noise reduction using the fine filter was applied to all z - stacks in all channels . The extended focus function in volocity was used to flatten z - stacks into a single image . For fluorescence intensity heat maps, single optical sections from z - stacks were separated into individual channels (16-bit grayscale images), and then heat maps were generated in imagej using the heatmap histogram plug - in (http://www.samuelpean.com/heatmap-histogram/ [in the public domain]). For tortuosity and cell neck width measurements, we evaluated single optical sections of single fibers stained with rhodamine - phalloidin to outline cell contours from 9 tmod1 and 9 tmod1 mice . Using imagej, we located the fiber cell edges using the find edges function and used the wand tool (mode: legacy; tolerance: 5.0) to measure the length of one of the curved edges of each fiber cell in pixels . Tortuosity was calculated as the contour length of the cell edge divided by the straight end - to - end distance along the fiber measured through the center of each fiber . Cell neck width across each fiber at the concave region was measured using the line tool . The average, standard error, and statistical significance were calculated and plotted in excel (microsoft, redmond, wa, usa). Statistical significance was determined using the student's t - test, and p values less than 0.01 were considered statistically significant . All animal procedures were performed in accordance with recommendations in the arvo statement for the use of animals in ophthalmic and vision research, in the guide for the care and use of laboratory animals by the national institutes of health, and under an approved protocol from the institutional animal care and use committees at the scripps research institute . Mixed - background tmod1 mice used in this study all contained a cardiac - restricted -myosin heavy chain (mhc) promoter - driven tmod1 transgene, as previously described . Genotyping was as described, and for brevity, mouse genotypes are referred to as tmod1 and tmod1 . Mixed - background mice carried an endogenous mutation in the bfsp2/cp49 gene leading to a loss of beaded intermediate filaments in the lens . We restored wild - type bfsp2/cp49 alleles to tmod1 mice by backcrossing with wild - type c57bl6 mice, as previously described . All mice used in this study were littermates that carried the mhc - tmod1 transgene and wild - type bsfp2/cp49 . Regarding rabbit polyclonal primary antibodies, anti - pan - fimbrin was a generous gift from paul matsudaira (national university of singapore), and anti - human tmod1 was prepared in our laboratory . With respect to mouse monoclonal primary antibodies, anti--actinin (nonsarcomeric, actn1) was from sigma - aldrich corp . Louis, mo, usa), anti - arp3 was from bd biosciences (612134; san jose, ca, usa), anti - ezrin from sigma - aldrich corp . Rat monoclonal primary antibody anti - n - cadherin was a generous gift from dietmar vestweber (max - planck - institute for molecular biomedicine). Secondary antibodies were alexa-488conjugated goat anti - rabbit (a11008; thermo fisher scientific, grand island, ny, usa), alexa-488conjugated goat anti - mouse (115 - 545 - 166, minimal cross - reaction; jackson immunoresearch, west grove, pa, usa), alexa-647conjugated goat anti - rat (112 - 605 - 167, minimal cross - reaction; jackson immunoresearch), and alexa-647conjugated goat anti - mouse igg (a21236; thermo fisher scientific). Rhodamine - phalloidin (r415, thermo fisher scientific) was used to stain f - actin, and hoechst 33258 (b2883; sigma - aldrich corp .) Three - month - old wild - type lenses were prepared for scanning electron microscopy (sem) as previously described . Briefly, lenses were removed from enucleated mouse eyes and immersed in 2.5% glutaraldehyde 2% formaldehyde in 0.1 m sodium cacodylate buffer at room temperature for 30 minutes to 1 hour . After critical point drying, lenses were split into quarters along the anterior posterior axis, yielding a freshly fractured surface that revealed a surface spanning a complete equatorial lens radius . Imaging was conducted with a phillips xl30 tmp scanning electron microscope (fei company, eindhoven, the netherlands). To observe interlocking protrusions and paddles along the short sides of fiber cells, freshly isolated lenses from 2-month - old tmod1 and tmod1 mice were fixed in 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer, ph 7.3, at room temperature for 48 to 72 hours . Each lens was properly oriented and fractured in an anterior posterior orientation with a sharp razor blade to expose the longitudinal features of the fiber cell short sides along the anterior, equatorial, and posterior regions of the lens . Lens halves were then postfixed in 1% aqueous oso4 for 1 hour at room temperature, dehydrated in graded ethanol, and dried in a samdri-795 critical point dryer (tousimis, inc ., rockville, md, usa). Lens halves were mounted on specimen stubs and coated with gold / palladium in a hummer 6.2 sputter coater (anatech, inc . Micrographs were taken with a jeol 820 scanning electron microscope at 10 kv (jeol, tokyo, japan). The lens nucleus was used as a reference to determine the location of images taken and to ensure that images were from comparable areas of different lenses (i.e., comparable regions were located based on measurements from the center outward). Freshly enucleated eyes were collected from 6-week - old mice . A small opening made at the corneal scleral junction facilitated fixative penetration . After fixation, samples were washed in pbs, cryoprotected in 30% sucrose, frozen in optimal cutting temperature (oct) medium (sakura finetek, torrance, ca, usa), and stored at 80c until sectioning . Frozen sections (12 m thick) were collected with a leica cm1950 cryostat (wetzlar, germany). Prolong gold antifade reagent (thermo fisher scientific) was used to mount the slides, and images were collected using a zeiss lsm780 confocal microscope (oberkochen, germany). Frozen sections with cross - sectionally oriented fiber cells near the lens equator were identified based on the thickness of the lens epithelium . Staining was repeated on three samples from different mice for each genotype, and representative data are shown . After fixation, lenses were cut into quarters using a sharp scalpel along the anterior posterior axis . Lens quarters were postfixed in 1% paraformaldehyde in pbs for 15 minutes at room temperature and then washed two times briefly with pbs . Lens quarters were then permeabilized and blocked using 3% normal goat serum, 3% bovine serum albumin, and 0.3% triton x-100 in pbs for 1 hour at room temperature . After blocking, then, samples were washed three times, 5 minutes per wash, with pbs . Lens quarters were incubated in secondary antibodies diluted in blocking solution for 3 hours at room temperature, followed by washing (four times, 5 minutes per wash) with pbs . Images and z - stacks with 0.2-m steps were collected of single fiber cells or small bundles of fibers from lens quarters using a zeiss lsm780 confocal microscope . Fiber cell morphology and cell location in relation to the bulk lens quarter were used to approximate the maturity of imaged fiber cells . Staining was repeated on at least three lenses from three different mice for each genotype, and representative data are shown . Z - stack confocal images were analyzed using volocity 6.3 (perkinelmer, waltham, wa, usa). Noise reduction using the fine filter was applied to all z - stacks in all channels . The extended focus function in volocity was used to flatten z - stacks into a single image . For fluorescence intensity heat maps, single optical sections from z - stacks were separated into individual channels (16-bit grayscale images), and then heat maps were generated in imagej using the heatmap histogram plug - in (http://www.samuelpean.com/heatmap-histogram/ [in the public domain]). For tortuosity and cell neck width measurements, we evaluated single optical sections of single fibers stained with rhodamine - phalloidin to outline cell contours from 9 tmod1 and 9 tmod1 mice . Using imagej, we located the fiber cell edges using the find edges function and used the wand tool (mode: legacy; tolerance: 5.0) to measure the length of one of the curved edges of each fiber cell in pixels . Tortuosity was calculated as the contour length of the cell edge divided by the straight end - to - end distance along the fiber measured through the center of each fiber . Cell neck width across each fiber at the concave region was measured using the line tool . Three measurements of cell neck width were averaged for each fiber cell . The average, standard error, and statistical significance were calculated and plotted in excel (microsoft, redmond, wa, usa). Statistical significance was determined using the student's t - test, and p values less than 0.01 were considered statistically significant . Classic electron microscopy studies have described the complex interface between differentiating and maturing lens fiber cells characterized by specialized membrane interdigitations, which is shown here by sem (figs . Are straight with small protrusions along the short sides and balls and sockets in the middle of the broad sides (fig . 1b, cells viewed from the short - side edge). As fiber cells differentiate, increased small protrusions appear along their short sides (fig . 1c, top - down view of the broad sides), with mature fiber cells developing elaborate large interlocking paddles decorated with small protrusions (fig . 1d, top - down view of the broad sides). In small bundles of tmod1 lens fibers fixed and stained with phalloidin, we observed that f - actin is enriched at the interfaces between cortical (fig . F - actin appears along the membrane of fiber cells and is present in regions of large paddles as well as small protrusions, outlining cell contours, but the precise location of f - actin with respect to the paddles or protrusions is unclear due to the complex geometry of the membrane interfaces . For clarity, a cartoon of mature fiber cells indicating the morphologic features of mature fiber cells (large paddles, small protrusions, valley between large paddles and base of small protrusions) that will be discussed below is shown in figure 1h . (a d) scanning electron microscopy (sem) at various depths in 3-month - old wild - type (wt) lenses . Boxed regions in (a) indicate the approximate location where (b d) higher - magnification images were obtained . (b d) cortical newly formed fiber cells (b) are straight, with balls and sockets along the broad sides and small protrusions along the vertices . As fiber cells differentiate (c), the cells remain straight and more small protrusions are formed along the cell vertices . Mature lens fibers (d) form large interlocking paddle domains decorated by small protrusions along the cell vertices . Single fibers are highlighted in green to show the changes in cell morphology as the fibers mature . Note that (d) is a different orientation of the same fiber cell shown in figure 3b of blankenship et al . (e g) confocal fluorescence microscopy of phalloidin staining of fiber cells in 6-week - old tmod1 lens fiber cell bundles (located at depths comparable to those in [b d]) reveals that f - actin is enriched in large paddle domains and small interlocking protrusions at the vertices of fiber cells . (h) diagram (not to scale) of mature lens fiber cells with large paddles (light blue shading), valleys between large paddles (red lines), small protrusions (green shading), and bases of small protrusions (yellow lines) along the short sides of the cell . Scale bars: 50 m (a); 6 m (b d); 4 m (e g). To better visualize individual fiber cell morphologies and f - actin organization at different stages of maturation located at different depths in the lens mouse lenses were microdissected, decapsulated, and fixed overnight at 4c; and after fixation, lenses were cut into quarters and postfixed before immunostaining . Single fiber cells as well as fiber cell bundles spontaneously detached from the bulk lens quarters during the immunostaining process, and segments up to 200 m long could be imaged by confocal microscopy . From our preparation, we observed three types of fiber cells: tightly apposed fiber cell bundles (fig . 2), and single lens fibers that are detached from neighboring cells (figs . Immunostaining of three neighboring fiber cell segments showed that f - actin is enriched in protrusions and paddles, and that the complex morphology of this ingenious 3d zipper is well preserved in these partially separated fibers (fig . Three - dimensional reconstruction of the lens fibers shows interlocking paddles and protrusions between adjacent cells (fig . While difficult to visualize in these images, each fiber cell has three sets of coordinated paddles and protrusions along the short sides, corresponding to the three vertices located on the short sides . Two - dimensional (2d) projections of single xz planes reveal normal hexagonal fiber cell shapes with enriched f - actin along the short sides near vertices, and less intense f - actin staining along the broad sides (figs . 2c) shows smooth f - actin staining along the cell membrane, while the xz projection through a region with paddles (fig . 2d) reveals f - actin rich regions of paddles and protrusions at their vertices (red asterisk). This is analogous to the view seen in a typical equatorial cross section through mature lens fibers that have undergone denucleation, 150 to 200 m from the lens capsule (fig . 2e, hexagonal cell body outlined with blue dashed lines and paddles and protrusions indicated by a red asterisk). An extended focus image of the flattened z - stack of the individual fiber cells clearly demonstrates the interlocking paddles and protrusions between adjacent cells (fig . 2 g, 2h) reveal slight gaps between the interlocking domains of adjacent cells (green and white cells in fig . 2 g, pink and white cells in fig . In favorable views of a fiber cell edge, f - actin appears more highly enriched in the small protrusions compared to paddles (fig . Note that due to the 3d nature of these complex interdigitations, single optical planes do not show all of the protrusions and paddles along the short sides of each cell . Our results indicate that our new immunostaining method for detached single fibers faithfully preserves fiber cell morphology observed in situ by sem (fig . 1), thus allowing detailed studies of the proteins required for the formation and/or stability of protrusions and paddles along fiber cell vertices . Confocal fluorescence microscopy images (2d single optical planes, extended focus flattened z - stack, 3d reconstruction and cross section) of f - actin in neighboring mature fiber cells in 6-week - old tmod1 lenses . 3d reconstruction of z - stacks through three neighboring fibers shows interlocking structures between adjacent cells (a) and coordinated paddle domains between cells of neighboring layers (b) (red arrows). Dashed boxes show the level where single xz planes in (c, d) are derived . A single 2d xz plane from the 3d reconstruction shows normal hexagonal fiber cell morphology (c, d) (hexagonal cell body outlined with blue dashed lines) with paddles and protrusions along the short side (d) (red asterisk). The f - actin staining pattern in dissociated fiber cells is similar to what is typically observed in cross sections through hexagonal mature lens fibers that have undergone denucleation (e) (hexagonal cell body outlined with blue dashed lines), demonstrating enriched f - actin along the short sides of these fibers and in regions of paddles and protrusions (red asterisk) with less intense f - actin signals along the broad sides . Extended focus image of the flattened z - stack shows f - actin rich interlocking paddles decorated by small protrusions along the short sides of the cells, creating a 3d zipper between adjacent cells (f). Single xy optical sections along the anterior posterior axis through the fiber cells (g, h), where the dashed lines are drawn through the xz plane in (c) show interlocking domains between fibers with some separation between the cells . (2d, single optical plane) of f - actin in fiber cells at various depths and sem in 6-week - old (a) and 2-month - old (c) tmod1 and tmod1 lenses . (a) diagrams of normal cortical, differentiating, and mature fiber cells are shown along the top . Tmod1 and tmod1 cortical and differentiating fiber cells are straight with small f - actin protrusions along their vertices . Tmod1 mature fibers have large paddle domains (asterisks) with f - actin rich small protrusions, while tmod1 fiber cells have f - actin positive protrusions, but very few paddles . (b) tmod1 mature fiber cells display a significant decrease in tortuosity (n = 9, * p <0.01), but the cell neck width was unaffected (double - headed arrows in [a]) between tmod1 and tmod1 lenses (n = 9). (c) low - magnification sem with boxed regions indicating the location of higher - magnification images . Comparable regions are identified by measuring outward from the nucleus at the center of the lens, since some peripheral cortical fibers are lost in the sample preparation procedure . Tmod1 lenses have rows of mature fiber cells with coordinated paddle protrusions that are decorated by smaller protrusions of equal size and spacing . Scale bars: 4 m (a); 1 mm (c) (low magnification left); 4 m (c) (high magnification). To investigate whether the tmod1-stablized actin cytoskeleton plays a role in the formation or stabilization of fiber cell interdigitations, we compared fiber cell morphologies and f - actin organization in single fiber cells from tmod1 and tmod1 lenses . We distinguished cortical, differentiating, and mature fiber segments by their unique morphologies (diagram in fig . 3a) and by observing the location of the single fiber with respect to depth in the bulk lens quarter . Single optical sections reveal that f - actin is highly enriched at the fiber cell membrane and in small protrusions in all fiber cells from both tmod1 and tmod1 lenses, including cortical, differentiating, and mature single fibers (fig . Tmod1 and tmod1 cortical and differentiating fibers are straight with numerous small protrusions along their vertices, and no obvious differences in f - actin or in cell morphologies are observed . Protrusions along cortical fibers appear finger - like, while protrusions along differentiating fibers have more obvious head and neck regions (fig . The tmod1 mature fiber cell, which has undergone denucleation (150250 m deep from the lens capsule), displays large paddle domains with small protrusions (fig . Unlike cortical and differentiating fibers, which are not affected by absence of tmod1, the tmod1 mature fiber cell lacks normal paddles despite retaining an overall undulating shape with abundant f - actin rich small protrusions . A useful metric to compare paddle formation between control and knockout mature fiber cells is the contour length of the fiber cell edge, or tortuosity, measured along the paddle contours relative to the overall fiber cell length . Indeed, tmod1 mature fiber cells had a statistically significant decrease in tortuosity compared to tmod1 mature fiber cells, consistent with a partial loss of large paddles (fig . 3b) to ensure that cells from a similar depth were being compared between tmod1 and tmod1 lenses and showed that there was no difference in the cell neck width . To validate the observations based on single fiber cell immunostaining, we visualized fiber cell edges by classic sem of half lenses to determine whether the loss of tmod1 affects lens fiber cell interdigitations and protrusion morphologies . This showed that tmod1 mature lens fibers are characterized by coordinated large paddle domains decorated by evenly spaced and sized small protrusions (0.51.5 m), as expected (fig . 3c). Similar to immunostained single fiber cells, the tmod1 mature lens fibers appear to have reduced paddles with irregularly sized and arranged smaller protrusions (fig . 3c), although the shapes of whole individual fiber cells are difficult to assess in these sem preparations . The ability to immunostain and visualize individual mature lens fiber cells provides an opportunity to investigate the actin cytoskeleton composition of protrusions versus paddles, and how loss of tmod1 and perturbations of actin - associated proteins could account for defective paddles but not small protrusions in tmod1 mature fiber cells . We hypothesized that tmod1 and a subset of actin - binding proteins (abps) would be associated with paddles but not small protrusions, and that loss of tmod1 would affect paddle - associated abps but not protrusion - associated abps, illuminating mechanisms underlying paddle formation . First, we investigated locations of tmod1 and 2-spectrin in tmod1 mature lens fiber cells as compared to tmod1 mature lens fibers by immunostaining and confocal microscopy . Z - stacks collected through single fibers were flattened to view and compare the morphology of tmod1 versus tmod1 cells (fig ., we observed that both tmod1 and 2-spectrin were excluded from the small protrusions and instead tended to be located along the contours of the paddle domains, where both tmod1 and 2-spectrin staining signals appeared in large bright puncta . We also observed small bright 2-spectrin puncta along the membrane of the broad sides (fig . 4a, center of cell), consistent with lens cross - section staining data in our previous study . In single optical sections through the cytoplasm of a tmod1 mature fiber, bright tmod1 and 2-spectrin puncta are located mainly in valleys between large paddle domains along the cell edges (i.e., regions of concave curvature) (figs . 4b, 4d, arrows). In contrast, in the tmod1 mature fiber cell with aberrant and attenuated paddle contours, the bright 2-spectrin puncta along cell edges are reduced in number and now mostly dispersed in the fiber cell cytoplasm, as seen in single optical sections through the middle of the cell (fig . A heat map of 2-spectrin fluorescence intensity shows increased cytoplasmic staining signals in the knockout mature fiber cell (fig . However, there were no obvious differences in the punctate 2-spectrin staining pattern along the broad sides of tmod1 cells as compared to tmod1 cells (fig . 2-spectrin puncta are also occasionally observed at the base of the f - actin rich small protrusions along the short sides (at the vertices) of the tmod1 and tmod1 fibers (figs . No changes in overall f - actin staining intensity or distribution were observed, mostly likely because absence of tmod1 does not affect morphology of small protrusions where most of the f - actin is located (fig . The presence of both tmod1 and 2-spectrin puncta in valleys between large paddles in tmod1 fiber cells supports the hypothesis that tmod1 plays a role in the formation or maintenance of large paddle domains by stabilizing a spectrin actin network normally located in these domains in mature fiber cells . Moreover, alterations in 2-spectrin puncta locations and distributions in tmod1 mature fibers are consistent with our previous results showing that the loss of tmod1 perturbs the spectrin network in lens fiber cells . Immunostaining of single mature fiber cells from 6-week - old tmod1 and tmod1 lenses for tmod1 (green), f - actin (red), and 2-spectrin (blue). The tmod1 fiber has f - actin rich large paddle domains and small protrusions along cell vertices . While the tmod1 fiber has very few paddles, f - actin rich small protrusions are still present . (b, d) single optical section (2d) from a z - stack, showing a section through the cytoplasm of the fiber cells, with enlargements . Tmod1 and 2-spectrin are enriched in puncta near the cell membrane in valleys between large paddle domains in the tmod1 fiber (arrows), and 2-spectrin is also enriched at the base of small protrusions in tmod1 and tmod1 fibers (arrowheads). The 2-spectrin staining signal appears diffuse and cytoplasmic (asterisks) with fewer membrane - associated puncta in the tmod1 fiber . (c) fluorescence intensity heat maps of 2-spectrin staining in tmod1 and tmod1 lens fibers show that 2-spectrin staining is more cytoplasmic (asterisks) in the tmod1 fiber . Scale bars: 4 m (a c); 2 m (d). Our new data show that tmod1 controls the morphology of large paddle domains in mature lens fibers, via stabilization of 2-spectrin associations at the base of paddle domains . These domains form the vertices between mature fiber cells visualized in lens cross sections and are associated with other abps, such as -actinin, arp3, and ezrin . Therefore, we investigated whether these abps are located in the small protrusions or large paddles and whether they are disrupted in tmod1 fibers . In mouse lenses, nonmuscle -actinin (actn1), a crosslinking protein for antiparallel actin filaments, has little or no staining signal in the cortical differentiating fibers, but is enriched at the short sides of mature fibers in a punctate pattern (fig . Bright tmod1 and -actinin puncta are colocalized in valleys between large paddles in the mature tmod1 lens fiber (figs . 5b, 5c, arrows), while in the absence of tmod1, -actinin is distributed discontinuously along the entire cell membrane, even in regions with the abnormal and attenuated paddles . Careful examination of the -actinin f - actin merged image reveals that the -actinin staining is now located at the bases of all the small protrusions (fig . We performed western blotting on soluble and insoluble lens protein samples to determine whether there was an increase in -actinin protein levels in knockout lenses . We found no change in the amount of -actinin in tmod1 lenses compared to the control (data not shown). These data suggest that in control fiber cells, tmod1 may restrict assembly of -actinin f - actin antiparallel bundles to the valleys between large paddles, promoting paddle morphogenesis . By contrast, in the absence of tmod1, there may be a global rearrangement of the f - actin, with increased -actinin on the membrane . (a) immunostaining of frozen lens section from 6-week - old tmod1 mice for -actinin (green) and f - actin (red). The staining signal is enriched on the short sides of mature fiber cells with a punctate pattern . (b, c) immunostaining of single mature fiber cells (2d, single optical plane) from 6-week - old tmod1 and tmod1 lenses for tmod1 (green), f - actin (red), and -actinin (blue). Similar to tmod1 and 2-spectrin, -actinin is enriched in large puncta in valleys between large paddle domains in the tmod1 fiber (arrows in [b, c]). In the tmod1 fiber enlargement shows that -actinin is enriched near the base of small protrusions in the tmod1 mature fiber cell (arrowheads in [c]). Scale bars: 20 m (a); 4 m (b); 2 m (c). Next, we immunostained lenses for ezrin, which links f - actin networks to the plasma membrane and fimbrin (aka plastin), a small crosslinker that bundles f - actin in a parallel orientation . Similar to previous reports, ezrin was detected only in lens fibers, where it was enriched on the short sides of cortical fibers, especially at vertices, with more uniform membrane staining in mature lens fibers as visualized in cross sections (fig . Cross sections, fimbrin was present in epithelial cells and was also associated with the membranes of both differentiating and mature fiber cells (fig . Ezrin staining was continuous and colocalized with f - actin along the cell membrane in both tmod1 and tmod1 mature lens fibers (fig . Ezrin also appeared to be enriched at the base of small protrusions as well as in the valleys between large paddles (fig . Fimbrin was restricted to puncta at the base of small protrusions in both control and knockout cells (figs . 6c, 6d, arrows) and was not enriched in the valleys between large paddles, unlike tmod1, 2-spectrin, and -actinin . Neither ezrin nor fimbrin displayed any significant changes in the absence of tmod1, suggesting that these f - actin networks are independent of tmod1 . (a) immunostaining of frozen lens section from 6-week - old tmod1 mice for ezrin (green) and f - actin (red). Ezrin is present along the membranes of lens fiber cells and enriched at the vertices of cortical fibers and along the short sides of mature fiber cells . (b) immunostaining of frozen lens section from 6-week - old tmod1 mice for fimbrin (green) and f - actin (red). (c, d) immunostaining of single mature fiber cells (2d, single optical plane) from 6-week - old tmod1 and tmod1 lenses for fimbrin (green), f - actin (red), and ezrin (blue). As expected, ezrin colocalizes with f - actin along the cell membrane and in interdigitations of tmod1 and tmod1 fibers . Interestingly, fimbrin is enriched in puncta near the base of small interlocking protrusions in tmod1 and tmod1 fibers (arrows in [c, d]). Enlargements show ezrin enrichment (arrowheads in [d]) at the base of small protrusions . Scale bars: 20 m (a, b); 4 m (c); 2 m (d). Immunostaining of single mature fiber cells (2d, single optical plane) from 6-week - old tmod1 and tmod1 lenses for arp3 (green), f - actin (red), and n - cadherin (blue). (a) n - cadherin is localized along the cell membrane in tmod1 and tmod1 fibers and appears enriched along the base of protrusions and valleys between large paddles . Arp3 is enriched in puncta (arrows) near the base of small interlocking protrusions (arrows) in tmod1 and tmod1 fibers . Arp3 is also found in valleys between large paddles in the tmod1 fiber (arrowheads). (b) enlargements show that weak arp3 staining extends into small protrusions (open triangles) in tmod1 and tmod1 fibers . Arp3 puncta at the base of small protrusions are often accompanied by enriched n - cadherin staining (arrows). Previous work hypothesized that arp2/3 may drive actin filament branching to push protrusions out of fiber cell membranes, while a pulling force on the membrane may be mediated by clathrin complexes along the membrane of the neighboring fiber . Arp2/3 initiates branched actin filament assembly from preexisting filaments, and activation of arp2/3 by wasp / wave family members and cortactin drives cadherin - directed actin assembly . We previously demonstrated that arp3 was enriched at the vertices of cortical fiber cells visualized in cross sections where it colocalized with n - cadherin . However, arp3 localization was not investigated in mature fibers, and thus we localized arp3 and n - cadherin in single mature fibers . Arp3 was enriched in puncta at the base of the small f - actin rich protrusions in both control and knockout mature fibers (fig . 7, arrows), and arp3 puncta were also found in valleys between large paddles in the tmod1 fiber (fig . Low - intensity arp3 staining can also be observed to extend into and fill small protrusions in both control and knockout fibers (fig . 7b, open triangles). N - cadherin staining signal was predominantly at the cell membrane with a discontinuous pattern in control and knockout cells (figs . 7a, 7b). This staining pattern is consistent with a recent study showing n - cadherin staining at the membranes in lens fiber cell bundles . We also observed that arp3 puncta at the base of small protrusions often coincide with areas of enriched n - cadherin staining (fig . These observations would be consistent with the original hypothesis that arp3 and n - cadherin play a role in assembly of the small f - actin rich protrusions in fiber cells . However, since loss of tmod1 does not greatly affect formation of small f - actin protrusions (figs . 37) nor alter arp3 or n - cadherin localization, fiber cell f - actin networks regulated by arp3 or n - cadherin do not appear to be associated with the tmod1-regulated f - actin networks that control paddle domain morphology . Classic electron microscopy studies have described the complex interface between differentiating and maturing lens fiber cells characterized by specialized membrane interdigitations, which is shown here by sem (figs . Are straight with small protrusions along the short sides and balls and sockets in the middle of the broad sides (fig . 1b, cells viewed from the short - side edge). As fiber cells differentiate, increased small protrusions appear along their short sides (fig . 1c, top - down view of the broad sides), with mature fiber cells developing elaborate large interlocking paddles decorated with small protrusions (fig . 1d, top - down view of the broad sides). In small bundles of tmod1 lens fibers fixed and stained with phalloidin, we observed that f - actin is enriched at the interfaces between cortical (fig . F - actin appears along the membrane of fiber cells and is present in regions of large paddles as well as small protrusions, outlining cell contours, but the precise location of f - actin with respect to the paddles or protrusions is unclear due to the complex geometry of the membrane interfaces . For clarity, a cartoon of mature fiber cells indicating the morphologic features of mature fiber cells (large paddles, small protrusions, valley between large paddles and base of small protrusions) that will be discussed below is shown in figure 1h . (a d) scanning electron microscopy (sem) at various depths in 3-month - old wild - type (wt) lenses . Boxed regions in (a) indicate the approximate location where (b d) higher - magnification images were obtained . (b d) cortical newly formed fiber cells (b) are straight, with balls and sockets along the broad sides and small protrusions along the vertices . As fiber cells differentiate (c), the cells remain straight and more small protrusions are formed along the cell vertices . Mature lens fibers (d) form large interlocking paddle domains decorated by small protrusions along the cell vertices . Single fibers are highlighted in green to show the changes in cell morphology as the fibers mature . Note that (d) is a different orientation of the same fiber cell shown in figure 3b of blankenship et al . (e g) confocal fluorescence microscopy of phalloidin staining of fiber cells in 6-week - old tmod1 lens fiber cell bundles (located at depths comparable to those in [b d]) reveals that f - actin is enriched in large paddle domains and small interlocking protrusions at the vertices of fiber cells . (h) diagram (not to scale) of mature lens fiber cells with large paddles (light blue shading), valleys between large paddles (red lines), small protrusions (green shading), and bases of small protrusions (yellow lines) along the short sides of the cell . Scale bars: 50 m (a); 6 m (b d); 4 m (e g). To better visualize individual fiber cell morphologies and f - actin organization at different stages of maturation located at different depths in the lens mouse lenses were microdissected, decapsulated, and fixed overnight at 4c; and after fixation, lenses were cut into quarters and postfixed before immunostaining . Single fiber cells as well as fiber cell bundles spontaneously detached from the bulk lens quarters during the immunostaining process, and segments up to 200 m long could be imaged by confocal microscopy . From our preparation, we observed three types of fiber cells: tightly apposed fiber cell bundles (fig . 2), and single lens fibers that are detached from neighboring cells (figs . Immunostaining of three neighboring fiber cell segments showed that f - actin is enriched in protrusions and paddles, and that the complex morphology of this ingenious 3d zipper is well preserved in these partially separated fibers (fig . Three - dimensional reconstruction of the lens fibers shows interlocking paddles and protrusions between adjacent cells (fig . While difficult to visualize in these images, each fiber cell has three sets of coordinated paddles and protrusions along the short sides, corresponding to the three vertices located on the short sides . Two - dimensional (2d) projections of single xz planes reveal normal hexagonal fiber cell shapes with enriched f - actin along the short sides near vertices, and less intense f - actin staining along the broad sides (figs . 2c) shows smooth f - actin staining along the cell membrane, while the xz projection through a region with paddles (fig . 2d) reveals f - actin rich regions of paddles and protrusions at their vertices (red asterisk). This is analogous to the view seen in a typical equatorial cross section through mature lens fibers that have undergone denucleation, 150 to 200 m from the lens capsule (fig . 2e, hexagonal cell body outlined with blue dashed lines and paddles and protrusions indicated by a red asterisk). An extended focus image of the flattened z - stack of the individual fiber cells clearly demonstrates the interlocking paddles and protrusions between adjacent cells (fig . 2 g, 2h) reveal slight gaps between the interlocking domains of adjacent cells (green and white cells in fig . 2 g, pink and white cells in fig . In favorable views of a fiber cell edge, f - actin appears more highly enriched in the small protrusions compared to paddles (fig . Note that due to the 3d nature of these complex interdigitations, single optical planes do not show all of the protrusions and paddles along the short sides of each cell . Our results indicate that our new immunostaining method for detached single fibers faithfully preserves fiber cell morphology observed in situ by sem (fig . 1), thus allowing detailed studies of the proteins required for the formation and/or stability of protrusions and paddles along fiber cell vertices . Confocal fluorescence microscopy images (2d single optical planes, extended focus flattened z - stack, 3d reconstruction and cross section) of f - actin in neighboring mature fiber cells in 6-week - old tmod1 lenses . 3d reconstruction of z - stacks through three neighboring fibers shows interlocking structures between adjacent cells (a) and coordinated paddle domains between cells of neighboring layers (b) (red arrows). Dashed boxes show the level where single xz planes in (c, d) are derived . A single 2d xz plane from the 3d reconstruction shows normal hexagonal fiber cell morphology (c, d) (hexagonal cell body outlined with blue dashed lines) with paddles and protrusions along the short side (d) (red asterisk). The f - actin staining pattern in dissociated fiber cells is similar to what is typically observed in cross sections through hexagonal mature lens fibers that have undergone denucleation (e) (hexagonal cell body outlined with blue dashed lines), demonstrating enriched f - actin along the short sides of these fibers and in regions of paddles and protrusions (red asterisk) with less intense f - actin signals along the broad sides . Extended focus image of the flattened z - stack shows f - actin rich interlocking paddles decorated by small protrusions along the short sides of the cells, creating a 3d zipper between adjacent cells (f). Single xy optical sections along the anterior posterior axis through the fiber cells (g, h), where the dashed lines are drawn through the xz plane in (c) show interlocking domains between fibers with some separation between the cells . (2d, single optical plane) of f - actin in fiber cells at various depths and sem in 6-week - old (a) and 2-month - old (c) tmod1 and tmod1 lenses . (a) diagrams of normal cortical, differentiating, and mature fiber cells are shown along the top . Tmod1 and tmod1 cortical and differentiating fiber cells are straight with small f - actin protrusions along their vertices . Tmod1 mature fibers have large paddle domains (asterisks) with f - actin rich small protrusions, while tmod1 fiber cells have f - actin positive protrusions, but very few paddles . (b) tmod1 mature fiber cells display a significant decrease in tortuosity (n = 9, * p <0.01), but the cell neck width was unaffected (double - headed arrows in [a]) between tmod1 and tmod1 lenses (n = 9). (c) low - magnification sem with boxed regions indicating the location of higher - magnification images . Comparable regions are identified by measuring outward from the nucleus at the center of the lens, since some peripheral cortical fibers are lost in the sample preparation procedure . Tmod1 lenses have rows of mature fiber cells with coordinated paddle protrusions that are decorated by smaller protrusions of equal size and spacing . Scale bars: 4 m (a); 1 mm (c) (low magnification left); 4 m (c) (high magnification). To investigate whether the tmod1-stablized actin cytoskeleton plays a role in the formation or stabilization of fiber cell interdigitations, we compared fiber cell morphologies and f - actin organization in single fiber cells from tmod1 and tmod1 lenses . We distinguished cortical, differentiating, and mature fiber segments by their unique morphologies (diagram in fig . 3a) and by observing the location of the single fiber with respect to depth in the bulk lens quarter . Single optical sections reveal that f - actin is highly enriched at the fiber cell membrane and in small protrusions in all fiber cells from both tmod1 and tmod1 lenses, including cortical, differentiating, and mature single fibers (fig . Tmod1 and tmod1 cortical and differentiating fibers are straight with numerous small protrusions along their vertices, and no obvious differences in f - actin or in cell morphologies are observed . Protrusions along cortical fibers appear finger - like, while protrusions along differentiating fibers have more obvious head and neck regions (fig . The tmod1 mature fiber cell, which has undergone denucleation (150250 m deep from the lens capsule), displays large paddle domains with small protrusions (fig . Unlike cortical and differentiating fibers, which are not affected by absence of tmod1, the tmod1 mature fiber cell lacks normal paddles despite retaining an overall undulating shape with abundant f - actin rich small protrusions . A useful metric to compare paddle formation between control and knockout mature fiber cells is the contour length of the fiber cell edge, or tortuosity, measured along the paddle contours relative to the overall fiber cell length . Indeed, tmod1 mature fiber cells had a statistically significant decrease in tortuosity compared to tmod1 mature fiber cells, consistent with a partial loss of large paddles (fig . 3b) to ensure that cells from a similar depth were being compared between tmod1 and tmod1 lenses and showed that there was no difference in the cell neck width . To validate the observations based on single fiber cell immunostaining, we visualized fiber cell edges by classic sem of half lenses to determine whether the loss of tmod1 affects lens fiber cell interdigitations and protrusion morphologies . This showed that tmod1 mature lens fibers are characterized by coordinated large paddle domains decorated by evenly spaced and sized small protrusions (0.51.5 m), as expected (fig . 3c). Similar to immunostained single fiber cells, the tmod1 mature lens fibers appear to have reduced paddles with irregularly sized and arranged smaller protrusions (fig . 3c), although the shapes of whole individual fiber cells are difficult to assess in these sem preparations . The ability to immunostain and visualize individual mature lens fiber cells provides an opportunity to investigate the actin cytoskeleton composition of protrusions versus paddles, and how loss of tmod1 and perturbations of actin - associated proteins could account for defective paddles but not small protrusions in tmod1 mature fiber cells . We hypothesized that tmod1 and a subset of actin - binding proteins (abps) would be associated with paddles but not small protrusions, and that loss of tmod1 would affect paddle - associated abps but not protrusion - associated abps, illuminating mechanisms underlying paddle formation . First, we investigated locations of tmod1 and 2-spectrin in tmod1 mature lens fiber cells as compared to tmod1 mature lens fibers by immunostaining and confocal microscopy . Z - stacks collected through single fibers were flattened to view and compare the morphology of tmod1 versus tmod1 cells (fig ., we observed that both tmod1 and 2-spectrin were excluded from the small protrusions and instead tended to be located along the contours of the paddle domains, where both tmod1 and 2-spectrin staining signals appeared in large bright puncta . We also observed small bright 2-spectrin puncta along the membrane of the broad sides (fig . 4a, center of cell), consistent with lens cross - section staining data in our previous study . In single optical sections through the cytoplasm of a tmod1 mature fiber, bright tmod1 and 2-spectrin puncta are located mainly in valleys between large paddle domains along the cell edges (i.e., regions of concave curvature) (figs . 4b, 4d, arrows). In contrast, in the tmod1 mature fiber cell with aberrant and attenuated paddle contours, the bright 2-spectrin puncta along cell edges are reduced in number and now mostly dispersed in the fiber cell cytoplasm, as seen in single optical sections through the middle of the cell (fig . A heat map of 2-spectrin fluorescence intensity shows increased cytoplasmic staining signals in the knockout mature fiber cell (fig . However, there were no obvious differences in the punctate 2-spectrin staining pattern along the broad sides of tmod1 cells as compared to tmod1 cells (fig . 2-spectrin puncta are also occasionally observed at the base of the f - actin rich small protrusions along the short sides (at the vertices) of the tmod1 and tmod1 fibers (figs . No changes in overall f - actin staining intensity or distribution were observed, mostly likely because absence of tmod1 does not affect morphology of small protrusions where most of the f - actin is located (fig . The presence of both tmod1 and 2-spectrin puncta in valleys between large paddles in tmod1 fiber cells supports the hypothesis that tmod1 plays a role in the formation or maintenance of large paddle domains by stabilizing a spectrin actin network normally located in these domains in mature fiber cells . Moreover, alterations in 2-spectrin puncta locations and distributions in tmod1 mature fibers are consistent with our previous results showing that the loss of tmod1 perturbs the spectrin network in lens fiber cells . Immunostaining of single mature fiber cells from 6-week - old tmod1 and tmod1 lenses for tmod1 (green), f - actin (red), and 2-spectrin (blue). The tmod1 fiber has f - actin rich large paddle domains and small protrusions along cell vertices . While the tmod1 fiber has very few paddles, f - actin rich small protrusions are still present . (b, d) single optical section (2d) from a z - stack, showing a section through the cytoplasm of the fiber cells, with enlargements . Tmod1 and 2-spectrin are enriched in puncta near the cell membrane in valleys between large paddle domains in the tmod1 fiber (arrows), and 2-spectrin is also enriched at the base of small protrusions in tmod1 and tmod1 fibers (arrowheads). The 2-spectrin staining signal appears diffuse and cytoplasmic (asterisks) with fewer membrane - associated puncta in the tmod1 fiber . (c) fluorescence intensity heat maps of 2-spectrin staining in tmod1 and tmod1 lens fibers show that 2-spectrin staining is more cytoplasmic (asterisks) in the tmod1 fiber . Scale bars: 4 m (a c); 2 m (d). Our new data show that tmod1 controls the morphology of large paddle domains in mature lens fibers, via stabilization of 2-spectrin associations at the base of paddle domains . These domains form the vertices between mature fiber cells visualized in lens cross sections and are associated with other abps, such as -actinin, arp3, and ezrin . Therefore, we investigated whether these abps are located in the small protrusions or large paddles and whether they are disrupted in tmod1 fibers . In mouse lenses, nonmuscle -actinin (actn1), a crosslinking protein for antiparallel actin filaments, has little or no staining signal in the cortical differentiating fibers, but is enriched at the short sides of mature fibers in a punctate pattern (fig . Bright tmod1 and -actinin puncta are colocalized in valleys between large paddles in the mature tmod1 lens fiber (figs . 5b, 5c, arrows), while in the absence of tmod1, -actinin is distributed discontinuously along the entire cell membrane, even in regions with the abnormal and attenuated paddles . Careful examination of the -actinin f - actin merged image reveals that the -actinin staining is now located at the bases of all the small protrusions (fig . We performed western blotting on soluble and insoluble lens protein samples to determine whether there was an increase in -actinin protein levels in knockout lenses . We found no change in the amount of -actinin in tmod1 lenses compared to the control (data not shown). These data suggest that in control fiber cells, tmod1 may restrict assembly of -actinin f - actin antiparallel bundles to the valleys between large paddles, promoting paddle morphogenesis . By contrast, in the absence of tmod1, there may be a global rearrangement of the f - actin, with increased -actinin on the membrane . (a) immunostaining of frozen lens section from 6-week - old tmod1 mice for -actinin (green) and f - actin (red). The staining signal is enriched on the short sides of mature fiber cells with a punctate pattern . (b, c) immunostaining of single mature fiber cells (2d, single optical plane) from 6-week - old tmod1 and tmod1 lenses for tmod1 (green), f - actin (red), and -actinin (blue). Similar to tmod1 and 2-spectrin, -actinin is enriched in large puncta in valleys between large paddle domains in the tmod1 fiber (arrows in [b, c]). In the tmod1 fiber enlargement shows that -actinin is enriched near the base of small protrusions in the tmod1 mature fiber cell (arrowheads in [c]). Scale bars: 20 m (a); 4 m (b); 2 m (c). Next, we immunostained lenses for ezrin, which links f - actin networks to the plasma membrane and fimbrin (aka plastin), a small crosslinker that bundles f - actin in a parallel orientation . Similar to previous reports, ezrin was detected only in lens fibers, where it was enriched on the short sides of cortical fibers, especially at vertices, with more uniform membrane staining in mature lens fibers as visualized in cross sections (fig . Cross sections, fimbrin was present in epithelial cells and was also associated with the membranes of both differentiating and mature fiber cells (fig . Ezrin staining was continuous and colocalized with f - actin along the cell membrane in both tmod1 and tmod1 mature lens fibers (fig . Ezrin also appeared to be enriched at the base of small protrusions as well as in the valleys between large paddles (fig . Fimbrin was restricted to puncta at the base of small protrusions in both control and knockout cells (figs . 6c, 6d, arrows) and was not enriched in the valleys between large paddles, unlike tmod1, 2-spectrin, and -actinin . Neither ezrin nor fimbrin displayed any significant changes in the absence of tmod1, suggesting that these f - actin networks are independent of tmod1 . (a) immunostaining of frozen lens section from 6-week - old tmod1 mice for ezrin (green) and f - actin (red). Ezrin is present along the membranes of lens fiber cells and enriched at the vertices of cortical fibers and along the short sides of mature fiber cells . (b) immunostaining of frozen lens section from 6-week - old tmod1 mice for fimbrin (green) and f - actin (red). (c, d) immunostaining of single mature fiber cells (2d, single optical plane) from 6-week - old tmod1 and tmod1 lenses for fimbrin (green), f - actin (red), and ezrin (blue). As expected, ezrin colocalizes with f - actin along the cell membrane and in interdigitations of tmod1 and tmod1 fibers . Interestingly, fimbrin is enriched in puncta near the base of small interlocking protrusions in tmod1 and tmod1 fibers (arrows in [c, d]). Enlargements show ezrin enrichment (arrowheads in [d]) at the base of small protrusions . Scale bars: 20 m (a, b); 4 m (c); 2 m (d). Immunostaining of single mature fiber cells (2d, single optical plane) from 6-week - old tmod1 and tmod1 lenses for arp3 (green), f - actin (red), and n - cadherin (blue). (a) n - cadherin is localized along the cell membrane in tmod1 and tmod1 fibers and appears enriched along the base of protrusions and valleys between large paddles . Arp3 is enriched in puncta (arrows) near the base of small interlocking protrusions (arrows) in tmod1 and tmod1 fibers . Arp3 is also found in valleys between large paddles in the tmod1 fiber (arrowheads). (b) enlargements show that weak arp3 staining extends into small protrusions (open triangles) in tmod1 and tmod1 fibers . Arp3 puncta at the base of small protrusions are often accompanied by enriched n - cadherin staining (arrows). Previous work hypothesized that arp2/3 may drive actin filament branching to push protrusions out of fiber cell membranes, while a pulling force on the membrane may be mediated by clathrin complexes along the membrane of the neighboring fiber . Arp2/3 initiates branched actin filament assembly from preexisting filaments, and activation of arp2/3 by wasp / wave family members and cortactin drives cadherin - directed actin assembly . We previously demonstrated that arp3 was enriched at the vertices of cortical fiber cells visualized in cross sections where it colocalized with n - cadherin . However, arp3 localization was not investigated in mature fibers, and thus we localized arp3 and n - cadherin in single mature fibers . Arp3 was enriched in puncta at the base of the small f - actin rich protrusions in both control and knockout mature fibers (fig . 7, arrows), and arp3 puncta were also found in valleys between large paddles in the tmod1 fiber (fig . Low - intensity arp3 staining can also be observed to extend into and fill small protrusions in both control and knockout fibers (fig . N - cadherin staining signal was predominantly at the cell membrane with a discontinuous pattern in control and knockout cells (figs . 7a, 7b). This staining pattern is consistent with a recent study showing n - cadherin staining at the membranes in lens fiber cell bundles . We also observed that arp3 puncta at the base of small protrusions often coincide with areas of enriched n - cadherin staining (fig . These observations would be consistent with the original hypothesis that arp3 and n - cadherin play a role in assembly of the small f - actin rich protrusions in fiber cells . However, since loss of tmod1 does not greatly affect formation of small f - actin protrusions (figs . 37) nor alter arp3 or n - cadherin localization, fiber cell f - actin networks regulated by arp3 or n - cadherin do not appear to be associated with the tmod1-regulated f - actin networks that control paddle domain morphology . We have developed a novel technique to immunostain single lens fiber cells while preserving their complex morphology; this new method has allowed us to conduct detailed investigations of the f - actin structures and abps required for the formation of the interdigitations at fiber cell vertices . Our study has shown that tmod1 is required for the formation of large paddles, but not small protrusions, between mature lens fiber cells . These subtle changes in fiber cell morphology are correlated with decreased stiffness in tmod1 lenses at low mechanical loads, providing the first evidence for the hypothesis that fiber cell interdigitations influence lens mechanical integrity . By contrast, the tmod1 lenses with reduced paddle domains between mature lens fibers remain transparent, indicating that formation of large paddles is not required for lens transparency . While recent studies have suggested that aqp0 or ephrin - a5 may be important for normal fiber cell interdigitations, aqp0 and mixed - background efna5 lenses have severe cataract phenotypes with fiber cell degeneration, which complicates interpretations and impedes further studies . Fortunately, the very mild phenotype in tmod1 lenses has given us an opportunity to dissect the mechanisms underlying the formation of small protrusions as compared to large paddles located at the vertices of mature lens fiber cells . (a) during fiber cell maturation, large paddle domains with small interlocking protrusions form along the vertices of hexagonal fiber cells . (b) tmod1 may stabilize the f - actin spectrin network as well as -actinin crosslinked antiparallel f - actin bundles in valleys between large paddles to maintain their structure . Formation of small protrusions may be facilitated by arp3-nucleated actin networks, and fimbrin - crosslinked parallel f - actin bundles at the base of protrusions, while ezrin may stabilize actin networks along the entire fiber cell membrane and n - cadherin promotes cell cell interactions . Our novel immunostaining protocol for single fiber cells has yielded a wealth of new information about the proteins that control f - actin organization in small protrusions and in large paddles at the vertices of lens fiber cells (fig . Actin network clearly play a key role in the formation and/or maintenance of the large paddle domains in mature lens fibers . Abnormal cytosolic localization of 2-spectrin in knockout single fibers extends our previous work showing that loss of tmod1 leads to disruptions in the lens fiber membrane skeleton by providing a molecular mechanism connecting the f - actin cytoskeleton to fiber cell morphology . Unlike loss of tmod1 alone, which leads to selective reduction of large paddle domains, we showed previously that combined loss of tmod1 and cp49, a beaded intermediate filament protein, leads to reduction in small protrusions at the vertices of mature fiber cells, suggesting that both actin and intermediate filaments are needed for normal formation or stabilization of these smaller interdigitations . Interestingly, -actinin is also found in foci in the valleys between large paddles where it colocalizes with tmod1 in mature tmod1 fiber cells, suggesting that tmod1 may cap the pointed ends of actin filaments crosslinked by spectrin and/or -actinin . -actinin belongs to a protein family that includes spectrin, dystrophin, and utrophin, and -actinin crosslinked f - actin bundles can interact with myosin ii to generate contractile forces . It is possible that -actinin bundled f - actin interacts with myosin ii to generate force to push and/or pull the fiber cell membrane to create the large paddles . Strikingly, in tmod1 mature fiber cells, the -actinin staining signal becomes more broadly localized along the fiber cell membrane in between the small protrusions . However, similar to 2-spectrin, we observe changes in the localization of -actinin without changes in the protein level . This implies that -actinin may be restricted to tmod1-capped f - actin in wild - type cells but become aberrantly distributed and abnormally crosslink other f - actin populations in the absence of tmod1 . Since tmod1 lenses have reduced levels of tropomyosin (tm), a stabilizing protein that binds along the side of f - actin and enhances tmod1 capping of actin pointed ends, and tm inhibits -actinin binding to f - actin, additional f - actin binding sites may now be available in knockout lens fibers for -actinin binding . It may also be significant that the localization of -actinin in the tmod1 mature fiber cell resembles that of n - cadherin in the tmod1 mature fiber cell . This suggests the possibility that expansion of -actinin crosslinked f - actin networks at n - cadherin mediated cell cell junctions may partially compensate for loss of spectrin actin networks, perhaps as a mechanical response, albeit being unable to assist fiber cells in forming large paddle domains . Our data in the lens parallel abnormal -actinin and f - actin staining patterns in tmod1 embryonic heart muscle cells that fail to assemble myofibrils . Further studies will be needed to define the interactions between tmod1 and -actinin associated f - actin, as well as the changes in -actinin and actin binding in the absence of tmod1 . This work also provides the first evidence that distinct abps may selectively control the formation of small protrusions as compared to large paddles in lens fiber cells . In addition to intestinal microvilli, fimbrin has been found in protrusions in other cell types, including stereocilia of the inner ear hair cells and microvilli of photoreceptors, taste receptor cells, nasal epithelial cells, and respiratory brush cells . The location of fimbrin in puncta at the base, but not throughout the small f - actin rich protrusions in single mature fiber cells, suggests that these structures are not analogous to the microvilli of other cells, where fimbrin is present along their entire length . Instead, fimbrin crosslinking of f - actin into rigid parallel filament bundles may help in stabilizing the small protrusions at their bases . Our data are also consistent with previous studies showing that the presence of fimbrin and -actinin is mutually exclusive due to the different spacings between f - actins when bundled by fimbrin or -actinin . While fimbrin is found in small puncta at the base of the small protrusions in mature fiber cells, ezrin is associated with f - actin along the entire membrane of mature fiber cells, including along the membranes of small protrusions . Immunoprecipitation experiments have shown that an actin anchorage complex consisting of ezrin periplakin periaxin desmoyokin (eppd) proteins is associated with moesin, spectrin, and plectin in the lens, suggesting an interaction between erm, eppd, and the spectrin actin networks . Decreased actin and spectrin protein levels and fiber cell membrane staining signals are observed in periaxin knockout lenses, further indicating that the eppd complexes may function to stabilize the spectrin our work strengthens the hypothesis that arp2/3 may drive the formation of small protrusions by nucleating branched f - actin networks . We had expected arp3 to be enriched with f - actin within the small protrusions; instead, we found that arp3 is predominantly enriched in bright puncta at the base of small protrusions, although dim, diffuse arp3 staining can be detected extending into small protrusions . This suggests that arp2/3 nucleation of branched f - actin networks occurs predominantly near the cell membrane at the base of protrusions and paddles, and to a lesser extent within the body of the protrusion, as originally proposed . Notably, location of arp3 at the base of small protrusions where n - cadherin is also located implies that arp2/3 may nucleate f - actin in n - cadherin cell contacts, as previously shown for e - cadherin at cell junctions in epithelial cells . Since arp3 staining is comparable between tmod1 and tmod1 mature lens fibers, tmod1 does not control arp2/3-driven f - actin branching, which is thus unlikely to play a role in formation or maintenance of the large paddle contours in mature lens fibers . We observe that tmod1, 2-spectrin, -actinin, fimbrin, and arp3 are localized to small puncta at the base of some but not all protrusions and/or paddles in mature fiber cells . There are several possible reasons why proteins are not consistently localized to all protrusions and/or paddles . First, since the protrusions and paddles are complex 3d structures, confocal imaging of single optical sections (as presented here) may miss staining signals from out - of - plane structures . Second, our fixation conditions may lead to variable epitope accessibility in the case of proteins that are components of dense f - actin cytoskeletal structures . Third, preparation of single fiber cells that dissociates fibers from tightly adhered neighboring cells may lead to small rips and tears along the membrane, leading to a loss in staining at some protrusions and paddles . However, based on staining of single fibers with wheat germ agglutinin (data not shown), plasma membrane continuity does not appear to be compromised by our preparation method . Finally, fourth, it is attractive to speculate that constant membrane remodeling occurs in lens fiber cells during differentiation and maturation, requiring dynamic changes in the actin cytoskeleton . This notion is supported by the presence of a pool of monomeric g - actin in the lens, consistent with ongoing f - actin polymerization and depolymerization in fiber cells . Actin cytoskeleton reorganization is a hallmark of normal lens fiber differentiation, and there is an increase in polymerized f - actin versus monomeric g - actin in newly formed lens fibers . Since our staining experiments capture a single snapshot in time of protein localization, we miss the dynamic changes that may remodel the membrane . In summary, we have developed a novel technique to immunostain single lens fiber cells and have shown that tmod1 stabilizes the spectrin actin network and -actinin crosslinked antiparallel f - actin bundles in the valleys between large paddles to maintain their structure . On the other hand, formation of small protrusions is likely facilitated by arp3-nucleated actin networks, as well as fimbrin - crosslinked parallel f - actin bundles, which do not depend on tmod1 . Thus, this work reveals specific abps required for the formation of large paddles between lens fibers, and suggests that large paddles between mature fiber cells are needed for lens mechanical integrity.
Bow hunter's syndrome (bhs) is caused by the compression of the dominant vertebral artery (va) against a fibrous band or osseous prominence by rotational head movement2,6,10), leading to ischemic insult in the vertebrobasilar territory8,14). Bhs presents as recurrent attacks of paroxysmal vertigo, nystagmus, and ataxia caused by head rotation5,8,10). Due to the rarity of this pathology recently, we encountered 2 cases, which we present herein along with our surgical approach . In these 2 cases, we were able to achieve successful decompression of the va; the two patients recovered completely without further clinical symptoms . A 50-year - old woman had been experiencing paroxysmal vertigo attacks for a 10 month period prior to visiting our institute . The patient's vertigo was aggravated when she turned her head to the left side . Scans, we observed no definitive abnormal findings except for hypoplastic left va in the neutral position (fig . 2a), but complete occlusion of right va was found at atlanto - axial level when the patient turned her head to the left side (fig . We decided on a surgical decompression of the left va at the cross transverse foramen of the first cervix as the surgical approach . We placed an adhesive, fibrous bandage between the left va and the transverse foramen . During the surgical procedure, after surgery, the patient's symptoms improved, and her neck three - dimensional angiography ct scans demonstrated complete decompression of the right vertebral artery (fig . 3, 4). A 42-year old woman reported severe vertigo, dizziness, right upper extremity tingling sensations and aggravated syncope when she turned her head to the right . Upon otolaryngological examination, she presented right beating nystagmus when her head was rotated to the right in the sitting or supine position . There were no abnormal findings except for hypoplasia of the posteroinferior cerebellar artery termination of the left va on magnetic resonance angiography . However, dynamic cerebroangiography showed no steno - occlusive lesions of left va in the neutral position (fig . 5a), but an occlusion of the left va was observed at the c1 - 2 level from right head rotation (fig . We selected a surgical approach and found an adhesion between the left va and transverse foramen . A 50-year - old woman had been experiencing paroxysmal vertigo attacks for a 10 month period prior to visiting our institute . The patient's vertigo was aggravated when she turned her head to the left side . Scans, we observed no definitive abnormal findings except for hypoplastic left va in the neutral position (fig . 2a), but complete occlusion of right va was found at atlanto - axial level when the patient turned her head to the left side (fig . We decided on a surgical decompression of the left va at the cross transverse foramen of the first cervix as the surgical approach . We placed an adhesive, fibrous bandage between the left va and the transverse foramen . During the surgical procedure, after surgery, the patient's symptoms improved, and her neck three - dimensional angiography ct scans demonstrated complete decompression of the right vertebral artery (fig . 3, 4). A 42-year old woman reported severe vertigo, dizziness, right upper extremity tingling sensations and aggravated syncope when she turned her head to the right . Upon otolaryngological examination, she presented right beating nystagmus when her head was rotated to the right in the sitting or supine position . There were no abnormal findings except for hypoplasia of the posteroinferior cerebellar artery termination of the left va on magnetic resonance angiography . No significant changes were observed with neck rotational positions . However, dynamic cerebroangiography showed no steno - occlusive lesions of left va in the neutral position (fig . 5a), but an occlusion of the left va was observed at the c1 - 2 level from right head rotation (fig . We selected a surgical approach and found an adhesion between the left va and transverse foramen . Bhs is syndrome characterized by symptoms such as dizziness, vertigo, and blurred vision when the patient rotates his neck . Bhs is defined as symptomatic vertebrobasilar insufficiency by a mechanical occlusion of the vertebral artery during head rotation9). However, because of the collateral blood flow through the contralateral va and the circle of willis, va occlusion does not cause symptoms in most individual cases11). Thus, symptomatic bhs is rare . Because of its rarity, no large - scale established protocols exist for its research, treatment, or diagnosis . The compressive occlusion of the va is occurs due to a variety of reasons, including the fibrous band tethering of the va at the transverse foramina of the c1-c2 junction, as in our cases, and at the point of dural penetration, which is located above the atlantooccipital membrane . A pathognomonic finding of bhs is the improvement of symptoms when the patient is in a neutral position, even after he claims to have dizziness or blackout when he turns his head to one side . When bhs is suspicious, considerable authors used digital subtraction angiography as the diagnostic modality1). In our cases, clinical symptoms were aggravated when patients turned their head to one side; symptoms were improved when the patients' heads returned to a neutral position . Furthermore, we conducted dynamic angiography to confirm occlusive va lesions toward a diagnosis of bhs . Because of the rarity of this pathology, there are no long term follow - up studies; only prospective studies exist for bhs treatment . Thus, there is no guideline to support the decision to proceed medically or operatively . Because of post - operative complications and the impact on quality of life, some authors have suggested medical treatment (anticoagulation, neck collar applied not to rotate the head, etc.12). However, other patients with bhs who were treated conservatively with medication required surgery because of repeated, aggravated symptoms; after surgery, these patients showed clinical improvement3). Patients who had vascular decompression for bhs at the atlanto - axial level had shown signs of improvement in several case reports1,4,7,13). As mentioned above, there is no definitive guideline for the diagnosis and treatment of bhs because of its rarity . The surgical decompression of mechanical va compressions constitutes an appropriate treatment with a good prognosis.
Malignant hematologic disorders (multiple myeloma, myelodysplasia, chronic lymphocytic leukemia, and hodgkin's and non - hodgkin's lymphoma) and idiopathic aplastic anemia that occur in the population with an incidence of 0.5 - 5/100,000 increase the surgical operative risk due to coagulation defects, changes of blood viscosity, immunosuppression, and bone marrow insufficiency (1). Pancytopenia associated with idiopathic aplastic anemia may pose an increased risk for postoperative bleeding and infection (2). Here, we report a patient with severe aortic valve insufficiency undergoing bioprosthetic aortic valve replacement who suffered from idiopathic aplastic anemia . A 66-yr - old man was admitted to the hospital because of shortness of breath . His dyspnea was classified by the new york heart association (nyha) as functional class iii . The patient's history included treatment for severe aplastic anemia with anti - thymocyte globulin and prednisone for 5 yr prior to admission . Echocardiography revealed reduced left ventricular function and severe aortic valve regurgitation (grade iv) with left ventricular end diastolic dimension measuring 87 mm . The presence of severe dyspnea and echocardiographically documented severe aortic valve insufficiency led us to schedule a surgery for elective aortic valve replacement . On admission, laboratory testing showed leukocytopenia (white blood cells 1,900/l), anemia (hemoglobin 6.7 mm / l), and thrombocytopenia (platelets 34,000/l). The differential blood count revealed 42.9% neutrophils (50 - 70%), 42.3% lymphocytes (25 - 40%), 6.9% monocytes (2 - 8%), and 4.3% eosinophils (2 - 4%). Preoperative preparation included two weeks of granulocyte colony - stimulating factor (neupogen 300 g, amgen, subcutaneously three times a week), transfusion of four units of packed red blood cells (rbcs), and ten units of platelet concentrations (pcs) one day before the surgical procedure resulting in a hemoglobin level of 9.8 mm / l, a leukocyte level of 10,300/l, and a platelet level of 120,000/l preoperatively . The patient received aortic valve replacement with a 21-mm bioprosthetic aortic valve (carpentier - edwards, edwards lifesciences, irvine, ca, u.s.a .) By a standard median sternotomy; 2 milrione units of aprotinin were added to the cardiopulmonary bypass circuit priming solution . On the second day after surgery, atrial fibrillation deveolped and was successfully converted with intravenous amiodarone . Antibiotic prophylaxis was given using ceftriaxon (rocephin, roche, basel, swiss) 2 g intravenously over 6 days . The patient was discharged from the intensive care unit 4 days after surgery and was discharged from the hospital postoperative on day 11 with a leukocyte count 4,300/l, hemoglobin of 6.2 mm / l, and a platelet count of 62,000/l . After the surgery, the patient received 5 units of rbcs and 20 units of pcs until discharge . Perioperative laboratory findings are summarized in table 1 . During the 6 months of follow - up, the patient did well with a functional class of nyha class i. the chest radiography at 3 months post surgery showed a decreased cardiac size . According to the definition of the severity of aplastic anemia (5), the patient's aplastic anemia could be categorized as non - severe aplastic anemia . Regardless of the severity of aplastic anemia, (1), two questions have to be considered when cardiac surgery is indicated in patients with a hematologic disorder causing pancytopenia . Given the increased operative risk and the potential benefit with regard to the life expectancy and quality of life, aggressive surgical treatment has to be compared with conservative treatment and the natural course of both, the hematologic disorder and the cardiac disease . Our patient suffered from severe dyspnea on exertion; the patient had stable idiopathic aplastic anemia and was under hematological supervision . Progressive deterioration of ventricular function was documented on serial echocardiographic examinations . Both symptomatic aortic regurgitation and impaired left ventricular function led us to the decision to perform aortic valve replacement for this patient . Another important issue for this specific subset of patients is perioperative management to decrease morbidity and mortality (1). Potential complications are caused by a decreased number or impaired function of blood cells; this condition is aggravated by the cell damaging properties of extracorporeal circulation . In addition to meticulous surgical hemostasis, substitution of various blood products is important to reduce the risk of bleeding complications . Total amount of the blood product transfused to this patient is regarded to be acceptable considering the higher risk of bleeding in these specific subset of patients . Aprotinin has been shown to decrease blood loss and reduce the use of blood products by at least 30%, in open heart surgery (4). Thoracic drainage in our patient was 785 ml, which was similar to that of other patients who required cardiac surgery with a variety of malignant hematological disorders (1, 3). Another important aspect of perioperative care in these patients is the increased risk for infections . It is of great importance to perform surgery in aseptic conditions and to avoid transmission of pathogenic microorganisms during the perioperative period . The patient received warfarin sodium 2 days after surgery, and inr was maintained between 1.5 to 2.0 . Follow up, we have noticed neither thrombotic nor hemorrhagic complications related to the anticoagulation therapy . (3), cardiac surgery with extracorporeal circulation is feasible in patients with idopathic aplastic anemia, and it was not associated with excessive complications that might be expected in a patient with this disorder.

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